Facilitates Chromatin Transcription Research Articles

The FAcilitates Chromatin Transcription complex regulates the ratio of glycolysis to oxidative phosphorylation in neural stem cells.

Defects in the FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone composed of SSRP1 and SUPT16H, are implicated in intellectual disability. Here, we reveal that the FACT complex promotes glycolysis and sustains the correct cell fate of neural stem cells/neuroblasts in the Drosophila 3rd instar larval central brain. We show that the FACT complex binds to the promoter region of the estrogen-related receptor (ERR) gene and positively regulates ERR expression. ERR is known to act as an aerobic glycolytic switch by upregulating the enzymes required for glycolysis. Dysfunction of the FACT complex leads to the downregulation of ERR transcription, resulting in a decreased ratio of glycolysis to oxidative phosphorylation (G/O) in neuroblasts. Consequently, neuroblasts exhibit smaller cell sizes, lower proliferation potential, and altered cell fates. Overexpression of ERR or suppression of mitochondrial oxidative phosphorylation in neuroblasts increases the relative G/O ratio and rescues defective phenotypes caused by dysfunction of the FACT complex. Thus, the G/O ratio, mediated by the FACT complex, plays a crucial role in neuroblast cell fate maintenance. Our study may shed light on the mechanism by which mutations in the FACT complex lead to intellectual disability in humans.

FEAR antiviral response pathway is independent of interferons and countered by poxvirus proteins.

The human facilitates chromatin transcription (FACT) complex is a chromatin remodeller composed of human suppressor of Ty 16 homologue (hSpt16) and structure-specific recognition protein-1 subunits that regulates cellular gene expression. Whether FACT regulates host responses to infection remained unclear. We identify a FACT-mediated, interferon-independent, antiviral pathway that restricts poxvirus replication. Cell culture and bioinformatics approaches suggest that early viral gene expression triggers nuclear accumulation of SUMOylated hSpt16 subunits required for the expression of E26 transformation-specific sequence-1 (ETS-1)-a transcription factor that activates virus restriction programs. However, biochemical studies show that poxvirus-encoded A51R proteins block ETS-1 expression by outcompeting structure-specific recognition protein-1 binding to SUMOylated hSpt16 and by tethering SUMOylated hSpt16 to microtubules. Furthermore, A51R antagonism of FACT enhances poxvirus replication in human cells and virulence in mice. Finally, we show that FACT also restricts rhabdoviruses, flaviviruses and orthomyxoviruses, suggesting broad roles for FACT in antiviral immunity. Our study reveals the FACT-ETS-1 antiviral response (FEAR) pathway to be critical for eukaryotic antiviral immunity and describes a unique mechanism of viral immune evasion.

Abstract B016: Targeting the epigenome through combined Facilitates Chromatin Transcription (FACT) and Bromodomain and Extra-Terminal Domain (BET) inhibition in Diffuse Midline Glioma (DMG)

