DOI: 10.6084/m9.figshare.19689322.v1
title: Additional file 1 of The steroid hormone estriol (E3) regulates epigenetic programming of fetal mouse brain and reproductive tract

description: 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&gt;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.