Supporting Information

Chakraborty et al. 10.1073/pnas.1612626113 SI Methods Cells were exposed to ambient or low oxygen tensions (0.5%) Animals and Tissue Collection. Holtzman Sprague–Dawley rats and 5% CO2 at 37 °C using a NAPCO Series 8000WJ incubator were acquired from Envigo. Animals were housed in an envi- (Thermo Scientific) for 24 h and then harvested for cell and ronmentally controlled facility with lights on from 0600 to 2000 molecular analyses. Pre-equilibration of culture medium to the hours and allowed free access to food and water. Virgin female low oxygen tension was not performed before the initiation of rats 8–10 wk of age were cohabited with adult males (>3moof the experiments. age) of the same strain. Mating was assessed by inspection of vaginal lavages. The presence of sperm in the vaginal lavage was DNA Microarray. Affymetrix 230 2.0 DNA microarray chips considered gd 0.5. Rat embryos were collected by flushing uteri (Affymetrix) were probed with cDNAs generated from rat TS with M2 medium (Millipore) at gd 4.5. cells grown under oxygen replete conditions versus low (0.5%) Pregnant rats were placed in a hypoxic [10.5% (vol/vol) oxygen] oxygen conditions or alternatively from dissected gd 13.5 metrial gas-regulated chamber (BioSpherix, Lacona, NY) from gd 6.5– gland tissue harvested from pregnant rats exposed to normoxic 13.5. Pregnant rats exposed to ambient conditions (normoxia) versus hypoxic [10.5% (vol/vol) oxygen] conditions between gd 6.5 served as controls. Rat placental tissues were collected on gd and 13.5. Each treatment group was repeated in triplicate. RNA 13.5. Placentation site dissections were performed as previously samples were hybridized to the Affymetrix 230 2.0 DNA micro- described (57). Tissues for histological analysis were frozen in array chip using the GeneChip Hybridization Oven 640 (Affy- dry-ice cooled heptane and stored at −80 °C. Tissue samples for metrix). Washing and staining of hybridized chips were conducted protein or RNA extraction were frozen in liquid nitrogen and using the GeneChip Fluidics Station 450 (Affymetrix). Chips were stored at −80 °C until processed. The University of Kansas scanned using the Affymetrix GeneChip Scanner 3000 (Affyme- Medical Center (KUMC) Animal Care and Use Committee trix) with autoloader by the KUMC Biotechnology Support Fa- approved protocols for the care and use of animals. cility. Hybridization signals were normalized with internal controls Paraffin-embedded human placental tissue and tissue micro- using the Mas5 algorithm in Expression Console (Affymetrix) and arrays containing placental tissue samples from preterm, term, fold change computed. Significant differences were determined by preeclampsia, and IUGR pregnancies were obtained from the paired two-tailed Student t tests. Microarray data were processed Research Centre for Women’s and Children’s Health Biobank for functional analysis using Ingenuity Pathway Analysis. Probe (Mount Sinai Hospital, Toronto, CA). Clinical details of the sets included in the analysis were restricted to those changing at tissue microarray are provided in Dataset S1. Tissue collections least 1.5-fold between group comparisons with signal strengths of were performed with consent and were approved by the Uni- ≥500 for the maximal value. versity of Toronto and the KUMC human research ethics review committees. RT-PCR. Total RNA was extracted from cells and tissues using TRIzol reagent (Invitrogen). cDNAs were synthesized from total Rat TS and Human Trophoblast Cell Cultures. Blastocyst-derived rat RNA (1 μg) for each sample using SuperScript 2 reverse tran- TS cells (32), mouse TS cells (58), Rcho-1 TS cells (59), and scriptase (Invitrogen), diluted five times with water, and sub- human trophoblast cells were used to evaluate the effects of low jected to qRT-PCR to estimate mRNA levels. Primers were oxygen on trophoblast cell behavior. Rat TS cells were cultured in designed using Beacon primer designer (Applied Biosystems). basal culture medium [RPMI 1640 (Cellgro), 20% (vol/vol) FBS Conventional RT-PCR and qRT-PCR primer sequences are (Sigma Aldrich), 100 μM 2-mercaptoethanol (Sigma-Aldrich), presented in Table S2. Conventional RT-PCR was performed for 1 mM sodium pyruvate (Cellgro), 50 μM penicillin, and 50 U/mL 30–35 cycles (94°C for 30 s; 55–60 °C for 30 s; 72 °C for 30 s). streptomycin (Cellgro)] supplemented with 70% rat embryonic Real-time PCR amplification of cDNAs for qRT-PCR was car- fibroblast conditioned medium, FGF4 (25 ng/mL; Sigma- ried out in a reaction mixture (20 μL) containing SYBR GREEN Aldrich), and heparin (1 μg/mL; Sigma-Aldrich). New rat TS cells PCR Master Mix (Applied Biosystems,) and primers (250 nM were established from WT and Mmp12-null blastocysts, char- each). Amplification and fluorescence detection were carried out acterized, and maintained based on procedures previously de- using the ABI 7500 real-time PCR system (Applied Biosystems). scribed (32). Sex chromosome composition of rat TS cells was Cycling conditions included an initial hold step (95 °C for 10 min) performed on genomic DNA using PCR for Kdm5c (X chromo- and 40 cycles of a two-step PCR (92 °C for 15 s and then 60 °C some) and Kdm5d (Y chromosome) genes (60). Primers can be for 1 min), followed by a dissociation step (95 °C for 15 s, 60 °C found in Table S2. Mouse TS cells were cultured under the same for 15 s, and then 95 °C for 15 s). The comparative cycle conditions with the substitution embryonic fibroblast conditioned threshold method was used for relative quantification of the medium from mouse instead of rat. Rcho-1 TS cells were main- amount of mRNA for each sample normalized to 18S RNA, and tained in basal culture medium without conditioned medium, 18S RNA was stable among the conditions and tissues tested. FGF4, and heparin. Human trophoblast cell lines (BeWo and JEG3) were obtained Immunohistochemistry. Immunocytochemical analyses were per- from the American Type Culture Collection and cultured in formed on 10-μm frozen tissue sections using indirect immuno- DMEM culture medium supplemented with 10% (vol/vol) FBS, fluorescence detection using goat anti-mouse IgG tagged with 50 μM penicillin, and 50 U/mL streptomycin (Cellgro). Primary Alexa 488 (Invitrogen; cat. no. A11029; 1:1,000 dilution), goat cultures of human trophoblast (20–22 wk of gestation) were anti-mouse IgG tagged with Alexa 568 (Invitrogen; cat. no. obtained from ScienCell and cultured according to the vendor’s A11031; 1:400 dilution), or goat anti-rabbit IgG tagged with recommendations. Term trophoblast cells were processed from cyanine 3 (Cy3; Jackson ImmunoResearch Laboratories; cat. no. placental villous tissue obtained with consent from the University 111-165-003; 1:250 dilution). Nuclei were visualized with DAPI of Kansas Hospital under a protocol approved by the KUMC (Molecular Probes). Primary antibodies to human KDM3A (No- Institutional Review Board. Trophoblast cells were processed vus; cat. no. NBP1-49601), mouse KDM3A (62), MMP12 (AbCam; according to the method of Kliman et al. (61). cat. no. ab66157), pan cytokeratin (Sigma-Aldrich; cat. no. F3418),

