Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10

Mediator and cohesin connect gene expression and chromatin architecture

Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator–cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.National Institutes of Health (U.S.) (Fellowship)Canadian Institutes of Health Research (Research Fellowship)National Institutes of Health (U.S.) (Grant R01 HG002668

Evidence for Transcript Networks Composed of Chimeric RNAs in Human Cells

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5′ and 3′ transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network

Isolation and characterization of human orthologs of yeast CCR4-NOT complex subunits

The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially com­plement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologousto, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4-NOT comple

Isolation and characterization of human orthologs of yeast CCR4–NOT complex subunits

The yeast CCR4–NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially complement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4–NOT complex

A high resolution protein interaction map of the yeast Mediator complex

Mediator is a large, modular protein complex remotely conserved from yeast to man that conveys regulatory signals from DNA-binding transcription factors to RNA polymerase II. In Saccharomyces cerevisiae, Mediator is thought to be composed of 24 subunits organized in four sub-complexes, termed the head, middle, tail and Cdk8 (Srb8-11) modules. In this work, we have used screening and pair-wise two-hybrid approaches to investigate protein–protein contacts between budding yeast Mediator subunits. The derived interaction map includes the delineation of numerous interaction domains between Mediator subunits, frequently corresponding to segments that have been conserved in evolution, as well as novel connections between the Cdk8 (Srb8-11) and head modules, the head and middle modules, and the middle and tail modules. The two-hybrid analysis, together with co-immunoprecipitation studies and gel filtration experiments revealed that Med31 (Soh1) is associated with the yeast Mediator that therefore comprises 25 subunits. Finally, analysis of the protein interaction network within the Drosophila Mediator middle module indicated that the structural organization of the Mediator complex is conserved from yeast to metazoans. The resulting interaction map provides a framework for delineating Mediator structure–function and investigating how Mediator function is regulated