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...gures S2 and S3). The most extreme example is found at the Hox clusters, which are known to have the lowest density of transposon-derived sequence in the mouse and human genomes (Lander et al., 2001; =-=Waterston et al., 2002-=-) and which have the largest Lys27 domains (up to 141 kb) in our sample. Most of the Lys27 domains contain long stretches (>10 kb) with little or no identifiable transposon-derived sequence. We define...

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...maintaining pluripotency. INTRODUCTION Epigenetic regulation of gene expression is mediated in part by posttranslational modifications of histone proteins, which in turn modulate chromatin structure (=-=Jenuwein and Allis, 2001-=-; Margueron et al., 2005). The core histones H2A, H2B, H3, and H4 are subject to dozens of different modifications, including acetylation, methylation, and phosphorylation. Histone H3 lysine 4 (Lys4) ...

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...sus 22% expected) (Figures S2 and S3). The most extreme example is found at the Hox clusters, which are known to have the lowest density of transposon-derived sequence in the mouse and human genomes (=-=Lander et al., 2001-=-; Waterston et al., 2002) and which have the largest Lys27 domains (up to 141 kb) in our sample. Most of the Lys27 domains contain long stretches (>10 kb) with little or no identifiable transposon-der...

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... to the reported binding sites of certain pluripotent TFs. A recent genomic analysis in human ES cells found that Oct4, Nanog, and Sox2 are frequently associated with developmentally important genes (=-=Boyer et al., 2005-=-). We mapped the Oct4, Nanog, and Sox2 binding sites reported in that study to orthologous positions in the mouse genome and examined their overlap with bivalent domains. About 50% of bivalent domains...

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...plified by in vitro transcription, converted into double-stranded cDNA with random primers, fragmented with DNase I, and end-labeled with biotin as described (Kapranov et al., 2002; Liu et al., 2003; =-=Cawley et al., 2004-=-; Bernstein et al., 2005). ChIP and control samples (5–10 mg) were hybridized to separate oligonucleotide arrays. Arrays were hybridized 16–18 hr at 45ºC, washed, stained, and scanned using an Affymet...

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...noncoding elements (HCNEs) in mammalian genomes cluster within 200 HCNE-rich genomic loci, which include all four Hox clusters (Nobrega et al., 2003; Bejerano et al., 2004; Lindblad-Toh et al., 2005; =-=Woolfe et al., 2005-=-). These regions tend to be gene-poor but are highly enriched for genes encoding transcription factors (TFs) implicated in embryonic development. These findings suggest that the HCNE-rich regions or t...

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...am. The expected density of CpG and transposable elements at methylated sites was determined from random intervals of the same size, anchored in nonrepetitive sequence. CpG islands were defined as in =-=Takai and Jones, 2002-=-. TEZs (transposon exclusion zones) were defined as regions satisfying one of two criteria: (1) regions of at least 10 kb without any transposable elements; (2) regions of at least 15 kb with no more ...

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...dies have revealed that the most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within 200 HCNE-rich genomic loci, which include all four Hox clusters (Nobrega et al., 2003; =-=Bejerano et al., 2004-=-; Lindblad-Toh et al., 2005; Woolfe et al., 2005). These regions tend to be gene-poor but are highly enriched for genes encoding transcription factors (TFs) implicated in embryonic development. These ...

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...odifications are catalyzed, respectively, by trithoraxand Polycomb-group proteins, which mediate mitotic inheritance of lineage-specific gene expression programs and have key developmental functions (=-=Ringrose and Paro, 2004-=-). Lys4 methylation positively regulates transcription by recruiting nucleosome remodeling enzymes and histone acetylases (Santos-Rosa et al., 2003; PrayGrant et al., 2005; Sims et al., 2005; Wysocka ...

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...Inc. 315schromatin immunoprecipitation (ChIP) and DNA microarrays. Initial studies in primary fibroblasts revealed thousands of genomic sites associated with Lys4 methylation (Bernstein et al., 2005; =-=Kim et al., 2005-=-). The vast majority show a ‘‘punctate’’ pattern, typically occurring at sites of 1-2 kb near promoters of active genes. Lys27 methylation is implicated in X chromosome inactivation and imprinting (Pl...

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...be pairs, each consisting of perfect match (PM) and single base mismatch (MM) 25-mer oligonucleotides, designed to interrogate the unique sequence in these regions at approximately 30 base intervals (=-=Kapranov et al., 2002-=-). DNA Amplification and Array Hybridization ChIP and control DNA samples were amplified by in vitro transcription, converted into double-stranded cDNA with random primers, fragmented with DNase I, an...

