Remodeling And Spacing Factor 1 Research Articles

RSF1 orchestrates p53 transcriptional activity by coordinating p300 acetyltransferase and FACT complex.

The transcriptional regulation of p53-dependent genes in response to DNA damage is critical for effective DNA repair and cell survival. We previously established that RSF1 (remodeling and spacing factor 1) is necessary for p53-dependent gene transcription in response to DNA strand breaks. Here, we further elucidate that the role of RSF1 in p53 regulation by demonstrating that its depletion results in a reduction in the acetylated-Lys(K)382 level of p53, which governs its transcriptional activity. RSF1 was co-precipitated with p300 acetyltransferase upon etoposide treatment. Chromatin immunoprecipitation assays on the upstream region of CDKN1A gene revealed reduced p300 and TBP accumulation, which were accompanied with low H3H27ac and H3K4me1 levels in RSF1 knockout cells. Moreover, RSF1 depletion led to a reduced accumulation of SSRP1 and SPT16, subunits of FACT complex at the promoter of CDKN1A gene. These findings suggest that RSF1 promotes p53-dependent p21 gene transcription by facilitating the accumulation of p300 acetyltransferase at the enhancer and FACT at the promoter region of CDKN1A gene, respectively.

Cryo-EM structure and functional analysis of the chromatin remodeler RSF.

The RSF complex belongs to the ISWI chromatin-remodeling family and is composed of two subunits: RSF1 (remodeling and spacing factor 1) and SNF2h (sucrose nonfermenting protein 2 homolog). The RSF complex participates in nucleosome spacing and assembly, and subsequently promotes nucleosome maturation. Although SNF2h has been extensively studied in the last few years, the structural and functional properties of the remodeler RSF1 still remain vague. Here, a cryo-EM structure of the RSF-nucleosome complex is reported. The 3D model shows a two-lobe architecture of RSF, and the structure of the RSF-nucleosome (flanked with linker DNA) complex shows that the RSF complex moves the DNA away from the histone octamer surface at the DNA-entry point. Additionally, a nucleosome-sliding assay and a restriction-enzyme accessibility assay show that the RSF1 subunit may cause changes in the chromatin-remodeling properties of SNF2h. As a `nucleosome ruler', the results of an RSF-dinucleosome binding affinity test led to the proposal that the critical distance that RSF `measures' between two nucleosomes is about 24 base pairs.

Recognition of H2AK119ub plays an important role in RSF1-regulated early Xenopus development.

Polycomb group (PcG) proteins are key regulators of gene expression and developmental programs via covalent modification of histones, but the factors that interpret histone modification marks to regulate embryogenesis are less studied. We previously identified Remodeling and Spacing Factor 1 (RSF1) as a reader of histone H2A lysine 119 ubiquitination (H2AK119ub), the histone mark deposited by Polycomb Repressive Complex 1 (PRC1). In the current study, we used Xenopus laevis as a model to investigate how RSF1 affects early embryonic development and whether recognition of H2AK119ub is important for the function of RSF1. We showed that knockdown of Xenopus RSF1, rsf1, not only induced gastrulation defects as reported previously, but specific targeted knockdown in prospective neural precursors induced neural and neural crest defects, with reductions of marker genes. In addition, similar to knockdown of PRC1 components in Xenopus, the anterior-posterior neural patterning was affected in rsf1 knockdown embryos. Binding of H2AK119ub appeared to be crucial for rsf1 function, as a construct with deletion of the UAB domain, which is required for RSF1 to recognize the H2AK119ub nucleosomes, failed to rescue rsf1 morphant embryos and was less effective in interfering with early Xenopus development when ectopically expressed. Furthermore, ectopic deposition of H2AK119ub on the Smad2 target gene gsc using a ring1a-smad2 fusion protein led to ectopic recruitment of RSF1. The fusion protein was inefficient in inducing mesodermal markers in the animal region or a secondary axis when expressed in the ventral tissues. Taken together, our results reveal that rsf1 modulates similar developmental processes in early Xenopus embryos as components of PRC1 do, and that RSF1 acts at least partially through binding to the H2AK119ub mark via the UAB domain during development.

LncRNA-PRLB Confers Paclitaxel Resistance of Ovarian Cancer Cells by Regulating RSF1/NF-κB Signaling Pathway.

