Epithelial-mesenchymal transition induced by tumor cell-intrinsic PD-L1 signaling predicts a poor response to immune checkpoint inhibitors in PD-L1-high lung cancer.

BACKGROUND: We investigated the role of tumor cell-intrinsic PD-L1 signaling in the epithelial-mesenchymal transition (EMT) in non-small-cell lung cancer (NSCLC) and the role of EMT as a predictive biomarker for immune checkpoint inhibitor (ICI) therapy. METHODS: PD-L1-overexpressing or PD-L1-knockdown NSCLC cells underwent RNA-seq and EMT phenotype assessment. Mouse lung cancer LLC cells were injected into nude mice. Two cohorts of patients with NSCLC undergoing ICI therapy were analyzed. RESULTS: RNA-seq showed that EMT pathways were enriched in PD-L1-high NSCLC cells. EMT was enhanced by PD-L1 in NSCLC cells, which was mediated by transforming growth factor-ß (TGFß). PD-L1 promoted the activation of p38-MAPK by binding to and inhibiting the protein phosphatase PPM1B, thereby increasing the TGFß production. Tumor growth and metastasis increased in nude mice injected with PD-L1-overexpressing LLC cells. In the ICI cohort, EMT signature was higher in patients with progressive disease than in those with responses, and EMT was significantly associated with poor survival in PD-L1-high NSCLC. In PD-L1-high NSCLC, EMT was associated with increased M2-macrophage and regulatory T-cell infiltrations and decreased cytotoxic T-cell infiltration. CONCLUSIONS: Tumor cell-intrinsic PD-L1 function contributes to NSCLC progression by promoting EMT. EMT may predict an unfavorable outcome after ICI therapy in PD-L1-high NSCLC.

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

The role of EZH1 and EZH2 in development and cancer.

Polycomb Repressive Complex 2 (PRC2) exhibits key roles in mammalian development through its temporospatial repression of gene expression. EZH1 or EZH2 is the catalytic subunit of PRC2 that mediates the mono-, di- and tri-methylation of histone H3 lysine 27 (H3K27me1/2/3), H3K27me2/me3 being a hallmark of facultative heterochromatin. PRC2 is a chromatinmodifying enzyme that is recruited to a limited number of "nucleation sites", spreads H3K27 methylation and fosters chromatin compaction. EZH1 and EZH2 exhibit differences in their expression patterns, levels of histone methyltransferase activity (HMT) in the context of PRC2, and DNA/nucleosome binding activity. This suggests that their roles in heterochromatin formation are disparate. Dysregulation of PRC2 activity leads to aberrant gene expression and is implicated in cancer and developmental diseases. In this review, we discuss the distinct function of PRC2/EZH1 and PRC2/EZH2 in the early and late developmental stages. We then discuss the cancers associated with PRC2/EZH1 and PRC2/EZH2. [BMB Reports 2022; 55(12): 595-601].

Techniques to Study Automethylation of Histone Methyltransferases and its Functional Impact.

The catalytic activity of histone methyltransferases is not restricted to histones but also includes noncanonical substrates. Increasing evidence shows that histone methyltransferases methylate themselves, and automethylation has emerged as a self-regulatory mechanism. Here, we introduce experimental procedures to identify automethylation sites of histone methyltransferases and to investigate the function of automethylation in a reconstituted biochemical system and in cellular contexts.

Structures of monomeric and dimeric PRC2:EZH1 reveal flexible modules involved in chromatin compaction.

Polycomb repressive complex 2 (PRC2) is a histone methyltransferase critical for maintaining gene silencing during eukaryotic development. In mammals, PRC2 activity is regulated in part by the selective incorporation of one of two paralogs of the catalytic subunit, EZH1 or EZH2. Each of these enzymes has specialized biological functions that may be partially explained by differences in the multivalent interactions they mediate with chromatin. Here, we present two cryo-EM structures of PRC2:EZH1, one as a monomer and a second one as a dimer bound to a nucleosome. When bound to nucleosome substrate, the PRC2:EZH1 dimer undergoes a dramatic conformational change. We demonstrate that mutation of a divergent EZH1/2 loop abrogates the nucleosome-binding and methyltransferase activities of PRC2:EZH1. Finally, we show that PRC2:EZH1 dimers are more effective than monomers at promoting chromatin compaction, and the divergent EZH1/2 loop is essential for this function, thereby tying together the methyltransferase, nucleosome-binding, and chromatin-compaction activities of PRC2:EZH1. We speculate that the conformational flexibility and the ability to dimerize enable PRC2 to act on the varied chromatin substrates it encounters in the cell.