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...ns unclear. Recent studies have revealed that the most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within 200 HCNE-rich genomic loci, which include all four Hox clusters (=-=Nobrega et al., 2003-=-; Bejerano et al., 2004; Lindblad-Toh et al., 2005; Woolfe et al., 2005). These regions tend to be gene-poor but are highly enriched for genes encoding transcription factors (TFs) implicated in embryo...

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...uson-Smith, 2005). Embryonic stem (ES) cell differentiation is accompanied by changes in chromatin accessibility at several key developmental genes, including a large-scale opening of the HoxB locus (=-=Chambeyron and Bickmore, 2004-=-; Perry et al., 2004). Furthermore, Polycomb-group proteins play an essential role in maintaining the pluripotent state of ES cells and show markedly reduced expression upon differentiation (O’Carroll...

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...eflect strong evolutionary pressure against the presence of transposon-derived sequence in these regions. Repetitive sequences are subject to repressive epigenetic modifications (Arnaud et al., 2000; =-=Lippman et al., 2004-=-; Martens et al., 2005), which might interfere with the function of the bivalent domains and thus be eliminated by selection. It has been reported previously that imprinted loci, while significantly d...

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...all genomic distribution of this repressive mark. Gene-specific and limited microarray studies have reported that Lys27 methylation tends to occur at punctate sites near promoters of repressed genes (=-=Cao and Zhang, 2004-=-; Kimura et al., 2004; Kirmizis et al., 2004; Koyanagi et al., 2005). Based on such studies, the distributions of Lys4 and Lys27 methylation have been thought to be nonoverlapping. A notable exception...

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...ion by promoting a compact chromatin structure (Francis et al., 2004; Ringrose et al., 2004). Various observations suggest that chromatin undergoes important alterations during mammalian development (=-=Delaval and Feil, 2004-=-; Margueron et al., 2005; Sado and Ferguson-Smith, 2005). Embryonic stem (ES) cell differentiation is accompanied by changes in chromatin accessibility at several key developmental genes, including a ...

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...05). The vast majority show a ‘‘punctate’’ pattern, typically occurring at sites of 1-2 kb near promoters of active genes. Lys27 methylation is implicated in X chromosome inactivation and imprinting (=-=Plath et al., 2003-=-; Umlauf et al., 2004). However, little is known about the overall genomic distribution of this repressive mark. Gene-specific and limited microarray studies have reported that Lys27 methylation tends...

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...elation is weaker in the differentiated cells; this is primarily due to loss of Lys4 methylation at 20%–35% of CpG islands (r phi = 0.40 for MLFs) (Table S4). We note that a recent genome-wide study (=-=Roh et al., 2005-=-) of histone H3 acetylation in T cells observed a correlation with CpG islands at a similar level to that seen in the differentiated cells examined here. Figure 5. Resolution of Bivalent Domains durin...

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...ro, 2004). Lys4 methylation positively regulates transcription by recruiting nucleosome remodeling enzymes and histone acetylases (Santos-Rosa et al., 2003; PrayGrant et al., 2005; Sims et al., 2005; =-=Wysocka et al., 2005-=-), while Lys27 methylation negatively regulates transcription by promoting a compact chromatin structure (Francis et al., 2004; Ringrose et al., 2004). Various observations suggest that chromatin unde...

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...emically defined media as described (Conti et al., 2005). These cells uniformly express nestin and Sox2 and upon growth factor withdrawal differentiate into neurons, astrocytes, and oligodendrocytes (=-=Brustle et al., 1999-=-; Conti et al., 2005). Chromatin Immunoprecipitation ChIP experiments for all cells except ES2 were carried out as described in Bernstein et al., 2005 and at http://www.upstate.com. Briefly, 5 10 7 ce...

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... 2004). Furthermore, Polycomb-group proteins play an essential role in maintaining the pluripotent state of ES cells and show markedly reduced expression upon differentiation (O’Carroll et al., 2001; =-=Silva et al., 2003-=-; Valk-Lingbeek et al., 2004). However, little is known about the overall structure of ES cell chromatin, how it is established, or how it contributes to the maintenance of pluripotency (Szutorisz and...

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...ed as real-time PCR template in (C). The sequential ChIP results indicate that, in ES cells, the Irx2 TSS is associated with chromatin marked by both Lys27 and Lys4 methylation. described previously (=-=Conti et al., 2005-=-). We focused on seven genes associated with bivalent domains in ES cells (Figure 5). These include three genes that are markedly induced during differentiation (Nkx2.2, Sox21, and Zfpm2), one that is...

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... serum and penicillin/streptomycin at 37º, 5% CO 2. ES1 cells were differentiated into pan-neural precursor cells through embryoid body formation for 4 days and selection in ITSFn media for 5–7 days (=-=Okabe et al., 1996-=-), and they were maintained in FGF2 and EGF2 (both from R&D Systems) containing chemically defined media as described (Conti et al., 2005). These cells uniformly express nestin and Sox2 and upon growt...

