The H3.3K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation

Abstract

Histone H3.3 is frequently mutated in tumors, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it is known that H3.3K36M changes the epigenetic landscape, its effects on gene expression dynamics remain unclear. Here, we use a synthetic reporter to measure the effects of H3.3K36M on silencing and epigenetic memory after recruitment of the ZNF10 Krüppel-associated box (KRAB) domain, part of the largest class of human repressors and associated with H3K9me3 deposition. We find that H3.3K36M, which decreases H3K36 methylation and increases histone acetylation, leads to a decrease in epigenetic memory and promoter methylation weeks after KRAB release. We propose a model for establishment and maintenance of epigenetic memory, where the H3K36 methylation pathway is necessary to maintain histone deacetylation and convert H3K9me3 domains into DNA methylation for stable epigenetic memory. Our quantitative model can inform oncogenic mechanisms and guide development of epigenetic editing tools.

Keywords: DNA methylation; H3.3K36M; H3K36me2; H3K36me3; KRAB; NSD1; SETD2; epigenetic memory; heterochromatin; oncohistone.

Figure 1.. H3.3K36M disrupts KRAB mediated epigenetic memory in HEK23T cells.

(A) Schematic of the synthetic reporter (EF1a promoter driving surface marker and citrine) integrated in the AAVS1 locus of HEK293T, to which rTetR-KRAB can be recruited with doxycycline (dox) to induce gene silencing in the presence or absence of H3.3K36M oncohistone. H3.3K36M is fused to an auxin-inducible degron (AID) and HA tag. (B) Representative Western blots against HA-tag (H3.3K36M), H3K36me2 and H3K36me3. Parent cells express only the reporter and rTetR-KRAB, while K36M cells express the reporter, rTetR-KRAB and H3.3K36M. IAA was removed for 48 hours and replaced with DMSO to allow accumulation of H3.3K36M. On the right, bar plots indicate quantification of blot band intensities normalized to H3 input (mean +/− STD of n=3 replicates). (C) CUT&RUN genome tracks measuring HA-tag (H3.3K36M) incorporation 80kb around the reporter in parental and H3.3K36M lines, +/− dox (1000 ng/ml) recruitment of KRAB for 6 days; total reads were normalized using E.coli DNA spike-in. (D) CUT&RUN genome tracks measuring H3.3K36M and total H3.3 at the reporter +/− IAA, normalized by total number of reads (counts per million). The EF1a promoter is shaded gray and not analyzed due to homology with endogenous EF1a promoters. (E) Quantification of normalized reads at the reporter for CUT&RUN tracks in D. (F) Flow cytometry time courses measuring the fraction of cells with Citrine OFF cells upon 6 days of KRAB recruitment (1000 ng/ml dox, dashed vertical line) and 14 days of memory (no dox). Shading represents SEM (n= 3 replicates). Statistical analysis was performed using Welch’s T-test. (G) Flow cytometry time course of low-dox KRAB recruitment (5ng/ml dox) in the absence of IAA in cells expressing H3.3K36M and parental cells. IAA (50μM) is added to partially degrade H3.3K36M. n= 3 replicates. Statistical analysis was performed using one-way ANOVA. (H) KRAB memory time course after low dox silencing (5ng/ml, shown in G): dox was removed and the percentage of cells Citrine OFF measured by flow cytometry for 14 days. (I) Bar plots quantifying memory data in H where the % cells with Citrine OFF at day 14 are normalized to the levels of silencing at dox removal (day 6 of silencing) for each cell line. n= 3 replicates. Statistical analysis was performed using one-way ANOVA

Figure 2.. KRAB mediated epigenetic memory and silencing speed is reduced in a H3.3K36M chondroblastoma model.

(A) Schematic describing engineering of TC28a2 chondroblastoma cell lines containing reporter and rTetR-KRAB. (B) Western blots and quantification of H3K36me2 and H3K36me3 (normalized by H3) in K36M clones . (C) Flow cytometry time course during 4 days of KRAB recruitment (1000ng/ml dox), with the fraction of Citrine silenced cells quantified over time for WT and H3.3K36M TC28a2 reporter clones (n=3 replicates). Statistical analysis performed using one-way ANOVA. (D) Flow cytometry time course during 4 days of KRAB recruitment with low dox (5ng/ml dox), as in C. (E) Flow cytometry time course measuring epigenetic memory in TC28a2 cells: percentage of cells with citrine OFF after 5 days of KRAB recruitment (1000ng/ml dox) and release (dox removal at day 0) over 18 days, normalized to the no dox control at each time point (n=2 replicates). (F) Normalized epigenetic memory from the timecouses in E by dividing memory (the %Citrine OFF cells at day 15) to the initial silencing (%OFF at day 0), n=2 replicates. (G) Flow cytometry time course measuring epigenetic memory after 6 days of KRAB recruitment (5ng/ml dox) in TC28a2 cells. The percentage of Citrine OFF cells is measured after dox removal at day 0 and normalized to the no dox control at each time point (n=3 biological replicates). Statistical analysis performed using one-way ANOVA. (H) Barplors quantifying normalized epigenetic memory as in F (n=3 replicates). (I) Schematic describing workflow to overexpress H3.3 WT or H3.3K36M in wildtype TC28a2 cells. (J) Epigenetic memory time course after 5 days of KRAB recruitment (1000ng/ml dox) and 18 days of dox release in WT TC28a2 cells transduced with lentivirus consisting of H3.3 or H3.3K36M overexpression. Shading represents standard deviation at each timepoint (n=4 replicates). Statistical analysis used Welch’s T-test. (K) Barplots quantifying normalized epigenetic memory from the timecouses in J by normalizing the %Citrine OFF cells at day 12 to silencing (%OFF) at day 0 (n=4 replicates). Statistical analysis used Welch’s T-test.

