Corepressor Function Research Articles

HIPK2 in Colon Cancer: A Potential Biomarker for Tumor Progression and Response to Therapies.

Colon cancer, one of the most common and fatal cancers worldwide, is characterized by stepwise accumulation of specific genetic alterations in tumor suppressor genes or oncogenes, leading to tumor growth and metastasis. HIPK2 (homeodomain-interacting protein kinase 2) is a serine/threonine protein kinase and a "bona fide" oncosuppressor protein. Its activation inhibits tumor growth mainly by promoting apoptosis, while its inactivation increases tumorigenicity and resistance to therapies of many different cancer types, including colon cancer. HIPK2 interacts with many molecular pathways by means of its kinase activity or transcriptional co-repressor function modulating cell growth and apoptosis, invasion, angiogenesis, inflammation and hypoxia. HIPK2 has been shown to participate in several molecular pathways involved in colon cancer including p53, Wnt/β-catenin and the newly identified nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2). HIPK2 also plays a role in tumor-host interaction in the tumor microenvironment (TME) by inducing angiogenesis and cancer-associated fibroblast (CAF) differentiation. The aim of this review is to assess the role of HIPK2 in colon cancer and the underlying molecular pathways for a better understanding of its involvement in colon cancer carcinogenesis and response to therapies, which will likely pave the way for novel colon cancer therapies.

Master corepressor inactivation through multivalent SLiM-induced polymerization mediated by the oncogene suppressor RAI2

While the elucidation of regulatory mechanisms of folded proteins is facilitated due to their amenability to high-resolution structural characterization, investigation of these mechanisms in disordered proteins is more challenging due to their structural heterogeneity, which can be captured by a variety of biophysical approaches. Here, we used the transcriptional master corepressor CtBP, which binds the putative metastasis suppressor RAI2 through repetitive SLiMs, as a model system. Using cryo-electron microscopy embedded in an integrative structural biology approach, we show that RAI2 unexpectedly induces CtBP polymerization through filaments of stacked tetrameric CtBP layers. These filaments lead to RAI2-mediated CtBP nuclear foci and relieve its corepressor function in RAI2-expressing cancer cells. The impact of RAI2-mediated CtBP loss-of-function is illustrated by the analysis of a diverse cohort of prostate cancer patients, which reveals a substantial decrease in RAI2 in advanced treatment-resistant cancer subtypes. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions.

Abstract 3023: Master corepressor inactivation through oncogene suppressor RAI2 mediated polymerization

Abstract Imbalanced transcription of a large number of genes can be caused at various levels, ranging from alterations in individual transcription factors to dysfunctional corepressor and coactivator complexes [1]. C-terminal binding proteins (CtBPs) are ubiquitous master transcriptional coregulators associated with the Polycomb Repressive Complex 2 (PRC2), with essential functions in the development and oncogenesis of various tumor entities, including breast cancer and prostate cancer (PC) [2,3]. As CtBPs bind to short linear PxDLS-like sequence motifs (SLiMs), the presence of such motifs in tandem on the putative metastasis suppressor RAI2 [4] suggested a previously undiscovered dual interaction with CtBPs. Here, we show that RAI2 induces CtBP polymerization through well-ordered filaments of stacked tetrameric CtBP layers, as illustrated by a high-resolution single particle cryo electron microscopy structure. These filaments are mirrored by RAI2-mediated CtBP nuclear foci and relief of CtBP corepressor function in RAI2-expressing cancer cells. Analyses of a diverse PC patient cohort revealed a substantial decrease in RAI2 in the aggressive subtypes, suggesting a pivotal role of RAI2 in the transition to androgen receptor signaling-independent progression. Taken together, our data demonstrate a previously unknown mechanism of multivalent short linear sequence motif-induced polymerization. As RAI2-like SLiM motifs are found in a wide range of organisms, including pathogenic viruses, our findings serve as a paradigm for diverse functional effects through multivalent interaction-mediated polymerization by disordered proteins in healthy and diseased conditions. The specific properties of these repeated interactions open up new therapeutic opportunities.

