Corepressor Binding Research Articles

Conformational Diversity of the Helix 12 of the Ligand Binding Domain of PPARγ and Functional Implications

Nuclear hormone receptors (NR) are transcription factors that activate gene expression in response to ligands. Structural and functional studies of the ligand binding domains (LBD) of NRs revealed that the dynamics of their C-terminal helix (H12) is fundamental for NR activity. H12 is rigid and facilitates binding of coactivator proteins in the agonist-bound LBD. In the absence of ligand, H12 exhibits increased flexibility. To provide a comprehensive picture of the H12 conformational equilibrium, extensive molecular dynamics simulations of the LBD of the PPARγ receptor in the presence or absence of ligand, and of coactivators and corepressor peptides, were performed. Free-energy profiles of the conformational variability of the H12 were obtained from more than four microseconds of simulations using adaptive biasing-force calculations. Our results demonstrate that, without ligand, multiple conformations of the H12 are accessible, including agonist-like conformations. We also confirm that extended H12 conformations are not accessible at ordinary temperatures. Ligand binding stabilizes the agonist H12 conformation relative to other structures, promoting a conformational selection. Similar effects are observed with coactivator association. The presence of corepressor peptides stabilizes conformations not allowed in the ligand-free, Rosiglitazone-bound or coactivator-bound LBDs. Corepressor binding, therefore, induces a conformational transition in the protein. Nevertheless, initial stages of corepressor dissociation could be induced by the ligand as it stabilizes the H12 in agonist form. Therefore, the present results provide a comprehensive picture of the H12 motions and their functional implications, with molecular resolution.

STUbL-mediated degradation of the transcription factor MATα2 requires degradation elements that coincide with corepressor binding sites.

The yeast transcription factor MATα2 (α2) is a short-lived protein known to be ubiquitylated by two distinct pathways, one involving the ubiquitin-conjugating enzymes (E2s) Ubc6 and Ubc7 and the ubiquitin ligase (E3) Doa10 and the other operating with the E2 Ubc4 and the heterodimeric E3 Slx5/Slx8. Although Slx5/Slx8 is a small ubiquitin-like modifier (SUMO)-targeted ubiquitin ligase (STUbL), it does not require SUMO to target α2 but instead directly recognizes α2. Little is known about the α2 determinants required for its Ubc4- and STUbL-mediated degradation or how these determinants substitute for SUMO in recognition by the STUbL pathway. We describe two distinct degradation elements within α2, both of which are necessary for α2 recognition specifically by the Ubc4 pathway. Slx5/Slx8 can directly ubiquitylate a C-terminal fragment of α2, and mutating one of the degradation elements impairs this ubiquitylation. Surprisingly, both degradation elements identified here overlap specific interaction sites for α2 corepressors: the Mcm1 interaction site in the central α2 linker and the Ssn6 (Cyc8) binding site in the α2 homeodomain. We propose that competitive binding to α2 by the ubiquitylation machinery and α2 cofactors is balanced so that α2 can function in transcription repression yet be short lived enough to allow cell-type switching.

Cutting edge: T follicular helper cell differentiation is defective in the absence of Bcl6 BTB repressor domain function.

T follicular helper (Tfh) cells are essential for germinal centers (GCs) and most long-term humoral immunity. Differentiation of Tfh cells depends on the transcriptional repressor B cell CLL/lymphoma 6 (Bcl6). Bcl6 mediates gene repression via the recruitment of corepressors. Currently, it is unknown how Bcl6 recruits corepressors to regulate gene expression of Tfh cells. In this article, we demonstrate, using a mutant form of Bcl6 with two BTB (bric-a-brac, tramtrack, broad-complex) mutations that abrogate corepressor binding, that the Bcl6 BTB domain is required for proper differentiation of Tfh and GC-Tfh cells in vivo. Importantly, we also observe a significant defect in GC B cell development. These results are consistent in multiple contexts, including a novel lymphocytic choriomeningitis virus nucleoprotein-specific TCR-transgenic mouse model. Taken together, these data suggest that the Bcl6 BTB domain is a key mediator of the differentiation of Tfh cells.

Molecular mechanisms of transcriptional control by Rev-erbα: An energetic foundation for reconciling structure and binding with biological function.

