Nuclear Receptor-mediated Transcription Research Articles

Selective effect of β-catenin on nuclear receptor-dependent hepatitis B virus transcription and replication

β-catenin regulates HBV transcription in cell culture and viral biosynthesis in vivo in the transgenic mouse model of chronic HBV infection. Therefore, it is important to understand which transcription factor activities are coactivated by β-catenin to enhance HBV biosynthesis. The effect of β-catenin expression in the context of nuclear receptor-mediated HBV transcription was evaluated initially in the human embryonic kidney cell line, HEK293T. Reporter gene and viral replication assays revealed that β-catenin can coactivate HBV transcription through some, most predominantly liver receptor homolog 1 (LRH1), but not all nuclear receptors known to activate viral biosynthesis. Similarly, β-catenin activated nuclear receptor-mediated HBV transcription and replication in the human hepatoma cell line, Huh7, primarily through its effect on the farnesoid X receptor α (FXRα). These data indicate that β-catenin can enhance nuclear receptor-mediated HBV biosynthesis, but the relative importance of various transcription factors is dependent upon the precise cellular environment.

An emerging link between LIM domain proteins and nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that partake in several biological processes including development, reproduction and metabolism. Over the last decade, evidence has accumulated that group 2, 3 and 4 LIM domain proteins, primarily known for their roles in actin cytoskeleton organization, also partake in gene transcription regulation. They shuttle between the cytoplasm and the nucleus, amongst other as a consequence of triggering cells with ligands of nuclear receptors. LIM domain proteins act as important coregulators of nuclear receptor-mediated gene transcription, in which they can either function as coactivators or corepressors. In establishing interactions with nuclear receptors, the LIM domains are important, yet pleiotropy of LIM domain proteins and nuclear receptors frequently occurs. LIM domain protein-nuclear receptor complexes function in diverse physiological processes. Their association is, however, often linked to diseases including cancer.

The actinin family proteins: biological function and clinical implications.

The alpha actinin (ACTN) family proteins are actin-bundling proteins that are evolutionarily conserved and possess several functional domains. Vertebrates including humans express four unique actinin genes that display distinct expression patterns and biochemical properties. Mutations in each of these actinins are associated with tissues-specific abnormalities. ACTN1 and ACTN4 are ubiquitously expressed and their major spliced isoforms harbor calcium-sensitive EF hands, while the expression of ACTN2 and ACTN3 are primarily restricted to muscle and are calcium-insensitive. Due to their ability to bind filamentous actin, ACTNs have been shown to regulate cytokinesis, cell adhesion, spreading, migration and signaling. In addition, a fraction of ACTN4 is localized in the nucleus where it interacts with transcription factors, histone modifying enzymes and chromatin remodeling proteins in order to stimulate transcription [1–3]. Notably, all ACTNs harbor an evolutionarily conserved motif,—LXXLL—(L, leucine, X, any amino acids), which is required for interaction with the nuclear receptor family of transcription factors [3]. Indeed, ACTN1 and ACTN2 have been shown to enhance nuclear receptor-mediated transcription [1, 4]. Thus, ACTNs join the list of several other cytoskeletal proteins that are capable of shuttling between the cytoplasm and the nucleus to control cell function [5]. In this thematic review series, we feature recent progress in our understanding of the biological and the pathological roles of the ACTNs and genetic lesions in this protein family that are associated with human diseases. Murphy and Young [6] first summarize genetic studies of actinin in invertebrates and cast an evolutionary perspective of this unique actin-binding protein family. They detail the similarities and differences among the 4 vertebrate ACTNs and provide an insightful discussion of actinin-related genetic disorders with primary focus on ACTN1, 2 and 3. For example, mutations in ACTN1 are associated with dominantly-inherited congenital macrothrombocytopenia (CMPT), a rare blood disorder, while mutations in ACTN2 are linked to hypertrophic cardiomyopathy (HCM). Interestingly, disease-associated ACTN1 and ACTN2 mutations are only found in the actin-binding domain, emphasizing the physiological significance of this functional domain. Surprisingly, ACTN3 is not essential and individuals with ACTN3 null alleles, due to a homozygous premature stop codon, are found in 16 % of world population. Interestingly, wild-type allele is found to be associated with better sprint and power performance in Caucasians. ACTN4 was initially identified in a screen for antigens to which antibodies strongly reacted in highly invasive breast cancer [7]. Honda reviews recent literature on the link between ACTN4 amplification, overexpression and spliced variants that predict metastatic potency in several types of tumors including breast, prostate, colon and lung [8]. He also summarizes evidence supporting a role of ACTN4 in transcriptional regulation by nuclear receptors and NF-κB and its link to tumorigenesis. Lastly, Honda suggests that ACTN4 may be a useful biomarker for evaluating treatment option in metastatic breast cancer. An elegant human genetics study has linked ACTN4 mutations to the kidney disorder focal segmental glomerular sclerosis (FSGS) [9]. This finding pioneered a novel concept that mutations in genes encoding cytoskeletal proteins in podocytes may be linked to glomerulopathy. Indeed, mutations of several cytoskeletal proteins have been subsequently found in patients with familial FSGS [10]. Interestingly, reduced expression of ACTN4 protein in kidney was also found in sporadic FSGS, minimal change disease and IgA nephropathy. Feng et al., thoroughly discusses the clinical presentation and biochemical and biophysical properties of disease causing ACTN4 mutations as well as related animal studies [11]. They further provide potential mechanisms underlying ACTN4-linked glomerulopathy. The physiological function of ACTN4 is unlikely limited to bundling actin and co-activating transcription. It is likely to also be a signaling molecule that transmits extracellular signalings to influence cell growth and differentiation. The intent of this thematic series is to summarize recent discoveries on actinin function and ACTN-related diseases and highlight future challenges in ACTN research.

