DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79

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 , 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 in mammals are decorated by H3K79 Unlike other known 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 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 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 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 | | | 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 (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. (E) Correlation analysis of gene expression levels between WT and respective DOT1L KO (Left) and DOT1L CI (Right) ESCs. Significantly down-regulated genes [compared with WT, adjusted P < 0.01, log2 fold change (log2FC) > |1|] are in blue, and up-regulated ones are in red. Unchanged genes are gray dots. The number of up- and down-regulated genes are indicated in the upper left and bottom-right corners, respectively. RPKM, reads per kilobase of transcript per million mapped reads.

27366 | www.pnas.org/cgi/doi/10.1073/pnas.2001075117 Cao et al. Downloaded by guest on September 28, 2021 (TSS) and gene bodies are largely unperturbed by DOT1L muta- The Differential Impact of DOT1L Deletion and SEC Inhibition on RNA tion (SI Appendix,Fig.S1H). These data demonstrate that neither Pol II Levels at Promoters. DOT1L has been linked to transcription DOT1L nor its catalytic activity plays a major role in the mainte- regulation mainly due to the enrichment of all three H3K79 nance of the naive pluripotent stem cell transcriptome. methylation marks at bodies of actively transcribed genes (Fig. 1D) and the observation that genes with higher levels of DOT1L Is Required for Establishing Transcription Signatures of Neural H3K79me2 tend to have higher transcription elongation rates Progenitor Cells and Neural Differentiation. DOT1L deletion leads (32, 33). Although removal of DOT1L has little effect on to embryonic lethality in mice (9), suggesting that DOT1L is genome-wide RNA Pol II distribution or transcript levels in required for lineage commitment during development. To ESCs (SI Appendix, Fig. S1H, and Fig. 1E), we noted that down- characterize the functions of DOT1L in cellular differentiation, regulated genes in DOT1L null NPCs have significantly higher we first performed embryoid body (EB) differentiation of WT, gene length, higher length of the first intron, and higher intron DOT1L KO, and DOT1L CI ESCs. Surprisingly, we did not numbers in comparison with up-regulated genes and all - observe deficiencies of EBs derived from DOT1L KO and CI coding genes (SI Appendix, Fig. S3 K–M). Together with the ESCs compared with WT cells (SI Appendix, Fig. S2A). RNA-seq observation that the DOT1 complex (DotCom) and SEC share analyses of WT and DOT1L mutant EBs further demonstrated subunits AF9 and ENL (17, 19), these data suggest a possible that the loss of DOT1L has little impact on gene up-regulation cooperation of these complexes in promoting productive tran- during EB differentiation (SI Appendix, Fig. S2B). These results scription elongation. To test this hypothesis, we utilized the re- prompted us to investigate the role of DOT1L in lineage specifi- cently reported SEC inhibitor KL-2 (28) and mutant ESCs cation. Previous studies indicate that DOT1L is highly expressed in generated here to dissect the relationship between SEC and the neural ectoderm and the optic vesicle in embryonic day 9.5 DOT1L in modulating transcription elongation. Consistent with (E9.5) to E12.5 mouse embryos, suggesting a potential role of previous findings (28), we found that KL-2 treatment for 6 h DOT1L and H3K79 methylation in neural development (9). We leads to a decrease in the global level of the SEC component thus differentiated DOT1L mutant ESCs toward neural progeni- AFF1 and an elevation of RNA Pol II levels around TSS in ESCs tor cells (NPCs) using a monolayer differentiation protocol which (SI Appendix, Fig. S4A and Fig. 3A), indicating that the inhibition bypasses EB generation (Fig. 2A) (29, 30). During the 17-d dif- of SEC increases promoter proximal pausing of Pol II in ESCs. ferentiation course, cells gradually lost the dome-shaped mor- Interestingly, despite both ENL and AF9 being shared subunits phology and adopted the spindle-shaped NPC morphology between SEC and DotCom, the protein