Rev-erbα and β are nuclear receptors that function as transcriptional repressors of genes involved in regulating circadian rhythms, glucose, and cholesterol metabolism and the inflammatory response. Given these key functions, Rev-erbs are important drug targets for treatment of a number of human pathologies, including cancer, heart disease, and type II diabetes. Transcriptional repression by the Rev-erbs involves direct competition with transcriptional activators for target sites, but also recruitment by the Rev-erbs of the NCoR corepressor protein. Interestingly, Rev-erbs do not appear to interact functionally with a very similar corepressor, Smrt. Transcriptional repression by Rev-erbs is thought to occur in response to the binding of heme, although structural, and ligand binding studies in vitro show that heme and corepressor binding are antagonistic. We carried out systematic studies of the ligand and corepressor interactions to address the molecular basis for corepressor specificity and the energetic consequences of ligand binding using a variety of biophysical approaches. Highly quantitative fluorescence anisotropy assays in competition mode revealed that the Rev-erb specificity for the NCoR corepressor lies in the first two residues of the β-strand in Interaction Domain 1 of NCoR. Our studies confirmed and quantitated the strong antagonism of heme and corepressor binding and significant stabilization of the corepressor complex by a synthetic ligand in vitro. We propose a model which reconciles the contradictory observations concerning the effects of heme binding in vitro and in live cells.

Differential effects of estrogen-dependent transactivation vs. transrepression by the estrogen receptor on invasiveness of HER2 overexpressing breast cancer cells

Estrogen (E2) supports breast cancer cell growth but suppresses invasiveness and both actions are antagonized by anti-estrogens. As a consequence, anti-estrogen treatment may increase the invasive potential of estrogen receptor (ER)+ tumor cell sub-populations that are endocrine resistant due to HER2 amplification. Either transactivation or transrepression by E2/ER could lead to both up- and down-regulation of many genes. Inhibition of the transactivation function of ER is adequate to inhibit E2-dependent growth. However, the impact of inhibiting E2-dependent transactivation vs. transrepression by ER on regulation of invasiveness by E2 is less clear. Here we dissect the roles of ER-mediated transactivation and transrepression in the regulation of invasiveness of ER+/HER2+ breast cancer cells by E2. Knocking down the general ER co-activators CBP and p300 prevented activation by E2 of its classical target genes but did not interfere with the ability of E2 to repress its direct target genes known to support invasiveness and tumor progression; there was also no effect on invasiveness or the ability of E2 to regulate invasiveness. On the other hand, overexpression of a co-repressor binding site mutant of ER (L372R) prevented E2-dependent transrepression but not transactivation. The mutant ER abrogated the ability of E2 to suppress invasiveness. E2 can partially down-regulate HER2 but knocking down HER2 below E2-regulated levels did not affect invasiveness or the ability of E2 to regulate invasiveness, although it did inhibit growth. Therefore, in ER+/HER2+ cells, the E2-dependent transrepression by ER rather than its transactivation function is critical for regulation of invasiveness and this is independent of HER2 regulation by E2. The findings suggest that selective inhibitors of transactivation by ER may be more beneficial in reducing tumor progression than conventional anti-estrogens that also antagonize E2-dependent transrepression.

Critical Role of a Glycine Residue in an Allosteric Switch

Thermodynamic coupling in proteins is ubiquitous but mechanistically poorly understood, particularly for systems in which the coupling occurs over large distances. In the E. coli biotin repressor the coupling between homodimerization and allosteric effector, bio-5′-AMP, is −4.0 kcal/mol. The two coupled processes occur at sites separated by 33A. Structural and thermodynamic studies indicate that the coupling is accompanied by disorder-to-order transitions at the two distant functional sites. Perturbations to the transition at the ligand binding site via alanine substitutions alter both ligand binding and coupled dimerization. Alanine substitutions in four loops in the dimerization surface yield a range of energetic consequences for BirA dimerization. For one of these variants, BirAG142A, the free energies of dimerization and corepressor binding are consistent with complete abolition of coupling. Structural studies of the variant indicate that the loss of coupling is accompanied by disruption of the disorder-to-order transitions at both functional surfaces. In this work the structural basis of coupling between the dimerization and ligand binding surfaces was further investigated by measuring the consequences of alanine substitutions distributed in three of the dimerization loops on corepressor binding. Isothermal titration calorimetry measurements indicate that, in contrast to BirAG142A, the variants, several of which dimerize very weakly, all bind to bio-5′-AMP with energetics indistinguishable from wild-type BirA. The results indicate that a single glycine residue serves as a switch for allosteric regulation of BirA.

