The transcriptional elongation factor CTR9 demarcates PRC2-mediated H3K27me3 domains by altering PRC2 subtype equilibrium.

CTR9 is the scaffold subunit in polymerase-associated factor complex (PAFc), a multifunctional complex employed in multiple steps of RNA Polymerase II (RNAPII)-mediated transcription. CTR9/PAFc is well known as an evolutionarily conserved elongation factor that regulates gene activation via coupling with histone modifications enzymes. However, little is known about its function to restrain repressive histone markers. Using inducible and stable CTR9 knockdown breast cancer cell lines, we discovered that the H3K27me3 levels are strictly controlled by CTR9. Quantitative profiling of histone modifications revealed a striking increase of H3K27me3 levels upon loss of CTR9. Moreover, loss of CTR9 leads to genome-wide expansion of H3K27me3, as well as increased recruitment of PRC2 on chromatin, which can be reversed by CTR9 restoration. Further, CTR9 depletion triggers a PRC2 subtype switch from the less active PRC2.2, to the more active PRC2.1 with higher methyltransferase activity. As a consequence, CTR9 depletion generates vulnerability that renders breast cancer cells hypersensitive to PRC2 inhibitors. Our findings that CTR9 demarcates PRC2-mediated H3K27me3 levels and genomic distribution provide a unique mechanism that explains the transition from transcriptionally active chromatin states to repressive chromatin states and sheds light on the biological functions of CTR9 in development and cancer.

Systematic identification of Ctr9 regulome in ERα-positive breast cancer.

BACKGROUND: We had previously identified Ctr9, the key scaffold subunit of the human RNA polymerase II (RNAPII) associated factor complex (PAFc), as a key factor regulating a massive ERα target gene expression and ERα-positive breast cancer growth. Furthermore, we have shown that knockdown of Ctr9 reduces ERα protein stability and decreases the occupancy of ERα and RNAPII at a few ERα-target loci. However, it remains to be determined whether Ctr9 controls ERα-target gene expression by regulating the global chromatin occupancy of ERα and RNAPII in the presence of estrogen. RESULTS: In this study, we determined the genome-wide ERα and RNAPII occupancy in response to estrogen reatment and/or Ctr9 knockdown by performing chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq). We found that loss of Ctr9 dramatically decreases the global occupancy of ERα and RNAPII, highlighting the significance of Ctr9 in regulating estrogen signaling in ERα-positive breast cancer cells. Combining this resource with previously published genomic data sets, we identified a unique subset of ERα and Ctr9 target genes, and further delineated the independent function of Ctr9 from other subunits in PAFc when regulating transcription. CONCLUSIONS: Our data demonstrated that Ctr9, independent of other PAFc subunits, controls ERα-target gene expression by regulating global chromatin occupancies of ERα and RNAPII.

CTR9 drives osteochondral lineage differentiation of human mesenchymal stem cells via epigenetic regulation of BMP-2 signaling.

Cell fate determination of human mesenchymal stem/stromal cells (hMSCs) is precisely regulated by lineage-specific transcription factors and epigenetic enzymes. We found that CTR9, a key scaffold subunit of polymerase-associated factor complex (PAFc), selectively regulates hMSC differentiation to osteoblasts and chondrocytes, but not to adipocytes. An in vivo ectopic osteogenesis assay confirmed the essentiality of CTR9 in hMSC-derived bone formation. CTR9 counteracts the activity of Enhancer Of Zeste 2 (EZH2), the epigenetic enzyme that deposits H3K27me3, in hMSCs. Accordingly, CTR9 knockdown (KD) hMSCs gain H3K27me3 mark, and the osteogenic differentiation defects of CTR9 KD hMSCs can be partially rescued by treatment with EZH2 inhibitors. Transcriptome analyses identified bone morphology protein-2 (BMP-2) as a downstream effector of CTR9. BMP-2 secretion, membrane anchorage, and the BMP-SMAD pathway were impaired in CTR9 KD MSCs, and the effects were rescued by BMP-2 supplementation. This study uncovers an epigenetic mechanism engaging the CTR9-H3K27me3-BMP-2 axis to regulate the osteochondral lineage differentiation of hMSCs.

Acetylation-dependent regulation of PD-L1 nuclear translocation dictates the efficacy of anti-PD-1 immunotherapy.

Immunotherapies that target programmed cell death protein 1 (PD-1) and its ligand PD-L1 as well as cytotoxic T-lymphocyte-associated protein 4 (CTLA4) have shown impressive clinical outcomes for multiple tumours. However, only a subset of patients achieves durable responses, suggesting that the mechanisms of the immune checkpoint pathways are not completely understood. Here, we report that PD-L1 translocates from the plasma membrane into the nucleus through interactions with components of the endocytosis and nucleocytoplasmic transport pathways, regulated by p300-mediated acetylation and HDAC2-dependent deacetylation of PD-L1. Moreover, PD-L1 deficiency leads to compromised expression of multiple immune-response-related genes. Genetically or pharmacologically modulating PD-L1 acetylation blocks its nuclear translocation, reprograms the expression of immune-response-related genes and, as a consequence, enhances the anti-tumour response to PD-1 blockade. Thus, our results reveal an acetylation-dependent regulation of PD-L1 nuclear localization that governs immune-response gene expression, and thereby advocate targeting PD-L1 translocation to enhance the efficacy of PD-1/PD-L1 blockade.