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Total: displaying 10 out of 60 results
  • Nuclear hubs built on RNAs and clustered organization of the genome.

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    Nuclear hubs built on RNAs and clustered organization of the genome.

    Curr Opin Cell Biol. 2020 Apr 04;64:67-76

    Authors: Smith KP, Hall LL, Lawrence JB

    Abstract
    RNAs play diverse roles in formation and function of subnuclear compartments, most of which are associated with active genes. NEAT1 and NEAT2/MALAT1 exemplify long non-coding RNAs (lncRNAs) known to function in nuclear bodies; however, we suggest that RNA biogenesis itself may underpin much nuclear compartmentalization. Recent studies show that active genes cluster with nuclear speckles on a genome-wide scale, significantly advancing earlier cytological evidence that speckles (aka SC-35 domains) are hubs of concentrated pre-mRNA metabolism. We propose the 'karyotype to hub' hypothesis to explain this organization: clustering of genes in the human karyotype may have evolved to facilitate the formation of efficient nuclear hubs, driven in part by the propensity of ribonucleoproteins (RNPs) to form large-scale condensates. The special capacity of highly repetitive RNAs to impact architecture is highlighted by recent findings that human satellite II RNA sequesters factors into abnormal nuclear bodies in disease, potentially co-opting a normal developmental mechanism.

    PMID: 32259767 [PubMed - as supplied by publisher]

  • Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation.

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    Silencing Trisomy 21 with XIST in Neural Stem Cells Promotes Neuronal Differentiation.

    Dev Cell. 2020 Feb 10;52(3):294-308.e3

    Authors: Czermiński JT, Lawrence JB

    Abstract
    The ability of XIST to dosage compensate a trisomic autosome presents unique experimental opportunities and potentially transformative therapeutic prospects. However, it is currently thought that XIST requires the natural context surrounding pluripotency to initiate chromosome silencing. Here, we demonstrate that XIST RNA induced in differentiated neural cells can trigger chromosome-wide silencing of chromosome 21 in Down syndrome patient-derived cells. Use of this tightly controlled system revealed a deficiency in differentiation of trisomic neural stem cells to neurons, correctible by inducing XIST at different stages of neurogenesis. Single-cell transcriptomics and other analyses strongly implicate elevated Notch signaling due to trisomy 21, thereby promoting neural stem cell cycling that delays terminal differentiation. These findings have significance for illuminating the epigenetic plasticity of cells during development, the understanding of how human trisomy 21 effects Down syndrome neurobiology, and the translational potential of XIST, a unique non-coding RNA.

    PMID: 31978324 [PubMed - in process]

  • Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic ...

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    Trisomy silencing by XIST normalizes Down syndrome cell pathogenesis demonstrated for hematopoietic defects in vitro.

    Nat Commun. 2018 12 05;9(1):5180

    Authors: Chiang JC, Jiang J, Newburger PE, Lawrence JB

    Abstract
    We previously demonstrated that an integrated XIST transgene can broadly repress one chromosome 21 in Down syndrome (DS) pluripotent cells. Here we address whether trisomy-silencing can normalize cell function and development sufficiently to correct cell pathogenesis, tested in an in vitro model of human fetal hematopoiesis, for which DS cellular phenotypes are best known. XIST induction in four transgenic clones reproducibly corrected over-production of megakaryocytes and erythrocytes, key to DS myeloproliferative disorder and leukemia. A contrasting increase in neural stem and iPS cells shows cell-type specificity, supporting this approach successfully rebalances the hematopoietic developmental program. Given this, we next used this system to extend knowledge of hematopoietic pathogenesis on multiple points. Results demonstrate trisomy 21 expression promotes over-production of CD43+ but not earlier CD34+/CD43-progenitors and indicates this is associated with increased IGF signaling. This study demonstrates proof-of-principle for this epigenetic-based strategy to investigate, and potentially mitigate, DS developmental pathologies.

    PMID: 30518921 [PubMed - indexed for MEDLINE]

  • Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs.

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    Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs.

    Cell Rep. 2017 12 26;21(13):3691-3699

    Authors: Wang F, McCannell KN, Bošković A, Zhu X, Shin J, Yu J, Gallant J, Byron M, Lawrence JB, Zhu LJ, Jones SN, Rando OJ, Fazzio TG, Bach I

    Abstract
    During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.

    PMID: 29281819 [PubMed - indexed for MEDLINE]

  • XIST RNA: a window into the broader role of RNA in nuclear chromosome architecture.

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    XIST RNA: a window into the broader role of RNA in nuclear chromosome architecture.

    Philos Trans R Soc Lond B Biol Sci. 2017 Nov 05;372(1733):

    Authors: Creamer KM, Lawrence JB

    Abstract
    XIST RNA triggers the transformation of an active X chromosome into a condensed, inactive Barr body and therefore provides a unique window into transitions of higher-order chromosome architecture. Despite recent progress, how XIST RNA localizes and interacts with the X chromosome remains poorly understood. Genetic engineering of XIST into a trisomic autosome demonstrates remarkable capacity of XIST RNA to localize and comprehensively silence that autosome. Thus, XIST does not require X chromosome-specific sequences but operates on mechanisms available genome-wide. Prior results suggested XIST localization is controlled by attachment to the insoluble nuclear scaffold. Our recent work affirms that scaffold attachment factor A (SAF-A) is involved in anchoring XIST, but argues against the view that SAF-A provides a unimolecular bridge between RNA and the chromosome. Rather, we suggest that a complex meshwork of architectural proteins interact with XIST RNA. Parallel work studying the territory of actively transcribed chromosomes suggests that repeat-rich RNA 'coats' euchromatin and may impact chromosome architecture in a manner opposite of XIST A model is discussed whereby RNA may not just recruit histone modifications, but more directly impact higher-order chromatin condensation via interaction with architectural proteins of the nucleus.This article is part of the themed issue 'X-chromosome inactivation: a tribute to Mary Lyon'.

    PMID: 28947659 [PubMed - indexed for MEDLINE]

  • Demethylated HSATII DNA and HSATII RNA Foci Sequester PRC1 and MeCP2 into Cancer-Specific Nuclear Bo...

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    Demethylated HSATII DNA and HSATII RNA Foci Sequester PRC1 and MeCP2 into Cancer-Specific Nuclear Bodies.

    Cell Rep. 2017 03 21;18(12):2943-2956

    Authors: Hall LL, Byron M, Carone DM, Whitfield TW, Pouliot GP, Fischer A, Jones P, Lawrence JB

    Abstract
    This study reveals that high-copy satellite II (HSATII) sequences in the human genome can bind and impact distribution of chromatin regulatory proteins and that this goes awry in cancer. In many cancers, master regulatory proteins form two types of cancer-specific nuclear bodies, caused by locus-specific deregulation of HSATII. DNA demethylation at the 1q12 mega-satellite, common in cancer, causes PRC1 aggregation into prominent Cancer-Associated Polycomb (CAP) bodies. These loci remain silent, whereas HSATII loci with reduced PRC1 become derepressed, reflecting imbalanced distribution of UbH2A on these and other PcG-regulated loci. Large nuclear foci of HSATII RNA form and sequester copious MeCP2 into Cancer-Associated Satellite Transcript (CAST) bodies. Hence, HSATII DNA and RNA have an exceptional capacity to act as molecular sponges and sequester chromatin regulatory proteins into abnormal nuclear bodies in cancer. The compartmentalization of regulatory proteins within nuclear structure, triggered by demethylation of "junk" repeats, raises the possibility that this contributes to further compromise of the epigenome and neoplastic progression.

    PMID: 28329686 [PubMed - indexed for MEDLINE]

  • SAF-A Requirement in Anchoring XIST RNA to Chromatin Varies in Transformed and Primary Cells.

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    SAF-A Requirement in Anchoring XIST RNA to Chromatin Varies in Transformed and Primary Cells.

    Dev Cell. 2016 10 10;39(1):9-10

    Authors: Kolpa HJ, Fackelmayer FO, Lawrence JB

    PMID: 27728783 [PubMed - indexed for MEDLINE]

  • Regulation of X-linked gene expression during early mouse development by Rlim.

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    Regulation of X-linked gene expression during early mouse development by Rlim.

    Elife. 2016 09 19;5:

    Authors: Wang F, Shin J, Shea JM, Yu J, Bošković A, Byron M, Zhu X, Shalek AK, Regev A, Lawrence JB, Torres EM, Zhu LJ, Rando OJ, Bach I

    Abstract
    Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos.

    PMID: 27642011 [PubMed - indexed for MEDLINE]

  • RNA as a fundamental component of interphase chromosomes: could repeats prove key?

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    RNA as a fundamental component of interphase chromosomes: could repeats prove key?

    Curr Opin Genet Dev. 2016 04;37:137-147

    Authors: Hall LL, Lawrence JB

    Abstract
    Beginning with the precedent of XIST RNA as a 'chromosomal RNA' (cRNA), there is growing interest in the possibility that a diversity of non-coding RNAs may function in chromatin. We review findings which lead us to suggest that RNA is essentially a widespread component of interphase chromosomes. Further, RNA likely contributes to architecture and regulation, with repeat-rich 'junk' RNA in euchromatin (ecRNA) promoting a more open chromatin state. Thousands of low-abundance nuclear RNAs have been reported, however it remains a challenge to determine which of these may function in chromatin. Recent findings indicate that repetitive sequences are enriched in chromosome-associated non-coding RNAs, and repeat-rich RNA shows unusual properties, including localization and stability, with similarities to XIST RNA. We suggest two frontiers in genome biology are emerging and may intersect: the broad contribution of RNA to interphase chromosomes and the distinctive properties of repeat-rich intronic or intergenic junk sequences that may play a role in chromosome structure and regulation.

