Tae Hoon Kim

A high-resolution map of active promoters in the human genome

In eukaryotic cells, transcription of every protein-coding gene begins with the assembly of an RNA polymerase II preinitiation complex (PIC) on the promoter. The promoters, in conjunction with enhancers, silencers and insulators, define the combinatorial codes that specify gene expression patterns. Our ability to analyse the control logic encoded in the human genome is currently limited by a lack of accurate information regarding the promoters for most genes. Here we describe a genome-wide map of active promoters in human fibroblast cells, determined by experimentally locating the sites of PIC binding throughout the human genome. This map defines 10,567 active promoters corresponding to 6,763 known genes and at least 1,196 un-annotated transcriptional units. Features of the map suggest extensive use of multiple promoters by the human genes and widespread clustering of active promoters in the genome. In addition, examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. These results provide a global view of the functional relationships among transcriptional machinery, chromatin structure and gene expression in human cells.

Analysis of the vertebrate insulator protein CTCF-binding sites in the human genome

Insulator elements affect gene expression by preventing the spread of heterochromatin and restricting transcriptional enhancers from activation of unrelated promoters. In vertebrates, insulators function requires association with the CCCTC-binding factor (CTCF), a protein that recognizes long and diverse nucleotide sequences. While insulators are critical in gene regulation, only a few have been reported. Here, we describe 13,804 CTCF-binding sites in potential insulators of the human genome, discovered experimentally in primary human fibroblasts. Most of these sequences are located far from the transcriptional start sites, with their distribution strongly correlated with genes. The majority of them fit to a consensus motif highly conserved and suitable for predicting possible insulators driven by CTCF in other vertebrate genomes. In addition, CTCF localization is largely invariant across different cell types. Our results provide a resource for investigating insulator function and possible other general and evolutionarily conserved activities of CTCF sites.

Global position analysis of the Pseudomonas aeruginosa quorum-sensing transcription factor LasR.

In Pseudomonas aeruginosa quorum sensing (QS), the transcriptional regulator LasR controls the expression of more than 300 genes. Several of these genes are activated indirectly via a second, subordinate QS regulator, RhlR. Conserved sequence elements upstream of individual other genes have been shown to bind LasR in vitro. To comprehensively identify all regions that are bound by LasR in vivo, we employed chromatin immunoprecipitation in conjunction with microarray analysis. We identified 35 putative promoter regions that direct the expression of up to 74 genes. In vitro DNA binding studies allowed us to distinguish between cooperative and non‐cooperative LasR binding sites, and allowed us to build consensus sequences according to the mode of binding. Five promoter regions were not previously recognized as QS‐controlled. Two of the associated transcript units encode proteins involved in the cold‐shock response and in Psl exopolysaccharide synthesis respectively. The LasR regulon includes seven genes encoding transcriptional regulators, while secreted factors and secretion machinery are the most over‐represented functional categories overall. This supports the notion that the core function of LasR is to co‐ordinate the production of extracellular factors, although many of its effects on global gene expression are likely mediated indirectly by regulatory genes under its control.

Conserved, developmentally regulated mechanism couples chromosomal looping and heterochromatin barrier activity at the homeobox gene A locus.

Establishment and segregation of distinct chromatin domains are essential for proper genome function. The insulator protein CCCTC-binding factor (CTCF) is involved in creating boundaries that segregate chromatin and functional domains and in organizing higher-order chromatin structures by promoting chromosomal loops across the vertebrate genome. Here, we investigate the insulation properties of CTCF at the human and mouse homeobox gene A (HOXA) loci. Although cohesin loading at the CTCF binding site is required for looping, we found that cohesin is dispensable for chromatin barrier activity at that site. Using mouse embryonic stem cells in both a pluripotent and differentiated neuronal progenitor state, we determined that embryonic stem cell pluripotency factor OCT4 antagonizes cohesin loading at the CTCF binding site. Loss of OCT4 in the committed and differentiated neuronal progenitor cells results in loading of cohesin and chromosome looping, which contributes to heterochromatin partitioning and selective gene activation across the HOXA locus. Our analysis reveals that chromatin barrier activity of CTCF is evolutionarily conserved and is responsible for the coordinated establishment of chromatin structure, higher-order architecture, and developmental expression of the HOXA locus.

|[beta]|-Catenin activates the growth factor endothelin-1 in colon cancer cells

Endothelin-1 (EDN1) is a growth factor that is frequently produced by cancer cells and plays a critical role in tumorigenesis. However, the molecular mechanism controlling the expression of EDN1 in cancers is unknown. Constitutive activation of β-catenin pathway is responsible for the initiation of the vast majority of colon cancers. Here we show that the EDN1 gene is directly regulated by β-catenin in colon cancer cells. A specific DNA element within the EDN1 promoter is required for activation, and is associated with β-catenins cognate DNA binding partner, TCF4, in vivo. Inhibition of β-catenin signaling results in lowered expression of EDN1, while enhancement of β-catenin signaling leads to further activation of the gene. Significantly elevated EDN1 expression occurs in 80% of primary human colon cancers, consistent with it being a direct target of β-catenin. Furthermore, EDN1 is able to rescue colon cancer cells from growth arrest and apoptosis resulting from inhibition of β-catenin signaling, implicating a key role of EDN1 in promoting the oncogenic function of β-catenin. These results indicate EDN1 overexpression as a major cause in colon cancers and reveal further details of the genetic programs responsible for tumorigenesis of colon cancers.