Arginine methyltransferases mediate an epigenetic ovarian response to endometriosis.

Endometriosis is associated with infertility and debilitating chronic pain. Abnormal epigenetic modifications in the human endometrium have recently been implicated in the pathogenesis of this condition. However, whether an altered epigenetic landscape contributes to pathological changes in the ovary is unknown. Using an established baboon endometriosis model, early-, and late-stage epigenetic changes in the ovary were investigated. Transcript profiling of key chromatin-modifying enzymes using pathway-focused PCR arrays on ovarian tissue from healthy control animals and at 3 and 15 months of endometriosis revealed dramatic changes in gene expression in a disease duration-dependent manner. Ingenuity Pathway Analysis indicated that transcripts for chromatin-remodeling enzymes associated with reproductive system disease and cancer development were abnormally regulated, most prominently the arginine methyltransferases CARM1, PRMT2, and PRMT8. Downregulation of CARM1 protein expression was also detected in the ovary, fully-grown oocytes and eutopic endometrium following 15 months of endometriosis. Sodium bisulfite sequencing revealed DNA hypermethylation within the PRMT8 promoter, suggesting that deregulated CpG methylation may play a role in transcriptional repression of this gene. These results demonstrate that endometriosis is associated with changes of epigenetic profiles in the primate ovary and suggest that arginine methyltransferases play a prominent role in mediating the ovarian response to endometriosis. Owing to the critical role of CARM1 in nuclear receptor-mediated transcription and maintenance of pluripotency in the cleavage stage embryo, our results suggest that epigenetic alterations in the ovary may have functional consequences for oocyte quality and the etiology of infertility associated with endometriosis.

A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and subsequent binding the activated AHR to xenobiotic response elements (XREs) on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT). In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs) at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism’s ability to respond on a phenotypic level to various stimuli within an inconstant environment.

Overexpression of G9a and MCM7 in oesophageal squamous cell carcinoma is associated with poor prognosis.

