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The Th2 cytokine interleukin 4 (IL4) promotes macrophage differentiation into alternative subtypes and plays important roles in physiology, in metabolic and inflammatory diseases, in cancer and in tissue regeneration. While the regulatory transcription factor networks governing IL4 signaling are already well-characterized, it is currently less understood which transcriptional coregulators are involved and how they operate mechanistically. In this study, we discover that G protein pathway suppressor 2 (GPS2), a core subunit of the HDAC3 corepressor complex assembled by SMRT and NCOR, represses IL4-dependent enhancer activation in mouse macrophages. Our genome-wide and gene-specific characterization revealed that, instead of directly repressing STAT6, chromatin-bound GPS2 cooperates with SMRT and NCOR to antagonize enhancer activation by lysine demethylase 1A (KDM1A, LSD1). Mechanistically, corepressor depletion increased KDM1A recruitment to enhancers linked to IL4-induced genes, accompanied by demethylation of the repressive histone marks H3K9me2/3 without affecting H3K4me1/2, the classic KDM1A substrates for demethylation in other cellular contexts. This in turn caused enhancer and gene activation already in the absence of IL4/STAT6 and sensitized the STAT6-dependent IL4 responsiveness of macrophages. Thus, our work identified with the antagonistic action of a GPS2-containing corepressor complex and the lysine demethylase KDM1A a hitherto unknown epigenetic corepressor-coactivator switching mechanism that governs alternative macrophage activation., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Eya1 is critical for establishing and maintaining nephron progenitor cells (NPCs). It belongs to a family of proteins called phosphatase-transcriptional activators but without intrinsic DNA-binding activity. However, the spectrum of the Eya1-centered networks is underexplored. Here, we combined transcriptomic, genomic and proteomic approaches to characterize gene regulation by Eya1 in the NPCs. We identified Eya1 target genes, associated cis-regulatory elements and partner proteins. Eya1 preferentially occupies promoter sequences and interacts with general transcription factors (TFs), RNA polymerases, different types of TFs, chromatin-remodeling factors with ATPase or helicase activity, and DNA replication/repair proteins. Intriguingly, we identified REST-binding motifs in 76% of Eya1-occupied sites without H3K27ac-deposition, which were present in many Eya1 target genes upregulated in Eya1-deficient NPCs. Eya1 copurified REST-interacting chromatin-remodeling factors, histone deacetylase/lysine demethylase, and corepressors. Coimmunoprecipitation validated physical interaction between Eya1 and Rest/Hdac1/Cdyl/Hltf in the kidneys. Collectively, our results suggest that through interactions with chromatin-remodeling factors and specialized DNA-binding proteins, Eya1 may modify chromatin structure to facilitate the assembly of regulatory complexes that regulate transcription positively or negatively. These findings provide a mechanistic basis for how Eya1 exerts its activity by forming unique multiprotein complexes in various biological processes to maintain the cellular state of NPCs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Revealing molecular mechanisms linked to androgen receptor activity can help to improve diagnosis and treatment of prostate cancer. Retinoic acid-induced 2 (RAI2) protein is thought to act as a transcriptional coregulator involved in hormonal responses and epithelial differentiation. We evaluated the clinical relevance and biological function of the RAI2 protein in prostate cancer., Methods: We assessed RAI2 gene expression in the Cancer Genome Atlas prostate adenocarcinoma PanCancer cohort and protein expression in primary tumors (n = 199) by immunohistochemistry. We studied RAI2 gene expression as part of a multimarker panel in an enriched circulating tumor cell population isolated from blood samples (n = 38) of patients with metastatic prostate cancer. In prostate cancer cell lines, we analyzed the consequences of androgen receptor inhibition on RAI2 protein expression and the consequences of RAI2 depletion on the expression of the androgen receptor and selected target genes., Results: Abundance of the RAI2 protein in adenocarcinomas correlated with the androgen receptor; keratins 8, 18, and 19; and E-cadherin as well as with an early biochemical recurrence. In circulating tumor cells, detection of RAI2 mRNA significantly correlated with gene expression of FOLH1, KLK3, RAI2, AR, and AR-V7. In VCaP and LNCaP cell lines, sustained inhibition of hormone receptor activity induced the RAI2 protein, whereas RAI2 depletion augmented the expression of MME, STEAP4, and WIPI1., Conclusions: The RAI2 protein functions as a transcriptional coregulator of the androgen response in prostate cancer cells. Detection of RAI2 gene expression in blood samples from patients with metastatic prostate cancer indicated the presence of circulating tumor cells., (© American Association for Clinical Chemistry 2022.)

