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2'-O-methylation is one of the most prevalent RNA modifications found in different RNA types. However, the identities of enzymes participating in the transfer of methyl groups are not well defined. To date, fibrillarin (FBL) is the only known small nucleolar ribonucleoprotein (snoRNP) 2'-O-methyltransferase. Whether other snoRNP 2'-O-methyltransferases exist and their functions in targeting RNAs to determine cell differentiation and function need to be elucidated. Here, we identify FBL-like protein 1 (FBLL1) as a 2'-O-methyltransferase and find its function in promoting neuronal differentiation. We show that FBLL1 is a key snoRNP complex enzyme that transfers methyl groups to substrate RNAs both in vitro and in vivo. Moreover, FBLL1 exhibits different 2'-O-methyltransferase site selectivity from FBL and tissue-specific distribution. FBLL1 is preferentially expressed in the brain, especially in human neuron cells, and promotes neuronal differentiation through 2'-O-methylation of GAP43 messenger RNA (mRNA). Knockdown of FBLL1, but not FBL, reduced 2'-O-methylation levels in GAP43 mRNA, decreased expression of GAP43 proteins, and eventually repressed neuronal differentiation. Our finding of neuron-specific FBLL1 adds insights into RNA modification in neurobiology and provides clues for understanding 2'-O-methylation in health and disease., Competing Interests: Competing interests statement:The authors declare no competing interest.

Oocyte loss, a natural process that accelerates as women approach their mid-30s, poses a significant challenge to female reproduction. Recent studies have identified DNA damage as a primary contributor to oocyte loss, but the mechanisms underlying DNA damage accumulation remain unclear. Here, we show that aged oocytes have a lower DNA repair capacity and reduced mobility of DNA damage sites compared to young oocytes. Incomplete DNA repair in aged oocytes results in defective chromosome integrity and partitioning, thereby compromising oocyte quality. We found that DNA repair proteins are arranged in spatially distinct DNA repair compartments that form during the late stages of oocyte growth, accompanied by changes in the activity of DNA repair pathways. We demonstrate alterations in these compartments with age, including substantial changes in the levels of key DNA repair proteins and a shift toward error-prone DNA repair pathways. In addition, we show that reduced cohesin levels make aged oocytes more vulnerable to persistent DNA damage and cause changes in DNA repair compartments. Our study links DNA damage accumulation in aged oocytes, a leading cause of oocyte loss, to cohesin deterioration and changes in the organization, abundance, and response of DNA repair machinery., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

SWI/SNF chromatin remodeling complex BAF subunit (SMARCB1)-deficient lung carcinoma is rare and may be suitable for immunotherapy. This article presents the case of an elderly male who presented with "a persistent dry cough for 3 months without obvious inducement", and was initially diagnosed with non-small cell lung carcinoma following bronchoscopic biopsy. Immunohistochemical staining for SMARCB1 (-), SMARCA4 (+), PD-L1(22C3) (tumor proportion score is 60%), Ki-67 (+) with a positive index of 60%. The final diagnosis is SMARCB1-deficient lung carcinoma. He was subsequently treated once with a combination of chemotherapy and immunotherapy, and has been followed up for 34 months. Currently, the patient continues to survive with tumor.

ATP-dependent chromatin remodeling complexes play crucial roles in various biological processes including enhancing local DNA accessibility, regulating gene transcription, and facilitating DNA replication and repair. Based on their functional structural domains, these complexes may be categorized into four families, including SWI/SNF, ISWI, CHD and INO80. Such families are vital factors for regulating gene expression and play pivotal roles in developmental processes. Variants of genes encoding the components of such complexes have been closely associated with human developmental disorders and neurodevelopmental genetic syndromes. This review has summarized the classification, fundamental functions and underlying mechanism of the ATP-dependent chromatin remodeling complexes, as well as common neurological genetic disorders due to variants of genes encoding the subunits of such complexes.

The chromatin-remodeling enzyme SMARCA5 plays a key role in DNA-templated events including transcription, DNA replication, and DNA repair. Loss of function of the SMARCA5 can cause neurodevelopmental disorder and Williams syndrome. However, the molecular mechanism underlying the regulation of SMARCA5 in prostate cancer remains largely elusive. Here, we report that the deubiquitinating enzyme USP3 directly interacts with SMARCA5 and removes K63-linked polyubiquitination of SMARCA5 to maintain its stability, which promotes DNA damage repair and chemotherapy resistance. Depletion of USP3 or SMARCA5 promoted PCa cells sensitive to docetaxel and overexpression of USP3 restored the cells resistance to docetaxel treatment in SMARCA5 silenced cells in vitro and vivo. Clinically, USP3 was significantly up-regulated in prostate cancer tissues and positively associated with SMARCA5 expression. Collectively, our findings uncover a novel molecular mechanism for the USP3-SMARCA5 axis in regulating DSB repair with an important role in chemotherapy response in human prostate cancers, highlighting that targeting USP3-SMARCA5 axis could be a valuable strategy to treat USP3/SMARCA5-overexpressing chemotherapy-resistant patients and improve drug treatment., (© 2024. The Author(s).)

During cell division, chromosomes build kinetochores that attach to spindle microtubules. Kinetochores usually form at the centromeres, which contain CENP-A nucleosomes. The outer kinetochore, which is the core attachment site for microtubules, is composed of the KMN network (Knl1c, Mis12c, and Ndc80c complexes) and is recruited downstream of CENP-A and its partner CENP-C. In C. elegans oocytes, kinetochores have been suggested to form independently of CENP-A nucleosomes. Yet kinetochore formation requires CENP-C, which acts in parallel to the nucleoporin MEL-28 ELYS . Here, we used a combination of RNAi and Degron-based depletion of CENP-A (or downstream CENP-C) to demonstrate that both proteins are in fact responsible for a portion of outer kinetochore assembly during meiosis I and are essential for accurate chromosome segregation. The remaining part requires the coordinated action of KNL-2 (ortholog of human M18BP1) and of the nucleoporin MEL-28 ELYS . Accordingly, co-depletion of CENP-A (or CENP-C) and KNL-2 M18BP1 (or MEL-28 ELYS ) prevented outer kinetochore assembly in oocytes during meiosis I. We further found that KNL-2 M18BP1 and MEL-28 ELYS are interdependent for kinetochore localization. Using engineered mutants, we demonstrated that KNL-2 M18BP1 recruits MEL-28 ELYS at meiotic kinetochores through a specific N-terminal domain, independently of its canonical CENP-A loading factor activity. Finally, we found that meiosis II outer kinetochore assembly was solely dependent on the canonical CENP-A/CENP-C pathway. Thus, like in most cells, outer kinetochore assembly in C. elegans oocytes depends on centromeric chromatin. However, during meiosis I, an additional KNL-2 M18BP1 and MEL-28 ELYS pathway acts in a non-redundant manner and in parallel to canonical centromeric chromatin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024.)

