Supporting Information

Bhatnagar et al. 10.1073/pnas.1413620111 SI Materials and Methods (University of Pennsylvania, Philadelphia), were cultured in Single-Nucleotide Primer Extension Assay. A single-nucleotide DMEM supplemented with 10% (vol/vol) FCS and 10% (vol/vol) primer extension (SNuPE) assay for Pgk1 was carried out using nonessential amino acids. Analysis of imprinted was per- a TaqMan SNP genotyping assay (Applied Biosystems) accord- formed using MEFs isolated from the C57BL/6 (CAST7) strain, ing to the manufacturer’s specifications. The following primers which contains 7 from the Mus castaneus (Cast) and reporters were used for the assay: 5′-CCGGCCAAAATT- strain in a C57BL/6 background, as previously described (1). GATGCTTTCC-3′,5′-CAGTCCCAAAAGCATCATTGACAT- Briefly, total RNA was extracted and cDNA synthesis was carried 3′,5′-CACTGTCCAAACTAGG-3′, and 5′-CACTGTCCACA- out as described above. For PCR amplification, the cDNA was CTAGG-3′. The data are plotted as the function of ΔRn for each added to Ready-To-Go PCR Beads (GE Life Sciences) together sample, which represents the reporter fluorescence for each allele with 0.3 μM -specific primers (Table S2). Expression of the (VIC/FAM) normalized to the passive reference dye. imprinted gene was analyzed by allele-specific restriction enzyme digestion (StcI for Ascl2,StuIforKcnq1ot1, MnlI for Peg3,and Imprinted Gene Analysis. Mouse embryonic fibroblasts (MEFs) FauI for Zim1), and digested PCR products were resolved by from strain C57BL6 (CAST 7), provided by M. Bartolomei polyacrylamide gel electrophoresis.

1. Rivera RM, et al. (2008) Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development. Hum Mol Genet 17(1):1–14.

A HAT selection (H4SV cells) B NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1 qRT-PCR (H4SV cells) 0.6 0.5 0.4 0.3

Pdpk1 Pygo1 Rnf165 Sox5 Stc1 Zfp426 1700001P01Rik 0.2 0.1 Knockdown efficiency 0 shRNA: Nf1 Stc1 Acvr1Aurka Layn Sox5 Dnmt1Fbxo8 Pdpk1Pygo1Rnf165 Zfp426 1700001 P01Rik C HAT selection (H4SV cells; 2nd shRNA) D NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1 0.9 qRT-PCR (H4SV cells; 2nd shRNA) 0.8 0.7 0.6 0.5 0.4 Pdpk1 Pygo1 Rnf165 Sox5 Stc1 Zfp426 1700001P01Rik 0.3 0.2

Knockdown efficiency 0.1 0 shRNA: Nf1 Stc1 Acvr1Aurka Layn Sox5 Dnmt1Fbxo8 Pdpk1Pygo1Rnf165 Zfp426 1700001 P01Rik

Fig. S1. shRNAs targeting an X-chromosome inactivation factor (XCIF) reactivate the inactive X chromosome (Xi)-linked Hprt gene and decrease mRNA levels of the targeted gene. (A) Bright-field images showing growth of the 13 XCIF KD H4SV cell lines following selection in hypoxanthine–aminopterin–thymidine (HAT) medium. (B) Quantitative real-time RT-PCR (qRT-PCR) analysis monitoring target gene expression in the 13 XCIF KD H4SV cell lines expressing the shRNA identified in the primary screen. For each gene, knockdown efficiency was determined relative to the level of target gene expression in the control cell line expressing a nonsilencing (NS) shRNA, which was set to 1. Error bars indicate SD. (C) Bright-field images showing growth of the 13 XCIF KD H4SV cell lines, expressing a second, unrelated shRNA to that shown in A, following selection in HAT medium. (D) qRT-PCR analysis monitoring target gene expression in the 13 XCIF KD H4SV cell lines expressing a second, unrelated shRNA to that shown in B. Error bars indicate SD.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 1of11 A RNA FISH (BMSL2 cells; 2nd shRNA) NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1

