Biology subjects

Stengel, K. R.

Publications and source records attributed to Stengel, K. R..

4 recordsLinked to original sources

bioRxiv2021

MTG16 (CBFA2T3) represses E protein-dependent transcription to regulate colonic secretory cell differentiation, epithelial regeneration, and tumorigenesis

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 disrupts MTG16:E protein complex formation (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 only 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/467178v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@21948borg.highwire.dtl.DTLVardef@9f60eeorg.highwire.dtl.DTLVardef@194b9fdorg.highwire.dtl.DTLVardef@13eb1a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2021

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, andRNA polymerase pause release at select gene targets

Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues1. Thus, it is difficult to achieve a mechanistic understanding of transcription factor function using traditional genetic deletion or RNAi methods, because these slow approaches make it challenging to distinguish direct from indirect transcriptional effects. Here, we used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO12-6 to define how the t(2;13)(q35;q14) disrupts normal gene expression programs to trigger cancer. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses, we identified a core transcriptional network that rapidly collapsed upon PAX3-FOXO1 degradation. Moreover, loss of PAX3-FOXO1 impaired RNA polymerase pause release and transcription elongation at regulated gene targets. The activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective and often only a single element within a complex super-enhancer was affected. In addition, fusion of the endogenous PAX3-FOXO1 with APEX2 identified proteins in close proximity with PAX3-FOXO1, including ARID1A and MYOD1. We found that continued expression of PAX3-FOXO1 was required to maintain chromatin accessibility and allow neighboring DNA binding proteins and chromatin remodeling complexes to associate with this small number of regulated enhancers. Overall, this work provides a detailed mechanism by which PAX3-FOXO1 maintains an oncogenic transcriptional regulatory network.

molecular biology↗

bioRxiv2020

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

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.

genetics↗

bioRxiv2020

Definition of a Small Core Transcriptional Circuit Regulated by AML1-ETO

Transcription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. While this seems to be a straight forward task, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies. We describe an approach utilizing CRISPR-based genome editing to insert a degron tag into the endogenous AML1-ETO locus of Kasumi-1 cells, as well as overexpression of a degradable AML1-ETO protein in CD34+ human cord blood cells, which is a an AML1-ETO-dependent pre-leukemia model. Upon addition of a small molecule proteolysis targeting chimera (PROTAC), the AML1-ETO protein was rapidly degraded in both systems. Furthermore, by combining rapid degradation with nascent transcript analysis (PRO-seq), RNA-seq and Cut&Run, we have defined the core AML1-ETO regulatory network, which consists of fewer than 100 direct gene targets. The ability of AML1-ETO to regulate this relatively small gene pool is critical for maintaining cells in a self-renewing state, and AML1-ETO degradation set off a cascade of transcriptional events resulting in myeloid differentiation.

genomics↗