Co-targeting JAK1/STAT6/GAS6/TAM signaling improves chemotherapy efficacy in Ewing sarcoma.

Ewing sarcoma is a pediatric bone and soft tissue tumor treated with chemotherapy, radiation, and surgery. Despite intensive multimodality therapy, ~50% patients eventually relapse and die of the disease due to chemoresistance. Here, using phospho-profiling, we find Ewing sarcoma cells treated with chemotherapeutic agents activate TAM (TYRO3, AXL, MERTK) kinases to augment Akt and ERK signaling facilitating chemoresistance. Mechanistically, chemotherapy-induced JAK1-SQ phosphorylation releases JAK1 pseudokinase domain inhibition allowing for JAK1 activation. This alternative JAK1 activation mechanism leads to STAT6 nuclear translocation triggering transcription and secretion of the TAM kinase ligand GAS6 with autocrine/paracrine consequences. Importantly, pharmacological inhibition of either JAK1 by filgotinib or TAM kinases by UNC2025 sensitizes Ewing sarcoma to chemotherapy in vitro and in vivo. Excitingly, the TAM kinase inhibitor MRX-2843 currently in human clinical trials to treat AML and advanced solid tumors, enhances chemotherapy efficacy to further suppress Ewing sarcoma tumor growth in vivo. Our findings reveal an Ewing sarcoma chemoresistance mechanism with an immediate translational value.

CHD4 and SMYD1 repress common transcriptional programs in the developing heart.

Regulation of chromatin states is essential for proper temporal and spatial gene expression. Chromatin states are modulated by remodeling complexes composed of components that have enzymatic activities. CHD4 is the catalytic core of the nucleosome remodeling and deacetylase (NuRD) complex, which represses gene transcription. However, it remains to be determined how CHD4, a ubiquitous enzyme that remodels chromatin structure, functions in cardiomyocytes to maintain heart development. In particular, whether other proteins besides the NuRD components interact with CHD4 in the heart is controversial. Using quantitative proteomics, we identified that CHD4 interacts with SMYD1, a striated muscle-restricted histone methyltransferase that is essential for cardiomyocyte differentiation and cardiac morphogenesis. Comprehensive transcriptomic and chromatin accessibility studies of Smyd1 and Chd4 null embryonic mouse hearts revealed that SMYD1 and CHD4 repress a group of common genes and pathways involved in glycolysis, response to hypoxia, and angiogenesis. Our study reveals a mechanism by which CHD4 functions during heart development, and a previously uncharacterized mechanism regarding how SMYD1 represses cardiac transcription in the developing heart.

SETD2 maintains nuclear lamina stability to safeguard the genome.

Histone methyltransferases play essential roles in the organization and function of chromatin. They are also frequently mutated in human diseases including cancer1. One such often mutated methyltransferase, SETD2, associates co-transcriptionally with RNA polymerase II and catalyzes histone H3 lysine 36 trimethylation (H3K36me3) - a modification that contributes to gene transcription, splicing, and DNA repair2. While studies on SETD2 have largely focused on the consequences of its catalytic activity, the non-catalytic functions of SETD2 are largely unknown. Here we report a catalysis-independent function of SETD2 in maintaining nuclear lamina stability and genome integrity. We found that SETD2, via its intrinsically disordered N-terminus, associates with nuclear lamina proteins including lamin A/C, lamin B1, and emerin. Depletion of SETD2, or deletion of its N-terminus, resulted in widespread nuclear morphology abnormalities and genome stability defects that were reminiscent of a defective nuclear lamina. Mechanistically, the N-terminus of SETD2 facilitates the association of the mitotic kinase CDK1 with lamins, thereby promoting lamin phosphorylation and depolymerization required for nuclear envelope disassembly during mitosis. Taken together, our findings reveal an unanticipated link between the N-terminus of SETD2 and nuclear lamina organization that may underlie how SETD2 acts as a tumor suppressor.