    PMID: 27218204 [PubMed - indexed for MEDLINE]

  • Unfolding the story of chromatin organization in senescent cells.

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    Unfolding the story of chromatin organization in senescent cells.

    Nucleus. 2015;6(4):254-60

    Authors: Swanson EC, Rapkin LM, Bazett-Jones DP, Lawrence JB

    Abstract
    Cell senescence, the permanent withdrawal of a cell from the cell cycle, is characterized by dramatic, cytological scale changes to DNA condensation throughout the genome. While prior emphasis has been placed on increases in heterochromatin, such as the formation of compact Senescent Associated Heterochromatin Foci (SAHF) structures, our recent findings showed that SAHF formation is preceded by the unravelling of constitutive heterochromatin into visibly extended structures, which we have termed Senescent Associated Distension of Satellites or SADS. Interestingly, neither of these marked changes in DNA condensation appear to be mediated by changes in canonical, heterochromatin-associated histone modifications. Rather, several observations suggest that these events may be facilitated by changes in LaminB1 levels and/or other factors that control higher-order chromatin architecture. Here, we review what is known about senescence-associated chromatin reorganization and present preliminary results using high-resolution microscopy techniques to show that each peri/centromeric satellite in senescent cells is comprised of several condensed domains connected by thin fibrils of satellite DNA. We then discuss the potential importance of these striking changes in chromatin condensation for cell senescence, and also as a model to provide a needed window into the higher-order packaging of the genome.

    PMID: 26107557 [PubMed - indexed for MEDLINE]

  • Spatial re-organization of myogenic regulatory sequences temporally controls gene expression.

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    Spatial re-organization of myogenic regulatory sequences temporally controls gene expression.

    Nucleic Acids Res. 2015 Feb 27;43(4):2008-21

    Authors: Harada A, Mallappa C, Okada S, Butler JT, Baker SP, Lawrence JB, Ohkawa Y, Imbalzano AN

    Abstract
    During skeletal muscle differentiation, the activation of some tissue-specific genes occurs immediately while others are delayed. The molecular basis controlling temporal gene regulation is poorly understood. We show that the regulatory sequences, but not other regions of genes expressed at late times of myogenesis, are in close physical proximity in differentiating embryonic tissue and in differentiating culture cells, despite these genes being located on different chromosomes. Formation of these inter-chromosomal interactions requires the lineage-determinant MyoD and functional Brg1, the ATPase subunit of SWI/SNF chromatin remodeling enzymes. Ectopic expression of myogenin and a specific Mef2 isoform induced myogenic differentiation without activating endogenous MyoD expression. Under these conditions, the regulatory sequences of late gene loci were not in close proximity, and these genes were prematurely activated. The data indicate that the spatial organization of late genes contributes to temporal regulation of myogenic transcription by restricting late gene expression during the early stages of myogenesis.

    PMID: 25653159 [PubMed - indexed for MEDLINE]

  • RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast.

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    RLIM is dispensable for X-chromosome inactivation in the mouse embryonic epiblast.

    Nature. 2014 Jul 03;511(7507):86-9

    Authors: Shin J, Wallingford MC, Gallant J, Marcho C, Jiao B, Byron M, Bossenz M, Lawrence JB, Jones SN, Mager J, Bach I

    Abstract
    In female mice, two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X chromosome. Later, around implantation, epiblast cells of the inner cell mass that give rise to the embryo reactivate the paternal X chromosome and undergo a random form of XCI (rXCI). Xist, a long non-coding RNA crucial for both forms of XCI, is activated by the ubiquitin ligase RLIM (also known as Rnf12). Although RLIM is required for triggering iXCI in mice, its importance for rXCI has been controversial. Here we show that RLIM levels are downregulated in embryonic cells undergoing rXCI. Using mouse genetics we demonstrate that female cells lacking RLIM from pre-implantation stages onwards show hallmarks of XCI, including Xist clouds and H3K27me3 foci, and have full embryogenic potential. These results provide evidence that RLIM is dispensable for rXCI, indicating that in mice an RLIM-independent mechanism activates Xist in the embryo proper.

    PMID: 24870238 [PubMed - indexed for MEDLINE]

  • Stable C0T-1 repeat RNA is abundant and is associated with euchromatic interphase chromosomes.

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    Stable C0T-1 repeat RNA is abundant and is associated with euchromatic interphase chromosomes.

    Cell. 2014 Feb 27;156(5):907-19

    Authors: Hall LL, Carone DM, Gomez AV, Kolpa HJ, Byron M, Mehta N, Fackelmayer FO, Lawrence JB

    Abstract
    Recent studies recognize a vast diversity of noncoding RNAs with largely unknown functions, but few have examined interspersed repeat sequences, which constitute almost half our genome. RNA hybridization in situ using C0T-1 (highly repeated) DNA probes detects surprisingly abundant euchromatin-associated RNA comprised predominantly of repeat sequences (C0T-1 RNA), including LINE-1. C0T-1-hybridizing RNA strictly localizes to the interphase chromosome territory in cis and remains stably associated with the chromosome territory following prolonged transcriptional inhibition. The C0T-1 RNA territory resists mechanical disruption and fractionates with the nonchromatin scaffold but can be experimentally released. Loss of repeat-rich, stable nuclear RNAs from euchromatin corresponds to aberrant chromatin distribution and condensation. C0T-1 RNA has several properties similar to XIST chromosomal RNA but is excluded from chromatin condensed by XIST. These findings impact two "black boxes" of genome science: the poorly understood diversity of noncoding RNA and the unexplained abundance of repetitive elements.

    PMID: 24581492 [PubMed - indexed for MEDLINE]

  • A long noncoding RNA mediates both activation and repression of immune response genes.

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    A long noncoding RNA mediates both activation and repression of immune response genes.

    Science. 2013 Aug 16;341(6147):789-92

    Authors: Carpenter S, Aiello D, Atianand MK, Ricci EP, Gandhi P, Hall LL, Byron M, Monks B, Henry-Bezy M, Lawrence JB, O'Neill LA, Moore MJ, Caffrey DR, Fitzgerald KA

    Abstract
    An inducible program of inflammatory gene expression is central to antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.

    PMID: 23907535 [PubMed - indexed for MEDLINE]

  • Translating dosage compensation to trisomy 21.

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    Translating dosage compensation to trisomy 21.

    Nature. 2013 Aug 15;500(7462):296-300

    Authors: Jiang J, Jing Y, Cost GJ, Chiang JC, Kolpa HJ, Cotton AM, Carone DM, Carone BR, Shivak DA, Guschin DY, Pearl JR, Rebar EJ, Byron M, Gregory PD, Brown CJ, Urnov FD, Hall LL, Lawrence JB

    Abstract
    Down's syndrome is a common disorder with enormous medical and social costs, caused by trisomy for chromosome 21. We tested the concept that gene imbalance across an extra chromosome can be de facto corrected by manipulating a single gene, XIST (the X-inactivation gene). Using genome editing with zinc finger nucleases, we inserted a large, inducible XIST transgene into the DYRK1A locus on chromosome 21, in Down's syndrome pluripotent stem cells. The XIST non-coding RNA coats chromosome 21 and triggers stable heterochromatin modifications, chromosome-wide transcriptional silencing and DNA methylation to form a 'chromosome 21 Barr body'. This provides a model to study human chromosome inactivation and creates a system to investigate genomic expression changes and cellular pathologies of trisomy 21, free from genetic and epigenetic noise. Notably, deficits in proliferation and neural rosette formation are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro also surmounts the major first step towards potential development of 'chromosome therapy'.

    PMID: 23863942 [PubMed - indexed for MEDLINE]

  • A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures...

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    A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures.

    Curr Protoc Hum Genet. 2013 Jan;Chapter 4:Unit 4.15

    Authors: Byron M, Hall LL, Lawrence JB

    Abstract
    Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results.

    PMID: 23315927 [PubMed - indexed for MEDLINE]

  • Heterochromatin instability in cancer: from the Barr body to satellites and the nuclear periphery.

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    Heterochromatin instability in cancer: from the Barr body to satellites and the nuclear periphery.

    Semin Cancer Biol. 2013 Apr;23(2):99-108

    Authors: Carone DM, Lawrence JB

    Abstract
    In recent years it has been recognized that the development of cancer involves a series of not only genetic but epigenetic changes across the genome. At the same time, connections between epigenetic regulation, chromatin packaging, and overall nuclear architecture are increasingly appreciated. The cell-type specific organization of heterochromatin, established upon cell differentiation, is responsible for maintaining much of the genome in a repressed state, within a highly compartmentalized nucleus. This review focuses on recent evidence that in cancer the normal packaging and higher organization of heterochromatin is often compromised. Gross changes in nuclear morphology have long been a criterion for pathologic diagnosis of many cancers, but the specific nuclear components impacted, the mechanisms involved, and the implications for cancer progression have barely begun to emerge. We discuss recent findings regarding distinct heterochromatin types, including the inactive X chromosome, constitutive heterochromatin of peri/centric satellites, and the peripheral heterochromatic compartment (PHC). A theme developed here is that the higher-order organization of satellites and the peripheral heterochromatic compartment may be tightly linked, and that compromise of this organization may promote broad epigenomic imbalance in cancer. Recent studies into the potential role(s) of the breast cancer tumor suppressor, BRCA1, in maintaining heterochromatin will be highlighted. Many questions remain about this new area of cancer epigenetics, which is likely more important in cancer development and progression than widely appreciated. We propose that broad, stochastic compromise in heterochromatin maintenance would create a diversity of expression profiles, and thus a rich opportunity for one or more cells to emerge with a selective growth advantage and potential for neoplasia.