Abstract Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable tumor that primarily arises in the brainstem of young children. The H3K27M driver histone mutation and resulting permissive chromatin indicates epigenetic targeting as a key therapeutic strategy against this aggressive cancer. One such epigenetic target is the Facilitates Chromatin Transcription (FACT) histone chaperone. FACT is targeted by the curaxin compound CBL0137 which is currently in phase I/II clinical trial for pediatric cancer patients (NCT04870944). This study aims to decipher the mechanism of CBL0137 to find effective combination therapies for the next round of clinical implementation. To this end, we applied a multi-omics approach by integrating epigenetic profiling with transcriptomic data sets (CUT&RUN, ATAC-seq and RNA-seq) to interrogate the therapeutic mechanism of CBL0137 in DMG. We then employed in vitro cytotoxicity assays and in vivo orthotopic patient-derived xenograft (PDX) pre-clinical models to test a novel, mechanistic-anchored epigenetic combination therapy. We found FACT to be enriched at the promoters of developmental genes, coinciding with regions of open chromatin and binding motifs of the core DMG regulatory transcription factors TCF12 and OLIG2. Furthermore, FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4, suggesting cooperation between FACT and BRD4 in DMG. Given that BRD4 is an established therapeutic target in DMG and other cancers, we investigated the combination of the FACT-inhibitor CBL0137 with the BET-inhibitor JQ1. Combination treatment was cytotoxic against DMG in vitro, with enhanced cytotoxicity observed against H3K27M-mutant cells. We demonstrated a significant survival extension in three independent orthotopic PDX mouse models treated with CBL0137 and JQ1 in combination. Mechanistically, CBL0137 and JQ1 decreased chromatin accessibility and transcription of genes involved in RNA regulatory processes, such as RNA methylation and splicing. Consistently, combination treatment led to increased intron retention, highlighting a possible link between chromatin disruption and the splicing machinery in DMG. Future work will investigate whether treatment-induced intron retention acts as a source of neoantigens, with the potential to enhance DMG immunogenicity. In sum, this study has identified a promising new epigenetic combination therapy – FACT + BET inhibition – while uncovering intriguing new insights into DMG epigenetics and pathobiology. Citation Format: Holly Holliday, Aaminah Khan, Nisitha Jayatilleke, Chelsea Mayoh, Samuel E. Ross, Yolanda Colino Sanguino, Anjana Gopalakrishnan, Anahid Ehteda, Benjamin Rayner, Maria Tsoli, David S. Ziegler. Targeting the epigenome through combined Facilitates Chromatin Transcription (FACT) and Bromodomain and Extra-Terminal Domain (BET) inhibition in Diffuse Midline Glioma (DMG) [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B016.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

Targeting FAcilitates Chromatin Transcription complex inhibits pleural mesothelioma and enhances immunotherapy

BackgroundDiffuse pleural mesothelioma (DPM) is an aggressive therapy-resistant cancer with unique molecular features. Numerous agents have been tested, but clinically effective ones remain elusive. Herein, we propose to use a small molecule CBL0137 (curaxin) that simultaneously suppresses nuclear factor-κB (NF-κB) and activates tumor suppressor p53 via targeting FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone critical for DNA repair.MethodsWe used DPM cell lines, murine models (xeno- and allo-grafts), plus DPM patient samples to characterize anti-tumor effects of CBL0137 and to delineate specific molecular mechanisms.ResultsWe verified that CBL0137 induced cell cycle arrest and apoptosis. We also discovered that DPM is a FACT-dependent cancer with overexpression of both subunits structure-specific recognition protein 1 (SSRP1), a poor prognosis indicator, and suppressor of Ty 16 (SUPT16H). We defined several novel uses of CBL0137 in DPM therapy. In combination with cisplatin, CBL0137 exhibited additive anti-tumor activity compared to monotherapy. Similarly, CBL0137 (systemic) could be combined with other novel agents like microRNA-215 (intrapleural) as a more effective regimen. Importantly, we established that CBL0137 induces immunogenic cell death that contributes to activating immune response pathways in DPM. Therefore, when CBL0137 is combined with dual immune checkpoint inhibitors DPM tumor growth is significantly suppressed.ConclusionsWe identified an unrecognized molecular vulnerability of DPM based on FACT dependency. CBL0137 alone and in several combinations with different therapeutics showed promising efficacy, including that of improved anti-tumor immunity. Overall, these preclinical findings suggest that CBL0137 could be ideally suited for use in DPM clinical trials.

Quantifying ATP-Independent Nucleosome Chaperone Activity with Single-Molecule Methods.

The dynamics of histone-DNA interactions govern chromosome organization and regulates the processes of transcription, replication, and repair. Accurate measurements of the energies and the kinetics of DNA binding to component histones of the nucleosome under a variety of conditions are essential to understand these processes at the molecular level. To accomplish this, we employ three specific single-molecule techniques: force disruption (FD) with optical tweezers, confocal imaging (CI) in a combined fluorescence plus optical trap, and survival probability (SP) measurements of disrupted and reformed nucleosomes. Short arrays of positioned nucleosomes serve as a template for study, facilitating rapid quantification of kinetic parameters. These arrays are then exposed to FACT (FAcilitates Chromatin Transcription), a non-ATP-driven heterodimeric nuclear chaperone known to both disrupt and tether histones during transcription. FACT binding drives off the outer wrap of DNA and destabilizes the histone-DNA interactions of the inner wrap as well. This reorganization is driven by two key domains with distinct function. FD experiments show the SPT16 MD domain stabilizes DNA-histone contacts, while the HMGB box of SSRP1 binds DNA, destabilizing the nucleosome. Surprisingly, CI experiments do not show tethering of disrupted histones, but increased rates of histone release from the DNA. SI experiments resolve this, showing that the two active domains of FACT combine to chaperone nucleosome reassembly after the timely release of force. These combinations of single-molecule approaches show FACT is a true nucleosome catalyst, lowering the barrier to both disruption and reformation.

