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Mounting findings have revealed the increasingly appreciated functional importance of Retinoblastoma binding protein (RBBP) family members in tumorigenesis. However, the biological function of RBBP4 in breast cancer, especially in the most malignant and aggressive subtype, i.e., triple-negative breast cancer (TNBC), remains to be elucidated.The present study was aimed at elucidating the role of RBBP4 in TNBC pathogenesis.The expression of RBBP4 in TNBC tissues and cell lines was examined and its oncogenic-related functions were verified by performing a series of in vitro and in vivo experiments.At the cellular and tissue level, a marked increase in the RBBP4 expression was observed. Functionally, RBBP4 knockdown dramatically inhibited the proliferation, invasion, and migration of TNBC cells in vitro. Further, mechanistically, RBBP4 downregulation regulated the inactivation of epithelial-mesenchymal transition (EMT) of TNBC cells. In vivo xenograft model in nude mice also validated these results.Collectively, our results showed that the inhibition of RBBP4 suppresses the malignant progression of TNBC cells by regulating EMT. Thus, RBBP4 could serve as a novel biomarker and target for TNBC diagnosis and treatment.

Retinoblastoma binding protein 4 (Rbbp4) is a component of transcription regulatory complexes that control cell cycle gene expression. Previous work indicated that Rbbp4 cooperates with the Rb tumor suppressor to block cell cycle entry. Here, we use genetic analysis to examine the interactions of Rbbp4, Rb, and Tp53 in zebrafish neural progenitor cell cycle regulation and survival.Rbbp4 is upregulated across the spectrum of human embryonal and glial brain cancers. Transgenic rescue of rbbp4 mutant embryos shows Rbbp4 is essential for zebrafish neurogenesis. Rbbp4 loss leads to apoptosis and γ-H2AX in the developing brain that is suppressed by tp53 knockdown or maternal zygotic deletion. Mutant retinal neural precursors accumulate in M phase and fail to initiate G0 gene expression. rbbp4; rb1 mutants show an additive effect on the number of M phase cells. In rbbp4 mutants, Tp53 acetylation is detected; however, Rbbp4 overexpression did not rescue DNA damage-induced apoptosis.Rbbp4 is necessary for neural progenitor cell cycle progression and initiation of G0 independent of Rb. Tp53-dependent apoptosis in the absence of Rbpb4 correlates with Tp53 acetylation. Together these results suggest that Rbbp4 is required for cell cycle exit and contributes to neural progenitor survival through the regulation of Tp53 acetylation.

Background RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). Methods shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. Results shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. Conclusions RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.

Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links associated with Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion decreased cellular viability and altered the expression of selected targets. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.

Summary Polycomb group (PcG) proteins exist in distinct multi-protein complexes and play a central role in silencing developmental genes, yet the underlying mechanisms remain elusive. Here, we show that deficiency of retinoblastoma binding protein 4 (RBBP4), a component of the Polycomb repressive complex 2 (PRC2), in embryonic stem cells (ESCs) leads to spontaneous differentiation into mesendodermal lineages. We further show that Rbbp4 and core PRC2 share an important number of common genomic targets, encoding regulators involved in early germ layer specification. Moreover, we find that Rbbp4 is absolutely essential for genomic targeting of PRC2 to a subset of developmental genes. Interestingly, we demonstrate that Rbbp4 is necessary for sustaining the expression of Oct4 and Sox2 and that the forced co-expression of Oct4 and Sox2 fully rescues the pluripotency of Rbbp4-null ESCs. Therefore, our study indicates that Rbbp4 links maintenance of the pluripotency regulatory network with repression of mesendoderm lineages., Graphical Abstract, Highlights • RBBP4 deficiency in ESCs leads to spontaneous differentiation into mesendodermal lineages • Rbbp4 binding sites in ESCs substantially overlap with PRC2 binding • Rbbp4 is absolutely essential for PRC2 chromatin occupancy • Rbbp4 is necessary for sustaining the expression levels of Oct4 and Sox2, Polycomb group (PcG) proteins play a critical role in silencing developmental genes, yet the underlying mechanisms remain elusive. Yikai et al. report that PcG protein Rbbp4 represses developmental gene expression by recruiting PRC2 to the genes' promoter regions, thereby maintaining stem cell self-renewal and identity.

Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.

RBBP4 is a subunit of the chromatin remodeling complexes known as Polycomb repressive complex 2 and histone deacetylase 1/2-containing complexes. These complexes are responsible for histone H3 lysine 27 methylation and deacetylation, respectively. How RBBP4 modulates the functions of these complexes remains largely unknown. We generated viable Rbbp4 mutant alleles in mouse embryonic stem cell lines by CRISPR-Cas9. The mutations disrupted Polycomb repressive complex 2 assembly and H3K27me3 establishment on target chromatin and altered histone H3 lysine 27 acetylation genome wide. Moreover, Rbbp4 mutant cells underwent dramatic changes in transcriptional profiles closely tied to the deregulation of H3K27ac. The alteration of H3K27ac due to RBBP4 dysfunction occurred on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 plays a central role in regulating histone H3 lysine 27 methylation and acetylation to modulate gene expression.

The scaffolding protein RbAp48 is part of several epigenetic regulation complexes and is overexpressed in a variety of cancers. In order to develop tool compounds for the study of RbAp48 function, we have developed peptide inhibitors targeting the protein–protein interaction interface between RbAp48 and the scaffold protein MTA1. Based on a MTA1‐derived linear peptide with low micromolar affinity and informed by crystallographic analysis, a bicyclic peptide was developed that inhibits the RbAp48/MTA1 interaction with a very low nanomolar K D value of 8.56 nM, and which showed appreciable stability against cellular proteases. Design included exchange of a polar amide cyclization strategy to hydrophobic aromatic linkers enabling mono‐ and bicyclization by means of cysteine alkylation, which improved affinity by direct interaction of the linkers with a hydrophobic residue on RbAp48. Our results demonstrate that stepwise evolution of a structure‐based design is a suitable strategy for inhibitor development targeting PPIs., Potent bicyclic peptide inhibitors of the RbAp48‐MTA1 interaction were developed by structure based stepwise optimization of the cyclization linker. The strategy exemplifies design of peptide derived inhibitors of protein–protein interactions involving large surface areas.