    PMID: 22722067 [PubMed - indexed for MEDLINE]

  • Paternal RLIM/Rnf12 is a survival factor for milk-producing alveolar cells.

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    Paternal RLIM/Rnf12 is a survival factor for milk-producing alveolar cells.

    Cell. 2012 Apr 27;149(3):630-41

    Authors: Jiao B, Ma H, Shokhirev MN, Drung A, Yang Q, Shin J, Lu S, Byron M, Kalantry S, Mercurio AM, Lawrence JB, Hoffmann A, Bach I

    Abstract
    In female mouse embryos, somatic cells undergo a random form of X chromosome inactivation (XCI), whereas extraembryonic trophoblast cells in the placenta undergo imprinted XCI, silencing exclusively the paternal X chromosome. Initiation of imprinted XCI requires a functional maternal allele of the X-linked gene Rnf12, which encodes the ubiquitin ligase Rnf12/RLIM. We find that knockout (KO) of Rnf12 in female mammary glands inhibits alveolar differentiation and milk production upon pregnancy, with alveolar cells that lack RLIM undergoing apoptosis as they begin to differentiate. Genetic analyses demonstrate that these functions are mediated primarily by the paternal Rnf12 allele due to nonrandom maternal XCI in mammary epithelial cells. These results identify paternal Rnf12/RLIM as a critical survival factor for milk-producing alveolar cells and, together with population models, reveal implications of transgenerational epigenetic inheritance.

    PMID: 22541433 [PubMed - indexed for MEDLINE]

  • XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome.

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    XIST RNA and architecture of the inactive X chromosome: implications for the repeat genome.

    Cold Spring Harb Symp Quant Biol. 2010;75:345-56

    Authors: Hall LL, Lawrence JB

    Abstract
    XIST RNA paints and induces silencing of one X chromosome in mammalian female cells, providing a powerful model to investigate long-range chromosomal regulation. This chapter focuses on events downstream from the spread of XIST RNA across the interphase chromosome, to consider how this large noncoding RNA interacts with and silences a whole chromosome. Several lines of evidence are summarized that point to the involvement of repeat sequences in different aspects of the X-inactivation process. Although the "repeat genome" comprises close to half of the human genome, the potential for abundant repeats to contribute to genome regulation has been largely overlooked and may be underestimated. X inactivation has the potential to reveal roles of interspersed and other repeats in the genome. For example, evidence indicates that XIST RNA acts at the architectural level of the whole chromosome to induce formation of a silent core enriched for nongenic and repetitive (Cot-1) DNA, which corresponds to the DAPI-dense Barr body. Expression of repeat RNAs may contribute to chromosome remodeling, and evidence suggests that other types of repeat elements may be involved in escape from X inactivation. Despite great progress in decoding the rest of the genome, we suggest that the repeat genome may contain meaningful but complex language that remains to be better studied and understood.

    PMID: 21447818 [PubMed - indexed for MEDLINE]

  • Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice.

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    Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice.

    Nature. 2010 Oct 21;467(7318):977-81

    Authors: Shin J, Bossenz M, Chung Y, Ma H, Byron M, Taniguchi-Ishigaki N, Zhu X, Jiao B, Hall LL, Green MR, Jones SN, Hermans-Borgmeyer I, Lawrence JB, Bach I

    Abstract
    Two forms of X-chromosome inactivation (XCI) ensure the selective silencing of female sex chromosomes during mouse embryogenesis. Imprinted XCI begins with the detection of Xist RNA expression on the paternal X chromosome (Xp) at about the four-cell stage of embryonic development. In the embryonic tissues of the inner cell mass, a random form of XCI occurs in blastocysts that inactivates either Xp or the maternal X chromosome (Xm). Both forms of XCI require the non-coding Xist RNA that coats the inactive X chromosome from which it is expressed. Xist has crucial functions in the silencing of X-linked genes, including Rnf12 (refs 3, 4) encoding the ubiquitin ligase RLIM (RING finger LIM-domain-interacting protein). Here we show, by targeting a conditional knockout of Rnf12 to oocytes where RLIM accumulates to high levels, that the maternal transmission of the mutant X chromosome (Δm) leads to lethality in female embryos as a result of defective imprinted XCI. We provide evidence that in Δm female embryos the initial formation of Xist clouds and Xp silencing are inhibited. In contrast, embryonic stem cells lacking RLIM are able to form Xist clouds and silence at least some X-linked genes during random XCI. These results assign crucial functions to the maternal deposit of Rnf12/RLIM for the initiation of imprinted XCI.

    PMID: 20962847 [PubMed - indexed for MEDLINE]

  • AURKB-mediated effects on chromatin regulate binding versus release of XIST RNA to the inactive chro...

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    AURKB-mediated effects on chromatin regulate binding versus release of XIST RNA to the inactive chromosome.

    J Cell Biol. 2009 Aug 24;186(4):491-507

    Authors: Hall LL, Byron M, Pageau G, Lawrence JB

    Abstract
    How XIST RNA strictly localizes across the inactive X chromosome is unknown; however, prophase release of human XIST RNA provides a clue. Tests of inhibitors that mimic mitotic chromatin modifications implicated an indirect role of PP1 (protein phosphatase 1), potentially via its interphase repression of Aurora B kinase (AURKB), which phosphorylates H3 and chromosomal proteins at prophase. RNA interference to AURKB causes mitotic retention of XIST RNA, unlike other mitotic or broad kinase inhibitors. Thus, AURKB plays an unexpected role in regulating RNA binding to heterochromatin, independent of mechanics of mitosis. H3 phosphorylation (H3ph) was shown to precede XIST RNA release, whereas results exclude H1ph involvement. Of numerous Xi chromatin (chromosomal protein) hallmarks, ubiquitination closely follows XIST RNA retention or release. Surprisingly, H3S10ph staining (but not H3S28ph) is excluded from Xi and is potentially linked to ubiquitination. Results suggest a model of multiple distinct anchor points for XIST RNA. This study advances understanding of RNA chromosome binding and the roles of AURKB and demonstrates a novel approach to manipulate and study XIST RNA.

    PMID: 19704020 [PubMed - indexed for MEDLINE]

  • Changing nuclear landscape and unique PML structures during early epigenetic transitions of human em...

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    Changing nuclear landscape and unique PML structures during early epigenetic transitions of human embryonic stem cells.

    J Cell Biochem. 2009 Jul 01;107(4):609-21

    Authors: Butler JT, Hall LL, Smith KP, Lawrence JB

    Abstract
    The complex nuclear structure of somatic cells is important to epigenomic regulation, yet little is known about nuclear organization of human embryonic stem cells (hESC). Here we surveyed several nuclear structures in pluripotent and transitioning hESC. Observations of centromeres, telomeres, SC35 speckles, Cajal Bodies, lamin A/C and emerin, nuclear shape and size demonstrate a very different "nuclear landscape" in hESC. This landscape is remodeled during a brief transitional window, concomitant with or just prior to differentiation onset. Notably, hESC initially contain abundant signal for spliceosome assembly factor, SC35, but lack discrete SC35 domains; these form as cells begin to specialize, likely reflecting cell-type specific genomic organization. Concomitantly, nuclear size increases and shape changes as lamin A/C and emerin incorporate into the lamina. During this brief window, hESC exhibit dramatically different PML-defined structures, which in somatic cells are linked to gene regulation and cancer. Unlike the numerous, spherical somatic PML bodies, hES cells often display approximately 1-3 large PML structures of two morphological types: long linear "rods" or elaborate "rosettes", which lack substantial SUMO-1, Daxx, and Sp100. These occur primarily between Day 0-2 of differentiation and become rare thereafter. PML rods may be "taut" between other structures, such as centromeres, but clearly show some relationship with the lamina, where PML often abuts or fills a "gap" in early lamin A/C staining. Findings demonstrate that pluripotent hES cells have a markedly different overall nuclear architecture, remodeling of which is linked to early epigenomic programming and involves formation of unique PML-defined structures.

    PMID: 19449340 [PubMed - indexed for MEDLINE]

  • Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells.

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    Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells.

    J Cell Biol. 2008 Jun 30;181(7):1055-63

    Authors: Mudhasani R, Zhu Z, Hutvagner G, Eischen CM, Lyle S, Hall LL, Lawrence JB, Imbalzano AN, Jones SN

    Abstract
    Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.

    PMID: 18591425 [PubMed - indexed for MEDLINE]

  • Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry...

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    Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains.