MS0621, a novel small-molecule modulator of Ewing sarcoma chromatin accessibility, interacts with an RNA-associated macromolecular complex and influences RNA splicing.

Ewing sarcoma is a cancer of children and young adults characterized by the critical translocation-associated fusion oncoprotein EWSR1::FLI1. EWSR1::FLI1 targets characteristic genetic loci where it mediates aberrant chromatin and the establishment of de novo enhancers. Ewing sarcoma thus provides a model to interrogate mechanisms underlying chromatin dysregulation in tumorigenesis. Previously, we developed a high-throughput chromatin-based screening platform based on the de novo enhancers and demonstrated its utility in identifying small molecules capable of altering chromatin accessibility. Here, we report the identification of MS0621, a molecule with previously uncharacterized mechanism of action, as a small molecule modulator of chromatin state at sites of aberrant chromatin accessibility at EWSR1::FLI1-bound loci. MS0621 suppresses cellular proliferation of Ewing sarcoma cell lines by cell cycle arrest. Proteomic studies demonstrate that MS0621 associates with EWSR1::FLI1, RNA binding and splicing proteins, as well as chromatin regulatory proteins. Surprisingly, interactions with chromatin and many RNA-binding proteins, including EWSR1::FLI1 and its known interactors, were RNA-independent. Our findings suggest that MS0621 affects EWSR1::FLI1-mediated chromatin activity by interacting with and altering the activity of RNA splicing machinery and chromatin modulating factors. Genetic modulation of these proteins similarly inhibits proliferation and alters chromatin in Ewing sarcoma cells. The use of an oncogene-associated chromatin signature as a target allows for a direct approach to screen for unrecognized modulators of epigenetic machinery and provides a framework for using chromatin-based assays for future therapeutic discovery efforts.

Regulation of EWSR1-FLI1 Function by Post-Transcriptional and Post-Translational Modifications.

Ewing sarcoma is the second most common bone tumor in childhood and adolescence. Currently, first-line therapy includes multidrug chemotherapy with surgery and/or radiation. Although most patients initially respond to chemotherapy, recurrent tumors become treatment refractory. Pathologically, Ewing sarcoma consists of small round basophilic cells with prominent nuclei marked by expression of surface protein CD99. Genetically, Ewing sarcoma is driven by a fusion oncoprotein that results from one of a small number of chromosomal translocations composed of a FET gene and a gene encoding an ETS family transcription factor, with ~85% of tumors expressing the EWSR1::FLI1 fusion. EWSR1::FLI1 regulates transcription, splicing, genome instability and other cellular functions. Although a tumor-specific target, EWSR1::FLI1-targeted therapy has yet to be developed, largely due to insufficient understanding of EWSR1::FLI1 upstream and downstream signaling, and the challenges in targeting transcription factors with small molecules. In this review, we summarize the contemporary molecular understanding of Ewing sarcoma, and the post-transcriptional and post-translational regulatory mechanisms that control EWSR1::FLI1 function.

CHD4 is recruited by GATA4 and NKX2-5 to repress noncardiac gene programs in the developing heart.

The nucleosome remodeling and deacetylase (NuRD) complex is one of the central chromatin remodeling complexes that mediates gene repression. NuRD is essential for numerous developmental events, including heart development. Clinical and genetic studies have provided direct evidence for the role of chromodomain helicase DNA-binding protein 4 (CHD4), the catalytic component of NuRD, in congenital heart disease (CHD), including atrial and ventricular septal defects. Furthermore, it has been demonstrated that CHD4 is essential for mammalian cardiomyocyte formation and function. A key unresolved question is how CHD4/NuRD is localized to specific cardiac target genes, as neither CHD4 nor NuRD can directly bind DNA. Here, we coupled a bioinformatics-based approach with mass spectrometry analyses to demonstrate that CHD4 interacts with the core cardiac transcription factors GATA4, NKX2-5, and TBX5 during embryonic heart development. Using transcriptomics and genome-wide occupancy data, we characterized the genomic landscape of GATA4, NKX2-5, and TBX5 repression and defined the direct cardiac gene targets of the GATA4-CHD4, NKX2-5-CHD4, and TBX5-CHD4 complexes. These data were used to identify putative cis-regulatory elements controlled by these complexes. We genetically interrogated two of these silencers in vivo: Acta1 and Myh11 We show that deletion of these silencers leads to inappropriate skeletal and smooth muscle gene misexpression, respectively, in the embryonic heart. These results delineate how CHD4/NuRD is localized to specific cardiac loci and explicates how mutations in the broadly expressed CHD4 protein lead to cardiac-specific disease states.

