Lyl1 and Scl Regulate Bcl2 Expression and Control Apoptosis at Different Stages of Hematopoietic Development.

Abstract 569

The Lymphoblastic Leukemia gene 1 (Lyl1) encodes a basic-helix-loop-helix (bHLH) transcription factor that is involved in T cell acute lymphoblastic leukemia via chromosomal translocations. Lyl1 has an almost identical bHLH motif to Scl, a known master regulator of hematopoiesis. In our previous work, we used a double knock-out mouse model to demonstrate functional redundancy between Lyl1 and Scl in adult hematopoietic stem cells (HSCs), where in the absence of both genes in adult mice, HSCs and progenitors undergo rapid apoptosis. Despite their common and overlapping functions, the two genes appear to also function at different aspects of hematopoietic development. Lyl1 knock-out mice exhibit a unique defect in B-cell development. This phenotype is recapitulated in vitro, where Lyl1 progenitors grown on a layer of OP9 cells and allowed to differentiate with the addition of cytokines fail to differentiate to B cells as well as wild type progenitors. Rescue assays, where Lyl1 was transduced using a retrovirus into Lyl1 knock-out progenitors and transplanted into lethally irradiated recipients, showed complete rescue of the B cell lineage. Forced expression of Scl into Lyl1 knock-out progenitors failed to rescue, giving rise to only about 5% of peripheral blood B cells. This demonstrates the distinct functions of the two genes in differentiated cells types, despite their functional redundancy in the HSCs. We further sought to dissect the functions and differences between Lyl1 and Scl based on their target genes. Using bioinformatics analysis and a literature search, we identified a number of genes that were candidates for Lyl1 transcriptional regulation; these interactions with Lyl1 and Scl were confirmed by chromatin immunoprecipitation. We found that Bcl2, and genes belonging to this family, are direct target genes of both Lyl1 and Scl in progenitor cells, and that they are downregulated in Lyl1 and Scl single knock-out mouse models. Since Bcl2 is critical for B cell survival, we hypothesized that the B cell deficiency in Lyl1 knock-out mice could be partly due to a loss of Bcl2. We performed rescue assays where Bcl2 was transduced using a retrovirus into Lyl1 knock-out progenitors which were transplanted into lethally irradiated recipient mice. Bcl2 overexpression rescued the B-cell defect in Lyl1 knock-out bone marrow. Since Bcl2 was identified as a target gene for Scl as well, we tested whether Bcl2 could also rescue the rapid apoptosis phenotype of the Lyl1/Scl double knock-out progenitors. Forced expression of Bcl2 in these cells, followed by deletion of the floxed Scl allele, resulted in a delayed onset of apoptosis, but incomplete rescue of the progenitor cell death. These findings distinguish the activities of Lyl1 and Scl in B cells and demonstrate a possible mechanism for their functions, through in part by their activation of the antiapoptotic gene Bcl2.