SAFB restricts contact domain boundaries associated with L1 chimeric transcription.

Long interspersed element-1 (LINE-1 or L1) comprises 17% of the human genome, continuously generates genetic variations, and causes disease in certain cases. However, the regulation and function of L1 remain poorly understood. Here, we uncover that L1 can enrich RNA polymerase IIs (RNA Pol IIs), express L1 chimeric transcripts, and create contact domain boundaries in human cells. This impact of L1 is restricted by a nuclear matrix protein scaffold attachment factor B (SAFB) that recognizes transcriptionally active L1s by binding L1 transcripts to inhibit RNA Pol II enrichment. Acute inhibition of RNA Pol II transcription abolishes the domain boundaries associated with L1 chimeric transcripts, indicating a transcription-dependent mechanism. Deleting L1 impairs domain boundary formation, and L1 insertions during evolution have introduced species-specific domain boundaries. Our data show that L1 can create RNA Pol II-enriched regions that alter genome organization and that SAFB regulates L1 and RNA Pol II activity to preserve gene regulation.

RAE1 promotes gastric carcinogenesis and epithelial-mesenchymal transition.

AIMS: The purpose of this study was to explore the role of RAE1 in the invasion and metastasis of gastric cancer (GC) cells. MATERIALS AND METHODS: RAE1 expression in GC cells was determined by reverse-transcription polymerase chain reaction (qRT-PCR) and Western blotting (WB). Cell models featuring RAE1 gene silencing and overexpression were constructed by lentiviral transfection; The proliferation, migration, and invasion ability of cells were detected by cell counting, colony formation assay, would healing assay, and transwell invasion and migration test. WB analysis of ERK/MAPK signaling pathway (ERK1/2, p-ERK1/2, c-Myc) and EMT-related molecules (ZEB1, E-cadherin, N-cadherin, and Vimentin). RESULTS: The expression level of RAE1 in GC was notably higher than in adjacent tissues. Elevated RAE1 expression correlated with an unfavorable prognosis for GC patients. Knockdown of RAE1, as compared to the control group, resulted in a significant inhibition of proliferation, migration, and invasion abilities in GC cell lines. Furthermore, RAE1 knockdown led to a substantial decrease in the expression of N-cadherin, vimentin, ZEB1, p-ERK1/2, and c-Myc proteins, coupled with a marked increase in E-cadherin expression. The biological effects of RAE1 in GC cells were effectively reversed by the inhibition of the ERK/MAPK signaling pathway using SCH772984. Additionally, RAE1 knockdown demonstrated a suppressive effect on GC tumor size in vivo. Immunohistochemistry (IHC) results revealed significantly lower expression of Ki-67 in RAE1 knockout mice compared to the control group. CONCLUSIONS: RAE1 promotes GC cell migration and invasion through the ERK/MAPK pathway and is a potential therapeutic target for GC therapy.

Long non-coding RNA MIDEAS-AS1 inhibits growth and metastasis of triple-negative breast cancer via transcriptionally activating NCALD.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with higher aggressiveness and poorer outcomes. Recently, long non-coding RNAs (lncRNAs) have become the crucial gene regulators in the progression of human cancers. However, the function and underlying mechanisms of lncRNAs in TNBC remains unclear. METHODS: Based on public databases and bioinformatics analyses, the low expression of lncRNA MIDEAS-AS1 in breast cancer tissues was detected and further validated in a cohort of TNBC tissues. The effects of MIDEAS-AS1 on proliferation, migration, invasion were determined by in vitro and in vivo experiments. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were carried out to reveal the interaction between MIDEAS-AS1 and MATR3. Luciferase reporter assay, Chromatin immunoprecipitation (ChIP) and qRT-PCR were used to evaluate the regulatory effect of MIDEAS-AS1/MATR3 complex on NCALD. RESULTS: LncRNA MIDEAS-AS1 was significantly downregulated in TNBC, which was correlated with poor overall survival (OS) and progression-free survival (PFS) in TNBC patients. MIDEAS-AS1 overexpression remarkably inhibited tumor growth and metastasis in vitro and in vivo. Mechanistically, MIDEAS-AS1 mainly located in the nucleus and interacted with the nuclear protein MATR3. Meanwhile, NCALD was selected as the downstream target, which was transcriptionally regulated by MIDEAS-AS1/MATR3 complex and further inactivated NF-κB signaling pathway. Furthermore, rescue experiment showed that the suppression of cell malignant phenotype caused by MIDEAS-AS1 overexpression could be reversed by inhibition of NCALD. CONCLUSIONS: Collectively, our results demonstrate that MIDEAS-AS1 serves as a tumor-suppressor in TNBC through modulating MATR3/NCALD axis, and MIDEAS-AS1 may function as a prognostic biomarker for TNBC.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

