Supplementary Material for: Impact of Thyroid Hormones on Estrogen Receptor α-Dependent Transcriptional Mechanisms in Ventromedial Hypothalamus and Preoptic Area

Elevated levels of thyroid hormones (TH) reduce estradiol (E2)-dependent female sexual behavior. E2 stimulates progesterone receptor <i>(Pgr)</i> and oxytocin receptor <i>(Oxtr)</i> within the ventromedial hypothalamus and preoptic area, critical hypothalamic nuclei for sexual and maternal behavior, respectively. Here, we investigated the impact of TH on E2-dependent transcriptional mechanisms in female mice. First, we observed that triiodothyronine (T₃) inhibited the E2 induction of <i>Pgr</i> and <i>Oxtr.</i> We hypothesized that differences in histone modifications and receptor recruitment could explain the influence of TH on E2-responsive <i>Pgr</i> and <i>Oxtr</i> expression. We observed that histone H3 acetylation (H3Ac) and methylation (H3K4me3) was gene and brain-region specific. We then analyzed the recruitment of estrogen receptor α (ERα) and TH receptor α (TRα) on the putative regulatory sequences of <i>Pgr</i> and <i>Oxtr</i>. Interestingly, T₃ inhibited E2-induced ERα binding to a specific <i>Pgr</i> enhancer site, whereas TRα binding was not affected, corroborating our theory that the competitive binding of TRα to an ERα binding site can inhibit ERα transactivation and the subsequent E2-responsive gene expression. On the <i>Oxtr</i> promoter, E2 and T₃ worked together to modulate ERα and TRα binding. Finally, the E2-dependent induction of cofactors was reduced by hypothyroidism and T₃. Thus, we determined that the <i>Pgr</i> and <i>Oxtr</i> promoter regions are responsive to E2 and that T₃ interferes with the E2 regulation of <i>Pgr</i> and <i>Oxtr</i> expression by altering the recruitment of receptors to DNA and changing the availability of cofactors. Collectively, our findings provide insights into molecular mechanisms of response to E2 and TH interactions controlling sex behavior in the hypothalamus.

甲状腺激素(thyroid hormones, TH)水平升高会降低依赖雌二醇(estradiol, E2)的雌性性行为表现。E2可在腹内侧下丘脑(ventromedial hypothalamus)与视前区(preoptic area)——二者分别是调控性行为与母性行为的关键下丘脑核团——内促进孕酮受体(progesterone receptor, <i>Pgr</i>)与催产素受体(oxytocin receptor, <i>Oxtr</i>)的表达。本研究以雌性小鼠为模型，探究TH对依赖E2的转录调控机制的影响。首先，我们观察到三碘甲状腺原氨酸(triiodothyronine, T₃)会抑制E2对<i>Pgr</i>与<i>Oxtr</i>的诱导表达。我们提出假说：组蛋白修饰模式与受体募集的差异，可解释TH对E2响应型<i>Pgr</i>及<i>Oxtr</i>表达的调控作用。研究发现，组蛋白H3乙酰化(histone H3 acetylation, H3Ac)与组蛋白H3赖氨酸4三甲基化(histone methylation, H3K4me3)的修饰特征具有基因与脑区特异性。随后，我们分析了雌激素受体α(estrogen receptor α, ERα)与甲状腺激素受体α(thyroid hormone receptor α, TRα)在<i>Pgr</i>及<i>Oxtr</i>的推定调控序列上的募集情况。有趣的是，T₃会抑制E2诱导的ERα与<i>Pgr</i>特定增强子位点的结合，而TRα的结合并未受到影响，这验证了我们的假说：TRα与ERα结合位点的竞争性结合可抑制ERα的反式激活，进而下调E2响应型基因的表达。在<i>Oxtr</i>启动子区域，E2与T₃协同调控ERα与TRα的结合。最终，依赖E2的辅因子诱导作用可因甲状腺功能减退与T₃处理而被削弱。综上，我们证实<i>Pgr</i>与<i>Oxtr</i>启动子区域可响应E2的调控，且T₃通过改变受体向DNA的募集以及辅因子的可获得性，干扰了E2对<i>Pgr</i>及<i>Oxtr</i>表达的调控。本研究结果为解析下丘脑内调控雌性性行为的E2与TH相互作用的分子机制提供了新的见解。