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ABSTRACT: Methylation regulates DNA by altering chromatin and limiting accessibility of transcription factors and RNA polymerase. In this way, DNA methylation controls gene expression and plays a role in ES cell regulation, tissue differentiation and the development of the organism. In abnormal circumstances methylation can also induce diseases and promote cancer progression. Chromatin remodeling proteins such as the SNF2 family member Lsh regulates genome-wide cytosine methylation patterns during mammalian development. Lsh promotes methylation by targeting and repressing repeat sequences that are imbedded in heterochromatin. Lsh also regulates cytosine methylation at unique loci. Alterations in histone modifications (such as H3K4me3, histone acetylation, H3K27me3 and H2Aub) can be associated with DNA methylation changes making Lsh-mediated cytosine methylation part of a larger epigenetic network defining gene expression and cellular differentiation during development. This article is part of a Special Issue entitled: Chromatin in time and space.
Biochimica et Biophysica Acta 02/2012; 1819(7):757-62. · 4.66 Impact Factor
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ABSTRACT: Inactivation of tumor suppressor genes plays an important role in tumorigenesis, and epigenetic modifications such as DNA methylation are frequently associated with transcriptional repression. Here, we show that gene silencing at selected genes with signs of DNA hypermethylation in breast cancer cells involves Pol II stalling. We studied several repressed genes with DNA hypermethylation within a region 1-kb upstream of the transcriptional start site that were upregulated after treatment with DNA demethylating agents, such as Azacytidine and several natural products. All those selected genes had stalled Pol II at their transcriptional start site and showed enhanced ser2 phosphorylated Pol II and elevated transcripts after drug treatment indicating successful elongation. In addition, a decrease of the epigenetic regulator LSH in a breast cancer cell line by siRNA treatment reduced DNA methylation and overcame Pol II stalling, whereas overexpression of LSH in a normal breast epithelial cell line increased DNA methylation and resulted in repression. Decrease of LSH was associated with reduced DNMT3b binding to promoter sequences, and depletion of DNMT3b by siRNA could release Pol II suggesting that DNMT3b is functionally involved. The release of paused Pol II was accompanied by a dynamic switch from repressive to active chromatin marks. Thus release of Pol II stalling can act as a mechanism for gene reactivation at specific target genes after DNA demethylating treatment in cancer cells.
Nucleic Acids Research 08/2011; 39(22):9508-20. · 8.03 Impact Factor
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Yongguang Tao,
Sichuan Xi,
Jigui Shan,
Alika Maunakea,
Anney Che,
Victorino Briones,
Eunice Y Lee,
Theresa Geiman,
Jiaqiang Huang,
Robert Stephens,
Robert M Leighty,
Keji Zhao, Kathrin Muegge
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ABSTRACT: DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5' end of genes. Furthermore, we demonstrate that loss of Lsh promotes--as well as prevents--cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.
Proceedings of the National Academy of Sciences 03/2011; 108(14):5626-31. · 9.68 Impact Factor
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Wenxian Zeng,
Claudia Baumann,
Anja Schmidtmann,
Ali Honaramooz,
Lin Tang,
Alla Bondareva,
Camila Dores,
Tao Fan,
Sichuan Xi,
Theresa Geiman,
Rahul Rathi,
Dirk de Rooij,
Rabindranath De La Fuente, Kathrin Muegge,
Ina Dobrinski
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ABSTRACT: Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.
Biology of Reproduction 02/2011; 84(6):1235-41. · 4.01 Impact Factor
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ABSTRACT: DNA cytosine methylation is an important epigenetic mechanism that is involved in transcriptional silencing of developmental genes. Several molecular pathways have been described that interfere with Pol II initiation, but at individual genes the molecular mechanism of repression remains uncertain. Here, we study the molecular mechanism of transcriptional regulation at Hox genes in dependence of the epigenetic regulator Lsh that controls CpG methylation at selected Hox genes. Wild type cells show a nucleosomal deprived region around the transcriptional start site at methylated Hox genes and mediate gene silencing via Pol II stalling. Hypomethylation in Lsh-/- cells is associated with efficient transcriptional elongation and splicing, in part mediated by the chromodomain protein Chd1. Dynamic modulation of DNA methylation in Lsh-/- and wild type cells demonstrates that catalytically active DNA methyltransferase activity is required for Pol II stalling. Taken together, the data suggests that DNA methylation can be compatible with Pol II binding at selected genes and Pol II stalling can act as alternate mechanism to explain transcriptional silencing associated with DNA methylation.