levels of ENL but not (Fig. 2A). RNA-seq analyses of NPCs and undifferentiated ESCs AF9 were down-regulated by SEC inhibition (SI Appendix, Fig. BIOCHEMISTRY showed that neural-related genes are significantly up-regulated S4A), indicating the dependency of ENL stability on the integrity (Fig. 2 B and C), supporting the success of NPC differentiation. of SEC. To precisely map the positions of RNA Pol II, we per- Although DOT1L mutant ESCs were able to differentiate into formed precision nuclear run-on and sequencing (PRO-seq) (34, NPC-like cells (SI Appendix,Fig.S3A), DOT1L deletion led to the 35), providing single-nucleotide resolution of Pol II occupancy. significant perturbation of 1,012 genes in NPCs (Fig. 2 D, Left). In Similar to Pol II ChIP sequencing (ChIP-seq) results, we ob- contrast, DOT1L CI NPCs have 225 significantly differentially served the elevation of Pol II occupancy at TSSs after KL-2 expressed genes in comparison with WT NPCs (Fig. 2 D, Right), treatment by PRO-seq (Fig. 3C). Such effects are more evident suggesting that CI cells have a milder defect in neural differenti- at TSSs of highly expressed genes (SI Appendix, Fig. S4 B and C), ation than DOT1L null cells. Moreover, down-regulated genes in consistent with SEC being the highly active elongation complex. DOT1L KO NPCs are enriched for genes important for neuron On the other hand, DOT1L deletion in ESCs has little impact on differentiation and brain development, including Nestin and Sox11 Pol II occupancy at TSSs of protein-coding genes regardless of (SI Appendix,Fig.S3B and E). Furthermore, ∼70% of genes SEC activity (Fig. 3 B and D,andSI Appendix, Fig. S4 B and C), down-regulated in DOT1L null NPCs are elevated during NPC suggesting that DOT1L is not a major regulator of Pol II pausing. differentiation (SI Appendix,Fig.S3C and G), further suggesting that DOT1L facilitates neural differentiation and the activation of SEC and DOT1L Coregulate the Poly(A)-Associated Transcription neural genes in NPCs. The expression levels of NPC marker genes Elongation Checkpoint. Although DOT1L deletion or catalytic such as Nestin, Sox11, Sox9,andCd117 are drastically reduced in inactivation has negligible effects on the distribution pattern of DOT1L KO cells post differentiation (SI Appendix,Fig.S3D and Pol II (SI Appendix, Fig. S1H), transcription elongation is more E). Interestingly, DOT1L deletion leads to a more substantial severely impaired by SEC inhibition in DOT1L KO cells com- down-regulation of these markers than DOT1L catalytic inacti- pared with WT or DOT1L CI ESCs as evidenced by RNA Pol II vation in NPCs (SI Appendix,Fig.S3D). As expected, the majority ChIP-seq and PRO-seq (Fig. 3 E–G). In DOT1L null cells with of misregulated genes in DOT1L CI NPCs overlaps with that in SEC inhibited, Pol II is prematurely terminated at Nop58 and DOT1L KO NPCs (SI Appendix,Fig.S3I and J); however, the Picalm genes with accumulated Pol II signals at gene bodies NPC transcriptome is much less perturbed by DOT1L catalytic (Fig. 3 E and F), suggesting that productive transcription elon- inactivation than deletion (Fig. 2D and SI Appendix,Fig.S3H). We gation is hampered. One of the major transcription elongation detected reduced RNA Pol II levels at the promoters of NPC checkpoints is located near the Poly(A) sites indicated by Pol II markers in DOT1L KO NPCs (SI Appendix,Fig.S3F), demon- accumulation at transcription termination sites (TTS) (36), strating that the reduction in RNA levels of neural genes is due to which ensures proper 3′ end processing. Interestingly, DOT1L the impairment in transcription. To further examine whether deletion but not catalytic inhibition led to a reduction of Pol II DOT1L has an impact on glial and neuronal lineage specification, levels near TTS in the presence of SEC inhibition (Fig. 3G and we differentiated ESCs using a previously established protocol SI Appendix, Fig. S4E), suggesting that this elongation check- (31). After the 27-d culture, we found that DOT1L KO ESCs are point is safeguarded by SEC and DOT1L in a partially redundant defective in generating glial cells as represented by the lack of manner. It is noteworthy that the expression levels of SEC GFAP-positive cells in comparison with WT and DOT1L CI cells, components are comparable in WT and DOT1L mutants and although the formation of neurons is largely unaffected by that the chromatin occupancy of AF10, one of the unique DOT1L deletion (Fig. 2E). Collectively, these results indicate that components of DotCom, is unperturbed after KL-2 treatment DOT1L is indispensable for the establishment of NPC transcrip- (SI Appendix, Fig. S4 D and F–G), suggesting that alternative tion signatures and glial cell specification, whereas its catalytic mechanisms, rather than compromised SEC levels in DOT1L activity fine-tunes the transcription outputs in NPC differentiation. null cells or the loss of DotCom recruitment when SEC is

