1. Supplemental Figure 1: Genomic region targeted in lemurs Schematic of the candidate human X inactivation center region is shown below the chromosome ideogram. Four PCR product probes were designed across this region within the CDX4, CHIC1 and XIST genes. Approximate location of the four probes used to screen both lemur BAC libraries is shown below the schematic. Numbers under probe pairs correspond to the primers used to generate the probes (see Supplemental Table 4). Probe A 3n7 Probe B 17n18 Probe C 19n20 Probe D 13n14

2. Supplemental Figure 2: The human XIC region is co-linear with both lemur XIC regions A miropeats analysis (Parsons, J.D., Brenner, S. and Bishop, M.J. 1992. Clustering cDNA Sequences, Comput. Applic. Biosci., 8, 461-466) comparing the repeatmasked human XIC sequence to the repeatmasked a) black lemur and b) ringtailed lemur XIC sequences, indicate that these sequences are co- linear. Black lines connecting the sequences correspond to regions of sequence similarity. Human Black lemur Ringtailed lemur a) b)

3. Supplemental Figure 3: Repeat content and alignment across the XIC Each page shows a 50kb window across the XIC region, enlarged from Figure 2. Colored bars representing sequence for each species are shown on a separate horizontal line. The red bars below the sequence indicate repetitive regions identified by RepeatMasker, which are color-coded depending on the repeat identified. Three ancestrally reconstructed sequences (H+C+O, C+C+O+R and Primate Ancestor) are indicated for comparison of which regions have been gained or lost throughout primate evolution. Below the aligned sequences are the exonic regions annotated based on the human and mouse gene structures. See attached File: SuppFig3.pdf

4. Supplemental Figure 4: Ancestrally reconstructed XIC confidence plots Confidence level for the a) structure and b) structure and predicted sequence for each pair of ancestral sequences. This representation indicates surface variation in 2D from one position into another and one ancestor into another. If the two ancestors have the same value for an interval, the color surface of that interval will be homogeneous and will correspond to the color labeled. If the two have different values, then a proportion will be computed for how far the two values are and each ancestor will have a colored portion. See Diallo, A.B., Makarenkov, V., and Blanchette, M. 2010. Ancestors 1.0: a web server for ancestral sequence reconstruction. Bioinformatics 26: 130-131.

5. Supplemental Figure 5: Ancestrally reconstructed XIST confidence plot Confidence level for the structure and predicted sequence for each pair of ancestral sequences across the XIST locus. This representation indicates surface variation in 2D from one position into another and one ancestor into another. If the two ancestors have the same value for an interval, the color surface of that interval will be homogeneous and will correspond to the color labeled. If the two have different values, then a proportion will be computed for how far the two values are and each ancestor will have a colored portion.

6. Mouse Black Lemur Ringtailed Lemur Macaque Marmoset Human Chimpanzee Orangutan Cow Dog Ancestral Primate Mouse Black Lemur Ringtailed Lemur Macaque Marmoset Human Chimpanzee Orangutan Cow Dog Ancestral Primate Mouse Black Lemur Ringtailed Lemur Macaque Marmoset Human Chimpanzee Orangutan Cow Dog Ancestral Primate Consensus Supplemental Figure 6: Alignment of XIST/Xist A repeat The human sequence corresponding to the A repeat [(hg19) chrX:73071816-73072238] was submitted to the program Tandem Repeats Finder V4.03 to identify A repeat monomers. Annotations using blue blocks were made in Geneious V5.3.4 (Biomatters Ltd) based on the human tandem repeat monomers identified within the larger XIST/Xist alignment. The orthologous regions in all other species are shown below.

7. Supplemental Figure 7: Primate XIST D Repeat Alignment The human sequence corresponding to (hg19) chrX:73063470-73066838 was submitted to the program Tandem Repeats Finder to identify D repeat monomers. Annotations of purple blocks were made in Geneious V5.3.4 (Biomatters, Ltd) based on the human tandem repeat monomers identified within the larger XIST/Xist alignment. The orthologous regions in all other species are shown below. The mouse, dog and cow D repeat regions have little sequence orthology to the primate D repeat regions and are therefore not shown in this short alignment window. Mouse Black Lemur Ringtailed Lemur Macaque Marmoset Human Chimpanzee Orangutan Cow Dog Ancestral Primate Consensus Mouse Black Lemur Ringtailed Lemur Macaque Marmoset Human Chimpanzee Orangutan Cow Dog Ancestral Primate Consensus Mouse Black Lemur Ringtailed Lemur Macaque Marmoset Human Chimpanzee Orangutan Cow Dog Ancestral Primate Consensus

