Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases.


Introduction
Chromatin-based transcriptional repression is mediated by macromolecular complexes containing proteins involved in chromatin writing, reading, erasing and remodeling activities.The combinatorial assembly of subunits with transcription factors affects cell-specific gene expression in response to developmental, physiological or environmental stimuli.Chromatin-repressive complexes control key pathways during embryonic development and adult life; as a consequence, deregulation or abnormalities in their components can lead to a wide range of pathological processes [1,2].The importance of chromatin-modifiers in development, cell differentiation and disease is well illustrated for three complexes containing the histone deacetylases HDAC1 and HDAC2: NuRD [3,4], Sin3A [5] and CoREST [6,7] (For reviews, see [2,[8][9][10]).By a proteomic approach, we found that the Bromo-Adjacent-Homology domain-containing 1 (BAHD1) protein co-purifies with HDAC1/2, together with heterochromatin proteins HP1 and KAP1 (or TRIM28) in human embryonic kidney (HEK) 293 cells, suggesting that BAHD1 is a core component of a novel HDAC1/2-associated complex [11].BAHD1 also interacts with the Methyl-CpG-binding protein MBD1 and the H3K9 methyltransferases (KMT) SETDB1 [12] and SUV39H1 [13] and acts as a repressor, pointing to a role of BAHD1 in heterochromatin-mediated transcriptional repression [12].In agreement with this, overexpression of BAHD1 in human cells induces large-scale chromatin condensation [12] and changes in the DNA methylation landscape [14].
BAHD1-associated heterochromatic domains lack acetyl histone H4 and partially overlap with HP1α, a marker of constitutive heterochromatin, and/or with H3 trimethylated at lysine 27 (H3K27me3), a marker of facultative heterochromatin [12].Furthermore, when overexpressed in human female cells, BAHD1 is enriched at the inactive X chromosome (Xi), a paradigm of facultative heterochromatin [12].A study in mouse embryonic stem cells (mESCs) recently reported that BAHD1, HDAC1 and HDAC2 are pulled-down by CDYL, a transcriptional co-repressor that may play a role in the maintenance of the Xi [15].Taken together, these data suggest that BAHD1 is a component of HDAC1/2-associated complexes involved in a variety of epigenetic mechanisms.
A single gene encodes BAHD1 in vertebrates and no ortholog is found in invertebrates or plants, suggesting that BAHD1 has vertebrate-specific functions.However, the low expression of BAHD1 in cell lines has hampered its functional characterization and, so far, the BAHD1 regulatory gene network is poorly characterized.We identified the insulin-like growth factor II (IGF2) transcript and its antisense transcript (IGF2AS) as BAHD1 targets in HEK293 embryonic cells [12].We also demonstrated that infection by a bacterial pathogen triggers BAHD1-mediated repression of Interferon-Stimulated Genes (ISGs) in epithelial cells [11].However, apart from bacterial infection, signals that control the expression and/or activity of BAHD1 are unknown.
The aim of the present study was to determine the physiological functions of BAHD1.We show that disruption of the Bahd1 gene in the mouse leads to a placental growth defect associated with low birth weight and neonatal death, hypocholesterolemia and decreased body fat in surviving adults.Proteomic studies of BAHD1-associated proteins identify MIER proteins as novel BAHD1 partners.Our extensive characterization of the transcriptome strongly suggests that BAHD1-MIER complexes repress genes involved in the control of steroid/lipid metabolism both in mouse and human cells.

Bahd1-deficient mice display hyporcholesterolemia, hypoglycemia and a lower body weight
We searched for tissue-specific expression of the BAHD1 gene by a survey of referenced transcriptome datasets (S1 Table ) and found that BAHD1 mRNA levels are low and do not vary much between tissues, when compared to a set of housekeeping or tissue-specific genes.This observation is consistent with the lack of detection of the endogenous BAHD1 protein in mammalian cultured cells and suggests that BAHD1 basal levels are low.In order to identify functions of BAHD1 in biological processes, we studied the physiological consequences of BAHD1 inactivation by performing a large-scale phenotyping of Bahd1 haplo-deficient (Bahd1 +/-) mice [11].The analysis was carried out on 18 heterozygous (HET) and 16 wild type (WT) littermates (results are detailed in S1 Text and S2 Table ).Mice were first fed a standard chow diet (CD) for 14 weeks and then a high fat/high carbohydrate diet (HFHCD) for 16 weeks.HET Bahd1 +/- mice did not show any morphological, sensory or cardiac abnormality and no change in bone density, body weight and fat, when compared to WT littermates, and the blood chemistry and hematology parameters were within the normal range.However, some slight and gender-specific modifications of glucose or cholesterol parameters were observed in HET mutants.At the age of 10 weeks, HET male mice displayed a significant hypoglycemia when compared to WT mice (Fig 1A).After the switch to HFHCD, blood levels of glucose remained lower in 30-weekold HET than in WT male mice, although difference did not then reach statistical significance (S2A Table ).In contrast to males, glycemia was not affected in female HET mice whatever the diet (Fig 1A) but female HET fed with HFHCD displayed a slight decrease in blood levels of total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) compared to WT littermates (Fig 1B).However, values were statistically significant only for LDL concentrations (S2B Table ).
We next crossed Bahd1-heterozygous mice to evaluate potential metabolism defects in the Bahd1-null context.Most Bahd1 -/-(KO) pups died within the first days of life, indicating that the Bahd1-null mutation leads to perinatal death.Only 5 Bahd1 -/-out of 200 genotyped pups (2.5%) survived birth.At six weeks, these 5 survivors, all males, displayed reduced sizes and weights compared to control littermates (

