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 with 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–/– colon in the azoxymethane/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.


Introduction
The colonic epithelium is a complex, self-renewing tissue composed of specialized cell types with diverse functions (1). Stem cells at the base of the colon crypt divide and differentiate into absorptive and secretory cells. Conversely, colonic epithelial regeneration in response to injury occurs through dedifferentiation of committed absorptive and secretory lineage cells (2)(3)(4). Secretory lineage dysregulation is implicated in 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 with a point mutation that attenuates MTG16:E protein interactions (Mtg16 P209T ), 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 -/colon in the azoxymethane/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.
Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor that regulates cell fate decisions by bridging transcription factors and chromatin modifiers to repress transcription of target loci (11)(12)(13)(14). Repression targets of MTG16 vary depending on the expression gradients of other components of the repression complex (14,15). 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.
In this study, we defined the topology of Mtg16 expression in the colon and discovered functions of MTG16 in the colonic epithelium. We used an MTG16:E protein uncoupling point mutant mouse model to demonstrate that the mechanism driving these phenotypes was repression of E protein transcription factors by MTG16. We tested the functional impact of this regulatory relationship in murine models of IBD and colitis-associated cancer (CAC) and correlated our observations with patient data. Overall, we demonstrate potentially novel, context-specific roles for MTG16 in colonic epithelial lineage allocation and protection from colitis that depend on its repression of E protein-mediated transcription.

MTG16 is expressed in goblet cells in human and murine colon.
To understand the topography of MTG16 expression in the human colon, we queried single-cell RNA-Seq (scRNA-Seq) data sets generated from 2 independent cohorts of normal human colon samples (10). Interestingly, we observed strong MTG16 expression in goblet cells ( Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.153045DS1). To understand if this expression pattern was evolutionarily conserved, we queried our inDrop scRNA-Seq generated from WT murine colon crypts (34,35) and similarly found that Mtg16 was expressed in goblet and enteroendocrine clusters ( Figure 1B and Supplemental Figure 1B). RNAscope for Mtg16 and Muc2, the predominant mucin expressed by colonic goblet cells (36), demonstrated strong colocalization in the murine colonic epithelium ( Figure 1C).
Loss of MTG16 distorts colonic secretory cell differentiation. We next assessed secretory lineage allocation in the Mtg16 -/homeostatic colon epithelium, which had not previously been investigated to our knowledge ( Figure 1, D-F). Mtg16 -/mice had fewer goblet and tuft cells but, unexpectedly, increased synaptophysin-positive (SYP + ) enteroendocrine cells per colon crypt ( Figure 1E). In contrast, and in agreement with prior studies (16,37), Mtg16 -/mice had fewer goblet and tuft cells, but no increase in enteroendocrine cells, in the SI (Supplemental Figure 2). These data implicated a colon-specific role for MTG16 in orchestrating cell type allocation within the secretory lineage.
Prior studies have demonstrated subtle differences in secretory cell differentiation along the length of the colon (36,(38)(39)(40)(41). Thus, we stratified our human scRNA-Seq data sets by proximal and distal colon and examined MTG16 expression. MTG16 was consistently expressed in goblet cells, but its expression pattern in other secretory cell types varied (Supplemental Figure 3). We next queried the Tabula Muris (42), a murine colon scRNA-Seq data set that specifies whether certain clusters originate from the proximal or distal colon, for Mtg16 expression. In the proximal colon, Mtg16 was enriched in all goblet cell clusters (Supplemental Figure 4, A and B). In the distal colon, Mtg16 was enriched in early progenitor cells still expressing the stem cell marker Lgr5 (Lgr5 + , amplifying, undifferentiated cells) and most goblet cells, except those located at the top of the crypt (Supplemental Figure 4, A and B). Bulk RNA-Seq of proximal and distal WT colon revealed higher Mtg16 expression in the proximal colon, potentially reflecting its higher goblet cell density (Supplemental Figure 4C). We confirmed this Mtg16 expression pattern by RNAscope (Supplemental Figure 5).
