The relative role of promoters, enhancers and other regulatory DNA elements in determining cell-type-specific gene expression is poorly understood. Now a chromatin-immunoprecipitation-based microarray (ChIP-chip) method has been used to generate maps of histone modifications at promoters and enhancers in several different human cell lines. The modification patterns at promoters are found to be largely invariant between cell types, whereas the patterns at most enhancers are specific to a single cell type and correlate with cell-type-specific gene expression. By generating maps of histone modifications at promoters and enhancers in several different human cell lines, it has been possible to determine that although modification patterns at promoters are largely invariant between cell types, the patterns at most enhancers are specific to a single cell type and correlate well with cell-type-specific gene expression. The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene1,2,3, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome4,5,6. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression.

Higher-order chromatin structure is emerging as an important regulator of gene expression. Although dynamic chromatin structures have been identified in the genome, the full scope of chromatin dynamics during mammalian development and lineage specification remains to be determined. By mapping genome-wide chromatin interactions in human embryonic stem (ES) cells and four human ES-cell-derived lineages, we uncover extensive chromatin reorganization during lineage specification. We observe that although self-associating chromatin domains are stable during differentiation, chromatin interactions both within and between domains change in a striking manner, altering 36% of active and inactive chromosomal compartments throughout the genome. By integrating chromatin interaction maps with haplotype-resolved epigenome and transcriptome data sets, we find widespread allelic bias in gene expression correlated with allele-biased chromatin states of linked promoters and distal enhancers. Our results therefore provide a global view of chromatin dynamics and a resource for studying long-range control of gene expression in distinct human cell lineages.

We have isolated and analyzed human CTCF cDNA clones and show here that the ubiquitously expressed 11-zinc-finger factor CTCF is an exceptionally highly conserved protein displaying 93% identity between avian and human amino acid sequences. It binds specifically to regulatory sequences in the promoter-proximal regions of chicken, mouse, and human c-myc oncogenes. CTCF contains two transcription repressor domains transferable to a heterologous DNA binding domain. One CTCF binding site, conserved in mouse and human c-myc genes, is found immediately downstream of the major P2 promoter at a sequence which maps precisely within the region of RNA polymerase II pausing and release. Gel shift assays of nuclear extracts from mouse and human cells show that CTCF is the predominant factor binding to this sequence. Mutational analysis of the P2-proximal CTCF binding site and transient-cotransfection experiments demonstrate that CTCF is a transcriptional repressor of the human c-myc gene. Although there is 100% sequence identity in the DNA binding domains of the avian and human CTCF proteins, the regulatory sequences recognized by CTCF in chicken and human c-myc promoters are clearly diverged. Mutating the contact nucleotides confirms that CTCF binding to the human c-myc P2 promoter requires a number of unique contact DNA bases that are absent in the chicken c-myc CTCF binding site. Moreover, proteolytic-protection assays indicate that several more CTCF Zn fingers are involved in contacting the human CTCF binding site than the chicken site. Gel shift assays utilizing successively deleted Zn finger domains indicate that CTCF Zn fingers 2 to 7 are involved in binding to the chicken c-myc promoter, while fingers 3 to 11 mediate CTCF binding to the human promoter. This flexibility in Zn finger usage reveals CTCF to be a unique "multivalent" transcriptional factor and provides the first feasible explanation of how certain homologous genes (i.e., c-myc) of different vertebrate species are regulated by the same factor and maintain similar expression patterns despite significant promoter sequence divergence.