Background: Long noncoding RNA (lncRNA)-PRLB (progression-associated lncRNA in breast cancer) has been identified to enhance the drug resistance of breast cancer cells. In this study, the authors explored PRLB effect in the paclitaxel (Tax) resistance of ovarian cancer cells and revealed the role of RSF1 (remodeling and spacing factor 1)/nuclear factor kappaB (NF-κB) signaling in this process. Materials and Methods: Tax resistance was established in CAOV3 and SKOV3 cell lines. The expressions of PRLB in Tax resistant tissues and cells of ovarian cancer were detected using the real time polymerase chain reaction assay. MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] and flow cytometry were used to detect cell survival and apoptosis. The RNA-binding protein immunoprecipitation (RIP) assay and/or the luciferase gene reporter assay were used to assess the cross talk among miR-150-5p and PRLB/RSF1. Results: PRLB expression was obviously enhanced in the Tax resistant ovarian cancer tissues and cells. Depletion of PRLB induced a significant decrease in the IC50 value of the CAOV3/Tax and SKOV3/Tax cells and increased cell apoptosis, as well as increased miR-150-5p expression through a direct binding. In addition, miR-150-5p upregulation decreased the luciferase activity of PRLB and RSF1, whereas this effect was abolished when the putative binding sites were mutated. And overexpression of RSF1 significantly rescued the effect of PRLB downregulation-caused decrease in the IC50 value and the increase in cell apoptosis and the decreased expressions of RSF1 and p-p65. Conclusion: This study reveals that knockdown of PRLB improves the sensitivity of ovarian cancer cells to Tax, at least in part, through inhibiting the activation of RSF1/NF-κB signaling through targeting miR-150-5p.

LncRNA NEAT1 Promotes the Progression of Gastric Cancer Through Modifying the miR-1224-5p/RSF1 Signaling Axis.

IntroductionThe therapy of patients with advanced phase gastric cancer is still a huge threat, with extremely imperfect therapies authorized. Even if the amassed indications have validated the significance of lncRNA in gastric cancer, few understandings are stated concerning nuclear paraspeckle assembly transcript 1 (NEAT1) practical functions and molecular mechanisms.MethodsIn this research, the expression of NEAT1 and miR-1224-5p in gastric cancer tissues was measured by qRT-PCR analysis, and the expression of remodeling and spacing factor 1 (RSF1) was measured by IHC assay. Then, the bioinformatics prediction software ENCORI was applied to envisage the assumed binding sites. The monitoring roles of NEAT1 or miR-1224-5p on the cell proliferation and migration capacity were verified by CCK-8, wound healing and transwell assay, correspondingly. The interactions among NEAT1, miR-1224-5p and RSF1 were investigated via luciferase analysis.ResultsOur findings revealed high expression levels of NEAT1, RSF1 and a decreased expression level of miR-1224-5p in gastric cancer. Upregulation of NEAT1 or knockdown of miR-1224-5p elevated gastric cancer cell proliferation, and migration. Bioinformatics and luciferase analyses simplified that NEAT1 directly cooperated with miR-1224-5p to weaken miR-1224-5p binding to the RSF1 3ʹ-UTR region. Likewise, the mechanical inquiries ratified that initiation of the miR-1224-5p/RSF1 regulatory loop by miR-1224-5p knockdown or overexpressed RSF1 validated the functions of NEAT1 in endorsing gastric cancer cell malignancy.DiscussionOur research initially validated that NEAT1 may regulate the expression of RSF1 competitive sponge to miR-1224-5p, contributed to the supervision of gastric cancer evolution, which exposed new brightness for diagnosis and therapy of gastric cancer.

Long Noncoding RNA SNHG6 Promotes Proliferation and Inhibits Apoptosis in Non-small Cell Lung Cancer Cells by Regulating miR-490-3p/RSF1 Axis.