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 1of13 and vimentin (Sigma-Aldrich; cat. no. V6630) were used in the were visualized by enhanced chemiluminescence according to the analyses. Negative controls were performed with normal rabbit manufacturer’s instructions (Amersham Biosciences). serum or isotype-specific control mouse IgG and did not exhibit positivereactivityinthetissuesections. Processed tissue sections Generation of the Mmp12 Mutant Rat Model. Targeted mutations were inspected and images recorded with a Leica MZFLIII ste- were generated using TALEN genome editing. TALEN con- reomicroscope equipped with a charge-coupled device (CCD) structs specific for the rat Mmp12 gene were designed, assem- camera (Leica). bled, and validated by the Genome Engineering Center, Washington University. Selected TALENs were targeted to exon In Situ Hybridization. Detection of Prl5a1 in situ hybridization was 2 of the Mmp12 gene (target sequence: TCTGAAAATAATG- performed as previously described (33). Ten-micrometer cry- CACACGTctcgatgtggagtgcctgATGTACAGCATCTTAGA; cor- osections were prepared and stored at −80 °C until used. A responding to nucleotides 281–300 and 319–335, NM_053963.2; plasmid containing a cDNA for PRL family 5, subfamily a, Fig. S1). Genome editing constructs were microinjected into the member 1 (Prl5a1) was used as a template to synthesize sense and cytoplasm of single-cell rat embryos. Injected embryos were antisense digoxigenin-labeled riboprobes according to the manu- transferred to oviducts of day 0.5 pseudopregnant rats. Offspring facturer’s instructions (Roche Molecular Biochemicals). Tissue were screened for mutations at specific target sites within the sections were air dried and fixed in ice cold 4% (wt/vol) para- Mmp12 gene. For initial screening, genomic DNA was purified formaldehyde in PBS. Prehybridization, hybridization, and de- from tail-tip biopsies using the E.Z.N.A. tissue DNA kit (Omega tection of alkaline phosphatase-conjugated antidigoxigenin were Bio-Tek). Potential mutations within 5,000 bp at each target locus performed. Detection of KDM3A, CDH1,andMMP12 transcripts were screened by PCR and precise boundaries of deletions de- was performed on paraffin-embedded human placenta tissue sec- termined by DNA sequencing (Genewiz). Founders were back- tions using the RNAScope 2-plex chromogenic assay (Advanced crossed with WT rats to demonstrate germ-line transmission. Two Cell Diagnostics), according to the manufacturer’s instructions. mutant rat strains possessing a 607- or 664-bp deletion including Probes were prepared to detect KDM3A (NM_018433.5; target all of exon 2 of the Mmp12 gene were characterized (Fig. S1). region: 450–1,443), CDH1 (NM_004360.3; target region: 263– Genotyping was performed using PCR on genomic DNA purified 1,255), MMP12 (NM_002426.4; target region: 295–1,598), POLR2A from tail-tip biopsies using the REDExtract-N-Amp tissue PCR (positive control, NM_000937.4; target region: 2,514–3,433), PPIB kit (Sigma-Aldrich) with specific sets of primers shown in (positive control, NM_000942.4; target region: 139–989); and DapB Table S2. The mutant Mmp12 rat model is available at the Rat (negative control, EF191515; target region: 414–862). Resource & Research Center (University of Missouri).