G6pdx Lamp2

Pdpk1 Pygo1 Rnf165 Sox5 Stc1Zfp426 1700001P01Rik

G6pdx Lamp2

NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1

Pgk1 Mecp2

Pdpk1 Pygo1 Rnf165 Sox5 Stc1Zfp426 1700001P01Rik

Pgk1 Mecp2

qRT-PCR (BMSL2 cells) B 3.0 Hprt 2.5 G6pdx

2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 Relative expression Relative expression 0 0 2.5 Mecp2 2.5 Pgk1

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5 Relative expression Relative expression 0 0 shRNA: NS shRNA: NS Nf1 Stc1 Nf1 Stc1 Acvr1Aurka Layn Sox5 Acvr1Aurka Layn Sox5 Dnmt1Fbxo8 Pdpk1Pygo1Rnf165 Zfp426 Dnmt1Fbxo8 Pdpk1Pygo1Rnf165 Zfp426 1700001 1700001 P01Rik P01Rik Pgk1 SNuPE assay (BMSL2 cells) X chromosome painting (BMSL2 cells) C 3.5 D 100 3.0 ∆Rn Pgk1b (Xa) 80 2 signals 2.5 ∆Rn Pgk1a (Xi) 1 signal 2.0 60 >2 signals 1.5 40 1.0 % nuclei

Rn (normalized to 20

∆ 0.5 non-template control) 0 0 shRNA: NS NS Nf1 Layn Layn Sox5Stc1 Acvr1 Sox5 Acvr1Aurka Fbxo8 Dnmt1 Pygo1 Zfp426 Dnmt1 Pdpk1Pygo1Rnf165 Zfp426 1700001P01Rik

Fig. S2. Additional RNA FISH images and control experiments related to Fig. 1. (A) Representative two-color RNA FISH images showing expression of G6pdx (red) and Lamp2 (green; Upper) and Pgk1 (red) and Mecp2 (green; Lower) in each of the 13 XCIF KD BMSL2 cell lines. DAPI staining is shown in blue. (B) qRT- PCR analysis monitoring expression of Hprt, G6pdx, Mecp2, and Pgk1 in each of the 13 XCIF KD BMSL2 cell lines. The results were normalized to that obtained with the NS shRNA, which was set to 1 (red line). A twofold increase in expression is indicated by the blue line. (C) In BMSL2 cells, the Xi and Xa encode two distinguishable Pgk1 alleles, Pgk1a and Pgk1b, respectively, which differ by a single-nucleotide polymorphism within the mRNA. Allele-specific expression of the Xi- and Xa-linked Pgk1 genes in six representative XCIF KD BMSL2 cell lines was analyzed using a SNuPE assay. The data are plotted as the function of ΔRn Legend continued on following page

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 2of11 for each sample, which represents the reporter fluorescence for each allele (VIC/FAM) normalized to the passive dye. The results show that, in each of the six XCIF KD BMSL2 cell lines, the Xi-linked Pgk1a gene was reactivated. (D) X-chromosome painting experiments in the 13 XCIF KD BMSL2 cell lines. The results show that the X-chromosome content of the XCIF KD BMSL2 cell lines was similar to that of the control BMSL2 cell line expressing a NS shRNA. Thus, the substantially increased biallelic expression of X-linked genes observed by RNA FISH in the XCIF KD cell lines cannot be explained by differences in X-chro- mosome number.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 3of11 A RNA FISH (Differentiated mouse ES cells) NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1

G6pdx Lamp2

Pdpk1 Pygo1 Rnf165 Sox5 Stc1Zfp426 1700001P01Rik

G6pdx Lamp2

NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1

Pgk1 Mecp2

Pdpk1 Pygo1 Rnf165 Sox5 Stc1Zfp426 1700001P01Rik

Pgk1 Mecp2

B X chromosome painting C 200 (Differentiated ES cells) 100 160 2 signals 40

80 1 signal expression 30 >2 signals 60 20

Fold increase 10 40 Eomes

in 0 % nuclei 20 700 400 0 50 shRNA: NS Nf1 40 Stc1 expression Acvr1Aurka Layn Sox5 Dnmt1Fbxo8 Pdpk1Pygo1Rnf165 Zfp426 30 1700001 P01Rik 20 Fold increase

Tcf7l2 Tcf7l2 10

in 0 200 160 80 60 expression 40

Fold increase 20 Cdx2

in 0

shRNA: NS Nf1 Stc1 Acvr1Aurka Layn Sox5 Dnmt1Fbxo8 Pdpk1Pygo1Rnf165 Zfp426 1700001 (Undiff ES) P01Rik