Histone methyltransferase G9a has been primarily understood as a co-repressor of gene expression, but it has been shown that G9a also positively regulates nuclear receptor-mediated transcription. MCM7, a critical component of the DNA replication licensing complex, is amplified and overexpressed in a variety of human malignancies. The objectives of the present study were to study the relationship between the expression of G9a and MCM7 and the pathological grade, clinical stage and prognosis of oesophageal squamous cell carcinoma (OSCC). We collected 139 formalin-fixed and paraffin-embedded tissues from patients with OSCC and surveyed them by tissue microarray-based immunohistochemical staining. Associations between the expression of MCM7 and G9a and clinicopathological parameters and prognosis of OSCC were examined. From tissue microarray immunohistochemistry staining results, we found that nuclear staining intensity for MCM7 and G9a was associated with histological grade (both P < 0.001), tumour depth (P = 0.050, 0.034), lymph node metastasis (P = 0.001, 0.009) and tumour stage (P < 0.001, =0.003). G9a expression was correlated with that of MCM7. G9a overexpression independently predicted poor cancer-specific survival in OSCC (hazard ratio 0.05, 95% confidence interval 0.006-0.417, P = 0.006) and MCM7 (hazard ratio 0.05, 95% confidence interval 0.013-0.441, P = 0.004). OSCC patients whose tumours showed double-positive expression of G9a and MCM7 (G9a(+) MCM7(+) ) had much shorter survival than those from either the G9a or MCM7 low expression groups (G9a(-) MCM7(-) , G9a(+) MCM7(-) , G9a(-) MCM7(+) ). MCM7 and G9a may serve as effective prognostic factors and could also be used as biomarkers for predicting various clinical outcomes of OSCCs in the Chinese population.

A Distinct Mechanism for Coactivator versus Corepressor Function by Histone Methyltransferase G9a in Transcriptional Regulation

Histone methyltransferase G9a has been understood primarily as a corepressor of gene expression, but we showed previously that G9a positively regulates nuclear receptor-mediated transcription in reporter gene assays. Here, we show that endogenous G9a contributes to the estradiol (E(2))-dependent induction of some endogenous target genes of estrogen receptor (ER)α in MCF-7 breast cancer cells while simultaneously limiting the E(2)-induced expression of other ERα target genes. Thus, G9a has a dual and selective role as a coregulator for ERα target genes. The ERα binding regions associated with the pS2 gene, which requires G9a for E(2)-induced expression, are transiently occupied by G9a at 15 min after beginning E(2) treatment, suggesting that G9a coactivator function is by direct interaction with ERα target genes. Transient reporter gene assays with deletion mutants of G9a demonstrated that domains previously associated with the corepressor functions of G9a (C-terminal methyltransferase domain, ankyrin repeat domain, and cysteine-rich domain) were unnecessary for G9a coactivator function in ERα-mediated transcription. In contrast, the N-terminal domain of G9a was necessary and sufficient for enhancement of ERα-mediated transcription and for E(2)-induced occupancy of G9a on ERα binding sites associated with endogenous target genes of ERα. In addition to a previously identified activation domain, this region contains a previously uncharacterized ligand-dependent ERα binding function, indicating how G9a is recruited to the target genes. Therefore, the coactivator and corepressor functions of G9a involve different G9a domains and different molecular mechanisms.

Retinoic acid influences neuronal migration from the ganglionic eminence to the cerebral cortex