BAHD1 is required for mouse placental and fetal growth
The high neonatal mortality rate of BAHD1-KO pups suggested that BAHD1 could have important functions during fetal life.In order to address this question, we examined embryos just before birth at the embryonic day 18.5 (E18.5).Bahd1 -/-fetuses were present at Mendelian frequency, had normal morphology, were alive and exhibited the breathing reflex.However, their weight was decreased by 30% (Fig 2A) when compared to WT or HET fetuses.In addition, Bahd1 -/-embryos exhibited a smaller placenta, with a reduction of 30% in the circumference and 55% of the area (Fig 2B ), when compared to placentas of WT or HET embryos.At an earlier stage (E16.5),Bahd1 -/-placentas were also smaller than Bahd1 +/+ ones (Fig 2B).Histology of placenta sections with hematoxylin and eosin (HE) staining showed that Bahd1 -/-placentas comprised the two fetal compartments (labyrinthine zone (Lz) and junctional zone (Jz)) and the maternal compartment (decidua basalis (Db)).However, the Lz surface area was significantly reduced and the Jz and Db were thinner in Bahd1 -/-placentas than in control littermates (Fig 2C).The junctional zone plays an important role in hormone synthesis, while the labyrinthine zone is critical for materno-foetal nutrient exchange [16,17].The Jz comprises fetal spongiotrophoblast cells and trophoblast glycogen cells (GCs).The exact origin and function of GCs is unknown, but they are believed to provide an important glucose supply for fetoplacental development.Evidence indicates that they differentiate early (E.6.5) in the ectoplacental cone at the origin of the Jz and migrate into the maternal decidua at about E12.5 [18,19].We used periodic acid-Shiff (PAS) staining to examine if GCs were altered by the Bahd1 null mutation.PAS staining confirmed the reduced thickness of Jz and Db in Bahd1 -/-relative to Bahd1 +/+ placentas at E16.5 and showed a severe reduction in the number of PAS-positive GCs both in the Jz and the Db (Fig 1D and S2A Fig).In contrast, histology of the fetal liver at E16.5 did not reveal any noticeable difference between Bahd1-KO and WT embryos (S2B Fig) .Together, these results indicate that BAHD1 is required for a normal placental development and intra-uterine fetal growth.BAHD1 mutants may die after birth from metabolic defects, or secondary to altered placental exchange prenatally, impairing the development of energy stores.
Expression of a set of steroid metabolism genes and imprinted genes is altered in Bahd1 -/-placentas To examine the consequence of BAHD1 deficiency on the placental transcriptome, we isolated RNA from Bahd1 -/-placentas at stages E16.5 (n = 6 per genotype) and at stage E18.5 (n = 3 per genotype) to perform a comparative microarray analysis using Affymetrix mouse arrays.The Bahd1-null mutation altered expression of 397 and 1396 genes (FDR-BH < 0.05) at E16.5 and at E18.5, respectively, with a much higher proportion of up-regulated genes (70-80%) than down-  ).We used the DAVID software to classify genes based on Gene Ontology (GO) [20] and highlight the most significant biological processes that could be altered by BAHD1 deficiency.At the E16.5 stage, the most significant gene cluster (P value < 5.10 −4 ) was a group of 16 genes involved in steroid metabolic processes (Fig 3B and S4A Table).Notably, 11 of these genes remained deregulated in Bahd1 -/- E18.5 placentas: 7 genes up-regulated (Apoc3, Atp8b1, Cyp11A1, Insig2, Osbpl5, Pbx1, VldlR) and 4 genes down-regulated (Fabp6, Hsd17b2, Hsd17b7, LepR) (S3 Table ).In addition, two steroid hormone receptor genes, the estrogen receptor alpha gene Esr1 and progesterone receptor gene Pgr, were up-regulated in Bahd1 -/-placentas, both in males and females embryos and at both gestational stages, as confirmed by Real time quantitative PCR (RT-qPCR) analysis (Fig 3C ).
Groups of genes involved in the response to oxidative stress, skeletal system development and blood vessel development were also over-represented in Bahd1-KO placentas (S4 Table ).Several of these genes are known to be involved in placental morphogenesis, such as Gja1/Cx43, Cxcl14, Mmp2, Mmp14, Runx1 (up-regulated) and Ada, Adm, Gcm1, Gjb5 (down-regulated).Altered expression of these genes in Bahd1-KO mice is consistent with an abnormal placentation.RT-qPCR analysis of a set of transcripts confirmed the transcriptome data (Fig 3C and 3D).
Several genes previously shown to be imprinted in the mouse during development or in the adult [21] were also up-regulated in Bahd1-KO placentas.Imprinted genes, expressed from only one of the parental alleles, play important functions during mammalian development, particularly in the placenta [22].The parental conflict hypothesis of imprinting proposes that maternally− or paternally−expressed imprinted genes restrict or increase the energy expenditure, respectively [23].We have previously shown that in human embryonic cells BAHD1 repressed IGF2 and IGF2AS [12], which are imprinted in many tissues.BAHD1 deficiency did not affect Igf2 expression in murine placenta, but, the paternally−expressed Igf2as and ten maternally −expressed putative imprinted (Ampd3, Ano1, Dcn, Gatm, Htra3, Osbpl5, Qpct, Tfpi2, Tnfrsf23 and Wt1) were up-regulated in Bahd1-deficient E16.5 placentas, while one, Gpr1, was down-regulated.RT-qPCR assays confirmed these results using several other imprinted genes as negative controls (Fig 3E).Most of these genes are imprinted only in the placenta.However, determining placental specific imprinting can be confounded by maternally derived placental tissue and there is controversy over the imprinted status of some of these genes [24].Strikingly, Htra3, Tfpi2, Ampd3, Gatm, Osbpl5, Qpct and Wnt1 were also consistently up-regulated in Bahd1-deficient E18.5 placentas (S3 Table ) and may be functionally implicated in the impaired placental growth.
Steroid metabolism genes are also altered in Bahd1 -/-mouse MEFs and BAHD1-overexpressing human cells Bahd1-KO fetuses exhibited a lower weight than Bahd1-WT.This phenotype could result from placental dysfunction but also from deregulation of BAHD1 target genes in intra-embryonic tissues.To address this point, we analyzed transcriptomes of mouse embryonic fibroblasts derived from Bahd1-WT and -KO embryos at stage E13.5 (n = 3 per genotype).The results of this analysis revealed 353 up-regulated and 139 down-regulated genes (FDR-BH < 0.05) in Bahd1-KO MEFs, when compared to Bahd1-WT MEFs (Fig 3A).We focused on up-regulated genes, as these likely contain those genes directly repressed by BAHD1.Strikingly, the most significant biological process associated with up-regulated genes in Bahd1-KO MEFs was sterol metabolism (Fig 3B).However, genes associated with this process were different to those affected in Bahd1-KO placentas (S4C Table ), suggesting that BAHD1-dependent gene networks are tissue-specific even though they may have a common function.
We next assessed whether BAHD1 has a similar role in repressing sterol metabolism genes in human cells, by comparing transcriptomes of HEK293 cells stably overexpressing BAHD1 (HEK-BAHD1) to that of parental isogenic cells (HEK-CT), in which the BAHD1 protein is undetectable [14].This analysis identified 1148 transcripts (FDR-BH < 0.05) that were down-regulated in cells constitutively expressing the BAHD1 repressor.Once again, GO term classification analysis of these transcripts highlighted sterol metabolism as the most significant biological process (Fig 3B and S4D Table).Genes involved in lipid and hexose metabolism also grouped as significant clusters.Although biological functions of BAHD1-associated chromatin repressive complexes are likely to be cell type-and species-specific, and a transcriptome will identify direct and indirect gene expression changes, BAHD1 bona fide target genes may be identified by comparing different biological systems (Fig 3F).Overlapping HEK-BAHD1 and Bahd1-KO E18.5 placenta transcriptomes identified 107 potential BAHD1 targets (S5A Table and Fig 3F), the most significant gene groups where those involved in the regulation of hormone levels (ALDH1A2, BACE2, CAMK2G, CRABP2, LY6E, SCARB1, SLC16A2) and lipid biosynthetic processes (ALDH1A2, CD81, EBP, ELOVL7, LASS4, LPL, LPCAT2, LTA4H, SCARB1)(S4E Table ).Overlapping HEK-BAHD1 and Bahd1-KO MEFs transcriptomes identified 44 potential BAHD1 target genes (S5B Table and Fig 3F), the most statistically significant group in term of biological processes including genes involved in sterol/steroid metabolism (DHCR24, HMGCS1, LDLR, NSDHL, SC4MOL, SREBF2; S4F Table ).Only six genes were altered consistently between all the three transcriptome datasets (S5C Table ).Using a tetracycline-inducible BAHD1 HEK293 line (HPT-BAHD1 cells, [11]), we confirmed that induction of BAHD1 expression (S3 Fig) is sufficient to repress five transcripts involved in lipid/steroid metabolism (HMGCS1, LDLR, NSDHL, CRABP2 and LASS4) (Fig 3G).Of note, no imprinted gene was found in this analysis, indicating that the effect of the Bahd1-null mutation on this gene category is specific to the placenta.Altogether, these findings show that changes in BAHD1 levels alter expression of gene networks controlling steroid/lipid metabolism and hormone signaling pathways, which are key players in the development of the placenta and in control of energy in the body.
We noticed that BAHD1 and MIER1 displayed conserved domains found in scaffolding proteins of other HDAC1/2 complexes: the metastasis-associated protein (MTA) subunits of NuRD [8,25] and RERE/atrophin-2, a transcriptional repressor belonging to a poorly characterized HDAC1/2 complex [26][27][28]  BAHD1 harbors a N-terminal proline-rich region with the highest density of prolines found between residues 239 to 361, a region termed the cPRR [31], and a C-terminal BAH domain that interacts with the N-terminal tail of histone H3 [12]   bound complex.This hypothesis is also supported by results from the Heard laboratory [15] showing that BAHD1, MIER1/2, HDAC1/2 and G9a co-immunoprecipitate with CDYL, a protein recruited to Xi in mouse ES cells and also found in our TAP assays (S6 Table ).