Morphometric Measurements. Morphological measurements of the shRNA Constructs and Production of Lentivirus. shRNA constructs depth of invasion and sizes of placental compartments were cloned into the pLKO.1 vector were obtained from Open Bio- performed with NIH Image J software as previously described systems. Several shRNAs were tested for each gene evaluated. (14, 15). The depth of intrauterine trophoblast cell invasion was shRNA sequences used in the analyses are provided in Table S3. used to determine an invasion index (distance of endovascular Lentiviral packaging vectors were purchased from Addgene and cytokeratin positive cell location relative to the trophoblast giant included pMDLg/pRRE (plasmid 12251), pRSV-Rev (plasmid cell layer of the chorioallantoic placenta/total distance from the 12253), and pMD2.G (plasmid 12259). Lentiviral particles were trophoblast giant cell layer to the outer mesometrial surface of the produced as previously reported (63). In brief, 293FT cells (In- uterus) and used in part as a surrogate for assessing trophoblast- vitrogen) were transiently transfected using Lipofectamine 2000 directed uterine spiral artery remodeling. The thickness of the (Invitrogen) with the following plasmids: shRNA containing junctional zone was estimated from cross-sectional area mea- transducing vector, packaging system plasmids (pMDLg/pRRE surements of vimentin immunostained placentation sites. Mea- and pRSV-Rev), and a VSVG envelope plasmid (pMD2.G). Af- surements were expressed as the ratio of junctional zone to ter transfection, cells were maintained in 50% DMEM with high labyrinth zone cross-sectional areas. Depth of intrauterine tro- glucose (Cellgro), 45% Opti-MEM I (Invitrogen), and 5% (vol/vol) phoblast cell invasion and chorioallantoic zone measurements FBS. Culture supernatants containing lentiviral particles were were all made from a histological plane at the center of each harvested every 24 h for 2–3 d. Supernatants were centrifuged placentation site perpendicular to the flat fetal surface of the to remove cell debris, filter sterilized, concentrated by ultracen- placenta. Sample sizes for the analyses were at least five placen- trifugation, and stored at −80 °C until used. tation sites from at least five different animals per treatment group. Ectopic Expression of KDM3A. A WT FLAG-tagged mouse Kdm3a Western Blot Analysis. KDM3A, ACTB, histone H3, histone H3K9 pcDNA3 expression vector (64) was generously provided by methylation modifications (monomethylated: H3K9me1, dime- Christopher Mack, University of North Carolina, Chapel Hill, thylated: H3K9me2, trimethylated: H3K9me3), and MMP12 NC. An enzymatically dead version of KDM3A was generated were monitored by Western blot analysis. Rat TS cell lysates were from full-length mouse Kdm3a (Open Biosystems-GE Dharma- prepared in RIPA buffer (10 mM Tris·HCl, pH 7.2, 1% Triton con) by introducing a point mutation within the catalytic domain X-100 or 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, (H1135Y) using PCR-based site directed mutagenesis and 150 mM NaCl, 5 mM EDTA, 1 mM sodium orthovanadate, 1 mM subcloned into pcDNA3. Empty and FLAG-tagged pcDNA3 phenylmethylsulfonyl fluoride, and 10 μg/mL aprotinin). Protein vectors expressing WT or mutant Kdm3a were transfected into concentrations were determined by the DC protein assay (Bio- rat TS cells using Lipofectamine 2000 (LifeTechnologies) trans- Rad). Proteins were separated by SDS/PAGE and transferred fection reagent according to manufacturer’s instructions. Cells onto nitrocellulose membranes. Immunoreactive proteins were were selected with Geneticin (LifeTechnologies) for 5 d before detected with rabbit antibodies to human KDM3A (Novus; cat. the initiation of the experiments. no. NBP1-49601), mouse KDM3A (62), ACTB (Sigma-Aldrich; cat. no. A1978), TFIID (Santa Cruz Biotechnology; cat. no. sc- Matrigel Invasion Assay. Invasive properties of rat TS cells were 56794), MMP12 (AbCam; cat. no. ab66157), and FLAG (Sigma- assessed as previously described (15). Rat TS cells were placed in Aldrich; cat. no. F3165). Antibodies to histone H3 and histone Matrigel invasion chambers (BD Biosciences) at a density of 2 × 104 H3K9 methylated isoforms were obtained from Abcam (histone cells per chamber. Cells were allowed to invade for 24 h. Mem- H3, cat. no. ab1791; H3K9me1, cat. no. ab9045; H3K9me2, cat. branes were collected and stained with Diff-Quick (Dade Behring). no. ab1220; H3K9me3, cat. no. ab8898). Immunoreactive proteins Invading cells were visualized by light microscopy and counted.