The ganglionic eminence contributes cells to several forebrain structures including the cerebral cortex, for which it provides GABAergic interneurons. Migration of neuronal precursors from the retinoic-acid rich embryonic ganglionic eminence to the cerebral cortex is known to be regulated by several factors, but retinoic acid has not been previously implicated. We found retinoic acid to potently inhibit cell migration in slice preparations of embryonic mouse forebrains, which was reversed by an antagonist of the dopamine-D(2) receptor, whose gene is transcriptionally regulated by retinoic acid. Histone-deacetylase inhibitors, which amplify nuclear receptor-mediated transcription, potentiated the inhibitory effect of retinoic acid. Surprisingly, when retinoic acid signalling was completely blocked with a pan-retinoic acid receptor antagonist, this also decreased cell migration into the cortex, implying that a minimal level of endogenous retinoic acid is necessary for tangential migration. Given these opposing effects of retinoic acid in vitro, the in vivo contribution of retinoic acid to migration was tested by counting GABAergic interneurons in cortices of adult mice with experimental reductions in retinoic acid signalling: a range of perturbations resulted in significant reductions in the numerical density of some GABAergic interneuron subpopulations. These observations suggest functions of retinoic acid in interneuron diversity and organization of cortical excitatory-inhibitory balance.

Measurement and Modulation of Nuclear Receptor-Mediated Transcription in Single Cells Using Light

Nuclear receptors coordinate a diverse range of responses in higher eukaryotes and are involved in a wide range of human diseases. The ligand-dependent nature of these transcription factors makes them appealing targets for therapeutic interventions, necessitating both a mechanistic molecular understanding of interactions between ligand and receptor and a quantitative understanding of how receptors control output from target genes. Here, we utilize single molecule observation of mRNA in living and fixed cells to develop a stochastic model of nuclear-receptor mediated gene activation, and we develop a new light-activation technology to turn on a single gene and follow dynamic synthesis of RNA from the active locus. We determined that nuclear-receptors control mRNA synthesis by frequency modulation of the active state. This mechanism reproduces the classic Hill dose response that has been observed for nuclear-receptor mediated transcription and also explains the variation or ‘noise’ in expression which we measure for our reporter gene. To quantitatively control this transcription with high temporal and spatial resolution, we developed a class of photolyzable reagents (“caged” ligands) which repress transcription in the caged form and activate transcription in the uncaged form. Using light-activation of the cage, we are able to turn on the transcription of single cells in confluent tissue culture. We expect that the ability to measure and modulate transcription at the single molecule level with light will be critical in understanding the function of nuclear-receptor mediated signaling in tissue and eventually in whole animals.

Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin

The carbon catabolite repressor protein 4 (Ccr4)–Negative on TATA (Not) complex controls gene expression at two levels. In the nucleus, it regulates the basal transcription machinery, nuclear receptor-mediated transcription and histone modifications. In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1. Not1 is the largest protein of the Ccr4–Not complex and serves as a scaffold for other subunits of the complex. Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs). Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1. Importantly, Not1 is required for the rapid decay of ARE-mRNAs, and TTP can recruit the Caf1 deadenylase only in presence of Not1. Thus, cytoplasmic Not1 provides a platform that allows a specific RNA binding protein to recruit the Caf1 deadenylase and thereby trigger decay of its target mRNAs.

Loss of Xenopus tropicalis EMSY causes impairment of gastrulation and upregulation of p53

EMSY interacts directly with BRCA2 and links the BRCA2 pathway to sporadic breast and ovarian cancer. It also interacts with BS69 and HP1b, both of which are involved in chromatin remodelling, and with NIF-1 and DBC-1 in the regulation of nuclear receptor-mediated transcription. Here we investigate the function of EMSY during amphibian development, and in doing so provide the first loss-of-function analysis of this protein. Injection of Xenopus tropicalis embryos with antisense morpholino oligonucleotides targeting XtEMSY impairs gastrulation movements, disrupts dorsal structures, and kills embryos by tailbud stages. Consistent with these observations, regional markers such as Xbra, Chd, Gsc, Shh, Sox3 and Sox17 are downregulated. In contrast to these regional markers, expression of p53 is upregulated in such embryos, and at later stages Bax expression is elevated and apoptotic cells can be detected. Our results demonstrate that EMSY has an essential role in development and they provide an in vivo loss-of-function model that might be used to explore the biochemical functions of this protein in more detail.

