RBBP7, regulated by SP1, enhances the Warburg effect to facilitate the proliferation of hepatocellular carcinoma cells via PI3K/AKT signaling.

BACKGROUND: Hepatocellular carcinoma (HCC) is characterized by aggressive progression and elevated mortality rates. This study aimed to investigate the regulatory effects of RBBP7 on HCC pathogenesis and the underlying mechanisms. METHODS: The expression and clinical feature of RBBP7 were evaluated using bioinformatics analysis and the assessment of clinical HCC samples. CCK8 and colony formation were employed to estimate cell proliferation function of RBBP7. Aerobic glycolysis levels of RBBP7 were evaluated by measuring ATP levels, lactic acid production, glucose uptake capacity, and the expression of relevant enzymes (PFKM, PKM2, and LDHA). The phosphorylation levels in PI3K/AKT signaling were measured by western blotting. The regulatory effect of transcription factors of specificity protein 1 (SP1) on RBBP7 mRNA expression was confirmed in dual-luciferase reporter assays and chromatin immunoprecipitation experiments. The proliferation- and glycolysis-associated proteins were assessed using immunofluorescence staining in vivo. RESULTS: We found that RBBP7 is expressed at high levels in HCC and predicts poor survival. Functional assays showed that RBBP7 promoted HCC proliferation and glycolysis. Mechanistically, it was demonstrated that RBBP7 activates the PI3K/AKT pathway, a crucial pathway in glycolysis, contributing to the progression of HCC. The outcomes of the dual-luciferase assay further confirmed that SP1 is capable of activating the promoter of RBBP7. CONCLUSIONS: RBBP7, which is up-regulated by SP1, promotes HCC cell proliferation and glycolysis through the PI3K/AKT pathway. The findings of this study suggest that RBBP7 is a potential biomarker for HCC.

X-linked RBBP7 mutation causes maturation arrest and testicular tumors.

Maturation arrest (MA) is a subtype of non-obstructive azoospermia, and male infertility is a known risk factor for testicular tumors. However, the genetic basis for many affected individuals remains unknown. Here, we identified a deleterious hemizygous variant of X-linked retinoblastoma-binding protein 7 (RBBP7) as a potential key cause of MA, which was also found to be associated with the development of Leydig cell tumors. This mutation resulted in premature protein translation termination, affecting the sixth WD40 domain of the RBBP7 and the interaction of the mutated RBBP7 with histone H4. Decreased BRCA1 and increased γH2AX were observed in the proband. In mouse spermatogonial and pachytene spermatocyte-derived cells, deprivation of rbbp7 led to cell cycle arrest and apoptosis. In Drosophila, knockdown of RBBP7/Caf1-55 in germ cells resulted in complete absence of germ cells and reduced testis size, whereas knockdown of RBBP7/Caf1-55 in cyst cells resulted in hyperproliferative testicular cells. Interestingly, male infertility caused by Caf1-55 deficiency was rescued by ectopic expression of wild-type human RBBP7 but not mutant variants, suggesting the importance of RBBP7 in spermatogenesis. Our study provides insights into the mechanisms underlying the co-occurrence of MA and testicular tumors and may pave the way for innovative genetic diagnostics of these 2 diseases.

FIT links c-Myc and P53 acetylation by recruiting RBBP7 during colorectal carcinogenesis.

Colorectal cancer (CRC) poses one of the most serious threats to human health worldwide, and abnormally expressed c-Myc and p53 are deemed the pivotal driving forces of CRC progression. In this study, we discovered that the lncRNA FIT, which was downregulated in CRC clinical samples, was transcriptionally suppressed by c-Myc in vitro and promoted CRC cell apoptosis by inducing FAS expression. FAS is a p53 target gene, and we found that FIT formed a trimer with RBBP7 and p53 that facilitated p53 acetylation and p53-mediated FAS gene transcription. Moreover, FIT was capable of retarding CRC growth in a mouse xenograft model, and FIT expression was positively correlated with FAS expression in clinical samples. Thus, our study elucidates the role of the lncRNA FIT in human colorectal cancer growth and provides a potential target for anti-CRC drugs.

