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1 Supporting Information hatnagar et al /pnas SI Materials and Methods Single-Nucleotide Primer Extension ssay. single-nucleotide primer extension (SNuPE) assay for was carried out using a TaqMan SNP genotyping assay (pplied iosystems) according to the manufacturer s specifications. The following primers and reporters were used for the assay: 5 -GGTT- GTGTTT-3, 5 -GTGTTTGT- 3, 5 -TGTTGG-3, and 5 -TGT- TGG-3. The data are plotted as the function of ΔRn for each sample, which represents the reporter fluorescence for each allele (VI/FM) normalized to the passive reference dye. Imprinted Gene nalysis. Mouse embryonic fibroblasts (MEFs) from strain 57L6 (ST 7), provided by M. artolomei (University of Pennsylvania, Philadelphia), were cultured in DMEM supplemented with 1% (vol/vol) FS and 1% (vol/vol) nonessential amino acids. nalysis of imprinted genes was performed using MEFs isolated from the 57L/6 (ST7) strain, which contains chromosome 7 from the Mus castaneus (ast) strain in a 57L/6 background, as previously described (1). riefly, total RN was extracted and cdn synthesis was carried out as described above. For PR amplification, the cdn was added to Ready-To-Go PR eads (GE Life Sciences) together with.3 μm gene-specific primers (Table S2). Expression of the imprinted gene was analyzed by allele-specific restriction enzyme digestion (StcI for scl2, StuI for Kcnq1ot1, MnlI for Peg3, and FauI for Zim1), and digested PR products were resolved by polyacrylamide gel electrophoresis. 1. Rivera RM, et al. (28) 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. HT selection (H4SV cells) cvr1 urka 171P1Rik HT selection (H4SV cells; 2nd shrn) cvr1 urka 171P1Rik Knockdown efficiency.3.1 shrn: D shrn: Knockdown efficiency cvr1 cvr1 urka urka qrt-pr (H4SV cells) qrt-pr (H4SV cells; 2nd shrn) 171 P1Rik 171 P1Rik Fig. S1. shrns targeting an X-chromosome inactivation factor (XIF) reactivate the inactive X chromosome (Xi)-linked Hprt gene and decrease mrn levels of the targeted gene. () right-field images showing growth of the 13 XIF KD H4SV cell lines following selection in hypoxanthine aminopterin thymidine (HT) medium. () Quantitative real-time RT-PR (qrt-pr) analysis monitoring target gene expression in the 13 XIF KD H4SV cell lines expressing the shrn 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 () shrn, which was set to 1. Error bars indicate SD. () right-field images showing growth of the 13 XIF KD H4SV cell lines, expressing a second, unrelated shrn to that shown in, following selection in HT medium. (D) qrt-pr analysis monitoring target gene expression in the 13 XIF KD H4SV cell lines expressing a second, unrelated shrn to that shown in. Error bars indicate SD. hatnagar et al. 1of11

2 RN FISH (MSL2 cells; 2nd shrn) cvr1 urka Lamp2 171P1Rik Lamp2 cvr1 urka 171P1Rik Relative expression Relative expression shrn: Rn (normalized to non-template control) cvr1 urka Hprt SNuPE assay (MSL2 cells) cvr1 qrt-pr (MSL2 cells) P1Rik Relative expression Relative expression shrn: Rn b (Xa) Rn a (Xi) D 1 % nuclei shrn: cvr1 urka X chromosome painting (MSL2 cells) cvr1 urka 171 P1Rik 171 P1Rik 2 signals 1 signal >2 signals Fig. S2. dditional RN FISH images and control experiments related to Fig. 1. () Representative two-color RN FISH images showing expression of (red) and Lamp2 (green; Upper) and (red) and (green; Lower) in each of the 13 XIF KD MSL2 cell lines. DPI staining is shown in blue. () qrt- PR analysis monitoring expression of Hprt,,, and in each of the 13 XIF KD MSL2 cell lines. The results were normalized to that obtained with the shrn, which was set to 1 (red line). twofold increase in expression is indicated by the blue line. () In MSL2 cells, the Xi and Xa encode two distinguishable alleles, a and b, respectively, which differ by a single-nucleotide polymorphism within the mrn. llele-specific expression of the Xi- and Xa-linked genes in six representative XIF KD MSL2 cell lines was analyzed using a SNuPE assay. The data are plotted as the function of ΔRn Legend continued on following page hatnagar et al. 2of11