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 2of13 ChIP. ChIP analysis was performed according to a previously blastocysts collected on gd 4.5 were treated with Pronase (Sigma- published procedure (42). Briefly, TS cells were grown in 150-mm Aldrich; 10 mg/mL for 10 min) to remove zonae pellucidae and dishes for 2 d in ambient conditions and then exposed to 0.5% incubated with concentrated lentiviral particles (750 ng of p24/mL) oxygen for 24 h. Cells were then fixed with 1% formaldehyde, and for 4.5 h. Transduced blastocysts were cultured ex vivo for purified nuclear lysates were sonicated using a Diagenode Bio- blastocyst outgrowth analysis or transferred to uteri of day 3.5 ruptor (Diagenode) to prepare DNA fragments at a size of ∼500 bp. Lysates were immunoprecipitated with 3 μg of appropriate pseudopregnant rats for subsequent in vivo evaluation of gene antibodies obtained from AbCam (ARNT, cat. no. ab2; histone knockdown and placentation site phenotypes. H3, cat. no. ab1791; H3K9me1, cat. no. ab9045; H3K9me2, cat. The blastocyst outgrowth analysis was performed as reported no. ab1220; H3K9me3, cat. no. ab8898; KDM3A, cat. no. before (33). Blastocysts were obtained from gd 4.5 rats by flushing NB100-77282) or with rabbit or mouse IgG (BD Biosciences). them from uteri with 0.1 mL DMEM (Life Technology). Embryos Immunoprecipitated chromatin fragments were eluted from aga- were cultured in basal culture medium at 37 °C in a 5% CO2 rose beads. DNA–protein interactions were reverse cross-linked incubator for 72 h to allow hatching from the zona pellucida. The and purified using a QiaQuick PCR purification kit (Qiagen). attached blastocysts were exposed to ambient or 0.5% oxygen for Purified DNA fragments were assessed by qPCR using primers 24 h in a NAPCO Series 8000WJ incubator (ThermoScientific) designed to putative regulatory regions of target or control genes and then fixed in 4% (wt/vol) paraformaldehyde solution. Out- (Table S4) and SYBRgreen PCR master mix (Applied Biosystems). growth surface areas were visualized using light microscopy and Putative regulatory regions were examined throughout 5 kb up- measured using NIH Image J software. stream and 1 kb downstream of the transcription start site for the In vivo analysis was based on assessing adaptations to hypoxia. target genes and at putative CpG islands. The presence of conserved hypoxia response elements (HREs) was determined On gd 6.5, rats were placed in a hypoxic [10.5% (vol/vol) oxygen] using the MultiTF program (https://multitf.dcode.org/). Rel- gas-regulated chamber until gd 13.5. Rat placental tissues were ative occupancy/enrichment was normalized to input samples collected on gd 13.5 for histologic, morphometric, and biochemical by use of the ΔΔCT method. analyses.

Ex Vivo Lentiviral Trophoblast shRNA Delivery and Analyses. Rat Statistical Analyses. Data analyses were performed using SigmaPlot embryos were transduced with lentiviral particles as previously 11.2 statistical software package (Systat Software). Specific details described (63) containing control or Kdm3a shRNAs. Briefly, of the analyses are presented in the figure legends.

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 3of13 Fig. S1. Effects of low oxygen culture conditions on TS cell numbers and TALEN targeting of exon 2 within the rat Mmp12 locus. (A) Low oxygen effects on TS cell numbers. Note that 24-h exposure to ambient (Amb) or low oxygen (0.5% O2) conditions showed similar growth responses (n = 4/group, Mann–Whitney test, *P < 0.05). (B) In situ hybridization analysis of Mmp12 transcripts in gd 13.5 placentation sites from pregnant rats exposed to ambient or hypoxia con- ditions. (Scale bar, 250 μm.) (C) Schematic representation of the rat Mmp12 gene and the TALEN target site within exon 2 (NC_005107.4). Diagrammatic organization of the MMP12 protein. (D) PCR-based identification of Mmp12 mutant founders (13 founders identified from 69 offspring). Founder numbers 3 and 69 were used for expansion and characterization. (E) DNA sequence analysis showing two founder strains possessing deletions of 607 bp (Δ607) or 664 bp (Δ664). (F) Mendelian ratios generated from breeding +/Δ607 males x +/Δ607 females (Left)and+/Δ664 males x +/Δ664 females (Right). (G) Assessment of postpartum day 1 neonatal weights of WT (+/+), Δ/Δ 607, and Δ/Δ664 pups, sexed at birth (WT: males, n = 39, females, n = 33; Δ/Δ 607: males, n = 44, females n = 23; Δ/Δ664: males, n = 44, females, n = 43 females). Different letters above bars signify differences among means (ANOVA with Dunnett’stest,P < 0.05).