Figure 3.. Loss of epigenetic memory by H3.3K36M is not due to reduced H3K9me3 deposition.

(A) Schematic describing magnetic cell separation after establishment of epigenetic memory. KRAB was recruited with dox (1000 ng/ml) for 2 days in parental HEK293T cells and 10 days in K36M HEK293T cells to generate ~50% OFF memory population after 14 days of release. This OFF population was sorted for each cell line, and silenced cells were propagated to assess maintenance of memory using flow cytometry 2 days and 20 days after sorting. (B) Flow cytometry distributions of Citrine reporter expression during KRAB recruitment in parental and K36M for no dox (D0), 2 days (D2) and 6 days (D6) with 1000 ng/ml dox. (C) CUT&RUN genome tracks for H3K9me3 during KRAB silencing for the same populations of cells shown in B. Read numbers are normalized by total number of reads and shown as counts per million. (D) Flow cytometry distributions of Citrine expression after enriching the stably silenced memory population by FACS 14 days after KRAB release. (E) CUT&RUN genome tracks for H3K9me3, normalized as in C, during KRAB memory corresponding to the sorted population of cells shown in D. (F) Left: H3K9me3 at the reporter and the surrounding 80kb domain around the reporter quantified from genome tracks at the time points shown in C&E (n=2 replicates). Right: H3K9me3 at a panel of control genes (n=2 replicates). (G) H3K9me3 (counts per million per kb) at the AAVS1 locus (PPP1R12C gene) and 80kb domain around the reporter over time in WT and K36M cells upon dox recruitment and release (dashed line). The release time point is the sorted memory population in D (n=2 replicates). (H) H3K9me3 at pEF promoter measured with CUT&RUN qPCR at different timepoints after KRAB recruitment and release normalized using the IGF2 gene. Primer binding regions upstream and downstream are marked in red. Data is the mean of 2 replicates with shading representing SEM. (I) A sequential scatter plot of H3K9me3 dynamics at the downstream pEF promoter region versus the fraction of Citrine silent cells from flow cytometry data in Fig. 1F. The Day 20 memory time point shows H3K9me3 for the sorted OFF cells in panel D. Percentages of cells OFF are the mean of 3 replicates +/− SEM. H3K9me3 levels are the mean of 2 biological replicates (CUT&RUN qPCR from 3H).

Figure 4.. H3.3K36M disrupts DNA methylation deposition after KRAB recruitment and release.

