The HMG-box module in FACT is critical for suppressing epigenetic variegation of heterochromatin in fission yeast.

Chromatin condensation state is the key for retrieving genetic information. High-mobility group protein (HMG) proteins exhibit DNA-binding and bending activities, playing an important role in the regulation of chromatin structure. We have shown that nucleosomes tightly packaged into heterochromatin undergo considerable dynamic histone H2A-H2B maintenance via the direct interaction between HP1/Swi6 and facilitate chromatin transcription (FACT), which is composed of the Spt16/Pob3 heterodimer and Nhp6. In this study, we analyzed the role of Nhp6, an HMG box protein, in the FACT at heterochromatin. Pob3 mutant strains showed derepressed heterochromatin-dependent gene silencing, whereas Nhp6 mutant strains did not show significant defects in chromatin regulation or gene expression, suggesting that these two modules play different roles in chromatin regulation. We expressed a protein fusing Nhp6 to the C-terminus of Pob3, which mimics the multicellular FACT component Ssrp1. The chromatin-binding activity of FACT increased with the number of Nhp6 fused to Pob3, and the heterochromatin formation rate was promoted more strongly. Furthermore, we demonstrated that this promotion of heterochromatinization inhibited the heterochromatic variegation caused by epe1+ disruption. Heterochromatic variegation can be observed in a variety of regulatory steps; however, when it is caused by fluctuations in chromatin arrangement, it can be eliminated through the strong recruitment of the FACT complex.

Hypoxia-inducible factor 1 recruits FACT and RNF20/40 to mediate histone ubiquitination and transcriptional activation of target genes.

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.

Transcriptional regulation of FACT involves Coordination of chromatin accessibility and CTCF binding.

Histone chaperone FACT (facilitates chromatin transcription) is well known to promote chromatin recovery during transcription. However, the mechanism how FACT regulates genome-wide chromatin accessibility and transcription factor binding has not been fully elucidated. Through loss-of-function studies, we show here that FACT component Ssrp1 is required for DNA replication and DNA damage repair and is also essential for progression of cell phase transition and cell proliferation in mouse embryonic fibroblast cells. On the molecular level, absence of the Ssrp1 leads to increased chromatin accessibility, enhanced CTCF binding, and a remarkable change in dynamic range of gene expression. Our study thus unequivocally uncovers a unique mechanism by which FACT complex regulates transcription by coordinating genome-wide chromatin accessibility and CTCF binding.

SFPQ promotes the proliferation, migration and invasion of hepatocellular carcinoma cells and is associated with poor prognosis.

Liver cancer is a prevalent type of tumor worldwide. CRISPR-Cas9 technology can be utilized to identify therapeutic targets for novel therapeutic approaches. In this study, our goal was to identify key genes related to the survival of hepatocellular carcinoma (HCC) cells by analyzing the DepMap database based on CRISPR-Cas9. We screened candidate genes associated with HCC cell survival and proliferation from DepMap and identified their expression levels in HCC from the TCGA database. To develop a prognostic risk model based on these candidate genes, we performed WGCNA, functional pathway enrichment analysis, protein interaction network construction, and LASSO analysis. Our findings show that 692 genes were critical for HCC cell proliferation and survival, and among them, 571 DEGs were identified in HCC tissues. WGCNA categorized these 584 genes into three modules, and the blue module consisting of 135 genes was positively linked to the tumor stage. Using the MCODE approach in Cytoscape, we identified ten hub genes in the PPI network, and through Cox univariate analysis and Lasso analysis, we developed a prognostic model consisting of three genes (SFPQ, SSRP1, and KPNB1). Furthermore, knocking down SFPQ inhibited HCC cell proliferation, migration, and invasion. In conclusion, we identified three core genes (SFPQ, SSRP1, and KPNB1) that are essential for the proliferation and survival of HCC cells. These genes were used to develop a prognostic risk model, and knockdown of SFPQ was found to inhibit the proliferation, migration, and invasion of HCC cells.

