Monday, May 13, 2024
5/13/2024
PROJECT SUMMARY Hematopoietic stem cells (HSC) maintain both a self-renewal capacity to persist throughout life and an ability to differentiate to downstream progenitors to generate blood cell lineages. Stem cells balance these fates through symmetric and asymmetric divisions, respectively. HSC self-renewal and symmetric division are necessary for rapid expansion of HSCs in fetal development as well as repopulation of bone marrow following stem cell transplantation. Recently, the transcriptional regulator Bclaf1 (Bcl2 associated transcription factor 1) was shown to support both fetal and adult HSC function. Bclaf1 is ubiquitously expressed in hematopoietic cells but its function in normal hematopoiesis has not been elucidated. We recently characterized BCLAF1 as a modulator of ETS-family transcription factor activity in developing B cells. Bclaf1 is highly expressed in HSCs, which also rely on ETS-family transcription factors for development and function, consistent with a potential role for BCLAF1 in HSC activities. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used murine models with germline loss of function and with selective deletion of BCLAF1 in hematopoietic cells. We find that Vav-cre:Bclaf1fl/fl mice have significantly reduced numbers of HSCs at embryonic day 17.5 (E17.5) that persists into adulthood, but does not exaggerate over time. To further characterize the function of BCLAF1 in HSCs in adult mice, we treated 8-week old Mx-cre:Bclaf1fl/fl mice with polyinosinic:polycytidylic acid (pIpC) to delete Bclaf1 in established adult HSCs. Loss of BCLAF1 did not alter HSC numbers in the adult bone marrow as compared to pIpC-treated Bclaf1fl/fl control mice. These findings demonstrate that BCLAF1 supports HSC development beginning in fetal populations but is not required for homeostasis of adult bone marrow HSCs. We performed competitive reconstitution assays with limited numbers of HSCs to investigate if BCLAF1 is required for HSC repopulation function. HSCs were sorted from fetal livers of BCLAF1-sufficient or -deficient mice and transplanted along with wild-type competitor bone marrow cells into lethally-irradiated recipient mice. Mice transplanted with Vav-cre:Bclaf1fl/fl HSCs had significantly reduced hematopoietic repopulation with lower percentage of donor-derived leukocytes and reduced percentage of donor HSCs at 16 weeks post-transplant than mice with Bclaf1fl/fl donor HSCs. This phenotype is consistent between fetal and adult donor HSCs from Vav-Cre:Bclaf1fl/fl mice. Additionally, single cell CITEseq reveals that BCLAF1-deficient HSCs have significantly upregulated AP-1 factors JUN and FOS. Collectively, our findings reveal that BCLAF1 is a novel regulator of fetal HSC development and HSC repopulation activity. On-going studies are investigating the mechanism of BCLAF1 function in HSCs and the activity of BCLAF1 as a transcription factor to regulate AP-1.
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