Due to these differences, we performed separate bulk RNA-Seq analyses of Mtg16 -/proximal and distal colon epithelial isolates. In both colon segments, goblet-associated genes were downregulated, and enteroendocrine-associated genes were upregulated, compared with WT ( Figure 1, G and H). However, goblet cell-associated gene downregulation was more pronounced in the proximal colon ( Figure 1G), while enteroendocrine cell-associated gene upregulation was more striking in the distal colon ( Figure  1H). Closer inspection of the distal Mtg16 -/colon revealed marked upregulation of class II bHLH transcription factors required for enteroendocrine cell differentiation (Neurog3 and Neurod1) and enteroendocrine cell hormone products (chromogranin A, Chga; Gcg; Sct) ( Figure 2A). We further investigated the distal colon using gene set enrichment analysis (GSEA) with custom gene sets derived from the literature (described in Supplemental Tables 3 and 4). GSEA of distal colon RNA-Seq demonstrated significant enrichment of all stages of enteroendocrine cells during differentiation except the earliest progenitor cells, in which expression of Neurog3, which is required for enteroendocrine cell differentiation (43)(44)(45)(46), had not yet been induced (45) ( Figure 2B). Concurrently, the goblet cell gene signature was significantly de-enriched in the absence of MTG16 ( Figure 2B).
Notably, Mtg16 -/versus WT RNA-Seq did not show significant differences in the stem cell genes Lgr5 and Ascl2, distinct from Mtg16 -/-SI (16). Additionally, GSEA using gene sets representing stem cells and other early progenitors (Supplemental Tables 3 and 4) did not demonstrate enrichment of any gene sets representing Lgr5 + crypt base columnar stem cells (CBCs) ( Figure 2C and Supplemental Figure 6A). We did observe enrichment of gene sets representing Bmi1 + and mTert + "reserve" stem cells ( Figure 2C), recently shown to be enteroendocrine cells expressing Chga (47). Although CHGA + cells were increased overall, they were not localized to the crypt base (including the +4/+5 cell position) ( Figure 2D). Altogether, these data indicate that MTG16 promotes goblet cell differentiation, at the expense of enteroendocrine cell differentiation, during colonic secretory lineage allocation.
MTG16-driven goblet cell differentiation is dependent on its repression of E protein-mediated transcription. We next sought to illuminate potential mechanisms by which MTG16 promotes the goblet lineage and restricts the enteroendocrine lineage. We and others have previously shown that MTG16 negatively regulates major transcription factor effectors of the WNT and Notch pathways in the hematopoietic system (15,48) and SI (16). Our RNA-Seq revealed upregulation of the Notch ligand Dll3 ( Figure  1H and Figure 2A). However, several WNT pathway inhibitors (Wif1, Cxxc4) and the Notch pathway inhibitor Neurl1a were also significantly upregulated ( Figure 2A). We thus performed GSEA for the canonical WNT and Notch pathways, as well as other pathways important in colonic epithelial homeostasis and differentiation (1). GSEA did not identify global canonical WNT or Notch pathway enrichment or de-enrichment in Mtg16 -/homeostatic colon crypts (Supplemental Figure 6B). We next investigated whether the target genes of known MTG16-interacting transcription factors, including E proteins (14,19,20), were dysregulated in Mtg16 -/colon crypts. As stated previously, E proteins coordinate lineage specification and differentiation by interacting with cell type-specific class II bHLH transcription factors (21). The E proteins E12, E47, and HEB are known to be expressed in SI crypts and adenomas (49), and E12 and E47 bind to NEUROD1 to induce transcription of secretin in the duodenum (50). Here, in Mtg16 -/colon crypts, we observed significant enrichment of all E protein gene signatures available in the Molecular Signatures Database (MSigDB) (51) ( Figure 2E and Supplemental Table 3). Gene sets associated with upregulation of Id1 and Id2, proteins that counteract E protein activity, were not enriched ( Figure 2E). These data demonstrated a strong induction of E protein-mediated transcription in the absence of repression by MTG16. Thus, we hypothesized that MTG16 drives goblet cell differentiation from secretory progenitors by inhibiting E protein-mediated transcription of key enteroendocrine genes.
