Activation function 1 of progesterone receptor is required for progesterone antagonism of oestrogen action in the uterus.

BACKGROUND: Progesterone receptor (PGR) is a master regulator of uterine function through antagonistic and synergistic interplays with oestrogen receptors. PGR action is primarily mediated by activation functions AF1 and AF2, but their physiological significance is unknown. RESULTS: We report the first study of AF1 function in mice. The AF1 mutant mice are infertile with impaired implantation and decidualization. This is associated with a delay in the cessation of epithelial proliferation and in the initiation of stromal proliferation at preimplantation. Despite tissue selective effect on PGR target genes, AF1 mutations caused global loss of the antioestrogenic activity of progesterone in both pregnant and ovariectomized models. Importantly, the study provides evidence that PGR can exert an antioestrogenic effect by genomic inhibition of Esr1 and Greb1 expression. ChIP-Seq data mining reveals intermingled PGR and ESR1 binding on Esr1 and Greb1 gene enhancers. Chromatin conformation analysis shows reduced interactions in these genes' loci in the mutant, coinciding with their upregulations. CONCLUSION: AF1 mediates genomic inhibition of ESR1 action globally whilst it also has tissue-selective effect on PGR target genes.

Estrogen markedly reduces circulating low-density neutrophils and enhances pro-tumoral gene expression in neutrophil of tumour-bearing mice.

BACKGROUND: Neutrophils are important for immune surveillance of tumour cells. Neutrophils may also be epigenetically programmed in the tumour microenvironment to promote tumour progression. In addition to the commonly known high-density neutrophils (HDN) based on their separation on density gradient, recent studies have reported the presence of high levels of low-density neutrophils (LDN) in tumour-bearing mice and cancer patients. We reported previously that estrogen promotes the growth of estrogen receptor α-negative mammary tumours in mice undergoing mammary involution through stimulating pro-tumoral activities of neutrophils in the mammary tissue. METHODS: Female BALB/cAnNTac mice at 7-8 weeks old were mated and bilateral ovariectomy was performed 2 days post-partum. At 24 h after forced-weaning of pups to induce mammary involution, post-partum female mice were injected with either E2V, or vehicle control on alternative days for 2-weeks. On 48 h post-weaning, treated female mice were inoculated subcutaneously with 4 T1-Luc2 cells into the 9th abdominal mammary gland. Age-matched nulliparous female was treated similarly. Animals were euthanized on day 14 post-tumour inoculation for analysis. To evaluate the short-term effect of estrogen, post-partum females were treated with only one dose of E2V on day 12 post-tumour inoculation. RESULTS: Estrogen treatment for 2-weeks reduces the number of blood LDN by more than 10-fold in tumour-bearing nulliparous and involuting mice, whilst it had no significant effect on blood HDN. The effect on tumour-bearing mice is associated with reduced number of mitotic neutrophils in the bone marrow and increased apoptosis in blood neutrophils. Since estrogen enhanced tumour growth in involuting mice, but not in nulliparous mice, we assessed the effect of estrogen on the gene expression associated with pro-tumoral activities of neutrophils. Whilst 48 h treatment with estrogen had no effect, 2-weeks treatment significantly increased the expression of Arg1, Il1b and Tgfb1 in both HDN and LDN of involuting mice. In contrast, estrogen increased the expression of Arg1 and Ccl5 in HDN and LDN of nulliparous mice. CONCLUSIONS: Prolonged estrogenic stimulation in tumour-bearing mice markedly hampered tumour-associated increase of LDN plausibly by inhibiting their output from the bone marrow and by shortening their life span. Estrogen also alters the gene expression in neutrophils that is not seen in tumour-free mice. The results imply that estrogen may significantly influence the tumour-modulating activity of blood neutrophils.

Ligand-activated progesterone receptor B activates transcription factor EB to promote autophagy in human breast cancer cells.

