Stengel, K. R. | bioRxiv Science

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↗