We next tested this hypothesis by selectively modulating MTG16-mediated repression of E protein transcriptional activity in vivo. To do so, we CRISPR-generated a mouse with a point mutation resulting in a proline-to-threonine substitution in MTG16 (Mtg16 P209T ) that attenuates MTG16:E2A and MTG16:HEB binding and functionally reduces MTG16-mediated repression of E protein transcriptional activity (52) (Supplemental Figure 7 WT controls, though tuft cell frequency per crypt was unchanged ( Figure 3, A-C). We next performed bulk RNA-Seq on Mtg16 T/T proximal and distal colon crypt isolates. In both colon segments, goblet cell genes were downregulated compared with WT, while enteroendocrine cell genes were upregulated compared with WT, in a pattern resembling that of Mtg16 -/colon ( Figure 3, D and E). Notably, the distal colon exhibited significant upregulation of Neurog3 ( Figure 3E). Additionally, GSEA of the overall  Table 3). Individual GSEA plot of the E47 target gene signature at right. NES, normalized enrichment score (ES); Tag %, the percentage of gene hits before (for positive ES) or after (for negative ES) the peak in the running ES, indicating the percentage of genes contributing to the ES. FDR q < 0.05 is considered significant.
enteroendocrine signature, key enteroendocrine progenitor genes, and Bmi1 + and mTert + stem cell signatures all largely phenocopied the Mtg16 -/distal colon (Figure 3, F and G, and Supplemental Tables 3 and  4). Confirming that the MTG16 P209T mutation functionally results in increased E protein-mediated transcription in the colonic epithelium, the gene sets representing E2A and HEB transcriptional targets were also enriched in Mtg16 T/T colonic epithelial isolates ( Figure 3H).  Tables 3 and 4). Tag %, the percentage of gene hits before (for positive ES) or after (for negative ES) the peak in the running ES, indicating the percentage of genes contributing to the ES. FDR q < 0.05 is considered significant.
MTG16 is upregulated in patients with active IBD. There has been considerable interest in the role of secretory cell lineage misallocation and dysfunction in the pathogenesis of IBD, which includes ulcerative colitis (UC) and Crohn's disease and is defined by continuous epithelial injury and regeneration (8,36,(53)(54)(55)(56)(57). Additionally, several large RNA-Seq studies of human UC biopsies have recently been published, including the Predicting Responses to Standardized Pediatric Colitis Therapy (PROTECT) study (58,59) and mucosal biopsies from patients with active UC, patients with UC in remission, and healthy controls (60). We found that MTG16 was upregulated in patients with UC in both RNA-Seq data sets ( Figure 5, A and B). MTG16 expression decreased with remission yet remained elevated compared with unaffected controls ( Figure 5B). Last, a microarray data set from pediatric colon biopsies (61) demonstrated upregulation of MTG16 in both UC and Crohn's colitis ( Figure 5C). Collectively, these data show that increased MTG16 expression is correlated with active injury and regeneration in IBD.
MTG16 contributes to colon crypt regeneration following DSS-induced colitis. As stated previously, MTG16 deficiency increases disease severity in DSS-induced colitis (17), a mouse model of IBD (schematic in Figure 5D) that recapitulates histologic features of human UC (62). We first assessed Mtg16 expression in DSS-induced colitis to determine whether it would recapitulate the increased MTG16 expression in human IBD. Because DSS treatment largely affects the distal colon (62), we compared distal WT colon crypt epithelial isolates after DSS-induced injury and regeneration with WT distal colon crypts at baseline. Indeed, Mtg16 expression was significantly increased in regenerating colon crypts ( Figure 5E), similar to human IBD. Thus, we posited that MTG16 expression may be critical for epithelial regeneration in colitis.
The cellular sources of intestinal repair have been a major focus of the field over the last several decades. Recently, Murata et al. demonstrated that any CBC progeny cell can dedifferentiate and repopulate ablated CBCs by inducing expression of Ascl2 (2). After CBC ablation, 238 significantly upregulated genes defined the dedifferentiating ASCL2 + cells compared with resting CBCs. Interestingly, Mtg16 was one of these few significantly upregulated genes (log 2 [fold change] = 2.05, P adj = 0.004) (2). Furthermore, ASCL2 ChIP-Seq data in the dedifferentiating population (2) yielded a peak at a second TSS of Mtg16, suggesting that ASCL2 induces Mtg16 expression during colonic epithelial regeneration ( Figure 5F).
