MTG16 regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors

Aberrant epithelial differentiation and regeneration contribute to colon pathologies, including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear. Here, we identified MTG16 as a central mediator of epithelial differentiation, promoting goblet and restraining enteroendocrine cell development in homeostasis and enabling regeneration following dextran sulfate sodium–induced (DSS-induced) colitis. Transcriptomic analyses implicated increased Ephrussi box–binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a mouse model homozygous for a point mutation that attenuates MTG16:E protein interactions (Mtg16P209T), we showed that MTG16 exerts control over colonic epithelial differentiation and regeneration by repressing E protein–mediated transcription. Mimicking murine colitis, MTG16 expression was increased in biopsies from patients with active IBD compared with unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16-null colon in the azoxymethane (AOM) /DSS-induced model of CAC, indicating that MTG16 protects from tumorigenesis through additional mechanisms. Collectively, our results demonstrate that MTG16, via its repression of E protein targets, is a key regulator of cell fate decisions during colon homeostasis, colitis, and cancer. Overall design: Mtg16-null mice (C57/BL6 background) and homozygous P209T (T/T) mice (C57/BL6 background) were bred using heterozygous x heterozygous breeding schemes in independent colonies. All mice were at least 8 weeks old. WT and Mtg16-null or WT and T/T mice were appropriately littermate-matched in each analysis. For baseline analyses, RNA-sequencing was performed on proximal (5 cm from cecum) and distal (5 cm from anus) murine colon crypt isolates (chelation and RNA extraction protocol described below) from untreated mice. Independent experiments and differential expression analyses: 2 Mtg16-null vs. 2 WT proximal homeostatic colon crypt isolates, 4 Mtg16-null vs. 4 WT homeostatic distal colon crypt isolates, 2 T/T vs. 2 WT homeostatic proximal colon crypt isolates, and 4 T/T vs. 3 WT homeostatic distal colon crypt isolates. As a secondary analysis, WT samples were pooled from these experiments to compare baseline gene expression in distal vs. proximal homeostatic colon. For analyses post-DSS injury-repair, mice were treated with DSS for 5 days and allowed to recover for 4 days as described below. RNA-sequencing was performed on distal colon crypts isolated following sacrifice. Independent experiments and differential expression analyses: 4 Mtg16-null vs. 3 WT distal colon crypt isolates post-DSS injury-repair, 3 T/T vs. 3 WT distal colon crypt isolates post-DSS injur-repair. As a secondary analysis, distal colon crypt isolates from WT mice matched to P209T mice post-DSS injury-repair were compared to homeostatic distal colon crypt isolates from WT mice matched to P209T mice. For AOM/DSS tumor analyses, mice were treated with AOM followed by 3 cycles of DSS injury repair as described below. Distal tumors were harvested at sacrifice. RNA-sequencing was performed on 3 T/T vs. 3 WT distal colon tumors.