Journal: Science advances

Article Title: DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

doi: 10.1126/sciadv.adf3980

Figure Lengend Snippet: Fig. 4. Erasure of H3K79 methylation promotes reprogramming by altering RNAPII dynamics. (A) Top: Reprogramming scheme. OCT4, SOX2, KLF4, and c-MYC (OSKM) are induced in MEFs with doxycycline (Dox), and DOT1Li or DMSO control. Transgene-dependent colonies express NANOG in doxycycline on days 6 to 8. Trans- gene-independent colonies maintain NANOG expression after doxycycline and DOT1Li removal. Bottom: Reprogramming data are presented as the change relative to the control condition (set to 1). Bars are the mean + SD (n = 3). **P < 0.01 and *P < 0.05 by unpaired two-tailed t test. (B) Metaplots of H3K79me1/2 at H3K79me2-shared genes (Fig. 1A). (C) RNAPII at H3K79me2-shared genes in day 4 control (C)– and DOT1Li (D)–treated cells as RNAPII heatmap (left). Middle: RNAPII metaplots centered at the TSS or genebody (GB). Right: TR. Data are the mean (n = 2). ****P < 0.0001 by unpaired two-tailed t test. (D) Immunoblot of RNAPII on day 4 reprogramming of cells treated with control (C), or DOT1Li (D), quantitated relative to tubulin. (E) Left: Flow cytometry EU-488 signal density plot of day 6 of reprogramming cells treated with control or DOT1Li. Outline = unreleased cells; shaded = cells paused and then released for 30 min in the presence of EU. Right: Fold increase of EU-488 signal after release, DRB sample set to 1. Data are the mean + SD (n = 3). *P < 0.05 by ratio paired two-tailed t test. (F) Reprogramming of cells treated with RNAPII inhibitor DRB, with and without DOT1Li, measured as transgene-dependent colonies. Colonies obtained in control condition set to 1. Bars are the mean + SD (n = 3). Not significant (n.s.) P > 0.05 by estimated marginal means on a mixed-effects model for a randomized complete block design with the Holm correction for multiple comparisons.

Article Snippet: Protein was transferred to a nitrocellulose membrane and blocked in 5% milk/PBS–0.1% Tween 20 for 30 min. Membranes were probed for 1 hour to overnight in primary antibody, including H3K79me1 (1:1000; Diagenode, C15410082 or Abcam, ab2886), H3K79me2 (1:1000; Active Motif, 39143), H3K79me3 (1:1000; Diagenode, C15410068), H3K9ac (1:1000; Active Motif, 39917), H3K23ac (1:1000; Active Motif, 39131), H3K27me3 (1:1000; Cell Signaling, 9733S), RNAPII RPB1 NTD D8L4Y (1:1000; Cell Signaling, 14958S), total H3 (1:3000; Cell Signaling, 3638S), and α-tubulin (1:3000; Cell Signaling, 3873).

Techniques: Methylation, Control, Expressing, Two Tailed Test, Western Blot, Flow Cytometry, Blocking Assay

Journal: Science advances

Article Title: DOT1L is a barrier to histone acetylation during reprogramming to pluripotency.

doi: 10.1126/sciadv.adf3980

Figure Lengend Snippet: Fig. 5. Low DOT1L activity enables higher histone acetylation. (A) Average fraction of peptides containing the specified modification determined by mass spectrom- etry of MEFs (blue) or ESCs (orange) treated with control (−) or DOT1Li (+) for 4 days. Significance determined relative to control treatment of the same cell type. Data are the mean + SD (n = 3 to 4). *P < 0.05 or not significant (n.s.) P > 0.05 by unpaired two-tailed t test. (B) Log2 fold change average peptide ratio heatmap of select mod- ifications in control (C)– and DOT1Li (D)–treated reprogramming cells relative to the fraction modified in ESCs. Data are the mean (n = 3 to 4). (C) Reprogramming scheme of transgene-dependent and transgene-independent NANOG+ colonies. (D, F, and G) Left: Schematic of histone modification targeting during reprogramming. Cells were treated with control (gray) or DOT1Li (black), with and without the specified small molecule—(D) EED inhibitor A395, (F) HDAC inhibitor SAHA, and (G) GCN5 inhibitor CPTH6—measured as transgene-dependent (middle) and transgene-independent (right) colonies. Colonies obtained in control condition set to 1. Bars are the mean + SD (n = 3). All unnormalized data are provided in fig. S7 and used for statistical testing. Not significant (n.s.) P > 0.05 and *P < 0.05 by estimated marginal means on a mixed- effects model for a randomized complete block design with the Holm correction for multiple comparisons. (E) Immunoblot 2 days after doxycycline and DOT1Li/SAHA removal (after 6 days of reprogramming) of H3K9ac, H3K23ac, and H3K79me2 in control or DOT1Li cells treated with SAHA or vehicle beginning on day 3 of reprogram- ming, relative to tubulin.