Background: Nonsmall cell lung cancer (NSCLC) is a malignant cancer type and has developed into the leading cause of cancer-related death worldwide. Small nucleolar RNA host gene 6 (SNHG6) has been identified as an oncogene in multiple cancers. However, the functions of SNHG6 in tumorigenesis and progression of NSCLC are still poorly understood. Materials and Methods: The expression of SNHG6, miR-490-3p, and remodeling and spacing factor 1 (RSF1) in NSCLC tumors and cells was measured by quantitative real-time polymerase chain reaction. The correlation between miR-490-3p and SNHG6 or RSF1 was analyzed by Pearson's correlation coefficient. Luciferase reporter assay was employed for verifying the interaction between miR-490-3p and SNHG6 or RSF1. Cell viability was examined by 3-(4, 5)-dimethylthiazole-2-y1)-2, 5-biphenyl tetrazolium bromide (MTT) assay. Cell apoptosis was evaluated by flow cytometry and Western blot, respectively. Protein expression of RSF1, Bcl-2, Bax, and cleaved caspase-3 (cleaved casp-3) was detected by Western blot assay. Xenograft mice models were established by subcutaneously injecting H460 cells stably transfected with sh-SNHG6 and sh-NC. Results: SNHG6 and RSF1 expression were upregulated, whereas miR-490-3p was downregulated in NSCLC tumors and cell lines compared with normal tissues and cells. Pearson's correlation coefficient analysis indicated that miR-490-3p was correlated with SNHG6 and RSF1 inversely. Then, luciferase reporter assay confirmed the interaction between miR-490-3p and SNHG6 or RSF1. More importantly, the rescue experiments clarified that miR-490-3p inhibitor could relieve SNHG6 silencing-mediated inhibition on proliferation and promotion on apoptosis in NSCLC. In addition, the authors discovered that SNHG6 promoted cell progression by regulating miR-490-3p/RSF1 axis. However, SNHG6 knockdown hindered tumor growth in vivo by regulating RSF1 by targeting miR-490-3p. Conclusion: The authors demonstrated that SNHG6 promoted proliferation and inhibits apoptosis in NSCLC by regulating miR-490-3p/RSF1 axis, representing promising targeted therapeutic strategies against NSCLC.

Chromatin-remodeling factor, RSF1, controls p53-mediated transcription in apoptosis upon DNA strand breaks

Remodeling and spacing factor 1 (RSF1), which is one of chromatin-remodeling factors, has been linked to the DNA damage response (DDR) and DNA repair. However, the biological consequence of RSF1 deficiency in DDR in vivo and its molecular mechanisms remain unknown. Because defective DDR is related to neuropathological phenotypes, we developed neural-specific Rsf1 knockout mice. Rsf1 deficiency did not result in any neuropathological abnormalities, but prevented neural apoptosis triggered by excessive DNA strand breaks during neurogenesis. Likewise, cell death was significantly reduced in RSF1 deficient human cell lines after DNA damage, and the global transcriptome of these cells revealed that the expressions of p53 downstream genes were significantly reduced upon DNA strand breaks. Inactivation of these genes resulted from decreased binding of p53/p300 complex and subsequent reduction of H3 acetylation at their promoters. Our data show that RSF1 is necessary for p53-dependent gene expression in response to DNA strand breaks via controlling the accessibility of p53/p300 complex to its target genes and contributes to the maintenance of cellular integrity.

Effects of RSF-1 on proliferation and apoptosis of breast cancer cells.

Effect of interference with chromatin remodeling and spacing factor-1 (RSF-1) on proliferation and apoptosis of breast cancer cells was investigated. MCF-7 and SKBR-3 cells were cultured in vitro and were divided into 3 groups: control group, negative siRNA control group (NC) and RSF-1 siRNA group. Western blot analysis was used to detect the expression of RSF protein after interference. Cell Counting Kit-8 (CCK-8) method was used to detect the effect of RSF-1 siRNA on cell proliferation. Plate cloning assay was used to detect the effect of RSF-1 siRNA on cell clone formation ability. Annexin V/PI double staining method was used to detect the effect of RSF-1 siRNA on cell apoptosis. Effect of RSF-1 siRNA on nuclear factor-κB (NF-κB) and its downstream signaling pathway were detected by western blot analysis. Western blot analysis showed that RSF-1 siRNA significantly downregulated the expression of RSF-1 protein in MCF-7 and SKBR-3 cells at 72 h after transfection (P<0.01). Cell proliferation assay showed that RSF-1 siRNA significantly reduced the proliferation ability and clone formation ability of MCF-7 and SKBR-3 cells compared with the control group (P<0.01). Annexin V/PI double staining assay results showed that compared with the control group, RSF-1 siRNA significantly increased the apoptosis rate of MCF-7 and SKBR-3 cells (P<0.01). Helenalin and Rsf-1 siRNA significantly reduced the expression levels of p-p65, Bcl-2, and XIAP proteins (P<0.01). Interfering with the expression of RSF-1, gene can effectively inhibit the proliferation of MCF-7 and SKBR-3 cells and promote their apoptosis. RSF-1 can be used as a potential new therapeutic target for the treatment of breast cancer.

Post-Translational Regulation of the RSF1 Chromatin Remodeler under DNA Damage.