MATR3-antisense LINE1 RNA meshwork scaffolds higher-order chromatin organization.

Long interspersed nuclear elements (LINEs) play essential roles in shaping chromatin states, while the factors that cooperate with LINEs and their roles in higher-order chromatin organization remain poorly understood. Here, we show that MATR3, a nuclear matrix protein, interplays with antisense LINE1 (AS L1) RNAs to form a meshwork via phase separation, providing a dynamic platform for chromatin spatial organization. MATR3 and AS L1 RNAs affect the nuclear localization of each other. After MATR3 depletion, the chromatin, particularly H3K27me3-modified chromatin, redistributes in the cell nuclei. Topologically associating domains (TADs) that highly transcribe MATR3-associated AS L1 RNAs show decreased intra-TAD interactions in both AML12 and ES cells. MATR3 depletion increases the accessibility of H3K27me3 domains adjacent to MATR3-associated AS L1, without affecting H3K27me3 modifications. Furthermore, amyotrophic lateral sclerosis (ALS)-associated MATR3 mutants alter biophysical features of the MATR3-AS L1 RNA meshwork and cause an abnormal H3K27me3 staining. Collectively, we reveal a role of the meshwork formed by MATR3 and AS L1 RNAs in gathering chromatin in the nucleus.

LncRNA15691 promotes T-ALL infiltration by upregulating CCR9 via increased MATR3 stability.

Our previous studies demonstrated that CCR9 plays an important role in several aspects of T-cell acute lymphoblastic leukemia progression and that CCR9 is a potential therapeutic target. However, the underlying mechanism that regulates CCR9 expression remains incompletely understood. In this study, bioinformatics analysis and validation in clinical samples revealed the lncRNA15691 to be positively correlated with CCR9 mRNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9high T-cell acute lymphoblastic leukemia cell lines. LncRNA15691, a previously uncharacterized lncRNA, was found to be located in both the cytoplasm and the nucleus via fluorescence in situ hybridization assay. In addition, lncRNA15691 upregulated the expression of CCR9 and was involved in T-cell acute lymphoblastic leukemia cell invasion. In vivo experiments showed that lncRNA15691 promoted leukemia cell homing/infiltration into the bone marrow, blood, and spleen, whereas the CCR9 ligand, CCL25, augmented the extramedullary infiltration of CCR9low leukemia cells overexpressing lncRNA15691 into blood, spleen, and liver. Subsequently, RNA protein pull-down assays, coupled with liquid chromatography-tandem mass spectrometry, were used to uncover potential lncRNA15691-interacting proteins, which were then validated by RNA immunoprecipitation. These mechanistic studies revealed that lncRNA15691 upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA. Collectively, our study revealed a novel mechanism of regulating CCR9 expression and implicated lncRNA15691 as a potential novel biomarker for T-cell acute lymphoblastic leukemia infiltration.

SAFB2 Inhibits the Progression of Breast Cancer by Suppressing the Wnt/ß-Catenin Signaling Pathway via NFAT5.

