An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation

Abstract

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of b-globinopathies (sickle cell disease and b-thalassemia) since it is a component of g-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 BAC transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of GMP-like cells, converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells (HSPC), coincident with induction of myeloid transcription factors (e.g. PU.1 and CEBPa). Finally, blocking the activity of transcription factors PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors, and that inhibition of the myeloid differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

Epigenetic and Transcriptional Control of Erythropoiesis

“…Dynamic changes in histone methylation also occur during erythropoiesis, with both histone methyltransferases, such as NSD1 ( Tauchmann et al, 2020 ) and SETD8, ( Malik et al, 2015 ; Malik et al, 2017 ; Myers et al, 2020 ; DeVilbiss et al, 2015 ), and histone demethylases, such as LSD1, ( Kerenyi et al, 2013 ; Yu et al, 2021 ), playing critical role in erythroid gene regulation and erythroid maturation. The distribution of well-characterized histone markers of activation and repression, including H3K4me3, H3K27me3, and H3K9me3 appear to be established at the time of erythroid commitment, and are relatively stable during terminal maturation.…”

Identification of novel γ-globin inducers among all potential erythroid druggable targets

“…HuDEP2 expansion and differentiation were described previously 10,11 . Human CD34+ HSPC erythroid differentiation were described previously 10,12 . HbF staining was modified from previous publication 13 .…”

“…This sgRNA pool was cloned into a LentiCRISPRv2 backbone, packaged into virus 8 , and then infected into human HuDEP2 erythroid cells at a multiplicity of infection (MOI) of 0.4. After puromycin selection, infected HuDEP2 cells were cultured under erythroid differentiation conditions for an additional 6 days 10 . At the end of this period, cells were subjected to intracellular HbF staining followed by flow cytometry 13 .…”

“…Finally, we asked whether pharmacological inhibition of PTEN could induce HbF under erythroid differentiation conditions in human CD34+ stem and progenitor cells (HSPC) 10 . In these experiments, the LSD1 inhibitor GSK690 was employed as a positive control for HbF induction 10 .…”

“…Finally, we asked whether pharmacological inhibition of PTEN could induce HbF under erythroid differentiation conditions in human CD34+ stem and progenitor cells (HSPC) 10 . In these experiments, the LSD1 inhibitor GSK690 was employed as a positive control for HbF induction 10 . Since EZH2 was previously identified as a candidate -globin corepressor 17 In summary, we identified here novel genes that regulate -globin expression, and showed that EZH and PTEN inhibitors significantly induce HbF.…”