Fig. S3. Additional RNA FISH images and control experiments related to Fig. 2. (A) Representative two-color RNA FISH images monitoring expression of G6pdx (green) and Lamp2 (red; Upper) and Pgk1 (green) Mecp2 (red; Lower) in the 13 XCIF KD ES cell lines following differentiation. DAPI staining is shown in blue. (B) X-chromosome painting experiments in the 13 XCIF KD ES cell lines following differentiation. (C) qRT-PCR analysis monitoring expression of Eomes, Tcf7l2, and Cdx2 in the 13 XCIF KD ES cell lines following treatment with RA. As a control, expression of each gene in undifferentiated ES cells is shown and was set to 1. Error bars indicate SD.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 4of11 A Xist RNA FISH NS Acvr1 Aurka Dnmt1 Fbxo8 Layn Nf1

Pdpk1 Pygo1 Rnf165 Sox5 Stc1 Zfp426 1700001P01Rik

BCqRT-PCR (H4SV cells) qRT-PCR (Differentiated mouse ES cells) 1.2 1.4 1.2 1.2 1.2 1.2 1.0 1.2 1.0 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.8 0.8 0.8 0.6 0.6 0.6 0.6 0.6 expression 0.6 expression expression expression expression expression 0.4 0.4 0.4 0.4 0.4 0.4 Tsix Tsix Xist Xist 0.2 0.2 0.2 Tsix 0.2 0.2 Dnmt1 0.2 Dnmt1 0 0 0 0 0 0 shRNA: shRNA: shRNA: shRNA: shRNA: shRNA: NS NS NS NS NS NS

Dnmt1-2 Dnmt1-2 Dnmt1-1Dnmt1-2 Dnmt1-1Dnmt1-2 Dnmt1-1Dnmt1-2 Dnmt1-1Dnmt1-2

Fig. S4. RNA FISH images and control experiments related to Fig. 3. (A) RNA FISH images. In each of the 13 XCIF KD ES cell lines following differentiation, the majority of cells that lost the typical Xist localization pattern lacked a detectable Xist signal (Fig. 3B). However, some cells that had lost the typical Xist lo- calization pattern contained two small Xist signals, reminiscent of undifferentiated ES cells. Examples of this latter localization pattern are shown here. Nuclear signals are indicated in red and denoted by arrowheads; DAPI staining is shown in blue. (B) qRT-PCR analysis monitoring expression of Xist (Left), Tsix (Center), and Dnmt1 (Right) in H4SV cells expressing a NS or one of two Dnmt1 shRNAs (Dnmt1-1 or Dnmt1-2). For Xist and Tsix expression, a second, unrelated Dnmt1 shRNA to that used in Fig. 3H. Expression was normalized to that obtained with the control NS shRNA, which was set to 1. Error bars indicate SD. (C) qRT-PCR analysis monitoring expression of Xist (Left), Tsix (Center), and Dnmt1 (Right) in differentiated ES cells expressing a NS shRNA or one of two Dnmt1 shRNAs (Dnmt1-1 or Dnmt1-2). Expression was normalized to that obtained with the control NS shRNA, which was set to 1. Error bars indicate SD.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 5of11 A Control OSU-03012 LY294002

Xist Mecp2

B Control GNE-317

C Control OSU-03012 After OSU-03012 removal

Xist Mecp2

LY294002 After LY294002 removal

Fig. S5. Additional RNA FISH images related to Fig. 4. (A and B) Two-color RNA FISH monitoring expression of Xist (red) and Mecp2 (green) in differentiated ES cells treated with DMSO (control), OSU-03012 (4 μM), or LY294002 (10 μM) (A), and in BMSL2 cells treated with DMSO or GNE-317 (5 μM) (B). The yellow boxes indicate cells with colocalizing Xist and Mecp2 signals; the white boxes indicate cells with biallelic expression of Mecp2 and complete loss of the Xist signal. (C) Two-color RNA FISH monitoring Xist (red) and Mecp2 (green) expression in BMSL2 cells treated with DMSO (control), OSU-03012 (2.5 μM), or LY294002 (8 μM), and at least 6 d following removal of the inhibitor. The white boxes indicate cells with biallelic expression of Mecp2.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 6of11 X chromosome painting A (Female MEFs) 100 2 signals 80 1 signal >2 signals 60