Background Our previous study demonstrated that RBBP4 was upregulated in colon cancer and correlated with poor prognosis of colon cancer and hepatic metastasis. However, the potential biological function of RBBP4 in colon cancer is still unknown. Aim To investigate the biological role and the potential mechanisms of RBBP4 in colon cancer progression. Methods Real-time polymerase chain reaction and western blot analysis were used to detect the expression of RBBP4 in colon cancer cell lines. The cell proliferation and viability of SW620 and HCT116 cells with RBBP4 knockdown was detected by Cell Counting Kit-8 and 5-ethynyl-2'-deoxyuridine staining. The transwell assay was used to detect the invasion and migration capabilities of colon cancer cells with RBBP4 knockdown. Flow cytometry apoptosis assay was used to detect the apoptosis of colon cancer cells. Western blotting analysis was used to detect the expression of epithelial-mesenchymal transition and apoptosis related markers in colon cancer. The nuclear translocation of β-catenin was examined by Western blotting analysis in colon cancer cells with RBBP4 knockdown. The TOPFlash luciferase assay was used to detect the effect of RBBP4 on Wnt/β-catenin activation. The rescue experiments were performed in colon cancer cells treated with Wnt/β-catenin activator LiCl and RBBP4 knockdown. Results We found that RBBP4 was highly expressed in colon cancer cell lines. The 5-ethynyl-2'-deoxyuridine assay showed that knockdown of RBBP4 significantly inhibited cell proliferation. RBBP4 inhibition reduced cell invasion and migration via regulating proteins related to epithelial-mesenchymal transition. Knockdown of RBBP4 significantly inhibited survivin-mediated apoptosis. Mechanistically, the TOPFlash assay showed that RBBP4 knockdown increased activity of the Wnt/β-catenin pathway. Meanwhile, RBBP4 knockdown suppressed nuclear translocation of β-catenin. With Wnt/β-catenin activator, rescue experiments suggested that the role of RBBP4 in colon cancer progression was dependent on Wnt/β-catenin pathway. Conclusion RBBP4 promotes colon cancer development via increasing activity of the Wnt/β-catenin pathway. RBBP4 may serve as a novel therapeutic target in colon cancer.

Retinoblastoma-binding protein 4 (RBBP4) (also known as chromatin-remodeling factor RBAP48) is an evolutionarily conserved protein that has been involved in various biological processes. Although a variety of functions have been attributed to RBBP4 in vitro, mammalian RBBP4 has not been studied in vivo. Here we report that RBBP4 is essential during early mouse embryo development. Although Rbbp4 mutant embryos exhibit normal morphology at E3.5 blastocyst stage, they cannot be recovered at E7.5 early post-gastrulation stage, suggesting an implantation failure. Outgrowth (OG) assays reveal that mutant blastocysts cannot hatch from the zona or can hatch but then arrest without further development. We find that while there is no change in proliferation or levels of reactive oxygen species, both apoptosis and histone acetylation are significantly increased in mutant blastocysts. Analysis of lineage specification reveals that while the trophoblast is properly specified, both epiblast and primitive endoderm lineages are compromised with severe reductions in cell number and/or specification. In summary, these findings demonstrate the essential role of RBBP4 during early mammalian embryogenesis.

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.

Aims Intervention for end-stage kidney disease (ESKD), which is associated with adverse prognoses and major economic burdens, is challenging due to its complex pathogenesis. The study was performed to identify biomarker genes and molecular mechanisms for ESKD by bioinformatics approach. Methods Using the Gene Expression Omnibus dataset GSE37171, this study identified pathways and genomic biomarkers associated with ESKD via a multi-stage knowledge discovery process, including identification of modules of genes by weighted gene co-expression network analysis, discovery of important involved pathways by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, selection of differentially expressed genes by the empirical Bayes method, and screening biomarker genes by the least absolute shrinkage and selection operator (Lasso) logistic regression. The results were validated using GSE70528, an independent testing dataset. Results Three clinically important gene modules associated with ESKD, were identified by weighted gene co-expression network analysis. Within these modules, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed important biological pathways involved in ESKD, including transforming growth factor-β and Wnt signalling, RNA-splicing, autophagy and chromatin and histone modification. Furthermore, Lasso logistic regression was conducted to identify five final genes, namely, CNOT8, MST4, PPP2CB, PCSK7 and RBBP4 that are differentially expressed and associated with ESKD. The accuracy of the final model in distinguishing the ESKD cases and controls was 96.8% and 91.7% in the training and validation datasets, respectively. Conclusion Network-based variable selection approaches can identify biological pathways and biomarker genes associated with ESKD. The findings may inform more in-depth follow-up research and effective therapy.

Background. The majority of lung cancers are adenocarcinomas, with the proportion being 40%. The patients are mostly diagnosed in the middle and late stages with metastasis and easy recurrence, which poses great challenge to the treatment and prognosis. Platinum-based chemotherapy is a primary treatment for adenocarcinoma, which frequently causes drug resistance. As a result, it is important to uncover the mechanisms of the chemoresponse of adenocarcinoma to platinum-based chemotherapy. Methods. The genes from the dataset GSE7880 were gathered into gene modules with the assistance of weighted gene coexpression network analysis (WGCNA), the gene trait significance absolute value (|GS|), and gene module memberships (MM). The genes from hub gene modules were calculated with a protein-protein interaction (PPI) network analysis in order to obtain a screening map of hub genes. The hub genes with both a high |GS| and MM and a high degree were selected. Furthermore, genes in the hub gene modules also went through a Gene Ontology (GO) functional enrichment analysis. Results. 11 hub genes in four hub gene modules (LY86, ACTR2, CDK2, CKAP4, KPNB1, RBBP4, SMAD4, MYL6, RPS27, TSPAN2, and VAMP2) were chosen as the significant hub genes. Through the GO function enrichment analysis, it was indicated that four modules were abundant in immune system functions (floralwhite), amino acid biosynthetic process (lightpink4), cell chemotaxis (navajowhite2), and targeting protein (paleturquoise). Four hub genes with the highest |GS| were verified by prognostic analysis.