PBRM1 Inactivation Promotes Upregulation of Human Endogenous Retroviruses in a HIF-Dependent Manner.

Clear cell renal cell carcinoma (ccRCC) is considered an immunotherapy-responsive disease; however, the reasons for this remain unclear. Studies have variably implicated PBRM1 mutations as a predictive biomarker of immune checkpoint blockade (ICB) response, and separate studies demonstrate that expression of human endogenous retroviruses (hERV) might be an important class of tumor-associated antigens. We sought to understand whether specific mutations were associated with hERV expression. Two large, annotated genomic datasets, TCGA KIRC and IMmotion150, were used to correlate mutations and hERV expression. PBRM1 mutations were consistently associated with increased hERV expression in primary tumors. In vitro silencing of PBRM1, HIF1A, and HIF2A followed by RNA sequencing was performed in UMRC2 cells, confirming that PBRM1 regulates hERVs in a HIF1α- and HIF2α-dependent manner and that hERVs of the HERVERI superfamily are enriched in PBRM1-regulated hERVs. Our results uncover a role for PBRM1 in the negative regulation of hERVs in ccRCC. Moreover, the HIF-dependent nature of hERV expression explains the previously reported ccRCC-specific clinical associations of PBRM1-mutant ccRCC with both a good prognosis as well as improved clinical outcomes to ICB. See related Spotlight by Labaki et al., p. 274.

Targeting disialoganglioside GD2 with chimeric antigen receptor-redirected T cells in lung cancer.

BACKGROUND: We explored whether the disialoganglioside GD2 (GD2) is expressed in small cell lung cancer (SCLC) and non-SCLC (NSCLC) and can be targeted by GD2-specific chimeric antigen receptor (CAR) T cells. METHODS: GD2 expression was evaluated in tumor cell lines and tumor biopsies by flow cytometry and immunohistochemistry. We used a GD2.CAR that coexpress the IL-15 to promote T-cell proliferation and persistence, and the inducible caspase 9 gene safety switch to ablate GD2.CAR-T cells in case of unforeseen toxicity. The antitumor activity of GD2.CAR-T cells was evaluated using in vitro cocultures and in xenograft models of orthotopic and metastatic tumors. The modulation of the GD2 expression in tumor cell lines in response to an epigenetic drug was also evaluated. RESULTS: GD2 was expressed on the cell surface of four of fifteen SCLC and NSCLC cell lines (26.7%) tested by flow cytometry, and in 39% of SCLC, 72% of lung adenocarcinoma and 56% of squamous cell carcinoma analyzed by immunohistochemistry. GD2 expression by flow cytometry was also found on the cell surface of tumor cells freshly isolated from tumor biopsies. GD2.CAR-T cells exhibited antigen-dependent cytotoxicity in vitro and in vivo in xenograft models of GD2-expressing lung tumors. Finally, to explore the applicability of this approach to antigen low expressing tumors, we showed that pretreatment of GD2low/neg lung cancer cell lines with the Enhancer of zeste homolog 2 inhibitor tazemetostat upregulated GD2 expression at sufficient levels to trigger GD2.CAR-T cell cytotoxic activity. CONCLUSIONS: GD2 is a promising target for CAR-T cell therapy in lung cancer. Tazemetostat treatment could be used to upregulate GD2 expression in tumor cells, enhancing their susceptibility to CAR-T cell targeting.

Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors.

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.

Radiation-Induced Phosphorylation of a Prion-Like Domain Regulates Transformation by FUS-CHOP.

Chromosomal translocations generate oncogenic fusion proteins in approximately one-third of sarcomas, but how these proteins promote tumorigenesis is not well understood. Interestingly, some translocation-driven cancers exhibit dramatic clinical responses to therapy, such as radiotherapy, although the precise mechanism has not been elucidated. Here we reveal a molecular mechanism by which the fusion oncoprotein FUS-CHOP promotes tumor maintenance that also explains the remarkable sensitivity of myxoid liposarcomas to radiation therapy. FUS-CHOP interacted with chromatin remodeling complexes to regulate sarcoma cell proliferation. One of these chromatin remodelers, SNF2H, colocalized with FUS-CHOP genome-wide at active enhancers. Following ionizing radiation, DNA damage response kinases phosphorylated the prion-like domain of FUS-CHOP to impede these protein-protein interactions, which are required for transformation. Therefore, the DNA damage response after irradiation disrupted oncogenic targeting of chromatin remodelers required for FUS-CHOP-driven sarcomagenesis. This mechanism of disruption links phosphorylation of the prion-like domain of an oncogenic fusion protein to DNA damage after ionizing radiation and reveals that a dependence on oncogenic chromatin remodeling underlies sensitivity to radiation therapy in myxoid liposarcoma. SIGNIFICANCE: Prion-like domains, which are frequently translocated in cancers as oncogenic fusion proteins that drive global epigenetic changes, confer sensitivity to radiation via disruption of oncogenic interactions.

SPOP and OTUD7A Control EWS-FLI1 Protein Stability to Govern Ewing Sarcoma Growth.

Chromosomal translocation results in development of an Ewing sarcoma breakpoint region 1-Friend leukemia integration 1 (EWS-FLI1) fusion oncogene in the majority of Ewing sarcoma. The persistent dependence of the tumor for this oncoprotein points to EWS-FLI1 as an ideal drug target. Although EWS-FLI1 transcriptional targets and binding partners are evaluated, the mechanisms regulating EWS-FLI1 protein stability remain elusive. Speckle-type POZ protein (SPOP) and OTU domain-containing protein 7A (OTUD7A) are identified as the bona fide E3 ligase and deubiquitinase, respectively, that control EWS-FLI1 protein turnover in Ewing sarcoma. Casein kinase 1-mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP activity to degrade EWS-FLI1. Opposing this process, OTUD7A deubiquitinates and stabilizes EWS-FLI1. Depletion of OTUD7A in Ewing sarcoma cell lines reduces EWS-FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in mice. Performing an artificial-intelligence-based virtual drug screen of a 4-million small molecule library, 7Ai is identified as a potential OTUD7A catalytic inhibitor. 7Ai reduces EWS-FLI1 protein levels and decreases Ewing sarcoma growth in vitro and in a xenograft mouse model. This study supports the therapeutic targeting of OTUD7A as a novel strategy for Ewing sarcoma bearing EWS-FLI1 and related fusions, and may also be applicable to other cancers dependent on aberrant FLI1 expression.

HDAC inhibition results in widespread alteration of the histone acetylation landscape and BRD4 targeting to gene bodies.

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and -independent dynamics of H3K36 methylation.

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively.

Accelerated aging among childhood, adolescent, and young adult cancer survivors is evidenced by increased expression of p16INK4a and frailty.