Aberrant scaffold attachment factor-B2 (SAFB2) expression is associated with several malignant tumors. In this study, we investigated how SAFB2 worked in the process of breast cancer as well as the underlying mechanism. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis were used to investigate the expression of SAFB2 and nuclear factor of activated T cells 5 (NFAT5). Cellular proliferative ability was detected with cell counting kit 8 (CCK8), colony formation and 5-Ethynyl-2'-deoxyuridine (EdU) staining assays. Cell apoptosis was measured via flow cytometry and western blotting analysis. Wound healing, transwell assays, and western blotting analysis were executed to estimate cell migration and invasion. The relationship between SAFB2 and NFAT5 was verified by RNA immunoprecipitation (RIP) assay and NFAT5 mRNA stability was examined with actinomycin (Act) D assay. Western blotting analysis also tested the expression of Wnt/ß-catenin signaling-associated proteins. As a result, SAFB2 was downregulated in breast cancer cell lines, while NFAT5 was highly expressed in most breast cancer cell lines. Overexpression of SAFB2 suppressed the proliferation, migration, and invasion while exacerbated the apoptosis of breast cancer cells. SAFB2 interacted with NFAT5 mRNA and declined the stability of NFAT5 mRNA. Overexpression of NFAT5 counteracted anti-proliferative, anti-metastatic and pro-apoptotic effects of SAFB2 in breast cancer cells. Mechanistically, SAFB2 overexpression inhibited the Wnt/ß-catenin signaling pathway, while this effect was partially eliminated by NFAT5. Collectively, SAFB2 hindered breast cancer development and inactivated Wnt/ß-catenin signaling via regulation of NFAT5, suggesting that SAFB2 might be a promising therapeutic target for breast cancer.

Short-term hypoxia triggers ROS and SAFB mediated nuclear matrix and mRNA splicing remodeling.

The cellular response to hypoxia, in addition to HIF-dependent transcriptional reprogramming, also involves less characterized transcription-independent processes, such as alternative splicing of the VEGFA transcript leading to the production of the proangiogenic VEGF form. We now show that this event depends on reorganization of the splicing machinery, triggered after short-term hypoxia by ROS production and intranuclear redistribution of the nucleoskeletal proteins SAFB1/2. Exposure to low oxygen causes fast dissociation of SAFB1/2 from the nuclear matrix, which is reversible, inhibited by antioxidant treatment, and also observed under normoxia when the mitochondrial electron transport chain is blocked. This is accompanied by altered interactions between SAFB1/2 and the splicing machinery, translocation of kinase SRPK1 to the cytoplasm, and dephosphorylation of RS-splicing factors. Depletion of SAFB1/2 under normoxia phenocopies the hypoxic and ROS-mediated switch in VEGF mRNA splicing. These data suggest that ROS-dependent remodeling of the nuclear architecture can promote production of splicing variants that facilitate adaptation to hypoxia.

Matr3 reshapes m6A modification complex to alleviate macrophage inflammation during atherosclerosis.

Atherosclerosis, characterized as the chronic inflammation of the arterial wall, is one of the leading causes of coronary artery disease (CAD), and macrophages are found to play essential roles in the initiation and progression of inflammation in atherosclerosis. N6-methyladenosine (m6A) modification, as the most abundant epi-transcriptomic modification in mRNA, is found to mediate the atherogenic inflammatory cascades in vascular endothelium. The detailed molecular mechanism of m6A methylation regulating inflammatory response during atherosclerosis is still not fully known. In this study, we find oxidized low-density lipoprotein (oxLDL) stimulation increases methyltransferases Mettl3 and Mettl14 expressions in macrophages, whereas the total m6A modification level in macrophages decreases under oxLDL stimulation. Matrin-3 (Matr3), an RNA binding protein, is identified to play a suppressive role on oxLDL-mediated macrophage inflammatory responses through inhibiting activation of pro-inflammatory signaling, mitogen-activated protein kinase (Mapk) by m6A-mediated mRNA decay via regulating the formation of Mettl3-Mettl14 complex. Moreover, we find that Matr3 expression decreases in the oxLDL-stimulated macrophages, and the peripheral blood-derived monocytes from patients with CAD, and overexpression of Matr3 significantly alleviates atherosclerosis development in vivo. Our study for the first time clarifies the role of Matr3 on macrophage inflammatory responses during atherosclerotic development, and supplies deep understanding on the relationship of m6A modification and inflammatory responses in atherosclerosis.

SRRM1 promotes the proliferation, migration, and invasion of hepatocellular carcinoma cells by regulating the JAK/STAT signaling pathway.

