Mechanisms of gene expression

National Institute Of Arthritis And Musculoskeletal And Skin Diseases

Investigators

Linked publications & trials

Abstract

Transcription is regulated by DNA elements known as enhancers, where transcription factors bind to specific DNA motifs. Before genes are turned on, enhancers become accessible; understanding the controlled accessibility and generation of active enhancers has been a longstanding interest of this lab. Highly regulated and dynamic gene expression in immune cells is essential for host defense and limiting immunopathology. Multiple factors contribute to activation and state-specific transcription including modulation of chromatin architecture. Nipped-B was identified through a Drosophila genetic screen as a key factor promoting long-range enhancer-promoter communication. Nipped-B-Like (NIPBL) haploinsufficiency in humans causes Cornelia DeLange syndrome (CdLS). Precisely how NIPBL contributes to gene expression is unclear. Our current work combines CRISPR-Cas9-mediated gene editing, high-resolution microscopy, genomics, and mass spectrometry to dissect the role of NIPBL in transcriptional regulation. Genome-wide binding profiles of NIPBL and its binding partner MAU2, revealed a substantial overlap with promoters, enhancers and super enhancers, mirroring profiles of the active transcription mark H3K27Ac in higher eukaryotes. Using alignment tools and AlphaFold predictions, we discovered that higher organisms have evolved a large intrinsically disordered region (IDR) at the N-terminus of NIPBL, in contrast to the conserved structured region at its C-terminus. Notably, the NIPBL IDR ranked within the top 1% of IDR lengths among the entire mouse proteome. Single-molecule tracking and confocal microscopy revealed that NIPBL IDR forms dynamic hubs, particularly around transcriptionally active regions, and contributes to increasing its chromatin residence time in cells. IDR-specific interactions include RNA-Polymerase II core complex subunits RPABC1 and RPABC5, transcription coactivator Mediator complex subunits MED1, MED14 and MED19, Integrator subunits, as well as transcription factors. Consequently, finely resolved temporal transcriptome profiling upon treatment with physiologically relevant stimulus (IFN) revealed either an absence or a significantly reduced magnitude of response genes in NIPBL-IDR-deleted B cells, as opposed to wildtype cells. Taken together, our findings reveal the importance of NIPBL in fine-tuning the magnitude of an induced gene response in B cells via an IDR-mediated hub formation and concentration of transcription machinery at regulatory elements. In addition to dissecting the functional evolution of NIPBL IDR, our findings discover a novel role for NIPBL in transcription regulation, distinct from its well-studied function in genome architecture. Future studies using a NIPBL haploinsufficiency mouse-model and CdLS patient lymphoblastoid cell lines will elucidate tissue-specific effects underlying disease etiology. Although the Mediator (MED) coactivator complex is well-appreciated in transcriptional regulation, the mechanisms underlying its role in activator-dependent transcription has been less clear. In this work, the modulation of metazoan MED interaction with RNA polymerase II (RNA Pol II) and antagonistic effects of the Mediator complex subunit 26 (MED26) subunit and the CDK8 kinase module (CKM) was investigated. Analysis revealed that the CKM blocks binding of the RNA Pol II carboxy-terminal domain (CTD), preventing RNA Pol II interaction. Structural findings revealed that PolII CTD interaction with MED is mediated by a large intrinsically disordered region (IDR) in MED13 that blocks MED26 and CTD interaction. Cells endure tens of thousands of DNA-damaging events daily. Consequently, defects in double-stranded break (DSB) repair promote genomic instabilities that lead to cancer, genetic disorders, immunological diseases, and developmental defects. Cohesin, a multi-subunit protein complex that generates architectural loops through chromatin extrusion, mediates both chromosome organization and DNA repair. This year we made advances in understanding how cohesin regulates these distinct processes. Specifically, we identified two discrete roles for cohesin in DNA repair in mammalian cells: 1. An intrinsic architectural function, which prevents long range interactions between DNA breaks, and 2. an architecture independent function triggered by ATM phosphorylation of SMC1 that accelerates DNA end joining. We found that these two activities function in a complementary fashion to minimize translocations and deletions associated with non-homologous end joining. One aspect of dynamic gene expression is discontinuous transcription, an evolutionarily conserved and fundamental feature of gene regulation. Analyses of bursting transcriptome-wide have focused on the role of cis-regulatory elements; however, the precise mechanisms underlying transcriptional bursting remain poorly understood and other factors that regulate this process remain elusive. This year we applied mathematical modeling to single-cell RNA sequencing data to infer bursting dynamics transcriptome-wide under multiple conditions to identify possible molecular mechanisms. We found that MED26 primarily regulates frequency, MYC regulates burst size, while cohesin and Bromodomain-containing protein 4 (BRD4) can modulate both. These results indicate that later steps in the initiation of transcriptional bursts are primary nodes for integrating gene networks in single cells. Based on the findings described, we sought to determine the relevance of transcriptional regulation and the impact on chemotherapeutic agents, cell senescence, and drug resistance. Specifically, we investigated the transcriptomic alterations induced by doxorubicin (DOX), a commonly used chemotherapeutic agent, in human colon cancer cells. Using single-cell RNA sequencing, we identified distinct cell populations characterized by differential expression of genes involved in cell cycle regulation and interferon (IFN) signaling. DOX-persisting proliferating cells exhibited upregulation of genes induced by the unphosphorylated form of ISGF3 (U‐ISGF3) transcription factor. Further analysis revealed that U‐ISGF3 drives the expression of target genes by modulating the number of mRNA produced per transcriptional burst, potentially contributing to chemoresistance. Importantly, we found high upregulation of HSH2D, a poor prognostic marker, in doxorubicin-resistant U-ISGF3-dependent cells. These findings provide insights into the molecular mechanisms underlying chemotherapy response and highlight potential targets for overcoming chemoresistance in colon cancer. Germinal Center (GC) B cell reactions are important for host defense, as well as diseases from autoimmunity to lymphoma. Given the complexities of transcriptional control in GC B cells, we sought to take an unbiased view. To identify molecular targets, we fluorescently labeled 47 key B cell genes and performed genome-wide sgRNA screens coupled to fluorescence-activated cell sorting to detect global activators and repressors of their gene expression. In total, we identified 4,440 independent gene regulators and established a complex regulatory network with 17,638 connections. Extensive network analysis revealed that Target of Rapamycin Complex 1 (TORC1) signaling serves as a central hub explaining, directly or indirectly, up to 40% of the hits in our dataset. We recapitulated the results from the screens using rapamycin, a potent TORC1 inhibitor. We detected a strong increase in immune response gene expression upon prolonged rapamycin treatment and further identified crosstalk between TORC1, glycogen synthase kinase 3 (GSK3), E3 ubiquitin ligases, and CTDSPL2 phosphatase. We discovered that a FOXO1 inhibitor is a potent inhibitor of GSK3A and GSK3B. These findings provide clues for treatment of diseases caused by dysregulated B cells. In addition, we have made efforts to understand the chromatin architecture of key cytokine genes including the extended interferon gamma (IFNG) and its long noncoding RNA (IFNGAS-1) and the extended IL4, IL13, and IL5 locus. We have generated multiple mouse lines to define the functionality of critical enhancer elements that are shared by mouse and humans.