Bum Kyu Lee

The accessible chromatin landscape of the human genome.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.

Heritable Individual-Specific and Allele-Specific Chromatin Signatures in Humans

Like Father, Like Mother, Like Child Transcriptional regulation is mediated by chromatin structure, which may affect the binding of transcription factors, but the extent of how individual-to-individual genetic variation affects such regulation is not well understood. Kasowski et al. (p. 232, published online 18 March) investigated the binding of two transcription factors across the genomes of human individuals and one chimpanzee. Transcription factor binding was associated with genomic features such as nucleotide variation, insertions and deletions, and copy number variation. Thus, genomic sequence variation affects transcription factor binding and may explain expression difference among individuals. McDaniell et al. (p. 235, published online 18 March) provide a genome-wide catalog of variation in chromatin and transcription factor binding in two parent-child trios of European and African ancestry. Up to 10% of active chromatin binding sites were specific to a set of individuals and were often inherited. Furthermore, variation in active chromatin sites showed heritable allele-specific correlation with variation in gene expression. An appreciable amount of variation in chromatin status and transcription factor binding has a genetic basis. The extent to which variation in chromatin structure and transcription factor binding may influence gene expression, and thus underlie or contribute to variation in phenotype, is unknown. To address this question, we cataloged both individual-to-individual variation and differences between homologous chromosomes within the same individual (allele-specific variation) in chromatin structure and transcription factor binding in lymphoblastoid cells derived from individuals of geographically diverse ancestry. Ten percent of active chromatin sites were individual-specific; a similar proportion were allele-specific. Both individual-specific and allele-specific sites were commonly transmitted from parent to child, which suggests that they are heritable features of the human genome. Our study shows that heritable chromatin status and transcription factor binding differ as a result of genetic variation and may underlie phenotypic variation in humans.

High-resolution genome-wide in vivo footprinting of diverse transcription factors in human cells.

Regulation of gene transcription in diverse cell types is determined largely by varied sets of cis-elements where transcription factors bind. Here we demonstrate that data from a single high-throughput DNase I hypersensitivity assay can delineate hundreds of thousands of base-pair resolution in vivo footprints in human cells that precisely mark individual transcription factor-DNA interactions. These annotations provide a unique resource for the investigation of cis-regulatory elements. We find that footprints for specific transcription factors correlate with ChIP-seq enrichment and can accurately identify functional versus nonfunctional transcription factor motifs. We also find that footprints reveal a unique evolutionary conservation pattern that differentiates functional footprinted bases from surrounding DNA. Finally, detailed analysis of CTCF footprints suggests multiple modes of binding and a novel DNA binding motif upstream of the primary binding site.

Cell-type specific and combinatorial usage of diverse transcription factors revealed by genome-wide binding studies in multiple human cells

Cell-type diversity is governed in part by differential gene expression programs mediated by transcription factor (TF) binding. However, there are few systematic studies of the genomic binding of different types of TFs across a wide range of human cell types, especially in relation to gene expression. In the ENCODE Project, we have identified the genomic binding locations across 11 different human cell types of CTCF, RNA Pol II (RNAPII), and MYC, three TFs with diverse roles. Our data and analysis revealed how these factors bind in relation to genomic features and shape gene expression and cell-type specificity. CTCF bound predominantly in intergenic regions while RNAPII and MYC preferentially bound to core promoter regions. CTCF sites were relatively invariant across diverse cell types, while MYC showed the greatest cell-type specificity. MYC and RNAPII co-localized at many of their binding sites and putative target genes. Cell-type specific binding sites, in particular for MYC and RNAPII, were associated with cell-type specific functions. Patterns of binding in relation to gene features were generally conserved across different cell types. RNAPII occupancy was higher over exons than adjacent introns, likely reflecting a link between transcriptional elongation and splicing. TF binding was positively correlated with the expression levels of their putative target genes, but combinatorial binding, in particular of MYC and RNAPII, was even more strongly associated with higher gene expression. These data illuminate how combinatorial binding of transcription factors in diverse cell types is associated with gene expression and cell-type specific biology.