Aberrant isoform expression of chromatin-associated proteins can induce epigenetic programs related to disease. The MDS1 and EVI1 complex locus (MECOM) encodes PRDM3, a protein with an N-terminal PR-SET domain, as well as a shorter isoform, EVI1, lacking the N-terminus containing the PR-SET domain (ΔPR). Imbalanced expression of MECOM isoforms is observed in multiple malignancies, implicating EVI1 as an oncogene, while PRDM3 has been suggested to function as a tumor suppressor through an unknown mechanism. To elucidate functional characteristics of these N-terminal residues, we compared the protein interactomes of the full-length and ΔPR isoforms of PRDM3 and its closely related paralog, PRDM16. Unlike the ΔPR isoforms, both full-length isoforms exhibited a significantly enriched association with components of the NuRD chromatin remodeling complex, especially RBBP4. Typically, RBBP4 facilitates chromatin association of the NuRD complex by binding to histone H3 tails. We show that RBBP4 binds to the N-terminal amino acid residues of PRDM3 and PRDM16, with a dissociation constant of 3.0 μM, as measured by isothermal titration calorimetry. Furthermore, high-resolution X-ray crystal structures of PRDM3 and PRDM16 N-terminal peptides in complex with RBBP4 revealed binding to RBBP4 within the conserved histone H3-binding groove. These data support a mechanism of isoform-specific interaction of PRDM3 and PRDM16 with the NuRD chromatin remodeling complex.

SUMMARY Precisely deciphering the molecular mechanisms of age-related memory loss is crucial to create appropriate therapeutic interventions. We have previously shown that the histone-binding protein RbAp48/Rbbp4 is a molecular determinant of Age-Related Memory Loss. By exploring how this protein regulates the genomic landscape of the hippocampal circuit, we find that RbAp48 controls the expression of BDNF and GPR158 proteins, both critical components of osteocalcin (OCN) signaling in the mouse hippocampus. We show that inhibition of RbAp48 in the hippocampal formation inhibits OCN’s beneficial functions in cognition and causes deficits in discrimination memory. In turn, disruption of OCN/GPR158 signaling leads to the downregulation of RbAp48 protein, mimicking the discrimination memory deficits observed in the aged hippocampus. We also show that activation of the OCN/GPR158 pathway increases the expression of RbAp48 in the aged dentate gyrus and rescues age-related memory loss., Graphical Abstract, In Brief Kosmidis et al. show how osteocalcin, a hormone produced by the bones, can ameliorate symptoms of age-related memory loss by upregulating the histone binding protein RbAp48.

The nucleosome remodeling and histone deacetylase (NuRD) complex is an essential multi-subunit protein complex that regulates higher-order chromatin structure. Cancers that use the alternative lengthening of telomere (ALT) pathway of telomere maintenance recruit NuRD to their telomeres. This interaction is mediated by the N-terminal domain of the zinc-finger protein ZNF827. NuRD–ZNF827 plays a vital role in the ALT pathway by creating a molecular platform for recombination-mediated repair. Disruption of NuRD binding results in loss of ALT cell viability. Here, we present the crystal structure of the NuRD subunit RBBP4 bound to the N-terminal 14 amino acids of ZNF827. RBBP4 forms a negatively charged channel that binds to ZNF827 through a network of electrostatic interactions. We identify the precise amino acids in RBBP4 required for this interaction and demonstrate that disruption of these residues prevents RBBP4 binding to both ZNF827 and telomeres, but is insufficient to decrease ALT activity. These data provide insights into the structural and functional determinants of NuRD activity at ALT telomeres.

Epigenetic regulation is mediated by protein complexes that couple recognition of chromatin marks to activity or recruitment of chromatin-modifying enzymes. Polycomb repressive complex 2 (PRC2), a gene silencer that methylates lysine 27 of histone H3, is stimulated upon recognition of its own catalytic product, and has been shown to be more active on dinucleosomes than H3 tails or single nucleosomes. These properties likely facilitate local H3K27me2/3 spreading causing heterochromatin formation and gene repression. Here, cryo-EM reconstructions of human PRC2 bound to bifunctional dinucleosomes show how a single PRC2, via interactions with nucleosomal DNA, positions the H3 tails of the activating and substrate nucleosome to interact with EED and the SET domain of EZH2, respectively. We show how the geometry of the PRC2-DNA interactions allows PRC2 to accommodate varying lengths of the linker DNA between nucleosomes. Our structures are the first to illustrate how an epigenetic regulator engages with a complex chromatin substrate.

Ash1 is a Trithorax group protein that possesses H3K36-specific histone methyltransferase activity, which antagonizes Polycomb silencing. Here we report the identification of two Ash1 complex subunits, Mrg15 and Nurf55. In vitro, Mrg15 stimulates the enzymatic activity of Ash1. In vivo, Mrg15 is recruited by Ash1 to their common targets, and Mrg15 reinforces Ash1 chromatin association and facilitates the proper deposition of H3K36me2. To dissect the functional role of Mrg15 in the context of the Ash1 complex, we identify an Ash1 point mutation (Ash1-R1288A) that displays a greatly attenuated interaction with Mrg15. Knock-in flies bearing this mutation display multiple homeotic transformation phenotypes, and these phenotypes are partially rescued by overexpressing the Mrg15-Nurf55 fusion protein, which stabilizes the association of Mrg15 with Ash1. In summary, Mrg15 is a subunit of the Ash1 complex, a stimulator of Ash1 enzymatic activity and a critical regulator of the TrxG protein function of Ash1 in Drosophila., Ash1 is an H3K36 methyltransferase, Trithorax group protein and is critical in antagonizing Polycomb silencing. Here, the authors purify the Drosophila Ash1 complex and identify Mrg15 and Nurf55 as subunits, finding that Mrg15 is recruited by Ash1 and reinforces Ash1 activity.

In eukaryotic cells, the organization of genomic DNA into chromatin regulates many biological processes, from the control of gene expression to the regulation of chromosome segregation. The proper maintenance of this structure upon cell division is therefore of prime importance during development for the maintenance of cell identity and genome stability. The chromatin assembly factor 1 (CAF-1) is involved in the assembly of H3-H4 histone dimers on newly synthesized DNA and in the maintenance of a higher order structure, the heterochromatin, through an interaction of its large subunit with the heterochromatin protein HP1a. We identify here a conserved domain in the large subunit of the CAF-1 complex required for its interaction with HP1a in the Drosophila fruit fly. Functional analysis reveals that this domain is dispensable for viability but participates in two processes involving heterochromatin: position-effect variegation and long range chromosomal interactions during meiotic prophase. Importantly, the identification in the large subunit of CAF-1 of a domain required for its interaction with HP1 allows the separation of its functions in heterochromatin-related processes from its function in the assembly of H3-H4 dimers onto newly synthesized DNA.

