Histone Demethylase LSD1 Is Required to Repress Hematopoietic Stem Cell Signatures in Mature Blood Cells to Permit Terminal Differentiation

Abstract 550

Lysine specific demethylase 1 (LSD1) is a demethylase that acts on mono- and dimethylated H3K4 (H3K4me1/2). Consistent with H3K4me2 (an active marker of transcription) as a substrate, LSD1 is part of a core complex with the co-repressor, CoREST and HDAC1/2. Previously our lab demonstrated that regulation of hematopoietic differentiation depends in part on the interaction of the growth factor independent transcription factors (= Gfi1 and Gfi1b) with the LSD1/CoREST/HDAC complex. We generated a conditional knock out mouse for LSD1 (LSD1^fl/fl) to study its roles in hematopoiesis. Inducible deletion of LSD1^fl/fl mice in all hematopoietic lineages with Mx-Cre resulted in severe neutropenia. Flow cytometric analysis showed that LSD1^fl/fl Mx-Cre mice lacked Gr-1^high Mac-1^high double positive mature neutrophilic granulocytes in the bone marrow and the peripheral blood; however, the frequency of Gr-1^dim Mac-1^high (mainly consisting of promyelocytes and myeloblasts but not mature neutrophils) increased in frequency. To reveal the mechanism responsible for the observed neutropenia, we performed global mRNA expression profiling and chromatin immunoprecipitation sequencing (ChIPSeq) for H3K4 methylation states in Gr-1^dim Mac-1^high cells from LSD1^fl/fl Mx-Cre and LSD1^fl/fl mice. Five hundred ninety-eight genes (412 up / 186 down; p≤0.01, 2-fold cutoff) were differentially expressed in the absence of LSD1. Although we did not detect changes in expression of established myeloid transcription factors, including Pu.1, C/EBPα, C/EBPε or Gfi1, gene set enrichment analysis (GSEA) of Gr-1^dim Mac-1^high cells from LSD1^fl/fl Mx-Cre using gene signatures for mature myeloid cells clearly showed that LSD1 deficient Gr-1^dim Mac-1^high cells failed to display a gene signature of differentiated myeloid cells (NES: 1.88; p≤0.003). Among the most highly upregulated genes, we observed genes highly expressed in hematopoietic stem and progenitor cells (HSPCs; i.e.: CD34 36.2-fold; HoxA9 26.3-fold; Sca-1 10.8-fold; Meis 1 2.6-fold). Therefore we performed GSEA using signatures from HSPCs (encompassing over 200 genes); the stem/progenitor gene set was highly significantly enriched (NES: −1.9; p<10⁻⁴) in LSD1 deficient Gr-1^dim Mac-1^high cells. Chromatin immunoprecipitation sequencing did not reveal any global changes in the amount of H3K4me2/3 histone methylation, however many genes critical for HSPCs, including Meis1 and the entire HoxA gene locus, where more strongly H3K4me2/3 marked than in control cells, which is in concord with the gene expression data.

To determine if LSD1 represses stem/progenitor genes in additional lineages, we analyzed the effects of LSD1 loss in erythroid cell development through breeding with EpoR-Cre. Wild type, as well as control embryos, were recovered at Mendalian ratios up to E12.5, but no live LSD1^fl/fl EpoR-Cre embryos were observed after E15.5. At E13.5, LSD1-deficient embryos were smaller and paler as compared to control embryos. Flow cytometry revealed a severe differentiation defect at the transition from pro-erythroblasts to basophilic erythroblasts, resulting in a paucity of more mature erythroid cells. To unravel molecular mechanisms responsible for this deficit, we performed gene expression profiling of wild type and knock out CD71⁺ c-kit⁺ Ter119^lo pro-erythroblasts. Again, we did not detect changes in the expression levels of established erythroid transcription factors, including Gata-1, Klf1, SCL/Tal1, NF-E1, Ldb1, Lmo2 or Myb. By GSEA analysis we observed that LSD1 deficient CD71⁺ c-kit⁺ Ter119^lo pro-erythroblasts displayed higher expression of the hematopoietic stem and progenitor cell gene signatures (NES: −2.4; p<10⁻⁴), a finding strikingly similar to the data in myeloid cells. Therefore, LSD1 is required in multiple hematopoietic lineages to repress stem/progenitor gene expression programs in maturing cells. We propose that repression of these early programs is essential for subsequent hematopoietic differentiation.