3 for each sample, which represents the reporter fluorescence for each allele (VI/FM) normalized to the passive dye. The results show that, in each of the six XIF KD MSL2 cell lines, the Xi-linked a gene was reactivated. (D) X-chromosome painting experiments in the 13 XIF KD MSL2 cell lines. The results show that the X-chromosome content of the XIF KD MSL2 cell lines was similar to that of the control MSL2 cell line expressing a shrn. Thus, the substantially increased biallelic expression of X-linked genes observed by RN FISH in the XIF KD cell lines cannot be explained by differences in X-chromosome number. hatnagar et al. 3of11

4 RN FISH (Differentiated mouse ES cells) cvr1 urka Lamp2 171P1Rik Lamp2 cvr1 urka 171P1Rik % nuclei shrn: X chromosome painting (Differentiated ES cells) cvr1 urka 171 P1Rik 2 signals 1 signal >2 signals shrn: Fold increase in Eomes expression Fold increase in Tcf7l2 expression Fold increase in dx2 expression (Undiff ES) cvr1 urka 171 P1Rik Fig. S3. dditional RN FISH images and control experiments related to Fig. 2. () Representative two-color RN FISH images monitoring expression of (green) and Lamp2 (red; Upper) and (green) (red; Lower) in the 13 XIF KD ES cell lines following differentiation. DPI staining is shown in blue. () X-chromosome painting experiments in the 13 XIF KD ES cell lines following differentiation. () qrt-pr analysis monitoring expression of Eomes, Tcf7l2, and dx2 in the 13 XIF KD ES cell lines following treatment with R. s a control, expression of each gene in undifferentiated ES cells is shown and was set to 1. Error bars indicate SD. hatnagar et al. 4of11

5 RN FISH cvr1 urka 171P1Rik expression 1..8 shrn: -2 qrt-pr (H4SV cells) Tsix expression shrn: -2 expression 1..8 shrn: -1-2 expression 1..8 qrt-pr (Differentiated mouse ES cells) -1-2 Tsix expression expression 1..8 shrn: shrn: shrn: -1-2 Fig. S4. RN FISH images and control experiments related to Fig. 3. () RN FISH images. In each of the 13 XIF KD ES cell lines following differentiation, the majority of cells that lost the typical localization pattern lacked a detectable signal (Fig. 3). However, some cells that had lost the typical localization pattern contained two small 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; DPI staining is shown in blue. () qrt-pr analysis monitoring expression of (Left), Tsix (enter), and (Right) in H4SV cells expressing a or one of two shrns (-1 or -2). For and Tsix expression, a second, unrelated shrn to that used in Fig. 3H. Expression was normalized to that obtained with the control shrn, which was set to 1. Error bars indicate SD. () qrt-pr analysis monitoring expression of (Left), Tsix (enter), and (Right) in differentiated ES cells expressing a shrn or one of two shrns (-1 or -2). Expression was normalized to that obtained with the control shrn, which was set to 1. Error bars indicate SD. hatnagar et al. 5of11

