Transcriptional dynamics driving differentiation programs in the murine intestine
Creators: name: Vemuri, Kiranmayi
Publisher: name: No Publisher Supplied
Year: 2024

description: The mammalian intestinal epithelium is one of the most rapidly self-renewing tissues in the body. It comprises of villi which primarily contain absorptive enterocytes, and crypts which harbor adult stem and progenitor cells. Understanding transcriptional regulation in intestinal differentiation is imperative for overall intestinal health but remains incomplete. Intestinal differentiation could also serve as a model to understand differentiation mechanisms of other tissues. Terminal differentiation requires a massive restructuring of the transcriptome. During intestinal differentiation, the expression patterns of nearly 4000 genes are altered as cells transition from progenitor cells in crypts to differentiated cells in villi. In this study, dynamic recruitment of RNA Polymerase II (Pol II) to gene promoters was identified as the primary driver of transcriptomic shifts during intestinal differentiation in vivo. Changes in enhancer-promoter looping interactions accompany dynamic Pol II recruitment and are dependent upon HNF4, a pro-differentiation transcription factor. Using genetic loss-of-function, ChIP-seq and IP mass spectrometry, this study shows that HNF4 collaborates with chromatin remodelers and loop-stabilizing proteins and facilitates Pol II recruitment at hundreds of genes pivotal to differentiation. This study also shows alternate mechanisms which drive differentiation gene expression. We find that pause-release of Pol II and post-transcriptional mRNA stability also regulate smaller subsets of differentially expressed genes. These studies provide insights into the mechanisms of differentiation in a renewing adult tissue.In addition, this thesis delves into the role of two transcription factors, the Estrogen Related Receptor alpha (ESRRA) and Castor Zinc Finger 1 (CASZ1), that govern specialized functions within the villus epithelium. The rising prevalence of obesity underscores the urgent need for new therapeutic strategies. Current treatments, such as lifestyle modifications, pharmacotherapy, and bariatric surgery, have limitations in terms of efficacy, accessibility, and sustainability. Understanding the genetic and molecular mechanisms of obesity can lead to targeted therapies that address specific metabolic dysfunctions. Studies have shown that germline loss of Esrra confers resistance to high-fat diet (HFD)-induced obesity. However, using germline Esrra mutants in most studies obscures the tissue-specific, spatial, and temporal aspects of ESRRA's involvement in diet-induced obesity. Among various tissues in the body, Esrra is highly expressed in the GI tract and kidney. This finding is significant because the intestine plays a central role in dietary lipid metabolism and is a major target of existing weight loss therapies, such as GLP-1 agonists and orlistat. Given the intestine's crucial role, we investigated whether intestinal ESRRA contributes to diet-induced obesity. By creating a conditional, intestine-specific Esrra mutant mouse model, we found that intestinal mutants did not exhibit the same resistance to obesity as seen in germline mutant animals, with the disparity particularly pronounced in males. A feeding experiment revealed that germline Esrra mutant mice consumed less food compared to control or intestinal Esrra mutants. This discovery is consistent with prior research indicating that knockdown of Esrra activity in the medial pre-frontal cortex decreases feeding and promotes resistance to HFD-induced obesity, implying that this resistance could stem from neurological impairments. These findings underscore a role for ESRRA in resisting HFD-induced obesity that is independent of the intestine. This study enhances our understanding of the tissue-specific modulation of ESRRA, providing crucial insights into efforts aimed at addressing the growing burden of metabolic diseases worldwide.Villus zonation refers to the phenomenon where enterocytes display distinct zonation patterns and undergo trans-differentiation as they migrate along the villus axis. Gene expression along the villus forms a continuum, with different genes and functions being enriched in specific zones along the villus. Hence, enterocytes at the base of the villus function differently compared to those at the tip. However, the regulation of villus zonation at the transcriptional level is incompletely understood. We present the transcription factor CASZ1 as a crucial regulator of villus zonation and its associated functions. CASZ1 is enriched in its expression at the villus tip and upon exposure to Bone Morphogenetic Protein 2 (BMP2), a crucial mediator of epithelial differentiation, CasZ1 expression is heightened in enteroids. Moreover, through the creation of an intestine-specific CasZ1 mutant mouse model, we demonstrate that loss of CasZ1 triggers an increase in BMP2 signaling and a decrease in BMP1 signaling. Our investigation into CASZ1's functions has unveiled its impact on a highly specific role at the villus tip: the breakdown of extracellular ATP (eATP) into adenosine within the intestinal lumen. The absence of CASZ1 disrupts the activity of enzymes crucial for eATP processing, leading to an accumulation of eATP. This accumulation could arise from irregularities in cell shedding or apoptosis at the villus tip, potentially triggering inflammation as eATP buildup is linked to an inflammatory phenotype. While our model does not exhibit a severe inflammatory phenotype, the loss of CASZ1 might predispose the intestine to inflammatory conditions, which could manifest following infection or injury. Further research is necessary to comprehensively grasp its effects, but CASZ1 likely plays a pivotal role in preserving the health and functionality of the intestinal epithelium. This study marks the first characterization of CASZ1 in the small intestinal epithelium, revealing it to be a BMP responsive locus and indicating its role in sustaining crucial functions of the differentiated villus epithelium.