Retinoic acid regulates bone morphogenic protein signal duration by promoting the degradation of phosphorylated Smad1

The proper function of the bone morphogenic protein (BMP) pathway during embryonic development and organ maintenance requires its communication with other signaling pathways. Unlike the well-documented regulation of the BMP pathway by FGF/MAPK and Wnt/GSK3 signals, cross-talk between BMP/Smad and retinoic acid (RA)/RA receptor (RAR) pathways is poorly understood. Here, we show that RA represses BMP signal duration by reducing the level of phosphorylated Smad1 (pSmad1). Through its nuclear receptor-mediated transcription, RA enhances the interaction between pSmad1 and its ubiquitin E3 ligases, thereby promoting pSmad1 ubiquitination and proteasomal degradation. This regulation depends on the RA-increased Gadd45 expression and MAPK activation. During the neural development in chicken embryo, the RA/RAR pathway also suppresses BMP signaling to antagonize BMP-regulated proliferation and differentiation of neural progenitor cells. Furthermore, this cross-talk between RA and BMP pathways is involved in the proper patterning of dorsal neural tube of chicken embryo. Our results reveal a mechanism by which RA suppresses BMP signaling through regulation of pSmad1 stability.

A muscle-specific knockout implicates nuclear receptor coactivator MED1 in the regulation of glucose and energy metabolism

As conventional transcriptional factors that are activated in diverse signaling pathways, nuclear receptors play important roles in many physiological processes that include energy homeostasis. The MED1 subunit of the Mediator coactivator complex plays a broad role in nuclear receptor-mediated transcription by anchoring the Mediator complex to diverse promoter-bound nuclear receptors. Given the significant role of skeletal muscle, in part through the action of nuclear receptors, in glucose and fatty acid metabolism, we generated skeletal muscle-specific Med1 knockout mice. Importantly, these mice show enhanced insulin sensitivity and improved glucose tolerance as well as resistance to high-fat diet-induced obesity. Furthermore, the white muscle of these mice exhibits increased mitochondrial density and expression of genes specific to type I and type IIA fibers, indicating a fast-to-slow fiber switch, as well as markedly increased expression of the brown adipose tissue-specific UCP-1 and Cidea genes that are involved in respiratory uncoupling. These dramatic results implicate MED1 as a powerful suppressor in skeletal muscle of genetic programs implicated in energy expenditure and raise the significant possibility of therapeutical approaches for metabolic syndromes and muscle diseases through modulation of MED1-nuclear receptor interactions.

The mammalian clock component PERIOD2 coordinates circadian output by interaction with nuclear receptors

Mammalian circadian clocks provide a temporal framework to synchronize biological functions. To obtain robust rhythms with a periodicity of about a day, these clocks use molecular oscillators consisting of two interlocked feedback loops. The core loop generates rhythms by transcriptional repression via the Period (PER) and Cryptochrome (CRY) proteins, whereas the stabilizing loop establishes roughly antiphasic rhythms via nuclear receptors. Nuclear receptors also govern many pathways that affect metabolism and physiology. Here we show that the core loop component PER2 can coordinate circadian output with the circadian oscillator. PER2 interacts with nuclear receptors including PPARalpha and REV-ERBalpha and serves as a coregulator of nuclear receptor-mediated transcription. Consequently, PER2 is rhythmically bound at the promoters of nuclear receptor target genes in vivo. In this way, the circadian oscillator can modulate the expression of nuclear receptor target genes like Bmal1, Hnf1alpha, and Glucose-6-phosphatase. The concept that PER2 may propagate clock information to metabolic pathways via nuclear receptors adds an important facet to the clock-dependent regulation of biological networks.

BTG1, a Gene Frequently Deleted in Pre-B ALL, Controls Glucocorticoid Receptor-Mediated Gene Expression.