40 % nuclei 20

0 Stc1+/+ Stc1-/- B RNA FISH (Mouse brain sections)

Xist Stc1+/+ Mecp2

Stc1-/- Xist Mecp2

Xist Stc1+/+ G6pdx

Xist Stc1-/- G6pdx

+ + − − Fig. S6. Control experiment and RNA FISH images related to Fig. 5. (A) X-chromosome painting experiments in female Stc1 / and Stc1 / MEFs. The results show that the X-chromosome content of Stc1−/− MEFs was similar to that of Stc1+/+ MEFs. Thus, the substantially increased biallelic expression of X-linked genes observed by RNA FISH in the Stc1−/− MEFs cannot be explained by differences in X-chromosome number. (B) Defective XCI in cortical neurons from brain sections of female Stc1−/− mice. Two-color RNA FISH monitoring expression of Xist (red) and Mecp2 or G6pdx (green) in cortical neurons from adjacent 5-μm − − + + brain sections of female Stc1 / and Stc1 / mice (n = 3 per genotype, stage P1). The boxed regions denote cells with two Mecp2 or G6pdx signals; the yellow boxes indicate cells with colocalizing Xist and Mecp2/G6pdx signals. All cells in the regions shown represent neurons that, based on anatomical landmarks, are present in posthybridized sections.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 7of11 A 12 B 11 10 4 WT 5 KO

0

log10 P-value 2 − -5 (log2 transformed FPKM)

Autosomeal gene expression -10 0 −10 −505 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Chromosome log2 KO:WT X−linked gene expression ratio

C Kcnq1ot1 Peg3 Ascl2 Zim1

Undig. 814 bp Undig. 487 bp Undig. 474 bp Undig. 490 bp

487 bp 474 bp FauI dig. 490 bp MnlI dig. SfcI dig. 601 bp StuI dig. shRNA: NS Nf1 Stc1 Acvr1Aurka Layn Sox5 shRNA: Dnmt1Fbxo8 Pdpk1Pygo1 Rnf165 Zfp426 NS Nf1 Layn Stc1 shRNA: Acvr1Aurka Fbxo8 Sox5 NS 213 bp Dnmt1 Pdpk1Pygo1 Rnf165 Zfp426 Nf1 Stc1 Acvr1Aurka Layn Sox5 Dnmt1Fbxo8 Pdpk1Pygo1 Rnf165 Zfp426 1700001P01Rik

shRNA: 1700001P01Rik NS Nf1 Layn Sox5Stc1 1700001P01Rik Acvr1AurkaDnmt1Fbxo8 Pdpk1Pygo1 Rnf165 Zfp426 Gene Expressed Restriction Digested paternal Digested maternal allele enzyme (Cast) allele (bp) (C57BL/6) allele (bp) 1700001P01Rik Kcnq1ot1 Paternal StuI 213, 601 814 Peg3 Paternal MnlI 487 110, 377 Ascl2 Maternal SfcI 207, 266 474 Zim1 Maternal FauI 236, 254 490

D E B cells (6 month old mice) Myeloid cells (2 month old mice) Myeloid cells (6 month old mice) qRT-PCR qRT-PCR 6 1.2 1.2 4 12.2%4 1.6% 4 12.2% 5 1.0 1.0 3 3 3 4 0.8 0.8 2 2 2 3 0.6 0.6

expression 1 1 1 knockdown knockdown

2 efficiency efficiency 0.4 0.4 0 0 0 Hprt Fold increase expression ratio Bmi1 1 Ezh2

in 0.2 0.2 -1 -1 -1 0 0 0 shRNA: shRNA: shRNA: X-linked gene log2 HET:WT -2 -2 -2 NS Bmi NS NS Ezh2 Ezh2 Bmi1 0 100 200 300 400 0 100 200 300 400 0 100 200 300 400 X-linked gene X-linked gene X-linked gene