Article Snippet: Protein was transferred to a nitrocellulose membrane and blocked in 5% milk/PBS–0.1% Tween 20 for 30 min. Membranes were probed for 1 hour to overnight in primary antibody, including H3K79me1 (1:1000; Diagenode, C15410082 or Abcam, ab2886), H3K79me2 (1:1000; Active Motif, 39143), H3K79me3 (1:1000; Diagenode, C15410068), H3K9ac (1:1000; Active Motif, 39917), H3K23ac (1:1000; Active Motif, 39131), H3K27me3 (1:1000; Cell Signaling, 9733S), RNAPII RPB1 NTD D8L4Y (1:1000; Cell Signaling, 14958S), total H3 (1:3000; Cell Signaling, 3638S), and α-tubulin (1:3000; Cell Signaling, 3873).

Techniques: Activity Assay, Modification, Control, Two Tailed Test, Blocking Assay, Western Blot

Journal: bioRxiv

Article Title: DOT1L induces RNAPII accumulation independent of its catalytic activity in pluripotent stem cells

doi: 10.1101/2025.08.08.668971

Figure Lengend Snippet: DOT1L overexpression increases homogeneity of NANOG (A) Percentage of NANOG-high cells in wild-type (WT) and catalytic mutant (CM) DOT1L-overexpressing serum/LIF ESCs after 24h Dox with FLAG-High (+) or -Low (-) populations. FLAG-Low populations were set to 1. Populations were gated as described in Supplementary Figure 3A. Bars represent median ± SD (n = 3 biological replicates; *p < 0.05; ns: not significant; ratio two-tailed paired t-test). (B) Relative Nanog mRNA expression measured by RT-qPCR in DOT1L-WT or -CM ESCs with (+) or without (-) 24h Dox induction (n = 3 biological replicates; ns: not significant; ratio two-tailed paired t-test). Controls were set to 1. (C) Quiescence assay by Hoechst33342 DNA content and Pyronin Y RNA staining showing gating of distinct cell cycle states (G0, G1, S, G2) in Control (left) and DOT1L-WT overexpressing 2i/LIF ESCs with (right) or without (left) 24h Dox induction. Representative flow cytometry plots are shown. (D) Relative c- Myc mRNA expression measured by RT-qPCR in WT and CM DOT1L-overexpressing ESCs with (+) or without (-) 24h Dox induction. Bars represent mean ± SD (n = 3 biological replicates; ns: not significant; paired two-tailed t-test). Controls were set to 1.

Article Snippet: Protein was transferred to a nitrocellulose membrane for 16hrs at 100mA and blocked in 5% milk/TBS–0.1% Tween 20 for 30 min. Membranes were probed for 1 hour to overnight in primary antibody, including H3K79me2 (1:1000; Active Motif, 39143), H3K9ac (1:1000; Active Motif, 39917), H3K27me3 (1:1000; Cell Signaling, 9733S and Active Motif, 39155), RNAPII RPB1 NTD D8L4Y (1:1000; Cell Signaling, 14958S), total H3 (1:3000; Cell Signaling, 3638S), and α-TUBULIN (1:3000; Cell Signaling, 3873), Ser2P (1:1000; Millipore, 04-1571), Ser5P (1:1000; Millipore, 04-1572), FLAG (1:3000; Sigma-Aldrich, F3165), DOT1L (1:1000, Abcam, ab239358), EZH2 (1:4000; BD Biosciences, 612667), NANOG (1:1000, Cell Signaling, D2A3#8822).

Techniques: Over Expression, Mutagenesis, Two Tailed Test, Expressing, Quantitative RT-PCR, Staining, Control, Flow Cytometry