6 ontrol OSU-312 LY2942 ontrol GNE-317 ontrol OSU-312 fter OSU-312 removal LY2942 fter LY2942 removal Fig. S5. dditional RN FISH images related to Fig. 4. ( and ) Two-color RN FISH monitoring expression of (red) and (green) in differentiated ES cells treated with DMSO (control), OSU-312 (4 μm), or LY2942 (1 μm) (), and in MSL2 cells treated with DMSO or GNE-317 (5 μm) (). The yellow boxes indicate cells with colocalizing and signals; the white boxes indicate cells with biallelic expression of and complete loss of the signal. () Two-color RN FISH monitoring (red) and (green) expression in MSL2 cells treated with DMSO (control), OSU-312 (2.5 μm), or LY2942 (8 μm), and at least 6 d following removal of the inhibitor. The white boxes indicate cells with biallelic expression of. hatnagar et al. 6of11

7 % nuclei X chromosome painting (Female MEFs) 2 signals 1 signal >2 signals 2 +/+ -/- RN FISH (Mouse brain sections) +/+ +/+ -/- -/- Fig. S6. ontrol experiment and RN FISH images related to Fig. 5. () X-chromosome painting experiments in female +/+ and / MEFs. The results show that the X-chromosome content of / MEFs was similar to that of +/+ MEFs. Thus, the substantially increased biallelic expression of X-linked genes observed by RN FISH in the / MEFs cannot be explained by differences in X-chromosome number. () Defective XI in cortical neurons from brain sections of female / mice. Two-color RN FISH monitoring expression of (red) and or (green) in cortical neurons from adjacent 5-μm brain sections of female / and +/+ mice (n = 3 per genotype, stage P1). The boxed regions denote cells with two or signals; the yellow boxes indicate cells with colocalizing and / signals. ll cells in the regions shown represent neurons that, based on anatomical landmarks, are present in posthybridized sections. hatnagar et al. 7of11

8 log1 P-value log2 KO:WT X linked gene expression ratio Kcnq1ot1 utosomeal gene expression (log2 transformed FPKM) Peg hromosome scl2 Zim1 WT KO Undig. 814 bp Undig. 487 bp Undig. 474 bp Undig. 49 bp StuI dig. shrn: D Fold increase in Hprt expression shrn: qrt-pr Ezh2 mi cvr1 urka 171P1Rik 1..8 shrn: Ezh2 knockdown efficiency Ezh2 MnlI dig. 61 bp 213 bp qrt-pr 1..8 shrn: mi1 knockdown efficiency shrn: Gene Kcnq1ot1 Peg3 scl2 Zim1 mi1 cvr1 urka 171P1Rik Expressed allele Paternal Paternal Maternal Maternal Restriction enzyme StuI MnlI SfcI FauI 487 bp SfcI dig. Digested paternal (ast) allele (bp) 213, , , 254 Digested maternal (57L/6) allele (bp) , bp FauI dig. shrn: cvr1 urka 171P1Rik E cells (6 month old mice) Myeloid cells (2 month old mice) Myeloid cells (6 month old mice) % 4 1.6% % log2 HET:WT X-linked gene expression ratio shrn: X-linked gene cvr1 urka 171P1Rik X-linked gene X-linked gene Fig. S7. dditional experiments and data analyses related to Fig. 6 and Discussion.() Volcano plot showing distribution of log 2 -transformed ratio of X-linked gene expression in MEFs isolated from / (KO) and +/+ (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 <.1. The results show that the similarity of X-linked gene expression between female +/+ and / MEFs was statistically significant. () ox plots displaying changes in autosomal gene expression [log 2 -transformed fragments per kilobase of exon per million fragments mapped (FPKM)] in / and +/+ 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. () XIFs are not required for repression of imprinted genes. Primary female mouse embryonic fibroblasts from the strain 57L/6 (ST7), which contains chromosome 7 from Mus castaneus (ast), were transduced with shrns against each of the XIFs and analyzed for allele-specific expression of four genes located on chromosome 7 that are either paternally expressed (Kcnq1ot1 and Peg3) or maternally expressed (scl2 and Zim1). Expression of the two alleles can be distinguished by allele-specific restriction enzyme digestion following gene-specific RT-PR. 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 XIF 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 XIF KD cell lines, all four genes displayed only the expected allele-specific expression pattern, indicating that the XIFs are not required for repression of the imprinted genes. (D) Requirement of Polycomb subunits EZH2 and MI1 for repression of the X-linked Hprt gene. (Left) qrt-pr analysis monitoring Hprt expression in MSL2 cells expressing an Ezh2 or mi1 shrn or, as a control, a shrn. (Right) qrt-pr analysis confirming target gene knockdown in mouse ES cells expressing an Ezh2 (Left) or mi1 (Right) shrn. Error bars indicate SD. (E) nalysis of available datasets from Yildirim et al. (1) showing the distribution of log 2 -transformed ratio of X-linked gene expression in hematopoietic cells from female heterozygous (HET) mutant mice and wild-type (WT) mice. The data were downloaded from Gene Expression Omnibus (GSE43961), normalized by RM, and filtered by detection above background (DG) (cutoff P value of <.1) using ioconductor package xps. The percentage of X-linked genes upregulated >1.5-fold is shown bp 1. Yildirim E, et al. (213) RN is a potent suppressor of hematologic cancer in mice. ell 152(4): hatnagar et al. 8of11