    J Cell Biol. 2007 Sep 10;178(6):951-64

    Authors: Smith KP, Byron M, Johnson C, Xing Y, Lawrence JB

    Abstract
    In myotonic dystrophy type 1 (DM1), triplet repeat expansion in the 3' untranslated region of dystrophia myotonica protein kinase (DMPK) causes the nuclear retention of mutant messenger RNA (mRNA). Although the DMPK gene locus positions precisely at the outer edge of a factor-rich SC-35 domain, the normal mRNA consistently accumulates within the domain, and this RNA is depleted upon transcriptional inhibition. In DM1, mutant transcripts detach from the gene but accumulate in granules that abut but do not enter SC-35 domains, suggesting that RNA entry into the domain is blocked. Despite their exclusion from these compartments, mutant transcripts are spliced. MBNL1 (muscleblind-like protein 1) is an alternative splicing factor that becomes highly concentrated with mutant RNA foci. Small interfering RNA-mediated knockdown of MBNL1 promotes the accumulation or entry of newly synthesized mutant transcripts in the SC-35 domain. Collectively, these data suggest that an initial step in the intranuclear path of some mRNAs is passage from the gene into an SC-35 domain and implicate these structures in postsplicing steps before export.

    PMID: 17846170 [PubMed - indexed for MEDLINE]

  • The disappearing Barr body in breast and ovarian cancers.

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    The disappearing Barr body in breast and ovarian cancers.

    Nat Rev Cancer. 2007 08;7(8):628-33

    Authors: Pageau GJ, Hall LL, Ganesan S, Livingston DM, Lawrence JB

    Abstract
    Interest has recently reawakened in whether loss of the heterochromatic X chromosome (Barr body) is prevalent in certain breast and ovarian cancers, and new insights into the mechanisms involved have emerged. Mitotic segregation errors commonly explain the loss of the inactive X chromosome (Xi), but compromise of Xi heterochromatin in some cancers may signal broader deficits of nuclear heterochromatin. The debated link between BRCA1 and Xi might reflect a general relationship between BRCA1 and heterochromatin, which could connect BRCA1 to both epigenetic and genetic instability. We suggest that heterochromatic instability is a common but largely unexplored mechanism, leading to widespread genomic misregulation and the evolution of some cancers.

    PMID: 17611545 [PubMed - indexed for MEDLINE]

  • BRCA1 does not paint the inactive X to localize XIST RNA but may contribute to broad changes in canc...

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    BRCA1 does not paint the inactive X to localize XIST RNA but may contribute to broad changes in cancer that impact XIST and Xi heterochromatin.

    J Cell Biochem. 2007 Mar 01;100(4):835-50

    Authors: Pageau GJ, Hall LL, Lawrence JB

    Abstract
    The BRCA1 tumor suppressor involved in breast and ovarian cancer is linked to several fundamental cell regulatory processes. Recently, it was reported that BRCA1 supports localization of XIST RNA to the inactive X chromosome (Xi) in women. The apparent cytological overlap between BRCA1 and XIST RNA across the Xi raised the possibility a direct role of BRCA1 in localizing XIST. We report here that BRCA1 does not paint the Xi or XIST territory, as do markers of Xi facultative heterochromatin. A smaller BRCA1 accumulation abuts Xi, although this is not exclusive to Xi. In BRCA1 depleted normal and tumor cells, or BRCA1 reconstituted cells, BRCA1 status does not closely correlate with XIST localization, however in a BRCA1 inducible system over-expression correlated strongly with enhanced XIST expression. We confirm frequent loss of an Xi in tumor cells. In addition to mitotic loss of Xi, we find XIST RNA expression or localization frequently become compromised in cultured breast cancer cells, suggesting Xi heterochromatin may not be fully maintained. We demonstrate that complex epigenetic differences between tumor cell subpopulations can have striking effects on XIST transcription, accumulation, and localization, but this does not strictly correlate with BRCA1. Although BRCA1 can have indirect effects that impact XIST, our results do not indicate a direct and specific role in XIST RNA regulation. Rather, regulatory factors such as BRCA1 that have broad effects on chromatin or gene regulation can impact XIST RNA and the Xi. We provide preliminary evidence that this may occur as part of a wider failure of heterochromatin maintenance in some cancers.

    PMID: 17146760 [PubMed - indexed for MEDLINE]

  • BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericent...

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    BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatin.

    J Cell Biol. 2006 Dec 04;175(5):693-701

    Authors: Pageau GJ, Lawrence JB

    Abstract
    Breast cancer-associated protein 1 (BRCA1) forms foci at sites of induced DNA damage, but any significance of these normal S-phase foci is unknown. BRCA1 distribution does not simply mirror or overlap that of replicating DNA; however, BRCA1 foci frequently abut sites of BrdU incorporation, mostly at mid-to-late S phase. Although BRCA1 does not overlap XIST RNA across the inactive X chromosome, BRCA1 foci position overwhelmingly in heterochromatic regions, particularly the nucleolar periphery where many centromeres reside. In humans and mice, including early embryonic cells, BRCA1 commonly associates with interphase centromere-kinetochore complexes, including pericentric heterochromatin. Proliferating cell nuclear antigen or BrdU labeling demonstrates that BRCA1 localizes adjacent to, or "paints," major satellite blocks as chromocenters replicate, where topoisomerase is also enriched. BRCA1 loss is often associated with proliferative defects, including postmitotic bridges enriched with satellite DNA. These findings implicate BRCA1 in replication-linked maintenance of centric/pericentric heterochromatin and suggest a novel means whereby BRCA1 loss may contribute to genomic instability and cancer.

    PMID: 17145961 [PubMed - indexed for MEDLINE]

  • Molecular anatomy of a speckle.

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    Molecular anatomy of a speckle.

    Anat Rec A Discov Mol Cell Evol Biol. 2006 Jul;288(7):664-75

    Authors: Hall LL, Smith KP, Byron M, Lawrence JB

    Abstract
    Direct localization of specific genes, RNAs, and proteins has allowed the dissection of individual nuclear speckles in relation to the molecular biology of gene expression. Nuclear speckles (aka SC35 domains) are essentially ubiquitous structures enriched for most pre-mRNA metabolic factors, yet their relationship to gene expression has been poorly understood. Analyses of specific genes and their spliced or mature mRNA strongly support that SC35 domains are hubs of activity, not stores of inert factors detached from gene expression. We propose that SC35 domains are hubs that spatially link expression of specific pre-mRNAs to rapid recycling of copious RNA metabolic complexes, thereby facilitating expression of many highly active genes. In addition to increasing the efficiency of each step, sequential steps in gene expression are structurally integrated at each SC35 domain, consistent with other evidence that the biochemical machineries for transcription, splicing, and mRNA export are coupled. Transcription and splicing are subcompartmentalized at the periphery, with largely spliced mRNA entering the domain prior to export. In addition, new findings presented here begin to illuminate the structural underpinnings of a speckle by defining specific perturbations of phosphorylation that promote disassembly or assembly of an SC35 domain in relation to other components. Results thus far are consistent with the SC35 spliceosome assembly factor as an integral structural component. Conditions that disperse SC35 also disperse poly(A) RNA, whereas the splicing factor ASF/SF2 can be dispersed under conditions in which SC35 or SRm300 remain as intact components of a core domain.

    PMID: 16761280 [PubMed - indexed for MEDLINE]

  • Word frequency analysis reveals enrichment of dinucleotide repeats on the human X chromosome and [GA...

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    Word frequency analysis reveals enrichment of dinucleotide repeats on the human X chromosome and [GATA]n in the X escape region.

    Genome Res. 2006 Apr;16(4):477-84

    Authors: McNeil JA, Smith KP, Hall LL, Lawrence JB

    Abstract
    Most of the human genome encodes neither protein nor known functional RNA, yet available approaches to seek meaningful information in the "noncoding" sequence are limited. The unique biology of the X chromosome, one of which is silenced in mammalian females, can yield clues into sequence motifs involved in chromosome packaging and function. Although autosomal chromatin has some capacity for inactivation, evidence indicates that sequences enriched on the X chromosome render it fully competent for silencing, except in specific regions that escape inactivation. Here we have used a linguistic approach by analyzing the frequency and distribution of nine base-pair genomic "words" throughout the human genome. Results identify previously unknown sequence differences on the human X chromosome. Notably, the dinucleotide repeats [AT]n, [AC]n, and [AG]n are significantly enriched across the X chromosome compared with autosomes. Moreover, a striking enrichment (>10-fold) of [GATA]n is revealed throughout the 10-Mb segment at Xp22 that escapes inactivation, and is confirmed by fluorescence in situ hybridization. A similar enrichment is found in other eutherian genomes. Our findings clearly demonstrate sequence differences relevant to the novel biology and evolution of the X chromosome. Furthermore, they implicate simple sequence repeats, linked to gene regulation and unusual DNA structures, in the regulation and formation of facultative heterochromatin. Results suggest a new paradigm whereby a regional escape from X inactivation is due to the presence of elements that prevent heterochromatinization, rather than the lack of other elements that promote it.

    PMID: 16533911 [PubMed - indexed for MEDLINE]

  • Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in ge...

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    Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands.

    Chromosoma. 2004 Dec;113(6):324-35

    Authors: Smith KP, Byron M, Clemson CM, Lawrence JB

    Abstract
    The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation.

    PMID: 15616869 [PubMed - indexed for MEDLINE]

  • The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin...

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    The 4q subtelomere harboring the FSHD locus is specifically anchored with peripheral heterochromatin unlike most human telomeres.