Chromatin remodeling factors are involved in many cellular processes such as transcription, replication, and DNA damage response by regulating chromatin structure. As one of chromatin remodeling factors, remodeling and spacing factor 1 (RSF1) is recruited at double strand break (DSB) sites and regulates ataxia telangiectasia mutated (ATM) -dependent checkpoint pathway upon DNA damage for the efficient repair. RSF1 is overexpressed in a variety of cancers, but regulation of RSF1 levels remains largely unknown. Here, we showed that protein levels of RSF1 chromatin remodeler are temporally upregulated in response to different DNA damage agents without changing the RSF1 mRNA level. In the absence of SNF2h, a binding partner of RSF1, the RSF1 protein level was significantly diminished. Intriguingly, the level of RSF1-3SA mutant lacking ATM-mediated phosphorylation sites significantly increased, and upregulation of RSF1 levels under DNA damage was not observed in cells overexpressing ATM kinase. Furthermore, failure in the regulation of RSF1 level caused a significant reduction in DNA repair, whereas reconstitution of RSF1, but not of RSF1-3SA mutants, restored DSB repair. Our findings reveal that temporal regulation of RSF1 levels at its post-translational modification by SNF2h and ATM is essential for efficient DNA repair.

MicroRNA-154 as a prognostic factor in bladder cancer inhibits cellular malignancy by targeting RSF1 and RUNX2.

Recent studies have demonstrated that microRNA-154 (miR-154) is involved in tumorigenesis, progression, invasion and metastasis in several types of human cancer. However, whether it plays a role in bladder cancer (BC) is unclear. The aim of the present study was to determine miR-154 levels in human BC tissues and investigate the correlation between miR-154 levels and clinicopathological characteristics as well as patient outcome. Using RT-qPCR, we found that the expression levels of miR-154 were significantly lower in BC tissues compared to adjacent normal tissues. We also demonstrated that downregulation of miR-154 was associated with advanced clinicopathological features and worse prognoses for patients with BC. Using a variety of integrated approaches, we demonstrated that both runt-related transcription factor 2 (RUNX2) and remodeling and spacing factor 1 (RSF1) were miR-154 targets. Notably, there was an inverse correlation between RSF1, RUNX2 and miR-154 expression in BC tissues. The biological functions of miR-154 were examined in vitro using Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays with T24 human bladder carcinoma cells transfected with miR-154 mimics or negative controls. These assays demonstrated that miR-154 significantly suppressed proliferation, migration and invasion of T24 cells (P<0.05). Furthermore, overexpression of RSF1 and RUNX2 rescued miR-154-induced inhibition of these aggressive behaviors. Our results indicated that miR-154, and its downstream targets RSF1 and RUNX2, are promising options for future BC therapies.

Role of remodeling and spacing factor 1 in histone H2A ubiquitination-mediated gene silencing

Posttranslational histone modifications play important roles in regulating chromatin-based nuclear processes. Histone H2AK119 ubiquitination (H2Aub) is a prevalent modification and has been primarily linked to gene silencing. However, the underlying mechanism remains largely obscure. Here we report the identification of RSF1 (remodeling and spacing factor 1), a subunit of the RSF complex, as a H2Aub binding protein, which mediates the gene-silencing function of this histone modification. RSF1 associates specifically with H2Aub, but not H2Bub nucleosomes, through a previously uncharacterized and obligatory region designated as ubiquitinated H2A binding domain. In human and mouse cells, genes regulated by RSF1 overlap significantly with those controlled by RNF2/Ring1B, the subunit of Polycomb repressive complex 1 (PRC1) which catalyzes the ubiquitination of H2AK119. About 82% of H2Aub-enriched genes, including the classic PRC1 target Hox genes, are bound by RSF1 around their transcription start sites. Depletion of H2Aub levels by Ring1B knockout results in a significant reduction of RSF1 binding. In contrast, RSF1 knockout does not affect RNF2/Ring1B or H2Aub levels but leads to derepression of H2Aub target genes, accompanied by changes in H2Aub chromatin organization and release of linker histone H1. The action of RSF1 in H2Aub-mediated gene silencing is further demonstrated by chromatin-based in vitro transcription. Finally, RSF1 and Ring1 act cooperatively to regulate mesodermal cell specification and gastrulation during Xenopus early embryonic development. Taken together, these data identify RSF1 as a H2Aub reader that contributes to H2Aub-mediated gene silencing by maintaining a stable nucleosome pattern at promoter regions.

Remodeling and spacing factor 1 (RSF1): a rising star in DNA repair.

Remodeling and spacing factor 1 (RSF1): a rising star in DNA repair.