Hypoxia-induced RBBP7 promotes esophagus cancer progression by inducing CDK4 expression.

Hypoxia-induced epigenetic regulation calls for more effective therapeutic targets for esophageal cancer. We used GEPIA and UALCAN databases to screen survival-related and cancer stage-associated genes. Eca109 and KYSE450 esophageal cancer cell lines were cultured under normoxia, hypoxia, or CoCl-induced hypoxia conditions, which were further transfected with plasmids expressing RB binding protein 7 (RBBP7), hypoxia-inducible factor 1 (HIF1)-α, or RBBP7 shRNA. Colony formation and MTT assays were used to detect cell proliferation. Tumor sphere formation and stemness marker detection were applied to assess cell stemness. RT-PCR and western blot analysis were used to detect the relative mRNA level and protein expression, respectively. Luciferase assay was utilized to detect the direct interaction between HIF1α and RBBP7. Up-regulated RBBP7 was identified as one of the most prominent survival-related genes, which is negatively correlated with the overall survival (OS), disease recurrence-free survival (DFS), and tumor stages. Hypoxia-induced HIF1α up-regulates RBBP7 expression, which promotes esophagus cancer cell viability, proliferation, and stemness with increased cyclin-dependent kinase 4 (CDK4) expression. Luciferase reporter assay verified that HIF1α transcriptionally regulates the expression of RBBP7. We conclude that hypoxia induces high expression of RBBP7 which is at least partially mediated by HIF1α, up-regulates the expression of downstream CDK4, and thereby promotes tumor progression in esophageal cancer cells.

Overlapping functions of RBBP4 and RBBP7 in regulating cell proliferation and histone H3.3 deposition during mouse preimplantation development.

Preimplantation development is critical for reproductive successes in mammals. Thus, it is important to understand how preimplantation embryogenesis is regulated. As a key event of preimplantation development, epigenetic reprogramming has been widely studied, yet how epigenetic complexes regulate preimplantation development remains largely unknown. Retinoblastoma binding protein 4 (RBBP4) and 7 (RBBP7) are integral components of epigenetic complexes including SIN3A, NuRD, and CoREST. Here, we demonstrate that double knockdown of Rbbp4 and 7, but not individually, causes embryonic lethality during the morula-to-blastocyst transition. Mechanistically, depletion of RBBP4 and 7 results in dysregulation of genes related to cell cycle, lineage development, and regulation of transcription, which is accompanied by cell cycle block, disrupted lineage specification and chromatin structure. Interestingly, RBBP4/7 depletion leads to a dramatic increase in H3.3 and H3K27ac abundance during morula-to-blastocyst transition. ChIP-seq analysis in early embryos and embryonic stem cells reveals enrichment of H3.3 at the promoter regions of RBBP4/7 target genes. In summary, our studies demonstrate the compensatory role of RBBP4/7 and reveal its potential mechanisms in preimplantation development.Summary sentence:RBBP4 and RBBP7 play a compensatory role in regulating cell proliferation, apoptosis, and histone H3.3 deposition during preimplantation development.

Identification of retinoblastoma binding protein 7 (Rbbp7) as a mediator against tau acetylation and subsequent neuronal loss in Alzheimer's disease and related tauopathies.

