Exploring transcriptional control of metabolism in human intestinal stem cells to inform tissue engineering
Creators: name: Dixit, Michelle
Publisher: name: The University of North Carolina at Chapel Hill University Libraries
Year: 2024

description: Introduction: Mitochondria are vital for intestinal stem cell (ISC) homeostasis, partially since they house oxidative phosphorylation, a major energy producing metabolic pathway. Studies discuss a Altered mitochondrial function is shown in diseases like inflammatory bowel disease (IBD), and many studies have explored these mechanisms by deleting mitochondrial  proteins. While these studies show mitochondrial dysfunction can directly induce disease phenotypes, by impairing mitochondria directly, these models pose challenges for identifying upstream transcription factor (TF) networks that regulate these defects physiologically. 
Early data and observations strongly suggest that SRY-Box Transcription Factor 9 (SOX9), a TF not known to directly regulate mitochondria, influences metabolism in human ISCs. SOX9, as a pioneer factor, is known to mediate TF environments by opening heterochromatin. Hence, we are using this platform to investigate the effects of SOX9 expression on ISC metabolism, and to explore identify other candidate TFs involved in mitochondrial regulation and dysfunction in human ISCs.

Methods: Mitochondrial activity was tracked using dyes for total and active mitochondria. Mitochondria and proteins involved in mitophagy were quantified in wildtype (WT) and SOX9 overexpression (SOX9OE) ISCs using confocal microscopy, western blots (WB), and quantitative polymerase chain reaction (qPCR). Multi-omic data (RNASeq and ATACseq) were used to choose TFs involved in mitochondrial regulation and had higher expression and binding motif accessibility in increased SOX9 expression. 

Results: Confocal microscopy showed SOX9OE cells had less total and active mitochondria, and increased LAMP1, an autophagy protein, than WT. qPCR performed on FACS-separated WT and SOX9OE cells revealed that SOX9OE cells expressed 65.9% of CO2 and 60.1% of D-Loop (mitochondrial DNA) compared to WT. WB showed SOX9OE cells had noticeably less of the protein TOMM20, a total mitochondria indicator, compared to WT cells. Multiomic data highlight 11 TFs with higher expression and target accessibility in SOX9OE ISCs.

Discussion: Mitochondrial activity appears to decrease with increasing SOX9 expression, as shown by the active mitochondria dye. This decrease is likely in part due to mitochondrial count as qPCR, WB, and confocal microscopy all show WT ISCs have higher total mitochondria than SOX9OE ISCs. Mitophagy also likely plays a role as increased LAMP1 is observed on or near mitochondria in SOX9OE cells, indicating that SOX9-driven mitophagy in SOX9OE cells likely contributes to their reduced count and activity compared to WT. As SOX9 expression is increased in subsets of IBD and mitochondrial repression is common in IBD, SOX9OE ISC monolayers may serve as a platform for observing metabolic changes that may lead to IBD. Based on multi-omic, data, and literature analyses, we have engineered constructs to analyze STAT1, FOXO4, IRF3 and BARX2 as potential TFs working downstream of SOX9 to affect mitochondrial function in ISCs and are currently engineering ISCs to overexpress these candidate factors.