The Sterile Alpha Motif Protein-1 Transcription Factor Controls Hematopoiesis through Modulation of H3K4 Methylation
Authors: Schaefer Meg , Dogiparthi Venkatasai Rahul , Zhou Yichao , Hewitt Kyle J 
Published: December 2024

Demethylation of the H3K4 mark by histone demethylases (HDMs) guides HSPCs into specific lineages by repressing developmental and lineage-specific genes. Recently, sterile alpha motif protein-1 (SAMD1) was found to promote H3K4 demethylation at specific sites - by coordinating lysine demethylase 1 (LSD1) activity. Since SAMD1 is highly expressed in HSPCs, interacts with and controls the function of known epigenetic writer proteins, and our data predicts that SAMD1 may control the expression of hematopoietic transcription factors (e.g., GATA2), we hypothesize that SAMD1 coordinates H3K4 methylation to control hematopoiesis. While LSD1 has established roles in hematopoiesis and erythropoiesis, SAMD1 function is not known. As SAMD1 and LSD1 are commonly upregulated in acute myeloid leukemia (AML), correlated with poor prognosis, and SAMD1 is required for the growth of the erythroleukemia cell line K562, understanding mechanisms may bring to light new therapeutic opportunities.
We tested our hypothesis using shRNA knockdown or CRISPR-Cas9 KO systems in human and mouse HSPCs. Following SAMD1 knockdown/knockout, we differentiated mouse lineage-depleted bone marrow or human CD34+ cells along the erythroid lineage. SAMD1 knockout increased the frequency of late erythroid progenitors (CD71-CD235a+) by 8-fold. Consistent with the observation that SAMD1 knockout increased erythropoiesis, an RNA-seq analysis identified a cohort of SAMD1 repressed genes were involved in heme metabolism (TRIM10 and TRAK2) and ROS pathways (GLRX2 and GLCM). Conversely, SAMD1-activated genes include those involved in hemostasis and platelet activity pathways (F2RL3 and FLNA) and signal transduction (MAPK3 and PIK3CG). To test whether SAMD1 regulation of signaling mediators alters cell signaling activity, we stimulated human erythroid progenitors with the Kit ligand stem cell factor (SCF). We observed a 2-fold decrease in phosphorylated ERK in the absence of SAMD1, confirming SAMD1's role in coordinating signaling and GATA2 transcription, a downstream target of the Kit signaling pathway. Altogether, these results indicate an inhibitory role for SAMD1 in differentiation towards the erythroid lineage.
We tested SAMD1-induced changes in H3K4 methylation in erythroid-differentiated human CD34+ cells. SAMD1 knockout increased H3K4me2 levels and decreased H3K4me3 levels compared to controls, reflecting a possible change in LSD1 activity. Global changes to LSD1 and SAMD1 occupancy and the presence of H3K4me2/3 are being examined in the human umbilical cord-derived erythroid progenitor 2 (HUDEP2) cell line in control versus SAMD1 knockout using CUT&RUN.
SAMD1 knockout is embryonic lethal, and no conditional knockout mouse currently exists. To clarify the role of SAMD1 in hematopoiesis, we conducted competitive transplant experiments in mice using shRNA knockdown HSCs. Samd1 knockdown versus control HSCs revealed an increase in HSC repopulation with 2.7-fold more CD45.2+ after 16 weeks. Additionally, the contribution of Samd1 knockdown cells in the peripheral blood was skewed towards the myeloid lineages, as we observed a 5.3-fold increase in the granulocyte and monocyte lineages compared to a 3.5-fold increase in the T and B cell lineages. These exciting results reveal new mechanisms that may be exploited to improve HSC expansion ex vivo. Currently, we are examining changes in gene expression of hematopoietic progenitor populations by scRNA-seq. Overall, we have identified a new transcription factor coordinating hematopoiesis potentially through maintenance of the HSPC pool. Linking Samd1 function to transcriptional mechanisms in hematopoiesis opens the door to translational avenues for studying the contribution of Samd1 in hematologic pathologies.