Corepressor SMRT Research Articles

SMRT-GPS2 corepressor pathway dysregulation coincides with obesity-linked adipocyte inflammation

Low-grade chronic inflammation is a major characteristic of obesity and results from deregulated white adipose tissue function. Consequently, there is interest in identifying the underlying regulatory mechanisms and components that drive adipocyte inflammation. Here, we report that expression of the transcriptional corepressor complex subunits GPS2 and SMRT was significantly reduced in obese adipose tissue, inversely correlated to inflammatory status, and was restored upon gastric bypass surgery-induced weight loss in morbid obesity. These alterations correlated with reduced occupancy of the corepressor complex at inflammatory promoters, providing a mechanistic explanation for elevated inflammatory transcription. In support of these correlations, RNAi-mediated depletion of GPS2 and SMRT from cultured human adipocytes promoted derepression of inflammatory transcription and elevation of obesity-associated inflammatory markers, such as IL-6 and MCP-1. Furthermore, we identified a regulatory cascade containing PPARγ and TWIST1 that controlled the expression of GPS2 and SMRT in human adipocytes. These findings were clinically relevant, because treatment of diabetic obese patients with pioglitazone, an antidiabetic and antiinflammatory PPARγ agonist, restored expression of TWIST1, GPS2, and SMRT in adipose tissue. Collectively, our findings identify alterations in a regulatory transcriptional network in adipocytes involving the dysregulation of a specific corepressor complex as among the initiating events promoting adipose tissue inflammation in human obesity.

Cross-talk between HER2 and MED1 Regulates Tamoxifen Resistance of Human Breast Cancer Cells

Despite the fact that most breast cancer patients have estrogen receptor (ER) α-positive tumors, up to 50% of the patients are or soon develop resistance to endocrine therapy. It is recognized that HER2 activation is one of the major mechanisms contributing to endocrine resistance. In this study, we report that the ER coactivator MED1 is a novel cross-talk point for the HER2 and ERα pathways. Tissue microarray analysis of human breast cancers revealed that MED1 expression positively correlates most strongly with HER2 status of the tumors. MED1 was highly phosphorylated, in a HER2-dependent manner, at the site known to be critical for its activation. Importantly, RNAi-mediated attenuation of MED1 sensitized HER2-overexpressing cells to tamoxifen treatment. MED1 and its phosphorylated form, but not the corepressors N-CoR and SMRT, were recruited to the ERα target gene promoter by tamoxifen in HER2-overexpressing cells. Significantly, MED1 attenuation or mutation of MED1 phosphorylation sites was sufficient to restore the promoter recruitment of N-CoR and SMRT. Notably, we found that MED1 is required for the expression of not only traditional E2-ERα target genes but also the newly described EGF-ERα target genes. Our results additionally indicated that MED1 is recruited to the HER2 gene and required for its expression. Taken together, these findings support a key role for MED1 in HER2-mediated tamoxifen resistance and suggest its potential usage as a therapeutic target to simultaneously block both ERα and HER2 pathways for the treatment of this type of endocrine resistant breast cancer.

Elevated nuclear expression of the SMRT corepressor in breast cancer is associated with earlier tumor recurrence

Silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), also known as nuclear corepressor 2 (NCOR2) is a transcriptional corepressor for multiple members of the nuclear receptor superfamily of transcription factors, including estrogen receptor-α (ERα). In the classical model of corepressor action, SMRT binds to antiestrogen-bound ERα at target promoters and represses ERα transcriptional activity and gene expression. Herein SMRT mRNA and protein expression was examined in a panel of 30 breast cancer cell lines. Expression of both parameters was found to vary considerably amongst lines and the correlation between protein and mRNA expression was very poor (R (2)=0.0775). Therefore, SMRT protein levels were examined by immunohistochemical staining of a tissue microarray of 866 patients with stage I-II breast cancer. Nuclear and cytoplasmic SMRT were scored separately according to the Allred score. The majority of tumors (67%) were negative for cytoplasmic SMRT, which when detected was found at very low levels. In contrast, nuclear SMRT was broadly detected. There was no significant difference in time to recurrence (TTR) according to SMRT expression levels in the ERα-positive tamoxifen-treated patients (P=0.297) but the difference was significant in the untreated patients (P=0.01). In multivariate analysis, ERα-positive tamoxifen-untreated patients with high nuclear SMRT expression (SMRT 5-8, i.e., 2nd to 4th quartile) had a shorter TTR (HR=1.94, 95% CI, 1.24-3.04; P=0.004) while there was no association with SMRT expression for ERα-positive tamoxifen-treated patients. There was no association between SMRT expression and overall survival for patients, regardless of whether they received tamoxifen. Thus while SMRT protein expression was not predictive of outcome after antiestrogen therapy, it may have value in predicting tumor recurrence in patients not receiving adjuvant tamoxifen therapy.

Wnt5a Controls Notch1 Signaling through CaMKII-mediated Degradation of the SMRT Corepressor Protein

Serine-threonine Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is the key component in noncanonical Wnt5a signaling and has been shown to regulate its signaling. In this study, we found that CaMKII induced by Wnt5a remarkably reduced the protein stability of the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT), a co-repressor of Notch signaling, through proteasomal degradation. Wnt5a was found to enhance Notch1 intracellular domain (Notch1-IC) transcription activity, which could be inhibited by treatment with KN93, a CaMKII inhibitor. The kinase activity of CaMKII was essential for the activation of Notch signaling. We also determined that CaMKII could enhance the association between Notch1-IC and RBP-Jk. Furthermore, the physical association between RBP-Jk and SMRT was substantially suppressed by CaMKII. We demonstrated that CaMKII directly bound and phosphorylated SMRT at Ser-1407, thereby facilitating SMRT translocation from the nucleus to the cytoplasm and proteasome-dependent degradation. These results suggest that CaMKII down-regulated the protein stability of SMRT through proteasomal degradation.

Altered corepressor SMRT expression and recruitment to target genes as a mechanism that change the response to androgens in prostate cancer progression

Androgen receptor (AR) is required for the development and progression of prostate cancer (CaP) from androgen-dependence to androgen-resistance. Both corepressors and coactivators regulate AR-mediated transcriptional activity, and aberrant expression or activity due to mutation(s) contributes to changes in AR function in the progression to androgen resistance acquired during hormonal ablation therapies. Primary culture of epithelial cells from androgen-dependent CWR22 and androgen-resistant CWR22R xenograft tumors were used to evaluate the effect of androgens on AR function, and the association with coactivators (SRC-1 and TIF-2) and corepressors (SMRT and NCoR). Both androgen-dependent CWR22 and androgen-resistant CWR22R cells expressed functional AR as the receptor bind ligand with high affinity and increased trafficking to the nuclei in the presence of androgens. However, in the presence of androgens, AR-mediated transcriptional activity in androgen-sensitive CWR22 cells was limited to a 2-fold increase, as compared to a 6-fold increase in androgen-resistance CWR22R cells. In androgen-sensitive CWR22 cells, immunoblot, confocal microscopy, and chromatin immunoprecipitation assays indicated that the androgen bound AR transcriptional initiation complex in the PSA promoter contained corepressor SMRT, resulting in limited receptor transcriptional activity. In contrast, increased AR-mediated transcriptional activity in the CWR22R cells was consistent with decreased expression and recruitment of the corepressors SMRT/NCoR, as well as increased recruitment of the coactivator TIF-2 to the receptor complex. Similar changes in the response to androgens were observed in the LNCaP/C4–2 model of androgen resistance prostate cancer. Thus, altered recruitment and loss of corepressors SMRT/NCoR may provide a mechanism that changes the response of AR function to ligands and contributes to the progression of the advanced stages of human prostate cancer.

