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MTG16 attenuates epithelial proliferation. (A) Mtg16 mRNA expression (adjusted to Gapdh) and protein levels in different colorectal cancer (CRC) lines with matched human CRC and adjacent normal sample (compared with normal, triplicate samples). NSB, nonspecific band. (B) Immunoblot analysis of MTG16 expression in HCT116 cells, cell growth by MTS assay (triplicate samples) and proliferation as determined by BrdU incorporation 48 hours after plating (vector = 7, MTG16 = 7). (C) Mtg16 mRNA expression (adjusted to GAPDH, triplicate samples), protein levels, change in cell growth by MTS assay (triplicate samples) and cell count (quadruplicate samples) method after 96 hours of siRNA treatment in young adult mouse colon (YAMC) cells using 3 individual siRNAs and 1 pooled siRNA. (D) MTG16 protein levels in different clones (2, 3, and 15) of YAMC cells after CRISPR-mediated knockout of Mtg16, cell growth by MTS assay (triplicate samples) and cellular proliferation by BrdU incorporation (5 samples/each). Each experiment repeated at least 2 times. One-way ANOVA and Newman-Keuls post-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Scr, scrambled; V, vector; ns, not significant.
Source publication
MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16-/- mice have increased intestinal proliferation and are more sensitive to inte...
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... higher proliferation was observed in Mtg16 -/-tumors, we tested whether MTG16 could modify the growth of established CRC cells. We noted decreases in both MTG16 messenger RNA and protein in HT29, HCT116, SW480, and Caco2 CRC lines, similar to those observed in primary CRC and far lower than in the normal colon ( Figure 5A). Interestingly, in the HCT116 line, Mtg16 message levels were similar to those observed in normal colon; however, protein expression was absent, suggesting posttranscriptional mechanisms regulating MTG16 expression in this line. ...
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... in the HCT116 line, Mtg16 message levels were similar to those observed in normal colon; however, protein expression was absent, suggesting posttranscriptional mechanisms regulating MTG16 expression in this line. Ectopi- cally expressing MTG16 in HCT116 cells attenuated proliferation as determined by both MTS and BrdU incorporation assays ( Figure 5B and Supplemental Figure 7). To test the impact of MTG16 loss in normal epithelial cells, we reduced MTG16 levels in the young adult mouse colon (YAMC; see ref. 24) cell line via RNA interference and then reproduced by deletion via CRISPR/Cas9-mediated genome editing. ...
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... test the impact of MTG16 loss in normal epithelial cells, we reduced MTG16 levels in the young adult mouse colon (YAMC; see ref. 24) cell line via RNA interference and then reproduced by deletion via CRISPR/Cas9-mediated genome editing. Interfer- ing with MTG16 expression using individual siRNAs as well as pooled siRNAs markedly reduced MTG16 protein levels and augmented cell growth ( Figure 5C). We observed variation in MTG16 knockdown effi- ciency when using different siRNAs. ...
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... the other hand, siRNA 12 and pooled siRNAs were most efficient in reducing MTG16 levels and signifi- cantly increased cellular growth as demonstrated by cell counts and the MTS assay. In confirmatory studies, CRISPR/Cas9-mediated genome editing with 2 gRNAs targeting exon 4 of MTG16 yielded multiple clones with markedly reduced MTG16 levels ( Figure 5D) and consequently increased proliferation ( Figure 5D). Surprisingly, although gene editing was confirmed by sequence analysis, we observed residual immunoreac- tivity in CRISPR-edited clones, which are likely due to MTG family member cross-reactivity. ...
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... the other hand, siRNA 12 and pooled siRNAs were most efficient in reducing MTG16 levels and signifi- cantly increased cellular growth as demonstrated by cell counts and the MTS assay. In confirmatory studies, CRISPR/Cas9-mediated genome editing with 2 gRNAs targeting exon 4 of MTG16 yielded multiple clones with markedly reduced MTG16 levels ( Figure 5D) and consequently increased proliferation ( Figure 5D). Surprisingly, although gene editing was confirmed by sequence analysis, we observed residual immunoreac- tivity in CRISPR-edited clones, which are likely due to MTG family member cross-reactivity. ...
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... tumors comprise a heterogeneous cellular environment, we probed a CRC tissue micro- array using high-resolution in situ hybridization to determine if epithelial expression of MTG16 was reduced. While MTG16 is uniformly expressed in the epithelium of normal crypts (Supplemental Figure 5), its expres- sion was reduced by varying degrees in the epithelial component of the tumor (Figure 9C). MTG16 protein level was also decreased in comparison with normal tissue ( Figure 9D and Supplemental Fig- ure 6). ...