BACKGROUND: To explore the regulatory effect of serine/arginine repetitive matrix 1 (SRRM1) on hepatocellular carcinoma (HCC) and its potential pathway. METHODS: SRRM1 level in the tissue was tested by western blot and immunohistochemistry. Cells proliferation, apoptosis, migration, and invasion were tested by EdU, flow cytometry, wound healing assay, and Transwell, respectively. The potential mechanism of SRRM1 was explored through GSEA enrichment analysis and GeneMANIA protein-protein interaction (PPI) network. Co-immunoprecipitation assay was used to detect PPI. Levels of Bcl-2, Bax, Cleaved caspase 3, E-cadherin, N-cadherin, Vimentin, p-JAK2, JAK2, p-STAT3, and STAT3 were tested by Western blot. SRRM1 was highly expressed in HCC tissues, and was related to the survival and prognosis of patients. RESULTS: SRRM1 is significantly upregulated in HCC tumour tissues and correlated with progression of HCC. Overexpression of SRRM1 accelerated the proliferation, migration and invasion of HCC cells, and inhibited cell apoptosis, but low expression of SRRM1 had the opposite effect. SRRM1 positively correlated with the expression of IL6ST (GP130) and activated the JAK/STAT signaling pathways. SRRM1 affected the level of key molecules p-JAK2, JAK2, p-STAT3, and STAT3 in the JAK/STAT pathway. CONCLUSION: SRRM1 played a significant role in the proliferation, migration, invasion and apoptosis of HCC, and promoted cancer by regulating the JAK/STAT signaling pathway.

Prognostic significance of MATR3 in stage I and II non-small cell lung cancer patients.

PURPOSE: Matrin 3 (MATR3) is a nuclear matrix protein involved in mRNA stabilization, nuclear retention of hyper-edited RNAs, and RNA splicing. The role of MATR3 in cancer is still unclear. The present study aimed to investigate expression levels and prognostic significance of MATR3 in stage I and II non-small cell lung cancer (NSCLC) patients. METHODS: We examined MATR3 protein immunohistochemically in tumoral and non-tumoral tissue sections from n = 67 NSCLC patients treated at hospital, and MATR3 mRNA from The Cancer Genome Atlas (TCGA) cohort with respect to valid prognostic and predictive features, as well as treatment outcome. RESULTS: Significantly higher immunohistochemical levels of MATR3 protein were found in tumor-adjacent tissue compared to cancer (p = 0.049). A decrease in MATR3 protein expression was found to be a significant independent adverse prognostic factor for patients overall survival (p = 0.007). By contrast, we observed higher MATR3 mRNA levels in tumoral tissue compared to control lung tissues (p < 0.001). Based on the TCGA dataset, we reported that high MATR3 mRNA level was significantly associated with worse OS of NSCLC patients (p < 0.001); however, it was not an independent prognostic marker (p = 0.156). The discrepancies in prognostic significance of MATR3 gene mRNA and protein levels imply a need for further investigation. CONCLUSION: In conclusion, the present study warrants further investigation into the biological and prognostic value of MATR3 as a potential prognostic marker in early-stage NSCLC patients.

RAE1 is a prognostic biomarker and is correlated with clinicopathological characteristics of patients with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a primary malignant tumor that accounts for approximately 90% of all cases of primary liver cancer worldwide. Microtubule alterations may contribute to the broad spectrum of resistance to chemotherapy, tumor development, and cell survival. This study aimed to assess the value of ribonucleic acid export 1 (RAE1), as a regulator of microtubules, in the diagnosis and prognosis of HCC, and to analyze its correlation with genetic mutations and pathways in HCC. RESULTS: The mRNA and protein levels of RAE1 were significantly elevated in HCC tissues compared with those in normal tissues. The high expression level of RAE1 was correlated with T stage, pathologic stage, tumor status, histologic grade, and alpha-fetoprotein level. HCC patients with a higher expression level of RAE1 had a poorer prognosis, and the expression level of RAE1 showed the ability to accurately distinguish tumor tissues from normal tissues (area under the curve (AUC) = 0.951). The AUC values of 1-, 3-, and 5-year survival rates were all above 0.6. The multivariate Cox regression analysis showed that RAE1 expression level was an independent prognostic factor for a shorter overall survival of HCC patients. The rate of RAE1 genetic alterations was 1.1% in HCC samples. Gene ontology and kyoto encyclopedia of genes and genomes pathway enrichment analyses indicated the co-expressed genes of RAE1 were mainly related to chromosome segregation, DNA replication, and cell cycle checkpoint. Protein-protein interaction analysis showed that RAE1 was closely correlated with NUP205, NUP155, NUP214, NUP54, and NXF1, all playing important roles in cell division and mitotic checkpoint. CONCLUSION: RAE1 can be a potential diagnostic and prognostic biomarker associated with microtubules and a therapeutic target for HCC.

