DNA-binding Factors Research Articles

Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara.

Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara. These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara. Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara. Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.

Abstract 3891: Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer

Abstract Prostate cancer (PCa) is the most diagnosed cancer among American men, which has traditionally been treated through hormone therapy. However, after hormone therapy many patients with PCa still develop a more aggressive stage of PCa, called metastasis castration resistant prostate cancer (mCRPC). The second- generation antiandrogens, such as enzalutamide or apalutamide, are used to competitively inhibit the androgen receptor (AR) signaling and achieved great clinical success. However, most of the mCRPC patients would develop resistance to the targeted therapy drugs within 6 months to 2 years after initial administration. Consequently, understanding the molecular mechanism of antiandrogen resistance, has become a critical endeavor to provide a greater benefit to patients. mCRPC is characterized by extensive heterogeneity of genomic copy number alterations, which may lead to antiandrogen resistance. TP53 and RB1 alterations have been reported to be dramatically enriched in mCRPC neuroendocrine cancer and we have previously found that inactivation of both TP53 and RB1 confers resistance to antiandrogen through lineage plasticity, where cancer cells can transdifferentiate from a luminal lineage to a mixture of basal and neuroendocrine lineages, which is no longer dependent on AR signaling. Despite these exciting discoveries, only 10% of patients have TP53 and RB1 loss and it is estimated that approximately 40% of patients develop antiandrogen resistance through an undiscovered mechanism.To identify more genomic alterations which confer antiandrogen resistance, we performed an in vivo library screening and identified some of the frequently depleted genes as top candidates mediating antiandrogen response, including the chromatin helicase DNA-binding factor (CHD1). Strikingly, the loss of CHD1 establishes an altered chromatin landscape and enables the activation of heterogenous resistant subclones to emerge, including clones with ectopic NR3C1/GR, POU3F2/BRN2, TBX2, and NR2F1. This work provided an innovative model to explain the dramatically increased tumor heterogeneity in therapy resistant prostate cancer and suggested potential druggable targets to overcome resistance. Building on those exciting results, we are now examining the underlying mechanism how the epigenetic rewiring in resistant tumor clones confer resistance and monitoring the evolution of those heterogenous resistant subclones. Furthermore, we are revealing the collaborative function of those four resistant driver genes in promoting metastasis to various distal organs through multidisplinary approaches including single cell transcriptomics and 3D organoid modeling. The completion of this study will not only add clarity to the mechanism of antiandrogen resistance but may also lead to the development of novel biomarker and therapeutic approaches to overcome resistance. Citation Format: Xiaong li, Su Deng, Julisa Gonzalez, Carla Rodriguez Tirado, Choushi Wang, Nickolas A. Johnson, Lauren Metang, Ping Mu. Epigenetic rewiring promotes antiandrogen resistance and metastasis via heterogenous oncogenic drivers in prostate cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3891.

Characterization of microRNAs and Target Genes in Musa acuminata subsp. burmannicoides, var. Calcutta 4 during Interaction with Pseudocercospora musae

Endogenous microRNAs (miRNAs) are small non-coding RNAs that perform post-transcriptional regulatory roles across diverse cellular processes, including defence responses to biotic stresses. Pseudocercospora musae, the causal agent of Sigatoka leaf spot disease in banana (Musa spp.), is an important fungal pathogen of the plant. Illumina HiSeq 2500 sequencing of small RNA libraries derived from leaf material in Musa acuminata subsp. burmannicoides, var. Calcutta 4 (resistant) after inoculation with fungal conidiospores and equivalent non-inoculated controls revealed 202 conserved miRNAs from 30 miR-families together with 24 predicted novel miRNAs. Conserved members included those from families miRNA156, miRNA166, miRNA171, miRNA396, miRNA167, miRNA172, miRNA160, miRNA164, miRNA168, miRNA159, miRNA169, miRNA393, miRNA535, miRNA482, miRNA2118, and miRNA397, all known to be involved in plant immune responses. Gene ontology (GO) analysis of gene targets indicated molecular activity terms related to defence responses that included nucleotide binding, oxidoreductase activity, and protein kinase activity. Biological process terms associated with defence included response to hormone and response to oxidative stress. DNA binding and transcription factor activity also indicated the involvement of miRNA target genes in the regulation of gene expression during defence responses. sRNA-seq expression data for miRNAs and RNAseq data for target genes were validated using stem-loop quantitative real-time PCR (qRT-PCR). For the 11 conserved miRNAs selected based on family abundance and known involvement in plant defence responses, the data revealed a frequent negative correlation of expression between miRNAs and target host genes. This examination provides novel information on miRNA-mediated host defence responses, applicable in genetic engineering for the control of Sigatoka leaf spot disease.