Abstract 1447: Retinoic acid-induced 2 driven polymerization of c-terminal binding proteins sustains epithelial differentiation in castration-resistant prostate cancer

Abstract Previously, we found that increased Retinoic acid-induced 2 (RAI2) protein concentration in primary tumors predicts early biochemical relapse of prostate cancer patients and described a co-repressor function of RAI2 related to androgen receptor activity (1). The presence of a non-canonical tandem ALDLS motif on RAI2 protein prompted the present investigation on a possible role of a dual interaction of RAI2 with C-terminal binding proteins (CtBPs) in relation to prostate cancer progression. We analyzed RAI2 gene expression as well as CtBP1 protein localization and constitution in circulating tumors cells (CTCs) isolated from metastatic prostate cancer patients by semi-quantitative PCR analysis and immune-fluorescence staining. We employed an integrated structural biology approach combining biophysical techniques like isothermal calorimetry, circular dichroism, size exclusion chromatography with high-resolution structural biology techniques including X-ray crystallography and single particle cryo-electron microscopy. We established RAI2-depleted VCaP cells and used this model for validation of the obtained results. RAI2 gene expression is significantly increased in CTCs isolated from patients with castration-resistant disease in comparison to CTCs isolated from patients with hormone-sensitive, aggressive or neuroendocrine variant prostate cancer. We detected CtBP1 protein foci in the nuclei of individual CTCs. We show that RAI2 induces CtBP polymerization. Using single particle cryo-electron microscopy, we obtained crucial insights into the structural organization of this filament. RAI2 inactivation in VCaP cells caused a significant reduction of both CtBP proteins, which is accompanied by the disappearance of CtBP protein aggregates in nuclei. In addition, we observed the induction of neuroendocrine features, marked by the induction of Synaptophysin and Chromogranin A, a higher nucleus to cytoplasm ratio and smaller cell size of RAI2-depleted cells. In summary, we report tandem motifs-induced polymerization of CtBP’s for the first time. We found that elevated RAI2 gene expression is a characteristic of castration-resistant prostate cancer. Because RAI2 depletion resulted in loss of CtBP aggregates and the induction of neuroendocrine traits, we conclude that RAI2-driven CtBP polymerization sustains epithelial differentiation in castration-resistant prostate cancer. These findings holds potential for the diagnosis of castration-resistant disease and inhibiting the progression from castration-resistant to neuroendocrine prostate cancer. 1. Besler K, Weglarz A, Keller L, von Amsberg G, Bednarz-Knoll N, Offermann A, et al. Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer. Clin Chem 2022;68:973-83 Citation Format: Stefan Werner, Nishit Goradia, Edukondalu Mullapudi, Lina Merkens, Sarah Greimeier, Aleksandra Węglarz, Gunhild von Amsberg, Harriet Wikman, Klaus Pantel, Matthias Wilmanns. Retinoic acid-induced 2 driven polymerization of c-terminal binding proteins sustains epithelial differentiation in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1447.

Expression Patterns and Corepressor Function of Retinoic Acid-induced 2 in Prostate Cancer.

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. 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. 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. 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.

The Biological Role of Cervical Cancer Suppressor 3 (CCS-3): Literature Review

CCS-3 is one of the isoforms of eEF1A1, the key protein of the translation elongation step. Discovered in 2006, it has been the subject of some studies on human cervical cancer cell lines and cervical cancer samples. These studies brought to identify its probable role as a transcriptional co-repressor but other functions are currently unknown. It has also been shown that it is able to inhibit cell growth and induce apoptosis in the tumor cells that downregulate it. Further studies could highlight its functions and its possible usefulness as a tumor marker. This review aims to brief the research about CCS-3 that has been carried out so far.

Genetic analysis argues for a coactivator function for the Saccharomyces cerevisiae Tup1 corepressor.

The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating-type genes. We report here a tup1(S649F) mutant that displays mating irregularities and an α-predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 occupancy changes in mutant vs wild type in both a and α cells. Increased Tup1(S649F) occupancy tended to occur upstream of upregulated genes, whereas locations with decreased occupancy usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including α2. We also found that large changes in mating-type-specific gene expression between a and α or between mutant and wild type were not easily explained by the range of Tup1 occupancy levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 occupancy upstream of the a-specific gene MFA2 and the α-specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with the identification of additional mating-related genes upregulated in the tup1(S649F) α strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated.

