Keji Zhao

High-resolution profiling of histone methylations in the human genome

Histone modifications are implicated in influencing gene expression. We have generated high-resolution maps for the genome-wide distribution of 20 histone lysine and arginine methylations as well as histone variant H2A.Z, RNA polymerase II, and the insulator binding protein CTCF across the human genome using the Solexa 1G sequencing technology. Typical patterns of histone methylations exhibited at promoters, insulators, enhancers, and transcribed regions are identified. The monomethylations of H3K27, H3K9, H4K20, H3K79, and H2BK5 are all linked to gene activation, whereas trimethylations of H3K27, H3K9, and H3K79 are linked to repression. H2A.Z associates with functional regulatory elements, and CTCF marks boundaries of histone methylation domains. Chromosome banding patterns are correlated with unique patterns of histone modifications. Chromosome breakpoints detected in T cell cancers frequently reside in chromatin regions associated with H3K4 methylations. Our data provide new insights into the function of histone methylation and chromatin organization in genome function.

Combinatorial patterns of histone acetylations and methylations in the human genome

Histones are characterized by numerous posttranslational modifications that influence gene transcription. However, because of the lack of global distribution data in higher eukaryotic systems, the extent to which gene-specific combinatorial patterns of histone modifications exist remains to be determined. Here, we report the patterns derived from the analysis of 39 histone modifications in human CD4+ T cells. Our data indicate that a large number of patterns are associated with promoters and enhancers. In particular, we identify a common modification module consisting of 17 modifications detected at 3,286 promoters. These modifications tend to colocalize in the genome and correlate with each other at an individual nucleosome level. Genes associated with this module tend to have higher expression, and addition of more modifications to this module is associated with further increased expression. Our data suggest that these histone modifications may act cooperatively to prepare chromatin for transcriptional activation.

Dynamic Regulation of Nucleosome Positioning in the Human Genome

The positioning of nucleosomes with respect to DNA plays an important role in regulating transcription. However, nucleosome mapping has been performed for only limited genomic regions in humans. We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Solexa high-throughput sequencing technique. We find that nucleosome phasing relative to the transcription start sites is directly correlated to RNA polymerase II (Pol II) binding. Furthermore, the first nucleosome downstream of a start site exhibits differential positioning in active and silent genes. TCR signaling induces extensive nucleosome reorganization in promoters and enhancers to allow transcriptional activation or repression. Our results suggest that H2A.Z-containing and modified nucleosomes are preferentially lost from the -1 nucleosome position. Our data provide a comprehensive view of the nucleosome landscape and its dynamic regulation in the human genome.

Genome-wide Mapping of HATs and HDACs Reveals Distinct Functions in Active and Inactive Genes

Histone acetyltransferases (HATs) and deacetylases (HDACs) function antagonistically to control histone acetylation. As acetylation is a histone mark for active transcription, HATs have been associated with active and HDACs with inactive genes. We describe here genome-wide mapping of HATs and HDACs binding on chromatin and find that both are found at active genes with acetylated histones. Our data provide evidence that HATs and HDACs are both targeted to transcribed regions of active genes by phosphorylated RNA Pol II. Furthermore, the majority of HDACs in the human genome function to reset chromatin by removing acetylation at active genes. Inactive genes that are primed by MLL-mediated histone H3K4 methylation are subject to a dynamic cycle of acetylation and deacetylation by transient HAT/HDAC binding, preventing Pol II from binding to these genes but poising them for future activation. Silent genes without any H3K4 methylation signal show no evidence of being bound by HDACs.

Global Mapping of H3K4me3 and H3K27me3 Reveals Specificity and Plasticity in Lineage Fate Determination of Differentiating CD4+ T Cells

Multipotential naive CD4(+) T cells differentiate into distinct lineages including T helper 1 (Th1), Th2, Th17, and inducible T regulatory (iTreg) cells. The remarkable diversity of CD4(+) T cells begs the question whether the observed changes reflect terminal differentiation with heritable epigenetic modifications or plasticity in T cell responses. We generated genome-wide histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) trimethylation maps in naive, Th1, Th2, Th17, iTreg, and natural Treg (nTreg) cells. We found that although modifications of signature-cytokine genes (Ifng, Il4, and Il17) partially conform to the expectation of lineage commitment, genes encoding transcription factors like Tbx21 exhibit a broad spectrum of epigenetic states, consistent with our demonstration of T-bet and interferon-gamma induction in nTreg cells. Our data suggest an epigenetic mechanism underlying the specificity and plasticity of effector and regulatory T cells and also provide a framework for understanding complexity of CD4(+) T helper cell differentiation.