Evidence indicates that tau hyper-phosphorylation and subsequent neurofibrillary tangle formation contribute to the extensive neuronal death in Alzheimer's disease (AD) and related tauopathies. Recent work has identified that increased tau acetylation can promote tau phosphorylation. Tau acetylation occurs at lysine 280 resulting from increased expression of the lysine acetyltransferase p300. The exact upstream mechanisms mediating p300 expression remain elusive. Additional work highlights the role of the epigenome in tau pathogenesis, suggesting that dysregulation of epigenetic proteins may contribute to acetylation and hyper-phosphorylation of tau. Here, we identify and focus on the histone-binding subunit of the Nucleosome Remodeling and Deacetylase (NuRD) complex: Retinoblastoma-Binding Protein 7 (Rbbp7). Rbbp7 chaperones chromatin remodeling proteins to their nuclear histone substrates, including histone acetylases and deacetylases. Notably, Rbbp7 binds to p300, suggesting that it may play a role in modulating tau acetylation. We interrogated Rbbp7 in post-mortem brain tissue, cell lines and mouse models of AD. We found reduced Rbbp7 mRNA expression in AD cases, a significant negative correlation with CERAD (neuritic plaque density) and Braak Staging (pathogenic tau inclusions) and a significant positive correlation with post-mortem brain weight. We also found a neuron-specific downregulation of Rbbp7 mRNA in AD patients. Rbbp7 protein levels were significantly decreased in 3xTg-AD and PS19 mice compared to NonTg, but no decreases were found in APP/PS1 mice that lack tau pathology. In vitro, Rbbp7 overexpression rescued TauP301L-induced cytotoxicity in immortalized hippocampal cells and primary cortical neurons. In vivo, hippocampal Rbbp7 overexpression rescued neuronal death in the CA1 of PS19 mice. Mechanistically, we found that increased Rbbp7 reduced p300 levels, tau acetylation at lysine 280 and tau phosphorylation at AT8 and AT100 sites. Collectively, these data identify a novel role of Rbbp7, protecting against tau-related pathologies, and highlight its potential as a therapeutic target in AD and related tauopathies.

Circular RNA hsa_circ_0006168 contributes to cell proliferation, migration and invasion in esophageal cancer by regulating miR-384/RBBP7 axis via activation of S6K/S6 pathway.

OBJECTIVE: Esophageal cancer (EC) ranks as the sixth leading cause of cancer-related mortality worldwide. Circular RNAs (circRNAs) are involved in the pathogenesis of different cancers. However, the regulatory mechanism of circ_0006168 in EC progression is still unclear. MATERIALS AND METHODS: The expression of circ_0006168, microRNA (miR)-384, and retinoblastoma binding protein 7 (RBBP7) in tumors and cells was measured by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The stability of circ_0006168 was analyzed after RNase R treatment. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay was conducted to evaluate cell viability. Transwell assay was applied to determine cell migration and invasion. Glucose consumption and lactate production were detected using glucose detection and lactic acid detection kits. The interaction between miR-384 and circ_0006168 or RBBP7 was certified by Dual-Luciferase reporter system. Protein expression of pyruvate kinase (PK), RBBP7, S6 ribosomal protein kinase (S6K), phosphorylated S6K (p-S6K), S6, phosphorylated S6 (p-S6) was analyzed by Western blot. RESULTS: Circ_0006168 and RBBP7 were over-expressed while miR-384 was low-expressed in EC tumors and cells. The repression of circ_0006168 attenuated cell proliferation, migration, invasion, and glycolysis in EC. Of note, circ_0006168 functioned as a sponge while RBBP7 acted as a target of miR-384 in EC. Rescue experiment revealed that miR-384 inhibitor abrogated circ_0006168 silencing-induced repression on cell proliferation, migration, and invasion in EC. Meanwhile, upregulation of RBBP7 restored the inhibition of miR-384 on EC cell progression. Moreover, circ_0006168 was able to improve RBBP7 level by interacting with miR-384. Also, circ_0006168 could activate S6K/S6 pathway by regulating RBBP7 expression. CONCLUSIONS: Abundance of circ_0006168 contributes to cell proliferation, migration, invasion, and glycolysis in EC by competitively sponging miR-384 to facilitate RBBP7 expression, representing prospective targets for EC therapy.

Retinoblastoma binding protein 7 is involved in Kiss1 mRNA upregulation in rodents.