Exclusive modifications of NuMA in malignant epithelial cells: A potential therapeutic mechanism.

NuMA (nuclear mitotic apparatus) protein is indispensable in the mitosis of human proliferating cells, both malignant and benign. The progression of mitosis requires stable spindles, which depend on the bipolar clustering of NuMA within the spindles. The phenanthridine PJ34 kills malignant epithelial cells during mitosis and targets NuMA. PJ34 treated healthy cells are not impaired. PJ34 exclusively blocks the post-translational modification of NuMA in a variety of malignant epithelial cells, but not in benign cells. This blockage of the post-translational modification of NuMA affects its protein-binding capacity, causing construction faults in the mitotic spindle poles of PJ34-treated cancer cells, leading to Mitotic Catastrophe cell death. PJ34 is a potent PARP1 inhibitor. Therefore its PARP independent exclusive cytotoxicity in human malignant cells, challenges the currently accepted notion that inhibition of PARP1 halts cancer by preventing DNA repair. Certain molecules that act as PARP1 inhibitors kill cancer cells by targeting other proteins and vital mechanisms.

Effects of intracellular calcium accumulation on proteins encoded by the major genes underlying amyotrophic lateral sclerosis.

The aetiology of Amyotrophic Lateral Sclerosis (ALS) is still poorly understood. The discovery of genetic forms of ALS pointed out the mechanisms underlying this pathology, but also showed how complex these mechanisms are. Excitotoxicity is strongly suspected to play a role in ALS pathogenesis. Excitotoxicity is defined as neuron damage due to excessive intake of calcium ions (Ca2+) by the cell. This study aims to find a relationship between the proteins coded by the most relevant genes associated with ALS and intracellular Ca2+ accumulation. In detail, the profile of eight proteins (TDP-43, C9orf72, p62/sequestosome-1, matrin-3, VCP, FUS, SOD1 and profilin-1), was analysed in three different cell types induced to raise their cytoplasmic amount of Ca2+. Intracellular Ca2+ accumulation causes a decrease in the levels of TDP-43, C9orf72, matrin3, VCP, FUS, SOD1 and profilin-1 and an increase in those of p62/sequestosome-1. These events are associated with the proteolytic action of two proteases, calpains and caspases, as well as with the activation of autophagy. Interestingly, Ca2+ appears to both favour and hinder autophagy. Understanding how and why calpain-mediated proteolysis and autophagy, which are physiological processes, become pathological may elucidate the mechanisms responsible for ALS and help discover new therapeutic targets.

Liprins in oncogenic signaling and cancer cell adhesion.

Liprins are a multifunctional family of scaffold proteins, identified by their involvement in several important neuronal functions related to signaling and organization of synaptic structures. More recently, the knowledge on the liprin family has expanded from neuronal functions to processes relevant to cancer progression, including cell adhesion, cell motility, cancer cell invasion, and signaling. These proteins consist of regions, which by prediction are intrinsically disordered, and may be involved in the assembly of supramolecular structures relevant for their functions. This review summarizes the current understanding of the functions of liprins in different cellular processes, with special emphasis on liprins in tumor progression. The available data indicate that liprins may be potential biomarkers for cancer progression and may have therapeutic importance.

Inner nuclear protein Matrin-3 coordinates cell differentiation by stabilizing chromatin architecture.