Autophagy is an evolutionary conserved, self-eating process that targets cellular constituents for lysosomal degradation. Transcription factor EB (TFEB) is a master regulator of autophagy by inducing the expression of genes involved in autophagic and lysosomal degradation. In breast cancer, ligand-activated progesterone receptor has been reported to influence cancer development by manipulating the autophagy pathway. However, understanding of the mechanism that underlies this autophagic response remains limited. Herein, we report that prolonged treatment with progestin R5020 upregulates autophagy in MCF-7 human breast cancer cells via a novel interplay between progesterone receptor B (PRB) and TFEB. R5020 upregulates TFEB gene expression and protein levels in a PRB-dependent manner. Additionally, R5020 enhances the co-recruitment of PRB and TFEB to each other to facilitate TFEB nuclear localization. Once in the nucleus, TFEB induces the expression of autophagy and lysosomal genes to potentiate autophagy. Together, our findings highlight a novel functional connection between ligand-activated PRB and TFEB to modulate autophagy in MCF-7 breast cancer cells. As breast cancer development is controlled by autophagy, the progestin-PRB-TFEB transduction pathway warrants future attention as a potential therapeutic target in cancer therapy.

Acetylation at lysine 183 of progesterone receptor by p300 accelerates DNA binding kinetics and transactivation of direct target genes.

The identification of lysine acetylation of steroid hormone receptors has previously been based on the presence of consensus motif (K/R)XKK. This study reports the discovery by mass spectrometry of a novel progesterone receptor acetylation site at Lys-183 that is not in the consensus motif. In vivo acetylation and mutagenesis experiments revealed that Lys-183 is a primary site of progesterone receptor (PR) acetylation. Lys-183 acetylation is enhanced by p300 overexpression and abrogated by p300 gene silencing, suggesting that p300 is the major acetyltransferase for Lys-183 acetylation. Furthermore, p300-mediated Lys-183 acetylation is associated with heightened PR activity. Accordingly, the acetylation-mimicking mutant PRB-K183Q exhibited accelerated DNA binding kinetics and greater activity compared with the wild-type PRB on genes containing progesterone response element. In contrast, Lys-183 acetylation had no influence on PR tethering effect on the nuclear factor κ-light chain enhancer of activated B cells (NFκB). Additionally, increases of Lys-183 acetylation by p300 overexpression or inhibition of deacetylation resulted in increases of Ser-294 phosphorylation levels. In conclusion, PR acetylation at Lys-183 by p300 potentiates PR activity through accelerated binding of its direct target genes without affecting PR tethering on other transcription factors. The effect may be mediated by enhancing Ser-294 phosphorylation.

Lysine methylation of progesterone receptor at activation function 1 regulates both ligand-independent activity and ligand sensitivity of the receptor.

Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity.

Protein arginine methyltransferase 6 enhances ligand-dependent and -independent activity of estrogen receptor α via distinct mechanisms.

Recent studies reported that protein arginine methyltransferase 6 (PRMT6) enhances estrogen-induced activity of estrogen receptor α (ERα) and dysfunction of PRMT6 is associated with overall better survival for ERα-positive breast cancer patients. However, it is unclear how PRMT6 promotes ERα activity. Here we report that PRMT6 specifically interacts with ERα at its ligand-binding domain. PRMT6 also methylates ERα both in vitro and in vivo. In addition to enhancing estrogen-induced ERα activity, PRMT6 over-expression up-regulates estrogen-independent activity of ERα and PRMT6 gene silencing in MCF7 cells inhibits ligand-independent ERα activation. More interestingly, the effect of PRMT6 on the ligand-independent ERα activity does not require its methyltransferase activity. Instead, PRMT6 competes with Hsp90 for ERα binding: PRMT6 and Hsp90 bindings to ERα are mutually exclusive and PRMT6 over-expression reduces ERα interaction with Hsp90. In conclusion, PRMT6 requires its methyltransferase activity to enhance ERα's ligand-induced activity, but its effect on ligand-independent activity is likely mediated through competing with Hsp90 for binding to the C-terminal domain of ERα. PRMT6-ERα interaction would prevent ERα-Hsp90 association. Since Hsp90 and associated chaperones serve to maintain ERα conformation for ligand-binding yet functionally inactive, inhibition of ERα-Hsp90 interaction would relieve ERα from the constraint of chaperone complex.