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 4of13 Fig. S2. Role of MMP12 in placental and fetal adaptations to hypoxia. (A) Fetal weights at gd 13.5 from WT and Mmp12 mutant (Δ/Δ607 and Δ/Δ664) rat pregnancies exposed to ambient (Amb) or hypoxia (Hyp) conditions (Ambient, WT: n = 36; Hypoxia, WT: n = 44; Ambient, Δ/Δ607: n = 49; Hypoxia, Δ/Δ607: n = 41; Ambient, Δ/Δ664: n = 35; Hypoxia, Δ/Δ664: n = 41; *P < 0.05). (B) Immunohistochemical analyses (pKRT) of gd 18.5 WT (+/+), Δ/Δ607, and Δ/Δ664 placentation sites. (C) Schematic representation of in vivo maternal exposure to hypoxia (10.5% O2) and analyses. WT and Mmp12 mutant conceptuses were generated by +/Δ607 male ×+/Δ607femalebreeding.(D) Effects of hypoxia (10.5% O2) on the numbers of viable and nonviable conceptuses from WT × WT and heterozygous × heterozygous breeding. Heterozygous × heterozygous pregnancies exposed to hypoxia exhibited a higher number of nonviable conceptuses than did WT × WT (+/+) pregnancies (n = 5/group; *P < 0.05). (E) Immunohistochemical analyses (pan cytokeratin, pKRT; MMP12; elastin) of placentation sites from WT (+/+)and Mmp12 mutant (Δ/Δ607) conceptuses exposed to hypoxic conditions. (F) Quantification of trophoblast cell invasion into the uterine mesometrial compartment (n = 5/group, Mann–Whitney test, *P < 0.05). (G) Genotyping of WT (WT-1) and Mmp12-null (Δ/Δ664–1andΔ/Δ664–2) TS cells for WT and Δ/Δ664 alleles (Top)and sex chromosome determination of WT and Mmp12-null TS cells (Bottom). WT-1 and Δ/Δ664–2 TS cells are X,Y and Δ/Δ664–1TScellsareX,X.(H) Conventional RT- PCR analysis of embryonic and trophoblast-specific markers in stem and differentiated WT-1, Δ/Δ664–1, and Δ/Δ664–2TScells.(I) Representative images of stem state colonies for WT-1, Δ/Δ664–1, and Δ/Δ664–2 TS cells. Data presented in A and D were analyzed with ANOVA and Student–Newman–Keuls test.

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 5of13 Fig. S3. KDM3A, hypoxia, and placental expression. (A) qRT-PCR analysis of known histone H3K9 demethylases in rat TS cells maintained in ambient con- ditions (white bars) or following 24-h exposure to 0.5% O2 (black bars). Statistical analysis: n = 3/group, not significant. (B) KDM3A expression in Rcho-1, mouse TS (mTS), and rat TS cells. (Left) Western blot analysis of KDM3A expression after Rcho-1 TS cells and mouse TS cells exposed to Amb or 0.5% O2 for 24 h. (Right) Western blot analysis for KDM3A in rat TS cell exposed to 0.5% O2 for various time intervals. ACTB was used as a loading control for Western blots. (C) ChIP analysis for HIF1B and histone H3 at a conserved hypoxia response element (HRE) within the Kdm3a promoter. Rat TS cells were infected with lentiviral vectors containing control (Ctrl) or Hif1b shRNAs and exposed to ambient (Amb) or 0.5% O2 culture conditions (n = 4, *P < 0.05). (D) ChIP analysis for HIF1B and histone

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Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 6of13 H3 at a conserved HRE within the Vegfa promoter. Rat TS cells were infected with lentiviral vectors containing Ctrl or Hif1b shRNAs and exposed to Amb or

0.5% O2 culture conditions (n = 4, *P < 0.05). (E) In vivo trophoblast KDM3A expression in response to maternal hypoxia. Rats were exposed to ambient or hypoxic (10.5% O2) conditions from gd 6.5 to 9.5 and euthanized, and placentation sites were dissected. Immunohistochemistry analysis (pan cytokeratin, pKRT; KDM3A) was performed on gd 9.5 placentation site tissue sections. White boxed areas shown in the upper images are presented in the lower panel.

(Scale bars, 100 μm.) (F) Effects of Amb or 0.5% O2 culture conditions on cell numbers for control shRNA (Ctrl shRNA)-, Kdm3a shRNA1-, and Kdm3a shRNA2- expressing TS cells (n = 4/group, *P < 0.05). (G) Effects of Kdm3a knockdown on hypoxia responsive transcripts in TS cells (n = 4, not significant). Data presented in B was analyzed with Mann–Whitney test and C, D, F, and G were analyzed with ANOVA and Student–Newman–Keuls test.

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 7of13 Fig. S4. Regulatory landscape associated with KDM3A target genes. (A) Western blot analysis for histone H3K9 methylation (monomethylation, me1; di- methylation, me2; trimethylation, me3) in lysates from rat TS cells exposed to ambient (Amb) or low oxygen (0.5% O2). (B) Immunolocalization of H3K9me1, H3K9me2, and H3K9me3 on gd 9.5 placentation sites from rats exposed to Amb or hypoxic (10.5% O2) conditions from gd 6.5 to 9.5. (C) Immunolocalization of H3K9me1, H3K9me2, and H3K9me3 on gd 13.5 placentation sites from rats exposed to Amb or 10.5% O2 from gd 6.5 to 13.5. (D) Schematic layout of the rat Mmp12 gene and location No. 2 (−920 to −765 bp upstream of TSS) within the 5′ flanking region surveyed by ChIP analysis in E and F. (E) ChIP analyses for histone H3K9 methylation and histone 3 (H3) at the Mmp12 locus in Amb and 0.5% O2 exposed TS cells. (F) ChIP analyses for histone H3K9 methylation and H3 at the Mmp12 locus in 0.5% O2 exposed TS cells treated with control (Ctrl) or Kdm3a shRNAs. (G) Schematic layout of the rat Mmp12 gene and location No. 3 (−414 to −276 bp upstream of TSS) within the 5′ flanking region surveyed by ChIP analysis in H and I.(H) ChIP analyses for histone H3K9 methylation and H3 at the Mmp12 locus in Amb and 0.5% O2 exposed TS cells. (I) ChIP analyses for histone H3K9 methylation and H3 at the Mmp12 locus in 0.5% O2 exposed TS cells treated with Ctrl or Kdm3a shRNAs. (J) Schematic layout of the gene desert location (Rnor_6.0, 44,746,608–44,746,715) surveyed by ChIP analysis in K and L.