    J Cell Biol. 2004 Oct 25;167(2):269-79

    Authors: Tam R, Smith KP, Lawrence JB

    Abstract
    This paper investigates the nuclear localization of human telomeres and, specifically, the 4q35 subtelomere mutated in facioscapulohumeral dystrophy (FSHD). FSHD is a common muscular dystrophy that has been linked to contraction of D4Z4 tandem repeats, widely postulated to affect distant gene expression. Most human telomeres, such as 17q and 17p, avoid the nuclear periphery to reside within the internal, euchromatic compartment. In contrast, 4q35 localizes at the peripheral heterochromatin with 4p more internal, generating a reproducible chromosome orientation that we relate to gene expression profiles. Studies of hybrid and translocation cell lines indicate this localization is inherent to the distal tip of 4q. Investigation of heterozygous FSHD myoblasts demonstrated no significant displacement of the mutant allele from the nuclear periphery. However, consistent association of the pathogenic D4Z4 locus with the heterochromatic compartment supports a potential role in regulating the heterochromatic state and makes a telomere positioning effect more likely. Furthermore, D4Z4 repeats on other chromosomes also frequently organize with the heterochromatic compartment at the nuclear or nucleolar periphery, demonstrating a commonality among chromosomes harboring this subtelomere repeat family.

    PMID: 15504910 [PubMed - indexed for MEDLINE]

  • c-Myc localization within the nucleus: evidence for association with the PML nuclear body.

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    c-Myc localization within the nucleus: evidence for association with the PML nuclear body.

    J Cell Biochem. 2004 Dec 15;93(6):1282-96

    Authors: Smith KP, Byron M, O'Connell BC, Tam R, Schorl C, Guney I, Hall LL, Agrawal P, Sedivy JM, Lawrence JB

    Abstract
    Definitive localization of c-Myc within the nucleus is important to fully understand the regulation and function of this oncoprotein. Studies of c-Myc distribution, however, have produced conflicting results. To overcome technical challenges inherent in c-Myc cytology, we use here three methods to visualize c-Myc and in addition examine the impact of proteasome inhibition. EYFP or HA-tagged Myc was reintroduced by stable transfection into myc null diploid rat fibroblasts, replacing endogenous Myc with tagged Myc expressed at or near normal levels. This tagged Myc is shown to functionally replace the endogenous Myc by restoration of normal cell morphology and growth rate. We were able to confirm key findings using antibodies to the endogenous c-Myc and/or its partner, Max. Contrary to some published reports, by all three methods the c-Myc protein in rat fibroblasts distributes predominantly throughout the nucleus in a dispersed granular pattern, avoiding the nucleolus. Importantly, however, several findings provide evidence for an unanticipated relationship between c-Myc and PML nuclear bodies, which is enhanced under conditions of proteasome inhibition. Evidence of Max concentration within PML bodies is shown both with and without proteasome inhibition, strengthening the relationship between PML bodies and Myc/Max. Some accumulation of Myc and Max in nucleoli upon proteasome inhibition is also observed, although co-localization of ubiquitin was only seen with PML bodies. This work provides a comprehensive study of c-Myc distribution and also presents the first evidence of a relationship between turnover of this oncoprotein and PML nuclear bodies, known to break down in certain cancers.

    PMID: 15503302 [PubMed - indexed for MEDLINE]

  • The cell biology of a novel chromosomal RNA: chromosome painting by XIST/Xist RNA initiates a remode...

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    The cell biology of a novel chromosomal RNA: chromosome painting by XIST/Xist RNA initiates a remodeling cascade.

    Semin Cell Dev Biol. 2003 Dec;14(6):369-78

    Authors: Hall LL, Lawrence JB

    Abstract
    X chromosome inactivation begins when a novel chromosomal RNA (cRNA) from the imprinted mouse Xist or human XIST locus coats or "paints" one X chromosome in cis and initiates a cascade of chromosome remodeling events. Molecular cytological studies have proven invaluable for understanding the distinctive cellular behavior of this singular RNA involved in chromosome structure and regulation. While the detailed mechanism of XIST/Xist (X-inactivation Specific Transcript) RNA function remains largely unknown, recent advances provide new insights into the complex cellular factors which impact the RNA's localization to the chromosome, as well as the early events of chromosome remodeling that follow painting by Xist RNA. Because chromatin changes can be directly visualized on a silenced chromosome, X chromosome inactivation provides an advantageous model to investigate genome-wide heterochromatin formation and maintenance, with wide-ranging implications for normal cells and disease.

    PMID: 15015744 [PubMed - indexed for MEDLINE]

  • Repositioning of muscle-specific genes relative to the periphery of SC-35 domains during skeletal my...

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    Repositioning of muscle-specific genes relative to the periphery of SC-35 domains during skeletal myogenesis.

    Mol Biol Cell. 2004 Jan;15(1):197-206

    Authors: Moen PT, Johnson CV, Byron M, Shopland LS, de la Serna IL, Imbalzano AN, Lawrence JB

    Abstract
    Previous studies have shown that in a given cell type, certain active genes associate with SC-35 domains, nuclear regions rich in RNA metabolic factors and excluded from heterochromatin. This organization is not seen for all active genes; therefore, it is important to determine whether and when this locus-specific organization arises during development and differentiation of specific cell types. Here, we investigate whether gene organization relative to SC-35 domains is cell type specific by following several muscle and nonmuscle genes in human fibroblasts, committed but proliferative myoblasts, and terminally differentiated muscle. Although no change was seen for other loci, two muscle genes (Human beta-cardiac myosin heavy chain and myogenin) became localized to the periphery of an SC-35 domain in terminally differentiated muscle nuclei, but not in proliferative myoblasts or in fibroblasts. There was no apparent change in gene localization relative to either the chromosome territory or the heterochromatic compartment; thus, the gene repositioning seemed to occur specifically with respect to SC-35 domains. This gene relocation adjacent to a prominent SC-35 domain was recapitulated in mouse 3T3 cells induced into myogenesis by introduction of MyoD. Results demonstrate a cell type-specific reorganization of specific developmentally regulated loci relative to large domains of RNA metabolic factors, which may facilitate developmental regulation of genome expression.

    PMID: 14617810 [PubMed - indexed for MEDLINE]

  • Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costame...

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    Archvillin, a muscle-specific isoform of supervillin, is an early expressed component of the costameric membrane skeleton.

    J Cell Sci. 2003 Jun 01;116(Pt 11):2261-75

    Authors: Oh SW, Pope RK, Smith KP, Crowley JL, Nebl T, Lawrence JB, Luna EJ

    Abstract
    The membrane skeleton protein supervillin binds tightly to both F-actin and membranes and can potentiate androgen receptor activity in non-muscle cells. We report that muscle, which constitutes the principal tissue source for supervillin sequences, contains a approximately 250 kDa isoform of supervillin that localizes within nuclei and with dystrophin at costameres, regions of F-actin membrane attachment in skeletal muscle. The gene encoding this protein, 'archvillin' (Latin, archi; Greek, árchos; 'principal' or 'chief'), contains an evolutionarily conserved, muscle-specific 5' leader sequence. Archvillin cDNAs also contain four exons that encode approximately 47 kDa of additional muscle-specific protein sequence in the form of two inserts within the function-rich N-terminus of supervillin. The first of these muscle-specific inserts contains two conserved nuclear targeting signals in addition to those found in sequences shared with supervillin. Archvillin, like supervillin, binds directly to radiolabeled F-actin and co-fractionates with plasma membranes. Colocalization of archvillin with membrane-associated actin filaments, non-muscle myosin II, and--to a lesser extent--vinculin was observed in myoblasts. Striking localizations of archvillin protein and mRNA were observed at the tips of differentiating myotubes. Transfected protein chimeras containing archvillin insert sequences inhibited myotube formation, consistent with a dominant-negative effect during early myogenesis. These data suggest that archvillin is among the first costameric proteins to assemble during myogenesis and that it contributes to myogenic membrane structure and differentiation.

    PMID: 12711699 [PubMed - indexed for MEDLINE]

  • Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained w...

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    Evidence that all SC-35 domains contain mRNAs and that transcripts can be structurally constrained within these domains.

    J Struct Biol. 2002 Oct-Dec;140(1-3):131-9

    Authors: Shopland LS, Johnson CV, Lawrence JB

    Abstract
    A fundamental question of mRNA metabolism concerns the spatial organization of the steps involved in generating mature transcripts and their relationship to SC-35 domains, nuclear compartments enriched in mRNA metabolic factors and poly A+ RNA. Because poly A+ RNA in SC-35 domains remains after transcription inhibition, a prevailing view has been that most or all SC-35 domains do not contain protein-encoding mRNAs but stable RNAs with nuclear functions and thus that these compartments do not have direct roles in mRNA synthesis or transport. However, the transcription, splicing, and transport of transcripts from a specific gene have been shown to occur in association with two of these 15-30 nuclear compartments. Here we show that virtually all SC-35 domains can contain specific mRNAs and that these persist in SC-35 domains after treatment with three different transcription-inhibitory drugs. This suggests perturbation of an mRNA transport step that normally occurs in SC-35 domains and is post-transcriptional but still dependent on ongoing transcription. Finally, even after several hours of transcription arrest, these transcripts do not disperse from SC-35 domains, indicating that they are structurally constrained within them. Our findings importantly suggest a spatially direct role for all SC-35 domains in the coupled steps of mRNA metabolism and transport.

    PMID: 12490161 [PubMed - indexed for MEDLINE]

  • Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of...

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    Unbalanced X;autosome translocations provide evidence for sequence specificity in the association of XIST RNA with chromatin.