Down-regulation of tripartite-motif containing 22 expression in breast cancer is associated with a lack of p53-mediated induction.

Tripartite-motif containing 22 (TRIM22) is a direct p53 target gene and inhibits the clonogenic growth of leukemic cells. Its expression in Wilms tumors is negatively associated with disease relapse. This study addresses if TRIM22 expression is de-regulated in breast carcinoma. Western blotting analysis of a panel of 10 breast cancer cell lines and 3 non-malignant mammary epithelial cell lines with a well-characterized TRIM22 monoclonal antibody showed that TRIM22 protein is greatly under-expressed in breast cancer cells as compared to non-malignant cell lines. Similarly, TRIM22 protein is significantly down-regulated in breast tumors as compared to matched normal breast tissues. Study of cell lines with methylation inhibitor and bisulfite sequencing indicates that TRIM22 promoter hypermethylation may not be the cause for TRIM22 under-expression in breast cancer. Instead, we found that TRIM22 protein level correlates strongly (R=0.79) with p53 protein level in normal breast tissue, but this correlation is markedly impaired (R=0.48) in breast cancer tissue, suggesting that there is some defects in p53 regulation of TRIM22 gene in breast cancer. This notion is supported by cell line studies, which showed that TRIM22 was no longer inducible by p53-activating genotoxic drugs in breast cancer cell lines and in a p53 null cell line H1299 transfected with wild type p53. In conclusion, this study shows that TRIM22 is greatly under-expressed in breast cancer. p53 dysfunction may be one of the mechanisms for TRIM22 down-regulation.

The local microenvironment instigates the regulation of mammary tetratricopeptide repeat domain 9A during lactation and involution through local regulation of the activity of estrogen receptor α.

Tetratricopeptide repeat domain 9A (TTC9A) belongs to a family of TTC9 proteins. Its induction by progesterone in breast cancer cells was associated with marked growth inhibition and induction of focal adhesion. TTC9A interacts specifically with actin-binding protein tropomyosin Tm5NM-1 which stabilizes actin filament and focal adhesion. However, the function of TTC9A is still obscure. This study exploited mice model to characterize the regulation of TTC9A gene expression during mammary development and explored possible mechanisms of TTC9A gene regulation. It was demonstrated that mammary TTC9A expression is distinctively down-regulated in gland undergoing functional differentiation (lactation) and up-regulated during involution. Furthermore, TTC9A expression during lactation and involution is regulated by the factors in the local microenvironment. This is illustrated with teat sealing model in which the teat sealed glands (undergoing involution) expressed significantly higher levels of TTC9A protein and mRNA than the contralateral non-sealed lactating glands. Importantly, this local induction of TTC9A expression upon involution coincided with the re-activation of estrogen receptor α (ERα). Together with the observation that TTC9A is a direct ERα target gene, we propose that the fall and rise of TTC9A levels during lactation and involution is caused by the changes of ERα activity that is in turn regulated by the factors in the microenvironment.

High Levels of Progesterone Receptor B in MCF-7 Cells Enable Radical Anti-Tumoral and Anti-Estrogenic Effect of Progestin.