Centromere function requires the presence of the histone H3 variant CENP-A in most eukaryotes. The precise localization and protein amount of CENP-A are crucial for correct chromosome segregation, and misregulation can lead to aneuploidy. To characterize the loading of CENP-A to non-centromeric chromatin, we utilized different truncation- and localization-deficient CENP-A mutant constructs in Drosophila melanogaster cultured cells, and show that the N-terminus of Drosophila melanogaster CENP-A is required for nuclear localization and protein stability, and that CENP-A associated proteins, rather than CENP-A itself, determine its localization. Co-expression of mutant CENP-A with its loading factor CAL1 leads to exclusive centromere loading of CENP-A whereas co-expression with the histone-binding protein RbAp48 leads to exclusive non-centromeric CENP-A incorporation. Mass spectrometry analysis of non-centromeric CENP-A interacting partners identified the RbAp48-containing NuRD chromatin remodeling complex. Further analysis confirmed that NuRD is required for ectopic CENP-A incorporation, and RbAp48 and MTA1-like subunits of NuRD together with the N-terminal tail of CENP-A mediate the interaction. In summary, our data show that Drosophila CENP-A has no intrinsic specificity for centromeric chromatin and utilizes separate loading mechanisms for its incorporation into centromeric and ectopic sites. This suggests that the specific association and availability of CENP-A interacting factors are the major determinants of CENP-A loading specificity.

Retinoblastoma binding protein 4 (RBBP4) plays an essential role in the development of multiple cancers. However, its relationship with prognosis in colon cancer and colon cancer hepatic metastasis has not been elucidated. The aim of this study was to explore the relationship between RBBP4 expression and prognosis of colon cancer patients and to evaluate RBBP4 as a new prognostic marker in these patients.Eighty colon cancer patients underwent surgical resection of the colon were enrolled. Among them, forty colon cancer patients suffered with hepatic metastasis. The colon cancer tissues, para-colon cancer tissues, and hepatic metastatic cancer tissues were collected from the pathological department for further analysis. The expression of RBBP4 proteins was examined by immunohistochemistry and correlated with clinicopathological parameters. The Cancer Genome Atlas (TCGA) database was used to validate the expression and explore its relationship with clinical characteristics.RBBP4 was up-regulated in the colon cancer tissues compared with the para-colon cancer tissues. The analysis of TCGA database verified the upregulation of RBBP4 in the colon cancer tissues and RBBP4 overexpression was correlated with nerve invasion and poor outcomes of chemotherapy. Moreover, the positive rate of RBBP4 expression in 40 colon cancer patients with hepatic metastasis was higher in the hepatic metastatic cancer tissues (39/40, 97.5%) than in the colon cancer tissues (26/40, 65.0%). Our clinicopathological analysis showed that RBBP4 expression was significantly correlated with vascular invasion, hepatic metastasis, and lymph node involvement (all P0.05). Additionally, the survival analysis demonstrated that RBBP4 over-expression was correlated with poor prognosis.RBBP4 was upregulated in the colon cancer. RBBP4 may be a novel predictor for poor prognosis of colon cancer and colon cancer hepatic metastasis.

The constitutively nuclear histone deacetylases (HDACs) 1, 2, and 3 erase acetyl marks on acetyllysine residues, alter the landscape of histone modifications, and modulate chromatin structure and dynamics and thereby crucially regulate gene transcription in higher eukaryotes. Nuclear HDACs exist as at least six giant multiprotein complexes whose nonenzymatic subunits confer genome targeting specificity for these enzymes. The deacetylase activity of HDACs has been shown previously to be enhanced by inositol phosphates, which also bridge the catalytic domain in protein–protein interactions with SANT (Swi3, Ada2, N-Cor, and TFIIIB) domains in all HDAC complexes except those that contain the Sin3 transcriptional corepressors. Here, using purified recombinant proteins, coimmunoprecipitation and HDAC assays, and pulldown and NMR experiments, we show that HDAC1/2 deacetylase activity in one of the most ancient and evolutionarily conserved Sin3L/Rpd3L complexes is inducibly up-regulated by inositol phosphates but involves interactions with a zinc finger motif in the Sin3-associated protein 30 (SAP30) subunit that is structurally unrelated to SANT domains, indicating convergent evolution at the functional level. This implies that this mode of regulation has evolved independently multiple times and provides an evolutionary advantage. We also found that constitutive association with another core subunit, Rb-binding protein 4 chromatin-binding factor (RBBP4), further enhances deacetylase activity, implying both inducible and constitutive regulatory mechanisms within the same HDAC complex. Our results indicate that inositol phosphates stimulate HDAC activity and that the SAP30 zinc finger motif performs roles similar to that of the unrelated SANT domain in promoting the SAP30–HDAC1 interaction and enhancing HDAC activity.

Human immunodeficiency virus (HIV) transcription is closely associated with chromatin remodeling. Retinoblastoma binding protein 4 (RBBP4) is a histone chaperone implicated in chromatin remodeling. However, the role of RBBP4 in HIV-1 infection and the underlying mechanism remain elusive. In the present study, we showed that RBBP4 plays a negative regulatory role during HIV-1 infection. RBBP4 expression was significantly increased in HIV-1-infected T cells. RBBP4 binds to the HIV-1 long terminal repeat (LTR)， represses HIV-1 LTR-mediated transcription through recruiting nuclear receptor subfamily 2 group F member 1(NR2F1) and histone deacetylase 1 and 2 (HDAC1/2) to HIV-1 LTR, and further controls local histone 3 (H3) deacetylation and chromatin compaction. Furthermore, the occupancy of RBBP4, HDAC1/2, and NR2F1 on LTR in HIV-latent J-lat cells was significantly higher than that in HIV-1-activated cells. In conclusion, our results establish RBBP4 as a new potent antiretroviral factor, which may provide theoretical basis for the treatment of HIV in the future.

