Loss of SMARCA4 Leads to an Impaired Hematopoiesis in Mice

Abstract

SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a) is the central ATPase containing enzyme in the SWI/SNF- (switch/sucrose non-fermentable) complex, which regulates chromatin accessibility and thereby influences gene transcription. By acting as a transcriptional activator or repressor in different cell types, SMARCA4 and the SWI/SNF complex have various functions. Our aim was to understand the function of SMARCA4 in healthy hematopoiesis in order to then apply our findings to different hematologic diseases in a second step. To investigate the role of SMARCA4 in haematopoiesis and hematopoietic stem cells, we created a transgenic Smarca4 knockout mouse model. An inducible knockout system was chosen since early embryonic knockout of SMARCA4, which is crucially involved in embryonic development leads to a lethal phenotype. The Cre/loxP system was used to induce homozygous Smarca4 knockout under the control of the hematopoiesis-specific promotor Mx-1. We then performed blood counts, bone marrow smears and RNA sequencing, including analysis of RNA signature of the different cell types in the bone marrow to decipher the composition of the bone marrow of Smarca 4 deficient mice. Blood count analysis showed that Smarca4 knockout mice have significantly lower leukocyte, reticulocyte, and thrombocyte counts. In addition, bone marrow showed lower colony forming capacity in colony formation assays compared to healthy cre-negative control mice. Bone marrow smears displayed aplasia of all cell lines in Smarca4 knockout mice with additional signs of dysplasia as hypolobulatedmegakaryocytes or nuclear blasts. RNA-sequencing revealed 749 upregulated and 520 downregulated genes with a log fold change ≥ +/- 1 in the bone marrow of Smarca4 knockout mice compared to cre-negative control mice. Relevant over represented pathways according to REACTOME data base included genes of generation of second messenger molecules, cell surface interactions at the vascular wall, and immunoregulatory interactions between lymphoid and non-lymphoid cells. Through cell type deconvolution (CIBERSORTx) of the bulk microarray gene expression data we estimated abundances for different cell types. Results revealed a decrease in granulocyte and macrophage progenitors (statistically significant) as well as in eosinophil progenitor cells, and in erythroid pre-colony forming units. Interestingly, both common myeloid and common lymphoid progenitors were increased. It seems as if the progenitor cells accumulate and take a different path when Smarca4 is knocked out in these cells (figure 2). We conclude that SMARCA4 is crucial for cell growth and differentiation of progenitor cells in the bone marrow during hematopoiesis since a Smarca4 knockout leads to differentiation arrest and accumulation of distinct progenitor cells and pancytopenia in peripheral blood. Figure 1: Abundances for 23 cell populations in Smarca4 knockout (KO) and wildtype (WT) samples were estimated using CIBERSORTx. Bubble plot showing Log2 fold changes of average abundances in KO vs WT with accumulation of distinct progenitor cells in the bone marrow of Smarca4 deficient mice. As underlying signature-matrix the haemopedia gene set was used. Bubbles with black borders indicate significance of difference between samples.