BAHD1 increases MIER1 nuclear translocation and recruitment to Xi.

<p><b>A.</b> Cytoplasmic, nuclear soluble or chromatin extracts from HPT-control or HPT-BAHD1 cells induced 30 h with doxycyline were analyzed by immunoblotting with BAHD1, MIER1, histone H3 or Tubulin-α antibodies. H3 and Tubulin were used as loading controls. <b>B.</b> Colocalization of Protein-C tagged-BAHD1 and MIER1 on heterochromatic Xi (pointed with arrows on the merged image) was examined by IF with Protein-C and MIER1 antibodies. Bar, 5 μm. <b>C.</b> Localization of BAHD1 and MIER1 in HPT-BAHD1 cells induced or not for 30 h with doxycycline. Enrichment on Xi was determined by IF with BAHD1 (top panels) or MIER1 (bottom panels) antibodies combined with Xist RNA FISH (red). DNA was stained with DAPI (blue in the merged image). Bars: 10 μm.</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

Loss or overexpression of BAHD1 alters expression of genes involved in steroid metabolism.

<p><b>A.</b> Number of genes up- and down-regulated in <i>Bahd1</i>-KO placentas or MEFs relative to WT counterparts. <b>B.</b> Gene ontology enrichment analysis of transcripts differentially expressed in <i>Bahd1</i>-KO relative to WT placentas and MEFs, and in human HEK-BAHD1 cells relative to HEK-CT cells. The enrichment score represents the negative logarithm of the <i>p</i>-value evaluating the significance of gene ontology terms for differentially expressed RNAs. The top 10 annotation clusters are listed as derived from the DAVID bioinformatics tool. <b>C.</b> Transcripts levels in <i>Bahd1</i>^−/− placentas relative to <i>Bahd1</i>^+/+ littermates at E16.5 (n = 6, with 3 males and 3 females for each genotype) or E18.5 (n = 3 for each genotype) were quantified by RT-qPCR. Values are normalized by <i>Gapdh</i>. <i>Ywhaz</i> is used as negative control. The differential expression in <i>Bahd1</i>^−/− placentas is shown (relative to that in <i>Bahd1</i>^+/+ = 1). <b>D.</b> Relative transcripts levels for several genes in <i>Bahd1</i>^−/− relative to <i>Bahd1</i>^+/+ placentas at E18.5 (n = 3 for each genotype). <b>E.</b> Relative transcripts levels for imprinted genes in <i>Bahd1</i>^−/− relative to <i>Bahd1</i>^+/+ placentas at E16.5 (n = 4 for each genotype). Values are normalized to <i>Hprt</i>, <i>ActB and Tuba1a</i>. <b>F</b>. Venn diagram depicting shared genes that are up-regulated in <i>Bahd1</i>-KO E18.5 placentas and MEFs and down-regulated in human HEK-BAHD1 cells. <b>G</b>. Relative transcripts levels for lipid metabolism genes in HPT-BAHD1 cells relative to control HPT-CT. <i>BAHD1</i> expression was induced with tetracycline for 30h. Data are expressed as mean ± SD (ns, non-significant; * <i>P</i> < 0.05; ** <i>P</i> < 0.005; *** <i>P</i> < 0.001).</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

Depletion or overexpression of BAHD1 in HEK293 cells induce epigenetic changes at <i>ESR1</i>.

<p><b>A. BAHD1 and MIER1/3 repress <i>ESR1</i> and <i>PGR</i></b>. HEK293-FT cells were transfected for 72 h with control or BAHD1, MIER1 or MIER3 siRNA. The levels of <i>BAHD1</i>, <i>MIER1</i>, <i>MIER3</i>, <i>ESR1</i>, <i>PGR</i> and <i>AR</i> transcripts were quantified by RT-qPCR. Data are expressed as mean ± SD (* <i>P</i><0.05; ** <i>P</i><0.005). <b>B.</b> Schematic representation of the proximal region of <i>ESR1</i> in chr6 of the human genome. <b>C.</b> BAHD1 binds the proximal region of <i>ESR1</i>. Amounts of DNA precipitated with BAHD1-TAP E1 eluates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.g004" target="_blank">Fig 4</a>) or with control TAP or inputs were quantified using qPCR with the primer sets indicated in B. The amount of DNA purified with BAHD1 was normalized to the amount precipitated in the control TAP, and to the <i>GAPDH</i> locus. Data are averages ± SD of qPCR triplicates, and representative of 2 independent TAP experiments. <b>D.</b> BAHD1 depletion alters the patterns of H3K9 acetylation and methylation at the <i>ESR1</i> locus. HEK293-FT cells were transfected with control or BAHD1 siRNA and enrichment of H3K9ac, H3K9me2 and H3K9me3 relative to IgG control at <i>ESR1</i> and <i>GAPDH</i> regions were estimated by ChIP-qPCR in BAHD1-depleted and control cells. The y-axis shows the relative fold change of ChIP enrichment in cells with BAHD1 siRNA over cells with control siRNA in Log2 ratios. Data are averages ± SD of two ChIP per antibody and representative of three biological replicates (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.s007" target="_blank">S6 Fig</a>). <b>E.</b> Overexpression of BAHD1 induces widespread DNA methylation at the <i>ESR1</i> locus. Bisulfite-modified genomic DNA of control HEK-CT cells and isogenic HEK-BAHD1 cells overexpressing BAHD1 were sequenced and analyzed for their DNA methylation status, as described in Libertini et al. (2015) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005898#pgen.1005898.ref014" target="_blank">14</a>]. 300bp-regions with reproducible gain of methylation in HEK-BAHD1 compared to HEK-CT DNA in two BS-seq replicates (i.e hypermethylated BAHD1-DMRs) were binned into 0.5Mb windows highlighting clusters of hyper-DMRs (shown as red bars). Contiguous clusters define BAHD1-associated domains (“Hyper-BADs”, shown as black boxes). BAHD1-DMRs are represented by black vertical lines in the track “hyper-DMRs”. Three hypermethylated BADs of chr6 are shown on the top, with the position of <i>ESR1</i> indicated by an arrow. A 1 Mb region encompassing the <i>Esr1</i> locus is magnified below, showing the high density of hypermethylated BAHD1-DMRs on the whole locus. CpG islands are indicated in green. The position of transcripts is shown below.</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