Histone chaperone SSRP1 is required for apoptosis inhibition and mitochondrial function in HCC via transcriptional promotion of TRAP1.

Epigenetic regulation contributes to human health and disease, especially cancer, but the mechanisms of many epigenetic regulators remain obscure. Most research is focused on gene regulatory processes, such as mRNA translation and DNA damage repair, rather than the effects on biological functions like mitochondrial activity and oxidative phosphorylation. Here, we identified an essential role for the histone chaperone structure-specific recognition protein 1 (SSRP1) in mitochondrial oxidative respiration in hepatocellular carcinoma, and found that SSRP1 suppression led to mitochondrial damage and decreased oxidative respiration. Further, we focused on TNF receptor-associated protein 1 (TRAP1), the only member of the heat shock protein 90 (HSP90) family, which directly interacts with selected respiratory complexes and affects their stability and activity. We confirmed that SSRP1 downregulation caused a decrease in TRAP1 expression at both the mRNA and protein levels. A chromatin immunoprecipitation assay also showed that SSRP1 could deposit in the TRAP1 promoter region, indicating that SSRP1 maintains mitochondrial function and reactive oxygen species levels through TRAP1. Additionally, rescue experiments and animal experiments confirmed the mechanism of SSRP1 and TRAP1 interaction. In summary, we identified a new mechanism that connects mitochondrial respiration and apoptosis, via SSRP1.

Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced stemness and cisplatin resistance in nasopharyngeal carcinoma side population cells through the miR-579-3p/SIRT1/SSRP1 axis.

OBJECTIVE: To explore the effects of exosomes loaded with circular RNA PARD3 on EBV-miR-BART4-induced stemness and resistance of cisplatin in nasopharyngeal carcinoma side population (NPC-SP) cells through the miR-579-3p/SIRT1/SSRP1 axis. METHODS: Sixty-five cancer tissues and 65 noncancerous tissues were collected from NPC patients or patients with rhinitis. The expressions of circPARD3, miR-579-3p, SIRT1, and SSRP1 were detected by qRT-PCR, western blot, or immunohistochemistry. In vivo tumor formation assay was performed in nude mice. Immunofluorescence and qRT-PCR were conducted for the determination of CD44 and CD133 expressions, and flow cytometry combined with Hoechst 33,342 dye efflux for identifying SP cells, CCK-8 and EdU assays for cell proliferation, and Transwell assay for migration and invasion. RESULTS: CircPARD3, SIRT1, and SSRP1 were upregulated while miR-579-3p was downregulated in NPC tissues and cells. CircPARD3 was positively correlated with the expressions of SIRT1 and SSRP1, and miR-579-3p was negatively correlated with circPARD3, SIRT1, and SSRP1. Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells, while miR-579-3p reversed the effect of exosomal circPARD3 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. Additionally, miR-579-3p suppressed EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells by regulating SIRT1. SIRT1 upregulated SSRP1 expression by catalyzing H3K4 methylation and down-regulation of SSRP1 reversed the effect of SIRT1 on EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells. CONCLUSION: Exosomes loaded with circPARD3 promoted EBV-miR-BART4-induced stemness and cisplatin resistance in NPC-SP cells through the miR-579-3p/SIRT1/SSRP1 axis. Graphical Headlights • EBV-miR-BART4 induces the stemness and resistance of NPC-SP cells. • CircPARD3 regulates SIRT1 by miR-579-3p. • SIRT1 regulates SSRP1 expression by histone methylation. • Exosomes loaded with circPARD3 promotes EBV-miR-BART4-induced NPC-SP cell stemness and resistance by the miR-579-3p/SIRT1/SSRP1 axis.

Mechanism of curaxin-dependent nucleosome unfolding by FACT.