Cao et al. PNAS | November 3, 2020 | vol. 117 | no. 44 | 27367 Downloaded by guest on September 28, 2021 Fig. 2. DOT1L is required for neural differentiation. (A) Differentiation strategy for generating NPCs from ESCs. Cell morphologies for each step are shown with phase-contrast images. (Scale bar, 100 μm.) (B) Correlation analysis of gene expression levels between ESCs and NPCs. Genes significantly up-regulated in NPCs are in red, whereas genes with significantly higher levels in ESCs are in blue. (C) analysis of significantly up-regulated genes in NPCs vs. ESCs. The top 300 genes with the highest log2FC (RPKM) values were used as the input for the analysis. (D) Correlation analysis of gene expression levels between WT and respective DOT1L KO (Left) and DOT1L CI (Right) NPCs. Significantly down-regulated genes (compared with WT, adjusted P < 0.01, log2FC > |1|) are in blue, and up-regulated ones are in red. The number of up- and down-regulated genes are red and blue, respectively. (E) Immunostaining analyses of WT, DOT1LKO, and DOT1L CI cells after 27 d of neural differentiation. Antibodies used are labeled on top of the images. Nuclei were stained with Hoechst 33342. (Scale bars, 50 μm.)

27368 | www.pnas.org/cgi/doi/10.1073/pnas.2001075117 Cao et al. Downloaded by guest on September 28, 2021 inhibited, underlie the effect of DOT1L in regulating productive H2B ubiquitinated suggest that the interaction of transcription elongation. H2Bub and DOT1L could stabilize the binding of DOT1L to nu- To investigate how transcription elongation is impaired by cleosomes (43–47). Intriguingly, such binding of DOT1L to ubiq- DOT1L and SEC loss-of-function, we analyzed our data focusing uitinated nucleosomes could in turn destabilize them by unwrapping on the 3′ end of genes. Indeed, KL-2 treatment caused defects of nucleosomal DNA (44), which could contribute to the catalytic- Pol II accumulation at the TTS of protein-coding genes in ESCs independent function of DOT1 in promoting transcription (Fig. 4 A and C), indicating the reduction of Pol II processivity elongation. under SEC inhibition. DOT1L deletion, rather than catalytic DOT1L null ESCs do not exhibit apparent transcription inactivation, further inhibited transcription elongation at TTS elongation defects; however, loss of DOT1L enhances the upon SEC inhibition (Fig. 4 B and D). Such effects are stronger processivity defects caused by SEC inhibition. This provides ex- at highly expressed genes (SI Appendix, Fig. S5), indicating that perimental evidence that DOT1L has a positive but catalytic- the productive elongation at active genes is a critical step for independent role in transcription elongation. It is possible that their transcription regulation and is governed by both SEC and proper expression of key genes of neural differentiation relies on DOT1L largely independently of H3K79 methyltransferase ac- an elevated transcription elongation rate, which may be impaired tivity. The impact of DOT1L on transcription elongation is fur- by DOT1L depletion alone, which could lead to diminished ex- ther supported by PRO-seq analyses. SEC inhibition leads to the pression of neural genes and an impairment of glial cell specifi- premature transcription termination at the 3′ end of genes cation. It is worth noting that ENL levels in ESCs are diminished (Fig. 4E). DOT1L KO led to a further reduction of Pol II at TTS by KL-2 treatment. ENL reductions could contribute to the en- upon KL-2 treatment while DOT1L catalytic inactivation had hanced elongation defect of combined SEC inhibition and limited impact on 3′ Pol II levels (Fig. 4F). Taken together, these DOT1L loss as it is a common subunit of both complexes. How- results argue that DOT1L, rather than its catalytic activity, par- ever, we