Fig 2: Dot1l directly interacts with Zc3h10 for its recruitment and activation of BAT gene program.(A) (Left) CoIP using αFlag for Zc3h10 or αHA for Dot1l after immunoprecipitation with either αHA- or αFlag, respectively, using lysates from HEK293FT cells transfected with Flag-Zc3h10 and HA-Dot1l. (Right) CoIP of endogenous Zc3h10 and Dot1l protein using BAT tissue of C57BL/6 using αZc3h10 and αDot1l. (B) Autoradiograph of GST pull-down using GST-Dot1l and 35S-labeled in vitro transcribed/translated Zc3h10. (C) CoIP using αFlag for Zc3h10 after immunoprecipitation with αHA for Dot1l using lysates from HEK293FT cells transfected with Flag-Zc3h10 and various HA-Dot1l constructs. (D) RT-qPCR and western blot analysis of Dot1l in various tissues from 10-week-old C57BL/6 mice (n = 5). (Bottom right) Quantification of intensity of protein bands, calculated by Dot1l/Gapdh. (E) (Left) RT-qPCR for indicated genes in the adipocyte fraction and SVF from BAT. (Right) RT-qPCR for Dot1l mRNA of BAT and iWAT from mice housed at either 30°C or 4°C (n = 5). (F) RT-qPCR for indicated genes during the course of BAT cell differentiation. (G) (Left) HEK293 cells were cotransfected with the −5.5 kb Ucp1-Luc promoter with Zc3h10 or Dot1l either together or individually (n = 5). (Right) Luciferase assay with HEK293 cells transfected with M55L-Dot1l or WT-Dot1l with the −5.5 kb Ucp1-Luc promoter with Zc3h10. (H) (Left) ChIP-qPCR for Zc3h10 and Dot1l enrichment at the −4.6 kb region of Ucp1 promoter using Flag or HA antibodies. Differentiated BAT cells were transduced with either Flag-Zc3h10 or HA-Dot1l. (Right) ReChIP-qPCR for Dot1l and Zc3h10 co-occupancy using chromatin from BAT cells that were transduced with Zc3h10 and/or Dot1l. Data are expressed as means ± standard errors of the means (SEM). *p<0.05, **p<0.01, ***p<0.001.

Fig 3: DOT1L overexpression (OE) in BJ-hTERT cells induces the SASP but not a senescence-associated cell cycle arrest or DNA damage accumulation; p-p65 is not affected by DOT1L OE.Related to Fig. 4. (A–F and I) BJ-hTERT cells were infected with retrovirus-expressing human DOT1L or empty vector control. (A) Immunoblot analysis of DOT1L. Vinculin was used as a loading control. One of three independent experimental replicates is shown. (B) H3K79me2 and H3K79me3 immunoblot analysis on chromatin fractions. Histone H3 was used as a loading control. One of three independent experimental replicates is shown. (C) IL1A mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD (n = 3). *, P < 0.0001 by Student’s t test. (D) IL6, IL1B, and CXCL8 mRNA expression was determined by RT-qPCR. One of three independent experimental replicates is shown. Data represent mean ± SD (n = 3). *, P < 0.0001 by Student’s t test. (E) Senescence-associated ß-galactosidase (SA-ß-Gal) activity. Shown are representative images from one of three independent experimental replicates. Scale bar, 10 µm. (F) Quantification of SA-ß-Gal in E. One of three independent experimental replicates is shown. Data represent mean ± SD (n = 3, where each dot represents >100 cells counted). Not significant by Student’s t test. (G–J) IMR90 cells were infected with retrovirus-expressing human DOT1L or empty vector control. (G) CDKN2A and CDKN1A mRNA expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD (n = 3). *, P < 0.005 by Student’s t test. (H) LMNB1 expression from RNA-seq. Three technical replicates from one experiment are shown. Data represent mean ± SD (n = 3). *, P < 0.0001 by Student’s t test. (I) Immunoblot analysis of p-p65 and total p65 in IMR90 and BJ-hTERT cells as indicated. ß-Actin was used as a loading control. One of three independent experimental replicates is shown. (J) CEBPB mRNA expression was determined by RT-qPCR. One of six independent experimental replicates is shown. Data represent mean ± SD (n = 3). *, P < 0.005 by Student’s t test.

Fig 4: HMGA2 downregulation contributes to the anticancer effects of DOT1L inhibition.a Lentiviral HMGA2 knockdown in Y79 cells determined by qRT-PCR (N = 2). b, c Immunoblots for the indicated proteins in Y79 shHMGA2 cells treated with 10 µM etoposide for 24 h (b) and in Weri-Rb1 shHMGA2 cells treated with 0.5 µM etoposide for 20 h (c). d Dose-response study for the IC50 of CPX in Y79 cells treated for 72 h by MTT assays. The IC50 is shown as the mean ± SD (N = 6). e, f MTT assays for relative cell viability and proliferation. Y79 cells were subjected to single or combined treatment of 0.5 µM CPX and 0.2 µM etoposide for 72 h to determine the relative cell viability (e) or up to 4 days to monitor the cell proliferation (f). The results are shown as the mean ± SD (N = 6). ****P < 0.0001. g Immunoblots for indicated proteins in Weri-Rb1 cells treated with 2 µM CPX and 0.5 µM etoposide for 20 h. h Immunostaining of DOT1L and HMGA2 in human RB. Serial sections of the indicated tumor tissues were immunostained for DOT1L and HMGA2. The same region of the serial sections (marked by a red square) was magnified on the right (×40). Arrowheads in ZOC-168 mark DOT1L+ HMGA2− tumor foci. Scale bar: 50 μm.

Fig 5: Cotreatment with a DOT1L inhibitor enhances the therapeutic efficacy of etoposide in orthotopic xenografts of RB.a Schematic of orthotopic xenograft study. b Retinal imaging to monitor tumor development on day 13 post-transplantation of Y79 cells. Xenografted eyes develop cloud-like tumors circled by dotted lines whereas uninjected eyes show a clear retinal view. ON optic nerve. c RB development in xenografted eyes indicated by an arrowhead. d Body weight changes of xenografted mice shown as the mean ± SEM. e Plot of average tumor areas in the indicated treatment groups. Data points on the plot represent the average tumor area per section per mouse (n = 17–19 mice per group), with the mean shown as the horizontal bar. The statistical analysis was performed by Mann–Whitney test (two-tailed). f Representative HE-stained images of tumor-burdened eyes from each treatment group. AC anterior chamber, L lens, ON optic nerve. g Quantification of apoptotic cells in xenografts after four rounds of the treatment. The percentage of apoptotic cells per eye was calculated by taking the average of TUNEL-positive nuclei/total nuclei from counting three random fields per eye. The statistical analysis was performed by Mann–Whitney test (two-tailed). h Representative TUNEL images for the indicated treatment groups. Scale bar: 50 µm.