The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies., (© 2024 Stoler-Barak et al.)

Pathogenic variants in the cohesin genes, NIPBL and SMC1A, both cause Cornelia de Lange syndrome (CdLS), a rare genetic disorder associated with developmental delay and intellectual disability. This study aimed to compare sleep behaviors across individuals with CdLS caused by a variant in NIPBL or SMC1A, and identify relationships between sleep and behavior functioning. A total of 31 caregivers of individuals with a variant in NIPBL (N = 22) or SMC1A (N = 9) completed questionnaires regarding their child's sleep behaviors, behavior regulation, attention, and autistic features (repetitive behaviors and social communication difficulties) as part of the Coordination of Rare Diseases (CoRDS) registry. Findings showed a trend of increased behavior regulation difficulties and repetitive behaviors in the NIPBL compared to the SMC1A participants. Both groups presented with a similar degree of attention, social communication, and sleep challenges. In the whole sample, sleep disturbance was strongly correlated with more behavior regulation difficulties, a relationship that was more robust in the NIPBL sample. In brief, study results support our prior observations of greater behavior difficulties among those with a variant in NIPBL as compared to SMC1A. Preliminary findings point to unique associations between sleep and behavior regulation in the NIPBL group, suggesting sleep interventions may yield differential effects on behavior management across variants., (© 2024 Wiley Periodicals LLC.)

Aurora-A is an essential cell-cycle kinase with critical roles in mitotic entry and spindle dynamics. These functions require binding partners such as CEP192 and TPX2, which modulate both kinase activity and localisation of Aurora-A. Here we investigate the structure and role of the centrosomal Aurora-A:CEP192 complex in the wider molecular network. We find that CEP192 wraps around Aurora-A, occupies the binding sites for mitotic spindle-associated partners, and thus competes with them. Comparison of two different Aurora-A conformations reveals how CEP192 modifies kinase activity through the site used for TPX2-mediated activation. Deleting the Aurora-A-binding interface in CEP192 prevents centrosomal accumulation of Aurora-A, curtails its activation-loop phosphorylation, and reduces spindle-bound TPX2:Aurora-A complexes, resulting in error-prone mitosis. Thus, by supplying the pool of phosphorylated Aurora-A necessary for TPX2 binding, CEP192:Aurora-A complexes regulate spindle function. We propose an evolutionarily conserved spatial hierarchy, which protects genome integrity through fine-tuning and correctly localising Aurora-A activity., Competing Interests: Disclosure and competing interests statement The authors declare no competing interests., (© 2024. The Author(s).)

Sister chromatid cohesion is mediated by the cohesin complex. In mitotic prophase cohesin is removed from chromosome arms in a Wapl- and phosphorylation-dependent manner. Sgo1-PP2A protects pericentromeric cohesion by dephosphorylation of cohesin and its associated Wapl antagonist sororin. However, Sgo1-PP2A relocates to inner kinetochores well before sister chromatids are separated by separase, leaving pericentromeric regions unprotected. Why deprotected cohesin is not removed by Wapl remains enigmatic. By reconstituting Wapl-dependent cohesin removal from chromatin in vitro, we discovered a requirement for Nek2a and Cdk1/2-cyclin A2. These kinases phosphorylate cohesin-bound Pds5b, thereby converting it from a sororin- to a Wapl-interactor. Replacement of endogenous Pds5b by a phosphorylation mimetic variant causes premature sister chromatid separation (PCS). Conversely, phosphorylation-resistant Pds5b impairs chromosome arm separation in prometaphase-arrested cells and suppresses PCS in the absence of Sgo1. Early mitotic degradation of Nek2a and cyclin A2 may therefore explain why only separase, but not Wapl, can trigger anaphase., (© 2024. The Author(s).)