Abstract 1383: Evolution of the NCOR1 and NCOR2/SMRT cistromes in prostate cancer progression

Abstract The archetypical co-repressors NCOR1 and NCOR2/SMRT exist in large complexes with histone deacetylases and significantly control multiple transcription factors (TFs), including nuclear receptors such as the VDR and AR. NCOR1 and NCOR2/SMRT are altered in cancer by mechanisms, including mutation, but it remains unclear how this disrupts their unique and shared functions either to commission or decommission enhancers through the genome. Previously, we established that altered co-repressor recruitment in prostate cancer (CaP) induces sustained and targeted H3K9me2 at target gene loci that drives increased CpG methylation. We propose that the oncogenic co-repressor cistromes drive CaP progression by inducing repressive histone modifications that in turn trigger methylated CpG regions at selective enhancers leading to stable and heritable changes to the transcriptome. Using a high throughput micro-fluidic Q-ChIP approach we examined NCOR1, NCOR2/SMRT, H3K9me2, VDR, AR, and RNA POL2 levels at 47 genomic regions distributed over 8 VDR and 10 AR target genes in 5 normal and malignant prostate cell line models, at two time points following either 1α,25(OH)2D3 (D3) or DHT treatment. Binding patterns of NCOR1, NCOR2/SMRT and H3K9me2 distinguished normal, androgen sensitive and castrate recurrent CaP models. Specifically, co-repressor binding was enriched on VDR and AR target genes in a gene-specific manner across the CaP models. For example, PDK2 and BMP2 are AR target genes that had significantly more NCOR2/SMRT binding and gain of CpG methylation in LNCaP C4-2 cells, compared to the isogenic LNCaP. For these targets, and the others examined, gene expression loss and increased CpG methylation was mirrored by the TCGA CaP data, and a more general positive correlation between NCOR2/SMRT and CpG methylation was identified on the genome-wide level in the TCGA CaP data. In LNCaP and LNCaP C4-2 cells genome-wide binding of NCOR1 and NCOR2/SMRT was examined by ChIP-seq to measure the unique and shared cistromes in the emergence of castrate recurrent phenotype. In the first instance, analyses of NCOR1 binding in LNCaP cells revealed very significant enrichment at several TFs including SRF and GATA family members; the most significantly enriched nuclear receptor was RARβ. Subsequent GO term enrichment analyses revealed these genes were involved in metabolic process and chromatin/gene silencing pathways. Finally, we examined NCOR1 and NCOR2/SMRT staining on a TMA of 480 men with CaP who underwent radical prostatectomy. In the first instance, the analysis has revealed that elevated NCOR2/SMRT staining intensity was significantly associated with time to treatment failure. These findings support the concept that NCOR1 and NCOR2/SMRT recruitment is significantly altered during CaP progression to drive selective, stable and heritable silencing of target genes for multiple transcription factors and is associated with poor outcome in CaP. Citation Format: Prashant K. Singh, Mark D. Long, Vineet K. Dhiman, Qianqian Zhu, Lara Sucheston-Campbell, Dominic Smiraglia, Moray J. Campbell. Evolution of the NCOR1 and NCOR2/SMRT cistromes in prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1383. doi:10.1158/1538-7445.AM2014-1383

Abstract 2111: Role of gene repression by estrogen in invasiveness of breast cancer cells

