Additional file 1 of The steroid hormone estriol (E3) regulates epigenetic programming of fetal mouse brain and reproductive tract

Additional file 1: Fig. S1 Prenatal exposure of estriol (E3) alters the global gene expression profile of the female offspring uteri. A) Grouping of altered genes in E3-treated animals compared to vehicle-treated groups according to biological functions using the Ingenuity (IPA) software. B) Pathway analyses using Ingenuity (IPA) software. Fig. S2 A, B, C, D) Prenatal exposure of E3 results in significant hypomethylation of over 2000 genes in the female offspring uteri. Grouping of altered genes in E3-treated compared to vehicle-treated groups according to A) biological functions and B) molecular functions using Ingenuity (IPA) software. C-D) Pathway analyses using Ingenuity (IPA) software. E, F) E3 treatment increases the numbers of ER-SUZ12-bound genes. Ishikawa cells were treated with/without E3, and ChIP (chromatin immunoprecipitation) was performed using an E) anti-estrogen receptor α (ERα) or F) ERβ antibody (First ChIP) and then with an anti-SUZ12 antibody (Re-ChIP). The bound DNA was then identified by sequencing. The Venn diagrams show the numbers of genes bound by the ER-SUZ12 complex with/without E3 treatment. Fig. S3) Prenatal exposure to E3 results in significant hypermethylation of over 2500 genes in the female offspring uteri. Grouping of altered genes in E3-treated compared to vehicle-treated groups according to A) biological functions and B) molecular functions using the Ingenuity (IPA) software. C-D) Pathway analyses using Ingenuity (IPA) software. Fig. S4 Prenatal exposure to E3 has no effect on learning or memory behavior. Eight weeks-old pregnant female CD-1 mice were treated with vehicle DMSO (CT) or E3. At 6 months after birth, the offspring were subjected to a water maze test and novel object recognition (NOR) task. A) During the day 2-5 of invisible platform tests, the E3 treated and CT mice exhibited a similar latency to escape onto the hidden platform. B-D) In the probe trial on the 6th day, the E3-treated offspring traveled into the third quadrant, where the hidden platform was previously placed. The B) passing time, C) time spent in the area and D) path length are similar between E3 and CT offspring. E) shows the time spent on two objects (initial object labeled A) at day 1, versus repeated object A and a new object B at day 3. The mice spent a longer amount of time on object B (new object) than object A (old object) at day 3. There was no difference in the time for either object A or B between CT and E3. Error bars: mean + SEM of the results, P>0.05 (Student t-test), N=24 for each CT and E3. Fig. S5 Pathway analysis of the anxiety related genes with altered expression in the E3-treated offspring. A) Pathway analysis of the anxiety related genes with altered expression in the Male and Female offspring cortex and verified by RT-PCR (Fig.5B-E), B) Pathway analysis of the anxiety related genes with altered expression in the female offspring hippocampus and verified by RT-PCR (Fig.5 A). Fig. S6. Prenatal exposure to E3 alters global gene expression in the adult offspring brains. Eight weeks-old pregnant female CD-1 mice were treated with vehicle DMSO (CT) or E3. 6 months after birth, the gene expression in the offspring brains was profiled by a whole genome mRNA microarray. The altered genes are grouped according to their biological functions in A) female hippocampus, C) male hippocampus, E) male cortex and G) female cortex. The altered genes that are related to the ERβ (also known as ESR2) signaling are shown in B) female hippocampus, , F) male cortex, H) female cortex, D) related to the ERα (also known as ESR1) signaling in male hippocampus. The data are analyzed using the Ingenuity (IPA) software.

附加文件1：图S1 产前暴露于雌三醇（estriol, E3）会改变雌性后代子宫的全局基因表达谱。A) 采用Ingenuity (IPA)软件，根据生物学功能对E3处理组与溶剂处理组中差异表达基因进行分组。B) 采用Ingenuity (IPA)软件进行通路分析。图S2 A、B、C、D) 产前暴露于E3会导致雌性后代子宫中超过2000个基因发生显著低甲基化。采用Ingenuity (IPA)软件，根据A) 生物学功能和B) 分子功能对E3处理组与溶剂处理组中的差异基因进行分组。C-D) 采用Ingenuity (IPA)软件进行通路分析。E、F) E3处理会增加ER-SUZ12结合基因的数量。将Ishikawa细胞用E3处理或不做处理，采用E) 抗雌激素受体α（estrogen receptor α, ERα）或F) 抗雌激素受体β（estrogen receptor β, ERβ）抗体进行染色质免疫共沉淀（chromatin immunoprecipitation, ChIP），随后使用抗SUZ12抗体进行二次免疫共沉淀（Re-ChIP）。通过测序鉴定结合的DNA。维恩图展示了E3处理与否时ER-SUZ12复合物结合的基因数量。图S3) 产前暴露于E3会导致雌性后代子宫中超过2500个基因发生显著高甲基化。采用Ingenuity (IPA)软件，根据A) 生物学功能和B) 分子功能对E3处理组与溶剂处理组中的差异基因进行分组。C-D) 采用Ingenuity (IPA)软件进行通路分析。图S4 产前暴露于E3对学习或记忆行为无影响。将8周龄的怀孕CD-1品系雌性小鼠用溶剂二甲基亚砜（dimethyl sulfoxide, DMSO）或E3处理。后代在出生6个月后接受水迷宫实验和新物体识别（novel object recognition, NOR）任务。A) 在隐匿平台测试的第2-5天，E3处理组与对照组（CT）小鼠到达隐藏平台的逃逸潜伏期无显著差异。B-D) 在第6天的探查试验中，E3处理组后代会进入先前放置隐藏平台的第三象限。B) 穿过该象限的时间、C) 在该区域停留的时间以及D) 运动路径长度在E3组与对照组之间均无显著差异。E) 展示了第1天小鼠在两个物体（初始标记为物体A）上的停留时间，与第3天小鼠在重复物体A和新物体B上的停留时间。第3天小鼠在新物体B上的停留时间长于物体A。对照组与E3处理组在物体A或B的停留时间上均无差异。误差棒表示均值±标准误（standard error of the mean, SEM），P>0.05（学生t检验，Student t-test），对照组与E3处理组每组样本量N=24。图S5 对E3处理后代中差异表达的焦虑相关基因进行通路分析。A) 对雌雄后代皮层中差异表达的焦虑相关基因进行通路分析，并通过逆转录聚合酶链式反应（reverse transcription PCR, RT-PCR）验证（图5B-E）；B) 对雌性后代海马体中差异表达的焦虑相关基因进行通路分析，并通过RT-PCR验证（图5A）。图S6 产前暴露于E3会改变成年后代大脑的全局基因表达。将8周龄的怀孕CD-1品系雌性小鼠用溶剂DMSO或E3处理。后代出生6个月后，通过全基因组mRNA微阵列对其大脑中的基因表达进行谱分析。采用Ingenuity (IPA)软件对差异基因进行分组：A) 雌性海马体、C) 雄性海马体、E) 雄性皮层以及G) 雌性皮层的差异基因按生物学功能分组；B) 雌性海马体、F) 雄性皮层、H) 雌性皮层中与ERβ（亦称ESR2）信号通路相关的差异基因；D) 雄性海马体中与ERα（亦称ESR1）信号通路相关的差异基因。