Kisspeptin, encoded by Kiss1, is essential for reproduction in mammals. Kiss1 expression is regulated by estrogen via histone acetylation in the Kiss1 promotor region. Thus, elucidation of histone modification factor(s) involved in the regulation of Kiss1 expression is required to gain further understanding of the mechanisms of its control. The RNA-seq analysis of isolated kisspeptin neurons, obtained from the arcuate nucleus (ARC) of female rats, revealed that Rbbp7, encoding retinoblastoma binding protein 7 (RBBP7), a member of histone modification and chromatin remodeling complexes, is highly expressed in the ARC kisspeptin neurons. Thus, the present study aimed to investigate whether RBBP7 is involved in Kiss1 expression. Histological analysis using in situ hybridization (ISH) revealed that Rbbp7 expression was located in several hypothalamic nuclei, including the ARC and the anteroventral periventricular nucleus (AVPV), where kisspeptin neurons are located. Double ISH for Rbbp7 and Kiss1 showed that a majority of kisspeptin neurons (more than 85%) expressed Rbbp7 mRNA in both the ARC and the AVPV of female rats. Further, Rbbp7 mRNA knockdown significantly decreased in vitro expression of Kiss1 in a mouse immortalized kisspeptin neuronal cell line (mHypoA-55). Estrogen treatment significantly decreased and increased Kiss1 mRNA levels in the ARC and AVPV of ovariectomized female rats, respectively, but failed to affect Rbbp7 mRNA levels in both the nuclei. Taken together, these findings suggest that RBBP7 is involved in the upregulation of Kiss1 expression in kisspeptin neurons of rodents in an estrogen-independent manner.

The RepID-CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation.

The spindle assembly checkpoint (SAC) prevents premature chromosome segregation by inactivating the anaphase promoting complex/cyclosome (APC/C) until all chromosomes are properly attached to mitotic spindles. Here we identify a role for Cullin-RING ubiquitin ligase complex 4 (CRL4), known for modulating DNA replication, as a crucial mitotic regulator that triggers the termination of the SAC and enables chromosome segregation. CRL4 is recruited to chromatin by the replication origin binding protein RepID/DCAF14/PHIP. During mitosis, CRL4 dissociates from RepID and replaces it with RB Binding Protein 7 (RBBP7), which ubiquitinates the SAC mediator BUB3 to enable mitotic exit. During interphase, BUB3 is protected from CRL4-mediated degradation by associating with promyelocytic leukemia (PML) nuclear bodies, ensuring its availability upon mitotic onset. Deficiencies in RepID, CRL4 or RBBP7 delay mitotic exit, increase genomic instability and enhance sensitivity to paclitaxel, a microtubule stabilizer and anti-tumor drug.

Molecular mechanism and role of microRNA-93 in human cancers: A study based on bioinformatics analysis, meta-analysis, and quantitative polymerase chain reaction validation.

INTRODUCTION: Currently, studies have shown that microRNA-93 (miR-93) can be an oncogene or a tumor suppressor in different kinds of cancers. The role of miR-93 in human cancers is inconsistent and the underlying mechanism on the aberrant expression of miR-93 is complicated. METHODS: We first conducted gene enrichment analysis to give insight into the prospective mechanism of miR-93. Second, we performed a meta-analysis to evaluate the clinical value of miR-93. Finally, a validation test based on quantitative polymerase chain reaction (qPCR) was performed to further investigate the role of miR-93 in pan-cancer. RESULTS: Gene Ontology (GO) enrichment analysis results showed that the target genes of miR-93 were closely related to transcription, and MAPK1, RBBP7 and Smad7 became the hub genes. In the diagnostic meta-analysis, the overall sensitivity, specificity, and area under the curve were 0.76 (0.64-0.85), 0.82 (0.64-0.92), and 0.85 (0.82-0.88), respectively, which suggested that miR-93 had excellent performance on the diagnosis for human cancers. In the prognostic meta-analysis, dysregulated miR-93 was found to be associated with poor OS in cancer patients. In the qPCR validation test, the serum levels of miR-93 were upregulated in breast cancer, breast hyperplasia, lung cancer, chronic obstructive pulmonary disease, nasopharyngeal cancer, hepatocellular cancer, gastric ulcer, endometrial cancer, esophageal cancer, laryngeal cancer, and prostate cancer compared with healthy controls. CONCLUSIONS: miR-93 could act as an effective diagnostic and prognostic factor for cancer patients. Its clinical value for cancer early diagnosis and survival prediction is promising.