The histone H2B Arg95 residue efficiently recruits the transcription factor Spt16 to mediate Ste5 expression of the pheromone response pathway

In yeast Saccharomyces cerevisiae, the immunosuppressant rapamycin inhibits the TORC1 kinase causing rapid alteration in gene expression and leading to G1 arrest. We recently reported the isolation and characterization from the histone mutant collection of a histone H2B R95A mutant that displays resistance to rapamycin. This mutant is defective in the expression of several genes belonging to the pheromone response pathway including STE5 encoding a scaffold protein that promotes the activation of downstream MAP kinases. Cells lacking Ste5 cannot arrest the cell cycle in response to rapamycin and as a consequence exhibit similar resistance to rapamycin as the H2B R95A mutant. Herein, we show that the H2B R95A mutation weakens the association of H2B with Spt16 a component of the FACT complex (FAcilitates Chromatin Transcription), and an essential factor that interacts with the histone H2A-H2B dimer to promote transcription and preserve chromatin integrity. From a collection of spt16 mutants, spt16 E857K and spt16-11 showed striking sensitivity to rapamycin as compared to the parent strain. spt16 E857K and spt16-11 expressed distinct forms of Ste5, while a suppressor mutation H2B A84D of the spt16-11 mutant prevents the expression of Ste5 and confers marked resistance to rapamycin. We interpret these findings to suggest that the Arg95 residue of histone H2B is required to recruit Spt16 to maintain the expression of STE5, which performs a role to arrest cells in the G1 phase in response to rapamycin.

Identification of curaxin as a potential new therapeutic for JAK2 V617F mutant patients.

Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms.

H2B ubiquitination recruits FACT to maintain a stable altered nucleosome state for transcriptional activation

Histone H2B mono-ubiquitination at lysine 120 (ubH2B) has been found to regulate transcriptional elongation by collaborating with the histone chaperone FACT (Facilitates Chromatin Transcription) and plays essential roles in chromatin-based transcriptional processes. However, the mechanism of how ubH2B directly collaborates with FACT at the nucleosome level still remains elusive. In this study, we demonstrate that ubH2B impairs the mechanical stability of the nucleosome and helps to recruit FACT by enhancing the binding of FACT on the nucleosome. FACT prefers to bind and deposit H2A-ubH2B dimers to form an intact nucleosome. Strikingly, the preferable binding of FACT on ubH2B-nucleosome greatly enhances nucleosome stability and maintains its integrity. The stable altered nucleosome state obtained by ubH2B and FACT provides a key platform for gene transcription, as revealed by genome-wide and time-course ChIP-qPCR analyses. Our findings provide mechanistic insights of how ubH2B directly collaborates with FACT to regulate nucleosome dynamics for gene transcription.

Structural basis for H2A–H2B recognitions by human Spt16

The human facilitates chromatin transcription (FACT) complex, consisting of Spt16 and SSRP1, is a versatile histone chaperone that can engage free H2A–H2B dimer and H3–H4 tetramer (or dimer), and partially unraveled nucleosome. The C-terminal domain of human Spt16 (hSpt16-CTD) is the decisive element for engaging H2A–H2B dimer and partially unraveled nucleosome. The molecular basis of the H2A–H2B dimer recognitions by hSpt16-CTD is not fully comprehended. Here, we present a high-resolution snapshot of the recognitions of the H2A–H2B dimer by hSpt16-CTD via an acidic intrinsically disordered (AID) segment, and reveal some distinct structural features of hSpt16-CTD as compared to the budding yeast Spt16-CTD.