cannot rule out that DOT1L has additional, unknown ticipates in the regulation of transcription regulation through noncatalytic functions that contribute to the observed defects. cooperating with SEC on safeguarding the Poly(A)-associated Although the transcriptional perturbation by DOT1L deletion elongation checkpoint. in ESCs is subtle, a more severe misregulation of transcription might be obtained upon acute degradation of DOT1L, as the Discussion DOT1L null cells may have adapted to the absence of DOT1L. In this study, we report H3K79 methylation independent roles of The 6-h treatment with KL-2 is less likely to have indirect effects, DOT1L in regulating neural differentiation and transcription but again, an acute degradation of 1 or 2 h could further mini- elongation. Furthermore, DOT1L’s contribution to transcription mize indirect effects of these complexes in promoting tran- BIOCHEMISTRY elongation appears to be redundant with SEC. Unlike previous scription elongation. Furthermore, elucidating the mechanisms reports (9, 12), we did not observe growth differences between underlying the role of DotCom in transcription elongation with DOT1L null and WT ESCs. We also did not observe defects of in vitro assays would be important to understand human diseases EB formation in DOT1L KO cells. It is noteworthy that all ESCs associated with defects in transcription elongation machineries described in our study are maintained in the naive pluripotent in the future. state with serum-free 2i/leukemia inhibitory factor (LIF) media In summary, our study defines catalytic-independent roles of (37), which is drastically different from serum/LIF conditions H3K79 methyltransferase DOT1L in cellular differentiation and used to culture ESCs in previous reports. Naive pluripotency is transcription elongation. Given that DOT1L has been implicated characterized by homogeneously expressed pluripotency genes in leukemogenesis, our study suggests that degrading DOT1L such as Nanog and Klf4, DNA hypomethylation, and reduced rather than solely inhibiting its catalytic activity could be in- bivalency (38–40). In addition, DOT1L loss did not trigger major strumental in cancer therapy. More importantly, the concept that transcriptional changes in ESCs or differentiated EBs, in line combining SEC inhibition and targeted DOT1L degradation with the finding that the developmental retardation of DOT1L further slows down RNA Polymerase II processivity (Fig. 4G) null embryos occurs during midgestation (9). EB differentiation may facilitate the development of therapy to treat transcription- generates a population of heterogeneous cells; thus it is possible addicted human developmental diseases and cancer (48). that the bulk-level RNA-seq technique could not detect tran- scriptional perturbation in a small group of cells. Single-cell Materials and Methods RNA sequencing would be a powerful tool to dissect the roles Antibodies. The following antibodies were used in this study: anti-DOT1L of DOT1L in regulating expression of lineage-specific genes [generated in house (17), 1: 500 for Western blotting]; anti-H3K79me1 (gen- during EB differentiation in the future. Consistent with erated in house, 1:1,000 for Western blotting, 1:100 for ChIP-seq); anti- DOT1L’s enrichment in neuroectoderm during mouse develop- H3K79me2 (Abcam 3594, 1 μg/mL for Western blotting); anti-H3K79me2 ment, many neural genes fail to properly express in DOT1L null (generated in house, 1:100 for ChIP-seq); anti-H3K79me3 (Abcam 2621, 1 μg/mL for Western blotting, 1:100 for ChIP-seq); anti-GFAP (Abcam 7260, cells during NPC differentiation, suggesting that DOT1L governs β the differentiation trajectory of neuroectoderm. Indeed, our 1:1,000 for immunostaining); anti- III tubulin/TUJ1 (Millipore MAB1637, 1:50 for immunostaining); anti-AFF1 (Bethyl A302-344A, 1:1,000 for Western blotting); neuronal and glial differentiation demonstrates impaired glial anti-AF9 (generated in house, 1:4,000 for Western blotting); anti-ENL (Cell Sig- cell specification in DOT1L KO cells. Interestingly, the catalytic naling Technology 14893, 1:1,000 for Western blotting); anti-RBBP5 (Bethyl activity of DOT1L is dispensable for both neuronal and glial A300-109A, 1:5,000 for Western blotting); anti-AF10 (Santa Cruz 53156, 1:100 for specification. ChIP-seq); and anti-RNA Pol II (Cell Signaling