DAXX displays complex biological functions. Remarkably, DAXX overexpression is a common feature in diverse cancers, which correlates with tumorigenesis, disease progression and treatment resistance. Structurally, DAXX is modular with an N-terminal helical bundle, a docking site for many DAXX interactors (e.g. p53 and ATRX). DAXX's central region folds with the H3.3/H4 dimer, providing a H3.3-specific chaperoning function. DAXX has two functionally critical SUMO-interacting motifs. These modules are connected by disordered regions. DAXX's structural features provide a framework for deciphering how DAXX mechanistically imparts its functions and how its activity is regulated. DAXX modulates transcription through binding to transcription factors, epigenetic modifiers, and chromatin remodelers. DAXX's localization in the PML nuclear bodies also plays roles in transcriptional regulation. DAXX-regulated genes are likely important effectors of its biological functions. Deposition of H3.3 and its interactions with epigenetic modifiers are likely key events for DAXX to regulate transcription, DNA repair, and viral infection. Interactions between DAXX and its partners directly impact apoptosis and cell signaling. DAXX's activity is regulated by posttranslational modifications and ubiquitin-dependent degradation. Notably, the tumor suppressor SPOP promotes DAXX degradation in phase-separated droplets. We summarize here our current understanding of DAXX's complex functions with a focus on how it promotes oncogenesis., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.

Cells have evolved DNA damage response (DDR) to repair DNA lesions and thus preserving genomic stability and impeding carcinogenesis. DNA damage induction is accompanied by transient transcription repression. Here, we describe a previously unrecognized role of chromodomain Y-like (CDYL1) protein in fortifying double-strand break (DSB)-induced transcription repression and repair. We showed that CDYL1 is rapidly recruited to damaged euchromatic regions in a poly (ADP-ribose) polymerase 1 (PARP1)-dependent, but ataxia telangiectasia mutated (ATM)-independent, manner. While the C-terminal region, containing the enoyl-CoA hydratase like (ECH) domain, of CDYL1 binds to poly (ADP-ribose) (PAR) moieties and mediates CDYL1 accumulation at DNA damage sites, the chromodomain and histone H3 trimethylated on lysine 9 (H3K9me3) mark are dispensable for its recruitment. Furthermore, CDYL1 promotes the recruitment of enhancer of zeste homolog 2 (EZH2), stimulates local increase of the repressive methyl mark H3K27me3, and promotes transcription silencing at DSB sites. In addition, following DNA damage induction, CDYL1 depletion causes persistent G2/M arrest and alters H2AX and replication protein A (RPA2) phosphorylation. Remarkably, the 'traffic-light reporter' system revealed that CDYL1 mainly promotes homology-directed repair (HDR) of DSBs in vivo. Consequently, CDYL1-knockout cells display synthetic lethality with the chemotherapeutic agent, cisplatin. Altogether, our findings identify CDYL1 as a new component of the DDR and suggest that the HDR-defective 'BRCAness' phenotype of CDYL1-deficient cells could be exploited for eradicating cancer cells harboring CDYL1 mutations.

Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.