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Citations
... MTGR1 (Cbfa2t2) is a member of the three-protein myeloid translocation gene (MTG) family of transcriptional co-repressors, which were originally identi ed in translocation fusion proteins driving acute myeloid leukemia 11 . MTGs, also including MTG16 and MTG8, serve as scaffolding proteins that orchestrate the formation of repression complexes containing histone deacetylases, other co-repressor proteins, and DNA binding factors, thereby modifying chromatin state of key loci 12 [13][14][15][16] . Mtgr1 −/− mice are also exquisitely sensitive to dextran sodium sulfate (DSS)-induced injury, with marked depletion of viable, regenerative crypts post-injury 17 . ...
Undifferentiated intestinal stem cells (ISCs), particularly those marked by Lgr5 , are crucial for maintaining homeostasis and resolving injury. Lgr5 + cells in the crypt base constantly divide, pushing daughter cells upward along the crypt axis, where they differentiate into a variety of specialized cell types. This process requires coordinated execution of complex transcriptional programs, which allow for the maintenance of undifferentiated stem cells while permitting differentiation of the wide array of intestinal cells necessary for homeostasis. Thus, disrupting these programs may negatively impact homeostasis and response to injury. Previously, members of the myeloid translocation gene (MTG) family have been identified as transcriptional co-repressors that regulate stem cell maintenance and differentiation programs in multiple organ systems, including the intestine. One MTG family member, myeloid translocation gene related 1 (MTGR1), has been recognized as a crucial regulator of secretory cell differentiation and response to injury. However, whether MTGR1 contributes to the function of ISCs has not yet been examined. Here, using Mtgr1 -/- mice, we have assessed the effects of MTGR1 loss on ISC biology and differentiation programs. Interestingly, loss of MTGR1 increased the total number of cells expressing Lgr5 , the canonical marker of cycling ISCs, suggesting higher overall stem cell numbers. However, expanded transcriptomic analyses revealed MTGR1 loss may instead promote stem cell differentiation into transit-amplifying cells at the expense of cycling ISC populations. Furthermore, ex vivo intestinal organoids established from Mtgr1 null were found nearly completely unable to survive and expand, likely due to aberrant ISC differentiation, suggesting that Mtgr1 null ISCs were functionally deficient as compared to WT ISCs. Together, these results identify a novel role for MTGR1 in ISC function and suggest that MTGR1 is required to maintain the undifferentiated state.
... MTG16 represses stem cell genes and pan-secretory genes in the homeostatic small intestine (SI) (16). We have previously shown that MTG16 deficiency leads to increased injury in dextran sulfate sodium-induced (DSS-induced) colitis (17) and tumor burden in azoxymethane (AOM)/DSS-induced inflammatory carcinogenesis and that these were epithelium-specific phenotypes (18). However, the precise mechanisms by which MTG16 controls colonic homeostasis, regeneration, and tumorigenesis remain unknown. ...
... We previously showed that Mtg16 -/mice have increased mortality, tumor burden, tumor grade, and tumor cell proliferation and apoptosis, as well as alterations in the intratumor immune environment in AOM/DSS-induced CAC (18). We next tested whether the mechanism driving these phenotypes was E protein dependent by treating Mtg16 T/T mice with AOM/DSS ( Figure 7C). ...
... We did not observe a difference in intratumor Ly6B.2 + cells (Supplemental Figure 8D), which we did not evaluate in the Mtg16 -/-AOM/DSS mouse study. We did, however, observe a subtle increase in apoptotic epithelial cells per tumor (Supplemental Figure 8B), similar to our prior findings in Mtg16 -/tumors (18). The epithelial phenotypes in Supplemental Figure 8, A-C, were recapitulated in tumoroids derived from distal, but not proximal, WT and Mtg16 T/T tumors (Supplemental Figure 9). ...
Aberrant epithelial differentiation and regeneration contribute to colon pathologies including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). MTG16 (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 (DSS)-induced colitis. Transcriptomic analyses implicated increased E box-binding transcription factor (E protein) activity in MTG16-deficient colon crypts. Using a novel mouse model with a point mutation that attenuates MTG16:E protein interactions (Mtg16P209T), we established 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 to unaffected controls. Finally, uncoupling MTG16:E protein interactions partially phenocopied the enhanced tumorigenicity of Mtg16-/- 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.
... Previously, we have observed MTG16 downregulation in tumors from the AOM/DSS CAC model, yet Kaiso expression in this model has not yet been reported [21]. Therefore, we next assessed Kaiso (Zbtb33) and MTG16 (Cbfa2t3) expression to determine if the trends observed in sporadic CRC were maintained in inflammatory tumorigenesis. ...
... Expression was normalized to GAPDH and represented as fold change over a nontargeted shRNA control. *P < 0.05, **P < 0.01, ****P < 0.0001; Student's t test (a, b, left and middle panels, c), Spearman correlation (a, b, right panels), or one-way ANOVA with Tukey's correction (d) scores, and morbidity [21]. We next aimed to determine whether these phenotypes were dependent on Kaiso expression and whether the inverse MTG16:Kaiso gene expression was relevant to inflammatory tumor formation [12]. ...