The widely reported conflicting effects of progestin on breast cancer suggest that the progesterone receptor (PR) has dual functions depending on the cellular context. Cell models that enable PR to fully express anti-tumoral properties are valuable for the understanding of molecular determinant(s) of the anti-tumoral property. This study evaluated whether the expression of high levels of PR in MCF-7 cells enabled a strong anti-tumoral response to progestin. MCF-7 cells were engineered to overexpress PRB by stable transfection. A single dose of Promegestone (R5020) induced an irreversible cell growth arrest and senescence-associated secretory phenotype in MCF-7 cells with PRB overexpression (MCF-7PRB cells) but had no effect on MCF-7 cells with PRA overexpression. The growth-arresting effect was associated with downregulations of cyclin A2 and B1, CDK2, and CDK4 despite an initial upregulation of cyclin A2 and B1. R5020 also induced an evident activation of Nuclear Factor κB (NF-κB) and upregulation of interleukins IL-1α, IL-1ß, and IL-8. Although R5020 caused a significant increase of CD24+CD44+ cell population, R5020-treated MCF-7PRB cells were unable to form tumorspheres and underwent massive apoptosis, which is paradoxically associated with marked downregulations of the pro-apoptotic proteins BID, BAX, PARP, and Caspases 7 and 8, as well as diminution of anti-apoptotic protein BCL-2. Importantly, R5020-activated PRB abolished the effect of estrogen. This intense anti-estrogenic effect was mediated by marked downregulation of ERα and pioneer factor FOXA1, leading to diminished chromatin-associated ERα and FOXA1 and estrogen-induced target gene expression. In conclusion, high levels of agonist-activated PRB in breast cancer cells can be strongly anti-tumoral and anti-estrogenic despite the initial unproductive cell cycle acceleration. Repression of ERα and FOXA1 expression is a major mechanism for the strong anti-estrogenic effect.

Activation function 1 of progesterone receptor is required for mammary development and regulation of RANKL during pregnancy.

Progesterone receptor (PGR) is a member of the nuclear receptor superfamily of transcription factors. It is critical for mammary stem cells expansion, mammary ductal branching and alveologenesis. The transcriptional activity of PGR is mainly mediated by activation functions AF1 and AF2. Although the discovery of AF1 and AF2 propelled the understanding of the mechanism of gene regulation by nuclear receptors, their physiological roles are still poorly understood. This is largely due to the lack of suitable genetic models. The present study reports gain or loss of AF1 function mutant mouse models in the study of mammary development. The gain of function mutant AF1_QQQ exhibits hyperactivity while the loss of function mutant AF1_FFF shows hypoactivity on mammary development. However, the involvement of AF1 is context dependent. Whereas the AF1_FFF mutation causes significant impairment in mammary development during pregnancy or in response to estrogen and progesterone, it has no effect on mammary development in nulliparous mice. Furthermore, Rankl, but not Wnt4 and Areg is a major target gene of AF1. In conclusion, PGR AF1 is a pivotal ligand-dependent activation domain critical for mammary development during pregnancy and it exerts gene specific effect on PGR regulated genes.

Dynamic localization of tripartite motif-containing 22 in nuclear and nucleolar bodies.

Tripartite motif-containing 22 (TRIM22) exhibits antiviral and growth inhibitory properties, but there has been no study on the localization and dynamics of the endogenous TRIM22 protein. We report here that TRIM22 is dramatically induced by progesterone in MDA-MB-231-derived ABC28 cells and T47D cells. This induction was associated with an increase in TRIM22 nuclear bodies (NB), and an even more prominent increase in nucleolar TRIM22 bodies. Distinct endogenous TRIM22 NB were also demonstrated in several other cell lines including MCF7 and HeLa cells. These TRIM22 NB resemble Cajal bodies, co-localized with these structures and co-immunoprecipitated with p80-coilin. However, IFNgamma-induced TRIM22 in HeLa and MCF7 cells did not form NB, implying the forms and distribution of TRIM22 are regulated by specific cellular signals. This notion is also supported by the observation that TRIM22 NB undergoes dynamic cell-cycle dependent changes in distribution such that TRIM22 NB started to form in early G0/G1 but became dispersed in the S-phase. In light of its potential antiviral and antitumor properties, the findings here provide an interesting gateway to study the relationship between the different forms and functions of TRIM22.

Delineation of critical amino acids in activation function 1 of progesterone receptor for recruitment of transcription coregulators.