Fig. S7. Additional experiments and data analyses related to Fig. 6 and Discussion.(A) Volcano plot showing distribution of log2-transformed ratio of X-linked − − + + gene expression in MEFs isolated from Stc1 / (KO) and Stc1 / (WT) embryos (n = 3 per genotype). The genes are plotted against negative transformed log of P value. The red circles represent genes with a more than twofold change in expression and P < 0.01. The results show that the similarity of X-linked gene +/+ −/− expression between female Stc1 and Stc1 MEFs was statistically significant. (B) Box plots displaying changes in autosomal gene expression [log2-trans- formed fragments per kilobase of exon per million fragments mapped (FPKM)] in Stc1−/− and Stc1+/+ MEFs. The boxed areas span the first to the third quartile. The whiskers represent 15th and 85th percentiles; samples falling outside these percentiles are shown as circles. (C) XCIFs are not required for repression of imprinted genes. Primary female mouse embryonic fibroblasts from the strain C57BL/6 (CAST7), which contains chromosome 7 from Mus castaneus (Cast), were transduced with shRNAs against each of the XCIFs and analyzed for allele-specific expression of four genes located on chromosome 7 that are either paternally expressed (Kcnq1ot1 and Peg3) or maternally expressed (Ascl2 and Zim1). Expression of the two alleles can be distinguished by allele-specific restriction enzyme digestion following gene-specific RT-PCR. The sizes of the undigested and digested bands are indicated, and the sizes of the predicted digested fragments are shown in the table (Lower). If knockdown of an XCIF results in reactivation of the normally silenced allele, a mixture of the maternal and paternal allele-specific digestion patterns would be observed. The results show that, in all 13 XCIF KD cell lines, all four genes displayed only the expected allele-specific expression pattern, indicating that the XCIFs are not required for repression of the imprinted genes. (D) Requirement of Polycomb subunits EZH2 and BMI1 for repression of the X-linked Hprt gene. (Left) qRT-PCR analysis monitoring Hprt expression in BMSL2 cells expressing an Ezh2 or Bmi1 shRNA or, as a control, a NS shRNA. (Right) qRT-PCR analysis confirming target gene knockdown in mouse ES cells expressing an Ezh2 (Left) or Bmi1 (Right) shRNA. Error bars indicate SD. (E)

Analysis of available datasets from Yildirim et al. (1) showing the distribution of log2-transformed ratio of X-linked gene expression in hematopoietic cells from female heterozygous (HET) Xist mutant mice and wild-type (WT) mice. The data were downloaded from Gene Expression Omnibus (GSE43961), normalized by RMA, and filtered by detection above background (DABG) (cutoff P value of <0.0001) using Bioconductor package xps. The percentage of X-linked genes up- regulated >1.5-fold is shown.

1. Yildirim E, et al. (2013) Xist RNA is a potent suppressor of hematologic cancer in mice. Cell 152(4):727–742.

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 8of11 Table S1. Summary of the 13 XCIFs Mouse gene Human gene Chromosome, symbol symbol Gene name mouse (human) Biological process

Acvr1 ACVR1 Activin A receptor, type 1 2 (2) Signal transduction Aurka AURKA Aurora kinase A 2 (20) Cell cycle regulation Dnmt1 DNMT1 DNA methyltransferase (cytosine-5) 1 9 (19) Chromatin modification Fbxo8 FBXO8 F-box 8 8 (4) Unknown/ubiquitin-dependent protein catabolic process Layn LAYN Layilin 9 (11) Unknown/receptor for hyaluronic acid Nf1 NF1 Neurofibromatosis 1 11 (17) Signal transduction Pdpk1 PDPK1 3-Phosphoinositide–dependent 17 (16) Signal transduction protein kinase 1 Pygo1 PYGO1 Pygopus 1 9 (15) Transcriptional regulation Rnf165 RNF165 Ring finger protein 165 18 (18) Unknown Sox5 SOX5 SRY-box containing gene 5 6 (12) Transcriptional regulation Stc1 STC1 Stanniocalcin 1 14 (8) Cell Zfp426 ZNF426 Zinc finger protein 426 9 (19) Transcriptional regulation 1700001P01Rik C17orf98 RIKEN cDNA 1700001P01 gene 11 (17) Unknown