Technology 14958, 1:100 for ChIP-seq). Our results that several NPC marker genes are mildly deregu- lated in DOT1L CI cells suggest that H3K79 methylation could ESC Culture, CRISPR/Cas9-Guided Gene Editing, and Differentiation. The v6.5 participate in fine-tuning the transcription program during neural ESCs (49) were grown in N2B27 media supplemented with two inhibitors and differentiation. Although we did not observe an instructive role of LIF as described previously (40). Briefly, cells were grown on plates coated H3K79 methylation in EB formation, NPC differentiation, or with 0.1% gelatin and passaged when they reach 60 to 75% confluency. All μ neuronal and glial specification, it is possible that H3K79 meth- KL-2 treatments were performed at 10 M of KL-2 for 6 h. Plasmids con- taining desired guide RNAs (gRNAs) were generated, and transfections were ylation functions in vivo in a tissue-specific manner, which is worth performed as previously described (50). Briefly, 20 μg of plasmids were investigating in the future. Recent reports have demonstrated transfected into ESCs using Nucleofector (Lonza). Single-cell clones were catalytic-independent functions of yeast DOT1 in promoting his- picked 10 d after transfection, and PCR was used for genotyping the clones. tone exchange, remodeling, and H2B ubiquitination For DOT1L KO ESCs, a plasmid carrying 500-bp homology arms flanking the (H2Bub) (41, 42). Independent structural analyses of DOT1L and inserted sequences including two loxP sites were cotransfected with the

Cao et al. PNAS | November 3, 2020 | vol. 117 | no. 44 | 27369 Downloaded by guest on September 28, 2021 Fig. 3. DOT1L deletion further impairs transcription elongation upon SEC inhibition. (A) Metagene analysis of Pol II occupancy at TSS of 22,219 protein- coding genes measured by ChIP-seq in ESCs treated with dimethylsulfoxide (DMSO) or KL-2 for 6 h. (B) Metagene analysis of Pol II occupancy at TSS of 22,219 protein-coding genes measured by ChIP-seq in WT and DOT1L KO ESCs treated with KL-2 for 6 h. (C) Metagene analysis of Pol II occupancy at TSS of 22,219 protein-coding genes measured by PRO-seq in ESCs treated with DMSO or KL-2 for 6 h. (D) Metagene analysis of Pol II occupancy at TSS of 22,219 protein-coding genes measured by PRO-seq in WT and DOT1L KO ESCs treated with KL-2 for 6 h. (E) Representative UCSC Genome Browser view of Pol II ChIP-seq in WT, DOT1L KO, and DOT1L CI cells treated with DMSO or KL-2 for the indicated genes. (F) Representative UCSC Genome Browser view of PRO- seq in WT, DOT1L KO, and DOT1L CI cells treated with DMSO or KL-2 for the indicated genes. (G) Metagene analysis of Pol II occupancy at TSS, gene bodies, and TTS of 22,219 protein-coding genes measured by PRO-seq in WT and DOT1L KO ESCs treated with KL-2 for 6 h. The length of each gene (from TSS to TTS) was normalized to 6 kb for plotting. Signals near TTS are highlighted by a pink box. (Inset) Zoomed-in view of the pink area.

27370 | www.pnas.org/cgi/doi/10.1073/pnas.2001075117 Cao et al. Downloaded by guest on September 28, 2021 BIOCHEMISTRY

Fig. 4. The poly(A)-associated transcription elongation checkpoint is regulated by SEC and DOT1L. (A) Metagene analysis of Pol II occupancy around TTS of 22,219 protein-coding genes measured by ChIP-seq in ESCs treated with DMSO or KL-2 for 6 h. (B) Metagene analysis of Pol II occupancy around TTS of 22,219 protein-coding genes measured by ChIP-seq in WT, DOT1L KO, and DOT1L CI ESCs treated with KL-2 for 6 h. (C) Metagene analysis of Pol II occupancy around TTS of 22,219 protein-coding genes measured by PRO-seq in ESCs treated with DMSO or KL-2 for 6 h. (D) Metagene analysis of Pol II occupancy around TTS of 22,219 protein-coding genes measured by PRO-seq in WT, DOT1L KO, and DOT1L CI ESCs treated with KL-2 for 6 h. (E) Heatmap depicting the log2 fold change in normalized PRO-seq signal (KL2/DMSO) within 10-bp bins from −1 κb to +50 kb relative to the TSS for WT ESCs. TTS are marked by the dashed line. (F) Heatmaps depicting the log2 fold change in normalized PRO-seq signal within 10-bp bins from −1 κb to +50 kb relative to the TSS for DOT1L KO/WT (Left) or DOT1L CI/WT (Right). (G) A model of catalytic-dependent and -independent function of DOT1L activity. DOT1L removal but not catalytic inactivation further lowers the elongation rate reduced by SEC inhibition.