BACKGROUND: Cellular senescence, measured by expression of the cell cycle kinase inhibitor p16INK4a , may contribute to accelerated aging in survivors of childhood, adolescent, and young adult cancer. The authors measured peripheral blood T-lymphocyte p16INK4a expression among pediatric and young adult cancer survivors, hypothesizing that p16INK4a expression is higher after chemotherapy and among frail survivors. METHODS: A cross-sectional cohort of young adult survivors and age-matched, cancer-free controls were assessed for p16INK4a expression and frailty. Newly diagnosed pediatric patients underwent prospective measurements of p16INK4a expression before and after cancer therapy. Frailty was measured with a modified Fried frailty phenotype evaluating sarcopenia, weakness, slowness, energy expenditure, and exhaustion. RESULTS: The cross-sectional cohort enrolled 60 survivors and 29 age-matched controls with a median age of 21 years (range, 17-29 years). The prospective cohort enrolled 9 newly diagnosed patients (age range, 1-18 years). Expression of p16INK4a was higher among survivors compared with controls (9.6 vs 8.9 log2 p16 units; 2-sided P = .005, representing a 25-year age acceleration in survivors) and increased among newly diagnosed patients from matched pretreatment to posttreatment samples (7.3-8.9 log2 p16 units; 2-sided P = .002). Nine survivors (16%) were frail and had higher p16INK4a expression compared with robust survivors (10.5 [frail] vs 9.5 [robust] log2 p16 units; 2-sided P = .055), representing a 35-year age acceleration among frail survivors. CONCLUSIONS: Chemotherapy is associated with increased cellular senescence and molecular age in pediatric and young adult cancer survivors. Frail survivors, compared with robust survivors, exhibit higher levels of p16INK4a , suggesting that cellular senescence may be associated with early aging in survivors.

Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple-Negative Breast Cancer.

Triple-negative breast cancers contain a spectrum of epithelial and mesenchymal phenotypes. SUM-229PE cells represent a model for this heterogeneity, maintaining both epithelial and mesenchymal subpopulations that are genomically similar but distinct in gene expression profiles. We identified differential regions of open chromatin in epithelial and mesenchymal cells that were strongly correlated with regions of H3K27ac. Motif analysis of these regions identified consensus sequences for transcription factors that regulate cell identity. Treatment with the MEK inhibitor trametinib induced enhancer remodeling that is associated with transcriptional regulation of genes in epithelial and mesenchymal cells. Motif analysis of enhancer peaks downregulated in response to chronic treatment with trametinib identified AP-1 motif enrichment in both epithelial and mesenchymal subpopulations. Chromatin immunoprecipitation sequencing (ChIP-seq) of JUNB identified subpopulation-specific localization, which was significantly enriched at regions of open chromatin. These results indicate that cell identity controls localization of transcription factors and chromatin-modifying enzymes to enhancers for differential control of gene expression. We identified increased H3K27ac at an enhancer region proximal to CXCR7, a G-protein-coupled receptor that increased 15-fold in expression in the epithelial subpopulation during chronic treatment. RNAi knockdown of CXCR7 inhibited proliferation in trametinib-resistant cells. Thus, adaptive resistance to chronic trametinib treatment contributes to proliferation in the presence of the drug. Acquired amplification of KRAS following trametinib dose escalation further contributed to POS cell proliferation. Adaptive followed by acquired gene expression changes contributed to proliferation in trametinib-resistant cells, suggesting inhibition of early transcriptional reprogramming could prevent resistance and the bypass of targeted therapy. IMPLICATIONS: We defined the differential responses to trametinib in subpopulations of a clinically relevant in vitro model of TNBC, and identified both adaptive and acquired elements that contribute to the emergence of drug resistance mediated by increased expression of CXCR7 and amplification of KRAS.

Genomes from bacteria associated with the canine oral cavity: A test case for automated genome-based taxonomic assignment.