Abstract More than 75% of breast tumors are estrogen receptor (ER)-positive and require estrogen for their growth and survival. Long-term adjuvant therapies that disrupt estrogen signaling, in particular tamoxifen and aromatase inhibitors, are a mainstay in breast cancer treatment. The principal focus of mechanistic studies evaluating the role of estrogen in breast cancer proliferation has been on gene activation by estrogen and its antagonism by anti-estrogens. Gene repression by estrogen and its antagonism by anti-estrogens have unclear physiological and therapeutic consequence in breast cancer. In this study, we found that estrogen-repressed genes but not estrogen-activated genes overlapped the gene overexpression signature of clinical progression of ductal carcinoma in situ to invasive ductal carcinoma. An extensive gene ontology analysis revealed that genes repressed by estrogen are enriched for functions known to collectively support breast cancer progression. In ER-positive breast cancer cells tamoxifen prevented gene repression by estrogen. In these cells, estrogen inhibited invasiveness in a manner that was opposed by tamoxifen. These effects of estrogen and tamoxifen on gene repression and invasiveness also occurred in Her2+/ER+ cells whose growth was hormone-independent and insensitive to tamoxifen. The effects were also independent of ErbB2 gene regulation by estrogen. Several ER co-repressors supported repression of the tumor progression genes by estrogen. Gene repression by estrogen was causally linked to inhibition of invasiveness as a co-repressor binding site mutation in ER that selectively disrupted the ability of estrogen to repress genes also prevented inhibition of invasiveness. Gene repression by estrogen involved ER binding at non-classical chromatin sites that was prevented by tamoxifen. The findings offer a mechanistic understanding of the invasive potential of ER+ breast cancer cells that have evaded growth inhibition during conventional anti-estrogen treatments. Citation Format: Mugdha Patki, Marcela d'Alincourt Salazar, Robert Trumbly, Manohar Ratnam. Role of gene repression by estrogen in invasiveness of breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2111. doi:10.1158/1538-7445.AM2014-2111

Abstract 2723: Breast cancer type 1 susceptibility protein mimetics induce specific estrogen receptor confirmations and inhibit MCF7 breast cancer cell proliferation

Abstract The human Estrogen Receptor (hER) is present in 70% of breast cancers, and dysregulation of hER signaling can lead to aberrant cell growth and proliferation. Known anti-estrogens used in treatments of breast cancer today, tamoxifen and raloxifene cause hER to undergo conformational changes and inhibit normal hER signaling by directly binding to the ligand binding pocket. However, often breast cancer cells become resistant to tamoxifen, and prolonged tamoxifen treatment can lead to endometrial cancers. Breast Cancer Type 1 Susceptibility protein (BRCA1) is a well characterized inhibitor of breast cancer cell growth. BRCA1 plays an important role in maintaining genomic stability through various DNA repair mechanisms. It is mutated in approximately 50% of breast cancer cases that are associated with heredity. While the majority of breast cancer cases are “sporadic” or unassociated with heredity, BRCA1 is expressed 30 to 40 % less in sporadic breast cancers. BRCA1 has also been shown to associate directly with hER, and inhibit transactivation of hER. This association and inhibition of hER transcriptional activity is reversed by cyclin D1. This, in conjunction with studies that have shown that cyclin D1 gene and protein levels are amplified and overexpressed respectively in breast cancers suggest that the normal regulation of hER by BRCA1 and cyclin D1 may be shifted in breast cancers to primarily regulation (activation) by cyclin D1 and other coactivators. A decrease in BRCA1 is associated with acquired tamoxifen resistance, suggesting that BRCA1 may play a critical role in maintaining breast cancer cells sensitivity to tamoxifen. The hER binding region of BRCA1 contains the known corepressor binding motif LXXIIXXXL (amino acids 78-95). Here we present a study of several small molecules identified based on pharmacophoric features of previously found conformation sensing BRCA1 peptides. BRCA1 mimetics induce unique ER conformations and inhibit breast cancer cell growth (MCF7). Further development of these compounds could lead to viable alternative breast cancer treatments as well as supplemental treatments in the anti-estrogen resistant cases. Acknowledgements: This research was supported via funds from the New York State, National Institutes of Health (5P20MD002725), through the NCMHD -RIMI program (RVR) and NSF LSAMP Bridge to Doctorate grant (BKA). Citation Format: Bethany Kristen Asare, Christ Ange Katche, Rajendram V. Rajnarayanan. Breast cancer type 1 susceptibility protein mimetics induce specific estrogen receptor confirmations and inhibit MCF7 breast cancer cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2723. doi:10.1158/1538-7445.AM2014-2723

Structural insights into selective agonist actions of tamoxifen on human estrogen receptor alpha.