Context: Necrolytic migratory erythema (NME) occurs in approximately 70% of patients with glucagonoma syndrome. Excessive stimulation of metabolic pathways by hyperglucagonemia, which leads to hypoaminoacidemia, contributes to NME pathogenesis. However, the molecular pathogenesis of glucagonoma and relationships between metabolic abnormalities and clinical symptoms remain unclear., Patient: A 53-year-old woman was referred to our hospital with a generalized rash and weight loss. NME was diagnosed by histopathological examination of skin biopsy tissue. Laboratory tests revealed diabetes, hyperglucagonemia, marked insulin resistance, severe hypoaminoacidemia, ketosis, and anemia. Enhanced computed tomography scans detected a 29-mm pancreatic hypervascular tumor, which was eventually diagnosed as glucagonoma. Preoperative treatment with octreotide long-acting release reduced the glucagon level, improved the amino acid profile, and produced NME remission. Surgical tumor excision normalized the metabolic status and led to remission of symptoms, including NME., Interventions: Whole-exome sequencing (WES) and subsequent targeted capture sequencing, followed by Sanger sequencing and pyrosequencing, identified biallelic alteration of death-domain associated protein (DAXX) with a combination of loss of heterozygosity and frameshift mutations (c.553_554del:p.R185fs and c.1884dupC:p.C629fs) in the glucagonoma. Consistently, immunohistochemistry confirmed near-absence of DAXX staining in the tumor cells. Tumor expression of glucagon and somatostatin receptor subtype 2 and 3 messenger RNA was markedly upregulated., Conclusions: This is a report of glucagonoma with biallelic inactivation of DAXX determined by WES. The tumor manifested as glucagonoma syndrome with generalized NME. This case showed the relationship between hypoaminoacidemia and NME status. Further investigations are required to elucidate the underlying mechanisms of NME onset and glucagonoma tumorigenesis.

The replicative immortality of human cancer cells is achieved by activation of a telomere maintenance mechanism (TMM). To achieve this, cancer cells utilise either the enzyme telomerase, or the Alternative Lengthening of Telomeres (ALT) pathway. These distinct molecular pathways are incompletely understood with respect to activation and propagation, as well as their associations with clinical outcomes. We have identified significant differences in the telomere repeat composition of tumours that use ALT compared to tumours that do not. We then employed a machine learning approach to stratify tumours according to telomere repeat content with an accuracy of 91.6%. Importantly, this classification approach is applicable across all tumour types. Analysis of pathway mutations that were under-represented in ALT tumours, across 1,075 tumour samples, revealed that the autophagy, cell cycle control of chromosomal replication, and transcriptional regulatory network in embryonic stem cells pathways are involved in the survival of ALT tumours. Overall, our approach demonstrates that telomere sequence content can be used to stratify ALT activity in cancers, and begin to define the molecular pathways involved in ALT activation.

Nuclear receptors (NRs) are transcription factors that bind to specific DNA sequences known as hormone response elements located upstream of their target genes. Transcriptional activity of NRs can be modulated by binding of the compatible ligand and transient interaction with cellular coregulators, functioning either as coactivators or as corepressors. Many coactivator proteins possess intrinsic histone acetyltransferase (HAT) activity that catalyzes the acetylation of specific lysine residues in histone tails and loosens the histone-DNA interaction, thereby facilitating access of transcriptional factors to the regulatory sequences of the DNA. Tat interactive protein 60 (TIP60), a member of the Mof-Ybf2-Sas2-TIP60 family of HAT protein, is a multifunctional coregulator that controls a number of physiological processes including apoptosis, DNA damage repair, and transcriptional regulation. Over the last two decades or so, TIP60 has been extensively studied for its role as NR coregulator, controlling various aspect of steroid receptor functions. The aim of this review is to summarize the findings on the role of TIP60 as a coregulator for different classes of NRs and its overall functional implications. We also discuss the latest studies linking TIP60 to NR-associated metabolic disorders and cancers for its potential use as a therapeutic drug target in future.

It has been shown that extremely low-frequency electromagnetic fields (ELFMF) affect regulation of cell fate and differentiation. Thus, the aim of this study was to investigate the role of ELFMFs in the enhancement of astrocytic differentiation. ELFMF exposure reduced the rate of proliferation and enhanced astrocytic differentiation. The ELFMF-treated cells showed increased levels of the astrocyte marker (GFAP), while those of the early neuronal marker (Nestin) and stemness marker (OCT3/4) were downregulated. The reactive oxygen species (ROS) level was observed to be significantly elevated after ELFMF exposure, which strengthens the modulatory role of SIRT1 and SIRT1 downstream molecules (TLE1, HES1, and MASH1) during astrocytic differentiation. After nicotinamide (5 mM) mediated inhibition of SIRT1, levels of TLE1, HES1, and MASH1 were examined; TLE1 was significantly upregulated and MASH1 was downregulated. These results suggest that ELFMFs induce astrocytic differentiation through activation of SIRT1 and SIRT1 downstream molecules.