Visualizing both nucleosome disassembly and reassembly during FACT chaperone activity.

The histone chaperone FACT (FAcilitates Chromatin Transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (strong site) interactions, altering the energy landscape of unwrapping and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling and DNA unwinding. Single molecule multi-channel fluorescence observes component histones as they disassemble under tension and are lost. Yet if tension is released, reassembly is also observed and surprisingly both loss and reassembly are accelerated by key domains of FACT. We resolve this ambiguity, noting that all histone-DNA kinetics are faster with FACT. Of the individual domains we studied, two were shown to influence nucleosome structure and dynamics with distinctly contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones, and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate how key FACT domains combine to catalyze nucleosome disassembly and reassembly.

Identifying the role of the FACT histone chaperone in epigenetics through single-molecule fluorescence imaging.

Modifications of histones in eukaryotic cells lead to reversible changes in gene expression without altering the genetic code itself. Such epigenetic modifications are necessary for the proper functioning of cellular processes like gene expression, DNA repair, and cell division. Modified histones directly affect chromatin dynamics and promote other regulation factors such as chromatin remodelers, including histone chaperones. Histone chaperones make functional DNA templates that maintain epigenetic information by remodeling the nucleosome during essential life processes, such as transcription and replication, and by avoiding histone aggregation. The facilitates chromatin transcription (FACT) histone chaperone includes two essential proteins, Pob3 and Spt16. Previously, we found that deleting Pob3 suppresses epigenetic inheritance, indicating that FACT plays an important role in epigenetic maintenance. Therefore, we engineered Schizosaccharomyces pombe (fission yeast) cell strains that express fusions of Spt16 or Pob3 to the fluorescent protein PAmCherry and investigated the dynamics of single proteins in the FACT complex in living S. pombe cells to identify the mechanism for this epigenetic inheritance suppression, including measuring changes in interactions during transcription and replication. By analyzing the motion of Spt16-PAmCherry single-molecule trajectories with nonparametric Bayesian statistics, we were able to distinguish three types of single-molecule motion: a fast (free) mobility state, an intermediate (transiently bound) mobility state, and a slow (bound) mobility state. We are assigning these biophysical states to specific biochemical processes by constructing Spt16 and Pob3 mutants with impaired histone protein binding properties, as well as drug-susceptible strains in which we can inhibit transcription and replication to identify changes in the FACT protein mobility. Overall, our live-cell single-molecule experiments reveal how the FACT complex interacts with histones and other chaperones and indicate a mechanism for epigenetic maintenance.

The FACT-targeted drug CBL0137 enhances the effects of rituximab to inhibit B-cell non-Hodgkin’s lymphoma tumor growth by promoting apoptosis and autophagy

BackgroundAggressive B-cell non-Hodgkin’s lymphoma (B-NHL) patients often develop drug resistance and tumor recurrence after conventional immunochemotherapy, for which new treatments are needed.MethodsWe investigated the antitumor effects of CBL0137. In vitro, cell proliferation was assessed by CCK-8 and colony formation assay. Flow cytometry was performed to analyze cell cycle progression, apoptosis, mitochondrial depolarization, and reactive oxygen species (ROS) production. Autophagy was detected by transmission electron microscopy and mGFP-RFP-LC3 assay, while western blotting was employed to detect proteins involved in apoptosis and autophagy. RNA-sequencing was conducted to analyze the transcription perturbation after CBL0137 treatment in B-NHL cell lines. Finally, the efficacy and safety of CBL0137, rituximab, and their combination were tested in vivo.ResultsCBL0137, a small molecule anticancer agent that has significant antitumor effects in B-NHL. CBL0137 sequesters the FACT (facilitates chromatin transcription) complex from chromatin to produce cytotoxic effects in B-NHL cells. In addition, we discovered novel anticancer mechanisms of CBL0137. CBL0137 inhibited human B-NHL cell proliferation by inducing cell cycle arrest in S phase via the c-MYC/p53/p21 pathway. Furthermore, CBL0137 triggers ROS generation and induces apoptosis and autophagy in B-NHL cells through the ROS-mediated PI3K/Akt/mTOR and MAPK signaling pathways. Notably, a combination of CBL0137 and rituximab significantly suppressed B-NHL tumor growth in subcutaneous models, consistent with results at the cellular level in vitro.ConclusionsCBL0137 has potential as a novel approach for aggressive B-NHL, and its combination with rituximab can provide new therapeutic options for patients with aggressive B-NHL.3a3Ge1vW8eA3YTTebu-sV5Video

