Probe A 3 n 7 Probe B 17
Probe A 3 n 7 Probe B 17 n 18 Probe C 19 n 20 Probe D 13 n 14 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 CDX 4, CHIC 1 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).
Human a) Black lemur Human b) Ringtailed lemur 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 c. DNA 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 colinear. Black lines connecting the sequences correspond to regions of sequence similarity.
See attached File: Supp. Fig 3. pdf Supplemental Figure 3: Repeat content and alignment across the XIC Each page shows a 50 kb 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 Repeat. Masker, 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.
Indel_HCOR 1525000 1485000 1445000 1405000 1365000 1325000 1285000 1245000 1205000 1165000 1125000 1085000 1045000 1005000 965000 925000 885000 845000 805000 765000 725000 685000 645000 605000 565000 525000 485000 445000 405000 365000 325000 285000 245000 205000 165000 125000 85000 45000 Indel_Primate Indel_HCOR 1525000 1485000 1445000 Primate 1405000 1365000 1325000 1285000 1245000 1205000 1165000 1125000 1085000 1045000 1005000 965000 925000 885000 845000 805000 765000 725000 685000 645000 605000 565000 525000 485000 445000 405000 365000 325000 285000 245000 205000 165000 125000 85000 45000 Indel_Primate 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 2 D 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.
Indel_HCOR Indel_Primate 64500 66500 68500 70500 2500 4500 6500 8500 10500 12500 14500 16500 18500 20500 22500 24500 26500 28500 30500 32500 34500 36500 38500 40500 42500 44500 46500 48500 50500 52500 54500 56500 58500 60500 62500 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 2 D 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.
Consensus Human Chimpanzee Orangutan Macaque Marmoset Ringtailed Lemur Black Lemur Ancestral Primate Mouse Dog Cow Supplemental Figure 6: Alignment of XIST/Xist A repeat The human sequence corresponding to the A repeat [(hg 19) chr. X: 73071816 -73072238] was submitted to the program Tandem Repeats Finder V 4. 03 to identify A repeat monomers. Annotations using blue blocks were made in Geneious V 5. 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.
Consensus Human Chimpanzee Orangutan Macaque Marmoset Ringtailed Lemur Black Lemur Ancestral Primate Mouse Dog Cow Supplemental Figure 7: Primate XIST D Repeat Alignment The human sequence corresponding to (hg 19) chr. X: 73063470 -73066838 was submitted to the program Tandem Repeats Finder to identify D repeat monomers. Annotations of purple blocks were made in Geneious V 5. 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.
Human Genomic Black Lemur BACCH 16 H 20 Black Lemur Genomic, PR 00254 Ringtailed Lemur BAC LB 223 D 18 Ringtailed Genomic, AG 7100 Bioline Hyperladder II Bioline Hyperladder I Negative control Black Lemur BAC CH 16 H 20 Black Lemur Genomic, PR 00254 Human BAC R 11 -216 E 22 Human Genomic 10 kb 6 kb 3 kb 2 kb 1 kb 0. 8 kb 0. 2 kb 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 JH 617 and JH 619 designed to regions flanking the human D repeat sequence in XIST exon 1. The human product was expected to be 3. 3 kb. The human XIST containing BAC (216 E 22) amplifies a robust product at just over 3 kb with a slight ladder of products below, while the black lemur XIST BAC (16 H 20) amplifies a product around 200 bp. To the right of the Hyperladders, a PCR assay was set up using primers designed to conserved sequences in both lemurs (JH 616 and JH 619). The ringtailed lemur XIST BAC (223 D 18) produces a ladder while the black lemur genomic and black lemur BAC both produce a 200 bp product indicating the absence of the intervening D repeat sequence in the black lemur.
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 (CH 273 -16 H 20) and c) immunostained with an antibody specific to H 3 K 4 me 2. d) Female black lemur image above e) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific black lemur BAC (CH 273 -16 H 20) and f) immunostained with an antibody specific to H 3 K 4 me 2. g) Male ringtailed lemur image above h) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific ringtailed lemur BAC (LB 2 -223 D 18) and i) immunostained with an antibody specific to H 3 K 4 me 2. j) Female ringtailed lemur image above k) DAPI stained metaphase chromosomes hybridized with an X chromosome-specific ringtailed lemur BAC (LB 2 -223 D 18) and l) immunostained with an antibody specific to H 3 K 4 me 2. These experiments indicate that female black and ringtailed lemurs have one X chromosome devoid of H 3 K 4 me 2 staining, suggestive of an inactive X chromosome.
Supplemental Table 1: XIST/Xist A Repeat Region Comparisons Species Compared to Human Chimpanzee Orangutan Rhesus Macaque Common Marmoset Ringtailed Lemur Black Lemur Ancestral Primate Mouse Dog Cow *Alignments were compared using Geneious V 5. 3. 4 Size 425 bp 421 bp 422 bp 420 bp 429 bp 443 bp 421 bp 431 bp 469 bp % ID to Human (423 bp) 99. 3% 97. 4% 96. 2% 93. 5% 82. 6% 90. 7% 57. 1% 52. 4% 62. 6%
Supplemental Table 2: Comparison of XIST D Repeat Monomer Consensus Sequences Species compared to Human Size (bp) %ID to Human (290 bp) Blast 2 Chimpanzee 293 94. 8 Orangutan 288 94. 5 Rhesus Macaque 289 97. 2 Marmoset 290 98. 3 Ringtailed Lemur 284 87. 6 Black Lemur absent n/a Ancestral Primate 185 94. 6 Consensus Sequences Generated by Tandem Repeats Finder (TRF) v 4. 03 prog used blastn blastn n/a blastn
Supplemental Table 3: Lemur Cell Line Information Common Name Species Source Catalog Number Sex Black lemur Eulemur macaco IPBIR PR 00254 Female Black lemur Eulemur macaco IPBIR PR 00266 Male Ringtailed lemur Lemur catta Coriell AG 07100 Female Ringtailed lemur Lemur catta IPBIR PR 00119 Male Coquerel's Sifaka Propithecus vereauxi coquereli IPBIR PR 00227 Female Coquerel's Sifaka Propithecus verreauxi coquereli IPBIR PR 00302 Male Gray Mouse lemur Microcebus murinus IPBIR PR 00275 Female Aye-aye Daubentonia madagascariensis IPBIR PR 1134 Female Aye-aye Daubentonia madagascariensis IPBIR PR 1017 Male Coriell Cell Repositories (http: //locus. umdnj. edu/) IPBIR is the Integrated Primate Biomaterials and Information Resource ( http: //ccr. coriell. org/Sections/Collections/IPBIR/? Ss. Id=18 )
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