Page 44 - Haematologica July

1122
Y. Zhou et al.
clearly indicated by globally elevated marks. The higher enrichment of pol II (Ser5P) and pol II (Ser2P) occupancy were also confirmed on Myc locus in Setd2Δ/Δ HSPCs. Setd2 and H3K36me3 generally mark the active genes; however, our surprising findings indicated that Setd2-H3K36me3 restrict the pol II elongation. To connect the Setd2 loss to enhanced elongation, we observed upregulations of Nsd1/2/3 after Setd2 knockout. There is much literature showing that NSDs could interact with BRD4, which could bridge to the SEC and DOT1l complex.31-34 Thus, we proposed a regulatory model in which there is a crosstalk between Setd2 and Nsds. Loss of Setd2 leads to the upreg- ulation of Nsds. Meanwhile, Nsds could interact with Brd4, SEC, and Dot1l complex to enhance the elongation, and results in the upregulation of a subset of target genes that regulate quiescence and differentiation of HSCs.
In summary, using our novel Setd2 conditional knock- out allele, we revealed unique roles of Setd2 in regulating quiescence and differentiation of HSCs. Our study not only provides us with a deeper understanding of Setd2 functions in HSCs, but also a better understanding of SETD2 functions during leukemic transformation and solid tumors. Along with using the KDM4 inhibitor to
restore the H3K36me3,43 inhibiting the elongation com- plex or downstream targets, could be effective for LOF mutation SETD2 in cancer and could also benefit bi-allele SETD2 mutant patients. Our data also indicate that GOF mutation of NSDs, such as NSD2 mutations or NSD1/3 translocations, might follow the same regulatory dysregu- lation as LOF of SETD2 on pol II elongation in leukemia and other cancers.
Acknowledgments
We would like to thank Cincinnati Children’s Research Flow Cytometry Core (RFCC), Cincinnati Children’s Veterinary Services, and CCHMC mouse core. We would also like to thank Damien Reynaud and George Mike Freudiger for their help on this project. This work was supported by grants from the Ministry of Science and Technology of China (2016YFA0100600) (to TC), the National Natural Science Foundation of China (81421002) (to TC), CAMS Initiative for Innovative Medicine (2016-I2M-1-017) (to TC), Leukemia Research Innovative Team of Zhejiang Province (2011R50015) (to JJ), National Natural Science Foundation of China (81370643-H0812) (to JJ), the CFK (to GH), National Institutes of Health (NIH) (R21CA187276) (to GH).
References
1. Kiel MJ, Yilmaz OH, Iwashita T, et al. SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell. 2005;121(7):1109-1121.
2. Orkin SH, Zon LI. Hematopoiesis: an evolv- ing paradigm for stem cell biology. Cell. 2008;132(4):631-644.
3. Dawson MA, Kouzarides T. Cancer epige- netics: from mechanism to therapy. Cell. 2012;150(1):12-27.
4. Oike T, Ogiwara H, Amornwichet N, Nakano T, Kohno T. Chromatin-regulating proteins as targets for cancer therapy. J Radiat Res. 2014;55(4):613-628.
5. Strahl BD, Grant PA, Briggs SD, et al. Set2 is a nucleosomal histone H3-selective methyl- transferase that mediates transcriptional repression. Mol Cell Biol. 2002;22(5):1298- 1306.
6. McDaniel SL, Strahl BD. Shaping the cellular landscape with Set2/SETD2 methylation. Cell Mol Life Sci. 2017;74(18):3317-3334.
7. Mao M, Fu G, Wu JS, et al. Identification of genes expressed in human CD34(+) hematopoietic stem/progenitor cells by expressed sequence tags and efficient full- length cDNA cloning. Proc Natl Acad Sci USA. 1998;95(14):8175-8180.
8. Wagner EJ, Carpenter PB. Understanding the language of Lys36 methylation at histone H3. Nat Rev Mol Cell Biol. 2012;13(2):115- 126.
9. Vougiouklakis T, Hamamoto R, Nakamura Y, Saloura V. The NSD family of protein methyltransferases in human cancer. Epigenomics. 2015;7(5):863-874.