Human FACT (FACT) is a multifunctional histone chaperone involved in transcription, replication and DNA repair. Curaxins are anticancer compounds that induce FACT-dependent nucleosome unfolding and trapping of FACT in the chromatin of cancer cells (c-trapping) through an unknown molecular mechanism. Here, we analyzed the effects of curaxin CBL0137 on nucleosome unfolding by FACT using spFRET and electron microscopy. By itself, FACT adopted multiple conformations, including a novel, compact, four-domain state in which the previously unresolved NTD of the SPT16 subunit of FACT was localized, apparently stabilizing a compact configuration. Multiple, primarily open conformations of FACT-nucleosome complexes were observed during curaxin-supported nucleosome unfolding. The obtained models of intermediates suggest "decision points" in the unfolding/folding pathway where FACT can either promote disassembly or assembly of nucleosomes, with the outcome possibly being influenced by additional factors. The data suggest novel mechanisms of nucleosome unfolding by FACT and c-trapping by curaxins.

Mir204 and Mir211 suppress synovial inflammation and proliferation in rheumatoid arthritis by targeting Ssrp1.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial hyperplasia. Mir204 and Mir211 are homologous miRNAs with the same gene targeting spectrum. It is known that Mir204/211 play an important role in protecting osteoarthritis development; however, the roles of Mir204/211 in RA disease have not been determined. In the present study, we investigated the effects and molecular mechanisms of Mir204/211 on synovial inflammation and hyperproliferation in RA. The effects of Mir204/211 on the inflammation and abnormal proliferation in primary fibroblast-like synoviocytes (FLSs) were examined by Mir204/211 gain-of-function and loss-of-function approaches in vitro and in vivo. We identified the structure-specific recognition protein 1 (Ssrp1) as a downstream target gene of Mir204/211 based on the bioinformatics analysis. We overexpressed Ssrp1and Mir204/211 in FLS to determine the relationship between Ssrp1 and Mir204/211 and their effects on synovial hyperplasia. We created a collagen-induced arthritis (CIA) model in wild-type as well as Mir204/211 double knockout (dKO) mice to induce RA phenotype and administered adeno-associated virus (AAV)-mediated Ssrp1-shRNA (AAV-shSsrp1) by intra-articular injection into Mir204/211 dKO mice. We found that Mir204/211 attenuated excessive cell proliferation and synovial inflammation in RA. Ssrp1 was the downstream target gene of Mir204/211. Mir204/211 affected synovial proliferation and decelerated RA progression by targeting Ssrp1. CIA mice with Mir204/211 deficiency displayed enhanced synovial hyperplasia and inflammation. RA phenotypes observed in Mir204/211 deficient mice were significantly ameliorated by intra-articular delivery of AAV-shSsrp1, confirming the involvement of Mir204/211-Ssrp1signaling during RA development. In this study, we demonstrated that Mir204/211 antagonize synovial hyperplasia and inflammation in RA by regulation of Ssrp1. Mir204/211 may serve as novel agents to treat RA disease.

CBL0137 impairs homologous recombination repair and sensitizes high-grade serous ovarian carcinoma to PARP inhibitors.