Cooperative action of separate interaction domains promotes high-affinity DNA binding of Arabidopsis thaliana ARF transcription factors

The signaling molecule auxin coordinates many growth and development processes in plants, mainly through modulating gene expression. Transcriptional response is mediated by the family of auxin response factors (ARF). Monomers of this family recognize a DNA motif and can homodimerize through their DNA-binding domain (DBD), enabling cooperative binding to an inverted binding site. Most ARFs further contain a C-terminal PB1 domain that is capable of homotypic interactions and mediating interactions with Aux/IAA repressors. Given the dual role of the PB1 domain, and the ability of both DBD and PB1 domain to mediate dimerization, a key question is how these domains contribute to DNA-binding specificity and affinity. So far, ARF-ARF and ARF-DNA interactions have mostly been approached using qualitative methods that do not provide a quantitative and dynamic view on the binding equilibria. Here, we utilize a DNA binding assay based on single-molecule Förster resonance energy transfer (smFRET) to study the affinity and kinetics of the interaction of several Arabidopsis thaliana ARFs with an IR7 auxin-responsive element (AuxRE). We show that both DBD and PB1 domains of AtARF2 contribute toward DNA binding, and we identify ARF dimer stability as a key parameter in defining binding affinity and kinetics across AtARFs. Lastly, we derived an analytical solution for a four-state cyclic model that explains both the kinetics and the affinity of the interaction between AtARF2 and IR7. Our work demonstrates that the affinity of ARFs toward composite DNA response elements is defined by dimerization equilibrium, identifying this as a key element in ARF-mediated transcriptional activity.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

How Deepbics Quantifies Intensities of Transcription Factor-DNA Binding and Facilitates Prediction of Single Nucleotide Variant Pathogenicity With a Deep Learning Model Trained On ChIP-Seq Data Sets.

The binding of DNA sequences to cell type-specific transcription factors is essential for regulating gene expression in all organisms. Many variants occurring in these binding regions play crucial roles in human disease by disrupting the cis-regulation of gene expression. We first implemented a sequence-based deep learning model called deepBICS to quantify the intensity of transcription factors-DNA binding. The experimental results not only showed the superiority of deepBICS on ChIP-seq data sets but also suggested deepBICS as a language model could help the classification of disease-related and neutral variants. We then built a language model-based method called deepBICS4SNV to predict the pathogenicity of single nucleotide variants. The good performance of deepBICS4SNV on 2 tests related to Mendelian disorders and viral diseases shows the sequence contextual information derived from language models can improve prediction accuracy and generalization capability.

The miR-20a/miR-92b Profile Is Associated with Circulating γδ T-Cell Perturbations in Mild Psoriasis.

Psoriasis vulgaris (PV) is an autoinflammatory dermatosis of unknown etiology. Current evidence suggests a pathogenic role of γδT cells, but the growing complexity of this population has made the offending subset difficult to pinpoint. The work on γδTCRint and γδTCRhi subsets, which express intermediate and high levels of γδTCR at their surface, respectively, is particularly scarce, leaving their inner workings in PV essentially unresolved. We have shown here that the γδTCRint/γδTCRhi cell composition and their transcriptom are related to the differential miRNA expression by performing a targeted miRNA and mRNA quantification (RT-qPCR) in multiplexed, flow-sorted γδ blood T cells from healthy controls (n = 14) and patients with PV (n = 13). A significant loss of miR-20a in bulk γδT cells (~fourfold decrease, PV vs. controls) largely mirrored increasing Vδ1-Vδ2- and γδintVδ1-Vδ2- cell densities in the bloodstream, culminating in a relative excess of γδintVδ1-Vδ2- cells for PV. Transcripts encoding DNA-binding factors (ZBTB16), cytokine receptors (IL18R1), and cell adhesion molecules (SELPLG) were depleted in the process, closely tracking miR-20a availability in bulk γδ T-cell RNA. Compared to controls, PV was also associated with enhanced miR-92b expression (~13-fold) in bulk γδT cells that lacked association with the γδT cell composition. The miR-29a and let-7c expressions remained unaltered in case-control comparisons. Overall, our data expand the current landscape of the peripheral γδT cell composition, underlining changes in its mRNA/miRNA transcriptional circuits that may inform PV pathogenesis.