Adaptor Protein Self-Assembly Drives the Control of a Cullin-RING Ubiquitin Ligase

The E3 ligases recruit substrate proteins for targeted ubiquitylation. Recent insights into the mechanisms of ubiquitylation demonstrate that E3 ligases can possess active regulatory properties beyond those of a simple assembly scaffold. Here, we describe the dimeric structure of the E3 ligase adaptor protein SPOP (speckle-type POZ protein) in complex with the N-terminal domain of Cul3 at 2.4Å resolution. We find that SPOP forms large oligomers that can form heteromeric species with the closely related paralog SPOPL. In combination, SPOP and SPOPL (SPOP-like) form a molecular rheostat that can fine-tune E3 ubiquitin ligase activity by affecting the oligomeric state of the E3 complex. We propose that adaptor protein self-assembly provides a graded level of regulation of the SPOP/Cul3 E3 ligase toward its multiple protein substrates.

Integrative Genomics Identifies the Corepressor SMRT as a Gatekeeper of Adipogenesis through the Transcription Factors C/EBPβ and KAISO

The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPβ-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.

Abstract 2918: Estrogen receptor coactivator MED1 in mammary progenitor/stem cell determination and tamoxifen resistance of breast cancer

Abstract Mediator has recently emerged as the central player to directly transduce signals from transcription factors to the transcription machinery. MED1 (also named TRAP220, DRIP205 and MED220) subunit of the Mediator complex has been shown to directly interact and mediate the transcription of nuclear receptors, in a ligand-dependent fashion, through two classical LxxLL motifs. We have recently generated MED1 LxxLL motifs mutant knockin mice and interestingly, found these mutations do not affect the overall survival and the development of most tissues. However, we did observe severe defects in pubertal mammary gland development of MED1-mutant mice. Consistent with this phenotype, expression of a number of known ERα-regulated genes was downregulated in MED1-mutant mammary epithelial cells and these genes could no longer respond to estrogen stimulation. Interestingly, along with the restricted expression of MED1 in luminal but not basal mammary epithelial cells, we observed a very significant increase in the luminal stem/progenitor cell population in the mammary glands of MED1 mutant mice. We have further crossed this MED1 mutant knockin mice with a MMTV-PyVT mouse model that produce mainly luminal type of mammary tumors. Our date indicate that, although MED1 mutations did not affect the time and number of tumors formed, it significantly impacted the tumor progression and cancer stem cell formation in this model. To further study the role of MED1 in human breast cancer, we examined MED1 protein expression by using human breast cancer tissue microarrays. Consistent with previously reports that MED1 coamplifies with HER2, we found MED1 protein expression level is correlated most strongly with HER2 status. Further study supports HER2 as a major upstream regulator responsible for MED1 phosphorylation at sites known to be critical for MED1 activation. Interestingly, we found an increased recruitment of phosphorylated MED1 but not corepressors SMRT or NcoR to endogenous ER target genes’ promoter by tamoxifen in HER2-overexpressing cells. Importantly, knockdown of MED1 significantly sensitized the otherwise resistant HER2 overexpressing cells to the tamoxifen treatment. These results support MED1 as a key convergence point between ER and HER2 signaling pathways in mediating endocrine resistance. In summary, our studies revealed new roles of MED1 in mediating ER functions in both mammary stem/progenitor cell determination and tamoxifen resistance of breast cancer, and support potential use of MED1 as a tissue-specific therapeutic target for the treatment of human breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2918. doi:1538-7445.AM2012-2918

Regulated Clearance of Histone Deacetylase 3 Protects Independent Formation of Nuclear Receptor Corepressor Complexes