Abstract 3458Poster Board III-346 BackgroundBy genome wide profiling we have found that about 10 % of pediatric pre-B ALL cases contain a (single copy) deletion of the B cell translocation gene 1 (BTG1) gene. BTG1 belongs to a family of potential tumor suppressor genes, which include BTG2, BTG3, TOB and TOB2. Proteins encoded by members of this gene family have been implicated in the induction of growth arrest or apoptosis in a variety of model systems. Moreover, BTG1 associates with and regulates the activity of the arginine methyl transferase PRMT1, a coactivator of nuclear receptor-mediated transcription. Hence we hypothesized that loss of BTG1 function, for instance due to deletion, may affect glucocorticoid induced therapy responses in ALL. ResultsUsing RNA interference, we find that loss of BTG1 expression decreases sensitivity of pre-B ALL cells to the apoptosis-inducing effects of synthetic GCs about 10,000 fold (Figure). This acquired GC resistance is accompanied by a greater than 10 fold reduction in GR protein expression as well as a (near complete) loss of GC-induced gene expression. Conversely, re-expression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression. By chromatin immunoprecipitations using anti PRMT1 antibodies we show that PRMT1 is recruited to the GR gene promoter in a BTG1-dependent manner, consistent with a role for this arginine methyl transferase in the regulation of GR-mediated gene expression. ConclusionsTogether, our results demonstrate the importance of the BTG1/PRMT1 complex in regulating GR-mediated gene expression and reveal how deregulation of the this complex can give rise to GC resistance. Targeting of these coactivators as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC based therapies in ALL as well as other hematological malignancies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Menin, a product of the MENI gene, binds to estrogen receptor to enhance its activity in breast cancer cells: possibility of a novel predictive factor for tamoxifen resistance

Multiple coactivator and corepressor complexes play an important role in endocrine processes and breast cancer; in particular, estrogen and estrogen receptor-alpha (ERalpha) promote the proliferation of breast cancer cells. Menin is a tumor suppressor encoded by Men1 that is mutated in the human-inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1); it also serves as a critical link in the recruitment of nuclear receptor-mediated transcription. Here, we show that menin expressed in breast cancer cell line MCF-7 is colocalized with ERalpha and functions as a direct coactivator of ER-mediated transcription in breast cancer cells. In MCF-7 cells, coexpression of menin and estrogen-response element-luciferase induced the activity of the latter in a hormone-dependent manner. Cells knocked down for ERalpha exhibited impaired ERE-luciferase activity induced by menin. Mammalian two-hybrid assay and GST pull-down assays indicated that menin could interact with the AF-2 domain of ERalpha. These results indicate that menin is a direct activator of ERalpha function. Tamoxifen inhibited the binding of menin to AF-2 in mammalian two-hybrid assay, but in menin-overexpressing clones, tamoxifen suppressed ERE-luciferase activity only to the levels of nontreated wild-type MCF-7. In a clinical study with 65 ER-positive breast cancer samples-all of which had been treated with tamoxifen for 2-5 years as adjuvant therapies--menin-positive tumors had a worse outcome than menin-negative ones. These indicated that menin can function as a transcriptional regulator of ERalpha and is a possible predictive factor for tamoxifen resistance.

Corepressors, nuclear receptors, and epigenetic factors on DNA: A tail of repression

The differential exposure to circulating steroid hormones during brain development can have lasting consequences on brain function and behavior; therefore, the tight control of steroid hormone action within the developing brain is necessary for the expression of appropriate sex-typical behavior patterns later in life. The restricted control of steroid hormone action at the level of the DNA can be accomplished through the recruitment of coregulatory complexes. Nuclear receptor action can either be enhanced by the recruitment of coactivator complexes or suppressed by the formation of corepressor complexes. Alternatively, the regulation of nuclear receptor-mediated gene transcription in the developing brain may involve a dynamic process of coactivator and corepressor function on DNA. It is likely that understanding how different combinations of coregulatory matrixes assembly on DNA will lead to further understanding of heterogeneous responses to nuclear receptor activation. We will discuss how coregulators influence gene transcription and repression, the role of chromatin-binding factors in the regulation of gene transcription, and their potential impact on brain development.