APE1/Ref-1 Inhibits Adipogenic Transcription Factors during Adipocyte Differentiation in 3T3-L1 Cells

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein involved in DNA repair and redox regulation. The redox activity of APE1/Ref-1 is involved in inflammatory responses and regulation of DNA binding of transcription factors related to cell survival pathways. However, the effect of APE1/Ref-1 on adipogenic transcription factor regulation remains unknown. In this study, we investigated the effect of APE1/Ref-1 on the regulation of adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, APE1/Ref-1 expression significantly decreased with the increased expression of adipogenic transcription factors such as CCAAT/enhancer binding protein (C/EBP)-α and peroxisome proliferator-activated receptor (PPAR)-γ, and the adipocyte differentiation marker adipocyte protein 2 (aP2) in a time-dependent manner. However, APE1/Ref-1 overexpression inhibited C/EBP-α, PPAR-γ, and aP2 expression, which was upregulated during adipocyte differentiation. In contrast, silencing APE1/Ref-1 or redox inhibition of APE1/Ref-1 using E3330 increased the mRNA and protein levels of C/EBP-α, PPAR-γ, and aP2 during adipocyte differentiation. These results suggest that APE1/Ref-1 inhibits adipocyte differentiation by regulating adipogenic transcription factors, suggesting that APE1/Ref-1 is a potential therapeutic target for regulating adipocyte differentiation.

Evolution of eukaryotic transcriptional activation domains.

Transcription factors activate gene expression in development, homeostasis, and disease by binding to DNA and recruiting coactivator complexes. Transcription factor DNA binding domains are conserved and structured, features that power statistical models for predicting them directly from amino acid sequence. Activation domains - the transcription factor regions that bind coactivators - are poorly conserved, intrinsically disordered, and remain difficult to predict from amino acid sequence.

PH dynamics regulates transcription factor-DNA binding.

Intracellular pH (pHi) dynamics regulates myriad cell behaviors, and we previously identified pH-sensitive proteins regulating cell migration, cytoskeleton and adhesion remodeling, and transformation through pH-dependent effects on protein-protein and protein-phospholipid binding. Whether pH regulates protein-nucleotide binding for established effects of pHi dynamics on gene expression, however, remains unknown. We identified at least 65 transcription factors in diverse families that have a conserved histidine, which in available structures forms hydrogen bonds with DNA nucleotides. With the ability of histidine to titrate in the cellular pH range, electrostatic principles of protein-nucleotide hydrogen bonds, we predicted that transcription factors with histidine-nucleotide hydrogen bonds could have pH regulated DNA binding selectivity. All FOX family transcription factors contain a conserved histidine in their DNA-binding domain (DBD) that in available structures forms hydrogen bonds with nucleotides. We confirmed pH regulates DNA binding of FOXM1 and FOXC2 DBDs using fluorescence anisotropy and a canonical FkhP DNA motif for binding to a thymine, with higher affinity at pH 7 compared with 7.5. This finding is consistent with thymine being a hydrogen bond acceptor and binding a protonated histidine. We confirmed the significance of histidine with a mutant FOXC2-His122K, with lysine mimicking a protonated residue, that had high affinity but pH-independent binding and a mutant FOXC2-H122N recurring in cancers that had low affinity pH-independent binding. We also confirmed pHi regulates FOXC2 activity in cells. Using MDA-MB-436 cancer cells with a high pHi of 7.7 and a luciferase assay we found increased transcriptional activity for an FkhP motif when pHi was lowered to 7.4 with EIPA, an inhibitor of the proton-extruding NHE1. Our data identify pH dynamics as a previously unrecognized regulator of DNA-binding selectivity for FOXM1 and FOXC2 with broad significance for transcription factors in diverse families with conserved histidines in the DBD.