BACKGROUND: High-grade serous ovarian carcinomas (HGSCs) are a heterogeneous subtype of epithelial ovarian cancers and include serous cancers arising in the fallopian tube and peritoneum. These cancers are now subdivided into homologous recombination repair (HR)-deficient and proficient subgroups as this classification impacts on management and prognosis. PARP inhibitors (PARPi) have shown significant clinical efficacy, particularly as maintenance therapy following response to platinum-based chemotherapy in BRCA-mutant or homologous recombination (HR)-deficient HGSCs in both the 1st and 2nd line settings. However, PARPi have limited clinical benefit in HR-proficient HGSCs which make up almost 50% of HGSC and improving outcomes in these patients is now a high priority due to the poor prognosis with ineffectiveness of the current standard of care. There are a number of potential lines of investigation including efforts in sensitizing HR-proficient tumors to PARPi. Herein, we aimed to develop a novel combination therapy by targeting SSRP1 using a small molecule inhibitor CBL0137 with PARPi in HR-proficient HGSCs. EXPERIMENTAL DESIGN: We tested anti-cancer activity of CBL0137 monotherapy using a panel of HGSC cell lines and patient-derived tumor cells in vitro. RNA sequencing was used to map global transcriptomic changes in CBL0137-treated patient-derived HR-proficient HGSC cells. We tested efficacy of CBL0137 in combination with PARPi using HGSC cell lines and patient-derived tumor cells in vitro and in vivo. RESULTS: We show that SSRP1 inhibition using a small molecule, CBL0137, that traps SSRP1 onto chromatin, exerts a significant anti-growth activity in vitro against HGSC cell lines and patient-derived tumor cells, and also reduces tumor burden in vivo. CBL0137 induced DNA repair deficiency via inhibition of the HR repair pathway and sensitized SSRP1-high HR-proficient HGSC cell lines and patient-derived tumor cells/xenografts to the PARPi, Olaparib in vitro and in vivo. CBL0137 also enhanced the efficacy of DNA damaging platinum-based chemotherapy in HGSC patient-derived xenografts. CONCLUSION: Our findings strongly suggest that combination of CBL0137 and PARP inhibition represents a novel therapeutic strategy for HR-proficient HGSCs that express high levels of SSRP1 and should be investigated in the clinic.

SSRP1 affects the growth and apoptosis of gastric cancer cells through AKT pathway.

Background: We aimed to determine the SSRP1's potential influence on the apoptosis and proliferation of gastric cancer (GC) cells and its regulatory mechanism. Methods: SSRP1 expression in GC cells and tissues was detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The interrelation between clinicopathological characteristics of GC patients and SSRP1 expression was analysed via x2 test, and the correlation between SSRP1 expression and overall survival rate was analysed using Kaplan-Meier survival analysis. After the knockdown of SSRP1 in AGS cells, the SSRP1 expression, colony formation ability, cell viability, cell cycle changes, apoptosis rate, and migration and invasion ability were detected through qRT-PCR, colony formation assay, CCK8 assay, flow cytometry and transwell test, respectively. Finally, the effects of down-regulation of SSRP1 on the expressions of phosphorylated-protein kinase B (p-AKT), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) were explored using Western blotting. Results: SSRP1 displayed a high expression in GC cells and tissues. SSRP1 expression was closely interrelated to the TNM stage, lymph node metastasis and tumour size. The survival rate of patients was markedly shorter in the high expression group than in the lower expression group. After the knockdown of SSRP1 in cells, the viability and colony formation ability of AGS cells were inhibited. In addition, the cell ratio in the G1 phase was increased, while that in the S phase declined, and the cell invasion and migration were obviously weakened. It was found from Western blotting that the knockdown of SSRP1 could evidently suppress the protein levels of Bcl-2 and p-AKT but promote the protein expression of Bax, indicating that silencing SSRP1 can inhibit the proliferative capacity and increase the number of GC cells through inactivating the AKT signalling pathway. Conclusions: SSRP1 rose up in GC tissues and cells. Reduction of SSRP1 can inhibit the proliferative capacity and increase the number of GC cells through inactivating the AKT signalling pathway.

FACT maintains nucleosomes during transcription and stem cell viability in adult mice.

Preservation of nucleosomes during replication has been extensively studied, while the maintenance of nucleosomes during transcription has gotten less attention. The histone chaperone FACT has a role in transcription elongation, although whether it disassembles or assembles nucleosomes during this process is unclear. To elucidate the function of FACT in mammals, we deleted the Ssrp1 subunit of FACT in adult mice. FACT loss is lethal, possibly due to the loss of the earliest progenitors in bone marrow and intestine, while more differentiated cells are not affected. Using cells isolated from several tissues, we show that FACT loss reduces the viability of stem cells but not of cells differentiated in vitro. FACT depletion increases chromatin accessibility in a transcription-dependent manner in adipose mesenchymal stem cells, indicating that nucleosomes are lost in these cells during transcription in the absence of FACT. We also observe activation of interferon (IFN) signaling and the accumulation of immunocytes in organs sensitive to FACT loss. Our data indicate that FACT maintains chromatin integrity during transcription in mammalian adult stem cells, suggesting that chromatin transcription in stem cells and differentiated cells is different.

