Biology subjects

Hiebert, S. W.

Publications and source records attributed to Hiebert, S. W..

3 recordsLinked to original sources

bioRxiv2021

Generalizing about GC (hypoxia): Dys- & Dat-InformaticaComment on Germinal center B cells selectively oxidize fatty acids for energy while conducting minimal glycolysis

Steadily accumulating evidence supports the concept that the outputs of immune responses are influenced by local nutrient and metabolite conditions or concentrations, as well as by the molecular programming of intermediary metabolism within immune cells. Humoral immunity and germinal center reactions are one setting in which these factors are under active investigation. Hypoxia has been highlighted as one example of how a particular nutrient is distributed in primary and secondary follicles during an antibody response, and how its sensors could impact the qualities of antibody output after immunization. Based on a bio-informatic analysis of mRNA levels in germinal center and other B cells, recently published work challenges the concept that there is any hypoxia or that it has any influence. In this perspective, we perform new analyses of published genomics data to explore potential sources of disparity and elucidate aspects of what on the surface might seem to be conflicting conclusions. In particular, the replicability and variance among data sets derived from different naive as well as germinal center B cells are considered. The results of the investigation highlight several broader issues that merit consideration, especially at a time of heightened focus on scientific reports in the realm of immunity and antibody responses. From one finding of this re-analysis, it is proposed that a standard should be expected in which the relationship of new data sets compared to prior "fingerprints" of cell types should be reported transparently to referees and readers. In light of the strong evidence for diversity in the constituencies within germinal centers elicited by protein immunization, it also is proposed that a core practice should be to avoid overly broad conclusions about germinal centers in general when experimental systems are subject to substantial constraints imposed by technical features.

immunology↗

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↗