Histone chaperones prevent promiscuous histone interactions before chromatin assembly. They guarantee faithful deposition of canonical histones and functionally specialized histone variants into chromatin in a spatial- and temporally-restricted manner. Here, we identify the binding partners of the primate-specific and H3.3-related histone variant H3.Y using several quantitative mass spectrometry approaches, and biochemical and cell biological assays. We find the HIRA, but not the DAXX/ATRX, complex to recognize H3.Y, explaining its presence in transcriptionally active euchromatic regions. Accordingly, H3.Y nucleosomes are enriched in the transcription-promoting FACT complex and depleted of repressive post-translational histone modifications. H3.Y mutational gain-of-function screens reveal an unexpected combinatorial amino acid sequence requirement for histone H3.3 interaction with DAXX but not HIRA, and for H3.3 recruitment to PML nuclear bodies. We demonstrate the importance and necessity of specific H3.3 core and C-terminal amino acids in discriminating between distinct chaperone complexes. Further, chromatin immunoprecipitation sequencing experiments reveal that in contrast to euchromatic HIRA-dependent deposition sites, human DAXX/ATRX-dependent regions of histone H3 variant incorporation are enriched in heterochromatic H3K9me3 and simple repeat sequences. These data demonstrate that H3.Y's unique amino acids allow a functional distinction between HIRA and DAXX binding and its consequent deposition into open chromatin., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Faithful transmission or restoration of epigenetic information such as repressive histone modifications through generations is critical for the maintenance of cell identity. We report here that chromodomain Y-like protein (CDYL), a chromodomain-containing transcription corepressor, is physically associated with chromatin assembly factor 1 (CAF-1) and the replicative helicase MCM complex. We showed that CDYL bridges CAF-1 and MCM, facilitating histone transfer and deposition during DNA replication. We demonstrated that CDYL recruits histone-modifying enzymes G9a, SETDB1, and EZH2 to replication forks, leading to the addition of H3K9me2/3 and H3K27me2/3 on newly deposited histone H3. Significantly, depletion of CDYL impedes early S phase progression and sensitizes cells to DNA damage. Our data indicate that CDYL plays an important role in the transmission/restoration of repressive histone marks, thereby preserving the epigenetic landscape for the maintenance of cell identity., (© The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.)

Aberrant activation of RAS/MAPK signaling is a driver of over one third of all human carcinomas. The homologous transcription factors ETS1 and ETS2 mediate activation of gene expression programs downstream of RAS/MAPK signaling. ETS1 is important for oncogenesis in many tumor types. However, ETS2 can act as an oncogene in some cellular backgrounds, and as a tumor suppressor in others, and the molecular mechanism responsible for this cell-type specific function remains unknown. Here, we show that ETS1 and ETS2 can regulate a cell migration gene expression program in opposite directions, and provide the first comparison of the ETS1 and ETS2 cistromes. This genomic data and an ETS1 deletion line reveal that the opposite function of ETS2 is a result of binding site competition and transcriptional attenuation due to weaker transcriptional activation by ETS2 compared to ETS1. This weaker activation was mapped to the ETS2 N-terminus and a specific interaction with the co-repressor ZMYND11 (BS69). Furthermore, ZMYND11 expression levels in patient tumors correlated with oncogenic versus tumor suppressive roles of ETS2. Therefore, these data indicate a novel and specific mechanism allowing ETS2 to switch between oncogenic and tumor suppressive functions in a cell-type specific manner., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A number of studies have demonstrated that various components of the ATRX/DAXX/Histone H3.3 complex are important for heterochromatin silencing at multiple genomic regions. We provide an overview of the individual components (ATRX, DAXX and/or H3.3) tested in each study and propose a model where the ATRX/DAXX chaperone complex deposits H3.3 to maintain the H3K9me3 modification at heterochromatin throughout the genome., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Amino-terminal enhancer of split (Aes) is a member of Groucho/Transducin-like enhancer (TLE) family. Aes is a recently found metastasis suppressor of colorectal cancer (CRC) that inhibits Notch signalling, and forms nuclear foci together with TLE1. Although some Notch-associated proteins are known to form subnuclear bodies, little is known regarding the dynamics or functions of these structures. Here, we show that Aes nuclear foci in CRC observed under an electron microscope are in a rather amorphous structure, lacking surrounding membrane. Investigation of their behaviour during the cell cycle by time-lapse cinematography showed that Aes nuclear foci dissolve during mitosis and reassemble after completion of cytokinesis. We have also found that heat shock cognate 70 (HSC70) is an essential component of Aes foci. Pharmacological inhibition of the HSC70 ATPase activity with VER155008 reduces Aes focus formation. These results provide insight into the understanding of Aes-mediated inhibition of Notch signalling., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