SSRP1 Worsens Malignant Melanoma Progression by Activating MAPKs Pathway.

OBJECTIVE: Structure-specific recognition protein 1 (SSRP1) is highly expressed in a variety of tumors and promotes cell proliferation and migration. Malignant melanoma is a highly malignant skin malignancy with low morbidity and high mortality. The role of SSRP1 in malignant melanoma is still unclear. Thus, this study is intended to investigate the role of SSRP1 in malignant melanoma and reveal the related mechanisms. METHODS: Western blots and immunohistochemistry assays were used to determine the expression of SSRP1 in benign nevi tissues and malignant melanoma tissues. The si-SSRP1 was used to knockdown the expression level of SSRP1 in A375 cells. Cell proliferation was assessed by MTT assay. Wound healing and Transwell assay were performed for detected cell migratory and invasive activities, respectively. Besides, the expression levels of epithelial-mesenchymal transition (EMT) markers and MAPKs signaling pathway were measured by western blot. RESULTS: The results showed that SSRP1 was highly expressed in malignant melanoma tissues and cells, and its expression in metastatic melanoma tissues was significantly higher than that in primary melanoma. Besides, high expression level of SSRP1 was accompanied with poor prognosis in malignant melanoma patients. SSRP1 knockdown inhibited the melanoma cell proliferation, migration, and invasion. Besides, SSRP1 knockdown inhibited the process of EMT by upregulating E-cadherin, and downregulating N-cadherin and vimentin. Further studies revealed that SSRP1 silencing affected MAPK signaling pathway and reduced its phosphorylation activity in melanoma cells. CONCLUSIONS: These results suggested that SSRP1 may promote the proliferation, migration, and invasion of melanoma cells through MAPK signaling pathway. SSRP1 is closely related to the malignancy of melanoma and may be a potential target for its clinical treatment.

APE1 and SSRP1 is overexpressed in muscle invasive bladder cancer and associated with poor survival.

BACKGROUND: Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) plays a critical role in DNA base excision repair (BER) pathway and has been reported to be overexpressed in multiple cancers. Previously, we have shown that histone chaperone FACT complex (Facilitates Chromatin Transcription, a heterodimer of SSRP1 and SPT16 proteins) facilitates the chromatin access and DNA repair function of APE1, and their expression levels are correlated with promoting drug resistance in cancer. FACT inhibitor has been introduced in phase I and II clinical trials for chemosensitization of advanced solid cancers. However, the expression profile and prognostic significance of APE1 and FACT complex in bladder cancer remains largely unknown. METHODS: Retrospectively, 69 bladder cancer samples were retrieved and submitted for immunohistochemical staining of APE1 and SSRP1. Expression profile including cytoplasmic and nuclear staining of APE1 and expression level of SSRP1 was examined and semi-quantified to render a H-score. The prognostic significance of APE1 and SSRP1 was evaluated by Kaplan-Meier survival analysis in our cohort and R2 database. RESULTS: APE1 expression is elevated in bladder cancer compared to normal adjacent tissues. Compared with low grade tumors, high grade tumors show a shift in the staining pattern including higher intensity and positive cytoplasmic staining. Carcinoma in situ has a similar staining pattern to high grade tumors. APE1 and SSRP1 staining intensity increases as tumor progresses with stage. There is a correlation between APE1 and SSRP1 staining in invasive bladder cancer (Spearman r = 0.5466, p < 0.0001). The increased expression of APE1 and SSRP1 is associated with poor survival in Kaplan-Meier analysis in our cohort and in R2-TCGA bladder cancer database. CONCLUSIONS: The expression levels of APE1 and SSRP1 are significantly elevated in bladder cancer as compared to normal adjacent tissues. APE1 correlates with SSRP1 expression in high grade tumors. Overexpression of APE1 and SSRP1 is associated with poor survival in bladder cancer. This suggests the usage of FACT inhibitor curaxins in muscle invasive bladder cancer to target FACT complex and APE1 to improve chemosensitization after further validation.