The activation functions AF1 and AF2 of nuclear receptors mediate the recruitment of coregulators in gene regulation. AF1 is mapped to the highly variable and intrinsically unstructured N terminal domain and AF2 lies in the conserved ligand binding domain. The unstructured nature of AF1 offers structural plasticity and hence functional versatility in gene regulation. However, little is known about the key functional residues of AF1 that mediates its interaction with coregulators. This study focuses on the progesterone receptor (PR) and reports the identification of K464, K481 and R492 (KKR) as the key functional residues of PR AF1. The KKR are monomethylated and function cooperatively. The combined mutations of KKR to QQQ render PR isoform B (PRB) hyperactive, whereas KKR to FFF mutations abolishes as much as 80% of PR activity. Furthermore, the hyperactive QQQ mutation rescues the loss of PR activity due to E911A mutation in AF2. The study also finds that the magnitudes of the mutational effect differ in different cell types as a result of differential effects on the functional interaction with coregulators. Furthermore, KKR provides the interface for AF1 to physically interact with p300 and SRC-1, and with AF2 at E911. Intriguingly, the inactive FFF mutant interacts strikingly stronger with both SRC-1 and AF2 than wt PRB. We propose a tripartite model to describe the dynamic interactions between AF1, AF2 and SRC-1 with KKR of AF1 and E911 of AF2 as the interface. An overly stable interaction would hamper the dynamics of disassembly of the receptor complex.

The activities of progesterone receptor isoform A and B are differentially modulated by their ligands in a gene-selective manner.

It is known that progesterone receptor (PR) isoform A (PR-A) and isoform B (PR-B) may mediate different effects of progesterone. The objective of this study was to determine if the functions of PR isoforms also vary in response to different PR modulators (PRM). The effects of 7 synthetic PRM were tested in MDA-MB-231 cells engineered to express PR-A, PR-B, or both PR isoforms. The effects of progesterone were similar in cells expressing PR-A or PR-B in which it inhibited growth and induced focal adhesion. On the other hand, synthetic PRM modulated the activity of the PR isoforms differently. RU486, CDB4124, 17alpha-hydroxy CDB4124 and VA2914 exerted agonist activities on cell growth and adhesion via PR-B. Via PR-A, however, these compounds displayed agonist effect on cell growth but induced stellate morphology which was distinct from the agonist's effect. Their dual properties via PR-A were also displayed at the gene expression level: the compounds acted as agonists on cell cycle genes but exhibited antagonistic effect on cell adhesion genes. Introduction of ERalpha by adenoviral vector to these cells did not change PR-A or PR-B mediated effect of PRM radically, but it causes significant cell rounding and modified the magnitudes of the responses to PRM. The findings suggest that the activities of PR isoforms may be modulated by different PRM through gene-specific regulatory mechanisms. This raises an interesting possibility that PRM may be designed to be PR isoform and cellular pathway selective to achieve targeted therapy in breast cancer.

Tetratricopeptide repeat domain 9A is an interacting protein for tropomyosin Tm5NM-1.

BACKGROUND: Tetratricopeptide repeat domain 9A (TTC9A) protein is a recently identified protein which contains three tetratricopeptide repeats (TPRs) on its C-terminus. In our previous studies, we have shown that TTC9A was a hormonally-regulated gene in breast cancer cells. In this study, we found that TTC9A was over-expressed in breast cancer tissues compared with the adjacent controls (P < 0.00001), suggesting it might be involved in the breast cancer development process. The aim of the current study was to further elucidate the function of TTC9A. METHODS: Breast samples from 25 patients including the malignant breast tissues and the adjacent normal tissues were processed for Southern blot analysis. Yeast-two-hybrid assay, GST pull-down assay and co-immunoprecipitation were used to identify and verify the interaction between TTC9A and other proteins. RESULTS: Tropomyosin Tm5NM-1 was identified as one of the TTC9A partner proteins. The interaction between TTC9A and Tm5NM-1 was further confirmed by GST pull-down assay and co-immunoprecipitation in mammalian cells. TTC9A domains required for the interaction were also characterized in this study. The results suggested that the first TPR domain and the linker fragment between the first two TPR domains of TTC9A were important for the interaction with Tm5NM-1 and the second and the third TPR might play an inhibitory role. CONCLUSION: Since the primary function of tropomyosin is to stabilize actin filament, its interaction with TTC9A may play a role in cell shape and motility. In our previous results, we have found that progesterone-induced TTC9A expression was associated with increased cell motility and cell spreading. We speculate that TTC9A acts as a chaperone protein to facilitate the function of tropomyosins in stabilizing microfilament and it may play a role in cancer cell invasion and metastasis.