Active adult stem cells maintain a bipotential state with progeny able to either self-renew or initiate differentiation depending on extrinsic signals from the surrounding microenvironment. However, the intrinsic gene regulatory networks and chromatin states that allow adult stem cells to make these cell fate choices are not entirely understood. Here we show that the transcription factor DNA Replication-related Element Factor (DREF) regulates adult stem cell maintenance in the Drosophila male germline. A temperature-sensitive allele of DREF described in this study genetically separated a role for DREF in germline stem cell self-renewal from the general roles of DREF in cell proliferation. The DREF temperature-sensitive allele caused defects in germline stem cell self-renewal but allowed viability and division of germline stem cells as well as cell viability, growth and division of somatic cyst stem cells in the testes and cells in the Drosophila eye. Germline stem cells mutant for the temperature sensitive DREF allele exhibited lower activation of a TGF-beta reporter, and their progeny turned on expression of the differentiation factor Bam prematurely. Results of genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, genetically antagonize the role of DREF in germline stem cell maintenance. Taken together, these data suggest that DREF contributes to intrinsic components of the germline stem cell regulatory network that maintains competence to self-renew., Author summary Many adult tissues are maintained throughout life by the dual ability of adult stem cells to produce progeny that either self-renew or differentiate to replace specialized cells lost to turnover or damage. Although signals from the surrounding microenvironment have been shown to regulate the choice between self-renewal and onset of differentiation, the intrinsic gene regulatory programs that set up and maintain this bipotential state are not well understood. In this report we describe antagonistic components of an intrinsic stem cell program important for maintaining the balance between self-renewal and differentiation in Drosophila male germline adult stem cell lineage. We identified a temperature-sensitive mutant in the transcription factor DNA Replication-related Element Factor (DREF) gene that disrupts the ability of germline stem cells to self-renew, but not stem cell viability, ability to divide or differentiate under the same conditions. DREF mutant germline stem cells showed defects in the TGF-beta signaling pathway, a pathway that is critical for maintaining the stem cell population. Genetic interaction analyses revealed that Mi-2 and Caf1/p55, components of the Nucleosome Remodeling and Deacetylase complex genetically antagonize the role of DREF in germline stem cell maintenance. We propose that DREF contributes to a transcriptional environment necessary for maintaining a bi-potential stem cell state able to properly respond to extrinsic niche signals.

Craniosynostosis, the premature ossification of cranial sutures, is a developmental disorder of the skull vault, occurring in approximately 1 in 2250 births. The causes are heterogeneous, with a monogenic basis identified in ~25% of patients. Using whole-genome sequencing, we identified a novel, de novo variant in BCL11B, c.7C>A, encoding an R3S substitution (p.R3S), in a male patient with coronal suture synostosis. BCL11B is a transcription factor that interacts directly with the nucleosome remodelling and deacetylation complex (NuRD) and polycomb-related complex 2 (PRC2) through the invariant proteins RBBP4 and RBBP7. The p.R3S substitution occurs within a conserved amino-terminal motif (RRKQxxP) of BCL11B and reduces interaction with both transcriptional complexes. Equilibrium binding studies and molecular dynamics simulations show that the p.R3S substitution disrupts ionic coordination between BCL11B and the RBBP4–MTA1 complex, a subassembly of the NuRD complex, and increases the conformational flexibility of Arg-4, Lys-5 and Gln-6 of BCL11B. These alterations collectively reduce the affinity of BCL11B p.R3S for the RBBP4–MTA1 complex by nearly an order of magnitude. We generated a mouse model of the BCL11B p.R3S substitution using a CRISPR-Cas9-based approach, and we report herein that these mice exhibit craniosynostosis of the coronal suture, as well as other cranial sutures. This finding provides strong evidence that the BCL11B p.R3S substitution is causally associated with craniosynostosis and confirms an important role for BCL11B in the maintenance of cranial suture patency.

Chromatin assembly factor 1 (CAF1), a histone chaperone that mediates the deposition of histone H3/H4 onto newly synthesized DNA, is involved in Notch signaling activation during Drosophila wing imaginal disc development. Here, we report another side of CAF1, wherein the subunits CAF1-p105 and CAF1-p180 (also known as CAF1-105 and CAF1-180, respectively) inhibit expression of Notch target genes and show this is required for proliferation of Drosophila ovarian follicle cells. Loss-of-function of either CAF1-p105 or CAF1-p180 caused premature activation of Notch signaling reporters and early expression of the Notch target Hindsight (Hnt, also known as Pebbled), leading to Cut downregulation and inhibition of follicle cell mitosis. Our studies further show Notch is functionally responsible for these phenotypes observed in both the CAF1-p105- and CAF1-p180-deficient follicle cells. Moreover, we reveal that CAF1-p105- and CAF1-p180-dependent Cut expression is essential for inhibiting Hnt expression in follicle cells during their mitotic stage. These findings together indicate a novel negative-feedback regulatory loop between Cut and Hnt underlying CAF1-p105 and CAF-p180 regulation, which is crucial for follicle cell differentiation. In conclusion, our studies suggest CAF1 plays a dual role to sustain cell proliferation by positively or negatively regulating Drosophila Notch signaling in a tissue-context-dependent manner.

Background Circular RNAs (circRNAs) are a novel type of noncoding RNAs and play important roles in tumorigenesis, including gastric cancer (GC). However, the functions of most circRNAs remain poorly understood. In our study, we aimed to investigate the functions of a new circRNA circ-DONSON in GC progression. Methods The expression of circ-DONSON in gastric cancer tissues and adjacent normal tissues was analyzed by bioinformatics method, qRT-PCR, Northern blotting and in situ hybridization (ISH). The effects of circ-DONSON on GC cell proliferation, apoptosis, migration and invasion were measured by using CCK8, colony formation, EdU, immunofluorescence (IF), FACS and Transwell assays. qRT-PCR and Western blotting were utilized to validate how circ-DONSON regulates SOX4 expression. ChIP, DNA fluorescence in situ hybridization (DNA-FISH) and DNA accessibility assays were used to investigate how circ-DONSON regulates SOX4 transcription. The interaction between circ-DONSON and NURF complex was evaluated by mass spectrum, RNA immunoprecipitation (RIP), pulldown and EMSA assays. Xenograft mouse model was used to analyze the effect of circ-DONSON on GC growth in vivo. Results Elevated expression of circ-DONSON was observed in GC tissues and positively associated with advanced TNM stage and unfavorable prognosis. Silencing of circ-DONSON significantly suppressed the proliferation, migration and invasion of GC cells while promoting apoptosis. circ-DONSON was localized in the nucleus, recruited the NURF complex to SOX4 promoter and initiated its transcription. Silencing of the NURF complex subunit SNF2L, BPTF or RBBP4 similarly attenuated GC cell growth and increased apoptosis. circ-DONSON knockdown inhibited GC growth in vivo. Conclusion circ-DONSON promotes GC progression through recruiting the NURF complex to initiate SOX4 expression. Electronic supplementary material The online version of this article (10.1186/s12943-019-1006-2) contains supplementary material, which is available to authorized users.