Tandem Affinity Purification and Mass-Spectrometric Analysis of FACT and Associated Proteins.

Isolation of a protein/complex is important for its biochemical and structural characterization with mechanistic insights. TAP (tandem affinity purification) strategy allows rapid isolation of cellular proteins/complexes with a high level of purity. This methodology involves an immuno-affinity-based purification followed by a conformation-based isolation to obtain a highly homogeneous protein/complex. Here, we describe the TAP-mediated isolation of endogenous FACT (facilitates chromatin transcription; a heterodimer), an essential histone chaperone associated with BER (base excision repair). However, it is not clearly understood how FACT regulates BER. Such knowledge would advance our understanding of BER with implications in disease pathogenesis, since BER is an evolutionarily conserved process that is linked to various diseases including ageing, neurodegenerative disorders, and cancers. Using isolated FACT by TAP methodology, one can study the mechanisms of action of FACT in BER. Further, isolated FACT can be used for studies in other DNA transactions such as transcription and replication, as FACT is involved in these processes. Furthermore, TAP-mediated isolation strategy can be combined with mass spectrometry to identify the protein interaction partners of FACT.

Human FACT subunits coordinate to catalyze both disassembly and reassembly of nucleosomes

The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.

Cross-regulome profiling of RNA polymerases highlights the regulatory role of polymerase III on mRNA transcription by maintaining local chromatin architecture

BackgroundMammalian cells have three types of RNA polymerases (Pols), Pol I, II, and III. However, the extent to which these polymerases are cross-regulated and the underlying mechanisms remain unclear.ResultsWe employ genome-wide profiling after acute depletion of Pol I, Pol II, or Pol III to assess cross-regulatory effects between these Pols. We find that these enzymes mainly affect the transcription of their own target genes, while certain genes are transcribed by the other polymerases. Importantly, the most active type of crosstalk is exemplified by the fact that Pol III depletion affects Pol II transcription. Pol II genes with transcription changes upon Pol III depletion are enriched in diverse cellular functions, and Pol III binding sites are found near their promoters. However, these Pol III binding sites do not correspond to transfer RNAs. Moreover, we demonstrate that Pol III regulates Pol II transcription and chromatin binding of the facilitates chromatin transcription (FACT) complex to alter local chromatin structures, which in turn affects the Pol II transcription rate.ConclusionsOur results support a model suggesting that RNA polymerases show cross-regulatory effects: Pol III affects local chromatin structures and the FACT-Pol II axis to regulate the Pol II transcription rate at certain gene loci. This study provides a new perspective for understanding the dysregulation of Pol III in various tissues affected by developmental diseases.

Therapeutic targeting the oncogenic driver EWSR1::FLI1 in Ewing sarcoma through inhibition of the FACT complex.

EWS/ETS fusion transcription factors, most commonly EWSR1::FLI1, drives initiation and progression of Ewing sarcoma (EwS). Even though direct targeting EWSR1::FLI1 is a formidable challenge, epigenetic/transcriptional modulators have been proved to be promising therapeutic targets for indirectly disrupting its expression and/or function. Here, we identified structure-specific recognition protein 1 (SSRP1), a subunit of the Facilitates Chromatin Transcription (FACT) complex, to be an essential tumor-dependent gene directly induced by EWSR1::FLI1 in EwS. The FACT-targeted drug CBL0137 exhibits potent therapeutic efficacy against multiple EwS preclinical models both in vitro and in vivo. Mechanistically, SSRP1 and EWSR1::FLI1 form oncogenic positive feedback loop via mutual transcriptional regulation and activation, and cooperatively promote cell cycle/DNA replication process and IGF1R-PI3K-AKT-mTOR pathway to drive EwS oncogenesis. The FACT inhibitor drug CBL0137 effectively targets the EWSR1::FLI1-FACT circuit, resulting in transcriptional disruption of EWSR1::FLI1, SSRP1 and their downstream effector oncogenic signatures. Our study illustrates a crucial role of the FACT complex in facilitating the expression and function of EWSR1::FLI1 and demonstrates FACT inhibition as a novel and effective epigenetic/transcriptional-targeted therapeutic strategy against EwS, providing preclinical support for adding EwS to CBL0137's future clinical trials.

