BCLAF1 Regulates Expression of AP-1 Genes and Fetal Hematopoietic Stem Cell Repopulation Activity

Hematopoietic stem cells (HSCs) balance differentiation and self-renewal to promote maintenance of mature blood cell lineages. The balance of these programs changes over the course of development to accommodate hematopoietic output needs. Fetal HSCs are highly proliferative whereas adult bone marrow HSCs are typically quiescent unless under stress conditions. The mechanisms that regulate HSC expansion during fetal hematopoietic development and adult HSC maintenance remain significant knowledge gaps. Bclaf1 (Bcl2-associated factor 1), a transcriptional regulator, is ubiquitously expressed in hematopoietic cells. We recently defined its function 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. Our objective in this study is to determine the molecular mechanism of BCLAF1 in regulation of fetal and adult HSCs.

To test whether BCLAF1 regulates HSC development and hematopoiesis, we used murine models of conditional hematopoietic and inducible deletion of Bclaf1. We find that Vav-Cre:Bclaf1^flox/flox mice, which have selective deletion of Bclaf1 in hematopoietic cells, have significantly reduced numbers of HSCs at E17.5, yet no difference in HSC emergence at E14.5. The reduced HSC numbers persist in adult Vav-Cre:Bclaf1^flox/flox mice; but there is no exacerbation with age. To further characterize the function of BCLAF1 in HSCs in adult mice, we treated adult (8-week old) Mx-Cre:Bclaf1^flox/flox mice with polyinosinic:polycytidylic acid (pIpC) to delete Bclaf1 in established HSCs. Loss of BCLAF1 did not alter HSC numbers in the adult bone marrow as compared to pIpC-treated Bclaf1^flox/flox control mice. These findings demonstrate that BCLAF1 supports fetal HSC development but is not required for maintenance of adult HSCs.

To determine if BCLAF1 is required for HSC repopulation capacity, we performed competitive reconstitution assays with limited numbers of HSCs. 20 HSCs were sorted from E17.5 fetal livers of Vav-Cre:Bclaf1^flox/flox or Bclaf1^flox/flox mice and transplanted along with wild-type competitor bone marrow cells into lethally-irradiated recipient mice. Compared to BCLAF1-sufficient HSCs, mice transplanted with fetal Vav-Cre:Bclaf1^flox/flox HSCs had significantly reduced hematopoietic repopulation with lower percentage of donor-derived leukocytes and reduced percentage of donor HSCs at 18 weeks post-transplant. All mature leukocyte populations (T cells, B cells, granulocytes, and monocytes) from Vav-Cre:Bclaf1^flox/flox donors were reduced consistent with aberrant HSC repopulating activity rather than a defect in a differentiation pathway. This repopulation defect was also present using adult Vav-Cre:Bclaf1^flox/flox donor HSCs. Interestingly, while induced deletion of BCLAF1 in adult Mx-Cre:Bclaf1^flox/flox mice does not alter HSC numbers, these BCLAF1-deficient HSCs are defective in reconstituting hematopoiesis following competitive transplantation. Notably, BCLAF1-deficient HSCs do not have abnormalities in 5-fluorouracil chemotherapy recovery, proliferation, cell death, or homing activity. Using Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of fetal HSCs, we find that loss of BCLAF1 results in reduction of a subpopulation of long-term HSCs and upregulation of the early response genes Jun, Fos, and Egr1. Collectively, these findings suggest BCLAF1 restrains activity of AP-1 factors and immediate early genes to preserve HSC self-renewal capacity and promote HSC expansion during both fetal development and repopulation of hematopoiesis after HSC transplant.

Bednarski:Sobi: Membership on an entity's Board of Directors or advisory committees; Horizon Therapeutics: Membership on an entity's Board of Directors or advisory committees; Prime Medicine: Membership on an entity's Board of Directors or advisory committees.