For cells to exit from pluripotency and commit to a lineage, the circuitry of a core transcription factor (CTF) network must be extinguished in an orderly manner through epigenetic modifications. However, how this choreographed epigenetic remodeling at active embryonic stem cell (ESC) genes occurs during differentiation is poorly understood. In this study, we demonstrate that C-terminal binding protein 2 (Ctbp2) regulates nucleosome remodeling and deacetylation (NuRD)-mediated deacetylation of H3K27 and facilitates recruitment of polycomb repressive complex 2 (PRC2)-mediated H3K27me3 in active ESC genes for exit from pluripotency during differentiation. By genomewide analysis, we found that Ctbp2 resides in active ESC genes and co-occupies regions with ESC CTFs in undifferentiated ESCs. Furthermore, ablation of Ctbp2 effects inappropriate gene silencing in ESCs by sustaining high levels of H3K27ac and impeding H3K27me3 in active ESC genes, thereby sustaining ESC maintenance during differentiation. Thus, Ctbp2 preoccupies regions in active genes with the NuRD complex in undifferentiated ESCs that are directed toward H3K27me3 by PRC2 to induce stable silencing, which is pivotal for natural lineage commitment., (© 2015 AlphaMed Press.)

We investigated DNA methylomes of pediatric B-cell acute lymphoblastic leukemias (B-ALLs) using whole-genome bisulfite sequencing and high-definition microarrays, along with RNA expression profiles. Epigenetic alteration of B-ALLs occurred in two tracks: de novo methylation of small functional compartments and demethylation of large inter-compartmental backbones. The deviations were exaggerated in lamina-associated domains, with differences corresponding to methylation clusters and/or cytogenetic groups. Our data also suggested a pivotal role of polycomb and CTBP2 in de novo methylation, which may be traced back to bivalency status of embryonic stem cells. Driven by these potent epigenetic modulations, suppression of polycomb target genes was observed along with disruption of developmental fate and cell cycle and mismatch repair pathways and altered activities of key upstream regulators., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Glial brain tumors cause considerable mortality and morbidity in children and adults. Innovative targets for therapy are needed to improve survival and reduce long-term sequelae. The aim of this study was to find a candidate tumor-promoting protein, abundantly expressed in tumor cells but not in normal brain tissues, as a potential target for therapy., Methods: In silico proteomics and genomics, immunohistochemistry, and immunofluorescence microscopy validation were performed. RNA interference was used to ascertain the functional role of the overexpressed candidate target protein., Results: In silico proteomics and genomics revealed pre-B-cell leukemia homeobox (PBX) interacting protein 1 (PBXIP1) overexpression in adult and childhood high-grade glioma and ependymoma compared with normal brain. PBXIP1 is a PBX-family interacting microtubule-binding protein with a putative role in migration and proliferation of cancer cells. Immunohistochemical studies in glial tumors validated PBXIP1 expression in astrocytoma and ependymoma but not in oligodendroglioma. RNAi-mediated PBXIP1-knockdown in glioblastoma cell lines strongly reduced proliferation and migration and induced morphological changes, indicating that PBXIP1 knockdown decreases glioma cell viability and motility through rearrangements of the actin cytoskeleton. Furthermore, expression of PBXIP1 was observed in radial glia and astrocytic progenitor cells in human fetal tissues, suggesting that PBXIP1 is an astroglial progenitor cell marker during human embryonic development., Conclusion: PBXIP1 is a novel protein overexpressed in astrocytoma and ependymoma, involved in tumor cell proliferation and migration, that warrants further exploration as a novel therapeutic target in these tumors.

