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. 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. Key publications in this area: 1- Ba et al. Nature, October 2020. In this manuscript we have shown in collaboration with Fred Alt (Harvard) that recombination of transcribed antibody gene segments is driven by the nuclear architectural protein CTCF. This activity ensures that B cells express a broad range of antibody receptors with which pathogens are recognized during infection. 2- Li et al. Nature, February 2021. Also in collaboration with Fred Alt, in this manuscript we showed that the orientation of the antibody gene locus is key to proper recombination (its inversion blocks joining). We also found that the architectural protein WAPL is depleted from B cells presumably to avoid its interference with recombination. 3- Zhao et al. Nature Communications, March 2021. In this report, in collaboration with Francisco Asturias (University of Colorado) we present a high-resolution structure of Mediator, a multi-subunit protein complex that activates the enzyme (RNA polymerase II) responsible for gene transcription. 4- Wu et al. Nature May 2021. In collaboration with Andre Nussenzweig (NCI) we have shown that neurons accumulate high levels of single-strand DNA breaks at enhancers, a feature that might explain why patients with single-strand break repair develop neurodegenerative diseases.