RADX Gene Variant May Predispose to Familial Asperger Syndrome.

Asperger syndrome (AS) is a pervasive developmental disorder characterized by general impairment in socialization, stereotypical behavior, defective adaptation to the social context usually without intellectual disability, and some high functioning areas related to memory and mathematics. Clinical criteria are not well defined and the etiology is heterogeneous and mostly unknown. Like in typical autism spectrum disorders (ASD), the genetic background plays a crucial role in AS, and often an almost mendelian segregation can be observed in some families. We performed a whole exome sequencing (WES) in three relatives of a family with vertical transmission of AS-ASD to identify variants in candidate genes segregating with the phenotype. Variant p.(Cys834Ser) in the RADX gene was the only one segregating among all the affected family members. This gene encodes a single-strand DNA binding factor, which mediates the recruitment of genome maintenance proteins to sites of replication stress. Replication stress and genome instability have been reported recently in neural progenitor cells derived from ASD patients, leading to a disruption of long neural genes involved in cell-cell adhesion and migration. We propose RADX as a new gene that when mutated could represent a predisposing factor to AS-ASD.

Transcription factor TOX maintains the expression of Mst1 in controlling the early mouse NK cell development.

Rationale: TOX is a DNA-binding factor required for the development of multiple immune cells and the formation of lymph nodes. However, the temporal regulation mode of TOX on NK cell development and function needs to be further explored. Methods: To investigate the role of TOX in NK cells at distinct developmental phases, we deleted TOX at the hematopoietic stem cell stage (Vav-Cre), NK cell precursor (CD122-Cre) stage and late NK cell developmental stage (Ncr1-Cre), respectively. Flow cytometry was used to detect the development and functional changes of NK cell when deletion of TOX. RNA-seq was used to assess the differences in transcriptional expression profile of WT and TOX-deficient NK cells. Published Chip-seq data was exploited to search for the proteins directly interact with TOX in NK cells. Results: The deficiency of TOX at the hematopoietic stem cell stage severely retarded NK cell development. To a less extent, TOX also played an essential role in the physiological process of NKp cells differentiation into mature NK cells. Furthermore, the deletion of TOX at NKp stage severely impaired the immune surveillance function of NK cells, accompanied by down-regulation of IFN-γ and CD107a expression. However, TOX is dispensable for mature NK cell development and function. Mechanistically, by combining RNA-seq data with published TOX ChIP-seq data, we found that the inactivation of TOX at NKp stage directly repressed the expression of Mst1, an important intermediate kinase in Hippo signaling pathway. Mst1 deficient at NKp stage gained the similar phenotype with Toxfl/flCD122Cre mice. Conclusion: In our study, we conclude that TOX coordinates the early mouse NK cell development at NKp stage by maintaining the expression of Mst1. Moreover, we clarify the different dependence of the transcription factor TOX in NK cells biology.

Genome-wide distribution of histone trimethylation reveals a global impact of bisphenol A on telomeric binding proteins and histone acetyltransferase factors: a pilot study with human and in vitro data