Rapid and Phosphoinositol-Dependent Binding of the SWI/SNF-like BAF Complex to Chromatin after T Lymphocyte Receptor Signaling

Lymphocyte activation is accompanied by visible changes in chromatin structure. We find that antigen receptor signaling induces the rapid association of the BAF complex with chromatin. PIP2, which is regulated by activation stimuli, is sufficient in vitro to target the BAF complex to chromatin, but it has no effect on related chromatin remodeling complexes containing SNF2L or hISWI. Purification and peptide sequencing of the subunits of the complex revealed beta-actin as well as a novel actin-related protein, BAF53. beta-actin and BAF53 are required for maximal ATPase activity of BRG1 and are also required with BRG1 for association of the complex with chromatin/matrix. This work indicates that membrane signals control the activity of the mammalian SWI/SNF or BAF complex and demonstrates a direct interface between signaling and chromatin regulation.

c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells

The c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism, and differentiation at the cellular, tissue, or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Mycs targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II, and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a nonlinear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature.

A clustering approach for identification of enriched domains from histone modification ChIP-Seq data

MOTIVATION Chromatin states are the key to gene regulation and cell identity. Chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-Seq) is increasingly being used to map epigenetic states across genomes of diverse species. Chromatin modification profiles are frequently noisy and diffuse, spanning regions ranging from several nucleosomes to large domains of multiple genes. Much of the early work on the identification of ChIP-enriched regions for ChIP-Seq data has focused on identifying localized regions, such as transcription factor binding sites. Bioinformatic tools to identify diffuse domains of ChIP-enriched regions have been lacking. RESULTS Based on the biological observation that histone modifications tend to cluster to form domains, we present a method that identifies spatial clusters of signals unlikely to appear by chance. This method pools together enrichment information from neighboring nucleosomes to increase sensitivity and specificity. By using genomic-scale analysis, as well as the examination of loci with validated epigenetic states, we demonstrate that this method outperforms existing methods in the identification of ChIP-enriched signals for histone modification profiles. We demonstrate the application of this unbiased method in important issues in ChIP-Seq data analysis, such as data normalization for quantitative comparison of levels of epigenetic modifications across cell types and growth conditions. AVAILABILITY http://home.gwu.edu/ approximately wpeng/Software.htm. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Chromatin Signatures in Multipotent Human Hematopoietic Stem Cells Indicate the Fate of Bivalent Genes during Differentiation

Histone modifications have been implicated in stem cell maintenance and differentiation. We have analyzed genome-wide changes in gene expression and histone modifications during differentiation of multipotent human primary hematopoietic stem cells/progenitor cells (HSCs/HPCs) into erythrocyte precursors. Our data indicate that H3K4me1, H3K9me1, and H3K27me1 associate with enhancers of differentiation genes prior to their activation and correlate with basal expression, suggesting that these monomethylations are involved in the maintenance of activation potential required for differentiation. In addition, although the majority of genes associated with both H3K4me3 and H3K27me3 in HSCs/HPCs become silent and lose H3K4me3 after differentiation, those that lose H3K27me3 and become activated after differentiation are associated with increased levels of H2A.Z, H3K4me1, H3K9me1, H4K20me1, and RNA polymerase II in HSCs/HPCs. Thus, our data suggest that gene expression changes during differentiation are programmed by chromatin modifications present at the HSC/HPC stage and provide a resource for enhancer and promoter identification.

Global analysis of the insulator binding protein CTCF in chromatin barrier regions reveals demarcation of active and repressive domains

Insulators are DNA elements that prevent inappropriate interactions between the neighboring regions of the genome. They can be functionally classified as either enhancer blockers or domain barriers. CTCF (CCCTC-binding factor) is the only known major insulator-binding protein in the vertebrates and has been shown to bind many enhancer-blocking elements. However, it is not clear whether it plays a role in chromatin domain barriers between active and repressive domains. Here, we used ChIP-seq to map the genome-wide binding sites of CTCF in three cell types and identified significant binding of CTCF to the boundaries of repressive chromatin domains marked by H3K27me3. Although we find an extensive overlapping of CTCF-binding sites across the three cell types, its association with the domain boundaries is cell-type-specific. We further show that the nucleosomes flanking CTCF-binding sites are well positioned. Interestingly, we found a complementary pattern between the repressive H3K27me3 and the active H2AK5ac regions, which are separated by CTCF. Our data indicate that CTCF may play important roles in the barrier activity of insulators, and this study provides a resource for further investigation of the CTCF function in organizing chromatin in the human genome.