Cao et al. PNAS | November 3, 2020 | vol. 117 | no. 44 | 27371 Downloaded by guest on September 28, 2021 gRNA plasmid. Cre recombinase was then transiently expressed in the loxP the NextSEq. 500 sequencer (Illumina) using single-end sequencing for 50 sites containing cells to remove the desired region. For generating DOT1L CI cycles. The Ceto modular pipeline (53) was used for generating bam files and ESCs, asymmetric single-stranded oligonucleotides were designed as de- genome browser tracks. Briefly, after BCL2FASTQ conversion, reads were scribed (51) and cotransfected as donors together with the gRNA plasmid. trimmed with Trimomatic, version 0.33 (54), aligned with Bowtie version The sequence of sgRNA used for generating the KO allele and the CI allele 1.1.2 [(55), ChIP-seq] or Tophat version 2.1.0 [(56), RNA-seq] to the mm9 was 5′-CACTGCCCAGGTCGACAAAC-3′. genome. Raw reads were normalized to total read counts per million (cpm) EB differentiation was performed using the hanging-drop method (31) and visualized in the UCSC Genome Browser as bigWig-formatted with adaptation as previously described (52). Briefly, hanging drops were coverage tracks. loaded on the lids of 15-cm plates and cultured for 6 d. NPC differentiation PRO-seq libraries were sequenced on the NextSEq. 500 sequencer using protocol was adapted from ref. 30. Briefly, cells were seeded on a gelati- single-end sequencing for 75 cycles, and after running BCL2FASTQ, reads nized dish in 2i/LIF media for a day. The two inhibitors and LIF were then were processed as described below. Adapters were removed from raw reads withdrawn from the media, and cells were kept in plain N2B27 media for 7 with cutadapt version 1.14 (57). Reads were trimmed from the 3′ end to d. Cells were then dissociated and seeded in ultra-low attachment plates 36 bp to remove low-quality bases using Trimmomatic version 0.33 (54) re- (Sigma) in N2B27 media containing 10 ng/mL epidermal growth factor (EGF) quiring a minimal read length of 16 bp. Reads were then mapped to the (Peprotech) and 10 ng/mL basic fibroblast growth factor (FGF2) (Peprotech) mouse genome (mm9) and fruit fly genome (dm3) using Bowtie version 1.1.2 for 3 d to form neurospheres. Floating aggregates (neurospheres) were then (55). Only uniquely mapped reads with up to two mismatches in the entire seeded on gelatinized plates in N2B27 containing 10 ng/mL EGF and 10 ng/ read were used for further analysis. The total number of mouse reads was mL FGF2 for 2 to 4 d. Attached cells were dissociated and seeded again on normalized by the number of Drosophila reads for each experiment. Nor- gelatinized plates in N2B27 containing 10 ng/mL EGF and 10 ng/mL FGF2. malized reads were then converted to single-nucleotide 3′ bigWig strand- Cells were passaged one more time and harvested for different assays. specific tracks by taking 5′ positions of the read using BEDtools genomecov Neural differentiation was performed as previously published (31). Briefly, version 2.17 (58) with the options -strand -bg -5. Strands were then swapped EBs were generated from ESCs using the hanging-drop method. EBs were to give the correct orientation with the 5′ end, now becoming the 3′ end of then treated with 1 μM all-trans retinoic acid for 2 d and reseeded on plates the read (59). coated with poly-L-ornithine (5 μg/mL) and laminin (5 μg/mL). EBs were cultured with NeuroCult NSC proliferation medium (StemCell Technologies) Data Analysis. Using Ceto (53), RNA-seq gene counts were computed by supplemented with 10 ng/mL FGF2 (Peprotech) for 21 d. Media was changed HTSeq. (60) and used as input for edgeR version 3.12.1 (61). Genes with cpm every 3 d. less than 1 were filtered out in each comparison. A Benjamini–Hochburg- adjusted P value threshold of 0.01 and a log2 cutoff of 1 were used to Immunofluorescence. Immunostaining was performed as previously described identify genes significantly differentially expressed in one experimental (31). Briefly, cells were fixed with 4% paraformaldehyde, permeabilized with condition relative to another. RNA-seq correlation plots were generated 0.2% TritonX-100 in phosphate-buffered saline (PBS), and blocked with 10% using cutsomized R scripts. Gene ontology analyses were performed using fetal bovine serum (FBS) in PBS. Cells were then stained with primary anti- Metascape (62). body diluted with 10% FBS