The intestinal crypt houses diverse proliferative cells including highly proliferative Transit Amplifying cells, less proliferative active intestinal stem cells (ISCs), and infrequently dividing quiescent ISCs. Evidence indicates ?plastic? properties are embodied in these cell types, but little is currently known about how these cells interconvert or decide to fully lineage commit. Studies from the Magness Lab found the transcription factor Sox9 to be expressed at different levels in crypt-based cells ranging from very high in non- or slowly-dividing cells (Sox9high), intermediate in aISCs (Sox9low), and lowest in TA cells (Sox9sublow). Sox9 affects proliferation rate: Sox9 knockout (Sox9KO) mice have hyperproliferation in crypts, and Sox9 over-expression (Sox9OE) blocks proliferation, which can be reversed when Sox9-levels return to endogenous levels. Preliminary data indicates that decreased proliferation upon Sox9OE does not a result from cell death or differentiation, instead showing more cells in the G1 cell cycle phase upon Sox9OE. The central hypothesis of this proposal is that Sox9 levels regulate proliferation in ISCs by modulating the length of the G1 cell cycle phase. If empirically supported, this concept has important implications for understanding mechanisms ISCs employ to make cell fate decisions and interconvert between active and reserve states.
All aims will use a novel strategy for transfecting cultured Sox9KO mouse and human ISCs with an inducible Sox9OE allele to allow for reintroduction of physiological levels of Sox9 expression in otherwise identical Sox9KO ISCs.
Aim 1 will explore the transcriptomic effects different Sox9 levels have on genes involved in the cell cycle.
Aim 2 will use the new two-reporter PIP-FUCCI construct to precisely quantify changes in cell cycle phase lengths induced by different Sox9 levels via live-imaging in freely growing cells.
Aim 3 will test whether Sox9 lengthens G1 phase by increasing time to the cell cycle restriction point and test whether this results from Sox9 inducing Rb1 expression, as indicated by preliminary data.
These aims will shed light on previously uncovered effects of how Sox9 regulates the cell cycle and will also give insight into a possible mechanism for how ISCs maintain their varied proliferation rates in homeostasis and injury. This work will be performed in the Magness Lab in the Center for Gastrointestinal Biology and Disease (CGIBD) at the University of North Carolina at Chapel Hill. The CGIBD is a well-established NIH-funded program known for cultivating the careers of young GI investigators. In this outstanding training environment, I will gain expertise in intestinal stem cell biology and learn new techniques in primary cell/organoid culture, single-cell biology/genomics, and bioinformatics. My career development training plan focuses on mentorship skills, grantsmanship, networking with the greater gastrointestinal, stem cell, and cell cycle communities, and didactic opportunities that will promote my path to independence as an academic scientist.
Renewal of the intestinal epithelium is an intestinal stem cell (ISC)-driven mechanism that relies on a critical balance of ISC proliferation and differentiation to maintain the ISC compartment and functionally diversify the epithelium. Levels of the Sox9 transcription factor differentially mark crypt cells with variable cell cycle lengths, and Sox9 regulates proliferation of crypt-based cells and is required for reserve ISC function following irradiation injury. This proposal seeks to investigate Sox9-dependent mechanisms regulating ISC cell cycle, which will have implications for better understanding ISC lineage fate decisions, radiosensitivity, and epithelial regeneration dynamics.
