HOXA10直接调控人子宫内膜间质细胞中p/CAF启动子的活性

目的：筛选与鉴定转录因子同源框蛋白（HOX）A10下游靶基因。方法：用Ad-HOXA10重组腺病毒感染人子宫内膜间质细胞,通过染色体免疫共沉淀（ChIP）方法,筛选HOXA10的下游靶基因;采用萤光素酶报告基因实验结合腺病毒介导的HOXA10过表达和小干扰RNA介导的基因沉默实验,分析HOXA10对下游靶基因的转录调控作用。结果：ChIP-PCR筛选并鉴定p/CAF（p300/CBP相关因子）为HOXA10直接作用的靶基因;过表达HOXA10抑制p/CAF启动子活性达60%;基因沉默内源性HOXA10的表达可以激活p/CAF启动子活性超过2倍以上。结论：p/CAF是HOXA10新的靶基因,HOXA10可能通过调节p/CAF的表达来调控子宫内膜的发育。     

Regulation of tryptophan 2,3-dioxygenase by HOXA10 enhances embryo viability through serotonin signaling

Tryptophan 2,3-dioxygenase (TDO) is expressed in endometrium and catabolizes tryptophan, a precursor in the biosynthesis of serotonin. Tryptophan metabolism is an important mechanism for regulation of serotonin levels. Preimplantation mouse embryos are known to express serotonin receptors, specifically the 5-HT1D and 5-HT7 serotonin receptor subtypes. Here we demonstrate that Hoxa10 regulates endometrial TDO expression and improves embryo viability through increased serotonin production. Transfection of pcDNA-Hoxa10 to the murine uterus increased total TDO expression. In vitro, epithelial cell TDO expression was decreased after transfection with Hoxa10. Decreased glandular TDO in response to HOXA10 may augment serotonin production by increasing tryptophan availability. Conversely, stromal TDO expression increased with constitutive Hoxa10 expression. In mice, epithelial serotonin was increased in response to constitutive expression of Hoxa10. Embryo quality was impaired after treatment with Hoxa10 antisense. Blockade of serotonin receptors 1D and 7 also resulted in impaired embryo development, indicating an essential role for Hoxa10 induction of TDO and subsequent serotonin production in embryo development. Transfection of pcDNA-TDO also decreased the number of T cells in the endometrial stroma. We have shown a novel mechanism by which HOXA10 regulates endometrial TDO expression. In the endometrial stroma, HOXA10 increases TDO mRNA, which may increase tryptophan catabolism, allowing for immune tolerance at the time of embryo implantation. In endometrial glands, HOXA10 decreases TDO mRNA leading to increased serotonin that in turn acts to promote normal embryo development.     

Regulation of HOXA2 gene expression by the ATP-dependent chromatin remodeling enzyme CHD8

Chromodomain, helicase, DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin remodeling enzyme that has been demonstrated to exist within a large protein complex which includes WDR5, Ash2L, and RbBP5, members of the Mixed Lineage Leukemia (MLL) histone modifying complexes. Here we show that CHD8 relocalizes to the promoter of the MLL regulated gene HOXA2 upon gene activation. Depletion of CHD8 enhances HOXA2 expression under activating conditions. Furthermore, depletion of CHD8 results in a loss of the WDR5/Ash2L/RbBP5 subcomplex, and consequently H3K4 trimethylation, at the HOXA2 promoter. These studies suggest that CHD8 alters HOXA2 gene expression and regulates the recruitment of chromatin modifying enzymes.

Structured summary

MINT-7542810: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with RbBP5 (uniprotkb:Q15291) by anti tag coimmunoprecipitation (MI:0007)MINT-7542794: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)MINT-7542820: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0915) with ASH2L (uniprotkb:Q9UBL3) by anti tag coimmunoprecipitation (MI:0007)MINT-7542769: CHD8 (uniprotkb:Q9HCK8) physically interacts (MI:0914) with RbBP5 (uniprotkb:Q15291), ASH2L (uniprotkb:Q9UBL3) and WDR5 (uniprotkb:P61964) by anti tag coimmunoprecipitation (MI:0007)     

Direct regulation of beta3-integrin subunit gene expression by HOXA10 in endometrial cells

