Page 39 - Haematologica July

Setd2 regulates hematopoietic stem cells
tion of SLAM-HSC is the sole reason to explain the com- plete loss of HSC functions. We used CD150 and CD48 to define SLAM-HSC, which remains heterogeneous, as reported in recent studies.20-22 We further analyzed quies- cent long-term HSCs with additional defined surface markers besides CD48 and CD150: CD135, CD34, CD201, and CD49b. The results showed that there was a dramatic reduction of CD201+CD49b- fraction and a sig- nificantly increased entry of CD201-CD49b- fraction in the CD48-CD150+CD135-CD34-LSK populations in Setd2Δ/Δ mice compared with the control (Figure 5A). It has been reported that CD201+ is an accurate marker in SLAM- HSCs to define the quiescent stem cell pool which is capa- ble of repopulation in bone marrow transplantation,23,24 and CD201+CD49b-CD135-CD34- SLAM-HSC population is reputed, as the “true HSC”, to have the longest-term self-renewal capacity in deep quiescence.25 Our data indi- cate that the real quiescent fractions are further reduced in the decreased SLAM-HSC population in Setd2Δ/Δ mice.
To explore the molecular mechanisms underlying HSC regulation by Setd2, we performed RNA-seq using LSKs from Setd2f/f and Setd2Δ/Δ mice. Unbiased Gene Set Enrichment Analysis (GSEA) revealed that the HSC, long- term HSC, and short-term HSC signature genes26 were all significantly down-regulated, while intermediate and late progenitor signature genes26 were significantly up-regulat- ed in Setd2Δ/Δ LSKs (Figure 5B), implying that immune-phe- notypically defined Setd2Δ/Δ HSCs lost the stem cell identi- ty and differentiated toward multipotent progenitors (MPP). Gene ontology analysis of top differentially expressed genes indicated that lineage development/dif- ferentiation-related genes were significantly up-regulated in Setd2Δ/Δ LSKs (Figure 5C and D, and Online Supplementary Table S3), including Gata1, Gata3, and Klf1, which are important in HSC differentiation toward myeloid and lymphoid lineages.
Collectively, loss of Setd2 could induce LT-HSCs to exit from quiescence and commit to differentiation, leading to the exhaustion of LT-HSCs, IT-HSCs, MPP1, and the dif- ferentiation to the MPP2, MPP3, and MPP4 populations (Figure 5E).
Δ/Δ
Setd2 HSPCs show increased Nsds and RNA Pol II
elongation associated phosphorylation changes
To understand whether loss of Setd2 and H3K36me3 would affect other H3K36-methyltransferases and subse- quent methylation states of H3K36, the expression levels of 4 other most closely related enzymes were assessed using LSK cells from BM. Our data showed that Ash1l was decreased, but Nsd1/2/3 were all significantly increased at both mRNA and protein levels (Figure 6A and Online Supplementary Figure S5A). Interestingly, when we over- expressed WT NSD2, or gain-of-function (GOF) mutant of NSD2 (E1099K) in a murine Mll-Af9 leukemia cell line, both WT NSD2 and the GOF of NSD2-E1099K showed similar H3K36me3/2 changes to LOF of Setd2 (Online Supplementary Figure S5B), which implies that Setd2 and Nsds actually antagonize each other’s function. LOF SETD2 and GOF NSD2 in human leukemia may result in similar transcriptional dysregulation. Indeed, we found that H3K36me1 and H3K36me2 were dramatically increased, correlated with the up-regulated Nsd1/2/3 (Figure 6B). In addition, H3K4me3 and H3K79me2 were also significantly increased, while H3K27me3 was slightly decreased (Figure 6B), which indicated the promoting
transcriptional elongation of RNA polymerase II. The sig- nificant increase in elongation-associated phosphorylation changes [RNA pol II (Ser5P) and pol II (Ser2P)] were fur- ther confirmed by immunoblotting (Figure 6C). Thus, we hypothesized that Setd2 knockout up-regulates the RNA pol II transcriptional elongation to activate a subset of genes, which could affect the identity and functions of HSCs.
Our bulk RNA-seq was performed with LSK cells due to limited cell numbers. Next we aimed to define a subset of genes that were up-regulated in Setd2Δ/Δ SLAM-HSCs. To identify some candidate genes, the expression profiles of Setd2 and transcriptional elongation related genes and complexes were checked using a published database.27 The results showed that BET family genes and some well- known super elongation regulating genes (Myc, Mycn, Myb etc.) were significantly up-regulated during HSC dif- ferentiation (Online Supplementary Figure S6). Thus, we performed RT-PCR on these up-regulated genes with sort- ed Setd2Δ/Δ SLAM-HSCs. The results showed the dramatic upregulation of Gata1, Gata3, and Klf1, which was consis- tent with our RNA-seq data (Figure 6D). At the same time, we noticed that Myc was also significantly up-regulated in Setd2Δ/Δ SLAM-HSCs (Figure 6D). Myc is well known to be very sensitive to RNA pol II promoter-proximal pausing and releasing from pausing by elongation changes. Importantly, the phenotypes of Setd2Δ/Δ HSCs recapitulat- ed the Myc overexpression situation. It has been reported that enforced expression of Myc in SLAM-HSCs promotes differentiation at the expense of self-renewal, inducing exit from quiescence, increased apoptosis, and failure to reconstitute BM in BMT assay,28 which phenocopies Setd2Δ/Δ HSCs. Also, we confirmed the dramatic upregula- tion of Gata1, Gata3, Klf1, and Myc at the protein level (Figure 6C).
As Myc is a well-studied gene, which regulates the entry and exit from stem cell quiescence during development, we first confirmed that the significant increase of Myc is due to enhanced RNA pol II elongation. ChIP-qPCR assays of Setd2, H3K36-related histone modifications, and Pol II were performed at the Myc locus using c-kit+ cells from Setd2f/f and Setd2Δ/Δ mice. The results showed that there was a significantly higher enrichment of pol II (Ser5P) and Pol II (Ser2P) occupancy along the whole gene body, promoter and enhancer regions of Myc in Setd2Δ/Δ HSPCs compared with control.29,30 Meanwhile, H3K36me2 occupancy also showed a significantly higher enrichment along the gene body, both promoter and enhancers, while the H3K36me1 mainly increased along the enhancer region. As expected, enrichment of Setd2 and H3K36me3 was dramatically reduced, especially at the gene body region (Figure 6E).
Δ/Δ
Setd2 deficiencies could be partially rescued
by super elongation complex-related inhibitors
Next, we tested whether the increased gene expres- sions, such as Myc, could be reversed by epigenetic drugs. The c-kit+ Setd2Δ/Δ cells were treated in an in vitro culture assay with super elongation complex (SEC) related inhibitors for 24 and 48 h: JQ1 (Brd4 inhibitor), EPZ-5676 (Dot1l inhibitor), and BAY 1143572 (p-TEFb/CDK9 inhibitor). The elevated H3K36me1/2 marks were not affected 48 h after treatment (Figure 7A), while the expres- sion levels of pol II (Ser2P), pol II (Ser5P), Gata1, Gata3, and Myc were significantly decreased in all 3 drug-treated
haematologica | 2018; 103(7)
1117