PLoS ONE 01/2010; 5(2):e9163. · 4.09 Impact Factor
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ABSTRACT: Development from separate parental germ cells through fertilization and proceeding to a fully functioning adult animal occurs through an intricate program of transcriptional and chromatin changes. Epigenetic alterations such as DNA methylation are an important part of this process. This review looks at the role of DNA methylation in early embryonic development, as well as how this epigenetic mark affects stem cell differentiation and tissue-specific gene expression in somatic cells.
Molecular Reproduction and Development 11/2009; 77(2):105-13. · 2.53 Impact Factor
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ABSTRACT: Transcriptional control of stem cell genes is a critical step in differentiation of embryonic stem cells and in reprogramming of somatic cells into stem cells. Here we report that Lsh, a regulator of repressive chromatin at retrotransposons, also plays an important role in silencing of stem cell-specific genes such as Oct4. We found that CpG methylation is gained during in vitro differentiation of several stem cell-specific genes (in 11 of 12 promoter regions) and thus appears to be a common epigenetic mark. Lsh depletion prevents complete silencing of stem cell gene expression and moreover promotes the maintenance of stem cell characteristics in culture. Lsh is required for establishment of DNA methylation patterns at stem cell genes during differentiation, in part by regulating access of Dnmt3b to its genomic targets. Our results indicate that Lsh is involved in the control of stem cell genes and suggest that Lsh is an important epigenetic modulator during early stem cell differentiation.
Stem Cells 08/2009; 27(11):2691-702. · 7.78 Impact Factor
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ABSTRACT: Histone modifications, specifically, methylation of lysine residues in histone 3 (H3), determine chromatin structure and accessibility for transcription. A recent study by Bernstein and Lander identified a unique pattern of H3 methylation at the promoter region of developmental genes in pluripotent stem cells. The pattern consists of the simultaneous presence of a silencing mark [methylation of Lys(27) on H3 (H3K27me)] and an activation mark (H3K4me). This bivalent mark seems associated with a poised state of transcription and resolves upon differentiation largely into either H3K27me or H3K4me. Skin fibroblasts that can be reverted in culture to the pluripotent stage show re-establishment of the bivalent chromatin pattern. This switch in chromatin states may be key to our understanding of developmental and tissue specific regulation of gene expression and may be therapeutically useful in regenerative medicine.
Molecular Interventions 03/2008; 8(1):15-8, 2. · 4.59 Impact Factor
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ABSTRACT: Establishment of chromosomal cytosine methylation and histone methylation patterns are critical epigenetic modifications required for heterochromatin formation in the mammalian genome. However, the nature of the primary signal(s) targeting DNA methylation at specific genomic regions is not clear. Notably, whether histone methylation and/or chromatin remodeling proteins play a role in the establishment of DNA methylation during gametogenesis is not known. The chromosomes of mouse neonatal spermatogonia display a unique pattern of 5-methyl cytosine staining whereby centromeric heterochromatin is hypo-methylated whereas chromatids are strongly methylated. Thus, in order to gain some insight into the relationship between global DNA and histone methylation in the germ line we have used neonatal spermatogonia as a model to determine whether these unique chromosomal DNA methylation patterns are also reflected by concomitant changes in histone methylation.
