Mechanisms of gene expression and recombination

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

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Abstract

Transcriptional regulation is the means whereby cells orchestrate expression of individual genes or group of genes. Recent studies with single cells have shown that, for the most part, transcription is not a continuous activity but it occurs in bursts. The amount of RNA produced for each gene is directly proportional to the amplitude and frequency of such bursts. How these two parameters are controlled is not totally clear, but it appears that DNA sequence information at promoters determine the amplitude, while burst frequencies rely on sequence information at enhancers. Much has been learned on transcriptional bursting through the study of individual genes. However such experiments are time consuming and do not provide a global view of gene expression in cell populations or tissues. To address this deficiency, this fiscal year we have developed a computer model to analyze single cell transcriptomes. This approach provides for the first time a comprehensive view of bursting in a cell population without the need to label individual genes with fluorescent molecules. Our goal moving forward is to use this strategy with cells deficient for proteins known to play key roles in transcription to determine whether they regulate bursting. Transcription is regulated by DNA elements known as enhancers, where transcription factors bind to specific DNA motifs. Before genes are turn on, enhancers become accessible through the removal of methyl groups on DNA. This activity has been shown to induce DNA damage. This fiscal year we have shown in collaboration with Andre Nussenzweig (NCI) that such damage in neurons may be responsible for the development of neurodegenerative diseases in humans. An important feature of transcription regulation is the combinatorial action of transcription factors at active regulatory DNA. This fiscal year, we explored transcription factor cooperativity in mammals by analyzing 500 mouse and human primary cells, combining an atlas of TF motifs, footprints, binding (ChIP-seq), transcriptomes (RNA-seq), and accessibility (ATAC-seq). We uncovered two transcription factor groups that colocalized with most expressed factors, forming stripes in hierarchical clustering maps. The first group included lineage-determining factors that occupied DNA elements broadly, consistent with their key role in tissue-specific transcription. The second one, dubbed universal stripe factors (USFs), comprised 30 SP, KLF, EGR, and ZBTB family members that recognized overlapping GC-rich sequences in all tissues analyzed. Knockouts and single-molecule tracking revealed that USFs imparted accessibility to colocalized partners and increased their residence time. Another important feature of transcription is its control of recombination in maturing B cells. In the bone marrow for instance, transcription facilitates the recombination of antibody variable genes to D and J segments to produce the V(D)J portion of antibodies. This fiscal year, our laboratory has continue exploring how transcription of these genes facilitate the physical pairing of V, D and J segments during recombination.