10. Zhang Y, Xie S, Zhou Y, et al. H3K36 histone methyltransferase Setd2 is required for murine embryonic stem cell differentiation toward endoderm. Cell Rep. 2014;8(6):1989- 2002.
11. Hu M, Sun XJ, Zhang YL, et al. Histone H3
lysine 36 methyltransferase Hypb/Setd2 is required for embryonic vascular remodeling. Proc Natl Sci USA. 2010;107(7):2956-2961.
12. Fahey CC, Davis IJ. SETting the Stage for Cancer Development: SETD2 and the Consequences of Lost Methylation. Cold Spring Harb Perspect Med. 2017;7(5).
13. Zhang J, Ding L, Holmfeldt L, et al. The genetic basis of early T-cell precursor acute lymphoblastic leukaemia. Nature. 2012;481 (7380):157-163.
14. Parker H, Rose-Zerilli MJ, Larrayoz M, et al. Genomic disruption of the histone methyl- transferase SETD2 in chronic lymphocytic leukaemia. Leukemia. 2016;30(11):2179- 2186.
15. Moffitt AB, Ondrejka SL, McKinney M, et al. Enteropathy-associated T cell lymphoma subtypes are characterized by loss of func- tion of SETD2. J Exp Med. 2017;214(5):1371-1386.
16. Zhu X, He F, Zeng H, et al. Identification of functional cooperative mutations of SETD2 in human acute leukemia. Nat Genet. 2014;46(3):287-293.
17. Speck NA, Iruela-Arispe ML. Conditional Cre/LoxP strategies for the study of hematopoietic stem cell formation. Blood Cells Mol Dis. 2009;43(1):6-11.
18. Kvasnicka HM, Beham-Schmid C, Bob R, et al. Problems and pitfalls in grading of bone marrow fibrosis, collagen deposition and osteosclerosis - a consensus-based study. Histopathology. 2016;68(6):905-915.
19. Cheng T, Rodrigues N, Shen H, et al. Hematopoietic stem cell quiescence main- tained by p21cip1/waf1. Science. 2000;287(5459):1804-1808.
20. Oguro H, Ding L, Morrison SJ. SLAM family markers resolve functionally distinct sub- populations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell. 2013;13(1):102-116.
21. Raaijmakers MH, Scadden DT. Divided within: heterogeneity within adult stem cell pools. Cell. 2008;135(6):1006-1008.
22. Wilson A, Laurenti E, Oser G, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 2008;135(6):1118-1129.
23. Balazs AB, Fabian AJ, Esmon CT, Mulligan RC. Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood. 2006;107(6):2317-2321.
24. Kent DG, Copley MR, Benz C, et al. Prospective isolation and molecular charac- terization of hematopoietic stem cells with durable self-renewal potential. Blood. 2009;113(25):6342-6350.
25. Schroeder T. Hematopoietic stem cell het- erogeneity: subtypes, not unpredictable behavior. Cell Stem Cell. 2010;6(3):203-207.
26. Ivanova NB, Dimos JT, Schaniel C, et al. A stem cell molecular signature. Science. 2002;298(5593):601-604.
27. Cabezas-Wallscheid N, Buettner F, Sommerkamp P, et al. Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy. Cell. 2017;169(5):807- 823 e819.
28. Wilson A, Murphy MJ, Oskarsson T, et al. c- Myc controls the balance between hematopoietic stem cell self-renewal and differentiation. Genes Dev. 2004;18(22): 2747-2763.
29. Herranz D, Ambesi-Impiombato A, Palomero T, et al. A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia. Nat Med. 2014;20(10):1130-1137.
30. Bahr C, von Paleske L, Uslu VV, et al. A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierar- chies. Nature. 2018;553(7689):515-520.
31. Zhang Q, Zeng L, Shen C, et al. Structural Mechanism of Transcriptional Regulator NSD3 Recognition by the ET Domain of BRD4. Structure. 2016;24(7):1201-1208.
32. Sarai N, Nimura K, Tamura T, et al. WHSC1 links transcription elongation to HIRA-
haematologica | 2018; 103(7)