Tamoxifen-an anti-estrogenic ligand in breast tissues used as a first-line treatment in estrogen receptor (ER)-positive breast cancers-is associated with the development of resistance followed by resumption of tumor growth in about 30% of cases. Whether tamoxifen assists in proliferation in such cases or whether any ligand-independent pathway to transcription exists is not fully understood; also, no ERα mutants have been detected so far that could lead to tamoxifen resistance. Using in silico conformational analysis of the ERα ligand binding domain (LBD), in the absence and presence of selective agonist (diethylstilbestrol; DES), antagonist (Faslodex; ICI), and selective estrogen receptor modulator (SERM; 4-hydroxy tamoxifen; 4-OHT) ligands, wehave elucidated ligand-responsive structural modulations of the ERα-LBD dimer in its agonist and antagonist complexes to address the issue of "tamoxifen resistance". DES and ICI were found to stabilize the dimer in their agonist and antagonist conformations, respectively. The ERα-LBD dimer without the presence of any bound ligand also led to a stable structure in agonist conformation. However, binding of 4-OHT to the antagonist structure led to a flexible conformation allowing the protein to visit conformations populated by agonists as was evident from principal component analysis and radius of gyration plots. Further, the relaxed conformations of the 4-OHT bound protein exhibited a diminished size of the co-repressor binding pocket in the LBD, thus signaling a partial blockage of the co-repressor binding motif. Thus, the ability of 4-OHT-bound ERα-LBD to assume flexible conformations visited by agonists and reduced co-repressor binding surface at the LBD provide crucial structural insights into tamoxifen-resistance that complement our existing understanding.

Allosteric Coupling via Communication of Distal Disorder-To-Order Transitions

Disorder-to-order transitions in proteins can play critical roles in regulating function. Corepressor bio-5'-AMP binding to the E. coli biotin repressor promotes homodimerization that is a prerequisite to sequence-specific DNA binding and resulting transcription repression. Folding of a loop segment comprised of residues 211-222 in the corepressor binding site is required to obtain the full −4.0 kcal/mole energetic coupling between bio-5'-AMP binding and dimerization. Several additional loops, some of which are disordered in the unliganded monomer but folded in the liganded dimer, participate directly in the homodimer interface. Alanine scanning mutagenesis has demonstrated the functional significance of these loops for dimerization. Replacement of a glycine at position 142 in loop 140-146 with alanine results in complete abolition of coupling of corepressor binding to dimerization. Consistent with solution measurements and in contrast to wild type BirA, the structure of this variant solved by x-ray crystallography reveals a monomeric liganded protein. Moreover, the alanine replacement results in adoption of an alternative conformation by the 140-146 loop. Furthermore, distinct from wild type corepressor-bound BirA, both a neighboring dimer interface loop comprised of residues 193-199 and the 211-222 loop that is located 33 Å away from the alanine replacement are disordered in the liganded variant. These results support a model for energetic coupling between ligand binding and dimerization in which distant disorder-to-order transitions are communicated through the folded protein core.

A Hybrid Mechanism of Action for BCL6 in B Cells Defined by Formation of Functionally Distinct Complexes at Enhancers and Promoters

The BCL6 transcriptional repressor is required for the development of germinal center (GC) B cells and diffuse large B cell lymphomas (DLBCLs). Although BCL6 can recruit multiple corepressors, its transcriptional repression mechanism of action in normal and malignant B cells is unknown. We find that in B cells, BCL6 mostly functions through two independent mechanisms that are collectively essential to GC formation and DLBCL, both mediated through its N-terminal BTB domain. These are (1) the formation of a unique ternary BCOR-SMRT complex at promoters, with each corepressor binding to symmetrical sites on BCL6 homodimers linked to specific epigenetic chromatin features, and (2) the "toggling" of active enhancers to a poised but not erased conformation through SMRT-dependent H3K27 deacetylation, which is mediated by HDAC3 and opposed by p300 histone acetyltransferase. Dynamic toggling of enhancers provides a basis for B cells to undergo rapid transcriptional and phenotypic changes in response to signaling or environmental cues.

