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23
A bivalent chromatin structure marks key developmental genes in embryonic stem cells, Cell 125
, 2006
"... The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by ex ..."
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The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed ‘‘bivalent domains,’ ’ consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.
Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BACarray CGH
- Nucleic Acids Res
, 2004
"... Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detec-tion of local one-copy-level variations. We report ..."
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Comparative genomic hybridization to bacterial artificial chromosome (BAC)-arrays (array-CGH) is a highly efficient technique, allowing the simultaneous measurement of genomic DNA copy number at hundreds or thousands of loci, and the reliable detec-tion of local one-copy-level variations. We report a genome-wide amplification method allowing the same measurement sensitivity, using 1 ng of starting genomic DNA, instead of the classical 1 mg usually necessary. Using a discrete series of DNA fragments, we defined theparametersadapted to themost faithful ligation-mediated PCR amplification and the limits of the technique. The optimized protocol allows a 3000-fold DNA amplification, retaining the quantita-tive characteristics of the initial genome. Validation of the amplification procedure, using DNA from 10 tumour cell lines hybridized to BAC-arrays of 1500 spots, showed almost perfectly superimposed ratios for the non-amplified and amplified DNAs. Correlation coefficientsof0.96and0.99wereobservedfor regions of low-copy-level variations and all regions, respect-ively (including in vivo amplified oncogenes). Finally, labelling DNA using two nucleotides bearing the same fluorophore led to a significant increase in reprodu-cibility and to the correct detection of one-copy gain or loss in>90 % of the analysed data, even for pseudotriploid tumour genomes.
An extended set of PRDM1/BLIMP1 target genes links binding motif type to dynamic repression
- Nucleic Acids Res
, 2010
"... The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP1) regulates gene ex-pression and cell fate. The DNA motif bound by BLIMP1 in vitro overlaps with that of interferon regu-latory factors (IRFs), which respond to inflamma-tory/immune signals. At such sites, BLIMP1 and IRFs ..."
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The transcriptional repressor B lymphocyte-induced maturation protein-1 (BLIMP1) regulates gene ex-pression and cell fate. The DNA motif bound by BLIMP1 in vitro overlaps with that of interferon regu-latory factors (IRFs), which respond to inflamma-tory/immune signals. At such sites, BLIMP1 and IRFs can antagonistically regulate promoter activity. In vitro motif selection predicts that only a subset of BLIMP1 or IRF sites is subject to antag-onistic regulation, but the extent to which antagon-ism occurs is unknown, since an unbiased assessment of BLIMP1 occupancy in vivo is lacking. To address this, we identified an extended set of promoters occupied by BLIMP1. Motif discov-ery and enrichment analysis demonstrate that multiple motif variants are required to capture BLIMP1 binding specificity. These are differentially associated with CpG content, leading to the obser-vation that BLIMP1 DNA-binding is methylation sen-sitive. In occupied promoters, only a subset of BLIMP1 motifs overlap with IRF motifs. Conversely, a distinct subset of IRF motifs is not enriched amongst occupied promoters. Genes linked to occupied promoters containing overlapping BLIMP1/IRF motifs (e.g. AIM2, SP110, BTN3A3) are shown to constitute a dynamic target set which is preferentially activated by BLIMP1 knock-down. These data confirm and extend the competitive model of BLIMP1 and IRF interaction.
DOT1L/KMT4 Recruitment and H3K79 Methylation Are Ubiquitously
, 2008
"... The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recru ..."
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The histone H3 lysine 79 methyltransferase DOT1L/KMT4 can promote an oncogenic pattern of gene expression through binding with several MLL fusion partners found in acute leukemia. However, the normal function of DOT1L in mammalian gene regulation is poorly understood. Here we report that DOT1L recruit-ment is ubiquitously coupled with active transcription in diverse mammalian cell types. DOT1L preferentially occupies the proximal transcribed region of active genes, correlating with enrichment of H3K79 di- and trimethylation. Furthermore, Dot1l mutant fibroblasts lacked H3K79 di- and trimethylation at all sites examined, indicating that DOT1L is the sole enzyme responsible for these marks. Importantly, we identified chromatin immunoprecipitation (ChIP) assay conditions necessary for reliable H3K79 methylation detection. ChIP-chip tiling arrays revealed that levels of all degrees of genic H3K79 methylation correlate with mRNA abundance and dynamically respond to changes in gene activity. Conversion of H3K79 monomethyl-ation into di- and trimethylation correlated with the transition from low- to high-level gene transcription. We also observed enrichment of H3K79 monomethylation at intergenic regions occupied by DNA-binding tran-scriptional activators. Our findings highlight several similarities between the patterning of H3K4 methylation
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, 2009
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Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): I. CIP Treatment of Samples and Tailing Reaction with Terminal Transferase
"... T7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from T7 phage. TLAD was designed for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate ..."
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T7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from T7 phage. TLAD was designed for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of
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"... ChIP-chip results. There are other potentially important factors that are not assessed here, and that from a practical standpoint are more difficult to systematically control and evaluate. These include the skill of the experimenter, the amount of starting material (chromatin, DNA, and antibody) use ..."
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ChIP-chip results. There are other potentially important factors that are not assessed here, and that from a practical standpoint are more difficult to systematically control and evaluate. These include the skill of the experimenter, the amount of starting material (chromatin, DNA, and antibody) used, the size of DNA fragments after shearing, the DNA labeling method, and the hybridization conditions. There have been several studies evaluating the performance of gene expression microarrays and analysis algorithms (Choe et al. 2005; Irizarry et al. 2005; MAQC Consortium 2006; Patterson et al. 2006). However, tiling arrays present distinct informatics and experimental challenges because large contiguous genomic regions are covered with high probe densities. Thus the results from the expression array spike-in experiments are not necessarily directly relevant to tiling-array experiments. One recent study
Rpd3p Relocation Brief Communication Mediates a Transcriptional Response to Rapamycin in Yeast
"... 3Harvard Biophysics Program repression, is catalyzed by enzymes called histone deacetylases (HDACs) [15–17]. To examine whether HDACs are required for the gene repression observed following treatment with rapamycin, we examined the transcriptional response to rapamycin in strains individ-ually delet ..."
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3Harvard Biophysics Program repression, is catalyzed by enzymes called histone deacetylases (HDACs) [15–17]. To examine whether HDACs are required for the gene repression observed following treatment with rapamycin, we examined the transcriptional response to rapamycin in strains individ-ually deleted for each known HDAC: RPD3, SIR2, HDA1, Harvard University HOS1, HOS2, and HOS3. Deletion of RPD3, but no other
unknown title
, 2006
"... Chromatin immunoprecipitation and high-density tiling microarrays: a generative model, methods for analysis, and methodology assessment in the absence of a “gold standard” by ..."
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Chromatin immunoprecipitation and high-density tiling microarrays: a generative model, methods for analysis, and methodology assessment in the absence of a “gold standard” by
