Supplementary Tables. Primers and probes. Target Primer / probe sequences Chemistry Human HBB F: AACTGTGTTCACTAGCAACCTCAAA

Transcription

1 Supplementary Tables Primers and probes Target Primer / probe sequences Chemistry H F: AACTGTGTTCACTAGCAACCTCAAA promoter R: ACAGGGCAGTAACGGCAGACT H Downstream Actb alpha (162) alpha (162.) (Pro) (16) (ex) FERDL 1 (222) FERDL 2 (218) FERDL (9) FERDL (91) Gata6 Hox7 HoxC Hbb Hbb ex1 P: CTCCTCAGGAGTCAGATGCACCATGGT F: GGCTGTTGCCAATGTGCAT R: TTCAAACCTTGGGAAAATACACTATATC P: TTTGCAGCCTCACCTTCTTTCATGGAGT F: CGGTGTGGGCATTTGATGA R: CGTCTGGTTCCCAATACTGTGTAC P: AAGATGGACCTAATACGGCTTTTAACACCCG F: AAGTCTTCCATCAGATAGCATTTGG R: GTCATGGACTCACAGTTTGTGTGA P: TGTCACAGTGAACCACGACCTCTAGGCC F: GGTGCGCGCATTCCTCTCCG R: CGACTCCAGCGGGATCGGGG P: CACTCGCTCGCCCGTGTGTT F: GGGCCGGCACTCTTCTG R: GGCCTTGACGTTGGTCTTGT P: CCCACAGACTCAGAGAGAACCCACCATG F: CCCACCCCTCACTCTGCTT R: TGCGGGAAGTAGGTCTTGGT P: TCCCCGCAGGATGTTCCTGTCC F: ACCTCCCCGCCGAGTTC R: AGGCTCCAGCTTAACGGTATTTG P: CAAGTTCCTGGCTTCTGTGAGCACCG F: GGAGAGGGCGTCTCGGGGAG R: TCCGGAGAGCTGCGTGGACA F: GGCACCTTCCTCCGCAGCTG R: GATCACCTACGCCCAGCGCC F: GAAGCTGATGCTGCACTTTAGATG R: ACGGACCACAGCTCCTTGTC P: AGGCACTGAATGAACACCACTGTGGC F: CAGTTACCCACTCCTTTCTGAAAGA R: GCTGAGAGGCCGCCTCTAGT P: AGAATCCTGGAATGGCACGGGTCC F: GGTTGGACGTGAGGTTTTGC R: GAAAGTGAGATGCCAGGAAAGG P: CATTGGTTTCCGGCACTATTTCCTAGATCC F: AGCTTCCTCGCACACCTTTTAC R: GGTCTTCTGAGCCTTGGAGTTG P: TTCCCCGAGGCAGGTGGGCT F: CTCTCAGCCTCAGGACCTGTCT R: GAGGTAGGCAGGATACCCAAGA P: CTGCTCATTCAGGAACCCTCTCTC F: GGTCATGTCATCACAGCTCCTTAT R: AATGGTCCTATGAGGGAAATATGG P: TGGGCAACAACATAACCTGCTAGGTTCC F: CCAATCTGCTCACACAGGATAGAG R: AGCAAATGTGAGGAGCAACTGA P: CCTTATATGCTCTGCCCTGGCTCCTG Syber green Syber green