The cohesin complex, encoded by SMC3, SMC1A, RAD21, and STAG2, is a critical regulator of DNA-looping and gene expression. Over a decade has passed since recurrent mutations affecting cohesin subunits were first identified in myeloid malignancies such as Acute Myeloid Leukemia (AML). Since that time there has been tremendous progress in our understanding of chromatin structure and cohesin biology, but critical questions remain because of the multiple critical functions the cohesin complex is responsible for. Recent findings have been particularly noteworthy with the identification of crosstalk between DNA-looping and chromatin domains, a deeper understanding of how cohesin establishes sister chromatid cohesion, a renewed interest in cohesin's role for DNA damage response, and work demonstrating cohesin's importance for Polycomb repression. Despite these exciting findings, the role of cohesin in normal hematopoiesis, and the precise mechanisms by which cohesin mutations promote cancer, remain poorly understood. This review discusses what is known about the role of cohesin in normal hematopoiesis, and how recent findings could shed light on the mechanisms through which cohesin mutations promote leukemic transformation. Important unanswered questions in the field, such as whether cohesin plays a role in HSC heterogeneity, and the mechanisms by which it regulates gene expression at a molecular level, will also be discussed. Particular attention will be given to the potential therapeutic vulnerabilities of leukemic cells with cohesin subunit mutations., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Aims: DEK::AFF2 squamous cell carcinoma is a recently described cancer entity, with 29 cases reported to date. Occasionally, these carcinomas appear deceptively indistinguishable; however, specific morphological and phenotypic features suggest the presence of this rearrangement. However, the prognostic value of this diagnosis remains unclear. We aimed to report a new case series with histological, molecular, and clinical features., Methods: We collected data from 15 patients and investigated their phenotypes, including the expression profiles of CK7, P63/P40, PDL1, AFF2, and P16, morphological features, and associated prognostic data. We analyzed these data along with the previously published data., Results: Most of these cases exhibited indicative morphological features, such as exophytic and endophytic papillary growth, nuclear monomorphism, and abundant neutrophil-rich inflammatory infiltrates. Immunohistochemical analysis revealed the expression of AFF2 and squamous cell markers in all the patients. Overexpression of P16 was not detected, whereas CK7 and PDL1 were expressed variably. In our study cohort, a 50% progression or recurrence rate, 25% lymph node metastasis, 17% distant metastasis, and 18% disease-related death were identified, with a short follow-up time., Conclusion: DEK::AFF2 squamous cell carcinoma incidence is probably underestimated. The low-grade appearance of these tumors sometimes limits their detection. The rates of recurrence and metastasis seem to be high despite an often bland morphology. We propose AFF2 immunohistochemistry as an effective tool, and a diagnostic algorithm has been established to support accurate diagnosis of these tumors., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Inactivating alterations in the SWItch/Sucrose NonFermentable (SWI/SNF) Chromatin Remodeling Complex subunits have been described in multiple tumor types. Recent studies focused on SMARC subunits of this complex to understand their relationship with tumor characteristics and therapeutic opportunities. To date, pancreatic cancer with these alterations has not been well studied, although isolated cases of undifferentiated carcinomas have been reported. Herein, we screened 59 pancreatic undifferentiated carcinomas for alterations in SWI/SNF complex-related (SMARCB1 [BAF47/INI1], SMARCA4 [BRG1], SMARCA2 [BRM]) proteins and/or genes using immunohistochemistry and/or next-generation sequencing. Cases with alterations in SWI/SNF complex-related proteins/genes were compared with cases without alterations, as well as with 96 conventional pancreatic ductal adenocarcinomas (PDAC). In all tumor groups, mismatch repair and PD-L1 protein expression were also evaluated. Thirty of 59 (51%) undifferentiated carcinomas had a loss of SWI/SNF complex-related protein expression or gene alteration. Twenty-seven of 30 (90%) SWI-/SNF-deficient undifferentiated carcinomas had rhabdoid morphology (vs 9/29 [31%] SWI-/SNF-retained undifferentiated carcinomas; P < .001) and all expressed cytokeratin, at least focally. Immunohistochemically, SMARCB1 protein expression was absent in 16/30 (53%) cases, SMARCA2 in 4/30 (13%), and SMARCA4 in 4/30 (13%); both SMARCB1 and SMARCA2 protein expressions were absent in 1/30 (3%). Five of 8 (62.5%) SWI-/SNF-deficient undifferentiated carcinomas that displayed loss of SMARCB1 protein expression by immunohistochemistry were found to have corresponding SMARCB1 deletions by next-generation sequencing. Analysis of canonical driver mutations for PDAC in these cases showed KRAS (2/5) and TP53 (2/5) abnormalities. Median combined positive score for PD-L1 (E1L3N) was significantly higher in the undifferentiated carcinomas with/without SWI/SNF deficiency compared with the conventional PDACs (P < .001). SWI-/SNF-deficient undifferentiated carcinomas were larger (P < .001) and occurred in younger patients (P < .001). Patients with SWI-/SNF-deficient undifferentiated carcinoma had worse overall survival compared with patients with SWI-/SNF-retained undifferentiated carcinoma (P = .004) and PDAC (P < .001). Our findings demonstrate that SWI-/SNF-deficient pancreatic undifferentiated carcinomas are frequently characterized by rhabdoid morphology, exhibit highly aggressive behavior, and have a negative prognostic impact. The ones with SMARCB1 deletions appear to be frequently KRAS wild type. Innovative developmental therapeutic strategies targeting this genomic basis of the SWI/SNF complex and the therapeutic implications of EZH2 inhibition (NCT03213665), SMARCA2 degrader (NCT05639751), or immunotherapy are currently under investigation., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Transcriptional silencing by RNAi paradoxically relies on transcription, but how the transition from transcription to silencing is achieved has remained unclear. The Cryptic Loci Regulator complex (CLRC) in Schizosaccharomyces pombe is a cullin-ring E3 ligase required for silencing that is recruited by RNAi. We found that the E2 ubiquitin conjugating enzyme Ubc4 interacts with CLRC and mono-ubiquitinates the histone H3K9 methyltransferase Clr4 SUV39H1 , promoting the transition from co-transcriptional gene silencing (H3K9me2) to transcriptional gene silencing (H3K9me3). Ubiquitination of Clr4 occurs in an intrinsically disordered region (Clr4 IDR ), which undergoes liquid droplet formation in vitro, along with Swi6 HP1 the effector of transcriptional gene silencing. Our data suggests that phase separation is exquisitely sensitive to non-coding RNA (ncRNA) which promotes self-association of Clr4, chromatin association, and di-, but not tri- methylation instead. Ubc4-CLRC also targets the transcriptional co-activator Bdf2 BRD4 , down-regulating centromeric transcription and small RNA (sRNA) production. The deubiquitinase Ubp3 counteracts both activities., (© 2024. The Author(s).)

Dysfunction of the ribosome manifests during cellular senescence and contributes to tissue aging, functional decline, and development of aging-related disorders in ways that have remained enigmatic. Here, we conducted a comprehensive CRISPR-based loss-of-function (LOF) screen of ribosome-associated genes (RAGs) in human mesenchymal progenitor cells (hMPCs). Through this approach, we identified ribosomal protein L22 (RPL22) as the foremost RAG whose deficiency mitigates the effects of cellular senescence. Consequently, absence of RPL22 delays hMPCs from becoming senescent, while an excess of RPL22 accelerates the senescence process. Mechanistically, we found in senescent hMPCs, RPL22 accumulates within the nucleolus. This accumulation triggers a cascade of events, including heterochromatin decompaction with concomitant degradation of key heterochromatin proteins, specifically heterochromatin protein 1γ (HP1γ) and heterochromatin protein KRAB-associated protein 1 (KAP1). Subsequently, RPL22-dependent breakdown of heterochromatin stimulates the transcription of ribosomal RNAs (rRNAs), triggering cellular senescence. In summary, our findings unveil a novel role for nucleolar RPL22 as a destabilizer of heterochromatin and a driver of cellular senescence, shedding new light on the intricate mechanisms underlying the aging process., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Spt5 is a well-conserved factor that manipulates multiple stages of transcription from promoter-proximal pausing (PPP) to termination. Recent studies have revealed an unexpected increase of antisense transcripts near promoters in cells expressing mutant Spt5. Here, we identify Spt5p-restricted intragenic antisense transcripts and their close relationship with sense transcription in yeast. We confirm that Spt5 CTR phosphorylation is also important to retain Spt5's facility to regulate antisense transcription. The genes whose antisense transcription is strongly suppressed by Spt5p share strong endogenous sense transcription and weak antisense transcription, and this pattern is conserved in humans. Mechanistically, we found that Spt5p depletion increased histone acetylation to initiate intragenic antisense transcription by altering chromatin structure. We additionally identified termination factors that appear to be involved in the ability of Spt5p to restrict antisense transcription. By unveiling a new role of Spt5 in finely balancing the bidirectionality of transcription, we demonstrate that Spt5-mediated suppression of DSIF complex regulated-unstable transcripts (DUTs) is essential to sustain the accurate transcription by RNA polymerase II., (© 2024. The Author(s).)

