Expression of candidate imprinted genes at E16.5 in the F2 generation and assessment of F1 germline imprints.

<p>(A) Schematic of the F2 generation, expression was assessed at E16.5. (B) There are no significant differences in the F2 hepatic expression of imprinted genes at E16.5. Error bars denote SEM. (C) At E16.5 CU individuals demonstrate a significant increased in placental expression of <i>Igf2P0</i> (One-way ANOVA, Bonferroni's multiple comparison post-test. P<0.05), while UU placentas significant up-regulate <i>Snrpn</i> (One-way ANOVA, Bonferroni's multiple comparison post-test. P<0.01). Per condition n≥24, 6 litters. Error bars denote SEM. (D, E) Methylation was assessed by pyrosequencing at the paternally methylated <i>H19</i> (A) and <i>Dlk1/Dio3</i> (B) germline ICRs. Sperm from both control and <i>in utero</i> undernourished males showed the expected hypermethylation in comparison to somatic tissues (liver). Controls: n = 12, 5 litters. Undernourished n = 11, 4 litters. Error bars denote SEM. (F, G) Pyrosequencing assessment of methylation at the maternally methylated <i>Peg3</i> (C) and <i>Snrpn</i> (D) germline DMRs shows that these regions are unmethylated in the sperm of both control and <i>in utero</i> undernourished males. Controls: n = 12, 5 litters Undernourished n = 11, 4 litters. Error bars denote SEM.</p

Characterisation of the E16.5 hepatic and placental transcriptome response to undernourishment.

<p>(A) Schematic of the experimental design: F1 generation: On pregnancy day 12.5, dams were randomly assigned to either control or undernutrition groups and food intake of undernutrition mothers was restricted to 50% that of controls. After delivery litter size was equalized to eight pups and dams received 9F chow <i>ad libitum</i>. Pups nursed freely and were weaned at 3 weeks onto 9F chow <i>ad libitum</i>. F2 generation: control and undernourished females from the F1 generation were mated at age 2 months with nonsibling control or undernourished males. After confirmation of pregnancy, females were caged individually and fed <i>ad libitum</i> throughout pregnancy to produce the four experimental F2 generation groups: CC – both parents are controls. CU – control dam, <i>in utero</i> undernourished sire; UC - <i>in utero</i> undernourished dam, control sire; UU - <i>in utero</i> undernourished dam, <i>in utero</i> undernourished sire. (B, C) Gene ontology (GO) analysis of the F1 undernourished hepatic transcriptome at E16.5: Functional enrichment analysis using (B) DAVID <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002605#pgen.1002605-Dennis1" target="_blank">[27]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002605#pgen.1002605-Huangda1" target="_blank">[28]</a> and (C) Ingenuity Pathway Analysis functional analysis tools. Among genes upregulated in the liver, both tools identify significant enrichment (after Benjamini-Hochberg correction for multiple testing) of gene groups associated with metabolism, particularly of lipids. Among genes downregulated in the liver, both tools identify significant enrichment of categories related to cell-cycle and the control of proliferation. (D) Transcriptome analysis of E16.5 undernourished versus control liver. Distribution of the ranked difference in gene expression according to FDR q-value. Lists of housekeeping genes and adult hepatic fasting-response genes curated from the literature were used as negative and positive controls respectively and their rankings shown. Imprinted genes most closely resemble randomly selected genes. (E) Transcriptome analysis of E16.5 undernourished versus control placenta; distribution of genes according to FDR q-value. The placental response to maternal undernourishment is undetermined, and is different to that of the liver, as fasting response genes are largely unperturbed. Therefore a network of genes involved in RNA post-transcriptional modification, identified as enriched in undernourished placenta by IPA analysis; was used as a positive control. Imprinted genes most closely resemble randomly selected genes.</p

<i>Bahd1</i>-knockout mice display decreased weight and fat mass and lower cholesterol, glucose and leptin levels.