Taxonomy for bacterial isolates is commonly assigned via sequence analysis. However, the most common sequence-based approaches (e.g. 16S rRNA gene-based phylogeny or whole genome comparisons) are still labor intensive and subjective to varying degrees. Here we present a set of 33 bacterial genomes, isolated from the canine oral cavity. Taxonomy of these isolates was first assigned by PCR amplification of the 16S rRNA gene, Sanger sequencing, and taxonomy assignment using BLAST. After genome sequencing, taxonomy was revisited through a manual process using a combination of average nucleotide identity (ANI), concatenated marker gene phylogenies, and 16S rRNA gene phylogenies. This taxonomy was then compared to the automated taxonomic assignment given by the recently proposed Genome Taxonomy Database (GTDB). We found the results of all three methods to be similar (25 out of the 33 had matching genera), but the GTDB approach required fewer subjective decisions, and required far less labor. The primary differences in the non-identical taxonomic assignments involved cases where GTDB has proposed taxonomic revisions.

Casein Kinase II Phosphorylation of Spt6 Enforces Transcriptional Fidelity by Maintaining Spn1-Spt6 Interaction.

Spt6 is a histone chaperone that associates with RNA polymerase II and deposits nucleosomes in the wake of transcription. Although Spt6 has an essential function in nucleosome deposition, it is not known whether this function is influenced by post-translational modification. Here, we report that casein kinase II (CKII) phosphorylation of Spt6 is required for nucleosome occupancy at the 5' ends of genes to prevent aberrant antisense transcription and enforce transcriptional directionality. Mechanistically, we show that CKII phosphorylation of Spt6 promotes the interaction of Spt6 with Spn1, a binding partner required for chromatin reassembly and full recruitment of Spt6 to genes. Our study defines a function for CKII phosphorylation in transcription and highlights the importance of post-translational modification in histone chaperone function.

PBRM1 bromodomains variably influence nucleosome interactions and cellular function.

Chromatin remodelers use bromodomains (BDs) to recognize histones. Polybromo 1 (PBRM1 or BAF180) is hypothesized to function as the nucleosome-recognition subunit of the PBAF chromatin-remodeling complex and is frequently mutated in clear cell renal cell carcinoma (ccRCC). Previous studies have applied in vitro methods to explore the binding specificities of the six individual PBRM1 BDs. However, BD targeting to histones and the influence of neighboring BD on nucleosome recognition have not been well characterized. Here, using histone microarrays and intact nucleosomes to investigate the histone-binding characteristics of the six PBRM1 BDs individually and combined, we demonstrate that BD2 and BD4 of PBRM1 mediate binding to acetylated histone peptides and to modified recombinant and cellular nucleosomes. Moreover, we show that neighboring BDs variably modulate these chromatin interactions, with BD1 and BD5 enhancing nucleosome interactions of BD2 and BD4, respectively, whereas BD3 attenuated these interactions. We also found that binding pocket missense mutations in BD4 observed in ccRCC disrupt PBRM1-chromatin interactions and that these mutations in BD4, but not similar mutations in BD2, in the context of full-length PBRM1, accelerate ccRCC cell proliferation. Taken together, our biochemical and mutational analyses have identified BD4 as being critically important for maintaining proper PBRM1 function and demonstrate that BD4 mutations increase ccRCC cell growth. Because of the link between PBRM1 status and sensitivity to immune checkpoint inhibitor treatment, these data also suggest the relevance of BD4 as a potential clinical target.

Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription.

Spt6 is an essential histone chaperone that mediates nucleosome reassembly during gene transcription. Spt6 also associates with RNA polymerase II (RNAPII) via a tandem Src2 homology domain. However, the significance of Spt6-RNAPII interaction is not well understood. Here, we show that Spt6 recruitment to genes and the nucleosome reassembly functions of Spt6 can still occur in the absence of its association with RNAPII. Surprisingly, we found that Spt6-RNAPII association is required for efficient recruitment of the Ccr4-Not de-adenylation complex to transcribed genes for essential degradation of a range of mRNAs, including mRNAs required for cell-cycle progression. These findings reveal an unexpected control mechanism for mRNA turnover during transcription facilitated by a histone chaperone.

CHD4 and the NuRD complex directly control cardiac sarcomere formation.

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.