Postmenopausal osteoporosis (PMOP), as well as its associated increased risk for fragility fracture, is one of the most disabling consequences of aging in women. This present study aimed to identify candidate genes that involve pathogenesis of PMOP and the therapeutic mechanism of Liuweidihuang (LWDH) pills on PMOP. We integrated microarray datasets of PMOP derived from the Gene Expression Omnibus (GEO) to screen differentially expressed genes (DEGs) between PMOP and normal controls as well as patients with PMOP and patients after treatment of LWDH pills. GO and KEGG enrichment analysis for DEGs were performed. The shared DEGs, associated with both the pathogenesis of PMOP and the therapeutic mechanism of LWDH, were further analyzed by protein-protein interaction (PPI) network. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to verify the DEGs obtained by our integrated analysis. Compared with normal controls, 1732 DEGs in PMOP were obtained with p

Retinoblastoma binding protein 4 (RBBP4) is a nuclear protein with four WD-repeat sequences and thus belongs to a highly conserved subfamily of proteins with such domains. This retinoblastoma-binding protein plays an important role in nucleosome assembly and histone modification, which influences gene transcription and regulates cell cycle and proliferation. Artemia sinica (brine shrimp) undergoes an unusual diapause process under stress conditions of high salinity and low temperature. However, the role of RBBP4 in diapause termination of embryo development in A. sinica remains unknown. Here, the full-length cDNA of the As-rbbp4 gene was obtained from A. sinica and found to contain 1411 nucleotides, including a 1281 bp open reading frame (ORF), 63 bp 5'-untranslated region (UTR) and a 67-bp 3'-UTR, which encodes a 427 amino acid (48 kDa) protein. Bioinformatic analysis indicated As-RBBP4 to be mainly located in the nucleus, with a theoretical isoelectric point of 4.79. Protein sequence domain analysis showed that As-RBBP4 is a conserved protein, especially in the WD40 domain. No specificity in expression of this gene was observed in tissues or organs by in situ hybridization. Real-time quantitative PCR and Western blot analyses of As-RBBP4 gene and protein expression, respectively, showed notably high levels at 10 h and a subsequent downward trend. Obvious trends in upregulation of As-RBBP4 were observed under conditions of low temperature and high salinity stress. As-E2F1 and As-CyclinE also presented similar trends as that of As-RBBP4 in Western blots. Analysis of the RBBP4 expression in early embryonic development of A. sinica indicated that this protein plays an important role in diapause termination and cell cycle regulation.

SummaryHere we provide evidence that RBBP4 modulates temozolomide (TMZ) sensitivity through coordinate regulation of two key DNA repair genes critical for recovery from TMZ-induced DNA damage: methylguanine-DNA-methyltransferase (MGMT) and RAD51. Disruption of RBBP4 enhanced TMZ sensitivity, induced synthetic lethality to PARP inhibition, and increased DNA damage signaling in response to TMZ. Moreover, RBBP4 silencing enhanced TMZ-induced H2AX phosphorylation and apoptosis in GBM cells. Intriguingly, RBBP4 knockdown suppressed the expression of MGMT, RAD51, and other genes in association with decreased promoter H3K9 acetylation (H3K9Ac) and increased H3K9 tri-methylation (H3K9me3). Consistent with these data, RBBP4 interacts with CBP/p300 to form a chromatin-modifying complex that binds within the promoter of MGMT, RAD51, and perhaps other genes. Globally, RBBP4 positively and negatively regulates genes involved in critical cellular functions including tumorigenesis. The RBBP4/CBP/p300 complex may provide an interesting target for developing therapy-sensitizing strategies for GBM and other tumors.

Summary Diverse accessory subunits are involved in the recruitment of polycomb repressive complex 2 (PRC2) to CpG island (CGI) chromatin. Here we report the crystal structure of a SUZ12-RBBP4 complex bound to fragments of the accessory subunits PHF19 and JARID2. Unexpectedly, this complex adopts a dimeric structural architecture, accounting for PRC2 self-association that has long been implicated. The intrinsic PRC2 dimer is formed via domain swapping involving RBBP4 and the unique C2 domain of SUZ12. MTF2 and PHF19 associate with PRC2 at around the dimer interface and stabilize the dimer. Conversely, AEBP2 binding results in a drastic movement of the C2 domain, disrupting the intrinsic PRC2 dimer. PRC2 dimerization enhances CGI DNA binding by PCLs in pairs in vitro, reminiscent of the widespread phenomenon of transcription factor dimerization in active transcription. Loss of PRC2 dimerization impairs histone H3K27 trimethylation (H3K27me3) on chromatin at developmental gene loci in mouse embryonic stem cells.

Background The discoidin domain receptor 1 (DDR1) is activated by collagens, upregulated in injured and fibrotic kidneys, and contributes to fibrosis by regulating extracellular matrix production, but how DDR1 controls fibrosis is poorly understood. DDR1 is a receptor tyrosine kinase (RTK). RTKs can translocate to the nucleus via a nuclear localization sequence (NLS) present on the receptor itself or a ligand it is bound to. In the nucleus, RTKs regulate gene expression by binding chromatin directly or by interacting with transcription factors. Methods To determine whether DDR1 translocates to the nucleus and whether this event is mediated by collagen-induced DDR1 activation, we generated renal cells expressing wild-type or mutant forms of DDR1 no longer able to bind collagen. Then, we determined the location of the DDR1 upon collagen stimulation. Using both biochemical assays and immunofluorescence, we analyzed the steps involved in DDR1 nuclear translocation. Results We show that although DDR1 and its natural ligand, collagen, lack an NLS, DDR1 is present in the nucleus of injured human and mouse kidney proximal tubules. We show that DDR1 nuclear translocation requires collagen-mediated receptor activation and interaction of DDR1 with SEC61B, a component of the Sec61 translocon, and nonmuscle myosin IIA and β-actin. Once in the nucleus, DDR1 binds to chromatin to increase the transcription of collagen IV, a major collagen upregulated in fibrosis. Conclusions These findings reveal a novel mechanism whereby activated DDR1 translates to the nucleus to regulate synthesis of profibrotic molecules.

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8(+) T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8(+) T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8(+) T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.