    Hum Mol Genet. 2002 Dec 01;11(25):3157-65

    Authors: Hall LL, Clemson CM, Byron M, Wydner K, Lawrence JB

    Abstract
    Whether XIST RNA is indifferent to the sequence content of the chromosome is fundamental to understanding its mechanism of chromosomal inactivation. Transgenic Xist RNA appears to associate with and inactivate an entire autosome. However, the behavior of XIST RNA on naturally occurring human X;autosome translocations has not been thoroughly investigated. Here, the relationship of human XIST RNA to autosomal chromatin is investigated in cells from two patients carrying X;autosome translocations in the context of almost complete trisomy for the involved autosome. Since trisomies of either 14 or 9 are lethal in early development, the lack of serious phenotypic consequences of the trisomy demonstrates that the translocated autosomes had been inactivated. Surprisingly, our analyses show that in primary fibroblasts from adult patients, XIST RNA does not associate with most of the involved autosome even though the bulk of it exhibits other hallmarks of inactivation beyond the region associated with XIST RNA. While results show that XIST RNA can associate with human autosomal chromatin to some degree, several observations indicate that this interaction may be unstable, with progressive loss over time. Thus, even where autosomal inactivation is selected for rather than against, there is a fundamental difference in the affinity of XIST RNA for autosomal versus X chromatin. Based on these results we propose that even autosomal chromatin that had been inactivated earlier in development may undergo a stepwise loss of inactivation hallmarks, beginning with XIST RNA. Hence compromised interaction with XIST RNA may be a primary cause of incomplete or unstable autosomal inactivation.

    PMID: 12444100 [PubMed - indexed for MEDLINE]

  • An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 c...

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    An ectopic human XIST gene can induce chromosome inactivation in postdifferentiation human HT-1080 cells.

    Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8677-82

    Authors: Hall LL, Byron M, Sakai K, Carrel L, Willard HF, Lawrence JB

    Abstract
    It has been believed that XIST RNA requires a discrete window in early development to initiate the series of chromatin-remodeling events that form the heterochromatic inactive X chromosome. Here we investigate four adult male HT-1080 fibrosarcoma cell lines expressing ectopic human XIST and demonstrate that these postdifferentiation cells can undergo chromosomal inactivation outside of any normal developmental context. All four clonal lines inactivated the transgene-containing autosome to varying degrees and with variable stability. One clone in particular consistently localized the ectopic XIST RNA to a discrete chromosome territory that exhibited striking hallmarks of inactivation, including long-range transcriptional inactivation. Results suggest that some postdifferentiation cell lines are capable of de novo chromosomal inactivation; however, long-term retention of autosomal inactivation was less common, which suggests that autosomal inactivation may confer a selective disadvantage. These results have fundamental significance for understanding genomic programming in early development.

    PMID: 12072569 [PubMed - indexed for MEDLINE]

  • Replication-dependent histone gene expression is related to Cajal body (CB) association but does not...

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    Replication-dependent histone gene expression is related to Cajal body (CB) association but does not require sustained CB contact.

    Mol Biol Cell. 2001 Mar;12(3):565-76

    Authors: Shopland LS, Byron M, Stein JL, Lian JB, Stein GS, Lawrence JB

    Abstract
    Interactions between Cajal bodies (CBs) and replication-dependent histone loci occur more frequently than for other mRNA-encoding genes, but such interactions are not seen with all alleles at a given time. Because CBs contain factors required for transcriptional regulation and 3' end processing of nonpolyadenylated replication-dependent histone transcripts, we investigated whether interaction with CBs is related to metabolism of these transcripts, known to vary during the cell cycle. Our experiments revealed that a locus containing a cell cycle-independent, replacement histone gene that produces polyadenylated transcripts does not preferentially associate with CBs. Furthermore, modest but significant changes in association levels of CBs with replication-dependent histone loci mimic their cell cycle modulations in transcription and 3' end processing rates. By simultaneously visualizing replication-dependent histone genes and their nuclear transcripts for the first time, we surprisingly find that the vast majority of loci producing detectable RNA foci do not contact CBs. These studies suggest some link between CB association and unusual features of replication-dependent histone gene expression. However, sustained CB contact is not a requirement for their expression, consistent with our observations of U7 snRNP distributions. The modest correlation to gene expression instead may reflect transient gene signaling or the nucleation of small CBs at gene loci.

    PMID: 11251071 [PubMed - indexed for MEDLINE]

  • Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: evidence for ...

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    Interactions of U2 gene loci and their nuclear transcripts with Cajal (coiled) bodies: evidence for PreU2 within Cajal bodies.

    Mol Biol Cell. 2000 Sep;11(9):2987-98

    Authors: Smith KP, Lawrence JB

    Abstract
    The Cajal (coiled) body (CB) is a structure enriched in proteins involved in mRNA, rRNA, and snRNA metabolism. CBs have been shown to interact with specific histone and snRNA gene loci. To examine the potential role of CBs in U2 snRNA metabolism, we used a variety of genomic and oligonucleotide probes to visualize in situ newly synthesized U2 snRNA relative to U2 loci and CBs. Results demonstrate that long spacer sequences between U2 coding repeats are transcribed, supporting other recent evidence that U2 transcription proceeds past the 3' box. The presence of bright foci of this U2 locus RNA differed between alleles within the same nucleus; however, this did not correlate with the loci's association with a CB. Experiments with specific oligonucleotide probes revealed signal for preU2 RNA within CBs. PreU2 was also detected in the locus-associated RNA foci, whereas sequences 3' of preU2 were found only in these foci, not in CBs. This suggests that a longer primary transcript is processed before entry into CBs. Although this work shows that direct contact of a U2 locus with a CB is not simply correlated with RNA at that locus, it provides the first evidence of new preU2 transcripts within CBs. We also show that, in contrast to CBs, SMN gems do not associate with U2 gene loci and do not contain preU2. Because other evidence indicates that preU2 is processed in the cytoplasm before assembly into snRNPs, results point to an involvement of CBs in modification or transport of preU2 RNA.

    PMID: 10982395 [PubMed - indexed for MEDLINE]

  • Tracking COL1A1 RNA in osteogenesis imperfecta. splice-defective transcripts initiate transport from...

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    Tracking COL1A1 RNA in osteogenesis imperfecta. splice-defective transcripts initiate transport from the gene but are retained within the SC35 domain.

    J Cell Biol. 2000 Aug 07;150(3):417-32

    Authors: Johnson C, Primorac D, McKinstry M, McNeil J, Rowe D, Lawrence JB

    Abstract
    This study illuminates the intra-nuclear fate of COL1A1 RNA in osteogenesis imperfecta (OI) Type I. Patient fibroblasts were shown to carry a heterozygous defect in splicing of intron 26, blocking mRNA export. Both the normal and mutant allele associated with a nuclear RNA track, a localized accumulation of posttranscriptional RNA emanating to one side of the gene. Both tracks had slightly elongated or globular morphology, but mutant tracks were cytologically distinct in that they lacked the normal polar distribution of intron 26. Normal COL1A1 RNA tracks distribute throughout an SC-35 domain, from the gene at the periphery. Normally, almost all 50 COL1A1 introns are spliced at or adjacent to the gene, before mRNA transits thru the domain. Normal COL1A1 transcripts may undergo maturation needed for export within the domain such as removal of a slow-splicing intron (shown for intron 24), after which they may disperse. Splice-defective transcripts still distribute thru the SC-35 domain, moving approximately 1-3 micrometer from the gene. However, microfluorimetric analyses demonstrate mutant transcripts accumulate to abnormal levels within the track and domain. Hence, mutant transcripts initiate transport from the gene, but are impeded in exit from the SC-35 domain. This identifies a previously undefined step in mRNA export, involving movement through an SC-35 domain. A model is presented in which maturation and release for export of COL1A1 mRNA is linked to rapid cycling of metabolic complexes within the splicing factor domain, adjacent to the gene. This paradigm may apply to SC-35 domains more generally, which we suggest may be nucleated at sites of high demand and comprise factors being actively used to facilitate expression of associated loci.

    PMID: 10931857 [PubMed - indexed for MEDLINE]

  • Seeking common ground in nuclear complexity.

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    Seeking common ground in nuclear complexity.

    J Cell Biol. 2000 Jul 10;150(1):F1-4

    Authors: Shopland LS, Lawrence JB

    PMID: 10893275 [PubMed - indexed for MEDLINE]

  • Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

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    Processing of endogenous pre-mRNAs in association with SC-35 domains is gene specific.

    J Cell Biol. 1999 Feb 22;144(4):617-29

    Authors: Smith KP, Moen PT, Wydner KL, Coleman JR, Lawrence JB

    Abstract
    Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript "trees" of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional "tracks" of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

    PMID: 10037785 [PubMed - indexed for MEDLINE]

  • Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-alpha t...

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    Identification of a nuclear matrix targeting signal in the leukemia and bone-related AML/CBF-alpha transcription factors.

    Proc Natl Acad Sci U S A. 1997 Jun 24;94(13):6746-51

    Authors: Zeng C, van Wijnen AJ, Stein JL, Meyers S, Sun W, Shopland L, Lawrence JB, Penman S, Lian JB, Stein GS, Hiebert SW

    Abstract
    Transcription factors of the AML (core binding factor-alpha/polyoma enhancer binding protein 2) class are key transactivators of tissue-specific genes of the hematopoietic and bone lineages. Alternative splicing of the AML-1 gene results in two major AML variants, AML-1 and AML-1B. We show here that the transcriptionally active AML-1B binds to the nuclear matrix, and the inactive AML-1 does not. The association of AML-1B with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal (NMTS), a 31 amino acid segment near the C terminus that is distinct from nuclear localization signals. A similar NMTS is present in AML-2 and the bone-related AML-3 transcription factors. Fusion of the AML-1B NMTS to the heterologous GAL4-(1-147) protein directs GAL4 to the nuclear matrix. Thus, the NMTS is necessary and sufficient to target the transcriptionally active AML-1B to the nuclear matrix. The loss of the C-terminal domain of AML-1B is a frequent consequence of the leukemia-related t(8;21) and t(3;21) translocations. Our results suggest this loss may be functionally linked to the modified interrelationships between nuclear structure and gene expression characteristic of cancer cells.