KRAB-ZFP Transcriptional Regulators Acting as Oncogenes and Tumor Suppressors: An Overview.

Krüppel-associated box zinc finger proteins (KRAB-ZFPs) constitute the largest family of transcriptional factors exerting co-repressor functions in mammalian cells. In general, KRAB-ZFPs have a dual structure. They may bind to specific DNA sequences via zinc finger motifs and recruit a repressive complex through the KRAB domain. Such a complex mediates histone deacetylation, trimethylation of histone 3 at lysine 9 (H3K9me3), and subsequent heterochromatization. Nevertheless, apart from their repressive role, KRAB-ZFPs may also co-activate gene transcription, likely through interaction with other factors implicated in transcriptional control. KRAB-ZFPs play essential roles in various biological processes, including development, imprinting, retroelement silencing, and carcinogenesis. Cancer cells possess multiple genomic, epigenomic, and transcriptomic aberrations. A growing number of data indicates that the expression of many KRAB-ZFPs is altered in several tumor types, in which they may act as oncogenes or tumor suppressors. Hereby, we review the available literature describing the oncogenic and suppressive roles of various KRAB-ZFPs in cancer. We focused on their association with the clinicopathological features and treatment response, as well as their influence on the cancer cell phenotype. Moreover, we summarized the identified upstream and downstream molecular mechanisms that may govern the functioning of KRAB-ZFPs in a cancer setting.

Salt-inducible kinase 1 maintains HDAC7 stability to promote pathologic cardiac remodeling.

Salt-inducible kinases (SIKs) are key regulators of cellular metabolism and growth, but their role in cardiomyocyte plasticity and heart failure pathogenesis remains unknown. Here, we showed that loss of SIK1 kinase activity protected against adverse cardiac remodeling and heart failure pathogenesis in rodent models and cardiomyocytes derived from human induced pluripotent stem cells. We found that SIK1 phosphorylated and stabilized histone deacetylase 7 (HDAC7) protein during cardiac stress, an event that is required for pathologic cardiomyocyte remodeling. Gain- and loss-of-function studies of HDAC7 in cultured cardiomyocytes implicated HDAC7 as a prohypertrophic signaling effector that can induce c-Myc expression, indicating a functional departure from the canonical MEF2 corepressor function of class IIa HDACs. Taken together, our findings reveal what we believe to be a previously unrecognized role for a SIK1/HDAC7 axis in regulating cardiac stress responses and implicate this pathway as a potential target in human heart failure.

Specific functions for Mediator complex subunits from different modules in the transcriptional response of Arabidopsis thaliana to abiotic stress

Adverse environmental conditions are detrimental to plant growth and development. Acclimation to abiotic stress conditions involves activation of signaling pathways which often results in changes in gene expression via networks of transcription factors (TFs). Mediator is a highly conserved co-regulator complex and an essential component of the transcriptional machinery in eukaryotes. Some Mediator subunits have been implicated in stress-responsive signaling pathways; however, much remains unknown regarding the role of plant Mediator in abiotic stress responses. Here, we use RNA-seq to analyze the transcriptional response of Arabidopsis thaliana to heat, cold and salt stress conditions. We identify a set of common abiotic stress regulons and describe the sequential and combinatorial nature of TFs involved in their transcriptional regulation. Furthermore, we identify stress-specific roles for the Mediator subunits MED9, MED16, MED18 and CDK8, and putative TFs connecting them to different stress signaling pathways. Our data also indicate different modes of action for subunits or modules of Mediator at the same gene loci, including a co-repressor function for MED16 prior to stress. These results illuminate a poorly understood but important player in the transcriptional response of plants to abiotic stress and identify target genes and mechanisms as a prelude to further biochemical characterization.