Precise control of gene expression during differentiation relies on the interplay of chromatin and nuclear structure. Despite an established contribution of nuclear membrane proteins to developmental gene regulation, little is known regarding the role of inner nuclear proteins. Here we demonstrate that loss of the nuclear scaffolding protein Matrin-3 (Matr3) in erythroid cells leads to morphological and gene expression changes characteristic of accelerated maturation, as well as broad alterations in chromatin organization similar to those accompanying differentiation. Matr3 protein interacts with CTCF and the cohesin complex, and its loss perturbs their occupancy at a subset of sites. Destabilization of CTCF and cohesin binding correlates with altered transcription and accelerated differentiation. This association is conserved in embryonic stem cells. Our findings indicate Matr3 negatively affects cell fate transitions and demonstrate that a critical inner nuclear protein impacts occupancy of architectural factors, culminating in broad effects on chromatin organization and cell differentiation.

Curcumin induced the cell death of immortalized human keratinocytes (HaCaT) through caspase-independent and caspase-dependent pathways.

Curcumin is a diketone compound found in turmeric. It is used as food additives and spices, and has anti-proliferation and anti-cancer properties. However, the effect of curcumin on human keratinocytes (KCs) is still unclear. In this study, curcumin dramatically inhibited the cell growth of immortalized human KCs (HaCaT) and arrested the cells at the G2/M phase, with an apoptosis rate of 33.95% after 24 µM curcumin treatment. HaCaT cells showed changes in typical apoptotic morphology and the configuration of nuclear matrix-intermediate filaments (NM-IFs) after treatment with curcumin. We identified 16 differentially expressed nuclear matrix (NM) proteins, including apoptosis inducing factor (AIF) and caspase 3, by 2-DE and MALDI-TOF/TOF mass spectrometry. The expression of AIF decreased in the mitochondria and increased in the nucleus. Immunofluorescence assays showed that AIF was released from the mitochondria to the nucleus. AIF silencing and caspase inhibitor (z-vad-fmk) both lead to HaCaT cells being insensitive to apoptosis induced by curcumin. Meanwhile, after curcumin treatment, mitochondrial membrane depolarization led to cytochrome c release from the mitochondria to the cytoplasm, and the ratio of Bax to Bcl-2 in HaCaT cells was also increased, which subsequently initiated the activation of caspase-3. These results suggest that curcumin-induced apoptosis of HaCaT cells occurs not only through the caspase-dependent pathway but also through the caspase-independent pathway. This discovery enhances the development and utilization of curcumin and provides possible evidence for the treatment of proliferative skin diseases, including skin cancer.

Subcellular dynamics of estrogen-related receptors involved in transrepression through interactions with scaffold attachment factor B1.

Estrogen-related receptor (ERR), a member of the nuclear receptor superfamily, consists of three subtypes (α, ß, γ) and has strong homology with estrogen receptor. No endogenous ligands have been identified for ERRs, but they play key roles in metabolic, hormonal, and developmental processes as transcription factors without ligand binding. Although subnuclear dynamics are essential for nuclear events including nuclear receptor-mediated transcriptional regulation, the dynamics of ERRs are poorly understood. Here, we report that ERRs show subcellular kinetic changes in response to diethylstilbestrol (DES), a synthetic estrogen that represses the transactivity of all three ERR subtypes, using live-cell imaging with fluorescent protein labeling. Upon DES treatment, all ERR subtypes formed discrete clusters in the nucleus, with ERRγ also displaying nuclear export. Fluorescence recovery after photobleaching analyses revealed significant reductions in the intranuclear mobility of DES-bound ERRα and ERRß, and a slight reduction in the intranuclear mobility of DES-bound ERRγ. After DES treatment, colocalization of all ERR subtypes with scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein, was observed in dot-like subnuclear clusters, suggesting interactions of the ERRs with the nuclear matrix. Consistently, co-immunoprecipitation analyses confirmed enhanced interactions between ERRs and SAFB1 in the presence of DES. SAFB1 was clarified to repress the transactivity of all ERR subtypes through the ERR-response element. These results demonstrate ligand-dependent cluster formation of ERRs in the nucleus that is closely associated with SAFB1-mediated transrepression. Taken together, the present findings provide a new understanding of the pathophysiology regulated by ERR/SAFB1 signaling pathways and their subcellular dynamics.

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress.

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.