An important step in transcriptional regulation by corepressors N-CoR and SMRT is the formation of a stable and active histone deacetylase 3 (HDAC3)-containing complex. Although N-CoR and SMRT are thought to bind HDAC3 competitively, multiple studies have shown that they do not interfere with the function of each other. How this functional independence is sustained under the competitive interaction is unclear. Here, we show that the coupling of corepressor expression with HDAC3 degradation allows cells to maintain a stable level of uncomplexed HDAC3, thereby preventing mutual interference in the assembly of N-CoR and SMRT complexes. The free uncomplexed HDAC3 is highly unstable. Unexpectedly, the rate of HDAC3 degradation is inversely correlated with the expression level of corepressors. Our results indicate that reducing one corepressor accelerates HDAC3 clearance, thus preventing an increase in complex formation between HDAC3 and the other corepressor. In addition, this study also indicates that the formation of a stable and active HDAC3-corepressor complex is a stepwise process in which the C terminus of HDAC3 plays a critical role at late steps of the assembly process.

Inhibitory effect of adiponectin on breast cancer cell growth: evidences of the crucial role of ERα expression

Adiponectin (adipo) has emerged as a possible link between obesity and breast cancer. Here, we demonstrated that adipo inhibited proliferation in ER‐negative cells, while stimulated growth in MCF‐7 cells. Adipo transactivated ERα in MCF‐7 cells and upregulated mRNA and protein content of the estrogen target gene cyclin D1 (CD1) involved in cell cycle progression. In contrast, in MDA‐MB‐231 cells adipo induced a downregulation of CD1 expression. The latter findings fitted with the opposite effects of adipo on CD1 promoter elicited in the two different cell lines. Mutagenesis studies and ChIP assays revealed that the Sp1 motif, present on CD1 promoter, was important for the modulatory role of adipo on CD1 promoter activity. In the presence of ERα, ChIP analysis revealed an increase in Sp1/ERα complex, concomitant with an enhanced Pol II and pCAF recruitment, addressing the involvement of an activator complex that mediated the adipo‐induced transcriptional activation of CD1. In contrast, in the absence of ERα, adipo exposure increased the recruitment of corepressors SMRT and NCoR and the association of HDAC‐1, that correlated with a significant decrease in Pol II on Sp1 site. Thus, we propose that the possible mechanism through which adipo may modulate CD1 expression affecting breast cancer growth is tightly related to the estrogen receptor dependency.The research has been supported by AIRC MFAG 6180.

The Bcl6-SMRT/NCoR Cistrome Represses Inflammation to Attenuate Atherosclerosis

Chronic inflammation is a hallmark of atherosclerosis, but its transcriptional underpinnings are poorly understood. We show that the transcriptional repressor Bcl6 is an anti-inflammatory regulator whose loss in bone marrow of Ldlr(-/-) mice results in severe atherosclerosis and xanthomatous tendonitis, a virtually pathognomonic complication in patients with familial hypercholesterolemia. Disruption of the interaction between Bcl6 and SMRT or NCoR with a peptide inhibitor invitro recapitulated atherogenic gene changes in mice transplanted with Bcl6-deficient bone marrow, pointing to these cofactors as key mediators of Bcl6 inflammatory suppression. Using ChIP-seq, we reveal the SMRT and NCoR corepressor cistromes, each consisting of over 30,000 binding sites with a nearly 50% overlap. While the complete cistromes identify a diversity of signaling pathways, the Bcl6-bound subcistromes for each corepressor are highly enriched for NF-κB-driven inflammatory and tissue remodeling genes. These results reveal that Bcl6-SMRT/NCoR complexes constrain immune responses and contribute to the prevention of atherosclerosis.

MiR-16 Targets Transcriptional Corepressor SMRT and Modulates NF-kappaB-Regulated Transactivation of Interleukin-8 Gene