CRL4BRBBP7 targets HUWE1 for ubiquitination and proteasomal degradation.

The E3 ubiquitin ligase HUWE1/Mule/ARF-BP1 plays an important role in diverse biological processes including DNA damage repair and apoptosis. Our previous study has shown that in response to DNA damage HUWE1 was downregulated in CUL4B-mediated ubiquitination and subsequent proteasomal degradation, and CUL4B-mediated regulation of HUWE1 was important for cell survival upon DNA damage. CUL4B is a core component of the CUL4B Ring ligase complexes containing ROC1, DDB1 and a DDB1-Cullin Associated Factors (DCAFs), the latter of which are DDB1-binding WD40 adaptors critical for substrate recognition and recruitment. However, the identity of DCAF in CRL4B that mediates degradation of HUWE1 remains elusive. Here we report that RBBP7 is the DCAF in the CRL4B complex bridging the DDB1-CUL4B-ROC1 to HUWE1. Loading of HUWE1 to the E3 ubiquitin ligase complex resulted in its polyubiquitination, and consequently its proteasome mediated degradation. Overexpression of RBBP7 promoted HUWE1 protein degradation, while depletion of RBBP7 stabilized HUWE1, and hence accelerated the degradation of MCL-1 and BRCA1, two substrates of HUWE1 that are critical in apoptosis and DNA damage repair. Taken together, these data reveal CRL4BRBBP7 is the E3 ligase responsible for the proteasomal degradation of HUWE1, and further provide a potential strategy for cancer therapy by targeting HUWE1 and the CUL4B E3 ligase complex.

Activation of TNF-α/NF-κB axis enhances CRL4BDCAF11 E3 ligase activity and regulates cell cycle progression in human osteosarcoma cells.

Cullin 4B, a member of the Cullins, which serve as scaffolds to facilitate the assembly of E3 ligase complexes, is aberrantly expressed in many cancers, including osteosarcoma. Recently, we observed that CUL4B forms the CRL4BDCAF11 E3 ligase, which specifically ubiquitinates and degrades the cyclin-dependent kinase (CDK) inhibitor p21Cip1 in human osteosarcoma cells. However, the underlying mechanisms regarding the aberrant expression of CUL4B and the upstream members of this signaling pathway are mostly unknown. In this study, we demonstrate that nuclear factor kappaB (NF-κB) is a direct modulator of CUL4B expression. The CUL4B promoter is responsive to several NF-κB subunits, including RelA, RelB, and c-Rel, but not to p50 or p52. Additional studies reveal that the tumor necrosis factor alpha (TNF-α)/NF-κB axis pathway is activated in human osteosarcoma cells. This activation causes both CUL4B and NF-κB subunits to become abundant in the nucleus of human osteosarcoma cells. The down-regulation of individual genes, including TNFR1, RelA, RelB, c-Rel, and CUL4B, or pairs of them, including TNFR1 + RelA, TNFR1 + RelB, TNFR1 + c-Rel, and RelA+CUL4B, has similar effects on cell growth inhibition, colony formation, cell invasion, and in vivo tumor formation, whereas the overexpression of CUL4B in these knockdown cells significantly reverses their phenotypes. The inhibition of the TNF-α/NF-κB pathway greatly attenuates CRL4BDCAF11 E3 ligase activity and causes the accumulation of p21Cip1 , thereby leading to cell cycle arrest at the S phase. Taken together, our results support a model in which the activation of the TNF-α/NF-κB axis contributes to an increase in CRL4BDCAF11 activity and a decrease in p21Cip1 protein levels, thereby controlling cell cycle progression in human osteosarcoma cells.