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...d ensure that each daughter chromosome would likely inherit a substantial proportion of the modified histones, which could then promote similar modification of new histones in the immediate vicinity (=-=Henikoff et al., 2004-=-; van Steensel, 2005). A fundamental issue that remains is to understand the mechanism by which the initial conditions are established in ES cells. The analysis here suggests that some of the answer c...

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...ses (Santos-Rosa et al., 2003; PrayGrant et al., 2005; Sims et al., 2005; Wysocka et al., 2005), while Lys27 methylation negatively regulates transcription by promoting a compact chromatin structure (=-=Francis et al., 2004-=-; Ringrose et al., 2004). Various observations suggest that chromatin undergoes important alterations during mammalian development (Delaval and Feil, 2004; Margueron et al., 2005; Sado and Ferguson-Sm...

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...INTRODUCTION Epigenetic regulation of gene expression is mediated in part by posttranslational modifications of histone proteins, which in turn modulate chromatin structure (Jenuwein and Allis, 2001; =-=Margueron et al., 2005-=-). The core histones H2A, H2B, H3, and H4 are subject to dozens of different modifications, including acetylation, methylation, and phosphorylation. Histone H3 lysine 4 (Lys4) and lysine 27 (Lys27) me...

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...ty show a ‘‘punctate’’ pattern, typically occurring at sites of 1-2 kb near promoters of active genes. Lys27 methylation is implicated in X chromosome inactivation and imprinting (Plath et al., 2003; =-=Umlauf et al., 2004-=-). However, little is known about the overall genomic distribution of this repressive mark. Gene-specific and limited microarray studies have reported that Lys27 methylation tends to occur at punctate...

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...e mark. Gene-specific and limited microarray studies have reported that Lys27 methylation tends to occur at punctate sites near promoters of repressed genes (Cao and Zhang, 2004; Kimura et al., 2004; =-=Kirmizis et al., 2004-=-; Koyanagi et al., 2005). Based on such studies, the distributions of Lys4 and Lys27 methylation have been thought to be nonoverlapping. A notable exception to the punctate pattern of histone modifica...

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... been reported previously that imprinted loci, while significantly depleted for short interspersed transposable elements (SINEs), are permissive to L1 long interspersed transposable elements (LINEs) (=-=Greally, 2002-=-). In contrast, we find that both classes of transposons tend to be excluded from regions associated with Lys27 methylation or bivalent domains in ES cells. The direct signal for Lys27 methylation rem...

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...sociated with trithorax-group proteins and low-level transcription. Remarkably, both of these activities appear to be required for subsequent gene activation during development (Orlando et al., 1998; =-=Schmitt et al., 2005-=-). By analogy, Lys4 methylation within bivalent domains and associated trithorax activities may keep silenced developmental genes poised in ES cells. Our analyses of differentiated cells suggest that ...

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...nts are nonetheless associated with trithorax-group proteins and low-level transcription. Remarkably, both of these activities appear to be required for subsequent gene activation during development (=-=Orlando et al., 1998-=-; Schmitt et al., 2005). By analogy, Lys4 methylation within bivalent domains and associated trithorax activities may keep silenced developmental genes poised in ES cells. Our analyses of differentiat...

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...NA samples were amplified by in vitro transcription, converted into double-stranded cDNA with random primers, fragmented with DNase I, and end-labeled with biotin as described (Kapranov et al., 2002; =-=Liu et al., 2003-=-; Cawley et al., 2004; Bernstein et al., 2005). ChIP and control samples (5–10 mg) were hybridized to separate oligonucleotide arrays. Arrays were hybridized 16–18 hr at 45ºC, washed, stained, and sca...

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...e functional significance of bivalent domains, we examined gene expression patterns across the three cell types with at least ten bivalent domains (ES cells, C2C12, and Neuro2a) (Mogass et al., 2004; =-=Tomczak et al., 2004-=-; Perez-Iratxeta et al., 2005). Within each cell type, we found that genes marked by Lys4 methylation tend to be expressed at significantly higher levels than those associated with Lys27 methylation (...

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...ion of this repressive mark. Gene-specific and limited microarray studies have reported that Lys27 methylation tends to occur at punctate sites near promoters of repressed genes (Cao and Zhang, 2004; =-=Kimura et al., 2004-=-; Kirmizis et al., 2004; Koyanagi et al., 2005). Based on such studies, the distributions of Lys4 and Lys27 methylation have been thought to be nonoverlapping. A notable exception to the punctate patt...