The signaling pathways associated with the Toll-like receptors (TLRs) and nuclear factor-kappaB (NF-κB) are essential to pro-inflammatory cytokine and chemokine expression, as well as initiating innate epithelial immune responses. The TLR/NF-κB signaling pathways must be stringently controlled through an intricate network of positive and negative regulatory elements. MicroRNAs (miRNAs) are non-coding small RNAs that regulate the stability and/or translation of protein-coding mRNAs. Herein we report that miR-16 promotes NF-κB-regulated transactivation of the IL-8 gene by suppression of the silencing mediator for retinoid and thyroid hormone receptor (SMRT). LPS stimulation activated miR-16 gene transcription in human monocytes (U937) and biliary epithelial cells (H69) through MAPK-dependent mechanisms. Transfection of cells with the miR-16 precursor promoted LPS-induced production of IL-8, IL-6, and IL-1α, without a significant effect on their RNA stability. Instead, an increase in NF-κB-regulated transactivation of the IL-8 gene was confirmed in cells following transfection of miR-16 precursor. Importantly, miR-16 targeted the 3′-untranslated region of SMRT and caused translational suppression of SMRT. LPS decreased SMRT expression via upregulation of miR-16. Moreover, functional manipulation of SMRT altered NF-κB-regulated transactivation of LPS-induced IL-8 expression. These data suggest that miR-16 targets SMRT and modulates NF-κB-regulated transactivation of the IL-8 gene.

SMRTε, a corepressor variant, interacts with a restricted subset of nuclear receptors, including the retinoic acid receptors α and β

The SMRT and NCoR corepressors bind to, and mediate transcriptional repression by, many nuclear receptors. Both SMRT and NCoR are expressed by alternative mRNA splicing, generating a series of structurally and functionally distinct corepressor “variants”. We report that a splice variant of SMRT, SMRTε, recognizes a restricted subset of nuclear receptors. Unlike the other corepressor variants characterized, SMRTε possesses only a single receptor interaction domain (RID) and exhibits an unusual specificity for a subset of nuclear receptors that includes the retinoic acid receptors (RARs). The ability of the single RID in SMRTε to efficiently interact with RARs appears to be enhanced by a recently recognized β-strand/β-strand interaction between corepressor and receptor. We suggest that alternative mRNA splicing of corepressors can restrict their function to specific nuclear receptor partnerships, and we propose that this may serve to customize the transcriptional repression properties of different cell types for different biological purposes.

Structure of HDAC3 bound to co-repressor and inositol tetraphosphate

SummaryHistone deacetylase enzymes (HDACs) are emerging cancer drug targets. They regulate gene expression by removing acetyl groups from lysine residues in histone tails resulting in chromatin condensation. The enzymatic activity of most class I HDACs requires recruitment to corepressor complexes. We report the first structure of an HDAC:corepressor complex - HDAC3 with the deacetylase-activation-domain (DAD) from the SMRT corepressor. The structure reveals two remarkable features. First the SMRT-DAD undergoes a large structural rearrangement on forming the complex. Second there is an essential inositol tetraphosphate molecule, Ins(1,4,5,6)P4, acting as an ‘intermolecular glue’ between the two proteins. Assembly of the complex is clearly dependent on the Ins(1,4,5,6)P4, which may act as a regulator – potentially explaining why inositol phosphates and their kinases have been found to act as transcriptional regulators. This mechanism for the activation of HDAC3 appears to be conserved in class I HDACs from yeast to man and opens novel therapeutic opportunities.

TNIP1, a Retinoic Acid Receptor Corepressor and A20-binding Inhibitor of NF-κB, Distributes to Both Nuclear and Cytoplasmic Locations

An increasingly wide range of functions, from repression of NF-κB signaling to protection from apoptosis, is being recognized for tumor necrosis factor α-induced protein 3-interacting protein 1 (TNIP1). The authors recently demonstrated TNIP1 interaction with and repression of liganded retinoic acid receptors, distinguishing it from the more typical NCoR and SMRT corepressors, which function only in the absence of ligand. To improve their understanding of TNIP1's roles in physiologic and pathologic events, the authors examined its distribution in normal and malignant human tissues and cultured cells. They found cytoplasmic and nuclear TNIP1 in normal skin keratinocytes as it colocalized with retinoic acid receptor α, one of the nuclear receptors it corepresses. Nuclear and cytoplasmic TNIP1 was also found in the malignant keratinocytes of squamous cell carcinomas. Compared to adjacent normal tissues of other organs, TNIP1 staining and distribution varied with increased levels in esophageal cancer and marked decreases in prostate cancer. The varying levels and distribution of TNIP1 in normal and disease state tissues could be expected to affect processes in which TNIP1 is involved, such as NF-κB and nuclear receptor signaling, possibly contributing to the disease course or response to therapies targeting these key players of cell growth and differentiation.