Relationship between BAHD1 and metabolism-associated transcription factors
We propose that BAHD1 and MIER are co-repressors that, like MTA subunits of NurD complexes [9], exert their function through association with sequence-specific DNA binding transcription factors (TFs).With the hypothesis that such TFs should regulate the same gene networks as BAHD1, we used the Ingenuity Pathway Analysis Upstream Regulator software to predict TFs responsible for differential gene expression in BAHD1-deficient or -overexpressing cells.This analysis identified seven TFs that were consistently found as upstream regulators of a set of BAH-D1-associated genes in all transcriptome datasets: ESR1, ESR2, EPAS1 (HIF2α), PPARG, FOS, TP53 and SP1 (S7 Table ), the latter being previously reported to associate with BAHD1 [12] and MIER1 [32].It is striking that these TFs have relevance both in placental development and regulation of genes involved in lipid/steroid metabolism ( [33][34][35][36] and other references in S1 Text).
Since ESR1 has been shown to bind MIER1 [37] and to cooperate with SP1 [38], we hypothesized that BAHD1-MIER could act as co-repressors for ESR1-mediated transcriptional regulation.Based on this assumption, BAHD1 should target the ESR1 gene itself, because ESR1 autoregulates its own expression [39].Accordingly, ESR1 was up-regulated in Bahd1-KO placentas (Fig 3C ), as well as in HEK293-FT cells depleted of BAHD1 with siRNA (Fig 6A).siRNA-mediated knockdown of individual MIER genes did not significantly change expression of ESR1 in HEK293-FT cells, but combined knockdown of MIER1 and MIER3 (MIER2 was undetectable) increased ESR1 expression (Fig 6A).We also found that expression of PGR (encoding the progesterone receptor), which has an ESR1 binding site in its proximal region [40] significantly increased in BAHD1-and MIER1/3-knockdown cells (Fig 6A ), whereas the androgen receptor transcript (AR) was unaffected.These results indicate that BAHD1 and MIER1/3 act as repressors for the steroid hormone receptor genes ESR1 and PGR.