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...nce of bivalent domains, we examined gene expression patterns across the three cell types with at least ten bivalent domains (ES cells, C2C12, and Neuro2a) (Mogass et al., 2004; Tomczak et al., 2004; =-=Perez-Iratxeta et al., 2005-=-). Within each cell type, we found that genes marked by Lys4 methylation tend to be expressed at significantly higher levels than those associated with Lys27 methylation (Figure 4). A good example is ...

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... association of Lys4 methylation with CpG islands may well be directly causal, inasmuch as the trithorax complexes that methylate Lys4 are reported to associate with CpG-rich DNA (Ayton et al., 2004; =-=Lee and Skalnik, 2005-=-). The strong association of Lys27 methylation with transposon-exclusion zonessmay instead reflect strong evolutionary pressure against the presence of transposon-derived sequence in these regions. Re...

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...em (ES) cell differentiation is accompanied by changes in chromatin accessibility at several key developmental genes, including a large-scale opening of the HoxB locus (Chambeyron and Bickmore, 2004; =-=Perry et al., 2004-=-). Furthermore, Polycomb-group proteins play an essential role in maintaining the pluripotent state of ES cells and show markedly reduced expression upon differentiation (O’Carroll et al., 2001; Silva...

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...odifications is evident at the Hox gene clusters: These loci contain large, cell type-specific Lys4 methylated regions, up to 60 kb in length, that overlay multiple Hox genes (Bernstein et al., 2005; =-=Guenther et al., 2005-=-). These regions likely reflect accessible chromatin domains established during embryonic development to maintain Hox gene expression programs (Chambeyron and Bickmore, 2004). However, the extent to w...

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...n zonessmay instead reflect strong evolutionary pressure against the presence of transposon-derived sequence in these regions. Repetitive sequences are subject to repressive epigenetic modifications (=-=Arnaud et al., 2000-=-; Lippman et al., 2004; Martens et al., 2005), which might interfere with the function of the bivalent domains and thus be eliminated by selection. It has been reported previously that imprinted loci,...

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... et al., 2003; Valk-Lingbeek et al., 2004). However, little is known about the overall structure of ES cell chromatin, how it is established, or how it contributes to the maintenance of pluripotency (=-=Szutorisz and Dillon, 2005-=-). Large-scale studies of mammalian chromatin have recently become possible with the combination of Cell 125, 315–326, April 21, 2006 ª2006 Elsevier Inc. 315schromatin immunoprecipitation (ChIP) and D...

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...nd limited microarray studies have reported that Lys27 methylation tends to occur at punctate sites near promoters of repressed genes (Cao and Zhang, 2004; Kimura et al., 2004; Kirmizis et al., 2004; =-=Koyanagi et al., 2005-=-). Based on such studies, the distributions of Lys4 and Lys27 methylation have been thought to be nonoverlapping. A notable exception to the punctate pattern of histone modifications is evident at the...

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...sequence. The strong association of Lys4 methylation with CpG islands may well be directly causal, inasmuch as the trithorax complexes that methylate Lys4 are reported to associate with CpG-rich DNA (=-=Ayton et al., 2004-=-; Lee and Skalnik, 2005). The strong association of Lys27 methylation with transposon-exclusion zonessmay instead reflect strong evolutionary pressure against the presence of transposon-derived sequen...

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...ility is that some of the HCNEs dictate chromosome conformation or nuclear localization in a manner that facilitates robust gene regulation and/or epigenetic switching (Chambeyron and Bickmore, 2004; =-=Kosak and Groudine, 2004-=-). Further studies will be needed to define bivalent domains and related features. It will be important to examine the entire genome in ES cells as well as to follow their fate during development and ...

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...7 methylation (Figure 4). A good example is the Ebf1 gene, which encodes a TF implicated in multiple differentiation pathways: In MEFs, MLFs, and myoblasts, it is expressed at relatively high levels (=-=Schraets et al., 2003-=-; Koli et al., 2004) and is associated with relatively large Lys4 sites (>5 kb), while in neuroblastoma cells it is expressed at an essentially undetectable level and is associated with an expansive L...

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...Francis et al., 2004; Ringrose et al., 2004). Various observations suggest that chromatin undergoes important alterations during mammalian development (Delaval and Feil, 2004; Margueron et al., 2005; =-=Sado and Ferguson-Smith, 2005-=-). Embryonic stem (ES) cell differentiation is accompanied by changes in chromatin accessibility at several key developmental genes, including a large-scale opening of the HoxB locus (Chambeyron and B...

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... gain insight into the functional significance of bivalent domains, we examined gene expression patterns across the three cell types with at least ten bivalent domains (ES cells, C2C12, and Neuro2a) (=-=Mogass et al., 2004-=-; Tomczak et al., 2004; Perez-Iratxeta et al., 2005). Within each cell type, we found that genes marked by Lys4 methylation tend to be expressed at significantly higher levels than those associated wi...