Alternative mRNA Splicing of Corepressors Generates Variants That Play Opposing Roles in Adipocyte Differentiation

The SMRT and NCoR corepressors partner with, and help mediate repression by, a wide variety of nuclear receptors and non-receptor transcription factors. Both SMRT and NCoR are expressed by alternative mRNA splicing, resulting in the production of a series of interrelated corepressor variants that differ in their tissue distribution and in their biochemical properties. We report here that different corepressor splice variants can exert opposing transcriptional and biological effects during adipocyte differentiation. Most notably, the NCoRω splice variant inhibits, whereas the NCoRδ splice variant promotes, adipogenesis. Furthermore, the ratio of NCoRω to NCoRδ decreases during adipogenic differentiation. We propose that this alteration in corepressor splicing helps convert the cellular transcriptional program from one that maintains the pre-adipocyte in an undifferentiated state to a new transcriptional context that promotes differentiation and helps establish the proper physiology of the mature adipocyte.

Oncogene Dosage Is a Major Determinant of Leukemogenic Transformation by AE9a

Abstract 1358Chromosomal translocation (8;21) results in the formation of the AML1-ETO (AE) leukemia-associated fusion protein. Roughly 70% of AE+ patient samples also harbor an alternatively spliced variant of AE, termed AE9a. AE9a promotes the leukemic transformation of mouse hematopoietic stem/progenitor cells without the need for secondary external cooperating oncogenes. By comparison, leukemia development in response to expression of full length AE requires one or more additional events. We extended these findings into a human cell system to determine whether AE9a is able to promote the transformation of human CD34+ cord blood (CB) cells to acute myeloid leukemia (AML). Transduction of CD34+ CB cells with AE9a promotes long-term culture potential, enhanced replating capacity, and expansion of CD34+ progenitor cells similar to full length AE. While we have not yet been able to generate AE9a leukemia in xenograft assays (despite several attempts), subcutaneous injection of AE9a cells promotes formation of a robust granulocytic sarcoma which is not seen using cells expressing full length AE. Several of our analyses have highlighted the importance of increased AE9a fusion protein expression in its enhanced function relative to full length AE, a concept that was also observed using the MLL-AF9 leukemia oncogene (Chen et. al, Cancer Cell, 2008). Indeed, flow cytometric sorting of freshly transduced AE or AE9a cultures based on high and low fusion protein expression revealed the importance of expression level for both AE and AE9a. High AE expression completely blocked colony formation and decreased cell expansion. These effects correlated with a dramatic upregulation of the cell cycle inhibitor p21. Conversely, high levels of AE9a did not upregulate p21 to the same degree, and colony formation and cell expansion were not as dramatically affected. In contrast, flow sort for cells expressing low levels of AE or AE9a had significantly less remarkable effects on cell function and p21 expression. Unexpectedly, AE9a-low cells eventually came to resemble the AE9a-high in terms of AE9a protein levels. These effects were not seen for full length AE sorted cultures. To determine whether similar effects were occurring in the murine AML model, we examined the effects of low and high expression levels of AE9a in mouse fetal liver cells. Corroborating our findings with the human cell model, AE9a-low samples promoted significantly less AML as compared to AE9a-high cells. This was not due to lack of engraftment of these cells or to loss of AE9a expression. Similar effects were found in vitro, where AE9a-low cells were unable to replate in methylcellulose assays while AE9a-high cells as well as AE-high and AE-low cells showed enhanced replating ability. Strikingly, the AML that did develop in mice transplanted with AE9a-low cells reverted to a very high level of AE9a protein expression, implying that increased expression of AE9a is essential for function. To better understand the mechanistic link between increased AE9a expression and AML development, we performed global gene expression analysis of murine progenitor cells expressing AE or AE9a (after three weeks of in vivo expansion). Analysis revealed a subset of genes that were highly repressed in AE9a expressing cells as compared to full length AE. Given that AE9a lacks the carboxy-terminal NHR3 and NHR4 domains of AE that interact with the NCoR and SMRT corepressors, the finding of significantly more repressed genes in AE9a cells is quite surprising. Network analysis has uncovered potential signaling pathways that may be involved in AE9a mediated transformation. Uncovering the mechanism of AE9a function could lead to a better understanding of t(8;21)-associated function in human AML. Disclosures:No relevant conflicts of interest to declare.