To confirm that the ASCL2-driven regeneration program described by Murata et al. was relevant to crypt regeneration following DSS-induced injury, we constructed a gene set consisting of the 238 genes upregulated in Ascl2 + regenerating cells (2) (Supplemental Tables 3 and 4), performed GSEA, and observed significant enrichment of this signature in WT distal colon crypts after DSS-induced regeneration ( Figure 5G). Conversely, this regeneration signature was significantly de-enriched in Mtg16 -/colon crypts following DSS-induced injury, despite enrichment of other gene signatures associated with intestinal epithelial regeneration ( Figure 5, H and I, and Supplemental Tables 3 and 4) (63-67). Together, these data implicate MTG16 as a component of ASCL2-driven regeneration and repair in the colon. Similar to our findings in colon homeostasis, E protein transcriptional signatures were enriched in post-DSS Mtg16 -/colon by GSEA ( Figure 5, J and K), leading us to hypothesize that the contribution of MTG16 to colon crypt regeneration occurs via repression of E protein-mediated transcription.
Attenuation of MTG16:E protein interactions disables epithelial regeneration following DSS-induced colitis. To test whether MTG16-dependent regeneration occurs via an E protein-dependent mechanism, we next analyzed colonic epithelial regeneration following DSS-induced injury ( Figure 6A) in Mtg16 T/T versus WT mice. Like Mtg16 -/mice, Mtg16 T/T mice developed worse colitis and poor regeneration measured by increased weight loss ( Figure 6B), decreased colon length ( Figure 6C), and increased histologic injury-regeneration score ( Figure 6D and Supplemental Table 2) compared with WT. Mtg16 T/T mice also exhibited increased extent of injury along the length of the Mtg16 T/T colon ( Figure 6E). Next, we injected mice with 5-ethynyl-2′-deoxyuridine (EdU) 1 hour prior to sacrifice following DSS injury and regeneration and found that Mtg16 T/T mice had fewer S-phase cells in injury-adjacent crypts (Figure 6F), indicating a proliferation defect in the crypts that actively regenerate the ulcerated colonic epithelium following DSS-induced colitis (68). Finally, like we observed in our Mtg16 -/analyses, RNA-Seq of Mtg16 T/T distal colon crypt epithelial isolates following DSS injury and regeneration was de-enriched for the ASCL2-mediated dedifferentiation signature despite the enrichment of other gene signatures associated with regeneration ( Figure 6, G and H, and Supplemental Tables 3 and 4). These data suggest that the effect of MTG16 loss on ASCL2-mediated colonic epithelial regeneration is directly or indirectly due to increased E protein transcription targets.
MTG16 expression is decreased in dysplasia. Because MTG16 expression was upregulated in IBD, we wondered whether MTG16 expression would be increased in IBD-driven colon cancer (CAC) compared with sporadic colorectal cancer (CRC). We previously observed decreased MTG16 expression in sporadic CRC and CAC compared with normal colon tissue (18) but had not directly compared expression levels of MTG16 between these different neoplastic origins. To do so, we first confirmed our previous findings in sporadic CRC using DESeq2 on raw counts generated by Rahman et al. (69) from The Cancer Genome Atlas (TCGA) (70) ( Figure 7A). We then analyzed MTG16 expression in bulk RNA-Seq of CRC and CAC tumors (71) and found no significant difference in MTG16 expression ( Figure 7B), indicating that MTG16 expression is reduced in colon dysplasia regardless of whether it is driven by IBD.

MTG16-mediated protection from CAC is partially dependent on MTG-mediated repression of E protein transcription factors.
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). Like Mtg16 -/mice, Mtg16 T/T mice did exhibit increased histologic injury, tumor multiplicity, and tumor size compared with WT (Figure 7, D-G). Interestingly, Mtg16 T/T mice had more tumors and larger tumors in the middle and proximal colon than their WT counterparts (Figure 7, H and I). This was consistent with DSS-induced injury extending further toward the proximal colon ( Figure 6E). These data suggest that this phenotype could be driven by increased proximal colon injury. However, unlike Mtg16 -/mice, Mtg16 T/T mice did not have significantly more weight loss ( Figure 7J Figure 8C), no difference in F4/80 + intratumor macrophages (Supplemental Figure 8E), a subtle decrease in tumor-infiltrating CD3 + T cells (Supplemental Figure 8F), and no decrease in infiltrating B220 + B cells (Supplemental Figure 8G). 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).  Finally, we performed RNA-Seq on distal colon tumors from littermate-and cage-matched WT and Mtg16 T/T mice to test whether Mtg16 T/T AOM/DSS tumors would exhibit the increased canonical WNT signaling tone that we previously demonstrated in Mtg16 -/-AOM/DSS tumors (18). On the contrary, RNA-Seq demonstrated differential expression of only 1 WNT-related gene, the WNT receptor Fzd9 (Figure 7N), and GSEA did not demonstrate enrichment of the canonical WNT signaling pathway (unpublished observation). We did observe increased expression of enteroendocrine-associated genes, potentially attributable to the epithelial phenotype at baseline ( Figure 7N). GSEA using the oncogenic signatures found in the MSigDB demonstrated deranged cell cycle control and KRAS signaling, though many conflicting gene sets were enriched (unpublished observation). Overall, these data indicate that Mtg16 T/T mice recapitulate few Mtg16 -/-AOM/DSS phenotypes, suggesting that MTG16 protects the colonic epithelium from tumorigenesis through additional mechanisms.