BAHD1 knockdown or overexpression induce epigenetic changes at the ESR1 locus
Human ESR1 forms a large and complex genetic unit that spans approximately 300 kb of chromosome 6 (chr6), of which 140 kb containing 8 protein-coding exons [41].It is transcribed from at least seven promoters into multiple transcripts that vary in their 5'-UTRs and whose expression is tissue-specific.To find out whether the BAHD1-associated complexes targets ESR1, we analyzed the DNA captured with BAHD1 and its associated partners in the TAP assays by using real-time PCR with primers targeting the proximal region of ESR1 at, or in the vicinity of ESR1-binding sites identified by ChIP-seq (Fig 6B, [40], ENCODE ChIP-seq data).To determine whether any change in the chromatin structure occur as a consequence BAHD1 binding at these sites, we tested the effect of BAHD1 depletion on histone H3 modifications at lysine 9 (H3K9) by using a ChIP-qPCR assay.We observed a reproducible increase in histone H3K9 acetylation and decrease in H3K9 dimethylation (H3K9me2) and trimethylation (H3K9me3) in cells with siRNA−induced knockdown of BAHD1 expression, relative to cells treated with control siRNA (Fig 6D and S6 Fig).This effect occurred at all sites of the ESR1 proximal region surveyed by PCR, but not at the GAPDH control site.This result strongly suggests that a BAHD1-associated complex containing HDACs and KMTs contributes to the epigenetic silencing of ESR1.
H3K9 methylation is often linked to DNA methylation and several BAHD1-associated partners are known to interact with DNA methyltransferases [42][43][44][45].To test whether BAHD1 level also affects DNA methylation patterns at ESR1, we exploited the DNA methylome datasets of HEK-CT and HEK-BAHD1 cells that we recently obtained by whole-genome bisulfite sequencing (BS-seq) [14].In this study, we found that BAHD1 overexpression in HEK293 cells stimulates de novo DNA methylation in autosomes at ~80,000 regions that become hypermethylated when compared to control cells.These "BAHD1-DMRs" group into large (0.3-6.5 Mb) chromosomal domains, termed BAHD1-Associated Domains (BADs, [14]).We observed that one such BAD was present on chr6 in a large region containing the ESR1 locus (Fig 6E).The high density of BAHD1-DMRs located at the ESR1 proximal and gene body regions suggests that BAHD1 overexpression stimulates large-scale epigenetic changes.Taken together, these results provide evidence that BAHD1-associated complexes induce histone and DNA modifications that shape repressive chromatin structures.

Developmental and physiological roles of BAHD1
The functions of BAHD1-mediated chromatin reorganization in developmental and physiological processes were hitherto unknown.Deletion of Bahd1 in mice does not lead to embryonic lethality or anatomical malformations in fetuses.However, murine Bahd1-KO placentas are small and display histomorphological alterations, including reduced surface area of the labyrinthine zone and a thinner junctional zone and decidua.The lack of trophoblast glycogen cells is particularly striking, suggesting a failure in the differentiation program.The growth restriction of Bahd1-KO fetuses is likely to be secondary to defective placental nutrient exchange resulting from these placental abnormalities.In addition, as there is substantial deregulation of metabolic genes in Bahd1-KO MEFs, the perinatal death of Bahd1-KO pups likely results from metabolic defects in embryos.The rare mice surviving beyond birth are males with a lower weight than controls, associated with decreased fat mass and lower levels of circulating cholesterol, glucose and leptin.In light of our finding that the transcriptional corepressor MIER1 is a key partner of BAHD1, it is striking that Mier1 null mice also show decreased body weight and reduced levels of circulating glucose and cholesterol (data from The Mouse Phenotyping Consortium and The Wellcome Trust Sanger Institute, references in S1 Text).This phenocopy is a strong argument in favor of BAHD1 and MIER1 cooperating to control the expression of genes involved in energy metabolism in somatic tissues.

BAHD1 and MIER proteins define a novel chromatin-repressive complex
Separate studies have shown that MIER1 binds to HDAC1/2, G9a and CDYL [27,28,46], while BAHD1 interacts with proteins involved in heterochromatin formation (e.g.HP1, MBD1) and its overexpression is sufficient to compact chromatin [12].We now bring evidence that BAHD1 and MIER act in partnership within a novel histone deacetylase complex involved in gene silencing.First, BAHD1 co-purifies with MIER, HDAC1/2, G9a and CDYL in very different cellular models: human HEK293 cells, as shown here, and mouse ES cells [15].Second, as in other subunits of HDAC1/2-associated complexes, MTAs of NuRD [8][9][10]25](Fig 4D ) and RERE [26][27][28] (Fig 4C ), a BAH domain is present in BAHD1 and HDAC-interacting ELM2-SANT domains are present in MIER [27,46].We found that a truncation of the BAH domain in BAHD1 abrogates BAHD1 coimmunoprecipitation with MIER1 and HDAC2.From this, we propose that MTA, RERE and BAHD1-MIER define related macromolecular complexes of the "NuRD superfamily" and to name "BAHD1 complexes" those containing a BAHD1 subunit, as BAHD1 has no homolog or isoform.MIER1, MIER2 and MIER3 could be incorporated into distinct BAHD1 complexes leading to functional redundancy and/or context-dependent functions, as described for MTA1, MTA2 and MTA3.MIER1 is the most abundant partner of BAHD1 in HEK293 cells and its nuclear localization, including at the Xi, depends on BAHD1 expression levels.This translocation is likely to have functional consequences, as MIER1 nuclear/cytoplasmic distribution varies with cell type and stage of differentiation [47,48].Bahd1-and Mier1-KO adult mice share common phenotypes but a prenatal growth restriction is not reported for Mier1-KO; hence, the BAHD1-associated regulatory gene network in embryonic tissues likely involves other partners than MIER1.