DLG1-AS1 is activated by MYC and drives the proliferation and migration of hepatocellular carcinoma cells through miR-497-5p/SSRP1 axis.

BACKGROUND: Long non-coding RNAs (lncRNAs) have been reported to be biological regulators in hepatocellular carcinoma (HCC). DLG1 antisense RNA 1 (DLG1-AS1) has been found to be up-regulated in cervical cancer. However, its function and underlying mechanism in HCC remains unknown. METHODS: DLG1-AS1 expression was assessed in HCC cells and normal cell by RT-qPCR. Luciferase reporter assay, RNA pull down assay and RIP assay were used to demonstrate the interaction between DLG1-AS1 and miR-497-5p. RESULTS: DLG1-AS1 was highly expressed in HCC cells. Silencing of DLG1-AS1 led to the inhibition of HCC cell growth and migration. Besides, MYC induced the transcriptional activation of DLG1-AS1. MYC could facilitate HCC cellular processes by up-regulating DLG1-AS1. MiR-497-5p could interact with DLG1-AS1 in HCC cells. Down-regulation of miR-497-5p could reverse the impacts of DLG1-AS1 silencing on HCC cells. SSRP1 expression could be positively regulated by DLG1-AS1 but was negatively regulated by miR-497-5p. Knockdown of DLG1-AS1 suppressed tumor growth in nude mice. CONCLUSIONS: DLG1-AS1 is activated by MYC and functions as an oncogene in HCC via miR-497-5p/SSRP1 axis.

Long intergenic noncoding RNA01134 accelerates hepatocellular carcinoma progression by sponging microRNA-4784 and downregulating structure specific recognition protein 1.

Dysregulation of long noncoding RNAs (lncRNAs) has been suggested to foster the carcinogenesis of hepatocellular carcinoma (HCC). To date, the role of long intergenic noncoding RNA01134 (LINC01134) in HCC have never been researched yet. Herein, we found that LINC01134 was highly expressed in HCC tissues in comparison with the matched normal liver tissues and increased LINC01134 expression correlated with shorter overall survival of patients with HCC. Additionally, we demonstrated LINC01134 downregulation significantly suppressed the proliferation ability and colony formation capacity of HCC cells. Furthermore, we revealed that LINC01134 functioned as a competitive endogenous RNA (ceRNA) for miR-4784 to upregulate structure-specific recognition protein 1 (SSRP1) in HCC cells. Meanwhile, miR-4784 inhibitor or restoration of SSRP1 could markedly attenuate the inhibitory effect of LINC01134 downregulation on HCC cells. Taken together, LINC01134 may promote the carcinogenesis of HCC at least partly via the miR-4784/SSRP1 axis. Therefore, LINC01134/miR-4784/SSRP1 axis should be developed as the promising therapeutic target for HCC.

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

The FACT Histone Chaperone: Tuning Gene Transcription in the Chromatin Context to Modulate Plant Growth and Development.