Histone deacetylase 3 preferentially binds and collaborates with the transcription factor RUNX1 to repress AML1–ETO–dependent transcription in t(8;21) AML

In up to 15% of acute myeloid leukemias (AMLs), a recurring chromosomal translocation, termed t(8;21), generates the AML1-eight-twenty-one (ETO) leukemia fusion protein, which contains the DNA-binding domain of Runt-related transcription factor 1 (RUNX1) and almost all of ETO. RUNX1 and the AML1-ETO fusion protein are coexpressed in t(8;21) AML cells and antagonize each other's gene-regulatory functions. AML1-ETO represses transcription of RUNX1 target genes by competitively displacing RUNX1 and recruiting corepressors such as histone deacetylase 3 (HDAC3). Recent studies have shown that AML1-ETO and RUNX1 co-occupy the binding sites of AML1-ETO-activated genes. How this joined binding allows RUNX1 to antagonize AML1-ETO-mediated transcriptional activation is unclear. Here we show that RUNX1 functions as a bona fide repressor of transcription activated by AML1-ETO. Mechanistically, we show that RUNX1 is a component of the HDAC3 corepressor complex and that HDAC3 preferentially binds to RUNX1 rather than to AML1-ETO in t(8;21) AML cells. Studying the regulation of interleukin-8 (IL8), a newly identified AML1-ETO-activated gene, we demonstrate that RUNX1 and HDAC3 collaboratively repress AML1-ETO-dependent transcription, a finding further supported by results of genome-wide analyses of AML1-ETO-activated genes. These and other results from the genome-wide studies also have important implications for the mechanistic understanding of gene-specific coactivator and corepressor functions across the AML1-ETO/RUNX1 cistrome.

KDM2B in polycomb repressive complex 1.1 functions as a tumor suppressor in the initiation of T-cell leukemogenesis.

KDM2B together with RING1B, PCGF1, and BCOR or BCORL1 comprise polycomb repressive complex 1.1 (PRC1.1), a noncanonical PRC1 that catalyzes H2AK119ub1. It binds to nonmethylated CpG islands through its zinc finger-CxxC DNA binding domain and recruits the complex to target gene loci. Recent studies identified the loss of function mutations in the PRC1.1 gene, BCOR and BCORL1 in human T-cell acute lymphoblastic leukemia (T-ALL). We previously reported that Bcor insufficiency induces T-ALL in mice, supporting a tumor suppressor role for BCOR. However, the function of BCOR responsible for tumor suppression, either its corepressor function for BCL6 or that as a component of PRC1.1, remains unclear. We herein examined mice specifically lacking the zinc finger-CxxC domain of KDM2B in hematopoietic cells. Similar to Bcor-deficient mice, Kdm2b-deficient mice developed lethal T-ALL mostly in a NOTCH1-dependent manner. A chromatin immunoprecipitation sequence analysis of thymocytes revealed the binding of KDM2B at promoter regions, at which BCOR and EZH2 colocalized. KDM2B target genes markedly overlapped with those of NOTCH1 in human T-ALL cells, suggesting that noncanonical PRC1.1 antagonizes NOTCH1-mediated gene activation. KDM2B target genes were expressed at higher levels than the others and were marked with high levels of H2AK119ub1 and H3K4me3, but low levels of H3K27me3, suggesting that KDM2B target genes are transcriptionally active or primed for activation. These results indicate that PRC1.1 plays a key role in restricting excessive transcriptional activation by active NOTCH1, thereby acting as a tumor suppressor in the initiation of T-cell leukemogenesis.