The yeast SWR1 complex catalyzes the exchange of histone H2A/H2B dimers in nucleosomes with Htz1/H2B dimers. We use cryoelectron microscopy to determine the structure of an enzyme-bound hexasome intermediate in the reaction pathway of histone exchange, in which an H2A/H2B dimer has been extracted from a nucleosome prior to the insertion of a dimer comprising Htz1/H2B. The structure reveals a key role for the Swc5 subunit in stabilizing the unwrapping of DNA from the histone core of the hexasome. By engineering a crosslink between an Htz1/H2B dimer and its chaperone protein Chz1, we show that this blocks histone exchange by SWR1 but allows the incoming chaperone-dimer complex to insert into the hexasome. We use this reagent to trap an SWR1/hexasome complex with an incoming Htz1/H2B dimer that shows how the reaction progresses to the next step. Taken together the structures reveal insights into the mechanism of histone exchange by SWR1 complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chromatin changes in response to estrogen and progesterone are well established in cultured cells, but how they control gene expression under physiological conditions is largely unknown. To address this question, we examined in vivo estrous cycle dynamics of mouse uterus hormone receptor occupancy, chromatin accessibility and chromatin structure by combining RNA-seq, ATAC-seq, HiC-seq and ChIP-seq. Two estrous cycle stages were chosen for these analyses, diestrus (highest estrogen) and estrus (highest progesterone). Unexpectedly, rather than alternating with each other, estrogen receptor alpha (ERα) and progesterone receptor (PGR) were co-bound during diestrus and lost during estrus. Motif analysis of open chromatin followed by hypoxia inducible factor 2A (HIF2A) ChIP-seq and conditional uterine deletion of this transcription factor revealed a novel role for HIF2A in regulating diestrus gene expression patterns that were independent of either ERα or PGR binding. Proteins in complex with ERα included PGR and cohesin, only during diestrus. Combined with HiC-seq analyses, we demonstrate that complex chromatin architecture changes including enhancer switching are coordinated with ERα and PGR co-binding during diestrus and non-hormone receptor transcription factors such as HIF2A during estrus to regulate most differential gene expression across the estrous cycle., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

Our understanding of heterochromatin nanostructure and its capacity to mediate gene silencing in a living cell has been prevented by the diffraction limit of optical microscopy. Thus, here to overcome this technical hurdle, and directly measure the nucleosome arrangement that underpins this dense chromatin state, we coupled fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between histones core to the nucleosome, with molecular editing of heterochromatin protein 1 alpha (HP1α). Intriguingly, this super-resolved readout of nanoscale chromatin structure, alongside fluorescence fluctuation spectroscopy (FFS) and FLIM-FRET analysis of HP1α protein-protein interaction, revealed nucleosome arrangement to be differentially regulated by HP1α oligomeric state. Specifically, we found HP1α monomers to impart a previously undescribed global nucleosome spacing throughout genome architecture that is mediated by trimethylation on lysine 9 of histone H3 (H3K9me3) and locally reduced upon HP1α dimerisation. Collectively, these results demonstrate HP1α to impart a dual action on chromatin that increases the dynamic range of nucleosome proximity. We anticipate that this finding will have important implications for our understanding of how live cell heterochromatin structure regulates genome function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Breast cancer is the most frequently diagnosed cancer worldwide, constituting 15% of cases in 2023. The predominant cause of breast cancer-related mortality is metastasis, and a lack of metastasis-targeted therapies perpetuates dismal outcomes for late-stage patients. By using meiotic genetics to study inherited transcriptional network regulation, we have identified, to the best of our knowledge, a new class of "essential expression-restricted" genes as potential candidates for metastasis-targeted therapeutics. Building upon previous work implicating the CCR4-NOT RNA deadenylase complex in metastasis, we demonstrate that RNA-binding proteins NANOS1, PUM2, and CPSF4 also regulate metastatic potential. Using various models and clinical data, we pinpoint Smarcd1 mRNA as a target of all three RNA-BPs. Strikingly, both high and low expression of Smarcd1 correlate with positive clinical outcomes, while intermediate expression significantly reduces the probability of survival. Applying the theory of "essential genes" from evolution, we identify 50 additional genes that require precise expression levels for metastasis to occur. Specifically, small perturbations in Smarcd1 expression significantly reduce metastasis in mouse models and alter splicing programs relevant to the ER+/HER2-enriched breast cancer. Identification subtype-specific essential expression-restricted metastasis modifiers introduces a novel class of genes that, when therapeutically "nudged" in either direction, may significantly improve late-stage breast cancer patients., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

DNA- and RNA-binding proteins (DRBPs) are versatile proteins capable of binding to both DNA and RNA molecules. In this study, we identified fibrillarin (FBL) as a key DRBP that is upregulated in liver cancer tissues vs. normal tissues and is correlated with patient prognosis. FBL promotes the proliferation of liver cancer cells both in vitro and in vivo. Mechanistically, FBL interacts with the transcription factor KHSRP, thereby regulating the expression of genes involved in glucose metabolism and leading to the reprogramming of glucose metabolism. Specifically, FBL and KHSRP work together to transcriptionally activate the glycolytic enzyme PFKFB4 by co-occupying enhancer and promoter elements, thereby further promoting liver cancer growth. Collectively, these findings provide compelling evidence highlighting the role of FBL as a transcriptional regulator in liver cancer cells, working in conjunction with KHSRP. The FBL/KHSRP-PFKFB4 regulatory axis holds potential as both a prognostic indicator and a therapeutic target for liver cancer. SIGNIFICANCE: A novel role of FBL in the transcriptional activation of PFKFB4, leading to glucose metabolism reprogramming in liver cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This study sheds light on the pivotal role of the oncoprotein DEK in B-cell lymphoma. We reveal DEK expression correlates with increased tumor proliferation and inferior overall survival in cases diagnosed with low-grade B-cell lymphoma (LGBCL). We also found significant correlation between DEK expression and copy number alterations in LGBCL tumors, highlighting a novel mechanism of LGBCL pathogenesis that warrants additional exploration. To interrogate the mechanistic role of DEK in B-cell lymphoma, we generated a DEK knockout cell line model, which demonstrated DEK depletion caused reduced proliferation and altered expression of key cell cycle and apoptosis-related proteins, including Bcl-2, Bcl-xL, and p53. Notably, DEK depleted cells showed increased sensitivity to apoptosis-inducing agents, including venetoclax and staurosporine, which underscores the therapeutic potential of targeting DEK in B-cell lymphomas. Overall, our study contributes to a better understanding of DEK's role as an oncoprotein in B-cell lymphomas, highlighting its potential as both a promising therapeutic target and a novel biomarker for aggressive LGBCL. Further research elucidating the molecular mechanisms underlying DEK-mediated tumorigenesis could pave the way for improved treatment strategies and better clinical outcomes for patients with B-cell lymphoma., (© 2024. The Author(s).)