8. Supplemental Figure 8: The XIST D repeat is absent from the black lemur genome. Primers were designed flanking the D repeat in XIST exon 1. To the left of the Hyperladders, a PCR assay was set up using primers JH617 and JH619 designed to regions flanking the human D repeat sequence in XIST exon 1. The human product was expected to be 3.3kb. The human XIST containing BAC (216E22) amplifies a robust product at just over 3kb with a slight ladder of products below, while the black lemur XIST BAC (16H20) amplifies a product around 200bp. To the right of the Hyperladders, a PCR assay was set up using primers designed to conserved sequences in both lemurs (JH616 and JH619). The ringtailed lemur XIST BAC (223D18) produces a ladder while the black lemur genomic and black lemur BAC both produce a 200bp product indicating the absence of the intervening D repeat sequence in the black lemur. Human GenomicHuman BAC R11-216E22Black Lemur Genomic, PR00254Black Lemur BAC CH16H20Negative controlBioline Hyperladder IBioline Hyperladder IIRingtailed Genomic, AG7100Ringtailed Lemur BAC LB223D18Human GenomicBlack Lemur Genomic, PR00254Black Lemur BACCH16H20 10kb 3kb 1kb 0.2kb 2kb 6kb 0.8kb

9. Supplemental Figure 9: Black and ringtailed lemurs have an inactive X chromosome a) Male black lemur image above b) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific black lemur BAC (CH273-16H20) and c) immunostained with an antibody specific to H3K4me2. d) Female black lemur image above e) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific black lemur BAC (CH273-16H20) and f) immunostained with an antibody specific to H3K4me2. g) Male ringtailed lemur image above h) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific ringtailed lemur BAC (LB2-223D18) and i) immunostained with an antibody specific to H3K4me2. j) Female ringtailed lemur image above k) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific ringtailed lemur BAC (LB2-223D18) and l) immunostained with an antibody specific to H3K4me2. These experiments indicate that female black and ringtailed lemurs have one X chromosome devoid of H3K4me2 staining, suggestive of an inactive X chromosome.

10. Supplemental Table 1: XIST/Xist A Repeat Region Comparisons Species Compared to HumanSize% ID to Human (423bp) Chimpanzee425bp99.3% Orangutan421bp97.4% Rhesus Macaque422bp96.2% Common Marmoset420bp93.5% Ringtailed Lemur429bp82.6% Black Lemur429bp82.6% Ancestral Primate443bp90.7% Mouse421bp57.1% Dog431bp52.4% Cow469bp62.6% *Alignments were compared using Geneious V5.3.4

11. Supplemental Table 2: Comparison of XIST D Repeat Monomer Consensus Sequences Species compared to HumanSize (bp)%ID to Human (290bp)Blast2 prog used Chimpanzee29394.8blastn Orangutan28894.5blastn Rhesus Macaque28997.2blastn Marmoset29098.3blastn Ringtailed Lemur28487.6blastn Black Lemurabsentn/a Ancestral Primate18594.6blastn Consensus Sequences Generated by Tandem Repeats Finder (TRF) v 4.03

12. Supplemental Table 3: Lemur Cell Line Information Common NameSpeciesSourceCatalog NumberSex Black lemurEulemur macaco macacoIPBIRPR00254Female Black lemurEulemur macaco macacoIPBIRPR00266Male Ringtailed lemurLemur cattaCoriellAG07100Female Ringtailed lemurLemur cattaIPBIRPR00119Male Coquerel's SifakaPropithecus vereauxi coquereliIPBIRPR00227Female Coquerel's SifakaPropithecus verreauxi coquereliIPBIRPR00302Male Gray Mouse lemurMicrocebus murinusIPBIRPR00275Female Aye-ayeDaubentonia madagascariensisIPBIRPR1134Female Aye-ayeDaubentonia madagascariensisIPBIRPR1017Male Coriell Cell Repositories (http://locus.umdnj.edu/) IPBIR is the Integrated Primate Biomaterials and Information Resource (http://ccr.coriell.org/Sections/Collections/IPBIR/?SsId=18)