Ecdysone- and NO-Mediated Gene Regulation by Competing EcR/Usp and E75A Nuclear Receptors during Drosophila Development

The Drosophila ecdysone receptor (EcR/Usp) is thought to activate or repress gene transcription depending on the presence or absence, respectively, of the hormone ecdysone. Unexpectedly, we found an alternative mechanism at work in salivary glands during the ecdysone-dependent transition from larvae to pupae. In the absense of ecdysone, both ecdysone receptor subunits localize to the cytoplasm, and the heme-binding nuclear receptor E75A replaces EcR/Usp at common target sequences in several genes. During the larval-pupal transition, a switch from gene activation by EcR/Usp to gene repression by E75A is triggered by a decrease in ecdysone concentration and by direct repression of the EcR gene by E75A. Additional control is provided by developmentally timed modulation of E75A activity by NO, which inhibits recruitment of the corepressor SMRTER. These results suggest a mechanism for sequential modulation of gene expression during development by competing nuclear receptors and their effector molecules, ecdysone and NO.

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.

In Cortical Neurons HDAC3 Activity Suppresses RD4-Dependent SMRT Export

The transcriptional corepressor SMRT controls neuronal responsiveness of several transcription factors and can regulate neuroprotective and neurogenic pathways. SMRT is a multi-domain protein that complexes with HDAC3 as well as being capable of interactions with HDACs 1, 4, 5 and 7. We previously showed that in rat cortical neurons, nuclear localisation of SMRT requires histone deacetylase activity: Inhibition of class I/II HDACs by treatment with trichostatin A (TSA) causes redistribution of SMRT to the cytoplasm, and potentiates the activation of SMRT-repressed nuclear receptors. Here we have sought to identify the HDAC(s) and region(s) of SMRT responsible for anchoring it in the nucleus under normal circumstances and for mediating nuclear export following HDAC inhibition. We show that in rat cortical neurons SMRT export can be triggered by treatment with the class I-preferring HDAC inhibitor valproate and the HDAC2/3-selective inhibitor apicidin, and by HDAC3 knockdown, implicating HDAC3 activity as being required to maintain SMRT in the nucleus. HDAC3 interaction with SMRT's deacetylation activation domain (DAD) is known to be important for activation of HDAC3 deacetylase function. Consistent with a role for HDAC3 activity in promoting SMRT nuclear localization, we found that inactivation of SMRT's DAD by deletion or point mutation triggered partial redistribution of SMRT to the cytoplasm. We also investigated whether other regions of SMRT were involved in mediating nuclear export following HDAC inhibition. TSA- and valproate-induced SMRT export was strongly impaired by deletion of its repression domain-4 (RD4). Furthermore, over-expression of a region of SMRT containing the RD4 region suppressed TSA-induced export of full-length SMRT. Collectively these data support a model whereby SMRT's RD4 region can recruit factors capable of mediating nuclear export of SMRT, but whose function and/or recruitment is suppressed by HDAC3 activity. Furthermore, they underline the fact that HDAC inhibitors can cause reorganization and redistribution of corepressor complexes.