AMPK promotes mitochondrial biogenesis and function by phosphorylating the epigenetic factors DNMT1, RBBP7, and HAT1.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) acts as a master regulator of cellular energy homeostasis by directly phosphorylating metabolic enzymes and nutrient transporters and by indirectly promoting the transactivation of nuclear genes involved in mitochondrial biogenesis and function. We explored the mechanism of AMPK-mediated induction of gene expression. We identified AMPK consensus phosphorylation sequences in three proteins involved in nucleosome remodeling: DNA methyltransferase 1 (DNMT1), retinoblastoma binding protein 7 (RBBP7), and histone acetyltransferase 1 (HAT1). DNMT1 mediates DNA methylation that limits transcription factor access to promoters and is inhibited by RBBP7. Acetylation of histones by HAT1 creates a more relaxed chromatin-DNA structure that favors transcription. AMPK-mediated phosphorylation resulted in the activation of HAT1 and inhibition of DNMT1. For DNMT1, this inhibition was both a direct effect of phosphorylation and the result of increased interaction with RBBP7. In human umbilical vein cells, pharmacological AMPK activation or pulsatile shear stress triggered nucleosome remodeling and decreased cytosine methylation, leading to increased expression of nuclear genes encoding factors involved in mitochondrial biogenesis and function, such as peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), transcription factor A (Tfam), and uncoupling proteins 2 and 3 (UCP2 and UCP3). Similar effects were seen in the aortas of mice given pharmacological AMPK activators, and these effects required AMPK2α. These results enhance our understanding of AMPK-mediated mitochondrial gene expression through nucleosome remodeling.

Expression, purification and characterization of the human MTA2-RBBP7 complex.

The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/ß and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.

The MTA1 subunit of the nucleosome remodeling and deacetylase complex can recruit two copies of RBBP4/7.

The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.

Liver X Receptor Agonist Modifies the DNA Methylation Profile of Synapse and Neurogenesis-Related Genes in the Triple Transgenic Mouse Model of Alzheimer's Disease.

The liver X receptor agonist, GW3965, improves cognition in Alzheimer's disease (AD) mouse models. Here, we determined if short-term GW3965 treatment induces changes in the DNA methylation state of the hippocampus, which are associated with cognitive improvement. Twenty-four-month-old triple-transgenic AD (3xTg-AD) mice were treated with GW3965 (50 mg/kg/day for 6 days). DNA methylation state was examined by modified bisulfite conversion and hybridization on Illumina Infinium Methylation BeadChip 450 k arrays. The Morris water maze was used for behavioral analysis. Our results show in addition to improvement in cognition methylation changes in 39 of 13,715 interrogated probes in treated 3xTg-AD mice compared with untreated 3xTg-AD mice. These changes in methylation probes include 29 gene loci. Importantly, changes in methylation status were mainly from synapse-related genes (SYP, SYN1, and DLG3) and neurogenesis-associated genes (HMGB3 and RBBP7). Thus, our results indicate that liver X receptors (LXR) agonist treatment induces rapid changes in DNA methylation, particularly in loci associated with genes involved in neurogenesis and synaptic function. Our results suggest a new potential mechanism to explain the beneficial effect of GW3965.

NKX6.1 functions as a metastatic suppressor through epigenetic regulation of the epithelial-mesenchymal transition.

