The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^

Proliferation index in ductal carcinoma in situ of the breast: Relation to estrogen, progesterone and HER2/neu receptors

Background. Ductal carcinoma in situ (DCIS) is the most prevalent precursor to invasive breast cancer (IBC), the second leading cause of death in women in the United States. The three most important prognostic markers for IBC are Estrogen receptor (ER), Progesterone receptor (PR) and HER2/neu. The four groups (IBC) defined as (1) ER and/or PR positive and HER2/neu negative, (2) ER and/or PR positive and HER2/neu positive (3) ER and/or PR negative and HER2/neu positive and (4) negative for all three of these receptors (Triple negative). However, they have not been well studied in DCIS. This is an exploratory study with a primary objective to examine the prevalence of ER, PR, and HER2/neu in DCIS, to explore if the defined groups of IBC occur in DCIS and to consider the biological relationship between these four groups and the proliferative activity of the tumor. A secondary goal of this study is to examine the relationship between grade and proliferative activity. Methods. Using immunohistochemistry, I have measured Ki-67, ER, PR and HER2/neu positivity for a series of cases of DCIS. Results. 20 ER and/or PR positive and HER2/neu negative (50%) with average PI of 0.05, 7 ER and/or PR positive and HER2/neu positive (17.5%) with average PI of 0.14, 10 ER and/or PR negative and HER2/neu positive (25%) with average PI of 0.18, and three triple negative (7.5%) with average PI of 0.18. ER and/or PR positive and HER2/neu positive group has the highest PI (p<0.001). Further, the ER and/or PR positive and HER2/neu positive group show a linear relationship between PI and average ER/PR positivity (R=0.6). PI increases with higher grades. Conclusion. PI appears to depend upon the average fraction of positive ER/PR tumor cells, possibly with a synergistic dependence when HER2/neu is positive. If ER/PR is negative, then both HER2/neu positive and the triple negative cases appear to cluster around an average PI that is higher than the average PI in HER2/neu negative ER/PR positive negative cases. In the triple negative tumors there must be another driver of proliferation.^

Estrogen-induced gene expression and patient survival in high-grade serous carcinoma of the ovary

Background. Assessment of estrogen receptor (ER) expression has inconsistent utility as a prognostic marker in epithelial ovarian carcinoma. In breast and endometrial cancers, the use of estrogen-induced gene panels, rather than ER expression alone, has shown improved prognostic capability. Specifically, over-expression of estrogen-induced genes in these tumors is associated with a better prognosis and signifies estrogen sensitivity that can be exploited with hormone antagonizing agents. It was therefore hypothesized that estrogen-induced gene expression in ovarian carcinoma would successfully predict outcomes and differentiate between tumors of varying estrogen sensitivities. Methods. Two hundred nineteen (219) patients with ovarian cancer who underwent surgery at M. D. Anderson between 2004 and 2007 were identified. Of these, eighty-three (83) patients were selected for inclusion because they had advanced stage, high-grade serous carcinoma of the ovary or peritoneum, had not received neoadjuvant chemotherapy, and had readily available frozen tissue for study. All patients had also received adjuvant treatment with platinum and taxane agents. The expression of seven genes known to be induced by estrogen in the female reproductive tract (EIG121, sFRP1, sFRP4, RALDH2, PR, IGF-1, and ER) was measured using qRT-PCR. Unsupervised cluster analyses of multiple gene permutations were used to categorize patients as high or low estrogen-induced gene expressors. QPCR gene expression results were then compared to ER and PR immunohistochemical (IHC) expression. Cox proportional hazards models were used to evaluate the effects of both individual genes and selected gene clusters on patient survival. Results. Median follow-up time was 38.7 months (range 1-68 months). In a multivariate model, overall survival was predicted by sFRP1 expression (HR 1.10 [1.02-1.19], p=0.01) and EIG121 expression (HR 1.28 [1.10-1.49], p<0.01). A cluster defined by EIG121 and ER was further examined because that combination appeared to reasonably segregate tumors into distinct groups of high and low estrogen-induced gene expressors. Shorter overall survival was associated with high estrogen-induced gene expressors (HR 2.84 [1.11-7.30], p=0.03), even after adjustment for race, age, body mass index, and residual disease at debulking. No difference in IHC ER or PR expression was noted between gene clusters. Conclusion. In sharp contrast to breast and endometrial cancers, high estrogen-induced gene expression predicts shorter overall survival in patients with high-grade serous ovarian carcinoma. An estrogen-induced gene biomarker panel may have utility as prognostic indicator and may be useful to guide management with estrogen antagonists in this population.^

