Abstract
A stem cell’s epigenome directs cell fate during development, homeostasis, and regeneration. Epigenetic dysregulation can lead to inappropriate cell fate decisions, aberrant cell function, and even cancer. The histone variant macroH2A has been shown to influence gene expression, guide cell fate, and safeguard against genotoxic stress. Interestingly, mice lacking functional macroH2A histones (hereafter referred to as macroH2A DKO) are viable and fertile; yet suffer from increased perinatal death and reduced weight and size compared to wildtype (WT). Here, we ask whether the ostensible reduced vigor of macroH2A DKO mice extends to intestinal stem cell (ISC) function during homeostasis, regeneration, and oncogenesis. Lgr5-eGFP-IRES-CreERT2 or Hopx-CreERT2::Rosa26-LSL-tdTomato ISC reporter mice or the C57BL/6J-Apcmin/J murine intestinal adenoma model were bred into a macroH2A DKO or strain-matched WT background and assessed for ISC functionality, regeneration and tumorigenesis. High-dose (12Gy) whole-body γ-irradiation was used as an injury model. We show that macroH2A is dispensable for intestinal homeostasis and macroH2A DKO mice have similar numbers of active crypt-base columnar ISCs (CBCs). MacroH2A DKO intestine exhibits impaired regeneration following injury, despite having significantly more putative reserve ISCs. DKO reserve ISCs disproportionately undergo apoptosis compared to WT after DNA damage infliction. Interestingly, a macroH2A DKO background does not significantly increase tumorigenesis in the Apcmin model of intestinal adenoma. We conclude that macroH2A influences reserve ISC number and function during homeostasis and regeneration. These data suggest macroH2A enhances reserve ISC survival after DNA damage and thus confers functional robustness to the intestinal epithelium.
Highlights
The intestinal epithelium is the most highly proliferative mammalian tissue
Our study demonstrates that the histone variant macroH2A, despite being dispensable during intestinal homeostasis and of limited overall influence on intestinal adenoma growth, bestows the intestinal stem cell (ISC) compartment with functional robustness, by providing resistance to genotoxic stress
We use Hopx-CreERT2 to mark reserve ISCs as we and others have shown this population to be molecularly and functionally overlapping with other reserve ISC markers including Bmi1-CreER and mTert-CreER, and single cell expression profiles indicate that the Hopx-CreERT2 population is more homogenous that the commonly used Bmi1-CreER marker.[4, 8,9,10, 12, 13]
Summary
Its rapid turnover and tremendous regenerative capacity following injury necessitate a robust and highly organized ISC compartment. ISCs are located within the intestinal crypt where they self-renew and produce progenitors, which in turn proliferate and terminally differentiate along the crypt-villus axis prior to being shed into the lumen. To accommodate this rapid turnover and respond to environmental cues, the intestine is served by at least two functionally distinct ISC populations, including the fast-cycling CBCs and slow-cycling reserve ISCs.[1]. CBCs are marked by expression of Wnt-responsive G-protein coupled receptor Lgr, are driven to actively proliferate by canonical Wnt pathway activity, strongly contribute to intestinal homeostasis[2, 3] and are ablated by γ-irradiation.[3,4,5,6,7] In contrast, reserve ISCs are rare, largely quiescent, radioresistant, and can be marked by CreER reporter genes inserted into the Bmi, or Hopx loci, as well as by transgenes driven by the mTert and Lrig promoters.[4, 8,9,10,11,12,13,14] Following DNA damage and CBC loss, reserve ISCs awaken en masse and play a critical role in epithelial regeneration–in part by producing CBCs.[4, 15, 16] Epigenetic mechanisms governing the identities of these two classes of ISCs have not been investigated
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