BS69/ZMYND11 reads and connects histone H3.3 lysine 36 trimethylation-decorated chromatin to regulated pre-mRNA processing

Abstract

BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.

Figure 1. BS69 shows a similar genomic distribution pattern as H3K36me3

(A) Genomic distribution of BS69 peaks (18,406) in HeLa cells. (B) Normalized tag density of BS69 and H3K36me3 along the transcription unit. Each gene body is represented from 0% (TSS) to 100% (Jorgensen et al.) on the X-axis. Normalized Tag density is plotted from 30% upstream of TSS to 30% downstream of TES. (C) Averaged exonic occupancies of BS69 and H3K36me3. The centers of exons are aligned at position 0 with 1kb of both upstream and downstream sequences included in the analysis. (D) Representatives of BS69, H3K36me3 and H3.3-FLAG ChIP-seq peaks in HeLa cells. Arrows denote TSS and transcription orientation. (E) and (F) Occupancies of H3K36me3 and BS69 at selected gene bodies (gb), but not promoters (pro), are reduced in SETD2 KD HeLa cells. Data are represented as mean ± SEM from 3 biological replicates (E, F), *P<0.05, **P<0.01. See also Figure S1.

Figure 2. BS69 binds H3.3K36me3 in vitro via its PWWP domain

(A) Schematic diagrams of human BS69 and mutants carrying PWWP deletion and point mutations denoted by red stars. (B) Recombinant GST-BS69_50-401 specifically recognizes H3.3K36me3 peptide in the biotinylated histone peptide pull-down assays. (C) Sequential ChIP using H3K36me3 and anti-FLAG antibodies in HeLa cells overexpressing H3.3-FLAG shows co-occupancy of H3K36me3 and H3.3-FLAG at indicated locales of BS69 target genes. (D) GST-BS69_50-401 FW point mutant (F293A, W294A) loses the ability to bind H3.3K36me3 in vitro. (E) and (F) MicroScale Thermophoresis analysis determined the Kd of GST-BS69_50-401 WT (E) interaction with H3.3K36me3 to be 50 μM. The Kd value of the interaction between the BS69 PWWP point mutant and H3.3K36me3 (F) was determined to be 277 μM. (G) HA-ChIP and q-PCR demonstrated that the ectopically expressed WT BS69 but not the BS69 PWWP deletion mutant binds the BS69 targets (identified through BS69 ChIP). (H) The binding of GST-BS69_50-401 to H3.3K36me3 peptide is abolished by phosphorylation at position S31. Data are represented as mean ± SEM from 3 biological replicates (C,G), *P<0.05, **P<0.01. See also Figure S2.

Figure 3. BS69 interacts with splicing factors

(A) Left panel: TAP purified FLAG-HA-BS69 protein complex was resolved by gradient SDS-PAGE and visualized by silver staining. The positions of the tagged BS69 (FH-BS69) as well as some of the RNA splicing factors are indicated on the right. Right panel: polypeptides identified by tandem mass spectrometry. (B) Reciprocal immunoprecipitation by EFTUD2 antibody incubated with FLAG immunoprecipitants purified from HeLa cells stably expressing FLAG-HA-BS69 brought down FLAG-HA-BS69 as well PRPF8 and SNRNP200. (C) Interaction between endogenous BS69 and EFTUD2 was confirmed by co-immunoprecipitation using HeLa nuclear extract. (D) Schematic representation of full length and various truncated forms of BS69. Amino acid positions are indicated. (E) and (F) Interactions between EFTUD2 and full length and various truncated forms of BS69 were determined by GST pull-down assays. Recombinant BS69 proteins and FLAG-EFTUD2 were purified from E. coli and insect cells, respectively. FL: full length. (G) and (H), Interactions between FLAG-BS69 and snRNAs were determined by Northern blotting (G) and RT-qPCR (H). Data are represented as mean ± SEM from 3 biological replicates, *P<0.05, **P<0.01; n.s., not significant. See also Figure S3, Table S1.

Figure 4. BS69 mainly regulates intro retention

(A) Summary of altered splicing (AS) events in HeLa cells upon BS69 KD detected by RNA-seq from three biological replicates. Two independent BS69 shRNAs were used. (B) Heatmap comparison of the mRNA levels of genes whose IR events are regulated by BS69 in the presence and absence of BS69. (C) RT-qPCR analyses of mRNA levels of six BS69-regulated IR genes in different cellular compartments. Data are represented as mean ± SEM from 3 biological replicates, *P<0.05, **P<0.01; n.s., not significant. See also Figure S4, Table S2.

Figure 5. BS69 regulates intron retention and exon skipping events by antagonizing EFTUD2 through physical interaction

(A) and (B) Wild type but not EFTUD2 interaction defective mutant (Δ556-562) rescued the alteration of IR (Intron Retention, panel A) and ES (Exon Skipping, panel B) caused by BS69 knockdown. (C) EFTUD2-interaction defective mutant of BS69 (BS69_Δ556-562aa) retains H3.3K36me3 binding ability at a comparable level as that of wildtype in histone peptide pull-down assay in vitro. (D) and (E) Knockdown of EFTUD2 caused an increase in IR (D) and an altered ratio of ES (panel E) of target genes in HeLa cells. Data are represented as mean ± SEM from 3 biological replicates (A, B, D, E), *P<0.05, **P<0.01; n.s., not significant. See also Figure S5.

Figure 6. Binding H3K36me3 is important for BS69 to regulate alternative splicing

(A) and (B) Knockdown of SETD2 by two independent shRNAs in HeLa cells caused a decrease in IR (A) and an altered ratio of ES (B) of BS69 target genes. (C) and (D) Wild type but not the PWWP FW point mutant rescued the alteration of IR (C) and ES (D) caused by BS69 knockdown. Data are represented mean ± SEM from 3 biological replicates, *P<0.05, **P<0.01; n.s., not significant. See also Figure S6.