ObjectiveTo assess the genetic and epigenetic effects promoted by Bisphenol A (BPA) exposure in adolescent males from the Spanish INMA-Granada birth cohort, and in human cells.MethodsDNA methylation was analysed using MEDIP. Repeat number variation in genomic DNA was evaluated, along with the analysis of H3K4me3 by using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Analyses were performed with material extracted from whole blood of the adolescents, complemented by in vitro assessments of human (HeLa) cells exposed to 10 nM BPA, specifically, immunofluorescence evaluation of protein levels, gene expression analysis and ChIP‒qPCR analysis.ResultsAdolescents in the high urinary BPA levels group presented a higher level of Satellite A (SATA) repetitive region copy numbers compared to those in the low BPA group and a tendency towards increase in telomere length. We also observed decreased DNA methylation at the promoters of the imprinted genes H19, KCNQ1, and IGF2; at LINE1 retroelements; and at the ARID2, EGFR and ESRRA and TERT genes. Genome-wide sequencing revealed increased H3K4me3 occupancy at the promoters of genes encoding histone acetyltransferases, telomeric DNA binding factors and DNA repair genes. Results were supported in HeLa cells exposed to 10 nM BPA in vitro. In accordance with the data obtained in blood samples, we observed higher H3K4me3 occupancy and lower DNA methylation at some specific targets in HeLa cells. In exposed cells, changes in the expression of genes encoding DNA repair factors (ATM, ARID2, TRP53) were observed, and increased expression of several genes encoding telomeric DNA binding factors (SMG7, TERT, TEN1, UPF1, ZBTB48) were also found. Furthermore, an increase in ESR1/ERa was observed in the nuclei of HeLa cells along with increased binding of ESR1 to KAT5, KMT2E and TERF2IP promoters and decreased ESR1 binding at the RARA promoter. The DNA damage marker p53/TP53 was also increased.ConclusionIn this pilot study, genome-wide analysis of histone trimethylation in adolescent males exposed to BPA revealed a global impact on the expression of genes encoding telomeric binding proteins and histone acetyltransferase factors with similar results in HeLa cells. Nevertheless, larger studies should confirm our findings.

AMPLIFICATION AND HIGHER EXPRESSION OF THE ZNF281 GENE IN PANCREATIC CANCER CORRELATES WITH POOR PROGNOSIS

Establishing precise gene expression patterns in different cell types is essential for the proper differentiation and development of multicellular organisms. The resulting level of transcription is determined both by the genetic nucleotide sequence of DNA and by epigenetic factors that modify chromatin. Epigenetic repressors of the Polycomb group (PcG) are regulatory proteins that repress gene transcription and maintain correct pattern of gene expression in multicellular organisms. PcG proteins form two main complexes: Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2) that possess ubiquitin ligase and histone methyltransferase enzymatic activities, respectively. Several mechanisms have been suggested to account for the recruitment of Polycomb proteins in mammals, one of which involves interactions with specific DNA-binding factors. While deregulation main PcG genes in cancer have been well documented, the role of PcG DNA-binding partners in oncology remains elusive. In the present study, we analyzed genomic and transcriptomic databases of clinical tumor samples (cBioPortal, TNMplot, KMplot) to evaluate clinical correlations of Polycomb-associated DNA-binding proteins. We found that amplifications and higher expression of the ZNF281 gene are often found in pancreatic cancer and correlate with poor prognosis of overall survival.

NetREX-CF integrates incomplete transcription factor data with gene expression to reconstruct gene regulatory networks

The inference of Gene Regulatory Networks (GRNs) is one of the key challenges in systems biology. Leading algorithms utilize, in addition to gene expression, prior knowledge such as Transcription Factor (TF) DNA binding motifs or results of TF binding experiments. However, such prior knowledge is typically incomplete, therefore, integrating it with gene expression to infer GRNs remains difficult. To address this challenge, we introduce NetREX-CF—Regulatory Network Reconstruction using EXpression and Collaborative Filtering—a GRN reconstruction approach that brings together Collaborative Filtering to address the incompleteness of the prior knowledge and a biologically justified model of gene expression (sparse Network Component Analysis based model). We validated the NetREX-CF using Yeast data and then used it to construct the GRN for Drosophila Schneider 2 (S2) cells. To corroborate the GRN, we performed a large-scale RNA-Seq analysis followed by a high-throughput RNAi treatment against all 465 expressed TFs in the cell line. Our knockdown result has not only extensively validated the GRN we built, but also provides a benchmark that our community can use for evaluating GRNs. Finally, we demonstrate that NetREX-CF can infer GRNs using single-cell RNA-Seq, and outperforms other methods, by using previously published human data.

Recruitment to Chromatin of (GA)n-Associated Factors GAF and Psq in the Transgenic Model System Depends on the Presence of Architectural Protein Binding Sites.