Fully grown oocytes in the ovary are arrested at prophase of meiosis I because of high levels of intraoocyte cAMP that maintain increased levels of cAMP-dependent protein kinase (PKA) activity. Following the luteinizing hormone surge at the time of ovulation, cAMP levels drop, resulting in a reduction in PKA activity that triggers meiotic resumption. Although much is known about the molecular mechanisms of how PKA activity fluctuations initiate the oocyte's reentry into meiosis, significantly less is known about the requirement for PKA activity in the oocyte after exit from the prophase I arrest. Here we show that although PKA activity decreases in the oocyte upon meiotic resumption, it increases throughout meiotic progression from the time of germinal vesicle breakdown (GVBD) until the metaphase II (MII) arrest. Blocking this meiotic maturation-associated increase in PKA activity using the pharmacological inhibitor H89 resulted in altered kinetics of GVBD, defects in chromatin and spindle dynamics, and decreased ability of oocytes to reach MII. These effects appear to be largely PKA specific because inhibitors targeting other kinases did not have the same outcomes. To determine potential proteins that may require PKA phosphorylation during meiosis, we separated oocyte protein extracts on an SDS-PAGE gel, extracted regions of the gel that had corresponding immune reactivity towards an anti-PKA substrate antibody, and performed mass spectrometry and microsequencing. Using this approach, we identified transducin-like enhancer of split-6 (TLE6)-a maternal effect gene that is part of the subcortical maternal complex-as a putative PKA substrate. TLE6 localized to the oocyte cortex throughout meiosis in a manner that is spatially and temporally consistent with the localization of critical PKA subunits. Moreover, we demonstrated that TLE6 becomes phosphorylated in a narrow window following meiotic resumption, and H89 treatment can completely block this phosphorylation when added prior to GVBD but not after. Taken together, these results highlight the importance of oocyte-intrinsic PKA in regulating meiotic progression after the prophase I arrest and offer new insights into downstream targets of its activity.

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.

Pancreatic neuroendocrine tumors (PanNETs) are complicated and often deadly neoplasms. A recent increased understanding of their molecular biology has contributed to expanded treatment options. DNA sequencing of samples derived from patients with PanNETs and rare genetic syndromes such as multiple endocrine neoplasia type 1 (MEN1) and Von Hippel-Lindau (VHL) syndrome reveals the involvement of MEN1, DAXX/ATRX, and the mammalian target of rapamycin (mTOR) pathways in PanNET tumorigenesis. Gene knock-out/knock-in studies indicate that inactivation of factors including MEN1 and abnormal PI3K/mTOR signaling uncouples endocrine cell cycle progression from the control of environmental cues such as glucose, leading to islet cell overgrowth. In addition, accumulating evidence suggests that further impairment of endothelial-endocrine cell interactions contributes to tumor invasion and metastasis. Recent phase III clinical trials have shown that therapeutic interventions, such as sunitinib and everolimus, targeting those signal transduction pathways improve disease-free survival rates. Yet, cure in the setting of advanced disease remains elusive. Further advances in our understanding of the molecular mechanisms of PanNETs and improved preclinical models will assist in developing personalized therapy utilizing novel drugs to provide prolonged control or even cure the disease.

Death domain-associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein-protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling.

The development of a neuron from a precursor cell comprises a complex set of steps ranging from regulation of the proliferative cycle through the acquisition of distinct morphology and functionality. How these processes are orchestrated is largely unknown. Using in utero manipulation of gene expression in the mouse embryonic cerebral cortex, we found that the transition between multipolar and bipolar stages of newborn cortical pyramidal neurons is markedly delayed by depletion of CoREST, a corepressor component of chromatin remodeling complexes. This profoundly affects the onset of their radial migration. The loss of CoREST function also perturbs the dynamics of neuronal precursor cell populations, transiently increasing the fraction of cells remaining in progenitor states, but not the acquisition of the neuronal glutamatergic fate of pyramidal cells. The function of CoREST in these processes appears to be independent of its best-known interactor, the RE-1 silencer of transcription/neural restrictive silencing factor, and requires the histone demethylase LSD1. This reveals the importance of epigenetic control in the execution of neural development programs, specifically in the cerebral cortex.