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The switch/sucrose non-fermentable (SWI/SNF) subfamily are evolutionarily conserved, ATP-dependent chromatin-remodelling complexes that alter nucleosome position and regulate a spectrum of nuclear processes, including gene expression, DNA replication, DNA damage repair, genome stability and tumour suppression. These complexes, through their ATP-dependent chromatin remodelling, contribute to the dynamic regulation of genetic information and the maintenance of cellular processes essential for normal cellular function and overall genomic integrity. Mutations in SWI/SNF subunits are detected in 25% of human malignancies, indicating that efficient functioning of this complex is required to prevent tumourigenesis in diverse tissues. During development, SWI/SNF subunits help establish and maintain gene expression patterns essential for proper cellular identity and function, including maintenance of lineage-specific enhancers. Moreover, specific molecular signatures associated with SWI/SNF mutations, including disruption of SWI/SNF activity at enhancers, evasion of G0 cell cycle arrest, induction of cellular plasticity through pro-oncogene activation and Polycomb group (PcG) complex antagonism, are linked to the initiation and progression of carcinogenesis. Here, we review the molecular insights into the aetiology of human malignancies driven by disruption of the SWI/SNF complex and correlate these mechanisms to their developmental functions. Finally, we discuss the therapeutic potential of targeting SWI/SNF subunits in cancer.

X chromosome inactivation triggers a dramatic reprogramming of transcription and chromosome architecture. However, how the chromatin organization of inactive X chromosome is established de novo in vivo remains elusive. Here, we identified an Xist-separated megadomain structure (X-megadomains) on the inactive X chromosome in mouse extraembryonic lineages and extraembryonic endoderm (XEN) cell lines, and transiently in the embryonic lineages, before Dxz4-delineated megadomain formation at later stages in a strain-specific manner. X-megadomain boundary coincides with strong enhancer activities and cohesin binding in an Xist regulatory region required for proper Xist activation in early embryos. Xist regulatory region disruption or cohesin degradation impaired X-megadomains in extraembryonic endoderm cells and caused ectopic activation of regulatory elements and genes near Xist, indicating that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. These data reveal stepwise X chromosome folding and transcriptional regulation to achieve both essential gene activation and global silencing during the early stages of X chromosome inactivation., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Aims/hypothesis: Strategies to augment functional beta cell mass include directed differentiation of stem cells towards a beta cell fate, which requires extensive knowledge of transcriptional programs governing endocrine progenitor cell differentiation in vivo. We aimed to study the contributions of the Brahma-related gene-1 (BRG1) and Brahma (BRM) ATPase subunits of the SWI/SNF chromatin remodelling complex to endocrine cell development., Methods: We generated mice with endocrine progenitor-specific Neurog3-Cre BRG1 removal in the presence of heterozygous (Brg1 Δendo ;Brm +/- ) or homozygous (double knockout: DKO Δendo ) BRM deficiency. Whole-body metabolic phenotyping, islet function characterisation, islet quantitative PCR and histological characterisation were performed on animals and tissues postnatally. To test the mechanistic actions of SWI/SNF in controlling gene expression during endocrine cell development, single-cell RNA-seq was performed on flow-sorted endocrine-committed cells from embryonic day 15.5 control and mutant embryos., Results: Brg1 Δendo ;Brm +/- mice exhibit severe glucose intolerance, hyperglycaemia and hypoinsulinaemia, resulting, in part, from reduced islet number; diminished alpha, beta and delta cell mass; compromised islet insulin secretion; and altered islet gene expression programs, including reductions in MAFA and urocortin 3 (UCN3). DKO Δendo mice were not recovered at weaning; however, postnatal day 6 DKO Δendo mice were severely hyperglycaemic with reduced serum insulin levels and beta cell area. Single-cell RNA-seq of embryonic day 15.5 lineage-labelled cells revealed endocrine progenitor, alpha and beta cell populations from SWI/SNF mutants have reduced expression of Mafa, Gcg, Ins1 and Ins2, suggesting limited differentiation capacity. Reduced Neurog3 transcripts were discovered in DKO Δendo endocrine progenitor clusters, and the proliferative capacity of neurogenin 3 (NEUROG3) + cells was reduced in Brg1 Δendo ;Brm +/- and DKO Δendo mutants., Conclusions/interpretation: Loss of BRG1 from developing endocrine progenitor cells has a severe postnatal impact on glucose homeostasis, and loss of both subunits impedes animal survival, with both groups exhibiting alterations in hormone transcripts embryonically. Taken together, these data highlight the critical role SWI/SNF plays in governing gene expression programs essential for endocrine cell development and expansion., Data Availability: Raw and processed data for scRNA-seq have been deposited into the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE248369., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation., (© 2024. The Author(s).)

Overnutrition has gradually become the primary causative factor in nonalcoholic fatty liver disease (NAFLD). However, how nutritional signals are integrated to orchestrate the transcriptional programs important for NAFLD progression remains poorly understood. We identified hepatic BAF60b as a lipid-sensitive subunit of the switch/sucrose nonfermentable chromatin-remodeling complex that is negatively associated with liver steatosis in mice and humans. Hepatic BAF60b deficiency promotes high-fat diet (HFD)-induced liver steatosis in mice, whereas transgenic expression of BAF60b in the liver attenuates HFD-induced obesity and NAFLD, both accompanied by a marked regulation of peroxisome proliferator-activated receptor γ (PPARγ) expression. Mechanistically, through motif analysis of liver assay for transposase-accessible chromatin sequencing and multiple validation experiments, we identified C/EBPβ as the transcription factor that interacts with BAF60b to suppress Pparγ gene expression, thereby controlling hepatic lipid accumulation and NAFLD progression. This work identifies hepatic BAF60b as a negative regulator of liver steatosis through C/EBPβ-dependent chromatin remodeling., (© 2024 by the American Diabetes Association.)

Heterochromatin, a key component of the eukaryotic nucleus, is fundamental to the regulation of genome stability, gene expression and cellular functions. However, the factors and mechanisms involved in heterochromatin formation and maintenance still remain largely unknown. Here, we show that insulin receptor tyrosine kinase substrate (IRTKS), an I-BAR domain protein, is indispensable for constitutive heterochromatin formation via liquid‒liquid phase separation (LLPS). In particular, IRTKS droplets can infiltrate heterochromatin condensates composed of HP1α and diverse DNA-bound nucleosomes. IRTKS can stabilize HP1α by recruiting the E2 ligase Ubc9 to SUMOylate HP1α, which enables it to form larger phase-separated droplets than unmodified HP1α. Furthermore, IRTKS deficiency leads to loss of heterochromatin, resulting in genome-wide changes in chromatin accessibility and aberrant transcription of repetitive DNA elements. This leads to activation of cGAS-STING pathway and type-I interferon (IFN-I) signaling, as well as to the induction of cellular senescence and senescence-associated secretory phenotype (SASP) responses. Collectively, our findings establish a mechanism by which IRTKS condensates consolidate constitutive heterochromatin, revealing an unexpected role of IRTKS as an epigenetic mediator of cellular senescence., (© 2024. The Author(s).)