Our results demonstrate that histone H3 tri-methylated at lysine 9 (H3K9me3), a hallmark of constitutive heterochromatin, as well as the chromatin remodeling protein ATRX remained associated with pericentric heterochromatin regions in spite of their extensive hypo-methylation. This suggests that in neonatal spermatogonia, chromosomal 5-methyl cytosine patterns are regulated independently of changes in histone methylation, potentially reflecting a crucial mechanism to maintain pericentric heterochromatin silencing. Furthermore, chromatin immunoprecipitation and fluorescence in situ hybridization, revealed that ATRX as well as H3K9me3 associate with Y chromosome-specific DNA sequences and decorate both arms of the Y chromosome, suggesting a possible role in heterochromatinization and the predominant transcriptional quiescence of this chromosome during spermatogenesis.
These results are consistent with a role for histone modifications and chromatin remodeling proteins such as ATRX in maintaining transcriptional repression at constitutive heterochromatin domains in the absence of 5-methyl cytosine and provide evidence suggesting that the establishment and/or maintenance of repressive histone and chromatin modifications at pericentric heterochromatin following genome-wide epigenetic reprogramming in the germ line may precede the establishment of chromosomal 5-methyl cytosine patterns as a genomic silencing strategy in neonatal spermatogonia.
BMC Molecular Biology 02/2008; 9:29. · 2.86 Impact Factor
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ABSTRACT: Polycomb-mediated repression and DNA methylation are important epigenetic mechanisms of gene silencing. Recent evidence suggests a functional link between the polycomb repressive complex (PRC) and Dnmts in cancer cells. Here we provide evidence that Lsh, a regulator of DNA methylation, is also involved in normal control of PRC-mediated silencing during embryogenesis. We demonstrate that Lsh, a SNF2 homolog, can associate with some Hox genes and regulates Dnmt3b binding, DNA methylation, and silencing of Hox genes during development. Moreover, Lsh can associate with PRC1 components and influence PRC-mediated histone modifications. Thus Lsh is part of a physiological feedback loop that reinforces DNA methylation and silencing of PRC targets.
Proceedings of the National Academy of Sciences 10/2007; 104(36):14366-71. · 9.68 Impact Factor
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ABSTRACT: Lymphoid specific helicase (Lsh) is a major epigenetic regulator that is essential for DNA methylation and transcriptional silencing of parasitic elements in the mammalian genome. However, whether Lsh is involved in the regulation of chromatin-mediated processes during meiosis is not known. Here, we show that Lsh is essential for the completion of meiosis and transcriptional repression of repetitive elements in the female gonad. Oocytes from Lsh knockout mice exhibit demethylation of transposable elements and tandem repeats at pericentric heterochromatin, as well as incomplete chromosome synapsis associated with persistent RAD51 foci and gammaH2AX phosphorylation. Failure to load crossover-associated foci results in the generation of non-exchange chromosomes. The severe oocyte loss observed and lack of ovarian follicle formation, together with the patterns of Lsh nuclear compartmentalization in the germ line, demonstrate that Lsh has a critical and previously unidentified role in epigenetic gene silencing and maintenance of genomic stability during female meiosis.
Nature Cell Biology 01/2007; 8(12):1448-54. · 19.49 Impact Factor
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ABSTRACT: Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh-/- embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.
The EMBO Journal 02/2006; 25(2):335-45. · 9.20 Impact Factor
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ABSTRACT: Methylation of cytosines controls a number of biologic processes such as imprinting and X chromosomal inactivation. DNA hypermethylation is closely associated with transcriptional silencing, while DNA hypomethylation is associated with transcriptional activation. Hypoacetylation of histones leads to compact chromatin with reduced accessibility to the transcriptional machinery. Methyl-CpG binding proteins can recruit corepressors and histone deacetylases; thus, the interplay between these epigenetic mechanisms regulates gene activation. Methylation has been implicated as an important mechanism during immune development, controlling VDJ recombination, lineage-specific expression of cell surface antigens, and transcriptional regulation of cytokine genes during immune responses. Aberrations in epigenetic machinery, either by genetic mutations or by somatic changes such as viral infections, are associated with early alterations in chronic diseases such as immunodeficiency and cancer.