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 9of11 Table S2. List of primers used for qRT-PCR and RT-PCR analysis, cDNA synthesis, ChIP assays, and mouse genotyping Forward primer, 5′→3′ Reverse primer(s), 5′→3′

qRT-PCR Actin TTGCCGACAGGATGCAGAA GCCGATCCACACGGAGTACTT Acvr1 (mouse) GGCCAGCAGTGTTTTTCTTC TTCCCCTGCTCATAAACCTG ACVR1 (human) TCAGGAAGTGGCTCTGGTCT CGTTTCCCTGAACCATGACT Aurka (mouse) TAGGATACTGCTTGTTACTT CCTCCAACTGGAGCTGTA AURKA (human) TGGAATATGCACCACTTGGA ACTGACCACCCAAAATCTGC Bmi1 AAATCAGGGGGTTGAAAAATCT GCTAACCACCAATCTTCCTTTG Cdx2 GCCAAGTGAAAACCAGGACAAAAGAC GCTGCTGTTGCTGCTGCTGCTTC Dnmt1 (mouse) GGAAGGCTACCTGGCTAAAGTCAAG ACTGAAAGGGTGTCACTGTCCGAC DNMT1 (human) GTGGGGGACTGTGTCTCTGT TGAAAGCTGCATGTCCTCAC Eomes CCTGGTGGTGTTTTGTTGTG TTTAATAGCACCGGGCACTC Ezh2 CTAATTGGTACTTACTACGATAACTTT ACTCTAAACTCATACACCTGTCTACAT Fbxo8 (mouse) GCTGAGCCATTTTCTTCTCG ATGATGGTTTCTGGCCACTC FBXO8 (human) CAAGGGTTGTGGAGAGTGGT ATGTCAATGCCTCCTTGGAC Gapdh ATGGCCTTCCGTGTTCCTAC ATAGGGCCTCTCTTGCTCAG G6pdx TCAAAGCACACGCCCTCTT TAGCGCACAGCCAGTTTCC Hprt AAGCTTGCTGGTGAAAAGGA TTGCGCTCATCTTAGGCTTT Layn (mouse) GCAAGGAGAGTGGATGGGTA ACTTGTGATGCTGTGCTTGC LAYN (human) CTACAGGCCGTGCTGCTG CTGACTAGCTGGCCTCCATC Mecp2 CATGGTAGCTGGGATGTTAGG GCAATCAATTCTACTTTAGAGCG Nf1 (mouse) GTAGCCACAGGTCCCTTGTC CTGAGAACAAGTACACAGAGAGTGA NF1 (human) AATTCTGCCTCTGGGGTTTT GCTGTTTCCTTCAGGAGTCG Oct4 CTCACCCTGGGCGTTCTCT AGGCCTCGAAGCGACAGA Pdpk1 (mouse) GGTCCAGTGGATAAGCGAAA TTTCTGCACCACTTGTGAGC PDPK1 (human) GACTCTTCCGTGCGTTCTTC GAGGAGAAAGGTGACCCACA Pgk1 ATGTCGCTTTCCAACAAGCTG GCTCCATTGTCCAAGCAGAAT Pygo1 (mouse) TAATGTCAGCGGAACAGGAC TTATCTGGGCTTCCGAGTTG PYGO1 (human) ATCCTGGCTTTGGAGGCTAT GTGGCCCAAAGTTAAAAGCA Rnf165 (mouse) ATGCCTCCAGCTACAGCCTA GCCCAATGCTAACTGAGAGC RNF165 (human) AGGGAGAGCTGGAAAAGGAG AGCCCTCCCTGGTTTAGTGT Sox5 (mouse) GTGGAAGAGGAGGAGAGTGAGA AAATTCCTCAGAGTGAGGCTTG SOX5 (human) AGGGACTCCCGAGAGCTTAG TTGTTCTTGTTGCTGCTTGG Stc1 (mouse) AAGTCATACAGCAGCCCAATCA CCAGAAGGCTTCGGACAAGTC STC1 (human) TGATCAGTGCTTCTGCAACC TCACAGGTGGAGTTTTCCAG Tcf7l2 AAAACAGCTCCTCCGATTCC TAAAGAGCCCTCCATCTTGC Tsix CAATCTCGCAAGATCCGGTGA (TSIX2F) TCAAGATGCGTGGATATCTCGG (P422R) Xist (nonstrand specific) CCCTGCTAGTTTCCCAATGA GGAATTGAGAAAGGGCACAA Xist (strand specific) GATGCCAACGACACGTCTGA (XIST2281F) AAGGACTCCAAAGTAACAATTCA (XIST2424R) XIST (human) ACGCTGCATGTGTCCTTAGTAGTC ATTTGGAGCCTCTTATAGCTGTTTG Zfp426 (mouse) ATGACCTTTCGCTCATGGAC GGCAAGCTTTGCTTTAGTGC ZNF426 (human) CTGAGGTGGGTGGATCACTT CTCTGCTTCCTGGGTTCAAG 1700001P01Rik (mouse) GCTGATGTCAACTGTTTCC CGCAGAATCTTCCACCCT C10orf98 (human) TCGGGCAAGGACAAAGATAC CGATGGCTATGAAGGGAAAA RT-PCR Mecp2 (first round) CCGATCTGTGCAGGAGACCG TGGGGTCCTCGGAGCTCTCGGGCT Mecp2 (second round) GACCCGGGAGACGGTCAGCA AGCTCTCGGGCTCAGGTGGAGGT Ascl2 TGAGCATCCCACCCCCCTA CCAAACATCAGCGTCAGTATAG Kncq1ot1 ATTGGGAACTTGGGGTGGAGGC GGCACACGGTATGAGAAAAGATTG Peg3 ATGCCCACTCCGTCAGCG GCTCATCCTTGTGAACTTTG Zim1 CTTCAAGCAGAGCACAAAGC GTGGCACACGAAAGGTTTCTC cDNA synthesis Xist AGAGCATTACAATTCAAGGCTC (XIST2688R) Tsix GATGCCAACGACACGTCTGA (TSIX2R) Gapdh TGTGAGGGAGATGCTCAGTG (GAPDR) ChIP Xist (promoter) TAAAGGTCCAATAAGATGTCAGAA GGAGAGAAACCACGGAAGAA Xist (exon 2) GTGCTCCTGCCTCAAGAAGAA GCACTCTTCACTCCTCTAAATCCAG Mouse genotyping Dnmt1+/+ CTTGGGCCTGGATCTTGGGGATC GGG CCAGTTGTGTGACTTGG Dnmt1−/− GGGAACTTCCTGACTAGGGG GGGCCAGTTGTGTGACTTGG + + Stc1 / AGCGCACGAGGCGGAACAAA AGAGAGCCGCTGTGAGGCGT − − Stc1 / AAAAGCCAGAGGTGCAAGAA TATGATCGGAATTCCTCGAC SRY TTGTCTAGAGAGCATGGAGGGCCATGTCAA CCACTCCTCTGTGACACTTTAGCCCTCCGA