TRIM24-REGULATED ESTROGEN RESPONSE IS DEPENDENT ON SPECIFIC HISTONE MODIFICATIONS IN BREAST CANCER CELLS

In this dissertation, I discovered that function of TRIM24 as a co-activator of ERα-mediated transcriptional activation is dependent on specific histone modifications in tumorigenic human breast cancer-derived MCF7 cells. In the first part, I proved that TRIM24-PHD finger domain, which recognizes unmethylated histone H3 lysine K4 (H3K4me0), is critical for ERα-regulated transcription. Therefore, when LSD1-mediated demethylation of H3K4 is inhibited, activation of TRIM24-regulated ERα target genes is greatly impaired. Importantly, I demonstrated that TRIM24 and LSD1 are cyclically recruited to estrogen responsive elements (EREs) in a time-dependent manner upon estrogen induction, and depletion of their expression exert corresponding time-dependent effect on target gene activation. I also identified that phosphorylation of histone H3 threonine T6 disrupts TRIM24 from binding to the chromatin and from activating ERα-regulated targets. In the second part, I revealed that TRIM24 depletion has additive effect to LSD1 inhibitor- and Tamoxifen-mediated reduction in survival and proliferation in breast cancer cells.

Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains

The 2.8-Å crystal structure of the complex formed by estradiol and the human estrogen receptor-α ligand binding domain (hERαLBD) is described and compared with the recently reported structure of the progesterone complex of the human progesterone receptor ligand binding domain, as well as with similar structures of steroid/nuclear receptor LBDs solved elsewhere. The hormone-bound hERαLBD forms a distinctly different and probably more physiologically important dimer interface than its progesterone counterpart. A comparison of the specificity determinants of hormone binding reveals a common structural theme of mutually supported van der Waals and hydrogen-bonded interactions involving highly conserved residues. The previously suggested mechanism by which the estrogen receptor distinguishes estradiol’s unique 3-hydroxy group from the 3-keto function of most other steroids is now described in atomic detail. Mapping of mutagenesis results points to a coactivator-binding surface that includes the region around the “signature sequence” as well as helix 12, where the ligand-dependent conformation of the activation function 2 core is similar in all previously solved steroid/nuclear receptor LBDs. A peculiar crystal packing event displaces helix 12 in the hERαLBD reported here, suggesting a higher degree of dynamic variability than expected for this critical substructure.

Crystallographic structures of the ligand-binding domains of the androgen receptor and its T877A mutant complexed with the natural agonist dihydrotestosterone

The structures of the ligand-binding domains (LBD) of the wild-type androgen receptor (AR) and the T877A mutant corresponding to that in LNCaP cells, both bound to dihydrotestosterone, have been refined at 2.0 Å resolution. In contrast to the homodimer seen in the retinoid-X receptor and estrogen receptor LBD structures, the AR LBD is monomeric, possibly because of the extended C terminus of AR, which lies in a groove at the dimerization interface. Binding of the natural ligand dihydrotestosterone by the mutant LBD involves interactions with the same residues as in the wild-type receptor, with the exception of the side chain of threonine 877, which is an alanine residue in the mutant. This structural difference in the binding pocket can explain the ability of the mutant AR found in LNCaP cells (T877A) to accommodate progesterone and other ligands that the wild-type receptor cannot.

Thyroid hormone and estrogen interact to regulate behavior.

Environmental perturbations that increase plasma thyroid hormone (T3) concentrations also profoundly affect female reproductive behavior and physiology. We explored whether these effects were mediated by interactions between T3 receptor (TR) and estrogen receptor (ER). This hypothesis was of interest because the half-site of a consensus T3 response element DNA sequence is identical to an ER response element (ERE), and TRs bind to a consensus ERE. Molecular data presented in the accompanying paper [Zhu, Y.-S., Yen, P.M., Chin, W.W.& Pfaff, D.W. (1996) Proc. Natl. Acad. Sci. USA 93, 12587-12592] demonstrate that TRs and ERs are both present in rat hypothalamic nuclear extracts and that both can bind to the promoter the hypothalamic gene preproenkephalin and that interations between liganded TRs and ERs affect preproenkephalin transcription. In this paper, we show that molecular interactions between TRs and ERs are sufficient to mediate environmental effects on estrogen-controlled reproductive behavior. Ovariectomized (OVX) rats treated with high doses of T3 showed significantly lower levels of lordosis behavior in response to estradiol benzoate (EB) compared with OVX females treated with EB alone. Conversely, thyroidectomized/OVX females treated with EB showed significantly greater levels of lordosis behavior compared with OVX females treated with EB, showing the effect of endogenous T3. Thyroid hormone interference with EB-induced behavior could not be explained by a reduction in plasma E2 concentrations or by a general reduction in responsiveness of EB-sensitive tissues. Moreover, numbers of hypothalamic ER-immunoreactive cells increased dramatically following T3 treatment. These data suggest that T3 may reduce EB-dependent sexual behavior through interactions between TR and ER in the nuclei of behaviorally relevant hypothalamic neurons, envisioning for the first time a functional consequence of interactions between two nuclear hormone receptors in brain. These results also open up the possibility of molecular interactions on DNA encoding environmental signals, a new field for the study of neuronal integration.

Estrogen and thyroid hormone interaction on regulation of gene expression.

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.

CREB binding protein acts synergistically with steroid receptor coactivator-1 to enhance steroid receptor-dependent transcription.