Annals of the New York Academy of Sciences 01/2006; 983(1):55 - 70. · 3.15 Impact Factor
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Kathrin Muegge
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ABSTRACT: Lymphoid-specific helicase (Lsh) is a crucial factor for normal embryonic development; targeted deletion of Lsh is lethal. Lsh belongs to a family of chromatin-remodeling proteins and is closely associated with pericentromeric heterochromatin. Lsh deficiency leads to abnormal heterochromatin organization, with a loss of DNA methylation, and an altered pattern of histone-tail acetylation and methylation. As a functional consequence of perturbed heterochromatin, aberrant reactivation of parasitic retroviral elements in the genome and abnormal mitosis with amplified centrosomes and genomic instability were observed. Thus, Lsh is a major epigenetic regulator crucial for normal heterochromatin structure and function.
Biochemistry and Cell Biology 09/2005; 83(4):548-54. · 2.67 Impact Factor
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ABSTRACT: Epigenetic regulation, such as DNA methylation plays an important role in the control of imprinting. Lsh, a member of the SNF2 family of chromatin remodeling proteins, controls DNA methylation in mice. To investigate whether Lsh affects imprinting, we examined CpG methylation and allelic expression of individual genes in Lsh-deficient embryos. We report here that loss of Lsh specifically alters expression of the Cdkn1c gene (also known as p57(Kip2)) but does not interfere with maintenance of imprints at the H19, Igf2, Igf2r, Zac1 and Meg9 genes. The reactivation of the silenced paternal Cdkn1c allele correlates closely with a loss of CpG methylation at the 5' DMR at the Cdkn1c promoter, whereas KvDMR1 and DMRs of other imprinted genes were not significantly changed. Chromatin immunoprecipitations demonstrate a direct association of Lsh with the 5' DMR at the Cdkn1c promoter, but not with Kv DMR1 or other imprinted loci. These data suggest that methylation of the 5' DMR plays an important role in the imprinting of the Cdkn1c gene. Furthermore, it suggests that Lsh is not required for maintenance of imprinting marks in general, but is only crucial for imprinting at distinct genomic sites.
Development 03/2005; 132(4):635-44. · 6.60 Impact Factor
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ABSTRACT: The genome is burdened with repetitive sequences that are generally embedded in silenced chromatin. We have previously demonstrated that Lsh (lymphoid-specific helicase) is crucial for the control of heterochromatin at pericentromeric regions consisting of satellite repeats. In this study, we searched for additional genomic targets of Lsh by examining the effects of Lsh deletion on repeat regions and single copy gene sequences. We found that the absence of Lsh resulted in an increased association of acetylated histones with repeat sequences and transcriptional reactivation of their silenced state. In contrast, selected single copy genes displayed no change in histone acetylation levels, and their transcriptional rate was indistinguishable compared to Lsh-deficient cells and wild-type controls. Microarray analysis of total RNA derived from brain and liver tissues revealed that <0.4% of the 15 247 examined loci were abnormally expressed in Lsh-/-embryos and almost two-thirds of these deregulated sequences contained repeats, mainly retroviral LTR (long terminal repeat) elements. Chromatin immunoprecipitation analysis demonstrated a direct interaction of Lsh with repetitive sites in the genome. These data suggest that the repetitive sites are direct targets of Lsh action and that Lsh plays an important role as 'epigenetic guardian' of the genome to protect against deregulation of parasitic retroviral elements.
Nucleic Acids Research 02/2004; 32(17):5019-28. · 8.03 Impact Factor
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ABSTRACT: The eukaryotic genome is packaged into distinct domains of transcriptionally active euchromatin and silent heterochromatin. A hallmark of mammalian heterochromatin is CpG methylation. Lsh, a member of the SNF2 family, is a major regulator of DNA methylation in mice and thus crucial for normal heterochromatin formation. In order to define the molecular function of Lsh, we examined its cellular localization and its association with chromatin. Our studies demonstrate that Lsh is an exclusively nuclear protein, and we define a nuclear localization domain within the N-terminal portion of Lsh. Lsh strongly associates with chromatin and requires the internal and C-terminal regions for this interaction. Lsh accumulates at pericentromeric heterochromatin, suggesting a direct role for Lsh in the methylation of centromeric DNA sequences and the formation of heterochromatin. In search of a signal that is responsible for Lsh recruitment to pericentromeric heterochromatin, we found that histone tail modifications were critical. Prolonged treatment with histone deacetylase inhibitors has been reported to disrupt higher-order heterochromatin organization, and this was accompanied by dissociation of Lsh from pericentromeric heterochromatin. These results are consistent with a model in which Lsh is recruited by intact heterochromatin structure and then assists in maintaining heterochromatin organization by establishing CpG methylation patterns.