RSF1 and Not Cyclin D1 Gene Amplification May Predict Lack of Benefit from Adjuvant Tamoxifen in High-Risk Pre-Menopausal Women in the MA.12 Randomized Clinical Trial

Most women with estrogen receptor expressing breast cancers receiving anti-estrogens such as tamoxifen may not need or benefit from them. Besides the estrogen receptor, there are no predictive biomarkers to help select breast cancer patients for tamoxifen treatment. CCND1 (cyclin D1) gene amplification is a putative candidate tamoxifen predictive biomarker. The RSF1 (remodeling and spacing factor 1) gene is frequently co-amplified with CCND1 on chromosome 11q. We validated the predictive value of these biomarkers in the MA.12 randomized study of adjuvant tamoxifen vs. placebo in high-risk premenopausal early breast cancer. Premenopausal women with node-positive/high-risk node-negative early breast cancer received standard adjuvant chemotherapy and then were randomized to tamoxifen (20 mg/day) or placebo for 5 yrs. Overall survival (OS) and relapse-free survival (RFS) were evaluated. Fluorescent in-situ hybridization was performed on a tissue microarray of 495 breast tumors (74% of patients) to measure CCND1 and RSF1 copy number. A multivariate Cox model to obtain hazard ratios (HR) adjusting for clinico-pathologic factors was used to assess the effect of these biomarkers on Os and RFS. 672 women were followed for a median of 8.4 years. We were able to measure the DNA copy number of CCND1 in 442 patients and RSF1 in 413 patients. CCND1 gene amplification was observed in 8.7% and RSF1 in 6.8% of these patients, preferentially in estrogen receptor-positive breast cancers. No statistically significant interaction with treatment was observed for either CCND1 or RSF1 amplification, although patients with high RSF1 copy number did not show benefit from adjuvant tamoxifen (HR = 1.11, interaction p = 0.09). Unlike CCND1 amplification, RSF1 amplification may predict for outcome in high-risk premenopausal breast cancer patients treated with adjuvant tamoxifen.

Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining

The cellular response to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in native chromatin requires a tight coordination between the activities of DNA repair machineries and factors that modulate chromatin structure. SMARCA5 is an ATPase of the SNF2 family of chromatin remodeling factors that has recently been implicated in the DSB response. It forms distinct chromatin remodeling complexes with several non-canonical subunits, including the remodeling and spacing factor 1 (RSF1) protein. Despite the fact that RSF1 is often overexpressed in tumors and linked to tumorigenesis and genome instability, its role in the DSB response remains largely unclear. Here we show that RSF1 accumulates at DSB sites and protects human cells against IR-induced DSBs by promoting repair of these lesions through homologous recombination (HR) and non-homologous end-joining (NHEJ). Although SMARCA5 regulates the RNF168-dependent ubiquitin response that targets BRCA1 to DSBs, we found RSF1 to be dispensable for this process. Conversely, we found that RSF1 facilitates the assembly of centromere proteins CENP-S and CENP-X at sites of DNA damage, while SMARCA5 was not required for these events. Mechanistically, we uncovered that CENP-S and CENP-X, upon their incorporation by RSF1, promote assembly of the NHEJ factor XRCC4 at damaged chromatin. In contrast, CENP-S and CENP-X were dispensable for HR, suggesting that RSF1 regulates HR independently of these centromere proteins. Our findings reveal distinct functions of RSF1 in the 2 major pathways of DSB repair and explain how RSF1, through the loading of centromere proteins and XRCC4 at DSBs, promotes repair by non-homologous end-joining.

RSF1 (remodeling and spacing factor 1)

Review on RSF1 (remodeling and spacing factor 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Identification of Proteins Interacting with Human SP110 During the Process of Viral Infections

Human SP110 plays an important role in resisting intracellular pathogens, and hence has become an important drug target for preventing intracellular pathogen diseases, such as tuberculosis, hepatic veno-occlusive disease, and intracellular cancers. Unfortunately, so far little is known about the interactions of SP110 with the other proteins in a cell, which is considered to be the key for revealing its action mechanism and mediated pathway. Using both the genetic and structural analyses as well as the segment-docking approach, we have identified two proteins: the human remodeling and spacing factor 1 (RSF1) and the activating transcription factor 7 interacting protein (ATF7IP). They are very likely interacting with human SP110 during the process of viral infections. Owing to the close relationship of RSF1 with the chromatin remodeling and ATF7IP with the chromatin formation, it is logical to infer that human SP110 may be involved in the chromatin remodeling and formation as well. These findings may provide useful insights into the development of new drugs for treating and preventing intracellular pathogen diseases.