Discussion
In this study, we investigated the mechanism underlying context-specific functions of the transcriptional corepressor MTG16. We identify what we believe are previously unappreciated roles for MTG16 as a critical regulator of colonic secretory cell differentiation and active colon regeneration. To define the mechanism underlying these phenotypes, we utilized a mouse model with a single point mutation in MTG16, MTG16 P209T , that attenuates direct binding between MTG16 and the E proteins E2A and HEB (52). Mtg16 T/T mice homozygous for this MTG16:E protein uncoupling mutation largely recapitulated Mtg16 -/lineage allocation and regeneration phenotypes, though the effect of MTG16 loss in inflammation-driven dysplasia appears to partially be due to increased E protein activity. Thus, these data indicate potentially new roles for MTG16:E protein complexes in colonic epithelial homeostasis, regeneration, and tumorigenesis.
First, we demonstrated a potentially novel role for MTG16 in colonic epithelial homeostasis. MTG16 has been shown to repress stem, goblet, and enteroendocrine cell genes to drive enterocyte differentiation in the SI (16). However, in accordance with numerous studies demonstrating important differences between SI and colon biology (2,36,(72)(73)(74)(75), Mtg16 -/colonic epithelial isolates did not exhibit increased stem cell transcriptional signatures or an overall decrease in secretory genes. Instead, we observed specific upregulation of E protein transcriptional signatures and the enteroendocrine lineage. This could be due to the fact that MTG16 repression targets, and its effects on them, vary in different tissues and cell types due to expression gradients of its binding partners (14). Another potential explanation for the difference between SI and colonic phenotypes is that SI secretory progenitor cells that express Neurog3, the class II bHLH transcription factor required for enteroendocrine cell differentiation (43,44), may still differentiate into goblet and Paneth cells, while colonic secretory progenitor cells that express Neurog3 are fully committed to the enteroendocrine lineage in the colon (46,73). Compared with WT, Mtg16 -/colon crypts demonstrated increased expression of Neurog3 by RNA-Seq, increased Neurog3 + secretory progenitor cells per colon crypt by RNAscope, and significant enrichment (FDR q < 0.05) of gene sets representing all enteroendocrine lineage cells starting at the Neurog3 + enteroendocrine progenitor stage. Importantly, expression of Neurog3 is transient and does not continue as enteroendocrine cells progress through differentiation (45). Thus, we believe the transcriptomic changes we observed by bulk RNA-Seq are not simply a reflection of the phenotypic decrease in differentiated enteroendocrine cells we observed by IHC for SYP and immunofluorescence staining for CHGA.