Molecular and Cellular Biology 01/2004; 23(23):8416-28. · 5.53 Impact Factor
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ABSTRACT: Methylation of histone tails and CpG methylation are involved in determining heterochromatin structure, but their cause and effect relationship has not been resolved as yet in mammals. Here we report that Lsh, a member of the SNF2 chromatin remodeling family, controls both types of epigenetic modifications. Lsh has been shown to be associated with pericentromeric heterochromatin and to be required for normal CpG methylation at pericentromeric sequences. Loss of Lsh, in Lsh-deficient mice, results in accumulation of di- and tri-methylated histone 3 at lysine 4 (H3-K4me) at pericentromeric DNA and other repetitive sequences. In contrast, di- or tri-methylation of H3-K9 and distribution of HP1 appear unchanged after Lsh deletion, suggesting independent regulatory mechanisms for H3-K4 or K9 methylation. Experimental DNA demethylation with 5'-azacytidine results in a similar increase of H3-K4me. These results support the model that loss of CpG methylation caused by Lsh deficiency antecedes elevation of H3-K4me. Thus, Lsh is crucial for the formation of normal heterochromatin, implying a functional role for Lsh in the regulation of transcription and mitosis.
The EMBO Journal 11/2003; 22(19):5154-62. · 9.20 Impact Factor
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ABSTRACT: Genomic hypomethylation and chromosomal instability are frequent characteristics of human cancer cells. Targeted deletion of Lsh leads to a global defect in genomic methylation, and Lsh-deficient mice die at birth with a reduced body weight. Here, we examine the growth pattern of embryonal fibroblasts derived from Lsh-/- mice. The absence of Lsh leads to a severe proliferative defect of fibroblasts with lower saturation density, early signs of senescence, and a lower frequency of immortalization. The impaired growth rate in vitro may be in part responsible for the small size of Lsh-deficient mice. In addition, Lsh-/- fibroblasts accumulated high centrosome numbers, formed multipolar spindles, displayed micronuclei formation, and elevated nuclear DNA content. A similar increase in centrosome abnormalities was observed when wild-type fibroblasts were treated with a DNA-demethylating agent, suggesting that genomic hypomethylation plays an important role in mitotic defects of Lsh-/- murine embryonal fibroblasts, possibly by altering chromatin structure. Because supernumerary centrosomes are a common feature in cancer cells, this Lsh-dependent pathway has the potential to contribute to genetic instability and chromosomal aberrations during tumor progression.
Cancer Research 09/2003; 63(15):4677-83. · 7.86 Impact Factor
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ABSTRACT: Methylation of cytosines controls a number of biologic processes such as imprinting and X chromosomal inactivation. DNA hypermethylation is closely associated with transcriptional silencing, while DNA hypomethylation is associated with transcriptional activation. Hypoacetylation of histones leads to compact chromatin with reduced accessibility to the transcriptional machinery. Methyl-CpG binding proteins can recruit corepressors and histone deacetylases; thus, the interplay between these epigenetic mechanisms regulates gene activation. Methylation has been implicated as an important mechanism during immune development, controlling VDJ recombination, lineage-specific expression of cell surface antigens, and transcriptional regulation of cytokine genes during immune responses. Aberrations in epigenetic machinery, either by genetic mutations or by somatic changes such as viral infections, are associated with early alterations in chronic diseases such as immunodeficiency and cancer.
Annals of the New York Academy of Sciences 04/2003; 983:55-70. · 3.15 Impact Factor