CtBP1 is involved in epithelial-mesenchymal transition and is a potential therapeutic target for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly invasive type of cancer. Metastasis is the leading cause of mortality of advanced HCC patients. In the metastasis cascade, cancer cells undergo epithelial-mesenchymal transition resulting in the loss of cell‑to‑cell adhesion, migration and invasion into the stroma. Loss of E-cadherin expression is a key molecular event in epithelial-mesenchymal transition through several regulatory mechanisms including epigenetic modification, regulation by inhibitory transcriptional factors and deletion of chromosome 16q24 locus. C-terminal binding protein1 (CtBP1) functions as a corepressor binding to several transcriptional factors and suppresses E-cadherin expression. We found that CtBP1 was upregulated in HCC when compared with paired normal liver tissues and was inversely correlated with E-cadherin expression in HCC by immunohistochemical assay using tissue array. Western blot analysis confirmed the results of the immunohistochemical assays. When CtBP1 was knocked down by siRNA in HepG2 cells (a human HCC cell line), E-cadherin was upregulated and the invasive ability of HepG2 cells was inhibited. In addition, following CtBP1 knockdown, the cell viability was decreased along with increased apoptosis rather than cell cycle arrest. These data suggest a pivotal role of CtBP1 in EMT of HCC, and its potential as a therapeutic target in human disease.

Recruitment of NCOR1 to VDR target genes is enhanced in prostate cancer cells and associates with altered DNA methylation patterns

The current study investigated transcriptional distortion in prostate cancer cells using the vitamin D receptor (VDR) as a tool to examine how epigenetic events driven by corepressor binding and CpG methylation lead to aberrant gene expression. These relationships were investigated in the non-malignant RWPE-1 cells that were 1α,25(OH)(2)D(3) responsive (RWPE-1) and malignant cell lines that were 1α,25(OH)(2)D(3) partially responsive (RWPE-2) and resistant (PC-3). These studies revealed that selective attenuation and repression of VDR transcriptional responses in the cancer cell lines reflected their loss of antiproliferative sensitivity. This was evident in VDR target genes including VDR, CDKN1A (encodes p21( (waf1/cip1) )) and GADD45A; NCOR1 knockdown alleviated this malignant transrepression. ChIP assays in RWPE-1 and PC-3 cells revealed that transrepression of CDKN1A was associated with increased NCOR1 enrichment in response to 1α,25(OH)(2)D(3) treatment. These findings supported the concept that retained and increased NCOR1 binding, associated with loss of H3K9ac and increased H3K9me2, may act as a beacon for the initiation and recruitment of DNA methylation. Overexpressed histone methyltransferases (KMTs) were detectable in a wide panel of prostate cancer cell lines compared with RWPE-1 and suggested that generation of H3K9me2 states would be favored. Cotreatment of cells with the KMT inhibitor, chaetocin, increased 1α,25(OH)(2)D(3)-mediated induction of CDKN1A expression supporting a role for this event to disrupt CDKN1A regulation. Parallel surveys in PC-3 cells of CpG methylation around the VDR binding regions on CDKN1A revealed altered basal and VDR-regulated DNA methylation patterns that overlapped with VDR-induced recruitment of NCOR1 and gene transrepression. Taken together, these findings suggest that sustained corepressor interactions with nuclear-resident transcription factors may inappropriately transform transient-repressive histone states into more stable and repressive DNA methylation events.

Dissecting and Circumventing the Requirement for RAM in CSL-Dependent Notch Signaling

The Notch signaling pathway is an intercellular communication network vital to metazoan development. Notch activation leads to the nuclear localization of the intracellular portion (NICD) of the Notch receptor. Once in the nucleus, NICD binds the transcription factor CSL through a bivalent interaction involving the high-affinity RAM region and the lower affinity ANK domain, converting CSL from a transcriptionally-repressed to an active state. This interaction is believed to directly displace co-repressor proteins from CSL and recruit co-activator proteins. Here we investigate the consequences of this bivalent organization in converting CSL from the repressed to active form. One proposed function of RAM is to promote the weak ANK:CSL interaction; thus, fusion of CSL-ANK should bypass this function of RAM. We find that a CSL-ANK fusion protein is transcriptionally active in reporter assays, but that the addition of RAM in trans further increases transcriptional activity, suggesting another role of RAM in activation. A single F235L point substitution, which disrupts co-repressor binding to CSL, renders the CSL-ANK fusion fully active and refractory to further stimulation by RAM in trans. These results suggest that in the context of a mammalian CSL-ANK fusion protein, the main role of RAM is to displace co-repressor proteins from CSL.