Next, we mechanistically probed the extent to which MTG16:E protein interactions drive MTG16-associated lineage allocation in the colon using a mouse model homozygous for an amino acid substitution in MTG16 (MTG16 P209T ) (52). Strikingly, with this single amino acid change, Mtg16 T/T mice recapitulated Mtg16 -/colonic secretory phenotypes, including increased enteroendocrine cells, increased Neurog3 expression by RNA-Seq, increased Neurog3 + progenitors, enrichment of gene sets representing all enteroendocrine lineage cells starting at the Neurog3 + enteroendocrine progenitor stage, and decreased goblet cells. Analysis of a publicly available SI ChIP-Seq data set (16) indicated MTG16 occupancy of an E box-rich region at the Neurog3 promoter. From these data, we believe that MTG16 prevents the differentiation of secretory progenitor cells toward the enteroendocrine cell lineage, enabling differentiation of the goblet cell lineage, by repressing E protein oligomers at the Neurog3 promoter. MTG16 could be repressing E protein homo-oligomers at this locus, as E2A homodimers have recently been found to promote neural transcriptional signatures (Supplemental Tables 3 and 4 and citations in the supplement). (G-K) Tag %, the percentage of gene hits before (for positive ES) or after (for negative ES) the peak in the running ES, indicating the percentage of genes contributing to the ES. FDR q < 0.05 is considered significant.   differentiation and increased Neurog3 expression and in pluripotent cells, even under nonpermissive conditions (31). MTG16 could also be repressing E protein:class II bHLH transcription factor hetero-oligomers activating transcription of the Neurog3 locus. One class II bHLH transcription factor known to bind to the Neurog3 promoter is atonal bHLH transcription factor 1 (ATOH1) (76). However, MTG16 has been shown to bind ATOH1 transcriptional targets involved in the differentiation of multiple secretory lineages, including Spdef, the transcription factor known for initiating goblet cell differentiation (16). It is possible that the enteroendocrine-promoting effect of increased E protein activity is amplified following increased induction of Neurog3. NEUROG3 induces transcription of Neurod1, the next class II bHLH transcription factor in the enteroendocrine transcription factor cascade (45,77), probably through heterodimerization with E proteins, which has been demonstrated in pancreatic endocrine cells (78,79). In turn, NEUROD1: E protein complexes induce transcription of other enteroendocrine transcription factors and hormones (50,77). On the contrary, SPDEF is not a bHLH transcription factor, has not been shown to bind to E proteins, and has not been shown to be repressed by MTG16. Additionally, complex regulatory relationships are at play in these transcription factor cascades, as Mtg16 itself has been shown to be a transcriptional target of E proteins in the hematopoietic system (1) and ATOH1 in both SI and colon (29).

JCI
Another transcription factor recently shown to be involved in SI and colonic secretory lineage allocation is retinoic acid receptor alpha (RARα). Jijon et al. demonstrated that Vil1-Cre RARα fl/fl mice develop increased goblet cells in the SI and decreased enteroendocrine cells in both the SI and colon (80). Recently, Steinauer et al. demonstrated that MTG16 regulates histone acetylation and chromatin accessibility of myeloid-specific enhancers to reduce expression of RARα target genes in human acute myeloid leukemia cells (81). This mechanism could explain increased colonic enteroendocrine cells in the setting of MTG16 loss but does not entirely explain the colonic goblet cell phenotype observed in our study. Additionally, GSEA of our bulk transcriptomic data did not reveal upregulation of RARα target genes or the RAR pathway in Mtg16 -/or Mtg16 T/T colonic epithelial isolates (unpublished observation). Studies have shown that RARα antagonizes E protein activity by inducing transcription of ID1 and ID2 in human acute promyelocytic leukemia cells treated with all-trans-retinoic acid (82) and ID1 and ID3 gene expression in normal human keratinocytes (83). Thus, it is possible that downstream effects of the RAR pathway counteract the effects of MTG16:E protein uncoupling in our bulk RNA-Seq data. Finally, another non-E protein-mediated pathway involved in secretory lineage allocation is the noncanonical, β-catenin-independent WNT/planar cell polarity (PCP) pathway, which may enable enteroendocrine cells to differentiate directly from "unipotentially primed" intestinal stem cells (84). The WNT/PCP pathway was mildly enriched (FDR q value = 0.11) in Mtg16 -/-, but not Mtg16 T/T , colon crypts. This could be responsible for the slight difference in magnitude between Mtg16 -/and Mtg16 T/T phenotypes. These are interesting areas of further investigation.