Steroid receptors are ligand-regulated transcription factors that require coactivators for efficient activation of target gene expression. The binding protein of cAMP response element binding protein (CBP) appears to be a promiscuous coactivator for an increasing number of transcription factors and the ability of CBP to modulate estrogen receptor (ER)- and progesterone receptor (PR)-dependent transcription was therefore examined. Ectopic expression of CBP or the related coactivator, p300, enhanced ER transcriptional activity by up to 10-fold in a receptor- and DNA-dependent manner. Consistent with this, the 12S E1A adenoviral protein, which binds to and inactivates CBP, inhibited ER transcriptional activity, and exogenous CBP was able to partially overcome this effect. Furthermore, CBP was able to partially reverse the ability of active ER to squelch PR-dependent transcription, indicating that CBP is a common coactivator for both receptors and that CBP is limiting within these cells. To date, the only other coactivator able to significantly stimulate receptor-dependent transcription is steroid receptor coactivator-1 (SRC-1). Coexpression of CBP and SRC-1 stimulated ER and PR transcriptional activity in a synergistic manner and indicated that these two coactivators are not functional homologues. Taken together, these data suggest that both CBP and SRC-1 may function in a common pathway to efficiently activate target gene expression.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Cloning of a novel receptor expressed in rat prostate and ovary.

We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium.

Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. Mutations or abnormal expression of AR in prostate cancer can play a key role in the process that changes prostate cancer from androgen-dependent to an androgen-independent stage. Using a yeast two-hybrid system, we were able to isolate a ligand-dependent AR-associated protein (ARA70), which functions as an activator to enhance AR transcriptional activity 10-fold in the presence of 10(-10) M dihydrotestosterone or 10(-9) M testosterone, but not 10(-6) M hydroxyflutamide in human prostate cancer DU145 cells. Our data further indicated that ARA70 Will only slightly induce the transcriptional activity of other steroid receptors such as estrogen receptor, glucocorticoid receptor, and progesterone receptor in DU145 cells. Together, these data suggest that AR may need a specific coactivator(s) such as ARA70 for optimal androgen activity.

Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif.

Elements responsible for dexamethasone responsiveness of CYP3A23, a major glucocorticoid-inducible member of the CYP3A gene family, have been identified. DNase I footprint analysis of the proximal promoter region revealed three protected sites (sites A, B, and C) within the sequence defined by -167 to -60. Mutational analysis demonstrated that both sites B and C were necessary for maximum glucocorticoid responsiveness and functioned in a cooperative manner. Interestingly, neither site contained a glucocorticoid responsive element. Embedded in site C was an imperfect direct repeat (5'-AACTCAAAGGAGGTCA-3'), showing homology to an AGGTCA steroid receptor motif, typically recognized by the estrogen receptor family, while site B contained an ATGAACT direct repeat; these core sequences were designated dexamethasone response elements 1 and 2 (DexRE-1 and -2), respectively. Neither element has previously been associated with a glucocorticoid-activated transcriptional response. Conversion of the DexRE-1 to either a perfect thyroid hormone or vitamin D3 responsive element further enhanced induction by dexamethasone. Gel-shift analysis demonstrated that glucocorticoid receptor did not associate with either DexRE-1 or -2; hence, glucocorticoid receptor does not directly mediate glucocorticoid induction of CYP3A23. These unusual features suggest an alternate pathway through which glucocorticoids exert their effects.

Ligand-dependent, transcriptionally productive association of the amino- and carboxyl-terminal regions of a steroid hormone nuclear receptor.

The estrogen receptor (ER), a 66-kDa protein that mediates the actions of estrogens in estrogen-responsive tissues, is a member of a large superfamily of nuclear hormone receptors that function as ligand-activated transcription factors. ER shares a conserved structural and functional organization with other members of this superfamily, including two transcriptional activation functions (AFs), one located in its amino-terminal region (AF-1) and the second located in its carboxyl-terminal, ligand-binding region (AF-2). In most promoter contexts, synergism between AF-1 and AF-2 is required for full ER activity. In these studies, we demonstrate a functional interaction of the two AF-containing regions of ER, when expressed as separate polypeptides in mammalian cells, in response to 17 beta-estradiol (E2) and antiestrogen binding. The interaction was transcriptionally productive only in response to E2, and was eliminated by point or deletion mutations that destroy AF-1 or AF-2 activity or E2 binding. Our results suggest a definitive mechanistic role for E2 in the activity of ER--namely, to alter receptor conformation to promote an association of the amino- and carboxyl-terminal regions, leading to transcriptional synergism between AF-1 and AF-2. The productive re assembly of two portions of ER expressed in cells as separate polypeptides demonstrates the evolutionarily conserved modular structural and functional organization of the nuclear hormone receptors. The ligand-dependent interaction of the two AF-containing regions of ER allows for the assembly of a complete activation function from two distinct regions within the same protein, providing a mechanism for hormonally regulated transcription.