Previously reported glucocorticoid response elements (GREs) in glucocorticoid-induced leucine zipper (GHz) and Kruppel-like factor 13 (Klf13) were used to design primers (Cruz-Topete et al.
Expression of Gilz, FK506 binding protein 5 (Fkbp5), Sgk1, Klf13, and Per1, and interleukin 13 receptor subunit alpha 2 (Il13ra2) and 11 [beta]-hydroxysteroid dehydrogenase II (11[beta]hsdII), classic target genes of GR signaling, was quantified by qRT-PCR.
Basal mRNA expression of Ppib, Gilz, Fkbp5, Sgk1, Klf13, Per1, IL13ra2, and 11[beta]hsdII was not different in adult mice exposed to genistein on PND1-5 compared with control mice (Figure 3B).
Attenuated induction of Gilz, Fkbp5, Sgk1, and Klf13 by glucocorticoids in the uterus of adult mice exposed to genistein during development suggested that the transcriptional response to glucocorticoids was altered, thus whole-genome microarray analysis was performed on uteri from adult control and genistein-exposed mice treated with vehicle or 1 mg/kg Dex for 4 h.
For this experiment, GR recruitment was evaluated for Gilz, Sgk1, and Klf13, which demonstrated blunted induction of mRNA expression by glucocorticoid treatment following neonatal genistein exposure, and Per1, which did not demonstrate differences in glucocorticoid-induced mRNA expression.
Indeed, enrichment of GR at GREs within classic glucocorticoid target genes Gilz, Sgk1, and Klf13 was reduced in the uterus of adult female mice following neonatal genistein exposure, which corresponded to blunted mRNA induction of these genes in response to glucocorticoids.
Such site-specific changes to the epigenetic landscape may have contributed to the latent differences in glucocorticoid responsiveness of Gilz, Sgk1, and Klf13 compared with Per1, 11[beta]-hsdII, and Il13ra2 in the uterus of adult mice following neonatal genistein exposure.
Notable upregulated genes were involved in neuron differentiation ( BTG2, NTNG2, DLL1, HES1, NR4A2, STAT3, and VEGF-A ), myeloid cell and osteoclast differentiation ( KLF10, TOB2, and ZFP36 ), cellular and macromolecular biosynthetic process ( KLF13
, MKL1, HES1, MYOG, NR4A1, NR4A2, STAT3, and MAF ), and skeletal system development (JUND, KLF10, NAB2, MYOG, and TIPARP ).
miRNA Target Partial target genes numbers miR-2070-3p 1352 SH3D21, BCL7C, ACTR3B, EPC1 miR-222 624 RGS6, HMG20A, RBM15, NFE2 miR-502-3p 462 CRTC1, FGD1, CCL8, STARD8 miR-6238 391 Mcph1, PDZK1 miR-7446-3p 414 KLF13
, SIAH2, TUB miR-7475-5p 517 LDB1, DVL3, PEG3, LRP1, LATS2, EFHD2 miR-125a-5p 2246 ESRRa, SENP2, BCL2L12, SREBP-1, ABCA2, NNMT miR-126 2438 TNKS2, PTPRU, RGS14, NAP1L5 miR-378e 1044 IGF1R, CACNB2, RASIP1, API5, SCD5, SLC25A29 miR-7930-3p 1793 CABIN1, PCDHA2, PLXNA4