(K) ChIP analyses for histone H3K9 methylation and H3 at a gene desert location in Amb and 0.5% O2 exposed TS cells. (L) ChIP analyses for histone H3K9 methylation and H3 at the gene desert locus in 0.5% O2 exposed TS cells treated with Ctrl or Kdm3a shRNAs. (M) Schematic layout of the rat Mmp12 gene and location No. 1 (−2,681 to −2,568 bp upstream of the TSS) within the 5′ flanking region surveyed by ChIP analysis in N.(N) In vivo ChIP analyses for KDM3A and

H3 in junctional zone tissue from gd 13.5 pregnant rats exposed to Amb or hypoxic (10.5% O2; Hyp) conditions. Statistical analyses: n = 4; control vs. hypoxia exposed TS cell experiments: Mann–Whitney test, *P < 0.05; shRNA experiments: ANOVA with Dunnett’s test vs. the control, *P < 0.05.

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 8of13 Fig. S5. Hypoxia responses of human trophoblast cells and protein and transcript localization in human placental tissues. (A) Effects of low oxygen (0.5% O2) on KDM3A transcript (qRT-PCR) and protein (Western blot) expression in BeWo and Jeg3 human trophoblast cells. Amb, ambient. (B and C) Effects of 0.5% O2 on MMP12, IL33, PPP1R3C, and PLOD2 expression in BeWo (B) and Jeg3 (C) human trophoblast cells. Statistical analysis for A–C: n = 3/group; Mann–Whitney test, *P < 0.0.5. (D) Effects of 0.5% O2 on invasive behavior of BeWo and Jeg3 human trophoblast cells. Images are representative Insets.(E) Quantification of invasion through Matrigel (Student t test, *P < 0.05). (F) First trimester (6 and 8 wk) and term human placental tissues were processed for immunohisto- chemistry using KDM3A and pan cytokeratin (pKRT) antibodies. (Scale bar, 25 μm.) (G) In situ hybridization analysis of first trimester human placental tissue, including positive [POLR2A (red) and PPIB (blue)] and negative (DapB) controls, and colocalization of KDM3A (blue, third image) and MMP12 (blue, fourth image) with CDH1 (red). (Scale bar, 100 μm.)

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 9of13 Fig. S6. KDM3A and MMP12 expression in diseased human placental tissues. (A) In situ hybridization localization of CDH1 (red) and KDM3A (blue), transcripts in replicate placenta sections from preterm, term, preeclampsia, and IUGR pregnancies (n = 6 for each). (Scale bar, 60 μm.) (B) In situ hybridization localization of CDH1 (red) and MMP12 (blue), transcripts in replicate placenta sections from preterm, term, preeclampsia, and IUGR pregnancies (n = 6 for each). (Scale bar, 60 μm.) The integrity and effectiveness of CDH1 and MMP12 probes were examined in first trimester placental tissue as a control.

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 10 of 13 Table S1. Effects of maternal hypoxia on metrial gland gene expression Microarray fold qRT-PCR ± SEM Protein Gene symbol Function change (Hyp/Amb) (Hyp/Amb)

TSH releasing hormone Trh Hormone 31.04 9.97 ± 1.162* Matrix metallopeptidase 10 Mmp10 ECM remodeler 8.75246 10.31 ± 1.415* HtrA serine peptidase 1 Htra1 Protease 4.78358 7.08 ± 0.883* Placenta-specific 1 Plac1 Unknown 4.45683 4.43 ± 0.459* Secretory leukocyte peptidase inhibitor Slpi Inhibitor of proteases 4.35631 3.92 ± 0.634* Bone morphogenetic protein 6 Bmp6 Growth factor 3.64587 9.92 ± 1.007* Cathepsin Q Ctsq Protease 3.60704 3.50 ± 0.371* Prolactin family 2, subfamily a, member 1 Prl2a1 Hormone 3.60029 5.62 ± 0.898* Integrin, β 6 Itgb6 Cell adhesion receptor/signaling 3.50875 2.54 ± 0.259* Prolactin family 4, subfamily a, member 1 Prl4a1 Hormone 3.3691 3.47 ± 0.457* Coagulation factor III (thromboplastin, F3 Cell surface glycoprotein/coagulation cascade 0.47 0.66 ± 0.0891* tissue factor) Epithelial cell adhesion molecule Epcam Cell adhesion molecule 0.29 0.4 ± 0.0809* WAP four-disulfide core domain 10 Wfdc10 Protease inhibitor 0.28 0.4 ± 0.0965* Insulin-like growth factor binding protein 1 Igfbp1 Growth factor binding protein 0.26 0.33 ± 0.0478*

*Mann–Whitney rank sum test (P < 0.05).