(A) Flow cytometry histograms of Citrine expression in WT and H3.3K36M HEK293T cells before KRAB recruitment, after 6 days of dox silencing at 1000ng/ml dox, and 14 days memory after dox removal (D20). Cells from these populations were collected for EMseq. (B) Top: Schematic of the AAVS1 reporter indicating regions for which DNA methylation is measured using EMseq. Bottom: %CpG methylation from EMseq plotted along the amplicon corresponding to the promoter region on the reporter upon KRAB recruitment and release at time points corresponding to populations shown in 4H (n=2 replicates). Shading represents standard deviation. (C) Top: Scatter plot measuring the time evolution of the mean %CpG methylation (n=2 replicates) at the promoter from EMseq (from Fig. 4H) and the mean %Citrine OFF from flow cytometry (n=3 replicates from Fig. 1F). Y-axis error bars represent the SEM of 3 replicates. Bottom: Scatter plot measuring the time evolution of the mean %CpG methylation (n=2 replicates) at the promoter from EMseq (from Fig. 4H) versus H3K9me3 levels at the pEF promoter measured from CUT&RUN qPCR (n=2 replicates from Fig. 3H). Y-axis error bars represent the SEM of 2 replicates. (D) Flow cytometry time courses measuring epigenetic memory in HEK293T cells after 2 days of 1000ng/ml dox-mediated KRAB recruitment and treatment with siRNA targeting DNMT3A and DNMT3B in WT or H3.3K36M cells starting at dox removal (n=3 replicates). The black bracket at day 14 indicates the difference in memory. (E) Barplots quantifying the difference in memory between the DNMT3A/B siRNA and control siRNA in WT and H3.3K36M cells at day 14 after dox removal (n=3 biological replicates). Statistical analysis performed using Welch’s T-test. (F) Schematic describing perturbations targeting H3K36 methyltransferases NSD1 or SETD2 in wildtype cells to phenocopy effects of the H3.3K36M oncohistone. (G) Left: Flow cytometry time course measuring epigenetic memory in HEK293T cells treated with siNSD1 or control siRNA upon KRAB release (n=2 replicates). Right: EMseq at the reporter promoter quantifying average %CpG methylation from siNSD1 and siControl treated cells at day 20. Shading represents SEM of 2 replicates. (H) Left: Flow cytometry time course measuring epigenetic memory in HEK293T cells treated with 2.5μM SETD2-IN-1 or DMSO for the first 6 days after KRAB release (n=2 replicates). Right: EMseq quantifying average %CpG methylation from SETD2-IN-1 treated cells and DMSO treated cells at the reporter promoter at day 20. Shading represents SEM of 2 replicates. (I) H3K36me3 from CUT&RUN qPCR during KRAB recruitment and release in parental (green) and H3.3K36M cells (orange) (n=2 replicates). Dashed line denotes dox removal. qPCR data for each sample was normalized to the endogenous TBP gene. (J) H3K27ac from CUT&RUN qPCR during KRAB recruitment and release, as in I. (K) Flow cytometry time course measuring KRAB epigenetic memory in HEK293T cells treated with 100nM of TSA or DMSO upon dox release for parental cells (green) and H3.3K36M cells (orange). Plots are means of 3 replicates. (L) Barplots comparing the difference in epigenetic memory at day 20 between DMSO and TSA treatments in parental and K36M cells in L. (M) Kinetic model describing steps associated with establishment and maintenance of epigenetic memory in parental and K36M cells. H3.3K36M cells (bottom) have reduced H3K36me3 and reduced ability to convert H3K9me3 into DNA methylation (thinner arrow).

Figure 5.. Chromatin spreading model that includes DNA methylation quantitatively predicts decreases in epigenetic memory caused by H3.3K36M.

(A) Schematic showing three nucleosome states: Active (A, orange: defines as H3Ac.), Repressive (R, pink: defined as H3K9me3) and Irreversibly Repressive (I, gray: defined as DNA methylation co-existing with H3K9me3). Example enzymes that mediate conversions between nucleosome states are indicated on the black arrows. KRAB recruitment (nucleation) increases the A to R transition rate (at the nucleosome where KRAB is recruited, blue arrow); dox concentration and recruitment time increase nucleation rate and duration respectively. (B) Typical time-evolution of the nucleosome array: 1) The system starts with all nucleosomes in the active state (orange), and upon dox addition KRAB nucleates H3K9me3 (pink). 2) H3K9me3 spreads from the nucleation site through reader-writer feedback between neighboring nucleosomes. 3) Spreading of repressing modifications can be counteracted (“erased”) by direct R to A transitions through histone turnover, removal of histone methylation, or deposition of histone acetylation. 4) Nucleosomes at the promoter can be irreversibly silenced (gray) by acquiring DNA methylation. (C) Left: Example of a cell with an irreversibly silenced reporter gene due to acquisition of I nucleosomes (gray) at the promoter. Right: Example of another cell whose reporter becomes reactivated after transient silencing. Zoomed in view of 20 nucleosomes. (D) Left: Experimental time-series data from HEK293T cells showing the fraction of silent cells during recruitment for 5 days (top) or 3 days (bottom) at different dox concentrations, as indicated in top panel legend (n=3 replicates). Dashed line denotes dox removal. Right: Simulation data showing the fraction of silent reporters (from 1000 simulations) for different nucleation rates that were kept constant for either 5 days (top) or 3 days (bottom), and set to zero for times to the right of the dashed line. (E) Schematic showing the kinetic model used for generating the memory landscapes of parental (WT) and oncohistone cells in F. Input variables are the nucleation rate and nucleation days. Oncohistone simulations were performed with the same parameters but with 75% of randomly chosen nucleosomes being blocked for R to I transitions to mimic the oncohistone effect on DNA methylation. (F) Memory-landscapes in parental (top) and H3.3K36M oncohistone cells (bottom) showing the fraction of silent cells at simulation day 14 as a function of nucleation-rate (in attempts/hour) and nucleation duration (in days). (G) Left: Simulation time courses performed with nucleation rates of 0.1 ℎ⁻¹ (transparent) and 10 ℎ⁻¹ (nontransparent) for 1 (top) and 6 days (bottom) for oncohistones (blue) and WT cells (mustard), respectively. Barplots show the fraction of silenced cells at memory day 14. Right: Experimental time courses from HEK293T cells performed with 10 ng/mL (transparent) and 1000 ng/mL (nontransparent) of dox for 1 (top) and 6 days (bottom) for oncohistones (blue) and parental WT cells (mustard), respectively. Barplots show the fraction of silenced cells at memory day 14.