Regulation of metabolism gene networks by BAHD1
Genes altered in Bahd1-KO placentas or embryonic fibroblasts are mainly up-regulated compared to wild type controls, consistent with BAHD1 acting as a repressor.The most significant biological process associated with these genes, and with those repressed in BAHD1-overexpressing human HEK293 cells, is steroid metabolism.This functional convergence is striking and consistent with the observed hypocholesterolemia and hypolipidaemia in Bahd1-KO mice.We observed that genes associated with this biological function in Bahd1-KO MEFs and HEK-BAHD1 cells (e.g.DHCR24, HMGCS1, LDLR, NSDHL, SREBF2, SC4MOL) mostly differ with those in Bahd1-KO murine placentas (e.g.Apoc3, Atp8b1, Cyp11A1, Insig2, Osbpl5, Pbx1, VldlR).Thus, like many other chromatin repressors [2], BAHD1 regulates distinct targets in different tissues and at different developmental stages.However, some of the BAHD1 bona fide target genes can be common to different biological systems, as exemplified by the steroid hormone receptor gene ESR1.BAHD1 binds the proximal region of human ESR1 and represses ESR1 both in human cells and murine placentas.In addition, changes in BAHD1 expression levels in HEK293 cells alter the patterns of H3K9 modifications and DNA methylation at the ESR1 locus, which is consistent with the interaction of BAHD1 with HDACs, KMTs, HP1 and MBD1 and the crosstalk between histone and DNA modifications [49].
It is worth mentioning that MTA1 [50] and SIN3a [39] have also been shown to represses ESR1 in other cell types, indicating that different HDAC complexes control the epigenetic silencing of ESR1.Recruitment of BAHD1 at specific sites of the genome might rely on the combinatorial assembly of BAHD1-MIER subunits with transcription factors.The product of ESR1 itself (ESR1/ERα) could be one such TF targeted by BAHD1 complexes because ESR1 autoregulates its own expression [39] and MIER1 has been shown to bind ESR1 [37].In line with this hypothesis, ESR1 is predicted to control several genes differentially expressed in BAHD1-KO tissues, such as PGR.In addition, ESR1 cooperates with SP1 [38], a TF known to bind BAHD1 and MIER1 [12,32], and which is also predicted to drive BAHD1-associated transcriptional changes.Our analysis also suggests that BAHD1 could act as a co-repressor with PPARγ and HIF2α, which like ESR1 are known to play roles in the regulation of energy metabolism and placental cell differentiation [33][34][35][36].This raises the plausible hypothesis that the BAHD1-associated chromatin complex could act as a transcriptional co-repressor in synergy with different TFs in the context of placental functions and lipid metabolism.

Role of BAHD1 in shaping the placental differentiation program
Placenta morphogenesis depends on the correct balance of cytotrophoblast proliferation and differentiation, into either syncytiotrophoblast involved in nutrient/gas exchange or invasive extravillous trophoblast involved in establishment of blood flow to the placenta.It is proposed that ESR1 controls the proliferation of estrogen-dependent cells, while ESR2 controls their maturation, hence trophoblast differentiation is associated with the transition from Esr1 to Esr2 expression [51,52].By maintaining unbalanced expression of Esr1 at an inappropriate time of the gestation, BAHD1 deficiency could disturb trophoblast differentiation.Pgr and Lepr genes are also deregulated in Bahd1-KO placentas; this should affect progesterone and leptin signaling, also important in placental development [53,54].Therefore, BAHD1 could play a role in trophoblast differentiation, in particular in the formation of glycogen-producing cells, by controlling hormone signaling in a timely manner, particularly in the junctional zone, an important endocrine region.Knockdown of Bahd1 in the mouse placenta results in up-regulation of several other genes that also have relevance to placental development.Among them, Htra3 and Tfpi2 are two confirmed imprinted genes expressed from the maternally inherited allele [24].Maternally expressed genes have been proposed to limit maternal resource provision; thus up-regulation of such genes is consistent with a placental growth restriction observed upon BAHD1 deficiency.In fact, HtrA3 and Tfpi2 are highly transcribed in the placenta and involved in the regulation of endothelial function, trophoblast migration and invasion [55][56][57][58].Further studies will be required to explore whether BAHD1 directly targets these genes and contributes to their imprinting.