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 11 of 13 Table S2. Primer sequences Symbol Gene ID/accession no. Forward primer Reverse primer Amplicon size (bp)

Conventional RT-PCR analyses Mmp12 (genotyping) 117033 TACCTCCCCCGATAAAGTGC GGGTGTGCATGTGTGTTTGT 976 Kdm5c (genotyping) 317432 TTTGTACGACTAGGCCCCAC CCGCTGCCAAATTCTTTGG 692 Kdm5d (genotyping) 100312983 TTGGTGAGATGGCTGATTCC CCGCTGCCAAATTCTTTGG 250 T NM_001106209 GCTCATCGGAACAACTCTCC CTCCGAGGCTAGACCAGTTG 91 Pou5f1 NM_013633 GAGGTGGAACCTGGAGAACA GCCGGTTACAGAACCACACT 122 Hnf4 NM_022180 GTGTCGTTACTGCAGGCTCA GCTGTCCTCGTAGCTTGACC 109 Cdx2 NM_023963 CCCTAGGAAGCCAAGTGAAA CTGCGGTTCTGAAACCAAAT 187 Prl3b1 NM_012535 CAAGTCCACCAGGCAAAATC CATGCACCGATACAGGACAC 311 Mmp12 NM_053963 TTGATGAACTGGACTCTG GAAGGTATCTGTAGGTAGG 757 qRT-PCR Ppp1r3c NM_001012072 TACCTGAACGACCATCTA AAAGTGTGGCAGAAAGTT Mmp12 NM_053963 GCTGGTTCGGTTGTTAGG GTAGTTACACCCTGAGCATAC Plod2 NM_175869 AGGAAGATCATTGCCCCTCT ATCTCTGATCGCAGCGTCTT Bhlhe40 NM_053328 TGAAGCGACACCTCAAAG GGTCTCAAGGGGTATGTTC Il33 NM_001014166 CAACGACCAATCTGTTAG CGGAGTAGCACCTTATCT Kdm3a NM_175764.2 CTGAGATTCCTGAGCAAGTTATTC AGCCGAAGACTGTTTACATCC Cd200 NM_031518 GTCTACTTGAATCTGATGTTA TGTAAATACTGAGGACCC Loxl2 NM_001106047 GCAGAGAAGACTTACAACC AGATGTGTGCTTCAGTTC Slc2a1 NM_138827 GCTCAGTGTCATCTTCATC ACCCTCTTCTTTCATCTCC Dpp3 NM_053748 AGAGTTACATTGGCTTCA CTTTGCACTCATGTCTTT Vegfa NM_031836 TTAGTGGTTTCAATGGTCTG GGTCCTGGCAAAGAGAAG Mmp9 NM_031055 TACTGCTGGTCCTTCTGA CCGTCCTTGAAGAAATGC Ndrg1 NM_001011991 CCCACTAAACTCATTCCT TCAGCATTCAGAACTCTAA Plau NM_013085 TTGGTTCAGACTGTGAGAT GCACTGTTCGTGAGAAAT Comt NM_012531 CTGACTTCCTGGCGTATG TTCTCCAAGCCGTCTACA Cul7 XM_002727163 CAAGTCATCAGACCTTCC TTCACATCCGTTTCCATC Cdh1 NM_031334 TTCTGAAGACTCCCGATT ATGGACGAGAAAACTGGT Ncoa3 XM_215947 AGTGTATCGTTTCTCATT TACATCCATTCTGTTCTC Kat6a NM_001100570 AATCTTGTATGCTATGGTTA TTATCCTGATGCTGAGTA Id1 NM_012797 CTGAACGGCGAGATCAGTG GGAGTCCATCTGGTTCCTCA Rbm3 NM_053696 ATATGGAAGTGGAAGATA TTGTCATAATTGTCTCTG Satb1 NM_001012129.2 GCCACTGGTATTTCCTAC TCCTTCTGACTTCCTGTT Ccdc86 NM_001006974.2 CAACGAGAGCAGTCATCCA TGGGATCACCTCCTCCTTAG Lin28 NM_001109269 AAACAAGTGTCAAACCAAGATTA GCCCTGTGGAAATAACCT Hand1 NM_021592 AGCAAGCGGAAAAGGGAGT CGGCCTCTTCTCACTGATTT Cdx2 NM_023963 CAGGAGGAAAGCTGAGTTGG TGCTGCTGTTGCAACTTCTT Egln1 XM_008772679.1 GACCTGTCACCTAACTGAG AATAGCATGAAGAGGTTTACAAA Ankrd37 NM_001108400 TGAGACAGAAGCGGAGTT TGCCCAACAAGACATCATC Tpbpa NM_172073 GCAAGAGCAGAAGGGTAAAGAAGG TTTCTATGTCGAGCTCCTCCTCCT Gjb3 NM_019240 TGTGAACCAGTACTCCACCGCATT GCTGCCTGGTGTTACAGTCAAAGT Ascl2 NM_031503 GATGGGGCAGATGTTTGACT GCAAGAAACGCAGGTAGGTC Tfeb NM_001025707 ACTCAGTTTCTCCTTATGC AGACAGGTCCATGAAGTA Gcm1 NM_017186 AGAGGCAAGAAGAGCCATGA TCCCTGACTTGGGATTTCAC Prl7b1 NM_153738 AACAATGCCTCTGGCCACTGC AGGCCATTGATGTGCTGAGACAGT Prl5a1 NM_138527 TCCACACCAGACATTCCAGA TTTCCAGGAAGCCAACATTC Phlda2 NM_001100521.1 AGAGACCCACGGGAGTAG AGCAGGTAACCAATATAAATACG Id2 NM_013060 GGACATCAGCATCCTGTCCT AAAAAGGAAAAAGTCCCCAAA Trh NM_013046.3 GGTGCTGCCTTAGACTCCTG TTCTCCCAAGTCTCCCCTCT Mmp10 NM_133514.1 AATTGTTCCTGCATGTGCTGTGGC TTCTGTGCTCAGGTGATGCTCTGT Htra1 NM_031721.1 GCGGGCAAAACAAATGTAAT GGCAGGGACAGATGTTGACT Plac1 NM_001024894.1 ACTATGCTTCCAAGGGCTCA CGCCCATGTTACTGCTAGGT Slpi NM_053372.1 GAATCCTGTTCCCATTCGTG TTCCCACACATACCCTCACA Bmp6 NM_013107.1 TTTGTGGAGGTGCCTTCTGT GTATGGCGTGGGAATGCTAT Ctsq NM_139262.1 TCATCCTCTGCTTGGGAGTT TCCACTCTTGCCATTGAACA Prl2a1 NM_053791.2 GCTTCCAAAACCCAGCAGTA AAGGATGGCAGGTTGTTCAC Itgb6 NM_001004263.1 TTGCTCCTCAAAGCTTGGTT CCGAGAGGTCCATGAGGTAA Prl4a1 NM_017036.2 GCTCCTGGATGCCATAAAGA TATTGGGCGATTGCAAGAAT F3 NM_013057.2 ACAAATGCACTGGAACCACA TCCCCATGAGTTCCAAAGAG Epcam NM_138541.1 ATTTGCTCCAAACTGGCATC TCGTCACACTCGGGATCATA Wfdc10 NM_001109461.1 GTCCAGCGAAAACGGAGATA GGCGTCTCCCACACACTATT Igfbp1 NM_013144.1 GCGGTAGTGCCTAGAACGAG TGGGATTCGATGAGGAAGTC Lsd1 NM_001130098.1 ATTATTATAGGCTCAGGTGTT CAGAAGTGTGACATCCAT

