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DOT1L-Controlled Cell-Fate Determination and Transcription Elongation Are Independent of H3K79 Methylation

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DOT1L-Controlled Cell-Fate Determination and Transcription Elongation Are Independent of H3K79 Methylation DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation Kaixiang Caoa,b,1, Michal Ugarenkoa,b, Patrick A. Ozarka,b, Juan Wanga,b, Stacy A. Marshalla,b, Emily J. Rendlemana,b, Kaiwei Lianga,b,2, Lu Wanga,b,c, Lihua Zoua,b,c, Edwin R. Smitha,b,c, Feng Yuea,b,c, and Ali Shilatifarda,b,c,3 aDepartment of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; bSimpson Querrey Center for Epigenetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; and cRobert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611 Edited by Shiv I. S. Grewal, National Institutes of Health, Bethesda, MD, and approved September 8, 2020 (received for review January 18, 2020) Actively transcribed genes in mammals are decorated by H3K79 Unlike other known lysine methyltransferases, DOT1L lacks a methylation, which is correlated with transcription levels and is SET domain and is structurally more similar to arginine methyl- catalyzed by the histone methyltransferase DOT1L. DOT1L is re- transferases (14–16). DOT1L is the core component of the DOT1- quired for mammalian development, and the inhibition of its containing multisubunit complex named DotCom that includes catalytic activity has been extensively studied for cancer therapy; the MLL translocation partners AF10, AF17, AF9, and ENL (17). however, the mechanisms underlying DOT1L’s functions in normal AF9 and ENL are also subunits—along with additional MLL development and cancer pathogenesis remain elusive. To dissect the translocation partners AFF1, AFF4, and ELL—of the super relationship between H3K79 methylation, cellular differentiation, elongation complex (SEC). and transcription regulation, we systematically examined the role SEC is a positive transcription elongation factor (P-TEFb)- of DOT1L and its catalytic activity in embryonic stem cells (ESCs). containing complex that is required for rapid transcriptional in- DOT1L is dispensable for ESC self-renewal but is required for estab- duction and expression of key leukemia genes (18–20). The ma- lishing the proper expression signature of neural progenitor cells, jority of P-TEFb in cells is sequestered by HEXIM1 and HEXIM2 while catalytic inactivation of DOT1L has a lesser effect. Further- in the 7SK small nuclear RNA-containing complex (21, 22). Once BIOCHEMISTRY more, DOT1L loss, rather than its catalytic inactivation, causes de- released from the 7SK-P-TEFb complex, P-TEFb can associate fects in glial cell specification. Although DOT1L loss by itself has no with SEC or BRD4 to form active complexes, phosphorylate the major defect in transcription elongation, transcription elongation serine 2 residues of the RNA Polymerase II C-terminal domain defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead (Pol II CTD), and facilitate the release of paused Pol II into gene DOT1L cells, revealing a role of DOT1L in promoting productive bodies (20, 23, 24). Multiple lines of evidence have pointed to a transcription elongation that is independent of H3K79 methylation. critical role for SEC in human diseases including MLL-rearranged Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional Significance elongation control. DOT1L is a histone methyltransferase that catalyzes histone transcription | chromatin | gene expression | epigenetics | cancer H3K79 methylation, a chromatin modification that is correlated with active transcription. Inhibition of DOT1L’s catalytic activity pigenetic landscapes of higher eukaryotes are implicated in has been studied for cancer therapy; however, the mechanisms Etranscription modulation, cell-fate determination, and diseases underlying its function in development and cancer pathogen- such as cancer. Posttranslational modifications of core histone esis remain elusive. To understand the catalytic-dependent and tails have been intensely investigated for the past few decades catalytic-independent functions of DOT1L, we generated cata- while the modifications of histone cores are relatively under- lytic dead and null DOT1L embryonic stem cells and found that DOT1L, but not its catalytic activity, is required for establishing studied. Lysine 79 methylation of histone H3 (H3K79) was the the proper expression signature of neural progenitor cells, first core histone modification identified outside the histone tail suggesting that DOTlL has biological functions that are inde- (1–4), the distribution of which is highly correlated with actively – pendent of its methyltransferase activity. We propose that the transcribed genes (5 8). DOT1L, the only enzyme catalyzing loss/degradation of DOT1L could be beneficial for cancer mono, di-, and trimethylation of H3K79 in metazoans, plays a therapeutics. critical role in embryogenesis and leukemia transformation (9–11), suggesting a potential function of H3K79 methylation in normal Author contributions: K.C. and A.S. designed research; K.C., M.U., S.A.M., and E.J.R. per- development and human disorders. formed research; K.C., K.L., L.W., and A.S. contributed new reagents/analytic tools; K.C., DOT1L null mouse embryos die around 10.5 d post coitum with P.A.O., J.W., L.Z., E.R.S., F.Y., and A.S. analyzed data; and K.C., E.R.S., and A.S. wrote the paper. developmental arrest and cardiac dilation, while DOT1L null The authors declare no competing interest. embryonic stem cells (ESCs) cultured in serum-containing media This article is a PNAS Direct Submission. maintain self-renewal capability despite having elongated telo- meres and reduced proliferation (9). An independent study showed Published under the PNAS license. 