An area of considerable interest in gastrointestinal biology is the relationship between epithelial differentiation and regeneration (4). Although it was long thought that dedicated Bmi1 + or mTert + "reserve" stem cells at the +4/+5 position replenished the stem cell compartment in intestinal epithelial regeneration, recent data indicate that these cells are enteroendocrine cells that can dedifferentiate into stem cells following injury (47). Recently, "committed" progenitors from both the secretory and absorptive lineages were demonstrated to share this ability through induction of ASCL2 (2). In this study, Ascl2 + dedifferentiating cells were isolated from the colonic epithelium following CBC ablation (2). Interestingly, these cells were not enriched for Clu or fetal epithelial genes (2), which were previously thought to enhance intestinal epithelial response to injury (63)(64)(65)(66)(67). Mtg16 was one of the few genes upregulated in these regenerating cells, and ChIP-Seq demonstrated an ASCL2 peak at a second TSS in the Mtg16 locus (2). Demonstrating relevance to DSS-induced colon regeneration, both Mtg16 and the Ascl2 + regenerating cell signature were upregulated in regenerating WT colon epithelium compared with baseline. MTG16 was also upregulated in multiple IBD patient cohorts and even decreased with remission, during which the colon epithelium is closer to homeostatic renewal. Finally, RNA-Seq and GSEA of Mtg16 -/and Mtg16 T/T colonic epithelial isolates following DSS-induced injury demonstrated enrichment of "revival" stem cell signatures and fetal epithelial signatures but de-enrichment of the Ascl2 + regenerating cell signature. These data indicate an active, E protein-mediated role for MTG16 in colon regeneration and provide a potential explanation for defective colonic epithelial regeneration in Mtg16 -/mice despite an apparent baseline enrichment of Bmi1 + and mTert + "reserve" stem cells. Future work is necessary to determine which E protein complexes and gene targets are repressed by MTG16 in Ascl2 + dedifferentiating cells. One possibility is that ASCL2-induced Mtg16 expression forms a negative feedback loop in which MTG16 attenuates stemness so that not all cells in the crypt dedifferentiate into Lgr5 + stem cells. This is supported by our previous work demonstrating that MTG16 negatively regulates major transcription factor effectors of the canonical WNT signaling pathway in the hematopoietic system (15), although we did not observe canonical WNT pathway activation by transcriptomic analyses of the homeostatic or regenerating Mtg16 -/colonic epithelia. Instead, ASCL2 could be inducing Mtg16 expression to prevent dedifferentiating cells from expressing Neurog3, reversing course, and differentiating toward the enteroendocrine lineage. Notably, although ASCL2 is a class II bHLH transcription factor, our study does not attempt to address whether MTG16 inhibits ASCL2:E protein complexes downstream of Ascl2-induced Mtg16 expression. These are interesting areas of future investigation.
Finally, we tested the role of MTG16:E protein interactions in inflammatory carcinogenesis. We previously showed that Mtg16 -/mice develop greater tumor burden than their WT counterparts with AOM/DSS treatment, and Mtg16 -/tumors exhibit increased WNT tone (18). Although we did observe increased histologic injury, tumor burden, and tumor size in Mtg16 T/T mice, Mtg16 T/T tumors did not recapitulate the higher grade of dysplasia observed in Mtg16 -/tumors or enrichment in the WNT signaling pathway. Thus, disabling one known function of MTG16 was not sufficient to fully replicate the Mtg16 -/phenotype. Indeed, our group recently showed that the effects of MTG16 loss on CAC development are dependent on Kaiso (ZBTB33) (85). Additionally, E proteins have been shown to be both antitumorigenic and protumorigenic in colon cancer via epithelium-intrinsic and -extrinsic effects, including attenuation of the canonical WNT signaling pathway (86)(87)(88). Future studies are necessary to determine additional MTG16 binding partners and repression targets responsible for suppressing carcinogenesis.
In conclusion, we discovered key functions of MTG16 in colonic secretory lineage allocation and regeneration following colitis dependent on its repression of E proteins. Confirming translational relevance, we determined that MTG16 is upregulated in patients with active IBD, reduced with restitution, and decreased in dysplasia. Thus, MTG16 may be a candidate biomarker for disease activity or a target to modulate differentiation and regeneration.