Roles of BAHD1 in diseases
With the importance of the placenta in the feto-maternal exchange processes, as well of steroid signaling in the body, alterations in the amounts or activity of BAHD1 may lead to various pathological processes.We previously identified a role for BAHD1 in infection of epithelial cells with the bacterial pathogen Listeria monocytogenes [11,31].The fact that BAHD1 is involved in placental function now opens the possibility that BAHD1 contributes to the fetoplacental step of listeriosis, as L. monocytogenes has a tropism for the placenta.More generally, a connection between BAHD1 and placenta-associated pathologies, such as Intrauterine Growth Restriction and Pre-Eclampsia, should be carefully examined.BAHD1-mediated regulation of ESR1 is also enticing as ESR1 is a key regulator in a variety of biological processes and ESR1 deregulation has been implicated in several diseases, including breast cancer [59].A shift from nuclear to cytoplasmic localization of MIER1 during breast cancer progression has been observed, suggesting that nuclear MIER1 contributes to the repression of genes involved in invasive breast carcinoma [37].Similar to chromatin-repressive complexes Polycomb and NuRD [9,60], BAHD1 could be implicated in the regulation of transcriptional events involved in diverse oncogenic pathways.Of note, insertion events in BAHD1 and MIER1 genes were identified in a screen for genes that cooperate with oncogenic KRAS (G12D) to accelerate tumorigenesis and promote progression in a mouse model of pancreatic ductal preneoplasia [61].Our data also implicate BAHD1 in mammalian metabolic regulation and several BAHD1 candidate target genes have been associated with metabolic diseases in humans.For instance, genetic polymorphisms in CRABP2 are associated with changes in plasma cholesterol levels [62], SNPs in LPL and LASS4 are associated with dyslipidemia [63][64][65], changes in expression levels of HMGCS1, LDLR and SC4MOL correlated with obesity-related type 2 diabetes and cardiovascular diseases [66].Together, these results indicate that dysfunction of BAHD1 complexes could promote aberrant epigenetic phenomena at the origin of different disorders.Detailed understanding of how and where BAHD1 complexes establish repressive chromatin states could be instrumental for the development of new strategies for selective treatment of metabolic disorders in the future.in S1 Text.All the data are expressed as mean ± SE.Statistical analysis were performed using a one way ANOVA tests followed by a Fischer's PLSD test with significance set at P<0.05.Ã P<0.05, ÃÃ P<0.01, ÃÃÃ P<0.0001.

Microarray processing and analysis
Total RNAs were extracted from Bahd1 +/+ and Bahd1 -/-placentas at E16.5 (n = 6/genotype) or E18.5 (n = 3/genotype), as well as from Bahd1 +/+ and Bahd1 -/-MEFs (n = 3/genotype) and HEK-CT and HEK-BAHD1 cells (n = 3/cell line) using RNeasy Kit (Qiagen), treated TURBO DNA-freeTM kit (Ambion).RNA concentration and integrity were tested with RNA quality was monitored on Agilent RNA Pico LabChips (Agilent Technologies, Palo Alto, CA). 100 ng of RNA per sample were used as templates for the synthesis of hybridization probes for Affymetrix GeneChip Microarrays (Genechip HuGene 1.0 ST for HEK-CT and HEK-BAHD1 cells; Mouse gene 1.0 for E16.5 placentas; Mouse Exon 1.0 ST for E18.5 placentas and MEFs).Hybridization was carried out with biological replicates according to the expression analysis technical manual with wash and stain kit (Affymetrix).Gene-level expression values were derived from the CEL file probe-level hybridization intensities using the model-based Robust Multichip Average algorithm (RMA) [67].RMA performs normalization, background correction and data summarization.An analysis is performed using the LPE test [68] and a p-value threshold of p<0.05 is used as the criterion for expression.The estimated false discovery rate (FDR) of this analyze was calculated using the Benjamini Hochberg approach in order to correct for multiple comparisons.Results were annotated using information provided by Affymetrix.Full data sets were reduced by discarding genes with "EST" and "unknown" annotation labels.To generate functional clusters of genes, we used the DAVID program (http://david.abcc.ncifcrf.gov,2015 version; [20]) for selected gene sets according to gene ontology (GO) of biological process categories.To search for transcriptional regulators driving the differential gene expression changes we used the Ingenuity Pathway Analysis Upstream Regulator software [69].Datasets have been deposited in Gene Expression Omnibus (GEO) and are accessible through accession number, as follows: GEO series GSE51868 (transcriptomes of HEK-CT and HEK-BAHD1); GSE53443 and GSE53442 (transcriptome of Bahd1-WT and Bahd1-KO placentas at E16.5 and at E18.5) and GSE73816 (transcriptome of Bahd1-WT and Bahd1-KO MEFs).

RNA extraction and quantification of transcript levels
Total RNA from HEK293-CT and HEK-BAHD1 cells, HEK293-FT cells treated 72h with siR-NAs, or from mouse placentas or MEFs was extracted using the RNeasy Kit (Qiagen), from three to six biological replicates.Genomic DNA was removed by treatment with TURBO DNA-freeTM kit (Ambion).cDNAs were generated from 1 to 2 μg total RNA using the RT2-HT first strand kit (Qiagen/SABiosciences). Quantitative Real-Time PCR was performed on Biorad MyiQ device (Biorad), using SsoFast Evagreen supermix (Biorad), as specified by the supplier.Each reaction was performed in triplicate.Data were analyzed by the ΔΔCt method.Target gene expression data were normalized to the relative expression of human GAPDH or mouse Gapdh (and Hprt, for imprinted genes) and YWHAZ was used as a control gene.Statistical significance of the difference in mean expression of genes was evaluated using the Student t test; a P value <0.05 was considered significant.Primer sets are provided in S1 Text.