Supt16 haploinsufficiency causes neurodevelopment disorder by disrupting MAPK pathway in neural stem cells.

Chromatin regulators constitute a fundamental means of transcription regulation, which have been implicated in neurodevelopment and neurodevelopment disorders (NDDs). Supt16, one of candidate genes for NDDs, encodes the large subunit of facilitates chromatin transcription. However, the underlying mechanisms remain poorly understood. Here, Supt16+/- mice was generated, modeling the neurodevelopment disorder. Abnormal cognitive and social behavior was observed in the Supt16 +/- mice. Simultaneously, the number of neurocytes in the cerebral cortex and hippocampus is decreased, which might be resulted from the impairment of mouse neural stem cells (mNSCs) in the SVZ. Supt16 haploinsufficiency affects the proliferation and apoptosis of mNSCs. As the RNA-seq and chromatic immunoprecipitation sequencing assays showed, Supt16 haploinsufficiency disrupts the stemness of mNSCs by inhibiting MAPK signal pathway. Thus, this study demonstrates a critical role of Supt16 gene in the proliferation and apoptosis of mNSCs and provides a novel insight in the pathogenesis of NDDs.

Differential Expression of Hub Genes and Activation of p53 by Anti-cancer Compound Curaxin CBL0137

Cancer is a global concern and there is a need for effective drugs. CBL0137 is a small water-soluble molecule and a new second-generation compound in the family of curaxins with potential anti-cancer activity. Curaxins in general, including CBL0137 intercalate into DNA, and act by targeting the histone chaperone ‘facilitates chromatin transcription’ (FACT) complex and have the potential to treat tumors by reducing the growth of cancer cells which is shown in a variety of cell lines and animal models. CBL0137 is found to activate the tumor suppressor gene p53. However, the mechanism of p53 activation is poorly understood. Utilizing bioinformatics analysis on available datasets of CBL0137 treated cancer cells of glioma, cervical and multiple myeloma, differentially expressed genes (DEGs) that may lead to the activation of p53 were examined. Three GEO datasets of cells treated with various concentrations of CBL0137 were analyzed, namely HSJD-DIPG007 (GSE153441), MM1.S (GSE117611) and HeLa S3 (GSE117611). The DEGs were identified based on p-values less than 0.05, logFC values greater than 1 and less than -1 and analyzed using GEO2R, Enrichr, and STRING, and data visualization was performed on Tableau. Compared to the controls, a total of 229, 1425, and 1005 genes were upregulated while 368, 2322, and 1673 genes were downregulated for HSJD-DIPG007, MM1.S and HeLa S3 datasets, respectively. Further collective analysis revealed a total of 38 common DEGs among the three datasets. Using Enrichr and STRING on these 38 DEGs, seven hub genes were obtained, SKP2, RGS16, CSRP2, CENPA, HJURP, DTL, and HEXIM1 with these possible mechanisms: inhibition of AKT phosphorylation by upregulated genes RGS16 and CSRP2, p300-mediated acetylation of p53 via SKP2, inhibition of MDM2 by DTL downregulation and HEXIM1 upregulation, and inhibition of AURKB via CENPA and HJURP downregulation. This study analyzed the three datasets and highlighted how these identified hub genes may play a role in leading to p53 activation by CBL0137. KEYWORDS: Curaxin; CBL0137; Differentially Expressed Genes; Cancer; p53; Glioblastoma; Cervical; Myeloma

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.

The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.