Wide-ranging functions of E2F4 in transcriptional activation and repression revealed by genome-wide analysis

The E2F family of transcription factors has important roles in cell cycle progression. E2F4 is an E2F family member that has been proposed to be primarily a repressor of transcription, but the scope of its binding activity and functions in transcriptional regulation is not fully known. We used ChIP sequencing (ChIP-seq) to identify around 16 000 E2F4 binding sites which potentially regulate 7346 downstream target genes with wide-ranging functions in DNA repair, cell cycle regulation, apoptosis, and other processes. While half of all E2F4 binding sites (56%) occurred near transcription start sites (TSSs), ∼20% of sites occurred more than 20 kb away from any annotated TSS. These distal sites showed histone modifications suggesting that E2F4 may function as a long-range regulator, which we confirmed by functional experimental assays on a subset. Overexpression of E2F4 and its transcriptional cofactors of the retinoblastoma (Rb) family and its binding partner DP-1 revealed that E2F4 acts as an activator as well as a repressor. E2F4 binding sites also occurred near regulatory elements for miRNAs such as let-7a and mir-17, suggestive of regulation of miRNAs by E2F4. Taken together, our genome-wide analysis provided evidence of versatile roles of E2F4 and insights into its functions.

Genome-wide Studies of CCCTC-binding Factor (CTCF) and Cohesin Provide Insight into Chromatin Structure and Regulation

Eukaryotic genomes are organized into higher order chromatin architectures by protein-mediated long-range interactions in the nucleus. CCCTC-binding factor (CTCF), a sequence-specific transcription factor, serves as a chromatin organizer in building this complex chromatin structure by linking chromosomal domains. Recent genome-wide studies mapping the binding sites of CTCF and its interacting partner, cohesin, using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) revealded that CTCF globally co-localizes with cohesin. This partnership between CTCF and cohesin is emerging as a novel and perhaps pivotal aspect of gene regulatory mechanisms, in addition to playing a role in the organization of higher order chromatin architecture.

Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection.

Understanding the molecular basis for phenotypic differences between humans and other primates remains an outstanding challenge. Mutations in non-coding regulatory DNA that alter gene expression have been hypothesized as a key driver of these phenotypic differences. This has been supported by differential gene expression analyses in general, but not by the identification of specific regulatory elements responsible for changes in transcription and phenotype. To identify the genetic source of regulatory differences, we mapped DNaseI hypersensitive (DHS) sites, which mark all types of active gene regulatory elements, genome-wide in the same cell type isolated from human, chimpanzee, and macaque. Most DHS sites were conserved among all three species, as expected based on their central role in regulating transcription. However, we found evidence that several hundred DHS sites were gained or lost on the lineages leading to modern human and chimpanzee. Species-specific DHS site gains are enriched near differentially expressed genes, are positively correlated with increased transcription, show evidence of branch-specific positive selection, and overlap with active chromatin marks. Species-specific sequence differences in transcription factor motifs found within these DHS sites are linked with species-specific changes in chromatin accessibility. Together, these indicate that the regulatory elements identified here are genetic contributors to transcriptional and phenotypic differences among primate species.

Production of Candida utilis biomass on molasses in different culture types

Three different types of aerobic fermentations were performed for the mass production of C. utilis as aquafeeds. From the best fermentation result of each culture type, the biomass yield and productivity were calculated to be 0.67 and 0.24 for batch, 0.51 and 1.95 for fed-batch with sigmoidal feeding strategy, and 0.36 g g −1 and 2.15 g − 1 l −1 h −1 for continuous cultures, respectively. The cultivation of C. utilis using chemicals for industrial use resulted in considerable reduction of production cost. The fed-batch fermentation was found to be the best culture type for mass production of C. utilis. The total production cost of C. utilis cultivated in the fed-batch fermentation was estimated to be US

Mass production of Rhodopseudomonas palustris as diet for aquaculture

2.76 per kg of dry cells. The total production cost is favorably comparable with the sale price of the commercial yeast product.

Dendritic cell fate is determined by BCL11A

Three different types of anaerobic fermentations were used for the mass production of the photosynthetic bacterium Rhodopseudomonas palustris as diet for aquaculture. The optimum agitation speed and malate concentration were 300 r.p.m. and 0.2% in the modified MYC medium, respectively. In batch fermentations of R. palustris, the maximum number of viable cells was 1.110 10 c.f.u. ml 1 with 2.65 g l 1 of DCW, and the maximum specific growth rate and biomass productivity were estimated to be 0.12 h 1 and 55 mg l 1 h 1 , respectively. Crude protein content of R. palustris was about 72‐74%. The composition of stearic acid and oleic acid of R. palustris was superior to those of Chlorella and yeasts, while that of other fatty acids tested was not. The amino acid profiles of the protein hydrolysate compared favorably with Food Agricultural Organization (FAO) guidelines. The biomass productivities from fed-batch experiments were found to be 50, 47 and 49 mg l 1 h 1 for linear, exponential, and sigmoidal feeding strategy, respectively. The maximum biomass productivity was found to be 112 mg l 1 h 1 in chemostat. Compared to growth in batch cultures, continuous fermentation yielded two times higher biomass productivity.