9 Table S1. Summary of the 13 XIFs Mouse gene symbol Human gene symbol Gene name hromosome, mouse (human) iological process cvr1 VR1 ctivin receptor, type 1 2 (2) Signal transduction urka URK urora kinase 2 (2) ell cycle regulation DNMT1 DN methyltransferase (cytosine-5) 1 9 (19) hromatin modification FXO8 F-box protein 8 8 (4) Unknown/ubiquitin-dependent protein catabolic process LYN Layilin 9 (11) Unknown/receptor for hyaluronic acid NF1 Neurofibromatosis 1 11 (17) Signal transduction PDPK1 3-Phosphoinositide dependent 17 (16) Signal transduction protein kinase 1 PYGO1 Pygopus 1 9 (15) Transcriptional regulation RNF165 Ring finger protein (18) Unknown SOX5 SRY-box containing gene 5 6 (12) Transcriptional regulation ST1 Stanniocalcin 1 14 (8) ell metabolism ZNF426 Zinc finger protein (19) Transcriptional regulation 171P1Rik 17orf98 RIKEN cdn 171P1 gene 11 (17) Unknown hatnagar et al. 9of11

10 Table S2. List of primers used for qrt-pr and RT-PR analysis, cdn synthesis, hip assays, and mouse genotyping Forward primer, 5 3 Reverse primer(s), 5 3 qrt-pr ctin TTGGGGTGG GGTGGGTTT cvr1 (mouse) GGGGTGTTTTTTT TTTGTTTG VR1 (human) TGGGTGGTTGGTT GTTTTGTGT urka (mouse) TGGTTGTTGTTTT TTGGGTGT URK (human) TGGTTGTTGG TGTTG mi1 TGGGGGTTGTT GTTTTTTTG dx2 GGTGGGG GTGTGTTGTGTGTGTT (mouse) GGGGTTGGTGTG TGGGGTGTTGTG DNMT1 (human) GTGGGGGTGTGTTTGT TGGTGTGTT Eomes TGGTGGTGTTTTGTTGTG TTTTGGGGT Ezh2 TTTGGTTTTGTTTT TTTTTGTTT (mouse) GTGGTTTTTTTG TGTGGTTTTGGT FXO8 (human) GGGTTGTGGGGTGGT TGTTGTTTGG Gapdh TGGTTGTGTTT TGGGTTTTGTG TGGTTT TGGGGTTT Hprt GTTGTGGTGGG TTGGTTTTGGTTT (mouse) GGGGGTGGTGGGT TTGTGTGTGTGTTG LYN (human) TGGGTGTGTG TGTGTGGTT TGGTGTGGGTGTTGG GTTTTTTTGGG (mouse) GTGGGTTTGT TGGGTGGGTG NF1 (human) TTTGTTGGGGTTTT GTGTTTTTGGGTG Oct4 TTGGGGTTTT GGTGGGG (mouse) GGTGTGGTGG TTTTGTTGTGG PDPK1 (human) GTTTGTGGTTTT GGGGGGTG TGTGTTTGTG GTTTGTGGT (mouse) TTGTGGGGG TTTTGGGTTGGTTG PYGO1 (human) TTGGTTTGGGGTT GTGGGTTG (mouse) TGTGTGT GTGTTGGG RNF165 (human) GGGGGTGGGGG