The transcription factor NKX6.1 (NK6 homeobox 1) is important in the development of pancreatic ß-cells and neurons. Although recent publications show that NKX6.1 is hypermethylated and downregulated during tumorigenesis, the function of NKX6.1 in carcinogenesis remains elusive. Here, we address the metastasis suppressor function of human NKX6.1 using cell, animal and clinical analyses. Our data show that NKX6.1 represses tumor formation and metastatic ability both in vitro and in vivo. Mechanistically, NKX6.1 suppresses cell invasion by inhibiting the epithelial-to-mesenchymal transition (EMT). NKX6.1 directly enhances the mRNA level of E-cadherin by recruiting BAF155 coactivator and represses that of vimentin and N-cadherin by recruiting RBBP7 (retinoblastoma binding protein 7) corepressor. Clinical cancer tumors with metastasis show low NKX6.1 protein expression coinciding with low E-cadherin and high vimentin expression. Our results demonstrate that NKX6.1 functions as an EMT suppressor by interacting with different epigenetic modifiers, making it a potential novel therapeutic option.

Rbbp7 Is Required for Uterine Stromal Decidualization in Mice.

Uterine stromal cells undergo extensive proliferation and differentiation during postimplantation development, a process known as decidualization. While a range of signaling molecules have been demonstrated to play essential roles in this event, its potential epigenetic regulatory mechanisms remain largely unknown. Retinoblastoma binding protein 7 (Rbbp7) is a protein reported as a core component of many histone modification and chromatin remodeling complexes. In the present study, our in situ hybridization and immunochemistry analysis first reveals a spatiotemporal expression of Rbbp7 in the uterus during the peri-implantation period. Observations of remarkable induction of Rbbp7 expression in uterine stromal cells in response to progesterone-nuclear receptor PR signaling point to its potential physiological significance during postimplantation uterine development. Employing a stealth RNA knockdown approach, combined with primary murine uterine stromal cell culture and an in vitro-induced decidualization model, we further demonstrate that Rbbp7 silencing compromises stromal cell decidualization via attenuating histone H4 acetylation and cyclin D3 expression. The results collectively suggest that Rbbp7 is a potentially functional player regulating normal histone acetylation modification and cyclin D3 expression in stromal cells during postimplantation decidual development.

RBBP4 regulates histone deacetylation and bipolar spindle assembly during oocyte maturation in the mouse.

During meiosis I (MI) in oocytes, the maturation-associated decrease of histone acetylation is critical for normal meiotic progression and accurate chromosome segregation. RBBP4 is a component of several different histone deacetylase containing chromatin-remodeling complexes, but RBBP4's role in regulating MI is not known. Depleting RBBP4 in mouse oocytes resulted in multipolar spindles at metaphase (Met) I with subsequent perturbed meiotic progression and increased incidence of abnormal spindles, chromosome misalignment, and aneuploidy at Met II. We attribute these defects to improper deacetylation of histones because histones H3K4, H4K8, H4K12, and H4K16 were hyperacetylated in RBBP4-depleted oocytes. Importantly, we show that RBBP4-mediated histone deacetylation is essential for regulating bipolar spindle assembly, at least partially, through promoting Aurora kinase (AURK) C function. To our knowledge, these results are the first to identify RBBP4 as a regulator of histone deacetylation during oocyte maturation, and they provide evidence that deacetylation is required for bipolar spindle assembly through AURKC.

Analysis of phosphorylation-dependent protein-protein interactions of histone h3.

Multiple posttranslational modifications (PTMs) of histone proteins including site-specific phosphorylation of serine and threonine residues govern the accessibility of chromatin. According to the histone code theory, PTMs recruit regulatory proteins or block their access to chromatin. Here, we report a general strategy for simultaneous analysis of both of these effects based on a SILAC MS scheme. We applied this approach for studying the biochemical role of phosphorylated S10 of histone H3. Differential pull-down experiments with H3-tails synthesized from l- and d-amino acids uncovered that histone acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7) are part of the protein network, which interacts with the unmodified H3-tail. An additional H3-derived bait containing the nonhydrolyzable phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited several isoforms of the 14-3-3 family and blocked the recruitment of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide new insights into the many functions of H3S10 phosphorylation. In addition, the outlined methodology is generally applicable for studying specific binding partners of unmodified histone tails.