Estrogen and progesterone regulate HOXA10 expression in the endometrium, where HOXA10 is necessary for implantation. The integrins are also involved in early embryo-endometrial interactions. Here we show that HOXA10 directly regulates beta3-integrin subunit expression in the endometrium, likely mediating the effect of sex steroids on beta3-integrin expression. beta3-Integrin expression was decreased in endometrium shown to have low HOXA10 expression. beta3-Integrin mRNA levels were increased in endometrial adenocarcinoma cells (Ishikawa) transfected with pcDNA3.1/HOXA10, and decreased in cells treated with HOXA10 antisense. Seven consensus HOXA10 binding sites were identified 5' of the beta3-integrin gene. Direct binding of HOXA10 protein to four sites was demonstrated by EMSA. Reporter gene expression increased in BT-20 cells cotransfected with pcDNA3.1/ HOXA10 and pGL3-promoter vector containing region F (encompassing all seven HOXA10 consensus sites). A 41-bp segment (Region A) showed highest affinity binding to HOXA10 protein. Increased reporter expression, equal in magnitude to that obtained with Region F, was obtained with Region A. HOXA10 protein binding within Region A was localized by deoxyribonuclease I footprinting. beta3-Integrin expression was directly up-regulated by HOXA10 through a 41-bp 5'-regulatory element. Sex steroids regulate the expression of endometrial beta3-integrin through a pathway involving HOXA10.     

Regulation of chemokine expression by IL-10 in lung inflammation

We have been interested in understanding the mechanisms regulating the inflammatory process underlying acute lung injury. The current studies have employed a model of acute lung inflammation in mice triggered by bacterial lipopolysaccharide. The development of this injury was associated with increased expression of the chemokines, MIP-1alpha and MIP-2, that coordinate recruitment of neutrophils to the lung. IL-10 is a potent, endogenous anti-inflammatory molecule that has been shown to decrease lung inflammation partly on the basis of TNF-alpha and IL-1beta inhibition. In these studies we tested the hypothesis that endogenous IL-10 modulates chemokine expression using the IL-10 knock-out mouse, and then explored the molecular mechanisms by which IL-10 might do so. The results demonstrate that significant elevations in both chemokines were observed in the absence of IL-10 and that these findings were associated with significant increases in lung neutrophil accumulation. In vitro studies defined two, gene-specific, mechanisms by which IL-10 regulated chemokine expression: mRNA destabilization and NF-kappaB inhibition. These results suggested that IL-10 is an important, endogenous regulator of chemokine expression in acute lung inflammation.     

同源盒基因A10编码蛋白的靶调节基因的筛选与鉴定

目的：筛选和鉴定同源盒基因A10编码蛋白（HOXA10）的靶调节基因。方法：以人子宫内膜细胞系AN3CA为实验对象,采用染色质免疫沉淀方法筛选HOXA10靶基因;采用平端克隆方法构建HOXA10靶基因库;采用DNA序列分析结合生物信息学方法鉴定HOXA10靶基因。结果：共获得含有HOXA10结合片段的克隆197个,选取插入片段大于100bp的质粒67个进行DNA序列分析,其中含有HOXA10结合序列TTAT的基因16个。结论：初步筛选出16个HOXA10候选靶基因,为进一步研究HOXA10的基因调节机理提供了新的思路。     

Androgen receptor expression in the rat prostate is down-regulated by dietary phytoestrogens

Background  

It is well established that the growth of the prostate gland is a hormone-dependent phenomenon involving both androgenic and estrogenic control. Proliferation of prostate cells is, at least in part, under control of estrogen receptor beta (ER-beta). Phytoestrogens bind ER-beta with high affinity and therefore may have antiproliferative effects in the prostate.     

MiR-135a functions as a tumor suppressor in epithelial ovarian cancer and regulates HOXA10 expression

The activation of homeobox A10 (HOXA10) has been proved to be an important event in epithelial ovarian carcinogenesis, yet its regulation in epithelial ovarian cancer (EOC) is still not fully understood. Here, we aimed to reveal the mechanism that a predicted target miRNA regulates HOXA10 expression and the association of its expression with progression of EOC. Here, by using computer-assisted algorithms from PicTar, TargetScan, and miRBase, we identified that the predicted target miRNA of HOXA10 was miR-135a. MiR-135a expression in EOC tissues and controls was measured with quantitative RT-PCR. The role of miR-135a and HOXA10 in the growth and survival of several EOC cell lines was determined with several in vitro approaches. We found that miR-135a expression was downregulated in an EOC patient cohort. Also, patients with low miR-135a expression had shorter overall survival and progression-free survival durations than those with high expression. Functional analysis of three EOC-derived cell lines (SKOV-3, HEY, and OVCAR-3) demonstrated that miR-135a directly regulated HOXA10 expression by targeting its 3′-UTR. Inhibition of HOXA10 expression with miR-135a mimics and HOXA10 siRNA consistently resulted in cell apoptosis with concomitant enhancement of caspase-3, increase of p53 expression and reduction of Bcl-2 expression, and also suppressed cell growth and adhesion. These findings suggest that ubiquitous loss of miR-135a expression is a critical mechanism for the overexpression of HOXA10 in EOC cells, which is implicated in epithelial ovarian carcinogenesis. Furthermore, miR-135a may be predictive of EOC prognosis.