Tandem affinity purification of BAHD1-associated proteins and DNA, nuclear protein and chromatin immunoprecipitations
The TAP-TAG protocol to purify the partners of His 6 -Protein-C-tagged BAHD1 in HPT-BAHD1 cell is described in [11].Modifications to this protocol, Mass spectrometry analysis and precipitation of the associated DNA are detailed in S1 Text.Immunoprecipitation (IP) of nuclear proteins was performed as described in [70] with the following modifications.Nuclear soluble and insoluble fractions were sonicated, mixed and incubated overnight at 4°C with 1-3μg of the indicated antibodies and then with Dynabeads Protein G (#10004D, Novex) at 4°C during 2h30.IPs were washed 4 times with washing buffer (Tris 20mM pH = 7,65, NaCl 150mM, 0,05% IGEPAL, 2,5% Glycerol, 0,5mM EDTA, 0,6mM DTT).Samples were resuspended in 1X Laemmli and boiled at 95°C for 10 min.Proteins were separated on 8-10% SDS-PAGE gels, transfered to nitrocellulose membrane, probed with primary and secondary antibodies and detected by chemiluminescence (SuperSignal West Femto Substrate #34094, ThermoFisher Scientific).Blots were visualized on Films (Amersham) or using a ChemiDoc MP Imaging system (Bio-Rad).ChIP of modified H3K9 were performed in three independent biological replicates, using the ChIP-IT Express Enzymatic Kit (Active Motif) according to manufacturers instructions.Briefly, HEK293-FT cells were grown in T150 flasks for 72h with either siRNA against BAHD1 or non-targeting control siRNA (up to 70-90% confluency).One flask was kept for RNA extraction and quantification of BAHD1 knockdown by RT-qPCR.Cells from other flasks were harvested and cross-linked with a final concentration of 1% formaldehyde (Sigma-Aldrich) for 10 minutes.Fixation was stopped with 0.125 M glycine and cells were washed twice with ice-cold PBS.Collected cells were lysed and nuclei pellet were resuspended in shearing cocktail and incubated for digestion for 10 minutes at 37°C.Shearing efficiency was tested by agarose gel electrophoresis and DNA concentration was quantified with a Nanodrop 2000 (ThermoFisher Scientific) to normalize the quantity of chromatin per ChIP.For each ChIP, 3 μg of antibody were used.10 μL of chromatin was kept as input and processed as ChIP samples.After washing and reverse crosslinking of precipitated samples, DNA was purified by two extractions with equal volumes of phenol:chloroform:isoamylalcohol (25:24:1, pH = 8), assisted by phase lock heavy gel tubes (5Prime), followed by ethanol precipitation.Pellets were washed once in 75% ethanol, then resuspended in 50 μL DNAse-free water.H3K9ac, H3K9me2 and H3K9me3 enrichment levels were measured by qPCR with primers matching in the ESR1 locus and the non-target control GAPDH region (primer sets are provided in S1 Text).A standard curve was generated using 10%, 1%, 0.1% and 0.01% of input DNA.We first determined the fold enrichment of the ChIP sample relative to the IgG sample and then the effect of BAHD1 knockdown on H3K9 modifications was calculated as the ratio of enrichment in cells treated with BAHD1 siRNA to that in cells treated with control siRNA (presented in Results of the double-affinity purification of HPC-tagged BAHD1 were validated by western blot analysis of inputs and eluates from the 1 st and 2 nd affinity purifications (E1 and E2) from chromatin extracts of HPT-BAHD1 cells ("BAHD1") or HPT-control cells (Control).Membranes were often cut in two pieces to detect proteins of different molecular weight, enabling to check sample loading with a common antibody (α-) (e.g.HDAC1, HDAC2, histones H3 or H4).However, this was not always feasible due to the similar molecular weight of a number of proteins of interest, resulting in overlapping hybridization signals in the same region of membranes.We thus had to perform several loadings of the same samples on distinct gels.A. Raw images of blots detecting HPC-tagged BAHD1 with Protein C antibodies.H4 was used as a loading control for inputs (same membrane, cut horizontally and probed with H4 antibodies).B. Raw images of blots detecting G9a with G9a antibodies.In Fig 4B, E1 and E2 lanes were cut to follow the order of samples used when probing with the other antibodies.FT indicates the column flow through.HDAC2 was used as a loading control (same membrane, cut horizontally).C. Raw images of blots detecting KAP1 with KAP1 antibodies.HDAC1 was used as a loading control (same membrane, cut horizontally).D. Raw images of blots detecting MIER1 with MIER1 antibodies.HDAC2 was used as a loading control (same membrane, cut horizontally).E. Raw images of blots detecting MBD1 with MBD1 antibodies.F. Raw images of blots for HDAC1 and HDAC2 using HDAC1 and HDAC2 antibodies, respectively.HDAC1 and HDAC2 having the same size, two different blots were performed.After IP of BAHD1-V5 (using BAHD1 antibodies) or MIER1 (using MIER1 antibodies), the membrane was first hybridized with V5 antibodies for detection of BAHD1-V5 (upper panel), then stripped and re-blotted with MIER1 antibodies (lower panel).The intense bands at the bottom of the lower panel are heavy chains of rabbit IgG used for the IP, and detected with rabbit secondary antibodies.Note that the BAHD1 antibody is poorly efficient in immunoprecipitating BAHD1-V5 (top panel, 3 rd lane), which can explain why MIER1 is not detected in the BAHD1 IP (bottom panel, 3 rd lane).B. Raw images of western blots used in Fig 4E (bottom panel) before cropping.After IP of BAHD1-V5 using V5 antibodies, the membranes were probed with V5-HRP (top) or MIER1 (bottom) antibodies.The background signal, which is visible in the IP control, is due to cross-reactivity with IgG and protein G affinity matrix used for the IP. C. IPs with IgG Mouse (IP M Ctrl), V5, HP1γ, IgG Rabbit (IP R Crtl) or HDAC2 antibodies were loaded on a membrane that was cut and probed with V5-HRP antibodies (top) or HP1γ antibodies.Note that the V5 immunoblot is the same as in B, before cropping and with a longer exposure time.D. After IP with HDAC2 antibodies, at the ICS.We are grateful to Alexis Gautreau for help in the TAP assays and to the Taplin Biological Mass Spectrometry facility for MS assays.
Fig 1C).After several months, they reached the length of Bahd1 +/+ littermates (Fig 1C and 1D), but kept a significant lower body weight (Fig 1E), characterized by reduced fat and lean mass (Fig 1F).Quantification of blood parameters highlighted lower levels of total cholesterol, HDL and LDL in plasma of Bahd1 -/-mice than in Bahd1 +/+ littermates at 3-5 months (S1A Fig), 7 and 18 months of age (Fig 1G and 1H).Glucose and leptin levels were also significantly lower in 18 month-old Bahd1 -/-mice than in Bahd1 +/+ littermates (Fig 1H).The other measured blood parameters were not significantly

