Computational study of Gene Regulatory Mechanisms
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Normal cell function is ensured by harmonious gene expression networks. Gene expression is orchestrated by multi-level modulation from DNA to proteins such as chromatin structure, transcription initiation, RNA processing, mRNA stability, translation, and post-translational modifications etc. Here we studied the role of three-dimensional (3D) organization of genome and post-transcriptional regulation, specifically, intron retention, on gene expression. With the advances in experimental techniques and next-generation sequencing, we can probe the 3D architecture of the genome in detail. The backbone of the 3D genome structure is to a large extent organized by architectural proteins, of which the CCCTC-binding factor (CTCF) is a critical component. However, how does gene expression changes upon loss of CTCF in terms of genome structure across different cell types is not well studied. Topologically associating domains (TADs) are the basic structures and functional units. CTCF are enriched at the TAD boundaries to maintain the structure. In chapter 1, we investigated how the expression of genes inside TADs changes after loss of CTCF. We found a general pattern that upon CTCF loss, up-regulated differentially expressed genes (DEGs) are relative enriched towards the center of TADs, whereas the down-regulated DEGs towards the boundaries. In systems with additional epigenomic data, we found similar pattern in differential chromatin accessibility (ChrAcc). Moreover, CTCF exhibited significant binding overlap with regions of decreased ChrAcc, whereas those with increased ChrAcc are enriched with different transcription factors (TFs), including those with cell-type specificity. Our findings suggest that dynamic changes in chromatin accessibility, transcription factor binding, and CTCF loss at TAD boundaries contribute to the distinct gene expression changes observed within TADs. Exhausted CD8 T (Tex) is always associated with chronic infections and cancer because they lose the effector function. With chronic viral infection, the immune system cannot eliminate the virus. The virus causes continuous stimulation of T cells, leading to their exhaustion/dysfunction. Tex has two major subsets that exhibit different expression levels of Tcf1, a master transcription factor in CD8 T cells. Tcf1+ TEX-prec cells that can maintain the Tex cell pool and Tcf1– TEX-eff cells that can fight viruses but do not persist. Whether TEX cell fate bifurcation is programmed by 3D genome topology and how much affected by CTCF are not studied yet. In chapter 2, we investigated how 3D genome organization programs TEX cell differentiation and the role of CTCF during the process. We demonstrated that TEX cell fate bifurcation is programmed early on the level of 3D genome topology by forming different Chromatin Interaction (ChrInt) hub that are in concordance with transcriptomic diversification between TEX-prec and TEX-eff cells. Further, through identifying ChrInt hubs we identified novel regulators in TEX-eff differentiation, including EZH2 and Id2. We demonstrated that CTCF prevents excessive activation of the exhaustion program and stemness genes. Altogether, for the first time, we demonstrated 3D genome organization programs TEX cell differentiation and the pivotal role of CTCF. Spliceosomopathies, a group of disorders caused by defects in the splicing machinery, frequently affect the craniofacial skeleton and limb. However, the molecular mechanism underlying this tissue-specific sensitivity remains unclear. Splicing factors are core components of splicing machinery. They are further controlled by post-translational modifications, among which arginine methylation is among the most frequent. To determine the splicing mechanisms in cranial neural crest cells (CNCCs), which give rise to the majority of craniofacial skeleton, we focused on an upstream regulator for splicing proteins responsible for arginine methylation, protein arginine methyltransferase one (PRMT1). PRMT1 is the highest expressing enzyme of the PRMT family in CNCCs and is important in craniofacial development. In chapter 3, we uncovered roles of PRMT1 in CNCCs in the regulation of intron retention, a type of alternative splicing where introns are retained in the mature mRNA sequence. Mandibular primordium of Prmt1-deficient embryos demonstrated an increase in intron-retaining mRNA of matrix genes, which triggered Nonsense-mediated decay, a process that finds and destroys defective mRNA transcripts by recognizing a premature termination codon, causing a reduction in matrix transcript expression. We further identified SFPQ as a substrate of PRMT1 that depends on PRMT1 for arginine methylation and protein expression in the developing craniofacial structures. Depletion of SFPQ in CNCCs phenocopied PRMT1 deletion in that matrix, Wnt signaling components and neuronal gene transcripts contained higher IR and exhibited lower expression. We further recognized gene length as a common feature among SFPQ-regulated genes in CNCCs. Altogether, these findings demonstrate that the PRMT1-SFPQ pathway modulates matrix Wnt signaling components and neuronal gene expression via intron retention in CNCCs during craniofacial development
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