Identification and Characterization of a Novel Nuclear Protein Complex Involved in Nuclear Hormone Receptor-mediated Gene Regulation

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of approximately 1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-alpha was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.

177. A MECHANISM UNDERLYING DISORDERS OF SEX DEVELOPMENT CAUSED BY DAX1 DUPLICATION

The DAX1 protein is an orphan nuclear hormone receptor expressed in developing and adult hypothalamic, pituitary, adrenal and gonadal tissues. In humans, duplication of the DAX1 gene at locus Xp21 causes Disorders of Sex Development (DSD), whereby XY individuals develop as females, due to the failure of testicular development. DAX1 acts as a co-factor for nuclear receptor-mediated transcription of steroidogenic genes. In mice, overexpression of a Dax1 transgene causes delayed testis cord formation, a milder phenotype than that seen in human (1). Exactly how DAX1 duplication interferes with typical testicular development is unclear but a ‘window' of DAX1 activity was proposed (2). In order to identify the mechanism of DAX1 action when overexpressed in the developing XY gonad, we have used both in vivo and in vitro approaches. We hypothesised that, when present in excess, DAX1 must repress the action of early testis-forming genes. We investigated the effect of Dax1 over expression, using the Dax1 transgenic mouse line, Dax1812 (1), on expression of Sox9, a critical testis-forming gene. Immunostaining of Dax1812 gonads revealed reduced Sox9 expression, suggesting excess Dax1 antagonises Sox9 upregulation during the early stages of sex determination. To determine whether antagonism of Sox9 was occurring at the transcriptional level we assessed the effect of excess Dax1 on the activity of the Testis-Specific Enhancer of Sox9 (TES), which drives Sox9 transcription in the developing XY gonad (3). In combination, the in vivo and in vitro evidence strongly suggests that Dax1, when present in excess, can repress Sox9 expression through TES and that this repression occurs through inhibition of Steroidogenic Factor-1 activity. With this work we have identified a potential mechanism for disruption of the male-specific sex determination pathway caused by DAX1 duplication and leading to DSD in XY individuals.

BTG1 Controls Glucocorticoid Sensitivity in Acute Lymphoblastic Leukemia by Regulating Nuclear Receptor-Mediated Transcription.

Glucocorticoids (GCs) are global regulators of gene transcription that act by binding to the glucocorticoid receptor, a ligand-activated transcription factor belonging to the nuclear receptor family. As synthetic GCs such as prednisolone or dexamethasone specifically induce cell cycle arrest and apoptosis in lymphoid cells, these drugs are widely used in the treatment of (childhood) acute lymphoblastic leukemia (ALL). GC resistance is a strong independent predictor of poor prognosis in the treatment of childhood ALL, but the underlying mechanisms remain poorly understood. In a previous study (Kuiper et al. Leukemia 2007) we determined by array-based SNP analysis that microdeletions affecting the anti-proliferative gene B-cell translocation gene 1 (BTG1) occurred in 4 out of 33 pre-B ALL cases. In our current study we show that prednisolone causes a 3-fold induction of BTG1 mRNA expression both in pre-B ALL cell lines and primary ALL, implicating BTG1 in cellular responses to GCs. Moreover, RNA-interference-mediated knockdown of BTG1 renders RS4;11 pre-B leukemia cells refractory to prednisolone-induced apoptosis. We show by genome-wide mRNA profiling that loss of BTG1 completely abrogates prednisolone-induced gene expression by downregulation of the glucocorticoid receptor NR3C1. Moreover, we present evidence showing that BTG1 is part of a nuclear receptor complex that regulates expression of NR3C1 and consequently GC responsiveness in ALL. In conclusion, we have identified BTG1 as an important determinant of GC sensitivity in ALL.