Abstract 923: FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells

Abstract Androgen receptor (AR) plays a critical function in driving prostate cancer (PCa) development and androgen deprivation therapy (ADT) is standard of care in treating PCa patients. Although patients generally response well, the cancer eventually relapse and progress to the castration-resistant stage of PCa (called CRPC), which can be further treated with more aggressive ADT. However, the tumors still develop resistance within a year and restored AR activity is found in the majority of relapsed tumors, indicating a pressing need for developing novel AR-targeted therapies. In PCa cells, AR activity is dependent on the chromatin binding of the pioneer factor, FOXA1. In particular, FOXA1 recognizes the H3K4me1,2 marked enhancers and its binding opens the chromatin for subsequent AR recruitment. Although the role of FOXA1 on AR signaling has been intensively studied, the mechanisms regulating FOXA1 binding to chromatin remain elusive. LSD1/KDM1A was initially identified as a transcription repressor through demethylation of H3K4me1,2. In contrast to its well-established corepressor function, LSD1 has been found to coactivate several transcription factors including AR. This AR coactivator function has been attributed to the phosphorylation of histone 3 on threonine 6 and threonine 11, which may switch LSD1 substrate specificity from H3K4me1,2 to the repressive mark H3K9me1,2. However, we have recently reported that the H3K4 demethylase activity of LSD1 persists at AR-regulated enhancer sites, including sites marked by H3T6ph, arguing against the coactivator function of LSD1 being generally controlled by this mechanism. Importantly, we have shown in a recent study that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 silencing impaired FOXA1 binding at AR-regulated enhancers, suggesting a role of LSD1 in regulating enhancer availability to AR. In the current study, we found that LSD1 inhibition globally disrupts FOXA1 chromatin binding prior to androgen stimulation and thus impairs further AR recruitment, resulting in the global inhibition of AR transcriptome. Using immunoprecipitation and mass-spectrometry assays, we discovered that LSD1 maintains FOXA1 binding by demethylating lysine K270, adjacent to its DNA binding domain. We further found that the expression of a methylation-deficient FOXA1 mutant (K270R) stabilizes AR chromatin binding and allows PCa cells resistant to enzalutamide treatment. Using a clinical tested LSD1 inhibitor, we have shown in vivothat LSD1 inhibition dramatically decreases PCa growth alone and in synergy with enzalutamide in AR/AR variants-positive CRPC models. These findings provide fundamental new insights into FOXA1, LSD1, and mechanisms that regulate the activity of AR in PCa cells. As LSD1 inhibitors are moving into the clinic, these new insights into LSD1 function strongly support LSD1 as a therapeutic target in CRPC. Citation Format: Shuai Gao, Sujun Chen, Dong Han, Wanting Han, Jude N. Owiredu, Steven P. Balk, Housheng H. He, Changmeng Cai. FOXA1 chromatin binding is regulated by LSD1-mediated methylation in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 923.

Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes to the regulation of hypoxia and estrogen signaling in breast cancer cells

BackgroundExosomes are small membrane-bound vesicles that contribute to tumor progression and metastasis by mediating cell-to-cell communication and modifying the tumor microenvironment at both local and distant sites. However, little is known about the predominant factors in exosomes that contribute to breast cancer (BC) progression. MTA1 is a transcriptional co-regulator that can act as both a co-activator and co-repressor to regulate pathways that contribute to cancer development. MTA1 is also one of the most up-regulated proteins in cancer, whose expression correlates with cancer progression, poor prognosis and increased metastatic potential.MethodsWe identified MTA1 in BC exosomes by antibody array and confirmed expression of exosome-MTA1 across five breast cancer cells lines. Ectopic expression of tdTomato-tagged MTA1 and exosome transfer were examined by fluorescent microscopy. CRISPR/Cas9 genetic engineering was implemented to knockout MTA1 in MCF7 and MDA-MB-231 breast cancer cells. Reporter assays were used to monitor hypoxia and estrogen receptor signaling regulation by exosome-MTA1 transfer.ResultsEctopic overexpression of tdTomato-MTA1 in BC cell lines demonstrated exosome transfer of MTA1 to BC and vascular endothelial cells. MTA1 knockout in BC cells reduced cell proliferation and attenuated the hypoxic response in these cells, presumably through its co-repressor function, which could be rescued by the addition of exosomes containing MTA1. On the other hand, consistent with its co-activator function, estrogen receptor signaling was enhanced in MTA1 knockout cells and could be reversed by addition of MTA1-exosomes. Importantly, MTA1 knockout sensitized hormone receptor negative cells to 4-hydroxy tamoxifen treatment, which could be reversed by the addition of MTA1-exosomes.ConclusionsThis is the first report showing that BC exosomes contain MTA1 and can transfer it to other cells resulting in changes to hypoxia and estrogen receptor signaling in the tumor microenvironment. These results, collectively, provide evidence suggesting that exosome-mediated transfer of MTA1 contributes to BC progression by modifying cellular responses to important signaling pathways and that exosome-MTA1 may be developed as a biomarker and therapeutic target for BC.