... We have previously determined Mtg16 −/− intestinal epithelium displays altered proliferation, apoptosis, and secretory lineage allocation at baseline and in the setting of AOM/DSS [21,23,24]. As these phenotypes likely contribute to the augmented injury response observed in Mtg16 −/− AOM/DSS-treated mice, we next analyzed the effect of concurrent MTG16;Kaiso loss in colonic tissue at the conclusion of the AOM/DSS protocol (Fig. 3). ...
The myeloid translocation gene family member MTG16 is a transcriptional corepressor that relies on the DNA-binding ability of other proteins to determine specificity. One such protein is the ZBTB family member Kaiso, and the MTG16:Kaiso interaction is necessary for repression of Kaiso target genes, such as matrix metalloproteinase-7. Using the azoxymethane and dextran sodium sulfate (AOM/DSS) murine model of colitis-associated carcinoma, we previously determined that MTG16 loss accelerates tumorigenesis and inflammation. However, it was unknown whether this effect was modified by Kaiso-dependent transcriptional repression. To test for a genetic interaction between MTG16 and Kaiso in inflammatory carcinogenesis, we subjected single and double knockout (DKO) mice to the AOM/DSS protocol. Mtg16−/− mice demonstrated increased colitis and tumor burden; in contrast, disease severity in Kaiso−/− mice was equivalent to wild-type controls. Surprisingly, Kaiso deficiency in the context of MTG16 loss reversed injury and pro-tumorigenic responses in the intestinal epithelium following AOM/DSS treatment, and tumor numbers were returned to near to wild-type levels. Transcriptomic analysis of non-tumor colon tissue demonstrated that changes induced by MTG16 loss were widely mitigated by concurrent Kaiso loss, and DKO mice demonstrated downregulation of metabolism and cytokine-associated gene sets with concurrent activation of DNA damage checkpoint pathways as compared with Mtg16−/−. Further, Kaiso knockdown in intestinal enteroids reduced stem- and WNT-associated phenotypes, thus abrogating the induction of these pathways observed in Mtg16−/− samples. Together, these data suggest that Kaiso modifies MTG16-driven inflammation and tumorigenesis and suggests that Kaiso deregulation contributes to MTG16-dependent colitis and CAC phenotypes.
BACKGROUND & AIMS
Notch signaling maintains intestinal stem cells (ISCs). When ISCs exit the niche, Notch signaling among early progenitor cells at position +4/5 regulates their specification toward secretory vs enterocyte lineages (binary fate). The transcription factor ATOH1 is repressed by Notch in ISCs; its de-repression, when Notch is inactivated, drives progenitor cells to differentiate along the secretory lineage. However, it is not clear what promotes transition of ISCs to progenitors and how this fate decision is established.
METHODS
We sorted cells from Lgr5-Gfp knock-in intestines from mice and characterized gene expression patterns. We analyzed Notch regulation by examining expression profiles (by quantitative reverse transcription PCR and RNAscope) of small intestinal organoids incubated with the Notch inhibitor DAPT, intestine tissues from mice given injections of the γ-secretase inhibitor dibenzazepine, and mice with intestine-specific disruption of Rbpj. We analyzed intestine tissues from mice with disruption of the RUNX1 translocation partner 1 gene (Runx1t1, also called Mtg8) or CBFA2/RUNX1 partner transcriptional co-repressor 3 (Cbfa2t3, also called Mtg16), and derived their organoids, by histology, immunohistochemistry, and RNA sequencing. We performed chromatin immunoprecipitation and sequencing analyses of intestinal crypts to identify genes regulated by MTG16.
RESULTS
The transcription co-repressors MTG8 and MTG16 were highly expressed by +4/5 early progenitors, compared with other cells along crypt–villus axis. Expression of MTG8 and MTG16 were repressed by Notch signaling via ATOH1 in organoids and intestine tissues from mice. MTG8- and MTG16-knockout intestines had increased crypt hyperproliferation and expansion of ISCs, but enterocyte differentiation was impaired, based on loss of enterocyte markers and functions. Chromatin immunoprecipitation and sequencing analyses showed that MTG16 bound to promoters of genes that are specifically expressed by stem cells (such as Lgr5 and Ascl2) and repressed their transcription. MTG16 also bound to previously reported enhancer regions of genes regulated by ATOH1, including genes that encode delta-like canonical Notch ligand and other secretory-specific transcription factors.
CONCLUSIONS
In intestine tissues of mice and human intestinal organoids, MTG8 and MTG16 repress transcription in the earliest progenitor cells to promote exit of ISCs from their niche (niche exit) and control the binary fate decision (secretory vs enterocyte lineage) by repressing genes regulated by ATOH1.