Sal-like 4 (SALL4) is a nuclear factor central to the maintenance of stem cell pluripotency and is a key component in hepatocellular carcinoma, a malignancy with no effective treatment. In cancer cells, SALL4 associates with nucleosome remodeling deacetylase (NuRD) to silence tumor-suppressor genes, such as PTEN. Here, we determined the crystal structure of an amino-terminal peptide of SALL4(1-12) complexed to RBBp4, the chaperone subunit of NuRD, at 2.7 Å, and subsequent design of a potent therapeutic SALL4 peptide (FFW) capable of antagonizing the SALL4-NURD interaction using systematic truncation and amino acid substitution studies. FFW peptide disruption of the SALL4-NuRD complex resulted in unidirectional up-regulation of transcripts, turning SALL4 from a dual transcription repressor-activator mode to singular transcription activator mode. We demonstrate that FFW has a target affinity of 23 nM, and displays significant antitumor effects, inhibiting tumor growth by 85% in xenograft mouse models. Using transcriptome and survival analysis, we discovered that the peptide inhibits the transcription-repressor function of SALL4 and causes massive up-regulation of transcripts that are beneficial to patient survival. This study supports the SALL4-NuRD complex as a drug target and FFW as a viable drug candidate, showcasing an effective strategy to accurately target oncogenes previously considered undruggable. NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore)

In the present study, the effects and underlying mechanism of RbAp48 on the radiosensitivity of AGS gastric cancer cells was investigated. Cell proliferation was determined with an MTT assay. Flow cytometry was performed to evaluate the cell cycle and apoptosis. Reverse transcription‑quantitative polymerase chain reaction and western blot analysis were performed to detect mRNA and protein expression, respectively, including RbAp48, phosphoinositide 3‑kinase (PI3K) and protein kinase B (Akt). The results revealed that radiation enhanced the expression level of RbAp48 in AGS cells, and that RbAp48 combined with radiation reduced AGS cell proliferation. In addition, RbAp48 combined with radiation resulted in G2 phase arrest and induced apoptosis via regulation of the PI3K/Akt pathway. In conclusion, it was demonstrated that overexpression of RbAp48 enhanced the radiosensitivity of AGS gastric cancer cells via suppression of PI3K/Akt pathway activity, suggesting that RbAp48 may hold potential as a gene therapeutic strategy in the future, aiding in the treatment of gastric cancer.

In this study, we used comparative genomics and developmental genetics to identify epigenetic regulators driving oncogenesis in a zebrafish retinoblastoma 1 (rb1) somatic-targeting model of RB1 mutant embryonal brain tumors. Zebrafish rb1 brain tumors caused by TALEN or CRISPR targeting are histologically similar to human central nervous system primitive neuroectodermal tumors (CNS-PNETs). Like the human oligoneural OLIG2+/SOX10+ CNS-PNET subtype, zebrafish rb1 tumors show elevated expression of neural progenitor transcription factors olig2, sox10, sox8b and the receptor tyrosine kinase erbb3a oncogene. Comparison of rb1 tumor and rb1/rb1 germline mutant larval transcriptomes shows that the altered oligoneural precursor signature is specific to tumor tissue. More than 170 chromatin regulators were differentially expressed in rb1 tumors, including overexpression of chromatin remodeler components histone deacetylase 1 (hdac1) and retinoblastoma binding protein 4 (rbbp4). Germline mutant analysis confirms that zebrafish rb1, rbbp4 and hdac1 are required during brain development. rb1 is necessary for neural precursor cell cycle exit and terminal differentiation, rbbp4 is required for survival of postmitotic precursors, and hdac1 maintains proliferation of the neural stem cell/progenitor pool. We present an in vivo assay using somatic CRISPR targeting plus live imaging of histone-H2A.F/Z-GFP fusion protein in developing larval brain to rapidly test the role of chromatin remodelers in neural stem and progenitor cells. Our somatic assay recapitulates germline mutant phenotypes and reveals a dynamic view of their roles in neural cell populations. Our study provides new insight into the epigenetic processes that might drive pathogenesis in RB1 brain tumors, and identifies Rbbp4 and its associated chromatin remodeling complexes as potential target pathways to induce apoptosis in RB1 mutant brain cancer cells. This article has an associated First Person interview with the first author of the paper., Summary: This study shows that chromatin remodelers that are overexpressed in a zebrafish model of RB1 mutant brain cancer are required for neural progenitor proliferation and survival, providing insight into potential mechanisms that drive tumor growth.

Postmenopausal osteoporosis (PMOP) is a major public health concern worldwide. The present study aimed to provide evidence to assist in the development of specific novel biomarkers for PMOP. Differentially expressed genes (DEGs) were identified between PMOP and normal controls by integrated microarray analyses of the Gene Expression Omnibus (GEO) database, and the optimal diagnostic gene biomarkers for PMOP were identified with LASSO and Boruta algorithms. Classification models, including support vector machine (SVM), decision tree and random forests models, were established to test the diagnostic value of identified gene biomarkers for PMOP. Functional annotations and protein‑protein interaction (PPI) network constructions were also conducted. Integrated microarray analyses (GSE56815, GSE13850 and GSE7429) of the GEO database were employed, and 1,320 DEGs were identified between PMOP and normal controls. An 11‑gene combination was also identified as an optimal biomarker for PMOP by feature selection and classification methods using SVM, decision tree and random forest models. This combination was comprised of the following genes: Dehydrogenase E1 and transketolase domain containing 1 (DHTKD1), osteoclast stimulating factor 1 (OSTF1), G protein‑coupled receptor 116 (GPR116), BCL2 interacting killer, adrenoceptor β1 (ADRB1), neogenin 1 (NEO1), RB binding protein 4 (RBBP4), GPR87, cylicin 2, EF‑hand calcium binding domain 1 and DEAH‑box helicase 35. RBBP4 (degree=12) was revealed to be the hub gene of this PMOP‑specific PPI network. Among these 11 genes, three genes (OSTF1, ADRB1 and NEO1) were speculated to serve roles in PMOP by regulating the balance between bone formation and bone resorption, while two genes (GPR87 and GPR116) may be involved in PMOP by regulating the nuclear factor‑κB signaling pathway. Furthermore, DHTKD1 and RBBP4 may be involved in PMOP by regulating mitochondrial dysfunction and interacting with ESR1, respectively. In conclusion, the findings of the current study provided an insight for exploring the mechanism and developing novel biomarkers for PMOP. Further studies are required to test the diagnostic value for PMOP prior to use in a clinical setting.