GTTGGTTTGTGT (mouse) GTGGGGGGGGGTGG TTTGGTGGGTTG SOX5 (human) GGGTGGGTTG TTGTTTTGTTGTGTTGG (mouse) GTTGGT GGGTTGGGT ST1 (human) TGTGTGTTTG TGGTGGGTTTTG Tcf7l2 GTTGTT TGGTTTTG Tsix TTGGTGGTG (TSIX2F) TGTGGTGGTTTGG (P422R) (nonstrand specific) TGTGTTTTG GGTTGGGGG (strand specific) GTGGGTTG (XIST2281F) GGTGTTT (XIST2424R) XIST (human) GTGTGTGTTTGTGT TTTGGGTTTTGTGTTTG (mouse) TGTTTGTTGG GGGTTTGTTTGTG ZNF426 (human) TGGGTGGGTGGTTT TTGTTTGGGTTG 171P1Rik (mouse) GTGTGTTGTTT GGTTTT 1orf98 (human) TGGGGGGT GTGGTTGGGG RT-PR (first round) GTTGTGGGGG TGGGGTTGGGTTGGGT (second round) GGGGGGGTG GTTGGGTGGTGGGGT scl2 TGGTT TGGTGTTG Kncq1ot1 TTGGGTTGGGGTGGGG GGGGTTGGGTTG Peg3 TGTGTGG GTTTTGTGTTTG Zim1 TTGGGG GTGGGGGTTTT cdn synthesis GGTTTTGGT (XIST2688R) Tsix GTGGGTTG (TSIX2R) Gapdh TGTGGGGGTGTGTG (GPDR) hip (promoter) TGGTTGTGTG GGGGGGG (exon 2) GTGTTGTGG GTTTTTTTG Mouse genotyping +/+ TTGGGTGGTTTGGGGT GGG GTTGTGTGTTGG / GGGTTTGTGGGG GGGGTTGTGTGTTGG +/+ GGGGGGG GGGGTGTGGGGT / GGGGTGG TTGTGGTTTG SRY TTGTTGGGTGGGGGTGT TTTGTGTTTGTG hatnagar et al. 1 of 11

11 Table S3. Oligo ID numbers for shrns obtained from Open iosystems/thermo Scientific Gene Oligo ID cvr1 urka mi1 Ezh2 171P1Rik V2MM_75565 V2MM_76215 V2MM_1885 V2MM_7199 V2MM_1594 V2MM_234 V2MM_46797 V2LMM_4317 V2MM_35988 V2MM_3422 V2MM_36526 V3LMM_49467 V2MM_13482 V2MM_21485 V2MM_19418 V2HS_7627 V2MM_75859 V2MM_72465 V2MM_1161 V2MM_1169 V2MM_ TRN V2MM_6385 V2HS_94936 V2MM_22454 V2MM_26886 TRN19921 V2MM_31994 TRN8516 V2MM_1177 V2MM_25788 Table S4. cdns used to prepare RN FISH probes Gene lone no.* Ref. clone RP23-13D21 1 Lamp2 clone RP Fosmid clone WI or WI1-1269o1 RP23-44E5 2 *Obtained from the P Resources enter. 1. Patrat, et al. (29) Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice. Proc Natl cad Sci US 16(13): Shin J, et al. (21) Maternal Rnf12/RLIM is required for imprinted X-chromosome inactivation in mice. Nature 467(7318): Other Supporting Information Files Dataset S1 (XLS) hatnagar et al of 11