Polycomb group (PcG) repressors and Trithorax group (TrxG) activators of transcription are essential for the proper development and maintenance of gene expression profiles in multicellular organisms. In Drosophila, PcG/TrxG proteins interact with DNA elements called PRE (Polycomb response elements). We have previously shown that the repressive activity of inactive PRE in transgenes can be induced by architectural protein-binding sites. It was shown that the induction of repression is associated with the recruitment of PcG/TrxG proteins, including the DNA-binding factors Pho and Combgap. In the present study, we tested the association of the two other PRE DNA-binding factors, GAF and Psq, with bxdPRE in the presence and absence of sites for architectural proteins. As a result, it was shown that both factors can be efficiently recruited to the bxdPRE only in the presence of adjacent binding sites for architectural proteins Su(Hw), CTCF, or Pita.

SMC complexes can traverse physical roadblocks bigger than their ring size.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use invitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.

IDHwt Glioblastomas Show Opposing Resistance Mechanisms Across Patients in Response to Standard Treatment

Abstract AIMS Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite aggressive treatment, a resistant tumour recurs in practically all patients. We therefore aimed to better understand the mechanisms driving this treatment resistance through investigating changes in gene expression across pairs of primary and recurrent GBM tumours. METHOD We generated or acquired bulk tumour RNA sequencing data for primary and first recurrent tumours from 107 patients who received standard treatment. Differential expression analysis between primary and recurrent samples found that the most dysregulated genes were involved in neurodevelopment and neurodifferentiation. We therefore used a publicly available ChIP-seq database to identify DNA binding factors for which binding sites are enriched in the promotors of genes with the largest expression changes from primary to recurrent. RESULTS Jumonji and AT-Rich Interacting Domain 2 (JARID2) was the most strongly enriched for binding to promotors of dysregulated genes. 65 patients showed an up-regulation and 42 showed a down-regulation of genes bound by this protein. The same set of JARID2 bound genes were found to be dysregulated in each direction, and correlated with the largest source of variation between samples in their response to treatment. Further enrichment analyses indicated that ‘Up’ responders may resist treatment through reduced proliferation and increased interaction with the tumour microenvironment, whereas ‘Down’ responders instead rely on a shift to mesenchymal cell states. CONCLUSION These results indicate that GBM tumours can be split into two subtypes that transcriptionally reprogramme in different directions through treatment and may benefit from different treatment approaches.

ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

BackgroundSWI/SNF (BAF) chromatin remodeling complexes regulate lineage-specific enhancer activity by promoting accessibility for diverse DNA-binding factors and chromatin regulators. Additionally, they are known to modulate the function of the epigenome through regulation of histone post-translational modifications and nucleosome composition, although the way SWI/SNF complexes govern the epigenome remains poorly understood. Here, we investigate the function of ARID1A, a subunit of certain mammalian SWI/SNF chromatin remodeling complexes associated with malignancies and benign diseases originating from the uterine endometrium.ResultsThrough genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements such as super-enhancers. ARID1A knockdown leads to H3.3 depletion and gain of canonical H3.1/3.2 at ARID1A-bound active regulatory elements, and a concomitant redistribution of H3.3 toward genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3, and ARID1A is required for CHD4 recruitment to H3.3. ZMYND8 interacts with CHD4 to suppress a subset of ARID1A, CHD4, and ZMYND8 co-bound, H3.3+ H4K16ac+ super-enhancers near genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas.ConclusionsThese studies demonstrate that ARID1A-containing BAF complexes are required for maintenance of the histone variant H3.3 at active regulatory elements, such as super-enhancers, and this function is required for the physiologically relevant activities of alternative chromatin remodelers.

RSC and GRFs confer promoter directionality by restricting divergent noncoding transcription.

The directionality of gene promoters-the ratio of protein-coding over divergent noncoding transcription-is highly variable. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription relative to protein-coding transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus to a decrease in promoter directionality. We find that RSC has a modest effect on nucleosome positioning upstream in promoters at the sites of divergent transcription. These promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 DNA-binding protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription relative to coding direction transcription. We propose that any DNA-binding factor, when stably associated with cryptic transcription start sites, forms a barrier which represses divergent transcription, thereby promoting promoter directionality.