    PMID: 9192636 [PubMed - indexed for MEDLINE]

  • Multifunctional compartments in the nucleus: insights from DNA and RNA localization.

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    Multifunctional compartments in the nucleus: insights from DNA and RNA localization.

    J Cell Biochem. 1996 Aug;62(2):181-90

    Authors: Clemson CM, Lawrence JB

    PMID: 8844398 [PubMed - indexed for MEDLINE]

  • U2 and U1 snRNA gene loci associate with coiled bodies.

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    U2 and U1 snRNA gene loci associate with coiled bodies.

    J Cell Biochem. 1995 Dec;59(4):473-85

    Authors: Smith KP, Carter KC, Johnson CV, Lawrence JB

    Abstract
    The coiled bodies are nuclear structures rich in a variety of nuclear and nucleolar components including snRNAs. We have investigated the possibility that coiled bodies may associate with snRNA genes and report here that there is a high degree of association between U2 and U1 genes with a subset of coiled bodies. As investigated in human HeLa cells grown in monolayer culture, about 75% of nuclei had at least one U2 gene associated with a coiled body, and 45% had at least one U1 locus associated. In another suspension-grown HeLa cell strain, 92% of cells showed associated of one or more U2 genes with coiled bodies. In contrast to the U2 and U1 gene associations, a locus closely linked to the U2 gene cluster appeared associated with a coiled body only in 10% of cells. Associated snRNA gene signals were repeatedly positioned at the edge of the coiled body. Thus, this associated was highly nonrandom and spatially precise. Our analysis revealed a much higher frequency of association for closely spaced "doublet" U2 gene signals, with over 80% of paired signals associated as opposed to 35% for single U2 signals. This finding, coupled with the fact that not all genes were associated in all cells, suggested the possibility of a cell-cycle-dependent, possibly S-phase, association. However, an analysis of S- and non-S-phase cells using BrdU incorporation or cell synchronization did not indicate an increased level of association in S-phase. These and other results suggested that a substantial fraction of paired U2 signals represented association of U2 genes on homologous chromosomes rather than only replicated DNA. Furthermore, triple label analysis showed that in a significant fraction of cells U1 and U2 genes were both associated with the same coiled body. U1 and U2 genes were closely paired in approximately 20% of cells, over 60% of which were associated with a readily identifiable coiled body. This finding raises the possibility that multiple genes of a particular class may be in association with each coiled body. Thus, the coiled body may be a dynamic structure which transiently interacts with or is formed by one or more specific genetic loci, possibly carrying out some function related to their expression.

    PMID: 8749717 [PubMed - indexed for MEDLINE]

  • Compartmentalization of specific pre-mRNA metabolism: an emerging view.

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    Compartmentalization of specific pre-mRNA metabolism: an emerging view.

    Hum Mol Genet. 1995;4 Spec No:1779-89

    Authors: Moen PT, Smith KP, Lawrence JB

    Abstract
    It is increasingly recognized that the mammalian interphase nucleus contains a number of non-membranous compartments in which macromolecules associated with different nuclear functions concentrate. This review focuses on the function of a major compartment consisting of domains highly enriched in pre-mRNA splicing components and poly (A) RNA, commonly identified by the splicing factor, SC-35. RNA synthesis, as judged interdomain space. However, uridine labels several types of nuclear RNA, only a fraction of which is pre-mRNA, and such studies cannot address the question of whether specific genes are transcribed in specific places. Similarly, interpretations of transcriptional inhibition studies are compromised by the global impact that inhibition has on nuclear structure and function, and by conflicting results. Localization of specific protein coding genes or RNAs circumvents these limitations. For several sequences studied thus far, a non-random relationship to SC-35 domains has been observed, with most, but not all, active genes encoding intron-containing pre-mRNAs showing a very high degree of association. In some cases this was directly demonstrated to be the site of transcription and processing. Consistent with earlier uridine incorporation studies, we have found that transcription occurs at the outer edge of the SC-35 domain, likely corresponding to the border of ultrastructures termed interchromatin granule clusters. These preliminary glimpses into gene localization strongly argue for a sequence-specific spatial association of some transcriptionally active genes with SC-35 domains, and suggest an integrated functional organization of the genome with these nuclear compartments enriched in splicing factors and poly (A) RNA.

    PMID: 8541878 [PubMed - indexed for MEDLINE]

  • Nuclear RNA tracks: structural basis for transcription and splicing?

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    Nuclear RNA tracks: structural basis for transcription and splicing?

    Trends Cell Biol. 1993 Oct;3(10):346-53

    Authors: Xing Y, Lawrence JB

    Abstract
    Knowledge of how the biochemical machineries governing metabolism and transport of several distinct classes of RNA may be organized and integrated into the structure of the nucleus remains very limited. Recent observations, including advances in the detection of specific nucleotide sequences directly within the nucleus, have heightened the long-standing interest in the structural organization of pre-mRNA transcription and processing.

    PMID: 14731904 [PubMed]

  • Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13.

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    Assignment of the nuclear mitotic apparatus protein NuMA gene to human chromosome 11q13.

    Genomics. 1993 Jul;17(1):222-4

    Authors: Sparks CA, Bangs PL, McNeil GP, Lawrence JB, Fey EG

    Abstract
    A monoclonal antibody that was specific for a nuclear matrix protein was obtained and used to screen a human lambda gt11 expression library. Several partial cDNA clones were isolated and sequenced. The sequence for this protein was shown to be identical to that of NuMA, a 236-kDa nuclear mitotic spindle apparatus protein. NuMA has been recently characterized by two independent studies, and is thought to be part of a family of proteins that is required for the completion of mitosis. In this report, the chromosomal localization and copy number of the NuMA gene are analyzed using cDNA clones. High-resolution in situ hybridization reveals a single pair of signals on sister chromatids of human chromosome 11 at band q13. Stringent Southern analysis of human genomic DNA resulted in simple restriction patterns. These results together indicate that the NuMA gene is present as a single copy on human chromosome 11q13.

    PMID: 8406455 [PubMed - indexed for MEDLINE]

  • A simple, rapid technique for precise mapping of multiple sequences in two colors using a single opt...

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    A simple, rapid technique for precise mapping of multiple sequences in two colors using a single optical filter set.

    Genet Anal Tech Appl. 1991 Apr;8(2):75-6

    Authors: Johnson CV, McNeil JA, Carter KC, Lawrence JB

    PMID: 2064822 [PubMed - indexed for MEDLINE]

  • Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

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    Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization.

    Genet Anal Tech Appl. 1991 Apr;8(2):41-58

    Authors: McNeil JA, Johnson CV, Carter KC, Singer RH, Lawrence JB

    Abstract
    The enormous potential of in situ hybridization derives from the unique ability of this approach to directly couple cytological and molecular information. In recent years, there has been a surge of success in powerful new applications, resulting from methodologic advances that bring the practical capabilities of this technology closer to its theoretical potential. A major advance has been improvements that enable, with a high degree of reproducibility and efficiency, precise visualization of single sequences within individual metaphase and interphase cells. This has implications for gene mapping, the analysis of nuclear organization, clinical cytogenetics, virology, and studies of gene expression. This article discusses the current state of the art of fluorescence in situ hybridization, with emphasis on applications to human genetics, but including brief discussions on studies of nuclear DNA and RNA organization, and on applications to clinical genetics and virology. Although a review of all of the literature in this field is not possible here, many of the major contributions are summarized along with recent work from our laboratory.

    PMID: 1712212 [PubMed - indexed for MEDLINE]

  • Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonst...

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    Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation.

    J Cell Biol. 1991 Mar;112(6):1055-63

    Authors: Xing YG, Lawrence JB

    Abstract
    Biochemical fractionation procedures previously shown to remove 95% of cellular protein, DNA, and phospholipid, were combined with fluorescence in situ hybridization to provide a critical evaluation of the retention and spatial preservation of specific primary transcripts within the chromatin-depleted nuclear substructure, operationally defined as the nuclear "matrix." This unique approach made it possible to directly address whether nuclear extraction procedures preserve, create, or destroy ribonucleoprotein filament structures. Comparison of nuclei before and after fractionation demonstrated that localized foci or "tracks" of specific nRNA are unambiguously retained in the nuclear matrix preparation. Two well-characterized nuclear fractionation procedures were used and three Epstein-Barr virus-infected cell types investigated, including latently and permissively infected cells carrying integrated or episomal genomes. The EBV primary transcripts as well as nucleolar RNA were preserved within the remaining nuclear substructure with unambiguous spatial and quantitative fidelity. Image processing and quantitative microfluorimetry, together with [3H]thymidine labeling of DNA, show that essentially 100% of the RNA signal intensity remained after removal of 85% of the DNA. That the native RNA distribution was unchanged was shown in other experiments in which the same individual nRNA tracks were examined before and after fractionation. Results conclusively demonstrate that the tight restriction of RNA to highly localized sites is independent of bulk DNA removal and of extensive extraction of proteins and phospholipids. Hence, this work provides direct visual evidence that the primary transcripts studied are localized via their binding to, or comprising part of, non-chromatin nuclear substructure.