The Drosophila Ash1 protein is a trithorax-group (trxG) regulator that antagonizes Polycomb repression at HOX genes. Ash1 di-methylates lysine 36 in histone H3 (H3K36me2) but how this activity is controlled and at which genes it functions is not well understood. We show that Ash1 protein purified from Drosophila exists in a complex with MRG15 and Caf1 that we named AMC. In Drosophila and human AMC, MRG15 binds a conserved FxLP motif near the Ash1 SET domain and stimulates H3K36 di-methylation on nucleosomes. Drosophila MRG15-null and ash1 catalytic mutants show remarkably specific trxG phenotypes: stochastic loss of HOX gene expression and homeotic transformations in adults. In mutants lacking AMC, H3K36me2 bulk levels appear undiminished but H3K36me2 is reduced in the chromatin of HOX and other AMC-regulated genes. AMC therefore appears to act on top of the H3K36me2/me3 landscape generated by the major H3K36 methyltransferases NSD and Set2. Our analyses suggest that H3K36 di-methylation at HOX genes is the crucial physiological function of AMC and the mechanism by which the complex antagonizes Polycomb repression at these genes., Highlighted Article: The trithorax group protein Ash1 and its regulator MRG15 form a multiprotein complex that maintains expression of HOX and other target genes by methylating histone H3 in their chromatin.

Summary Developmentally regulated accessory subunits dictate PRC2 function. Here, we report the crystal structures of a 120 kDa heterotetrameric complex consisting of Suz12, Rbbp4, Jarid2, and Aebp2 fragments that is minimally active in nucleosome binding and of an inactive binary complex of Suz12 and Rbbp4. Suz12 contains two unique structural platforms that define distinct classes of PRC2 holo complexes for chromatin binding. Aebp2 and Phf19 compete for binding of a non-canonical C2 domain of Suz12; Jarid2 and EPOP occupy an overlapped Suz12 surface required for chromatin association of PRC2. Suz12 and Aebp2 progressively block histone H3K4 binding to Rbbp4, suggesting that Rbbp4 may not be directly involved in PRC2 inhibition by the active H3K4me3 histone mark. Nucleosome binding enabled by Jarid2 and Aebp2 is in part accounted for by the structures, which also reveal that disruption of the Jarid2-Suz12 interaction may underlie the disease mechanism of an oncogenic chromosomal translocation of Suz12.

Non-coding RNAs and mRNAs have been implicated in replication, pathogenesis and host response in HIV infection. However, the impact of long intergenic non-coding RNAs (lincRNAs) on HIV-1 and HIV-2 infection is not known. In this study, we have analyzed expression profiles of lincRNAs and mRNAs in monocyte derived macrophages (MDMs) infected with HIV-1/HIV-2 using microarrays. Our study identified many differentially expressed lincRNAs and mRNAs in MDMs infected with HIV-1/HIV-2 compared to uninfected MDMs. Genes involved in glutathione metabolism and lysine degradation were differentially regulated only in HIV-1 infected MDMs. In HIV-2 infected MDMs, CUL 2, SFRS9, and RBBP4 genes were differentially expressed. Furthermore, we found that plasma levels of lincRNA: chr2: 165509129-165519404 and lincRNA: chr12: 57761837-57762303 were better indicators of HIV-1 infection while lincRNA: chr10:128586385-128592960, XLOC_001148 and lincRNA: chr5:87580664-87583451, were better indicators of HIV-2 infection. In summary, our study has demonstrated that there is substantial alteration in lincRNA and mRNA expression in response to HIV-1/HIV-2 infection. These differentially expressed lincRNAs and mRNAs could serve as prognostic and diagnostic biomarkers of HIV infection and help in the identification of new targets for therapy.

// Wei Wang 1,2 , Yongju Lu 1 , Paul M. Stemmer 3 , Xiangmin Zhang 1 , Yongyi Bi 2 , Zhengping Yi 1 and Fei Chen 1 1 Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, USA 2 School of Public Health, Wuhan University, Wuhan, Hubei, P.R. China 3 The Proteomics Core and Institute of Environmental Health Sciences, School of Medicine, Wayne State University, Detroit, MI, USA Correspondence to: Fei Chen, email: // Keywords : mdig, DNA repair, NHEJ, XRCC5, XRCC6 Received : June 08, 2015 Accepted : July 03, 2015 Published : July 22, 2015 Abstract To investigate how mineral dust-induced gene (mdig, also named as mina53, MINA, or NO52) promotes carcinogenesis through inducing active chromatin, we performed proteomics analyses for the interacting proteins that were co-immunoprecipitated by anti-mdig antibody from either the lung cancer cell line A549 cells or the human bronchial epithelial cell line BEAS-2B cells. On SDS-PAGE gels, three to five unique protein bands were consistently observed in the complexes pulled-down by mdig antibody, but not the control IgG. In addition to the mdig protein, several DNA repair or chromatin binding proteins, including XRCC5, XRCC6, RBBP4, CBX8, PRMT5, and TDRD, were identified in the complexes by the proteomics analyses using both Orbitrap Fusion and Orbitrap XL nanoESI-MS/MS in four independent experiments. The interaction of mdig with some of these proteins was further validated by co-immunoprecipitation using antibodies against mdig and its partner proteins, respectively. These data, thus, provide evidence suggesting that mdig accomplishes its functions on chromatin, DNA repair and cell growth through interacting with the partner proteins.

The NuRD complex is a conserved transcriptional coregulator that contains both chromatin-remodeling and histone deacetylase activities. Mutations of PHF6 are found in patients with Börjeson-Forssman-Lehmann syndrome, T-cell acute lymphoblastic leukemia, or acute myeloid leukemia. Recently, PHF6 was identified to interact with the NuRD complex, and this interaction is mediated by the RBBP4 component. However, little is known about the molecular basis for the interaction. Here, we present the crystal structure of the complex of the NuRD subunit RBBP4 bound to the PHF6 peptide (residues 162-170). The PHF6 peptide binds to the top surface of the RBBP4 β-propeller. A pair of positively charged residues of the PHF6 peptide insert into the negatively charged pocket of RBBP4, which is critical for the interaction between PHF6 and RBBP4. Corresponding PHF6 mutants impair this interaction in vitro and in vivo. Structural comparison shows that the PHF6-binding pocket overlaps with FOG1 and histone H3 on RBBP4/Nurf55, but it is distinct from the pocket recognizing histone H4, Su(z)12, and MTA1. We further show that the middle disordered region (residues 145-207, containing the RBBP4-binding motif) is sufficient for the transcriptional repression mediated by PHF6 on the GAL4 reporter, and knockdown of RBBP4 diminished the PHF6-mediated repression. Our RBBP4-PHF6 complex structure provides insights into the molecular basis of PHF6-NuRD complex interaction and implicates a role for PHF6 in chromatin structure modulation and gene regulation.