1Present address: Department of Biochemistry, School of Medicine, Case Western Reserve that DOT1L knockdown by short hairpin RNA led to significant University, Cleveland, OH 44106. proliferation defects of ESCs only under differentiation conditions 2Present address: Medical Structural Biology Research Center, Wuhan University, 430071 (12), supporting the notion that DOT1L facilitates cellular differ- Wuhan, Hubei, People’s Republic of China. entiation. Furthermore, DOT1L inhibition enhanced the conver- 3To whom correspondence may be addressed. Email: [email protected]. sion efficiency of induced pluripotent stem cells from fibroblasts This article contains supporting information online at https://www.pnas.org/lookup/suppl/ (13), suggesting that DOT1L and H3K79 methylation are major doi:10.1073/pnas.2001075117/-/DCSupplemental. roadblocks of somatic cell reprogramming. First published October 19, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2001075117 PNAS | November 3, 2020 | vol. 117 | no. 44 | 27365–27373 Downloaded by guest on September 28, 2021 leukemia (19, 25), HIV (26, 27), and high-MYC–expressing solid sequencing of genomic DNA and complementary DNA (cDNA) tumors (24, 28). The existence of common subunits between SEC (SI Appendix,Fig.S1A and B). The morphology and growth rate of and DotCom, together with the observation that the bodies of both KO and CI mutant cells are comparable to wild-type (WT) active genes are decorated by H3K79 methylation, suggests that cells (SI Appendix,Fig.S1C and D). Furthermore, RNA sequencing DOT1L may play a role in regulating transcription elongation and (RNA-seq) results confirmed the removal of exon 5 in DOT1L KO disorders related to it. Here, we aim to elucidate the functions of cell lines (SI Appendix,Fig.S1E). We noted that the deletion of DOT1L and H3K79 methylation in cellular differentiation and exon 5 leads to a reduction in RNA levels of DOT1L (SI Appendix, transcription elongation. Unexpectedly, our results reveal that Fig. S1E), suggesting that the stability of the exon 5 null transcript is DOT1L harbors H3K79 methyltransferase-activity-independent impaired. Western blotting results indicated that DOT1L is unde- functions in modulating neural differentiation and productive tectable in KO cells, while its levels in CI cells are comparable to transcription elongation. that in WT ESCs (Fig. 1C). All three types of H3K79 methylation are abolished in respective DOT1L KO and CI cells (SI Appendix, Results Fig. S1 F and G and Fig. 1D), indicating the lack of in vivo catalytic DOT1L and H3K79 Methylation Are Dispensable for ESC Self-Renewal. activity of the catalytic mutant DOT1L in CI ESCs. Consistent with To investigate the role of DOT1L and its catalytic activity in tran- previous results, H3K79 methylation is enriched at genes with scription regulation, we first generated DOT1L knockout (KO) higher RNA Polymerase II (Pol II) levels (SI Appendix,Fig.S1H ESCs by deleting exon 5 of the DOT1L gene, which generates a and I and Fig. 1D). To examine if the loss of DOT1L or its activity frameshift mutant DOT1L with premature termination (9) has any impact on the ESC transcriptome, we performed differ- (Fig. 1A). DOT1L catalytic inactive (CI) ESCs were derived by ential gene expression analyses of RNA-seq data from WT, DOT1L mutating the catalytic pocket Gly163 and Ser164 of the endogenous KO, and DOT1L CI ESCs. We found that a limited number of DOT1L as suggested previously (1, 14) (Fig. 1B). The genotypes of genes were altered by DOT1L deletion or catalytic inactivation mutant cell lines were validated by PCR genotyping and Sanger (Fig. 1E). In addition, RNA Pol II levels at transcription start sites Fig. 1. DOT1L deletion has limited impact on gene expression in ESCs. (A) Schematic representation of the generation of the DOT1L KO allele. (B) Sequences of WT (Top) and CI DOT1L alleles at exon 5. Mutated bases and amino acid residues are in red. PAM sequences are in orange. (C) Western blotting of total cell lysates of WT, DOT1L KO, and DOT1L CI ESCs. Antibodies used are labeled on the left. (D) Heatmaps showing the occupancy of H3K79me1 (Left), H3K79me2 (Middle), and H3K79me3 (Right) in WT, DOT1L KO, and DOT1L CI ESCs at the 7,362 RNA Pol II-positive genes of ESCs. The profiles are sorted by descending occupancy of Pol II in WT ESCs. cpm, counts per million mapped reads.
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