in PBS, washed with 0.2% TritonX-100 in PBS, The 7,362 genes used for ChIP-seq analysis were selected as follows. Genes stained with fluorescent secondary antibody and Hoechst dye diluted with filtered from an mm9 TxDb were required to be Ensembl protein-coding 10% FBS in PBS in the dark, and washed again with 0.2% TritonX-100 in PBS. genes that were also RefSeq validated. The best transcript with the high- Coverslips were mounted to glass slides using Fluoromount-G (Thermo est coverage in the TSS region from 0 to +100 bp was selected, after which Fisher) and sealed with nail polish. Images were collected using the Nikon the genes were required to be at least 1 kb in length, have a minimal dis- confocal microscope A1 located at the imaging center of Northwestern tance of 1 kb to the nearest gene, and have a maximum cpm in the TSS University. region of at least 1. Genes were then organized in a descending order based on their maximum signals at the TSS. All ChIP-seq heat maps were generated RNA-seq, ChIP-seq, and PRO-seq. RNA- and ChIP-seq experiments and library using ngsplot version 2.47 (63), and metagene plots were generated using generation were performed following previously published protocols (50). deepTools version 3.1.1 (64), where signal is grouped into 10-bp bins in For RNA-seq, cells were lysed with TRIzol reagent (Thermo Fisher), and RNA figures depicting signal relative to the TSS or TTS. AF10 peaks were called ’ was purified according to the manufacturer s instructions. RNA was further using model-based analysis of ChIP-seq (MACS) v1.4.2 with default param- treated with RNase free DNase I (Sigma), and the treated RNA was purified eters (65) with sonicated input DNA as the control. with an RNeasy mini kit (Qiagen). RNA-seq libraries were prepared using a PRO-seq metagene plots and heat maps were generated using deepTools TruSeq-stranded total RNA sample preparation kit (Illumina). For ChIP-seq, version 3.1.1, where signal is grouped into 10-bp bins in figures depicting ESCs were fixed with 1% formaldehyde for 10 min. Chromatin was sheared signal relative to the TSS and in 50-bp bins in figures depicting signal relative using an E220 focused ultrasonicator (Covaris). Sheared chromatin was to the TTS. mixed with antibody and protein A/G beads (Santa Cruz Biotechnology) and For RNA-seq box plot analyses in SI Appendix, Figs. S3 and S4, Student’s incubated overnight at 4 °C. Immunoprecipitated DNA was purified and t test and Wilcoxon signed-rank test were performed to calculate the sta- submitted for library preparation. ChIP-seq libraries were generated with a tistical significance. Sample size and P values are reported in the figures and KAPA HTP library preparation kit (KAPA Biosystems) following the figure legends. manufacturer’s instruction. PRO-seq was performed as previously described (28, 35). In brief, 0.5 million Drosophila S2 cell nuclei were spiked into 10 million ESC nuclei. Data Availability. All sequencing data generated by this study have been Nuclear run-on assays were then performed, and RNA was fragmented. deposited in the Gene Expression Omnibus under accession number Biotinylated RNA was purified by streptavidin beads M-280 (Thermo Fisher), GSE134083 (66). 5’ cap removed, the 5’ hydroxyl was repaired, and ligated with adaptors. After reverse transcription, cDNA was amplified using Phusion Hot Start II ACKNOWLEDGMENTS. We thank the members of the A.S. laboratory for DNA polymerase (ThermoFisher). DNA Libraries were size selected by helpful discussions; Fei X. Chen for insightful suggestions; and Nicole Ethen for illustrations in Fig. 4G. K.C. was supported by the NIH Pathway to PippinHT (Sage). Independence Award from the Eunice Kennedy Shriver National Institute of RNA-seq libraries were prepared using a TruSeq-stranded total RNA Child Health and Human Development (Grant K99HD094906). E.R.S. is sup- sample preparation kit (Illumina), and ChIP-seq libraries were prepared with ported by the Research Specialist Award from the National Cancer Institute a KAPA HTP library preparation kit (KAPA Biosystems) following the man- (NCI) (Grant R50CA211428). This study was supported in part by Grants ufacturer’s instructions. ChIP-seq and RNA-seq libraries were sequenced on R01CA214035 and R35CA197569 from the NCI (to A.S.).

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