The C-terminal WD40 repeats on the TOPLESS co-repressor function as a protein–protein interaction surface

The two predicted WD40 propellers on TOPLESS function as protein-protein interaction domains. The 1st WD40 propeller mediates interaction with RAV1, and the 2nd WD40 propeller mediates interaction with VRN5. The TOPLESS/TOPLESS-RELATED (TPL/TPR) co-repressor family proteins are known to interact with a wide variety of proteins including transcription factors, Mediator subunits, histone deacetylases, and histone tails. Through these interactions, TPL/TPR act to repress transcription in an increasingly diverse array of plant pathways. Proteins that bind TPL/TPR typically contain one or more Repression Domains (RDs) that mediate the interaction. For example, the well-characterized Ethylene response factor-associated Amphiphilic Repression (EAR) motif is known to facilitate interaction by binding the TOPLESS Domain (TPD) located in the N-terminus. Here we show that in yeast two-hybrid assays, the non-EAR protein, Related to ABI3/VP1-1 (RAV1), binds a novel region located within the first nine WD40-repeats of TPL. Protein modeling and in silico analysis suggest that these nine WD40 repeats may form the first of two WD40 propellers located on C-terminus of TPL. The interaction between RAV1 and the 1st WD40 propeller is conserved with another RAV family member, TEMPRANILLO1 (TEM1) and is mediated by the B3 Repression Domain (BRD) located on both RAV1 and TEM1. Also, the predicted 2nd WD40 propeller was shown in yeast cells to bind Vernalization 5 (VRN5), which contains several unconfirmed partial RDs. Furthermore, we demonstrate that the 1st WD40 propeller of TPL can form a complex with RAV1 both in yeast and in Arabidopsis protoplasts.

Epigenetic reactivation of LINE-1 retrotransposon disrupts NuRD corepressor functions and induces oncogenic transformation in human bronchial epithelial cells.

Long interspersed nuclear element‐1 (LINE‐1 or L1) reactivation is linked to poor prognosis in non‐small‐cell lung carcinoma (NSCLC), but the molecular bases of this response remain largely unknown. In this report, we show that challenge of human bronchial epithelial cells (HBECs) with the lung carcinogen, benzo(a)pyrene (BaP), shifted the L1 promoter from a heterochromatic to euchromatic state through disassembly of the nucleosomal and remodeling deacetylase (NuRD) complex. Carcinogen challenge was also associated with partial displacement of constituent proteins from the nuclear to the cytoplasmic compartment. Disruption of NuRD corepression by genetic ablation or carcinogen treatment correlated with accumulation of L1 mRNA and proteins. Mi2β bound directly to the L1 promoter to effect retroelement silencing, and this response required the DNA‐ and ATPase‐binding domains of Mi2β. Sustained expression of L1 in HBECs was tumorigenic in a human–SCID mouse xenograft model, giving rise to tumors that regressed over time. Together, these results show that functional modulation of the NuRD constituent proteins is a critical molecular event in the activation of L1 retrotransposon. L1 expression creates a microenvironment in HBECs that is conducive to neoplasia and malignant transformation.

15 DISRUPTION OF FOXP3-EZH2 INTERACTION BY GENETIC MUTATION OR SIGNALING-INDUCED INACTIVATION REPRESENTS A NOVEL PATHOBIOLOGICAL MECHANISM IN INFLAMMATORY BOWEL DISEASE

