SETD5 Modulates Hematopoietic Stem Cells Homeostasis By Mediating RNA Polymerase II Pausing in Cooperation with HCF-1

Abstract

Stem cells maintain their self-renewal and differentiation through tight regulation of gene expression patterns at the transcriptional and epigenetic levels. SETD5 is a member of SET domain-containing histone lysine methyltransferase family. Its mutations were identified as the genetic causes of neurodevelopmental disorders, although SETD5 has been shown to lack methyltransferase activity in several studies. Deletion of Setd5 resulted in embryonic lethality at E10.5-11.5 with reduced cell number of CD41 + early hematopoietic cells in the blood island, while the role of SETD5 in adult stem cell, especially in hematopoietic stem cells (HSC) remains unexplored.In this study, by using Vav-Cre and Mx1-Cre mediated conditional knockout murine models, we explored the role of Setd5 in hematopoiesis. We found that Setd5 deficiency led to an enhanced accumulation of HSC in both Setd5 CKO (Vav-Cre; Setd5 fl/fl) and Setd5 IKO (Mx1-Cre; Setd5 fl/fl with pIpC treatment) mice. Cell cycle analysis revealed that higher proportions of SLAM-HSC and LSK + cells underwent active cycling in both Setd5 CKO and Setd5 IKO mice. However, limiting dilution assay revealed a significant ~4-fold decrease of functional HSCs in Setd5 CKO mice, while competitive serial transplantation assays exhibited a progressive decrease in repopulation capacity in Setd5 CKO than that of Setd5 fl/fl. While a progressive decrease in PB chimerism was observed in Setd5 IKO recipients via competitive transplantation assay, myeloid lineage reconstitution was increased, implicating a differentiation bias towards myeloid lineage at the expense of lymphoid lineage of Setd5 IKO HSCs. Thus, our phenotypical studies revealed that Setd5 deficiency impaired the homeostasis of the numbers and function of HSCs.To dissect the underlying mechanism, single cell transcriptome was performed in Setd5 CKO and Setd5 fl/fl control LSK +s using Smart-seq2 method. LSK +s were grouped into 5 clusters with distinct transcriptional features. Significantly, LT stem-like cluster revealed a disruption of LT-HSC and quiescence signatures in Setd5 CKO group, accompanied with an elevated S/G2/M cell cycle signature. When these cells were further projected onto Nestorowa's data (Nestorowa et al, Blood, 2016), Setd5 CKO LSK +s were found to be largely deviated from the core HSC territory toward more differentiated progenitor state. Consistent results were also observed in the bulk RNA-seq of Setd5 CKO and Setd5 IKO SLAM-HSCs. These data indicated that both transcriptional- and immunophenotype-defined LT-HSCs lost the long-term stem cell signatures due to Setd5 deficiency.Although SETD5 has a SET domain, no obvious changes were observed in a majority of histone methylation in Setd5 KO hematopoietic cells. Co-IP assay revealed that SETD5 could interact with HDAC3, PAF1 and HCF-1 complex. SETD5 ChIP-seq revealed that SETD5 could bind to E2F-responsive promoters and regulated the transcription of E2F targets. HCF-1 was reported to interact with E2F and regulate cell cycle entry. HCF-1 and PAF1 was recently found to associate with multiple transcription initiation and elongation complexes to regulate Pol II pausing. We thus investigated whether SETD5 regulates E2F targets via its association with HCF-1, and whether it also regulates E2F targets transcription via modulating Pol II pausing. By analyzing the genome-wide distribution of Pol II in control and Setd5 CKO c-Kit + cells by ChIP-Seq, we found an increase of Pol II occupancy upon SETD5 deficiency. Deletion of Setd5 lead to a significant decrease of pausing index on average which represent a transcription elongation state. Inhibition of the transition of paused Pol II in Setd5 CKO c-Kit + cells with BAY 1143572 (p-TEFb/CDK9 inhibitor) or JQ1 (Brd4 inhibitor) significantly suppressed S phases entry in Setd5 CKO LSK + cells, indicating that increased transition of paused Pol II to elongation mediated by Setd5 depletion could be a main cause for the exit of quiescence stage of Setd5 CKO HSC cells.In summary, we here demonstrate a critical role of Setd5 in HSC maintenance and provide a new insight into the mechanism of Setd5 in regulating HSC quiescence by pausing Pol II-mediated differential expression of cell cycle genes via HCF-1-SETD5-PAF1-Pol II axis.MK.L and YJ.C contributed equally to this work.Corresponding authors: WP.Y and YJ.C.Disclosures: No relevant conflicts of interest to declare. DisclosuresNo relevant conflicts of interest to declare.