HDAC inhibitors induce transcriptional repression of high copy number genes in breast cancer through elongation blockade

Treatment with histone deacetylase inhibitors (HDACI) results in potent cytotoxicity of a variety of cancer cell types, and these drugs are used clinically to treat hematological tumors. They are known to repress the transcription of ERBB2 and many other oncogenes, but little is known about this mechanism. Using global run-on sequencing (GRO-seq) to measure nascent transcription, we find that HDACI cause transcriptional repression by blocking RNA polymerase II elongation. Our data show that HDACI preferentially repress the transcription of highly expressed genes as well as high copy number genes in HER2+ breast cancer genomes. In contrast, genes that are activated by HDACI are moderately expressed. We analyzed gene copy number in combination with microarray and GRO-seq analysis of expression level, in normal and breast cancer cells to show that high copy number genes are more likely to be repressed by HDACI than non-amplified genes. The inhibition of transcription of amplified oncogenes, which promote survival and proliferation of cancer cells, might explain the cancer-specific lethality of HDACI, and may represent a general therapeutic strategy for cancer.

Histone Deacetylases Positively Regulate Transcription through the Elongation Machinery

Transcription elongation regulates the expression ofxa0many genes, including oncogenes. Histone deacetylase (HDAC) inhibitors (HDACIs) block elongation, suggesting that HDACs are involved in gene activation. To understand this, we analyzed nascent transcription and elongation factor binding genome-wide after perturbation of elongation with small molecule inhibitors. We found that HDACI-mediated repression requires heat shock protein 90 (HSP90) activity. HDACIs promote the association of RNA polymerase II (RNAP2) and negative elongation factor (NELF), a complex stabilized by HSP90, at the same genomic sites. Additionally, HDACIs redistribute bromodomain-containing protein 4 (BRD4), a key elongation factor involved in enhancer activity. BRD4 binds to newly acetylated sites, and its occupancy at promoters and enhancers is reduced. Furthermore, HDACIs reduce enhancer activity, as measured by enhancer RNA production. Therefore, HDACs are required for limiting acetylation in gene bodies and intergenic regions. This facilitates the binding of elongation factors to properly acetylated promoters and enhancers for efficient elongation.

ChIP-chip for genome-wide analysis of protein binding in mammalian cells.

ChIP‐chip combines chromatin immunoprecipitation (ChIP) with microarrays (chip) to determine protein‐DNA interactions occurring in living cells. The high throughput nature of this method makes it an ideal approach for identifying transcription factor targets or chromatin modification sites along the genome. UNIT 21.9 describes a protocol for analysis of protein‐DNA interactions in yeast cells. This unit introduces an alternative protocol developed for mammalian cells. Curr. Protoc. Mol. Biol. 79:21.13.1‐21.13.22.

Oct-1 Regulates IL-17 Expression by Directing Interchromosomal Associations in Conjunction with CTCF in T Cells

Interchromosomal associations can regulate gene expression, but little is known about the molecular basis of such associations. In response to antigen stimulation, naive Txa0cells can differentiate into Th1, Th2, and Th17 cells expressing IFN-γ, IL-4, and IL-17, respectively. We previously reported that in naive Txa0cells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sitesxa0cooperatively, and CTCF deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF.

Genomic imprinting at a boundary element flanking the SDHD locus

Germline mutations in SDHD, a mitochondrial complex II (succinate dehydrogenase) subunit gene at chromosome band 11q23, cause highly penetrant paraganglioma (PGL) tumors when transmitted through fathers. In contrast, maternal transmission rarely, if ever, leads to tumor development. The mechanism underlying this unusual monogenic tumor predisposition pattern is poorly understood. Here, we describe identification of imprinted methylation within an alternative promoter for a large intergenic non-coding RNA located at a distant gene desert boundary flanking SDHD. Methylation at this site primarily occurs within two consecutive HpaII restriction enzyme sites in a tissue-specific manner, most commonly in the adrenal gland. Informative fetal tissues and PGL tumors demonstrate maternal allelic hypermethylation. While a strong binding site for the enhancer-blocking protein CTCF within the alternative promoter shows no evidence of methylation, hyper-methylated adrenal tissues show increased binding of the chromatin-looping factor cohesin relative to the hypo-methylated tissues. These results suggest that the differential allelic methylation we observe at this locus is associated with altered chromatin architectures. These results provide molecular evidence for imprinting at a boundary element flanking the SDHD locus and suggest that epigenetic suppression of the maternal allele is the underlying mechanism of the imprinted penetrance of SDHD mutations.