Prenatal Iron Deficiency and Choline Supplementation Interact to Epigenetically Regulate Jarid1b and Bdnf in the Rat Hippocampus into Adulthood

Abstract

Early-life iron deficiency (ID) causes long-term neurocognitive impairments and gene dysregulation that can be partially mitigated by prenatal choline supplementation. The long-term gene dysregulation is hypothesized to underlie cognitive dysfunction. However, mechanisms by which iron and choline mediate long-term gene dysregulation remain unknown. In the present study, using a well-established rat model of fetal-neonatal ID, we demonstrated that ID downregulated hippocampal expression of the gene encoding JmjC-ARID domain-containing protein 1B (JARID1B), an iron-dependent histone H3K4 demethylase, associated with a higher histone deacetylase 1 (HDAC1) enrichment and a lower enrichment of acetylated histone H3K9 (H3K9ac) and phosphorylated cAMP response element-binding protein (pCREB). Likewise, ID reduced transcriptional capacity of the gene encoding brain-derived neurotrophic factor (BDNF), a target of JARID1B, associated with repressive histone modifications such as lower H3K9ac and pCREB enrichments at the Bdnf promoters in the adult rat hippocampus. Prenatal choline supplementation did not prevent the ID-induced chromatin modifications at these loci but induced long-lasting repressive chromatin modifications in the iron-sufficient adult rats. Collectively, these findings demonstrated that the iron-dependent epigenetic mechanism mediated by JARID1B accounted for long-term Bdnf dysregulation by early-life ID. Choline supplementation utilized a separate mechanism to rescue the effect of ID on neural gene regulation. The negative epigenetic effects of choline supplementation in the iron-sufficient rat hippocampus necessitate additional investigations prior to its use as an adjunctive therapeutic agent.

Keywords: Bdnf; Jarid1b; choline; epigenetics; gene expression; histone modification; iron deficiency.

Figure 1

Graphical diagram of experimental groups. Abbreviations: Gestational day (G), iron-deficient group (ID), iron-deficient with choline supplementation group (IDCh), iron-sufficient group (IS), iron-sufficient with choline supplementation group (ISCh), and postnatal day (P).

Figure 2

Iron deficiency alters expression and enzymatic activity of hippocampal JARID1 in the ID rats. (A–C) Hippocampal Jarid1b RNA expression in IS, ID, ISCh and IDCh groups at P0 (A), P15 (B) and P65 (C) quantified by Real-time quantitative PCR(RT-qPCR). (D) Hippocampal JARID1B protein expression in IS and ID groups at P15 and P65 measured with Western blot. Representative Western blot image of JARID1B at P15 is shown. (E) Hippocampal JARID1 activity in IS and ID groups at P0, P15 and P65 assessed with enzyme-linked immunosorbent assay. All data were normalized to the age-matched IS group. Tx: Treatment (Supplementation). Values are mean ± SEM; n = 5–6/group; * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Iron deficiency and choline supplementation alter rat hippocampal Jarid1b regulation. (A–C) HDAC1 enrichment at the Jarid1b promoter in the 4 groups at P0 (A), P15 (B), and P65 (C). (D–F) H3K9ac enrichment at the Jarid1b promoter in the 4 groups at P0 (D), P15 (E), and P65 (F). (G–I) Phosphorylated cAMP response element-binding protein (pCREB) enrichment at the Jarid1b promoter in the 4 groups at P0 (G), P15 (H), and P65 (I). All data were assessed with quantitative chromatin immunoprecipitation (qChIP) and normalized to the age-matched IS group. Tx: Treatment (Supplementation). Values are mean ± SEM, n = 5–6/group, * p < 0.05, ** p < 0.01.

Figure 4

Iron deficiency and choline supplementation alter hippocampal histone H3K9 methylation. (A–C) Expression levels of G9a in the 4 groups at P0 (A), P15 (B) and P65 (C). (D–F) Expression levels of Suv39h1 in the 4 groups at P0 (D), P15 (E) and P65 (F). (G–I) H3K9me3 enrichment at the Jarid1b promoter in the 4 groups at P0 (G), P15 (H) and P65 (I). All data were assessed with RT-qPCR or qChIP and normalized to the age-matched IS group. Tx: Treatment (Supplementation). Values are mean ± SEM; n = 5–6/group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 5

Iron deficiency and choline supplementation induce epigenetic changes at the hippocampal Bdnf4 promoter. (A,B) Enrichment levels of JARID1B, H3K4me3, H3K9me3 and upstream stimulatory factor 1(USF1) at the Bdnf4 promoter in P15 (A) and P65 (B) IS and ID groups. (C,D) H3K9ac enrichment at the Bdnf4 promoter in the 4 groups at P15 (C) and P65 (D). (E,F) pCREB enrichment at the Bdnf4 promoter in the 4 groups at P15 (E) and P65 (F). All data were assessed by qChIP and normalized to the age-matched IS group. Tx: Treatment (Supplementation). Values are mean ± SEM; n = 5–6/group; * p < 0.05, ** p < 0.01.

Figure 6

Iron deficiency and choline supplementation induce epigenetic changes at the hippocampal Bdnf6 promoter. (A,B) Enrichment levels of JARID1B, H3K4me3 and USF1 at the Bdnf6 promoter in P15 (A) and P65 (B) IS and ID groups. (C,D) HDAC1 enrichment at the Bdnf6 promoter in the 4 groups at P15 (C) and P65 (D). (E,F) H3K9ac enrichment at the Bdnf6 promoter in the 4 groups at P15 (E) and P65 (F). (G,H) H3K9me3 enrichment at the Bdnf6 promoter in the 4 groups at P15 (G) and P65 (H). (I,J) pCREB enrichment at the Bdnf6 promoter in the 4 groups at P15 (I) and P65 (J). All data were assessed by qChIP and normalized to the age-matched IS group. Tx: Treatment (Supplementation). Values are mean ± SEM; n = 5–6/group; * p < 0.05, ** p < 0.01, **** p < 0.0001.