Transducer-like enhancer of split 1 (TLE1) is overexpressed in synovial sarcomas. We investigated TLE1 expression by immunohistochemical analysis in a well-characterized series of synovial sarcomas and other mesenchymal tumors most commonly considered in the differential diagnosis. Whole tissue sections of 212 tumors were evaluated: 73 synovial sarcomas (23 biphasic, 28 monophasic, 22 poorly differentiated), 47 malignant peripheral nerve sheath tumors (MPNSTs), 49 solitary fibrous tumors (SFTs), 20 fibrosarcomatous variants of dermatofibrosarcoma protuberans, and 23 Ewing sarcomas/primitive neuroectodermal tumors (PNETs). All monophasic and poorly differentiated SSs and Ewing sarcoma/PNETs were previously confirmed to harbor t(X;18) and EWSR1 gene rearrangements, respectively. In total, 60 (82%) of 73 synovial sarcomas were positive for TLE1, including 18 biphasic (78%), 22 monophasic (79%), and 20 poorly differentiated (91%) tumors. Of the other tumors, only 7 MPNSTs (15%) and 4 SFTs (8%) were positive for TLE1, most of which showed only weak staining. TLE1 is a sensitive and specific marker for synovial sarcoma and can be helpful to distinguish synovial sarcoma from histologic mimics, particularly if moderate or strong staining is observed. In this study, only a small subset of MPNSTs and SFTs showed limited staining for TLE1.

Growth Factor Independent-1B (Gfi-1B) is a transcriptional repressor that plays critical roles in the control of erythropoiesis and megakaryopoiesis. Gfi-1B expression was described to be repressed by an autoregulatory feedback control loop. Here, we show that Gfi-1 transcription is positively regulated early after induction of erythroid differentiation and remains highly active to late erythroblasts. Using chromatin immunoprecipitation assays in CD34+ cells from human cord blood, we found that Gfi-1 and GATA-2 in immature progenitors and then Gfi-1B and GATA-1 in erythroblasts are bound to the Gfi-1B promoter as well as to the promoter of c-myc, a known Gfi-1B target gene. Surprisingly, this Gfi-1/GATA-2-Gfi-1B/GATA-1 switch observed at erythroblast stages is associated to an increase in the Gfi-1B transcription whereas it triggers repression of c-myc transcription. Accordingly, analysis of chromatin modification patterns shows that HDAC, CoREST, and LSD1 are recruited to the c-myc promoter leading to appearance of repressive chromatin marks. In contrast, the Gfi-1B promoter remains associated with a transcriptionally active chromatin configuration as highlighted by an increase in histone H3 acetylation and concomitant release of the LSD1 and CoREST corepressors. The repressive function of Gfi-1B therefore depends on the nature of the proteins recruited to the target gene promoters and on chromatin modifications. We conclude that Gfi-1B behaves as a lineage-affiliated gene with an open chromatin configuration in multipotent progenitors and sustained activation as cells progress throughout erythroid differentiation.

Specific wiring of gene-regulatory networks is likely to underlie much of the phenotypic difference between species, but the extent of lineage-specific regulatory architecture remains poorly understood. The essential vertebrate transcriptional repressor REST (RE1-Silencing Transcription Factor) targets many neural genes during development of the preimplantation embryo and the central nervous system, through its cognate DNA motif, the RE1 (Repressor Element 1). Here we present a comparative genomic analysis of REST recruitment in multiple species by integrating both sequence and experimental data. We use an accurate, experimentally validated Position-Specific Scoring Matrix method to identify REST binding sites in multiply aligned vertebrate genomes, allowing us to infer the evolutionary origin of each of 1,298 human RE1 elements. We validate these findings using experimental data of REST binding across the whole genomes of human and mouse. We show that one-third of human RE1s are unique to primates: These sites recruit REST in vivo, target neural genes, and are under purifying evolutionary selection. We observe a consistent and significant trend for more ancient RE1s to have higher affinity for REST than lineage-specific sites and to be more proximal to target genes. Our results lead us to propose a model where new transcription factor binding sites are constantly generated throughout the genome; thereafter, refinement of their sequence and location consolidates this remodeling of networks governing neural gene regulation.

The CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis, and it is bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone methylations and the association of enzymatic activities. Two novel results emerged: (i) H3-H4 are present on core promoters under active conditions, with the expected cohort of 'positive' modifications; H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show that NF-Y is important for H3K36me3 deposition and for elongation, not recruitment of Pol II; (ii) H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Functional siRNA inactivation and treatment with Tranylcypromine determined that KDM1 (LSD1) plays a positive role in transcription, specifically of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. These data are the first in vivo indication of a crucial interplay between core histones and 'deviant' histone-fold such as NF-Y, leading to fine-tuning of histone methylations.

Genomic integration by the Streptomyces bacteriophage C31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the C31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34(+) haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of C31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in C31 integrase transfected A549 lung than Jurkat T cells. When the C31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the C31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of C31 integrase activity.

The control of gene expression in neural stem cells is key to understanding their developmental and therapeutic potential, yet we know little of the transcriptional mechanisms that underlie their differentiation. Recent evidence has implicated the RE1 silencing transcription factor (REST) in neuronal differentiation. However, the means by which REST regulates transcription in neural stem cells remain unclear. Here, we show that REST recruits distinct corepressor platforms in neural stem cells. REST is able to both silence and repress neuronal genes in embryonic hippocampal neural stem cells by creating a chromatin environment that contains both repressive local epigenetic signature (characterized by low levels of histones H4 and H3K9 acetylation and elevated dimethylation of H3K9) and H3K4 methylation, which are characteristic of gene activation. Furthermore, inhibition of REST function leads to activation of several neuron-specific genes but does not lead to overt formation of mature neurons, supporting the notion that REST regulates part, but not all, of the neuronal differentiation program.

The exosome is a complex of 3'-5' exoribonucleases and RNA-binding proteins, which is involved in processing or degradation of different classes of RNA. Previously, the characterization of purified exosome complexes from yeast and human cells suggested that C1D and KIAA0052/hMtr4p are associated with the exosome and thus might regulate its functional activities. Subcellular localization experiments demonstrated that C1D and KIAA0052/hMtr4p co-localize with exosome subunit PM/Scl-100 in the nucleoli of HEp-2 cells. Additionally, the nucleolar accumulation of C1D appeared to be dependent on PM/Scl-100. Protein-protein interaction studies showed that C1D binds to PM/Scl-100, whereas KIAA0052/hMtr4p was found to interact with MPP6, a previously identified exosome-associated protein. Moreover, we demonstrate that C1D, MPP6 and PM/Scl-100 form a stable trimeric complex in vitro. Knock-down of C1D, MPP6 and KIAA0052/hMtr4p by RNAi resulted in the accumulation of 3'-extended 5.8S rRNA precursors, showing that these proteins are required for rRNA processing. Interestingly, C1D appeared to contain RNA-binding activity with a potential preference for structured RNAs. Taken together, our results are consistent with a role for the exosome-associated proteins C1D, MPP6 and KIAA052/hMtr4p in the recruitment of the exosome to pre-rRNA to mediate the 3' end processing of the 5.8S rRNA.

It is increasingly common to combine Microarray and Quantitative Trait Loci data to aid the search for candidate genes responsible for phenotypic variation. Workflows provide a means of systematically processing these large datasets and also represent a framework for the re-use and the explicit declaration of experimental methods. In this article, we highlight the issues facing the manual analysis of microarray and QTL data for the discovery of candidate genes underlying complex phenotypes. We show how automated approaches provide a systematic means to investigate genotype-phenotype correlations. This methodology was applied to a use case of resistance to African trypanosomiasis in the mouse. Pathways represented in the results identified Daxx as one of the candidate genes within the Tir1 QTL region. Subsequent re-sequencing in Daxx identified a deletion of an amino acid, identified in susceptible mouse strains, in the Daxx-p53 protein-binding region. This supports recent experimental evidence that apoptosis could be playing a role in the trypanosomiasis resistance phenotype. Workflows developed in this investigation, including a guide to loading and executing them with example data, are available at http://workflows.mygrid.org.uk/repository/myGrid/PaulFisher/.

Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.