Supplemental Figure 1: Genomic region targeted in lemursSchematic 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 A3n7

Probe B17n18

Probe C19n20

Probe D13n14

Supplemental Figure 2: The human XIC region is co-linear with both lemur XIC regionsA 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.

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Supplemental Figure 3: Repeat content and alignment across the XICEach 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

Supplemental Figure 4: Ancestrally reconstructed XIC confidence plotsConfidence 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.

Supplemental Figure 5: Ancestrally reconstructed XIST confidence plotConfidence 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.

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Supplemental Figure 6: Alignment of XIST/Xist A repeatThe 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.

Supplemental Figure 7: Primate XIST D Repeat AlignmentThe 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.

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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.

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Supplemental Figure 9: Black and ringtailed lemurs have an inactive X chromosomea) 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.

Supplemental Table 1: XIST/Xist A Repeat Region Comparisons

Species Compared to Human Size % ID to Human (423bp)Chimpanzee 425bp 99.3%Orangutan 421bp 97.4%

Rhesus Macaque 422bp 96.2%Common Marmoset 420bp 93.5%Ringtailed Lemur 429bp 82.6%

Black Lemur 429bp 82.6%Ancestral Primate 443bp 90.7%

Mouse 421bp 57.1%Dog 431bp 52.4%Cow 469bp 62.6%

*Alignments were compared using Geneious V5.3.4

Supplemental Table 2: Comparison of XIST D Repeat Monomer Consensus Sequences

Species compared to Human Size (bp) %ID to Human (290bp) Blast2 prog usedChimpanzee 293 94.8 blastnOrangutan 288 94.5 blastn

Rhesus Macaque 289 97.2 blastnMarmoset 290 98.3 blastn

Ringtailed Lemur 284 87.6 blastnBlack Lemur absent n/a n/a

Ancestral Primate 185 94.6 blastnConsensus Sequences Generated by Tandem Repeats Finder (TRF) v 4.03

Supplemental Table 3: Lemur Cell Line Information

Common Name Species Source Catalog Number Sex

Black lemur Eulemur macaco macaco IPBIR PR00254 Female

Black lemur Eulemur macaco macaco IPBIR PR00266 Male

Ringtailed lemur Lemur catta Coriell AG07100 Female

Ringtailed lemur Lemur catta IPBIR PR00119 Male

Coquerel's Sifaka Propithecus vereauxi coquereli IPBIR PR00227 Female

Coquerel's Sifaka Propithecus verreauxi coquereli IPBIR PR00302 Male

Gray Mouse lemur Microcebus murinus IPBIR PR00275 Female

Aye-aye Daubentonia madagascariensis IPBIR PR1134 Female

Aye-aye Daubentonia madagascariensis IPBIR PR1017 Male

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)