Association between body image dissatisfaction and poor quality of life and depression among patients with hematopoietic stem cell transplantation.

PURPOSE: This study aims to evaluate the association between body image dissatisfaction and quality of life and depression among patients after hematopoietic stem cell transplantation (HSCT). METHODS: We conducted a cross-sectional survey at three university-based HSCT outpatient clinics and the Korea Blood Cancer Association. We assessed the body image using the body image scale; quality of life and depression were measured using the World Health Organization Quality of Life-BREF and the Patient Health Questionnaire 9, respectively. Univariate and multivariate linear regression models were used to find an association between body image, quality of life, and depression. RESULTS: Among 163 study participants, 71.8% were male, and the mean age of the participants was 48.3 (SD = 11.2). Over 70% of the participants reported that they felt less physically and sexually attractive due to HSCT, and 39.3% of the patients were dissatisfied with their body image. In fully adjusted models, patients with dissatisfied body image had significantly poorer quality of life (- 13.68, 95% confidence interval [CI] = - 18.16, - 9.21). Moreover, patients with body image dissatisfaction were 8.59 times (95% CI = 3.79, 19.48) more likely to have depressive symptoms than patients without it. CONCLUSION: The majority of HSCT patients experienced body image dissatisfaction, which was significantly associated with poor quality of life and depression. It would be essential to evaluate body image after HSCT and provide appropriate interventions for preventing further psychological consequences.

H1 histones control the epigenetic landscape by local chromatin compaction.

H1 linker histones are the most abundant chromatin-binding proteins1. In vitro studies indicate that their association with chromatin determines nucleosome spacing and enables arrays of nucleosomes to fold into more compact chromatin structures. However, the in vivo roles of H1 are poorly understood2. Here we show that the local density of H1 controls the balance of repressive and active chromatin domains by promoting genomic compaction. We generated a conditional triple-H1-knockout mouse strain and depleted H1 in haematopoietic cells. H1 depletion in T cells leads to de-repression of T cell activation genes, a process that mimics normal T cell activation. Comparison of chromatin structure in normal and H1-depleted CD8+ T cells reveals that H1-mediated chromatin compaction occurs primarily in regions of the genome containing higher than average levels of H1: the chromosome conformation capture (Hi-C) B compartment and regions of the Hi-C A compartment marked by PRC2. Reduction of H1 stoichiometry leads to decreased H3K27 methylation, increased H3K36 methylation, B-to-A-compartment shifting and an increase in interaction frequency between compartments. In vitro, H1 promotes PRC2-mediated H3K27 methylation and inhibits NSD2-mediated H3K36 methylation. Mechanistically, H1 mediates these opposite effects by promoting physical compaction of the chromatin substrate. Our results establish H1 as a critical regulator of gene silencing through localized control of chromatin compaction, 3D genome organization and the epigenetic landscape.

Association between sexuality knowledge and sexual dysfunction in hematopoietic stem cell transplantation patients and their partners.

OBJECTIVE: To evaluate the association of sexual knowledge with sexual desire, sexual activity, and sexual satisfaction in hematopoietic stem cell transplantation (HSCT) patients and partners, and their willingness to participate in sexual education. METHODS: This is a multi-center survey. Patients were eligible if they had received HSCT. Patients' current sexual partners were invited to the study unless they had limitations on sexual activity. Sexual desire, activity and satisfaction was assessed using the Sexual Activity Questionnaire. Sexual knowledge, experience of information seeking, sexual counseling or education, and willingness of participate in sexual education were assessed using questionnaire. RESULTS: Of 151 participants, 61.8 % had experience of receiving counseling about their sexual issues after HSCT. Compared to the lower sexual knowledge group, participants with higher sexual knowledge reported to be 1.91 times more sexually active with 3.04 times higher sexual desire. Among the participants, 79.4 % of participants had the willingness to receive sexual education after HSCT and preferred to receive sexual education from sexual education specialists CONCLUSIONS: Higher sexual knowledge was associated with higher sexual desire, sexual activity, and sexual satisfaction. PRACTICE IMPLICATIONS: Sexual education should be provided to patients and their partners after HCST by trained experts for HSCT patient's sexual life.

LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells.

FACT (facilitates chromatin transcription) is a protein complex that allows RNA polymerase II (RNAPII) to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. We identified two proteins, lens epithelium-derived growth factor (LEDGF) and hepatoma-derived growth factor 2 (HDGF2), each containing two high mobility group A (HMGA)-like AT-hooks and a methyl-lysine reading Pro-Trp-Trp-Pro (PWWP) domain that binds to H3K36me2 and H3K36me3.LEDGF and HDGF2 colocalize with H3K36me2/3 at genomic regions containing active genes. In myoblasts, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease, while HDGF2 levels are maintained. Moreover, HDGF2 is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation.

Automethylation of PRC2 promotes H3K27 methylation and is impaired in H3K27M pediatric glioma.

The histone methyltransferase activity of PRC2 is central to the formation of H3K27me3-decorated facultative heterochromatin and gene silencing. In addition, PRC2 has been shown to automethylate its core subunits, EZH1/EZH2 and SUZ12. Here, we identify the lysine residues at which EZH1/EZH2 are automethylated with EZH2-K510 and EZH2-K514 being the major such sites in vivo. Automethylated EZH2/PRC2 exhibits a higher level of histone methyltransferase activity and is required for attaining proper cellular levels of H3K27me3. While occurring independently of PRC2 recruitment to chromatin, automethylation promotes PRC2 accessibility to the histone H3 tail. Intriguingly, EZH2 automethylation is significantly reduced in diffuse intrinsic pontine glioma (DIPG) cells that carry a lysine-to-methionine substitution in histone H3 (H3K27M), but not in cells that carry either EZH2 or EED mutants that abrogate PRC2 allosteric activation, indicating that H3K27M impairs the intrinsic activity of PRC2. Our study demonstrates a PRC2 self-regulatory mechanism through its EZH1/2-mediated automethylation activity.

PRC2 is high maintenance.

As the process that silences gene expression ensues during development, the stage is set for the activity of Polycomb-repressive complex 2 (PRC2) to maintain these repressed gene profiles. PRC2 catalyzes a specific histone posttranslational modification (hPTM) that fosters chromatin compaction. PRC2 also facilitates the inheritance of this hPTM through its self-contained "write and read" activities, key to preserving cellular identity during cell division. As these changes in gene expression occur without changes in DNA sequence and are inherited, the process is epigenetic in scope. Mutants of mammalian PRC2 or of its histone substrate contribute to the cancer process and other diseases, and research into these aberrant pathways is yielding viable candidates for therapeutic targeting. The effectiveness of PRC2 hinges on its being recruited to the proper chromatin sites; however, resolving the determinants to this process in the mammalian case was not straightforward and thus piqued the interest of many in the field. Here, we chronicle the latest advances toward exposing mammalian PRC2 and its high maintenance.

Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma.

A methionine substitution at lysine-27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits polycomb repressive complex 2 (PRC2) in a dominant-negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found that this interaction on chromatin is transient, with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-containing chromatin, suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to H3K27M, leading to the failure to spread H3K27me from PRC2 recruitment sites and consequently abrogating PRC2's ability to establish H3K27me2-3 repressive chromatin domains. In turn, levels of polycomb antagonists such as H3K36me2 are elevated, suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.

Capturing the Onset of PRC2-Mediated Repressive Domain Formation.

Polycomb repressive complex 2 (PRC2) maintains gene silencing by catalyzing methylation of histone H3 at lysine 27 (H3K27me2/3) within chromatin. By designing a system whereby PRC2-mediated repressive domains were collapsed and then reconstructed in an inducible fashion in vivo, a two-step mechanism of H3K27me2/3 domain formation became evident. First, PRC2 is stably recruited by the actions of JARID2 and MTF2 to a limited number of spatially interacting "nucleation sites," creating H3K27me3-forming Polycomb foci within the nucleus. Second, PRC2 is allosterically activated via its binding to H3K27me3 and rapidly spreads H3K27me2/3 both in cis and in far-cis via long-range contacts. As PRC2 proceeds further from the nucleation sites, its stability on chromatin decreases such that domains of H3K27me3 remain proximal, and those of H3K27me2 distal, to the nucleation sites. This study demonstrates the principles of de novo establishment of PRC2-mediated repressive domains across the genome.

Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin.

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.

Distinct Stimulatory Mechanisms Regulate the Catalytic Activity of Polycomb Repressive Complex 2.

The maintenance of gene expression patterns during metazoan development is achieved, in part, by the actions of polycomb repressive complex 2 (PRC2). PRC2 catalyzes mono-, di-, and trimethylation of histone H3 at lysine 27 (H3K27), with H3K27me2/3 being strongly associated with silenced genes. We demonstrate that EZH1 and EZH2, the two mutually exclusive catalytic subunits of PRC2, are differentially activated by various mechanisms. Whereas both PRC2-EZH1 and PRC2-EZH2 are able to catalyze mono- and dimethylation, only PRC2-EZH2 is strongly activated by allosteric modulators and specific chromatin substrates to catalyze trimethylation of H3K27 in mouse embryonic stem cells (mESCs). However, we also show that a PRC2-associated protein, AEBP2, can stimulate the activity of both complexes through a mechanism independent of and additive to allosteric activation. These results have strong implications regarding the cellular requirements for and the accompanying adjustments in PRC2 activity, given the differential expression of EZH1 and EZH2 upon cellular differentiation.

Satisfaction with sexual activity and sexual dysfunction in hematopoietic stem cell transplantation survivors and their partners: a couple study.

Sexual dysfunction is a common long-term complication of hematopoietic stem cell transplantation (HSCT). We assessed the extent to which HSCT survivors and their partners agree on the importance of and satisfaction with sexual activity and causes of sexual dysfunction, using a cross-sectional survey. Ratings of the importance of sexual activity were significantly higher in survivors than those of partners (2.57 vs. 2.14, P < 0.01). More survivors (48.4%) tried to discuss about sexuality with their partners than partners themselves (23.1%, P < 0.01). Male survivors were more likely to be sexually active than female survivors (odds ratio [OR] 5.04, 95% CI 1.85, 13.74). While 23.3 and 38% of male survivors and partners reported "rejection of partners" as a cause of sexual dysfunction, only 13.3% and none of female partners and survivors pointed this as a cause of sexual dysfunction respectively. There was poor concordance between survivors and partners in attitudes toward sexuality, satisfaction with sexual activity, and causes of sexual dysfunction. Couples who considered adequate sexual activity important were more likely to be sexually active than those who did not (OR 5.53, 95% CI 1.18, 25.89). Our study highlights the need for providing information and counselling about sexuality both to survivors and partners.

GSK-3ß Homolog Rim11 and the Histone Deacetylase Complex Ume6-Sin3-Rpd3 Are Involved in Replication Stress Response Caused by Defects in Dna2.

Lagging strand synthesis is mechanistically far more complicated than leading strand synthesis because it involves multistep processes and requires considerably more enzymes and protein factors. Due to this complexity, multiple fail-safe factors are required to ensure successful replication of the lagging strand DNA. We attempted to identify novel factors that are required in the absence of the helicase activity of Dna2, an essential enzyme in Okazaki-fragment maturation. In this article, we identified Rim11, a GSK-3ß-kinase homolog, as a multicopy suppressor of dna2 helicase-dead mutant (dna2-K1080E). Subsequent epistasis analysis revealed that Ume6 (a DNA binding protein, a downstream substrate of Rim11) also acted as a multicopy suppressor of the dna2 allele. We found that the interaction of Ume6 with the conserved histone deacetylase complex Sin3-Rpd3 and the catalytic activity of Rpd3 were indispensable for the observed suppression of the dna2 mutant. Moreover, multicopy suppression by Rim11/Ume6 requires the presence of sister-chromatid recombination mediated by Rad52/Rad59 proteins, but not vice versa. Interestingly, the overexpression of Rim11 or Ume6 also suppressed the MMS sensitivity of rad59Δ. We also showed that the lethality of dna2 helicase-dead mutant was attributed to checkpoint activation and that decreased levels of deoxynucleotide triphosphates (dNTPs) by overexpressing Sml1 (an inhibitor of ribonucleotide reductase) rescued the dna2 mutant. We also present evidence that indicates Rim11/Ume6 works independently but in parallel with that of checkpoint inhibition, dNTP regulation, and sister-chromatid recombination. In conclusion, our results establish Rim11, Ume6, the histone deacetylase complex Sin3-Rpd3 and Sml1 as new factors important in the events of faulty lagging strand synthesis.