Tetrabromobisphenol-A disrupts thyroid hormone receptor alpha function in vitro: Use of fluorescence polarization to assay corepressor and coactivator peptide binding

Thyroid hormone receptors (TRs) recruit corepressor or coactivator factors to the promoters of target genes to regulate their transcription. Corepressors such as nuclear hormone receptor corepressor (NCoR) are recruited by unliganded TRs, whereas coactivators such as steroid receptor coactivator-2 (SRC2) are recruited when triiodothyronine (T3) is bound to TRs. These coregulator proteins interact with the ligand binding domain (LBD) of TRs via short, conserved peptide sequences that can be used to probe the conformational changes induced in TR LBD by TR ligands. Recombinant LBD of the human TRα1 isoform (hTRα1 LBD) was produced as a fusion with glutathione S-transferase, and used to develop assays based on fluorescence polarization to quantify the binding of either NCoR- or SRC2-derived fluorescent peptides to the hTRα1 LBD. The optimum concentrations of recombinant hTRα1 LBD, and of peptide probes were adjusted in order to produce the greatest possible T3-dependent signal variations in fluorescence polarization. Under these conditions, T3 induced a dose-dependent decrease in NCoR peptide binding, and a reciprocal dose-dependent increase in SRC2 peptide binding, in both cases at similar 50%-effective doses. The TR agonists triiodothyroacetic acid and thyroxine were also effective in preventing NCoR peptide binding and increasing SRC2 peptide binding, whereas reverse-triiodothyronine was less efficient and the biologically inactive thyronine had no effect on either process. These experiments validate cell-free assays based on the use of binding of corepressor or coactivator peptide probes, as measured by fluorescence polarization, for investigating the conformational changes of TRα1 LBD induced by potentially TR-interfering compounds. Both these methods were used to elucidate the mechanism of the disrupting effects of tetrabromobisphenol-A (TBBPA) on the hTRα1 LBD conformation related to the transcriptional activity of the receptor. TBBPA is a flame retardant that is released into the environment, and is a suspected disrupter of thyroid homeostasis. The present results indicate that TBBPA did indeed interfere with the ability of the hTRα1 LBD to bind both NCoR and SRC2. TBBPA behaved similarly to T3 in promoting the release of NCoR from LBD, whereas it failed to promote LBD interactions with SRC2. However, it did reduce the T3-induced interactions between LBD and the coactivator peptide. This study therefore suggests that TBBPA in the micromolar range can affect the regulation of transcription by both the apo- and the holo-TRα1, with potential disruption of the expression of genes that are either up- or down-regulated by T3.

Structural basis of coactivation of liver receptor homolog-1 by β-catenin

We report the three-dimensional structure of a β-catenin armadillo repeat in complex with the liver receptor homolog-1 (LRH-1) ligand binding domain at 2.8Å resolution as the first structure of β-catenin in complex with any nuclear receptor. The surface of β-catenin that binds LRH-1 partly overlaps defined contact sites for peptide segments of β-catenin partners, including T-cell factor-4. The surface of LRH-1 that engages β-catenin is comprised of helices 1, 9, and 10 and is distinct from known interaction surfaces of LRH-1, including corepressor and coactivator binding sites. Targeted mutagenesis of amino acids forming both sides of the LRH-1/β-catenin interface reveals that they are essential for stable interactions between these proteins in solution. The LRH-1 binding site in β-catenin is also required for association with androgen receptor, providing evidence that the observed LRH-1/β-catenin interaction may be prototypic.

Prolonged Membrane Depolarization Enhances Midbrain Dopamine Neuron Differentiation via Epigenetic Histone Modifications

