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Setd2 regulates hematopoietic stem cells
Histone H3K36 methylation is one of the most promi- nent epigenetic modifications that are associated with gene activation. In yeast, Set2 is the sole H3K36 methyl- transferase, which is responsible for all three methyla- tion events and can interact with RNA polymerase II (RNA Pol II).5 Set2 contains several conserved domains. One of them is the SET domain, which is the catalytic domain for H3K36 methylations. Another important domain is the SRI domain, which binds to serine 2 (Ser2) and serine 5 (Ser5) doubly phosphorylated carboxyl ter- minal domain (CTD) repeats of RNA Pol II.6 The human ortholog of Set2, SETD2, was first isolated from human CD34+ hematopoietic stem/progenitor cells (HSPCs).7 SETD2 mainly works as H3K36 tri-methyltransferase, while H3K36me1 and H3K36me2 are catalyzed by other methyltransferases. To date, 7 other HMT enzymes have been reported to methylate H3K36, including NSD1, NSD2, NSD3, and ASH1L.8 NSD1/2/3 and ASH1L can methylate H3K36 to generate H3K36me1 and H3K36me2. The NSDs have been reported as oncogenic drivers in many cancers including leukemia. Furthermore, NSDs could regulate WNT, MYC, and NF- κB to affect various physiological or pathological processes.9
It has been reported that Setd2 is required for murine embryonic stem cells (mESCs) differentiation toward endoderms and endoderm development during murine embryonic development,10 while Setd2-/- resulted in embryonic lethality at E10.5-11.5.11 SETD2 was identi- fied as a tumor suppressor, as loss-of-function (LOF) mutations of SETD2 have been found in many human cancers, including leukemia and lymphoma.12-15 Previously, we have reported that there are SETD2 muta- tions in 6% of acute leukemia with 22% enriched in MLL-rearranged leukemia.16 However, the roles of SETD2 in adult HSPCs and normal hematopoiesis have not been fully studied. To understand the mechanisms of how Setd2 regulates the normal hematopoiesis, by using a novel conditional knockout model, we revealed a unique and critical role of Setd2 in regulating quiescence and differentiation of adult HSCs through restricting NSDs/SEC mediated RNA polymerase II elongation.
Methods
Animals
Setd2f/f (B6, CD45.2) mice were generated by Cincinnati Children’s Hospital Transgenic Core. Vav1-Cre, Mx1-Cre, Tie2- Cre mice were purchased from Jackson Laboratory. All mice were housed in the rodent barrier facility at Cincinnati Children’s Hospital Medical Center (CCHMC).
Small molecular inhibitors treatment
The CD117 positive selection of bone marrow (BM) cells was performed using magnetic CD117 microbeads (Miltenyi 130- 091-224) following the manufacturer's instructions. The CD117 positive fractions were cultured in medium (Stemspan+100 ng/mL SCF+100 ng/mL TPO) and treated with JQ1 500 nM, EPZ-5676 1uM, BAY 1143572 400 nM for 24 and 48 hours (h). The inhibitors were from the following companies: JQ1 (Sigma- Aldrich, SML0974), EPZ-5676 (Selleckchem, S7062), BAY 1143572 (MedChem Express, HY-12871).
Details of the methods used are available in the Online Supplementary Appendix.
Results
Generating a novel Setd2 conditional knockout allele Setd2 is involved in the ESCs differentiation and vascu- lar formation during embryonic development. Setd2-/- mice are embryonic lethal.10 Thus, we generated a Setd2 condi- tional knockout allele by inserting two LoxP sites flanking Setd2 exon6, which encodes part of the SET domain. Deletion of exon 6 could result in frame-shift and trigger nonsense-mediated decay (NMD) of the mutant mRNA transcript (Online Supplementary Figure S1A). Three Cre transgenic lines were used: Tie2-Cre, Mx1-Cre, and Vav1- Cre. Tie2-Cre mice display Cre activities in both endothe- lial cells and hematopoietic cells.17 However, we were unable to develop any Setd2f/f/Tie2-Cre mice by intercross- ing Setd2f/w/Tie2-Cre mice with Setd2f/f mice in multiple lit- ters (Online Supplementary Table S1), while polyinosinic- polycytidylic acid (pIpC) induced Setd2f/f/Mx1-Cre mice and Setd2f/f/Vav1-Cre mice are viable and fertile. Thus, we focused on Mx1-Cre and Vav1-Cre alleles to achieve
Setd2f/f deletion in the hematopoietic system.
First, to confirm Setd2 deletion, the mice were geno- typed using tail tissue and peripheral blood by genomic PCR (Online Supplementary Figure S1B and C). The LoxP insertion and Setd2 deletion were confirmed in Setd2f/f and Setd2f/f/Vav1-Cre mice. Subsequently, the Setd2 expres- sions were confirmed to be dramatically decreased at both mRNA and protein levels in Setd2f/f/Vav1-Cre mice and pIpC induced Setd2f/f/Mx1-Cre mice BM cells (Figure 1A, Online Supplementary Figure S1D, and data not shown). Consistent with the role of Setd2 in regulating H3K36 methylation, global H3K36me3 was significantly reduced
in BM cells of Setd2 knockout mice (Figure 1A).
Setd2Δ/Δ mice showed leukopenia, anemia, erythroid dysplasia, increased thrombopoiesis, and mild
BM fibrosis
Setd2f/f/Vav1-Cre mice are born small and pale. When the circulating blood count (CBC) was checked at eight weeks, they showed leukopenia, macrocytic anemia, and increased platelet count compared to the control litter- mates (Figure 1B-D). To exclude the possibility that the phenotype is contingent on deletion early in fetal hematopoiesis, we induced excision in 6- to 10-week old Setd2f/f/Mx1-Cre mice with pIpC injection. We found sim- ilar phenotypes two weeks after pIpC injection (Online Supplementary Figure S2A-C).
Consistent with peripheral blood (PB) phenotype, the Setd2Δ/Δ mice, both Setd2f/f/Vav1-Cre and Setd2f/f/Mx1-Cre models, had 30% fewer nucleated BM cells, enlarged spleens, and obviously shrunken thymuses (Figure 1E-G and Online Supplementary Figure S2D), which were also confirmed by pathology. There are significantly increased erythroblasts and mature megakaryocytes in Setd2Δ/Δ mice BM compared to the controls (Online Supplementary Table S2). Notably, the percentage of erythroblasts gradually increases with aging and could even reach up to 80% in some mice (data not shown). Erythroid dysplasia could also be observed. Compared with the round nuclei of ery- throblasts in control mice, erythroblasts in Setd2Δ/Δ mice showed frequent multi-nucleation, nuclear budding, nuclear fragments, and more cells in mitosis (Figure 1H). However, no obvious dysplasia was observed in other myeloid lineages or megakaryocytes. In addition to ery- throid dysplasia, the histology showed increased reticulin
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