Myb Controls Intestinal Stem Cell Genes and Self-Renewal

Abstract

Rapid advances have been made in the understanding of how the highly proliferative gastrointestinal tract epithelium is regulated under homeostasis and disease. The identification of putative intestinal stem cell (ISC) genes and the ability to culture ISC capable of generating all four lineages plus the architecture of small intestinal (SI) crypts has been transformative. Here, we show that transcription factor Myb governs ISC gene expression, particularly Lgr5. Lgr5 is associated with cells that have the capacity to generate all cell lineages in SI organoid cultures and colorectal cancer cells, which overexpress Myb. Furthermore, Wnt signaling and Myb cooperate in maximal Lgr5 promoter activation while hypomorphic Myb (plt4/plt4) mice have decreased Lgr5 expression. After ionizing radiation (IR), ISC genes are elevated; but in plt4/plt4 mice, this response is substantially subdued. ISC genes bmi-1 and olfm4 are expressed at subnormal levels in plt4/plt4 mice, and bmi-1 is induced with IR to half the level in mutant mice. dcamkl-1 and olfm4 failed to recover after IR in both wild-type (wt) and mutant mice. Although not considered as an ISC gene, cyclinE1 is nevertheless used to assist cells in the emergence from a quiescent state (an expectation of ISC following IR) and is overexpressed after IR in wt mice but does not change from a very low base in plt4/ plt4 mice. Self-renewal assays using organoid cultures and inducible Myb knockout studies further highlighted the dependence of ISC on Myb consistent with role in other stem cell-containing tissues.

Defective Myb Function Ablates Cyclin E1 Expression and Perturbs Intestinal Carcinogenesis

“…We have previously shown that cell-cycle re-entry, progression, and proliferation appear retarded within the colonic crypts of Myb Plt4/Plt4 hypomorphic mutant mice, which correlated with a significant reduction in Ccne1 expression (14,15). These data suggested that the cell-cycle defects observed in the Myb hypomorphs may be due, in part, to reduced Ccne1 expression.…”

“…The propagation and derivation of CT26, MC38, YAMC, and NIH3T3 cell lines have been described in detail elsewhere (14). Lentiviral small hairpin RNA particles (CyclinE1; NM007633/ TRCN0000077777) were purchased from Sigma.…”

“…Protocols have been described in detail previously (14). Primers used in the chromatin immunoprecipitation (ChIP) analyses are described elsewhere (Supplementary Table S2).…”

“…pCMV E2F-1 was a gift of Dr Patrick Humbert (PMCC). Protocols for CAT reporter assays and qRT-PCR analysis have been described previously (14). Primers used for qRT-PCR are described (Supplementary Table S2).…”

“…As a control, we used an IgY antibody directed against glutathione S-transferase (GST). Both were affinity purified on antigen columns and have been characterized previously (14). Binding of Myb correlated with active chromatin (AcH3K27 mark) in WT crypts ( Fig.…”