Chakraborty et al. www.pnas.org/cgi/content/short/1612626113 12 of 13 Table S2. Cont. Symbol Gene ID/accession no. Forward primer Reverse primer Amplicon size (bp)

Kdm4a NM_001107966.1 AGCCTTCCCTTCCCAGATTA CGAGGCGTCCTTCTACAGAC Kdm4b NM_001044236.2 ACAGCTGGGGTAGCTGGTCT CTTGCTCCTGGCCTTAGTTG Kdm4c NM_001106663.3 TGACACCAAGCTCTCAGGAA AACGTGACTCCTTGCCTGTT Kdm4d NM_001079712.1 TAGCCCCACCATATCCTCTG GGACTGGGACTGAAGCTCTG Kdm3b XM_008772060.1 AAAGGAAAGAAGAAGAGA CATTACTGTCTACTTCAC Kdm3c NM_001191719.1 GTTACTTAGTCACATTCCT TTCCTCTCCAATTCTTCT MMP12 (human) NM_002426.5 TGGTTTTTGCCCGTGGAGCTCAT GAATGGCCAATCTCGTGAACAGCA IL33 (human) NM_001199640.1 GGCTGAGAATTACCATACAAGG AGTGTTTTTCAGATGGGATGA PLOD2 (human) NM_182943.2 GCGTTCTCTTCGTCCTCATC CATGAAGCTCCAGCCTTTTC KDM3A (human) NM_001146688.1 ACTCTTCAAGTCAACTGT ATGTCACAAGGTTATCGT PPP1R3C (human) NM_005398.5 CATGTTGGGCAAGGAAAAGT CCCACTCCTCtGACTTGAGC 18S rRNA M11188 GCAATTATTCCCCATGAACG GGCCTCACTAAACCATCCAA

Table S3. shRNA sequences Target shRNA sequence

Control shRNA CCTAAGGTTAAGTCGCCCTCGCTCTAGCGAGGGCGACTTAACCTTAGG Hif1b shRNA GAGAAGTCAGAAGGTCTCTTTTCTCGAGTAAAGAGACCTTCTGACTTCTC Kdm3a shRNA-1 CATGATCAGAGCTGGTATTTACTCGAGTAAATACCAGCTGATCATG Kdm3a shRNA-2 CTTCGAAGTTTCATTGGATTTCTCGAGAAATCCAATGAAACTTCGAAG KDM3A shRNA (human) CTTTGATTGTGAAGCATTTACTCGAGTAAATGCTTCACAATCAAAGCT

Table S4. ChIP primer sequences Target gene Forward primer Reverse primer

Mmp12 region 1 TCCTGGGCCTCTGACTCTTA AGTCAGCTGCCTGGAACATT Mmp12 region 2 AAGGATTCGTGTATTCTAGCA ACGCCATTAGCAGAGTTT Mmp12 region 3 TGCTGAGTCATTTTGCAAGC ATCCCACCACAAGCAGAGTC Il33 region 1 TGACTTCCCAGACGGGTAAC CCATTATTCACCAGGCAAGC Ppp1r3c region 1 TTGGATACACAACGCAAGGA GTGGGTCTGTGCAGAGTTGA Kdm3a HRE AGGATCATGAGCGCCTATTG TCTAACTGCGTACGCCTGAA Vegfa HRE GCCAGACTCCACAGTGCATA TTCCCAGGGGAGAAGAATTT Gene desert GGGGGATAATGATTGCAAAA GCGTGGACAGAGATCTAGGC

Other Supporting Information Files

Dataset S1 (XLS)

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