Tumor cell heterogeneity defines therapy responsiveness in neuroblastoma (NB), a cancer derived from neural crest cells. NB consists of two primary subtypes: adrenergic and mesenchymal. Adrenergic traits predominate in NB tumors, while mesenchymal features becomes enriched post-chemotherapy or after relapse. The interconversion between these subtypes contributes to NB lineage plasticity, but the underlying mechanisms driving this phenotypic switching remain unclear. Here, we demonstrate that SWI/SNF chromatin remodeling complex ATPases are essential in establishing an mesenchymal gene-permissive chromatin state in adrenergic-type NB, facilitating lineage plasticity. Targeting SWI/SNF ATPases with SMARCA2/4 dual degraders effectively inhibits NB cell proliferation, invasion, and notably, cellular plasticity, thereby preventing chemotherapy resistance. Mechanistically, depletion of SWI/SNF ATPases compacts cis-regulatory elements, diminishes enhancer activity, and displaces core transcription factors (MYCN, HAND2, PHOX2B, and GATA3) from DNA, thereby suppressing transcriptional programs associated with plasticity. These findings underscore the pivotal role of SWI/SNF ATPases in driving intrinsic plasticity and therapy resistance in neuroblastoma, highlighting an epigenetic target for combinational treatments in this cancer., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Bosma arhinia microphthalmia syndrome (BAMS, OMIM #603457) is a rare autosomal dominant disorder caused by heterozygous variation in the SMCHD1 gene on chromosome 18p11. Clinically, it is characterized by microphthalmia, absence or hypoplasia of nose, choanal atresia, anosmia, palatal abnormalities, hypogonadotropic hypogonadism, and cryptorchidism. Here we report a Brazilian patient with a likely pathogenic variation in SMCHD1 gene (c.1418A>T; p.Glu473Val) presenting hemiarhinia associated with short stature and hypogonadotropic hypogonadism. Due to the clinical variability of BAMS, we considered that hemiarhinia, without microphthalmia, in the present case, can be considered a mild form of BAMS and could be considered for screening of SMCHD1 gene variation., (© 2024 Wiley Periodicals LLC.)

This study determines the functional role of the plant ultraviolet-B radiation (UV-B) photoreceptor, UV RESISTANCE LOCUS 8 (UVR8) under natural conditions using a large-scale 'synchronized-genetic-perturbation-field-experiment'. Laboratory experiments have demonstrated a role for UVR8 in UV-B responses but do not reflect the complexity of outdoor conditions where 'genotype × environment' interactions can mask laboratory-observed responses. Arabidopsis thaliana knockout mutant, uvr8-7, and the corresponding Wassilewskija wild type, were sown outdoors on the same date at 21 locations across Europe, ranging from 39°N to 67°N latitude. Growth and climatic data were monitored until bolting. At the onset of bolting, rosette size, dry weight, and phenolics and glucosinolates were quantified. The uvr8-7 mutant developed a larger rosette and contained less kaempferol glycosides, quercetin glycosides and hydroxycinnamic acid derivatives than the wild type across all locations, demonstrating a role for UVR8 under field conditions. UV effects on rosette size and kaempferol glycoside content were UVR8 dependent, but independent of latitude. In contrast, differences between wild type and uvr8-7 in total quercetin glycosides, and the quercetin-to-kaempferol ratio decreased with increasing latitude, that is, a more variable UV response. Thus, the large-scale synchronized approach applied demonstrates a location-dependent functional role of UVR8 under natural conditions., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Topologically associating domain (TAD) boundaries are the flanking edges of TADs, also known as insulated neighborhoods, within the 3D structure of genomes. A prominent feature of TAD boundaries in mammalian genomes is the enrichment of clustered CTCF sites often with mixed orientations, which can either block or facilitate enhancer-promoter (E-P) interactions within or across distinct TADs, respectively. We will discuss recent progress in the understanding of fundamental organizing principles of the clustered CTCF insulator codes at TAD boundaries. Specifically, both inward- and outward-oriented CTCF sites function as topological chromatin insulators by asymmetrically blocking improper TAD-boundary-crossing cohesin loop extrusion. In addition, boundary stacking and enhancer clustering facilitate long-distance E-P interactions across multiple TADs. Finally, we provide a unified mechanism for RNA-mediated TAD boundary function via R-loop formation for both insulation and facilitation. This mechanism of TAD boundary formation and insulation has interesting implications not only on how the 3D genome folds in the Euclidean nuclear space but also on how the specificity of E-P interactions is developmentally regulated., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Long non-coding RNA (lncRNA) is associated with a large number of tumor cellular functions together with chemotherapy resistance in a variety of tumors. LINC00963 was identified to regulate the malignant progression of various cancers. However, whether LINC00963 affects drug resistence in esophageal squamous cell carcinoma (ESCC) and the relevant molecular mechanisms have never been reported. This study aims to investigate the effect of LINC00963 on cisplatin resistance in ESCC. After detecting the level of LINC00963 in human esophageal squamous epithelial cells (HET-1 A), ESCC cells (TE-1) and cisplatin resistant cells of ESCC (TE-1/DDP), TE-1/DDP cell line and nude mouse model that interfered with LINC00963 expression were established. Then, the interaction among LINC00963, miR-10a, and SKA1 was clarified by double luciferase and RNA immunoprecipitation (RIP) assays. Meanwhile, the biological behavior changes of TE-1/DDP cells with miR-10a overexpression or SKA1 silencing were observed by CCK-8, flow cytometry, scratch, Transwell, and colony formation tests. Finally, the biological function of the LINC00963/SKA1 axis was elucidated by rescue experiments. LINC00963 was upregulated in TE-1 and TE-1/DDP cell lines. LINC00963 knockdown inhibited SKA1 expression of both cells and impaired tumorigenicity. Moreover, LINC00963 has a target relationship with miR-10a, and SKA1 is a target gene of miR-10a. MiR-10a overexpression or SKA1 silencing decreased the biological activity of TE-1/DDP cells and the expression of SKA1. Furthermore, SKA1 overexpression reverses the promoting effect of LINC00963 on cisplatin resistance of ESCC. LINC00963 regulates TE-1/DDP cells bioactivity and mediates cisplatin resistance through interacting with miR-10a and upregulating SKA1 expression., Competing Interests: Declarations Ethical Approval This study was approved by Shandong Provincial Hospital Affiliated to Shandong University Laboratory Animal Ethics Committee (No. 2019-020). Consent to Participate All authors have their consent to participate. Consent for Publication All authors have their consent to publish their work. Competing Interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Polycomb group (PcG) proteins are essential gene repressors in higher eukaryotes. However, how PcG proteins mediate transcriptional regulation of specific genes remains unknown. LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), as a component of Polycomb Repression Complexes (PRC), epigenetically mediates several plant developmental processes together with PcG proteins. We observed physical interaction between MYB73 and LHP1 in vitro and in vivo. Genetic analysis indicated that myb73 mutants showed slightly late flowering, and the lhp1-3 myb73-2 double mutant exhibited delayed flowering and downregulated FT expression compared to lhp1-3. Chromatin immunoprecipitation and yeast one-hybrid assays revealed that MYB73 preferentially binds to the FT promoter. Additionally, our protoplast transient assays demonstrated that MYB73 activates to the FT promoter. Interestingly, the LHP1-MYB73 interaction is necessary to repress the FT promoter, suggesting that the LHP1-MYB73 interaction prevents FT activation by MYB73 in Arabidopsis. Our results show an example in which a chromatin regulator affects transcriptional regulation by negatively regulating a transcription factor through direct interaction., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin-mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter-TAD loop formation. Based on the reanalysis of Nano-C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read-through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter-TAD loops is restricted though. We speculate that the DNA-encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer-promoter loop formation and other genomic processes., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Activated mTORC2/AKT signaling plays a role in hepatocellular carcinoma (HCC). Research has shown that TSC/mTORC1 and FOXO1 are distinct downstream effectors of AKT signaling in liver regeneration and metabolism. However, the mechanisms by which these pathways mediate mTORC2/AKT activation in HCC are not yet fully understood. Amplification and activation of c-MYC are key molecular events in HCC. In this study, we explored the roles of tuberous sclerosis complex/mTORC1 (TSC/mTORC1) and FOXO1 as downstream effectors of mTORC2/AKT1 in c-MYC-induced hepatocarcinogenesis. Using various genetic approaches in mice, we found that manipulating the FOXO pathway had a minimal effect on c-MYC-induced HCC. In contrast, loss of mTORC2 inhibited c-MYC-induced HCC, an effect that was completely reversed by ablation of TSC2, which activated mTORC1. Additionally, we discovered that p70/RPS6 and 4EBP1/eIF4E acted downstream of mTORC1, regulating distinct molecular pathways. Notably, the 4EBP1/eIF4E cascade is crucial for cell proliferation and glycolysis in c-MYC-induced HCC. We also identified centromere protein M (CENPM) as a downstream target of the TSC2/mTORC1 pathway in c-MYC-driven hepatocarcinogenesis, and its ablation entirely inhibited c-MYC-dependent HCC formation. Our findings demonstrate that the TSC/mTORC1/CENPM pathway, rather than the FOXO cascade, is the primary signaling pathway regulating c-MYC-driven hepatocarcinogenesis. Targeting CENPM holds therapeutic potential for treating c-MYC-driven HCC.