FACT is a heterodimeric histone chaperone consisting of the SSRP1 and SPT16 proteins and is conserved among eukaryotes. It interacts with the histones H2A-H2B and H3-H4 as well as with DNA. Based on in vitro and in vivo studies mainly in yeast and mammalian cells, FACT can mediate nucleosome disassembly and reassembly and thus facilitates in the chromatin context DNA-dependent processes including transcription, replication and repair. In plants, primarily the role of FACT related to RNA polymerase II transcription has been examined. FACT was found to associate with elongating Arabidopsis RNA polymerase II (RNAPII) as part of the transcript elongation complex and it was identified as repressor of aberrant intragenic transcriptional initiation. Arabidopsis mutants depleted in FACT subunits exhibit various defects in vegetative and reproductive development. Strikingly, FACT modulates important developmental transitions by promoting expression of key repressors of these processes. Thus, FACT facilitates expression of DOG1 and FLC adjusting the switch from seed dormancy to germination and from vegetative to reproductive development, respectively. In the central cell of the female gametophyte, FACT can facilitate DNA demethylation especially within heterochromatin, and thereby contributes to gene imprinting during Arabidopsis reproduction. This review discusses results particularly from the plant perspective about the contribution of FACT to processes that involve reorganisation of nucleosomes with a main focus on RNAPII transcription and its implications for diverse areas of plant biology.

Histone chaperone FACT complex mediates oxidative stress response to promote liver cancer progression.

OBJECTIVE: Facilitates Chromatin Transcription (FACT) complex is a histone chaperone participating in DNA repair-related and transcription-related chromatin dynamics. In this study, we investigated its oncogenic functions, underlying mechanisms and therapeutic implications in human hepatocellular carcinoma (HCC). DESIGN: We obtained HCC and its corresponding non-tumorous liver samples from 16 patients and identified FACT complex as the most upregulated histone chaperone by RNA-Seq. We further used CRISPR-based gene activation and knockout systems to demonstrate the functions of FACT complex in HCC growth and metastasis. Functional roles and mechanistic insights of FACT complex in oxidative stress response were investigated by ChIP assay, flow cytometry, gene expression assays and 4sU-DRB transcription elongation assay. Therapeutic effect of FACT complex inhibitor, Curaxin, was tested in both in vitro and in vivo models. RESULTS: We showed that FACT complex was remarkably upregulated in HCC and contributed to HCC progression. Importantly, we unprecedentedly revealed an indispensable role of FACT complex in NRF2-driven oxidative stress response. Oxidative stress prevented NRF2 and FACT complex from KEAP1-mediated protein ubiquitination and degradation. Stabilised NRF2 and FACT complex form a positive feedback loop; NRF2 transcriptionally activates the FACT complex, while FACT complex promotes the transcription elongation of NRF2 and its downstream antioxidant genes through facilitating rapid nucleosome disassembly for the passage of RNA polymerase. Therapeutically, Curaxin effectively suppressed HCC growth and sensitised HCC cell to sorafenib. CONCLUSION: In conclusion, our findings demonstrated that FACT complex is essential for the expeditious HCC oxidative stress response and is a potential therapeutic target for HCC treatment.

SSRP1 influences colorectal cancer cell growth and apoptosis via the AKT pathway.

Colorectal cancer is one of the most common cancers worldwide with a high incidence rate. Therefore, the molecular basis of colorectal tumorigenesis and evolution must be clarified. Structure-specific recognition protein 1 (SSRP1) is involved in transcriptional regulation, DNA damage repair, and cell cycle regulation and has been confirmed to be highly expressed in various tumor tissues, including colorectal cancer. However, the role of SSRP1 in the development of colorectal cancer remains unclear. Therefore, this study explored the role of SSRP1 in the occurrence and development of colorectal cancer. Using bioinformatics databases, including samples from the Cancer Genome Atlas (TCGA), we confirmed high SSRP1 expression in human colorectal adenocarcinoma tissues. We demonstrated that SSRP1 knockdown via small interfering RNA significantly inhibited the proliferation of colorectal cancer cells and promoted apoptosis through the AKT signaling pathway, suppressing the invasion and migration of colorectal cancer cells in vitro and in vivo. In conclusion, this study demonstrated that SSRP1 silencing influenced the proliferation and apoptosis of colorectal cancer cells via the AKT signaling pathway.

A Role for FACT in RNA Polymerase II Promoter-Proximal Pausing.

FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.