Physical and functional interactions between nucleosomes and Rad27, a critical component of DNA processing during DNA metabolism.

Highly conserved eukaryotic histones are polybasic proteins that package DNA into nucleosomes, a building block of chromatin, allowing extremely long DNA molecules to form compact and discrete chromosomes. The histone N-terminal tails that extend from the nucleosome core act as docking sites for many proteins through diverse post-translational modifications, regulating various DNA transactions. In this report, we present evidence that the nucleosomes can positively regulate the enzymatic activity of Rad27 (yeast Fen1), a major processing enzyme important for Okazaki fragment in eukaryotes. We found that individual histones, histone octamers, and nucleosomes are able to stimulate Rad27 in a manner dependent on the N-terminal tails of histones. Kinetic analyses suggest that an increase in catalytic efficiency of Rad27 was mainly due to increased affinity between DNA substrates and Rad27. It appears that the physical interaction in vivo between histones and Rad27 results in the enrichment of Rad27 in the vicinity of chromatin, increasing the availability of Rad27 for various DNA metabolisms. These results indicate that nucleosomes are not a mere structural component of chromatin, but an active regulator of DNA metabolisms that serves to ensure the efficient and faithful processing of structural intermediates arising during DNA transactions.

Homodimeric PHD Domain-containing Rco1 Subunit Constitutes a Critical Interaction Hub within the Rpd3S Histone Deacetylase Complex.

Recognition of histone post-translational modifications is pivotal for directing chromatin-modifying enzymes to specific genomic regions and regulating their activities. Emerging evidence suggests that other structural features of nucleosomes also contribute to precise targeting of downstream chromatin complexes, such as linker DNA, the histone globular domain, and nucleosome spacing. However, how chromatin complexes coordinate individual interactions to achieve high affinity and specificity remains unclear. The Rpd3S histone deacetylase utilizes the chromodomain-containing Eaf3 subunit and the PHD domain-containing Rco1 subunit to recognize nucleosomes that are methylated at lysine 36 of histone H3 (H3K36me). We showed previously that the binding of Eaf3 to H3K36me can be allosterically activated by Rco1. To investigate how this chromatin recognition module is regulated in the context of the Rpd3S complex, we first determined the subunit interaction network of Rpd3S. Interestingly, we found that Rpd3S contains two copies of the essential subunit Rco1, and both copies of Rco1 are required for full functionality of Rpd3S. Our functional dissection of Rco1 revealed that besides its known chromatin-recognition interfaces, other regions of Rco1 are also critical for Rpd3S to recognize its nucleosomal substrates and functionin vivo. This unexpected result uncovered an important and understudied aspect of chromatin recognition. It suggests that precisely reading modified chromatin may not only need the combined actions of reader domains but also require an internal signaling circuit that coordinates the individual actions in a productive way.

Nucleosome contact triggers conformational changes of Rpd3S driving high-affinity H3K36me nucleosome engagement.

The Rpd3S histone deacetylase complex utilizes two subunits, Eaf3 and Rco1, to recognize nucleosomes methylated at H3K36 (H3K36me) with high affinity and strong specificity. However, the chromobarrel domain of Eaf3 (CHD) that is responsible for H3K36me recognition only binds weakly and with little specificity to histone peptides. Here, using deuterium exchange mass spectrometry (DXMS), we detected conformational changes of Rpd3S upon its contact with chromatin. Interestingly, we found that the Sin3-interacting domain of Rco1 (SID) allosterically stimulates preferential binding of Eaf3 to H3K36-methylated peptides. This activation is tightly regulated by an autoinhibitory mechanism to ensure optimal multivalent engagement of Rpd3S with nucleosomes. Lastly, we identified mutations at the interface between SID and Eaf3 that do not disrupt complex integrity but severely compromise Rpd3S functions in vitro and in vivo, suggesting that the nucleosome-induced conformational changes are essential for chromatin recognition.