Estrogen reprograms the activity of neutrophils to foster protumoral microenvironment during mammary involution.

Epidemiological studies have indicated increased risk for breast cancer within 10 years of childbirth. Acute inflammation during mammary involution has been suggested to promote this parity-associated breast cancer. We report here that estrogen exacerbates mammary inflammation during involution. Microarray analysis shows that estrogen induces an extensive proinflammatory gene signature in the involuting mammary tissue. This is associated with estrogen-induced neutrophil infiltration. Furthermore, estrogen induces the expression of protumoral cytokines/chemokines, COX-2 and tissue-remodeling enzymes in isolated mammary neutrophils and systemic neutrophil depletion abolished estrogen-induced expression of these genes in mammary tissue. More interestingly, neutrophil depletion diminished estrogen-induced growth of ERα-negative mammary tumor 4T1 in Balb/c mice. These findings highlight a novel aspect of estrogen action that reprograms the activity of neutrophils to create a pro-tumoral microenvironment during mammary involution. This effect on the microenvironment would conceivably aggravate its known neoplastic effect on mammary epithelial cells.

In silico modelling of hormone response elements.

BACKGROUND: An important step in understanding the conditions that specify gene expression is the recognition of gene regulatory elements. Due to high diversity of different types of transcription factors and their DNA binding preferences, it is a challenging problem to establish an accurate model for recognition of functional regulatory elements in promoters of eukaryotic genes. RESULTS: We present a method for precise prediction of a large group of transcription factor binding sites - steroid hormone response elements. We use a large training set of experimentally confirmed steroid hormone response elements, and adapt a sequence-based statistic method of position weight matrix, for identification of the binding sites in the query sequences. To estimate the accuracy level, a table of correspondence of sensitivity vs. specificity values is constructed from a number of independent tests. Furthermore, feed-forward neural network is used for cross-verification of the predicted response elements on genomic sequences. CONCLUSION: The proposed method demonstrates high accuracy level, and therefore can be used for prediction of hormone response elements de novo. Experimental results support our analysis by showing significant improvement of the proposed method over previous HRE recognition methods.

Establishing a statistic model for recognition of steroid hormone response elements.

Identification of hormone response elements (HREs) is essential for understanding the mechanism of hormone-regulated gene expression. To date, there has been a lack of effective bioinformatics tools for recognition of specific HRE such as Progesterone Response Elements (PRE). In this paper, a comprehensive survey and comparison of in silico methods is conducted for establishing a more accurate statistic model. Homogeneity of steroid HRE is analyzed and a reliable training dataset is constructed through extensive searching for experimentally validated response elements from more than 150 literature sources. Based on the observation that the verified HREs carry di-nucleotide preservation in comparison with uniform nucleotide distributions, both mono and di-nucleotide Position Weight Matrices are computed to extract the statistic pattern of the positions. It is followed by the sequence transition pattern recognition using a specifically designed profile Hidden Markov Model. Reciprocal combination of the statistic and transition patterns significantly improves the performance of the model in terms of higher sensitivity and specificity. Upon acquisition of the putative response elements in the promoter areas of vertebrate genes, a qualitative scheme is applied to assess the probability for each gene to be a hormone primary target. Using >650 records of experimentally validated steroid hormone response elements, a high sensitivity level of 73% and high specificity level of one prediction per 8.24 kb is reached, allowing this model to be used for further prediction of primary target genes through the analysis of their upstream promoters, for human or other vertebrate genomes of interest. Additional documents, supplementary data and the web-based program developed for response elements prediction are freely available for academic research at . Submission of putative gene promoter regions for recognition of potential regulatory PREs can be as long as 5 kb.

Genome-wide methylation analysis identified sexually dimorphic methylated regions in hybrid tilapia.

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms.

Tetratricopeptide repeat domain 9A negatively regulates estrogen receptor alpha activity.

Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.