While FOXP3+ regulatory T cell (Treg) dysfunction has been linked to human inflammatory bowel disease (IBD), molecular mechanisms for disease pathophysiology are unclear. The transcription factor FOXP3 via histone methyltransferase EZH2 suppresses inflammation in mice. Here, we tested the hypothesis that a physical FOXP3-EZH2 protein interaction is essential for gene co-repressive function critical for human Treg physiological functions. Disease-relevant human FOXP3 mutations by site-directed mutagenesis, T cell isolation from human blood donors and lamina propria of Crohn’s disease (CD) patients, Proximity Ligation Assay (PLA), confocal microscopy, co-immunoprecipitation (co-IP), Dual Luciferase Reporter (DLR) Assay, Chromatin-immunoprecipitation (ChIP), RT-PCR, Immunofluorescence and Fluorescence-activated Cell Sorting. PLA, confocal imaging and co-IP studies revealed that constitutive FOXP3-EZH2 interaction was uniquely abrogated by early-onset IBD-associated FOXP3 cysteine 232 mutation in contrast to IPEX-FOXP3 variants. Furthermore, FOXP3-C232 mutant displayed impaired repression of IL17 and IFNG genes via DLR and ChIP-PCR respectively, indicative of compromised Treg-physiologic function. Exploring a generalizable mechanism, IL-6 impaired FOXP3-EZH2 interaction in a manner that correlated with increased tyrosine phosphorylation of FOXP3. Notably, IL-6-induced effects were reversed by a clinically available JAK1/2 inhibitor ruxolitinib. Remarkably, in lamina propria-derived CD4+ T cells, reduced FOXP3-EZH2 complex was a feature of CD patients in contrast to non-CD control subjects. Destabilized FOXP3-EZH2 protein interaction and consequent impairment of EZH2-mediated co-repressor function may drive the pathological characteristics of intestinal inflammation. Our study indicates potential therapeutic approaches and mechanisms for improving Treg function during IBD pathogenesis via stabilizing FOXP3-EZH2 interaction.

NCOA5 promotes proliferation, migration and invasion of colorectal cancer cells via activation of PI3K/AKT pathway.

The nuclear receptor coactivator 5 (NCOA5) displays both coactivator and corepressor functions. Previous studies showed that alteration of NCOA5 participates in carcinogenesis and progression. However, its roles in colorectal cancer (CRC) remain unknown. Herein, we demonstrated that expression of NCOA5 in human CRC tissues was notably higher than that in adjacent tissues, which significantly correlated with clinicopathological features such as length of tumor, regional lymph node staging and cancer staging. Knockdown of NCOA5 markedly suppressed proliferation, migration and invasion of SW620 high malignant CRC cells. Silencing of NCOA5 also inhibited in vivo growth of SW620 CRC subcutaneously xenografted tumors in athymic BALB/c nude mice. Meanwhile, Overexpression of NCOA5 facilitated these processes in SW480 low malignant CRC cells. Furthermore, knockdown of NCOA5 induced cell cycle G1 phase arrest in SW620 cells, whereas overexpression of NCOA5 promoted G1 to S phase transition in SW480 cells. Mechanistic studies revealed that NCOA5 upregulated phospho-protein kinase B (p-PKB/AKT), Cyclin D1 and matrix metalloproteinase 9 (MMP9) as well as downregulated P27 in CRC cells. Notably, PI3K inhibitor LY294002 obviously attenuated the effects of NCOA5 on p-AKT, Cyclin D1, P27 and MMP9. Moreover, LY294002 and knockdown of Cyclin D1 or MMP9 remarkably blocked the tumor-promoting activity of NCOA5. Collectively, NCOA5 promoted CRC cell proliferation, migration and invasion by upregulating Cyclin D1 and MMP9 while downregulating P27 to a great extent via activating PI3K/AKT signaling pathway. These findings suggested that NCOA5 exhibits an oncogenic effect in human CRC and represents a novel therapeutic target for CRC.

Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression

Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions.

Corepressor diversification by alternative mRNA splicing is species specific.

BackgroundSMRT and NCoR are corepressor paralogs that help mediate transcriptional repression by a variety of transcription factors, including the nuclear hormone receptors. The functions of both corepressors are extensively diversified in mice by alternative mRNA splicing, generating a series of protein variants that differ in different tissues and that exert different, even diametrically opposite, biochemical and biological effects from one another.ResultsWe report here that the alternative splicing previously reported for SMRT appears to be a relatively recent evolutionary phenomenon, with only one of these previously identified sites utilized in a teleost fish and a limited additional number of the additional known sites utilized in a bird, reptile, and marsupial. In contrast, extensive SMRT alternative splicing at these sites was detected among the placental mammals. The alternative splicing of NCoR previously identified in mice (and shown to regulate lipid and carbohydrate metabolism) is likely to have arisen separately and after that of SMRT, and includes an example of convergent evolution.ConclusionsWe propose that the functions of both SMRT and NCoR have been diversified by alternative splicing during evolution to allow customization for different purposes in different tissues and different species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0781-2) contains supplementary material, which is available to authorized users.