ALBERT

Description: ARID1A is a key component of ATP-dependent SWI/SNF complex involved in chromatin remodeling. Chromatin remodeling mediated by SWI/SNF complex is crucial for gene expression and affects a broad range of biological processes including hematopoietic development. ARID1A is frequently mutated across several solid tumors as well as hematopoietic malignancies, including Burkitt's lymphoma, diffuse large B-cell lymphoma and acute promyelocytic leukemia. Nevertheless, function of ARID1A in adult hematopoiesis and implications of its deficiency in development and progression of hematopoietic diseases has not been explored. In this study, we used a murine model of ARID1A deficiency to establish its essential function in maintaining normal hematopoietic development. Germline loss of Arid1a is embryonic lethal; therefore, we generated mice with deletion of Arid1a specifically in the hematopoietic compartment using Vav-iCre and Mx1-Cre transgenic mice. Arid1afl/fl;Vav-iCre+ mice occurred at a lower than expected frequency, suggesting some perinatal mortality. For the Mx1-Cre model, Arid1a exon 9 was excised by administrating poly(I:C) to adult mice and hematopoiesis was evaluated using flow cytometry. An increase in both percentage and absolute number of long-term hematopoietic stem cells (LTHSCs) defined as Lin-Sca1+Kit+CD34-FLT3- or Lin-Sca1+Kit+CD48-CD150+ occurred in the bone marrow using both models of Arid1a deficiency. RNA-sequencing of sorted LTHSCs from Arid1a KO bone marrow revealed dysregulated expression of several genes involved in cell cycle, G2/M checkpoint and related pathways. In vivo BrdU incorporation assays showed a substantially lower proportion of quiescent hematopoietic stem cells in Arid1a deficient bone marrow. To assess the reconstitution ability of ARID1A deficient HSCs, sorted KO or WT LTHSCs were transplanted into irradiated congenic recipient mice in competitive repopulation assays. Proportion of donor-derived cells in recipients transplanted with KO cells was strikingly lower compared to wild-type cells, suggesting poor reconstitution ability of Arid1a KO LTHSCs. Also, differentiation of both myeloid and lymphoid lineages was impaired in Arid1a KO mice compared to WT controls. To investigate the mechanism of perturbed differentiation of the myeloid and erythroid lineages, RNA-Seq was performed on sorted CMPs, GMPs and MEPs from WT and Arid1a KO BM. Our analysis showed significant decrease in expression of several transcription factors (Runx1, Gata2, Cebpa), which play a crucial role in lineage differentiation. To determine how Arid1a deficiency alters chromatin accessibility in myeloid precursors, Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) was performed on sorted Lin-Kit+ BM cells from both Arid1a KO and WT mice. A global reduction in open chromatin in Arid1a KO cells was noted compared to WT cells. A substantial overlap occurred between down regulated genes (RNA-seq) and reduced chromatin accessibility in Arid1a KO myeloid progenitors. Motifs for PU.1, RUNX1, GATA and CEBPA were significantly enriched in loci with reduced ATAC-seq signals in Arid1a KO cells. Our findings demonstrate an indispensable function of Arid1a in hematopoietic development and underline the importance of precise chromatin dynamics maintained by ARID1A-containing SWI/SNF complex in hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Description: Mutations in genes encoding components of the RNA splicing machinery constitute the leading class of genetic alterations in myelodysplastic syndromes (MDS). Somatic inactivating alterations of splicing factor, ZRSR2, are observed in ~10% of MDS patients. Mutations/loss of ZRSR2 in human myeloid cells causes aberrant splicing, primarily impairing splicing of the U12-type introns. However, the precise role of ZRSR2 in splicing and hematopoietic development needs further exploration. To understand the function of ZRSR2 in hematopoietic development and splicing, we generated Zrsr2 knockout mice. RNA-Seq of sorted myeloid precursor populations (common myeloid progenitors, granulocyte monocyte progenitors and megakaryocyte erythrocyte progenitors) revealed aberrant retention of U12-type introns in ZRSR2-deficient murine cells, similar to our previous observation in ZRSR2 mutant MDS bone marrow cells (Madan et al, Nat Commun 2015). However, despite complete loss of ZRSR2 in our mouse model, the effect on splicing of U12-type introns was modest compared to the ZRSR2 mutant MDS bone marrow and ZRSR2 knockdown human AML cells (K562 and TF1). Moreover, our comprehensive analyses of hematopoietic compartment in ZRSR2-deficient (Zrsr2Δ/Y) compared to WT (Zrsr2+/Y) male mice, indicated that ZRSR2 was dispensable for hematopoietic differentiation. We did not observe any significant difference in peripheral blood counts, bone marrow cellularity, proportion of hematopoietic stem cells (HSCs) and myeloid precursors (CMP, GMP, MEP) in the bone marrow of both young (7-10 weeks) and old (〉1 year) mice of either genotypes. Competitive and non-competitive reconstitution assays also demonstrated that loss of ZRSR2 does not affect repopulation potential of HSCs. Collectively, our data demonstrate that ZRSR2 is not essential for hematopoietic development in mice. Further, to understand the underlying determinant for our unexpected observation that deletion of Zrsr2 in mice did not impact hematopoietic development and affected modestly splicing of U12-type introns, we investigated the role of a closely-related homolog, ZRSR1, in splicing of U12-type introns. We utilized shRNA-mediated silencing of ZRSR1 in murine myeloid precursors (Lin−Kit+ bone marrow cells), and assessed splicing using RNA-Sequencing. Wildtype myeloid cells expressing Zrsr1 shRNA did not display significant mis-splicing of the U12-type introns. However, knockdown of Zrsr1 in ZRSR2-deficient myeloid cells exacerbated mis-splicing of the U12-introns. This suggested that ZRSR1 contributes to regulation of U12-spliceosome in murine hematopoietic cells. Increased mis-splicing of U12-type introns in ZRSR1/ZRSR2-deficient cells was validated using quantitative RT-PCR. Further to verify the compensatory role of murine ZRSR1 in splicing of U12-type introns, we used CRISPR/Cas9 technology to generate 32D cells lacking either one or both ZRSR proteins. Quantitative RT-PCR analysis showed notable aberrant retention of U12-type introns in Zrsr1/Zrsr2 double-deficient 32D cells compared to either WT or single KO cells. We also demonstrated that aberrant retention of U12-type introns of MAPK9 and MAPK14 caused by deficiency of ZRSR proteins led to their reduced protein expression in both human and murine myeloid cells. Taken together, our study highlights functional role of murine ZRSR1 in splicing of U12-type introns in murine hematopoietic cells where it can partially compensate for the deficiency of ZRSR2. Therefore, deficiency of ZRSR2 alone is insufficient to impact extensively RNA splicing in mice, and further studies with concurrent deficiency of ZRSR1 and ZRSR2 are warranted to replicate complete loss of ZRSR activity. Disclosures Chng: Novartis: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.