Mtg16-dependent repression of E protein activity is required for early lymphopoiesis

Abstract

The ETO/MTG family of transcriptional co-repressors play a key role in adult stem cell functions in various tissues. These factors are commonly found in complex with E proteins such as E2A, HEB, and Lyl1 as well as PRDM14 and BTB/POZ domain factors. Structural studies identified a region in the first domain of MTGs that is conserved in the Drosophila homologue Nervy (Nervy Homology Domain-1, or NHR1) that is essential for ETO/MTG8 to inhibit E protein-dependent transcription. The Cancer Genome Atlas (TCGA) identified cancer associated single nucleotide variants (SNVs) near the MTG16:E protein contact site. We tested these SNVs using sensitive yeast two-hybrid association assays, which suggested that only P209T significantly affected E protein binding. We then used CRISPR-Cas9 and homology directed DNA repair to insert P209T and a known inactivating mutation, F210A, into NHR1 of Mtg16 in the germ line of mice. These mice developed normally, but in competitive bone marrow transplantation assays, the F210A-containing stem cells failed to contribute to lymphopoiesis, while P209T mutant cells were reduced in mature T cell populations. High content fluorescent activated analytical flow cytometry assays identified a defect in the multi-potent progenitor to common lymphoid progenitor transition during lymphopoiesis. These data indicate that the cancer associated changes are likely benign polymorphisms, and the MTG:E protein association is required for lymphopoiesis, but less important for myelopoiesis and stem cell functions.

MTG16 (CBFA2T3) regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors

“…MTG16 was also upregulated in active IBD, characterized by repeated cycles of injury and regeneration, and likely contributes to murine colon regeneration following DSS-induced injury. To investigate the mechanism underlying these phenotypes, we utilized a novel mouse model with a single point mutation in MTG16, Mtg16 P209T , that disrupts MTG16 binding to the E proteins E2A and HEB 38 . Mtg16 T/T mice homozygous for this MTG16:E protein uncoupling mutation largely recapitulated Mtg16 -/lineage allocation and regeneration phenotypes, although the effect of MTG16 loss in dysplasia appears to only partially be due to increased E protein activity.…”

“…Mtg16 P209T mutant mice were CRISPR-generated as recently described 38 . Littermate-matched wild type (WT) and Mtg16 -/and littermate-matched WT and homozygous Mtg16 P209T mutant (Mtg16 T/T ) mice were bred using heterozygous x heterozygous breeding schemes and housed in the same facility throughout the duration of the experiments.…”

“…the SI. Transcriptional corepressors, including MTG16, regulate differentiation and cell-fate decisions [25][26][27][28][37][38][39]79,80 . Prior studies have established that Mtg16 -/mice have fewer goblet cells in the SI but retain normal numbers of enteroendocrine cells 33,81 .…”

“…We tested this hypothesis in vivo by selectively disrupting the MTG16:E protein interface. To do so, we leveraged a CRISPR-generated mouse with a point mutation resulting in a proline-to-threonine substitution in MTG16 (Mtg16 P209T ) that impairs MTG16:E2A and MTG16:HEB binding and transcriptional repression 38 . We then characterized colonic secretory cell populations in homozygous Mtg16 P209T mutant (Mtg16 T/T ) mice compared to WT littermatematched controls (Fig.…”

“…One family of transcription factors repressed by MTG16 are basic helix-loop-helix (bHLH) transcription factors 25,28,[37][38][39] . Ubiquitously expressed (class I) and tissue-specific (class II) bHLH transcription factors homo-and heterodimerize to control stem cell dynamics, lineage allocation, and differentiation [40][41][42] .…”