    PMID: 1705562 [PubMed - indexed for MEDLINE]

  • Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in ...

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    Distribution of myosin heavy chain mRNA in embryonic muscle tissue visualized by ultrastructural in situ hybridization.

    Dev Biol. 1991 Jan;143(1):58-67

    Authors: Pomeroy ME, Lawrence JB, Singer RH, Billings-Gagliardi S

    Abstract
    We have localized myosin heavy chain (MHC) mRNAs in cells of intact embryonic chick muscle using high resolution in situ hybridization. Blocks of muscle were aldehyde-fixed prior to detergent treatment and hybridized with a biotinated cDNA probe, followed by colloidal gold-labeled antibodies, before embedment. Labeling was determined to represent MHC mRNA by extensive quantitative comparisons of electron micrographs from experimental and four different types of control samples. MHC mRNA was localized primarily to peripheral regions of 14-day chick pectoral muscle cells, where the majority of developing myofibrils were found. MHC mRNAs were consistently associated with the nonmyofibrillar cytoskeletal filaments which had diameters ranging from 4 to 10 nm. They were often oriented parallel to the longitudinal axis of the cell. The resolution of the ultrastructural approach allowed us to demonstrate that the mRNA molecules visualized were not directly associated with myofilaments, suggesting that nascent chains read from those messages do not assemble directly into myofilaments simultaneous with translation.

    PMID: 1985024 [PubMed - indexed for MEDLINE]

  • Interphase and metaphase resolution of different distances within the human dystrophin gene.

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    Interphase and metaphase resolution of different distances within the human dystrophin gene.

    Science. 1990 Aug 24;249(4971):928-32

    Authors: Lawrence JB, Singer RH, McNeil JA

    Abstract
    Fluorescence in situ hybridization makes possible direct visualization of single sequences not only on chromosomes, but within decondensed interphase nuclei, providing a potentially powerful approach for high-resolution (1 Mb and below) gene mapping and the analysis of nuclear organization. Interphase mapping was able to extend the ability to resolve and order sequences up to two orders of magnitude beyond localization on banded or unbanded chromosomes. Sequences within the human dystrophin gene separated by less than 100 kb to 1 Mb were visually resolved at interphase by means of standard microscopy. In contrast, distances in the 1-Mb range could not be ordered on the metaphase chromosome length. Analysis of sequences 100 kb to 1 Mb apart indicates a strong correlation between interphase distance and linear DNA distance, which could facilitate a variety of gene-mapping efforts. Results estimate chromatin condensation up to 1 Mb and indicate a comparable condensation for different cell types prepared by different techniques.

    PMID: 2203143 [PubMed - indexed for MEDLINE]

  • Detection of HIV-1-infected cells from patients using nonisotopic in situ hybridization.

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    Detection of HIV-1-infected cells from patients using nonisotopic in situ hybridization.

    Blood. 1989 Nov 01;74(6):2295-301

    Authors: Singer RH, Byron KS, Lawrence JB, Sullivan JL

    Abstract
    We have demonstrated that a sensitive, nonisotopic in situ hybridization (ISH) assay can be used to detect HIV-infected cells from seropositive, asymptomatic individuals. Our assay is based on the detection of a biotinated HIV DNA probe hybridized to human immunodeficiency virus (HIV)-infected peripheral blood lymphocytes (PBL) using streptavidin and alkaline phosphatase to identify positive cells. This assay is rapid in that it can be performed within a day and is sensitive enough to unambiguously identify a rare, single, positive cell. Patient samples derived from HIV-seropositive hemophiliacs and HIV-seropositive infants were analyzed before and after coculture with normal PBL. The same samples were investigated using a Dupont P24 antigen-capture kit. It was found that ISH always detected the same positive samples as antigen capture, often in shorter times of coculture. In situ hybridization detected over half of our HIV-infected hemophilia patient population as virus positive, whereas the antigen capture assay detected less than one fourth as virus positive. In situ hybridization detected positive cells directly, without coculture, in 12 out of 35 (34%) hemophiliacs and in three out of eight (37%) infants. The speed, sensitivity, and confidence of ISH and nonisotopic detection indicates that it will be useful as a tool for clinical research and diagnosis.

    PMID: 2804364 [PubMed - indexed for MEDLINE]

  • Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label...

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    Ultrastructural visualization of cytoskeletal mRNAs and their associated proteins using double-label in situ hybridization.

    J Cell Biol. 1989 Jun;108(6):2343-53

    Authors: Singer RH, Langevin GL, Lawrence JB

    Abstract
    We have been able to visualize cytoskeletal messenger RNA molecules at high resolution using nonisotopic in situ hybridization followed by whole-mount electron microscopy. Biotinated cDNA probes for actin, tubulin, or vimentin mRNAs were hybridized to Triton-extracted chicken embryo fibroblasts and myoblasts. The cells were then exposed to antibodies against biotin followed by colloidal gold-conjugated antibodies and then critical-point dried. Identification of mRNA was possible using a probe fragmented to small sizes such that hybridization of several probe fragments along the mRNA was detected as a string of colloidal gold particles qualitatively and quantitatively distinguishable from nonspecific background. Extensive analysis showed that when eight gold particles were seen in this iterated array, the signal to noise ratio was greater than 30:1. Furthermore, these gold particles were colinear, often spiral, or circular suggesting detection of a single nucleic acid molecule. Antibodies against actin, vimentin, or tubulin proteins were used after in situ hybridization, allowing simultaneous detection of the protein and its cognate message on the same sample. This revealed that cytoskeletal mRNAs are likely to be extremely close to actin protein (5 nm or less) and unlikely to be within 20 nm of vimentin or tubulin filaments. Actin mRNA was found to be more predominant in lamellipodia of motile cells, confirming previous results. These results indicate that this high resolution in situ hybridization approach is a powerful tool by which to investigate the association of mRNA with the cytoskeleton.

    PMID: 2738094 [PubMed - indexed for MEDLINE]

  • Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybri...

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    Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization.

    Cell. 1989 May 05;57(3):493-502

    Authors: Lawrence JB, Singer RH, Marselle LM

    Abstract
    Use of in situ hybridization optimized for fluorescent detection of nuclear RNA has revealed a striking localization of specific viral RNAs within nuclei of cells latently infected with EBV. Several hundred kb of specific transcripts is sharply restricted to a small region of the nucleus, frequently in a curvilinear "track". Detection of nuclear RNA was evidenced by hybridization without denaturation, sensitivity to RNAase, inhibition by actinomycin D, and specificity of transcribed sequences. Results indicate that RNA "tracks" extend from an internal genome into the nuclear periphery, and that RNA transport may be coupled to transcription. Localized nRNA is apparent for other viral sequences, different lymphoblastoid cell lines, nuclei prepared by two different methods, and an abundant, nonviral transfected sequence. Implications for understanding nuclear organization and the investigation of gene expression are discussed.

    PMID: 2541917 [PubMed - indexed for MEDLINE]

  • Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridiza...

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    Temporal resolution and sequential expression of muscle-specific genes revealed by in situ hybridization.

    Dev Biol. 1989 May;133(1):235-46

    Authors: Lawrence JB, Taneja K, Singer RH

    Abstract
    The expression of muscle-specific mRNAs was analyzed directly within individual cells by in situ hybridization to chicken skeletal myoblasts undergoing differentiation in vitro. The probes detected mRNAs for sarcomeric myosin heavy chain (MHC) or the skeletal, cardiac, and beta isoforms of actin. Precise information as to the expression of these genes in individual cells was obtained and correlated directly with analyses of cell morphology and interactions, cell cycle stage, and immunofluorescence detection of the corresponding proteins. Results demonstrate that mRNAs for the two major muscle-specific proteins, myosin and actin, are not synchronously activated at the time of cell fusion. The mRNA for alpha-cardiac actin (CAct), known to be the predominant embryonic actin isoform in muscle, is expressed prior to cell fusion and prior to the expression of any isoform of muscle MHC mRNA. MHC mRNA accumulates rapidly immediately after fusion, whereas skeletal actin mRNA is expressed only in larger myofibers. Single cells expressing CAct mRNA have a characteristic short bipolar morphology, are in terminal G1, and do not contain detectable levels of the corresponding protein. In a pattern of expression reciprocal to that of CAct mRNA, beta-actin mRNA diminishes to low or undetectable levels in myofibers and in cells of the morphotype which expresses CAct mRNA. Finally, the intracellular distribution of mRNAs for different actin isoforms was compared using nonisotopic detection of isoform-specific oligonucleotide probes. This work illustrates a generally valuable approach to the analysis of cell differentiation and gene expression which directly integrates molecular, morphological, biochemical, and cell cycle information on individual cells.

    PMID: 2651181 [PubMed - indexed for MEDLINE]

  • Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two...

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    Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line.

    Cell. 1988 Jan 15;52(1):51-61

    Authors: Lawrence JB, Villnave CA, Singer RH

    Abstract
    Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.

    PMID: 2830981 [PubMed - indexed for MEDLINE]

  • Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization.

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    Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization.

    Proc Natl Acad Sci U S A. 1988 Jan;85(2):463-7

    Authors: Lawrence JB, Singer RH, Villnave CA, Stein JL, Stein GS

    Abstract
    We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs.

    PMID: 3422437 [PubMed - indexed for MEDLINE]