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 10 of 11 Table S3. Oligo ID numbers for shRNAs obtained from Open Biosystems/Thermo Scientific Gene Oligo ID

Acvr1 V2MM_75565 V2MM_76215 Aurka V2MM_188005 V2MM_71909 Bmi1 V2MM_10594 V2MM_2034 Dnmt1 V2MM_46797 V2LMM_43170 Ezh2 V2MM_35988 V2MM_30422 Fbxo8 V2MM_36526 V3LMM_494067 Layn V2MM_130482 V2MM_214085 Nf1 V2MM_194180 V2HS_76027 Pdpk1 V2MM_75859 V2MM_72465 Pygo1 V2MM_110610 V2MM_110609 Rnf165 V2MM_172866 TRCN0000135474 Sox5 V2MM_6385 V2HS_94936 Stc1 V2MM_22454 V2MM_26886 TRCN0000109921 Zfp426 V2MM_31994 TRCN0000085016 1700001P01Rik V2MM_100177 V2MM_205788

Table S4. cDNAs used to prepare RNA FISH probes Gene Clone no.* Ref.

G6pdx BAC clone RP23-13D21 1 Lamp2 BAC clone RP24-173A8 1 Mecp2 Fosmid clone WI1-894A5 or WI1-1269o10 Pgk1 BAC RP23-404E5 Xist — 2

*Obtained from the BACPAC Resources Center.

1. Patrat C, et al. (2009) Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice. Proc Natl Acad Sci USA 106(13):5198–5203. 2. Shin J, et al. (2010) Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice. Nature 467(7318):977–981.

Other Supporting Information Files

Dataset S1 (XLS)

Bhatnagar et al. www.pnas.org/cgi/content/short/1413620111 11 of 11