Fig 3 .
Fig 3. Loss or overexpression of BAHD1 alters expression of genes involved in steroid metabolism. A. Number of genes up-and down-regulated in Bahd1-KO placentas or MEFs relative to WT counterparts.B. Gene ontology enrichment analysis of transcripts differentially expressed in Bahd1-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 p-value (Fig 4C).BAHD1, MTA and RERE share a Bromo-Adjacent Homology (BAH) domain, which is known to promote protein-protein interactions and binding to nucleosomes[25,29,30], while MIER1, MTA and RERE proteins contain juxtaposed ELM2 and SANT domains that recruit HDACs[25].Thus, BAHD1 and MIER1 could cooperate to fulfill a function similar to that of MTA and RERE in other HDAC1/2 complexes (Fig 4D).To investigate this hypothesis, we performed a series of co-immunoprecipitation experiments on HEK293-FT cells transiently expressing tagged-versions of BAHD1 from plasmid vectors, since endogenous BAHD1 is undetectable in HEK293 cells.Reciprocal co-IP assays with nuclear extracts confirmed association of endogenous MIER1 with V5-tagged BAHD1 in HEK293-FT cells and their co-immunoprecipitation with HDAC2 and HP1γ (Fig 4E and S5 Fig).Likewise, BAHD1 tagged with the fluorescent protein citrine (YFPc-BAHD1) pulled-down HDAC2, MIER1 and HP1γ (Fig 4F, lane 1).
(Fig 4C).To assess the requirement of specific protein domains in BAHD1 for productive interaction with MIER and HDACs, we expressed truncated forms of YFPc-BAHD1 in HEK293-FT cells.As shown in Fig 4Fand S5EFig, the deletion mutant YFPc-BAHD1-ΔcPRR lacking residues 239 to 361 co-immunoprecipitated with HDAC2, MIER1 and HP1γ, as the full-length YFPc-BAHD1.In contrast, the deletion mutant YFPc-BAHD1-ΔBAH lacking residues 592 to 780 encompassing the BAH 624-780 domain failed to pull-down HDAC2 and MIER1, while retaining the ability to bind HP1γ.We conclude that BAHD1 interaction with MIER1 and HDAC2 requires the integrity of the BAH domain of BAHD1.We also found that varying BAHD1 amounts has an impact on MIER1 subcellular localization.First, MIER1 shifted from cytosolic to chromatin-bound fraction in response to induction of BAHD1 expression in HPT-BAHD1 cells (Fig5A).Second, microscopy experiments showed that BAHD1 overexpression increased MIER1 nuclear staining and induced enrichment of MIER1 at the heterochromatic inactive X chromosome (Xi) (Fig 5Band 5C).This effect was not due to changes in MIER1 transcript levels (S3 Fig).The fact that BAHD1 levels dictate the localization of MIER1 strongly suggests that BAHD1 and MIER1 form a stable chromatin-

Fig 5 .
Fig 5. BAHD1 increases MIER1 nuclear translocation and recruitment to Xi. A. Cytoplasmic, 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. 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.C. 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.doi:10.1371/journal.pgen.1005898.g005

Fig 6 .
Fig 6.Depletion or overexpression of BAHD1 in HEK293 cells induce epigenetic changes at ESR1. A. BAHD1 and MIER1/3 repress ESR1 and PGR.HEK293-FT cells were transfected for 72 h with control or BAHD1, MIER1 or MIER3 siRNA.The levels of BAHD1, MIER1, MIER3, ESR1, PGR and AR transcripts were quantified by RT-qPCR.Data are expressed as mean ± SD (* P<0.05; ** P<0.005).B. Schematic representation of the proximal region of ESR1 in chr6 of the human genome.C. BAHD1 binds the proximal region of ESR1.Amounts of DNA precipitated with BAHD1-TAP E1 eluates (see Fig 4) 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 GAPDH locus.Data are averages ± SD of qPCR triplicates, and representative of 2 independent TAP experiments.D. BAHD1 depletion alters the patterns of H3K9 acetylation and methylation at the ESR1 locus.HEK293-FT cells were transfected with control or BAHD1 siRNA and enrichment of H3K9ac, H3K9me2 and H3K9me3 relative to IgG control at ESR1 and GAPDH 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 S6 Fig).E. Overexpression of BAHD1 induces widespread DNA methylation at the ESR1 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) [14].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 ESR1 indicated by an arrow.A 1 Mb region encompassing the Esr1 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.doi:10.1371/journal.pgen.1005898.g006 Fig 6 and S6 Fig as a Log2 ratio).delineate decidua basalis (Db) and junctional zone (Jz).B. Histological analysis and Bahd1 +/+ and Bahd1 −/− E16.5 livers with hematoxylin and eosin (HE) staining.Magnification 20-fold.Squared regions highlight hepatic cells.(TIF) S3 Fig. Expression of BAHD1, IGF2 and MIER1 transcripts in response to induction of BAHD1 in HPT-BAHD1 cells.RT-qPCR quantification of transcript levels in HPT-BAHD1 cells relative to HPT-Control cells, grown 72h with doxycycline (n = 3 for each condition).Values are normalized by GAPDH.Data are expressed as mean ± SD (ns, non-significant; ÃÃÃ P<0.001).(TIF) S4 Fig. Raw images for the western blots used in Fig 4B.
G. Raw images of the blots detecting HP1γ with HP1γ antibodies.(TIF) S5 Fig. Co-immunoprecipitation (IP) assays of BAHD1 with several partners.A. Raw images of western blots used in Fig 4E (top panel) before cropping.IP Ctrl corresponds to the use of control IgG in IP assays.