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1 , coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1 - sz , stn1 - sz and cdc13 - sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1 - sz , stn1 - sz and cdc13 - sz mutants.

Purpose: DEK::AFF2 fusion-associated squamous cell carcinoma (DEK::AFF2 SCC), also reported in the literature as low-grade papillary sinonasal (Schneiderian) carcinoma (LGPSC), is a rare, primarily bland-appearing, but locally aggressive neoplasm. Morphologically, these tumors can closely resemble sinonasal papilloma (SP), especially on small or limited biopsy, often leading to misdiagnosis. DEK::AFF2 SCC is devoid of the underlying mutually exclusive EGFR or KRAS driver mutations of SP, suggesting it may represent a distinct unique entity., Methods: In this study, we conducted a retrospective search of "unusual" SP reported either as atypical, dysplastic, or suspicious for malignant transformation at our institution in the last 13 years (2010-2023), to identify potential cases of DEK::AFF2 SCC., Results: Of the 201 SP cases during this time period, 30 "unusual" SP cases were identified. On morphologic review of these 30 cases, 6 were worrisome for DEK::AFF2 SCC and were selected for AFF2 immunohistochemical stain (IHC), of which 3 cases were positive. All 3 AFF2 IHC positive cases were also positive for DEK::AFF2 fusion by fluorescence in situ hybridization (FISH), thereby, confirming IHC results., Conclusions: This study highlights that AFF2 IHC can be an invaluable surrogate marker to FISH in identifying DEK::AFF2 SCC in challenging cases to avoid misdiagnosis. Detailed clinical and pathologic data were collected to gain a better understanding of this emerging challenging entity. A literature review was performed to enrich our knowledge of DEK::AFF2 SCC., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Partitioning of repressive from actively transcribed chromatin in mammalian cells fosters cell-type-specific gene expression patterns. While this partitioning is reconstructed during differentiation, the chromatin occupancy of the key insulator, CCCTC-binding factor (CTCF), is unchanged at the developmentally important Hox clusters. Thus, dynamic changes in chromatin boundaries must entail other activities. Given its requirement for chromatin loop formation, we examined cohesin-based chromatin occupancy without known insulators, CTCF and Myc-associated zinc-finger protein (MAZ), and identified a family of zinc-finger proteins (ZNFs), some of which exhibit tissue-specific expression. Two such ZNFs foster chromatin boundaries at the Hox clusters that are distinct from each other and from MAZ. PATZ1 was critical to the thoracolumbar boundary in differentiating motor neurons and mouse skeleton, while ZNF263 contributed to cervicothoracic boundaries. We propose that these insulating activities act with cohesin, alone or combinatorially, with or without CTCF, to implement precise positional identity and cell fate during development., Competing Interests: Declaration of interests D.R. was a co-founder of Constellation Pharmaceuticals and Fulcrum Therapeutics. Currently, D.R. has no affiliation with either company., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here, we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression program at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C, promoter-capture Hi-C, CUT&Tag for H3K27ac and RNA-seq. We find that genes involved in axonal regeneration form long-range, complex chromatin loops, and that cohesin is required for the full induction of the regenerative transcriptional program. Importantly, loss of cohesin results in disruption of chromatin architecture and severely impaired nerve regeneration. Complex enhancer-promoter loops are also enriched in the human fetal cortical plate, where the axonal growth potential is highest, and are lost in mature adult neurons. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent long-range promoter interactions in nerve regeneration., Competing Interests: Competing interests statement:The authors declare no competing interest.

Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, although the specific mechanisms are not fully understood. Here, we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos. We found that depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. Knockdown of CSR-1 does not affect mRNA and protein levels of the centromeric histone H3 variant and CENP-A homolog HCP-3 but does increase the localization of HCP-3 and some kinetochore proteins to the mitotic chromosomes. Such elevation of HCP-3 chromatin localization depends on EGO-1, which is an upstream factor in the CSR-1 RNA interference (RNAi) pathway, and PIWI domain activity of CSR-1. Our results suggest that CSR-1 restricts the level of HCP-3 at the holocentromeres, prevents erroneous kinetochore assembly and thereby promotes accurate chromosome segregation. Our work sheds light on the role of CSR-1 in regulating deposition of HCP-3 on chromatin and centromere function in embryos., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

SWI/SNF chromatin remodeling complexes play a key role in gene transcription as epigenetic regulators and are typically considered to act as tumor suppressors in cancers. Compared to other cancer-related components of the SWI/SNF complex, research on SMARCC2, a component of the initial BAF core, has been relatively limited. This study aimed to elucidate the role of SMARCC2 in breast cancer by employing various in vitro and in vivo methods including cell proliferation assays, mammosphere formation, and xenograft models, complemented by RNA-seq, ATAC-seq, and ChIP analyses. The results showed that SMARCC2 silencing surprisingly led to the suppression of breast tumorigenesis, indicating a pro-tumorigenic function for SMARCC2 in breast cancer, which contrasts with the roles of other SWI/SNF subunits. In addition, SMARCC2 depletion reduces cancer stem cell features of breast cancer cells. Mechanistic study showed that SMARCC2 silencing downregulated the oncogenic Ras-PI3K signaling pathway, likely by directly regulating the chromatin accessibility of the enhancers of the key genes such as PIK3CB. Together, these results expand our understanding of the SWI/SNF complex's role in cancer development and identify SMARCC2 as a promising new target for breast cancer therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

SMCHD1 is an epigenetic regulatory protein known to modulate the targeted repression of large chromatin domains. Diminished SMCHD1 function in muscle fibers causes Facioscapulohumeral Muscular Dystrophy (FSHD2) through derepression of the D4Z4 chromatin domain, an event which permits the aberrant expression of the disease-causing gene DUX4. Given that SMCHD1 plays a broader role in establishing the cellular epigenome, we examined whether loss of SMCHD1 function might affect muscle homeostasis through additional mechanisms. Here we show that acute depletion of SMCHD1 results in a DUX4-independent defect in myoblast proliferation. Genomic and transcriptomic experiments determined that SMCHD1 associates with enhancers of genes controlling cell cycle to activate their expression. Amongst these cell cycle regulatory genes, we identified LAP2 as a key target of SMCHD1 required for the expansion of myoblasts, where the ectopic expression of LAP2 rescues the proliferation defect of SMCHD1-depleted cells. Thus, the epigenetic regulator SMCHD1 can play the role of a transcriptional co-activator for maintaining the expression of genes required for muscle progenitor expansion. This DUX4-independent role for SMCHD1 in myoblasts suggests that the pathology of FSHD2 may be a consequence of defective muscle regeneration in addition to the muscle wasting caused by spurious DUX4 expression., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Beyond its essential roles in ensuring faithful chromosome segregation and genomic stability, the human Smc5/6 complex acts as an antiviral factor. It binds to and impedes the transcription of extrachromosomal DNA templates; an ability which is lost upon integration of the DNA into the chromosome. How the complex distinguishes among different DNA templates is unknown. Here we show that, in human cells, Smc5/6 preferentially binds to circular rather than linear extrachromosomal DNA. We further demonstrate that the transcriptional process, per se, and particularly the accumulation of DNA secondary structures known to be substrates for topoisomerases, is responsible for Smc5/6 recruitment. More specifically, we find that in vivo Smc5/6 binds to positively supercoiled DNA. Those findings, in conjunction with our genome-wide Smc5/6 binding analysis showing that Smc5/6 localizes at few but highly transcribed chromosome loci, not only unveil a previously unforeseen role of Smc5/6 in DNA topology management during transcription but highlight the significance of sensing DNA topology as an antiviral defense mechanism., (© 2024. The Author(s).)

In this issue of Molecular Cell, Sahu et al. 1 find that shielding heterochromatin from SWI/SNF chromatin remodelers is essential to maintain and epigenetically propagate pre-existing heterochromatin domains, whereas SWI/SNF action protects facultative heterochromatic regions from premature silencing., Competing Interests: Declaration of interests C.L.P. is a member of the advisory board of Molecular Cell., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80., (© 2024. The Author(s).)

Homology search is a central step of DNA double-strand break (DSB) repair by homologous recombination (HR). How it operates in cells remains elusive. We developed a Hi-C-based methodology to map single-stranded DNA (ssDNA) contacts genome-wide in S. cerevisiae, which revealed two main homology search phases. Initial search conducted by short Rad51-ssDNA nucleoprotein filaments (NPFs) is confined in cis by cohesin-mediated chromatin loop folding. Progressive growth of stiff NPFs enables exploration of distant genomic sites. Long-range resection drives this transition from local to genome-wide search by increasing the probability of assembling extensive NPFs. DSB end-tethering promotes coordinated search by opposite NPFs. Finally, an autonomous genetic element on chromosome III engages the NPF, which stimulates homology search in its vicinity. This work reveals the mechanism of the progressive expansion of homology search that is orchestrated by chromatin organizers, long-range resection, end-tethering, and specialized genetic elements and that exploits the stiff NPF structure conferred by Rad51 oligomerization., Competing Interests: Declaration of interests A patent, “A METHOD FOR IDENTIFYING GENOMIC INTERACTIONS,” that covers aspects of the ssHi-C methodology has been submitted on September 11, 2023 by the Centre National de la Recherche Scientifique under PCT application number PCT/FR2023/051384, with A.D. and A.P. as listed inventors., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dysregulated biomolecular condensates, formed through multivalent interactions among proteins and nucleic acids, have been recently identified to drive tumorigenesis. In acute myeloid leukemia (AML), condensates driven by RNA-binding proteins alter transcriptional networks. Yang and colleagues performed a CRISPR screen and identified fibrillarin (FBL) as a new driver in AML leukemogenesis. FBL depletion caused cell cycle arrest and death in AML cells, with minimal impact on normal cells. FBL's phase separation domains are essential for pre-rRNA processing, influencing AML cell survival by regulating ribosome biogenesis and the translation of oncogenic proteins like MYC. Therapeutically, the chemotherapeutic agent CGX-635 targets FBL, inducing its aggregation, impairing pre-rRNA processing, and reducing AML cell survival. This highlights FBL's phase separation as a therapeutic vulnerability in AML. These findings suggest that targeting the phase separation properties of RNA-binding proteins could offer a novel and effective strategy for AML treatment. Further research into condensate dynamics in cancer and development of condensate-modulating drugs holds significant promise for future cancer therapies., (©2024 American Association for Cancer Research.)