Methods
Animal models. Mtg16 -/mice were previously generated and backcrossed to a C57BL/6J background (89). Mtg16 P209T mutant mice were generated by the Vanderbilt Genome Editing Resource (VGER) using CRISPR/Cas9-mediated dsDNA cleavage followed by homology-directed repair. Briefly, 50 ng/μL guide RNA (5′ TTTGTTATCCCTTTTCTGAAGG 3′), 200 ng/μL of 119 bp single-stranded donor DNA (5′ GTTTCATGCCAAGCTCCAGGAAGCCACCAACTTTCCACTGAGGCCGTTTGTTAT-AACTTTTCTGAAAGTAAGCGAAAGCAGCACCTTCCAGGCAACAGGGATGGTGTACA-CAAAGGCCC 3′), and 100 ng/μL Cas9 mRNA were microinjected into the cytoplasm of C57BL/6J mouse embryos. Embryos were implanted into the oviduct of pseudopregnant female mice. The resulting pups were screened for successful editing by PCR. PCR for the WT Mtg16 allele was performed using the primers SWH1432 (5′ AGCACCTGCCCCCGGCGTGCG 3′) and SWH1438 (5′ TTGCCT-GGAAGGTGCTGCTTTCGCTTACCTTCAGAAAAGGG 3′). PCR for the Mtg16 P209T mutant allele was performed using the primers SWH1432 and SWH1435 (5′ TTGCCTGGAAGGTGCTGCTTTC-GCTTACTTTCAGAAAAGTT 3′). Both reactions were performed using the following thermal cycle: 95°C for 5 minutes initial denaturation; 35 cycles of 95°C for 30 seconds, 61°C for 1 minute, and 72°C for 5 minutes; and 72°C for 10 minutes final extension (representative reactions in Supplemental Figure  7, A and B; full, uncropped gels available in online supplemental material). On-target edits in potential founders were validated by genomic sequencing of the donor DNA sequence and at least 200 bp of flanking genomic DNA. Founders were crossed with WT C57BL/6J mice to minimize any remaining off-target effects. Expression of full-length MTG16 (MTG16 P209T ) in Mtg16 T/T mice was confirmed by immunoblot (Supplemental Methods and Supplemental Figure 7C; full, uncropped gels available in online supplemental material). Littermate-matched WT and Mtg16 -/and WT and homozygous Mtg16 P209T mutant (Mtg16 T/T ) mice were bred in independent mouse colonies using heterozygous × heterozygous breeding schemes, housed in the same facility with a 12-hour light/12-hour dark cycle, and JCI Insight 2022;7(10):e153045 https://doi.org/10.1172/jci.insight.153045 pathologist blinded to genotype. Distal colon crypts were collected as described above for RNA isolation and RNA-Seq as described below. For EdU experiments, mice were i.p. injected with 50 mg/kg EdU (NE08701, Biosynth Carbosynth) in PBS 1 hour prior to sacrifice. Mice were weighed daily throughout all experiments.
Inflammatory carcinogenesis. Mice were prepared as described above and injected i.p. with 10 mg/kg AOM (A5486, MilliporeSigma) followed by three 4-day cycles of 3% (w/v) DSS (DB001, TdB Labs), each followed by 16 days of recovery. Mice were weighed daily. At sacrifice, colons were dissected, flushed with PBS, splayed longitudinally, and imaged using a Nikon SMZ1270 dissecting microscope. Tumors were identified by vascular, nondimpled appearance from these images in conjunction with direct visualization and measured macroscopically in 2 perpendicular dimensions using calipers. Tumor volume was calculated using the previously validated formula (W 2 × L)/2, where width (W) was the shorter caliper measurement, and length (L) was the longer caliper measurement (99). Representative tumors from each colon were dissected, incubated in RNAlater (R0901, MilliporeSigma) at 4°C for 24 hours, and stored at -80°C. Additional representative tumors were used to generate tumoroids (Supplemental Methods). Histologic injury and regeneration were assessed (using the scoring system in Supplemental Table 2). Grade of dysplasia was evaluated as previously described (100) by a gastrointestinal pathologist blinded to genotype.
Statistics. Statistical analyses of human CAC and sporadic CRC samples were performed using limma (v3.34.9) (110). Statistical analyses of all other bulk RNA-Seq were performed using DESeq2 (106). Statistical analyses of microarray data were performed using limma in Geo2R (97). P adj < 0.05 was considered significant. Statistical analyses for GSEA were performed using the GSEA program (v4.1.0). FDR (q value) < 0.05 was considered significant. All other statistical analyses (nonparametric, 2-tailed comparisons between 2 or more groups) were performed in GraphPad Prism (v9.0.1) as indicated in each figure legend. Data are displayed as arithmetic mean ± SEM unless otherwise noted. P < 0.05 was considered significant.
Study approval. All animal experiments were carried out in accordance with protocols approved by the Vanderbilt IACUC (Nashville, Tennessee, USA). Human CRC and CAC studies (71) were approved