<p><b>A-B.</b> Plasma levels of glucose (A) or of total cholesterol, HDL and LDL (B) in <i>Bahd1</i> wild-type (WT) and -heterozygous (HET) male (top) or female (bottom) mice, 10-week-old fed a chow diet (CD) or 30-week-old fed 14 weeks CD followed by 16 weeks HFHC (n = 8-10/group. <b>C.</b> Representative macroscopic images of <i>Bahd1</i>-WT and -knockout (KO) mice at 6 weeks and 9 months. <b>D.</b> Body length at 9 months. <b>E</b>. Body weight curve of <i>Bahd1-</i>WT and KO mice between the ages of 7 to 17 months (n = 5/group). <b>F.</b> Quantification of body fat mass and lean by QNMR analysis on 15 month-old animals (n = 4/group). <b>G.</b> Plasma levels of glucose, total cholesterol, HDL, insulin, leptin and adiponectin in 16-hours fasted 7-month-old WT and KO mice fed a normal diet (n = 4/group). <b>H.</b> Plasma levels of the same parameters in the same animals one year later (18 month-old). All data are expressed as the mean ± SE (* <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i>< 0.005).</p

BAHD1 binds to MIER and HDACs.

<p><b>A-B.</b> TAP-MS purification of the His₆-Protein-C-BAHD1-associated complex. Solubilized chromatin extracts (“Inputs”) from HPT-CT cells (“Control”) or HPT-BAHD1 cells (“BAHD1”) were processed on anti–protein C affinity matrix (E1) and nickel-Sepharose (E2). Eluted fractions were separated by SDS-PAGE and analyzed by Mass Spectrometry. <b>A</b>. Silver staining of E2. <b>B</b>. Immunoblots of inputs E1 and E2, using antibodies against the specified proteins (for G9a, also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.s005" target="_blank">S4 Fig</a>). <b>C.</b> Schematic representation of MTA1, RERE, MIER1 and BAHD1. ELM2, SANT, BAH domains, cPRR region and amino-acid numbers are indicated. <b>D.</b> Schematic diagrams of NurD and BAHD1 co-repressor complexes. Different protein paralogs can be part of distinct complexes. <b>E-F.</b> Nuclear extracts from HEK293-FT cells expressing BAHD1-V5 or YFPc-BAHD1, YFPc-BAHD1- ΔcPRR, YFPc-BAHD1- ΔBAH were used in immunoprecipitations (IP) assays with MIER1 or V5 antibodies or IgG control (<b>E</b>) or YFP antibodies. Vertical lines indicate cropping sites in original blots (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.s006" target="_blank">S5 Fig</a>). (<b>F</b>). Eluted fractions were separated by SDS-PAGE and analyzed by immunoblot with the indicated antibodies (α-). In (F), the MIER1 blot was stripped and reprobed with YFP antibodies (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.s006" target="_blank">S5 Fig</a> for a replicate experiment).</p

BAHD1 plays a role in fetal and placental growth.

<p><b>A.</b> Representative images of <i>Bahd1</i>^+/+ and <i>Bahd1</i>^−/− fetuses at E18.5 and quantification of fetus weight (on the right, * <i>P</i> < 0.05). <b>B.</b> Representative images of <i>Bahd1</i>^−/− and <i>Bahd1</i>^+/+ placentas at E18.5 and E16.5. Scale bar, 200 mm. Quantification of areas and circumferences of E18.5 placentas are shown on the right (<i>Bahd1</i>^+/+, n = 4; <i>Bahd1</i>^+/-, n = 11; <i>Bahd1</i>^-/-; n = 7). Data are expressed as mean ± SD (*<i>P</i> < 0.05). <b>C.</b> Histological analysis of <i>Bahd1</i>^−/− and <i>Bahd1</i>^+/+ placentas at E18.5 and E16.5. Placentas were collected, fixed and subjected to hematoxylin and eosin (HE) staining. A representative image is shown. Scale bar: 1 mm. <b>D.</b> Periodic acid-Shiff (PAS) staining of the same E16.5 placentas as in C, at three different scales. Scale bar, 1 mm (top), 250μm (bottom), 60 μm (squared region). High magnification of vacuolated glycogen cells GCs (arrowheads) in the junctional zone is shown in squared regions. Ms, mesometrial triangle; Db: decidua basalis; Jz junctional zone; Lz, labyrinthine zone.</p