Understanding midbrain dopamine (DA) neuron differentiation is of importance, because of physiological and clinical implications of this neuronal subtype. We show that prolonged membrane depolarization induced by KCl treatment promotes DA neuron differentiation from neural precursor cells (NPCs) derived from embryonic ventral midbrain (VM). Interestingly, the depolarization-induced increase of DA neuron yields was not abolished by L-type calcium channel blockers, along with no depolarization-mediated change of intracellular calcium level in the VM-derived NPCs (VM-NPCs), suggesting that the depolarization effect is due to a calcium-independent mechanism. Experiments with labeled DA neuron progenitors indicate that membrane depolarization acts at the differentiation fate determination stage and promotes the expression of DA phenotype genes (tyrosine hydroxylase [TH] and DA transporter [DAT]). Recruitment of Nurr1, a transcription factor crucial for midbrain DA neuron development, to the promoter of TH gene was enhanced by depolarization, along with increases of histone 3 acetylation (H3Ac) and trimethylation of histone3 on lysine 4 (H3K4m3), and decreases of H3K9m3 and H3K27m3 in the consensus Nurr1 binding regions of TH promoter. Depolarization stimuli on differentiating VM-NPCs also induced dissociation of methyl CpG binding protein 2 and related repressor complex molecules (repressor element-1 silencing transcription factor corepressor and histone deacetylase 1) from the CpG sites of TH and DAT promoters. Based on these findings, we suggest that membrane depolarization promotes DA neuron differentiation by opening chromatin structures surrounding DA phenotype genes and inhibiting the binding of corepressors, thus allowing transcriptional activators such as Nurr1 to access DA neuron differentiation gene promoter regions.

Dominant-negative mechanism of leukemogenic PAX5 fusions

PAX5 encodes a master regulator of B-cell development. It fuses to other genes associated with acute lymphoblastoid leukemia (ALL). These fusion products are potent dominant-negative (DN) inhibitors of wild-type PAX5 resulting in a blockade of B-cell differentiation. Here, we show that multimerization of PAX5 DNA-binding domain (DBD) is necessary and sufficient to cause extremely stable chromatin binding and DN-activity. ALL-associated PAX5-C20S results from fusion of the N-terminal region of PAX5 including its paired DBD, to the C-terminus of C20orf112, a protein of unknown function. We report that PAX5-C20S is a tetramer which interacts extraordinarily stably with chromatin as determined by fluorescence recovery after photobleaching (FRAP) in living cells. Tetramerization, stable chromatin-binding and DN-activity all require a putative five-turn amphipathic α-helix at the C-terminus of C20orf112, and does not require potential co-repressor binding peptides elsewhere in the sequence. In vitro, the monomeric PAX5 DBD and PAX5-C20S binds a PAX5-binding site with equal affinity when it is at the center of an oligonucleotide too short to bind to more than one PAX5 DBD. But PAX5-C20S binds the same sequence with tenfold higher affinity than the monomeric PAX5 DBD when it is in a long DNA molecule. We suggest that the increased affinity results from interactions of one or more of the additional DBDs with neighboring non-specific sites in a long DNA molecule, and that this can account for the increased stability of PAX5-C20S chromatin binding compared to wt PAX5, resulting in DN-activity by competition for binding to PAX5-target sites. Consistent with this model, the ALL-associated PAX5 fused to ETV6 or the multimerization domain of ETV6 SAM results in stable chromatin binding and DN-activity. In addition, PAX5 DBD fused to artificial dimerization, trimerization, and tetramerization domains result in parallel increases in the stability of chromatin binding and DN-activity. Our studies suggest that oncogenic fusion proteins that retain the DBD of the transcription factor and the multimerization sequence of the partner protein can act in a DN fashion by multimerizing and binding avidly to gene targets preventing the normal transcription factor from binding and inducing expression of its target genes. Inhibition of this multimeriztion may provide a novel therapeutic approach for cancers with this or similar fusion proteins.

Identification and characterization of a functional zebrafish smrt corepressor (ncor2)

The retinoic acid receptors (RARs or rars) and the thyroid hormone receptors are members of the steroid receptor superfamily that interact with their DNA response elements (for RARs: retinoic acid response elements or RAREs) in the regulatory regions of promoters in the absence of their ligand. In this ligand minus configuration, it has been suggested that the RAR provides a binding site for a corepressor (SMRT or N-CoR) that also brings in other proteins to repress the gene. In the presence of the ligand, the receptor goes through an allosteric change eliminating the corepressor binding site and providing a coactivator binding site. In this manuscript we describe the isolation of the zebrafish corepressor, smrt. We show that its association with the zebrafish rar aa is sensitive to retinoic acid and that the corepressor mRNA is present in 8 cell zebrafish embryos — a time at which the embryonic genome is not active. We suggest that this rar–corepressor complex may be part of an embryonic, epigenetic switch that keeps retinoic acid responsive genes off before retinoic becomes available to the embryo.