2 Hbb alpha -1 alpha -26 alpha --12 promoter ex1 ex intermediate KcnJ bisulfite Olfr21 bisulfite Sorbs1 bisulfite Kank1 bisulfite Stox2 bisulfite Fat1 qpcr KcnJ qpcr Olfr21 qpcr F: TGTTAGAGGCCATCAACTATGTCTTTC R: AGCACCAAAAGGGATAGGTTAGAGA P: TTCCATCTGTCTCCCCACCTTCCCTC F: TTCTGACCTCACCTCAGCTAAGC R: TGTGTGGGCAGAGGACACA P: TCTTCCTCCTCTGAGAATCCGCCATG F: GTACAGAGTCAGAAAGGAAAGGACAA R: GGCAAATAAACAGGATGGGAAA P: TGGTACCACTGATTAGGACCTCTGACGCTG F: AACCCTGACTCAAAACAACAAAGTAA R: GGTTTCTGAGTTTCCTTATCTGCAA P: ACCATCATCGCCCAGAGCAGAAGG F: TGACCAAGGTAGGAGGATACTAACTTCT R: TTGCCCGGACACACTTCTTAC P: CCCAAACTGCCATCACTGGAGACGTAGT F: TGGAGGGCATATAAGTGCTACTTG R: TGCTTTTGTCTTCCCCAGAGA P: TGCAGGTCCAAGACACTTCTGATTCTGACA F: AGGCATCAGGGTGTCCACTTT R: CACAGAGGCAAGGAATTTATCCA P: TTCACCCCCGCGGTGCATG F: GCTTCCACTCTGGTCATTCTCTAGAC R: GGTGACTGCCTTGCAAATAGG P: CAGGCCTAACCCATCTGGATCTGAGCAG F1: CACCACCACCATCATCAGGAAG R1: TAAGTTGGGAGGCAAACGGG F2: GGGATGGTTTGGAGAACTGCTAC R2: CTGGTATGAACGCCTCACTTCC F1: TTATGTTTGTATTGGGGG R1: TCTCTCATTTTCTCTATC F2: GTAGTATGTGGTGGTAGG R2: CTTCCATTCCTCTCTTTC F1: TGAGCATTCCCGCCTTTACG R1: ATGGTCCCTACCCACAACCTTG F2: CATTGTGCTAAGTGCCTGTTGGTC R2: GAAACGCAAATACCCTTGACTCC F1: TAAGAAGAGAAAAGGTAG R1: CTAAACTCTAATACCACC F2: AGTTGAAAAGTTAGAGGG R2: CTCTCCATCTCTTCCTCC F1: AGTAGTTTTTTTTGGGGG R1: ACTAACCAATTTCTCTAC F2: ATTTTTTTTATTGTGGTG R2: CTCTACCCAATTTCCTCC F: GCCTCCGACGGCAGGTTCAC R: TGCGGAGTAGGCGTCTTGGGT F: GTTGCGGCGTTTCCGCCTG R: GGTCATGCACGGACACAACCGA F: ACCAGCGAGGAGGTGGACAGG R: CCGTAGGCGTAGATGCCGCG Nested PCR Nested PCR Nested PCR Nested PCR Nested PCR Syber green Syber green Syber green Table S1: Primers and probes employed in this study

3 Supplementary figures Figure S1 Chromatin state at the tested DNA fragments in human ES cells and unmodified H ChIP (A-) Comparison of chromatin state at human and mouse α globin loci in ES cells (Data obtained from Ernst et al. 211 and Ku et al. 28). The location of duplicated homology blocs is indicated. α globin genes are highlighted (red). Data is displayed as mapped reads per 1 million. (C-D) High resolution view of chromatin state at human and mouse α globin genes. (E) Chromatin state at the DNA fragment encompassing the human FERDL gene in human ES cells. (F) Chromatin state at the DNA fragment encompassing the human H gene in human ES cells. (G-I) ChIP was performed with an antibody to unmodified H for cell lines for which HA2, FERDL and H fragments were inserted using the RMCE system. Data represents the results of at least two biological replicates (separately derived cell lines). qpcr amplicons are labelled as in Figure 2. Data are expressed percentage of input material as mean +/- S.D. Figure S2 ase composition of tested DNA fragments (A-) CpG density (A) and G/C content () in a sliding bp window were plotted for the sequence corresponding to the tested HA2 subfragments labelled as in Figure. (C-D) CpG density and G/C content plotted for the DNA test fragment including the human FERDL gene. (E-F) CpG density and G/C content plotted for the DNA test fragment including the human H gene. Note that the H fragment is displayed here in the orientation in which it appears in the RMCE system; this is on the opposite strand to its endogenous locus.

4 Figure S Comparison of recruitment at conserved versus nonconserved CpG islands in mouse and human Identical data to that presented in Figure is displayed here as scatter plots. (A) Peak CpG density in a bp window in the human and mouse genome was plotted for the corresponding human and mouse genomic regions illustrated in Figure. The dashed line indicates identity. () Peak density in a bp window was plotted for corresponding human and mouse genomic regions illustrated in Figure. Regions for which CpG density is conserved in the mouse are indicated in green and those for which it has been eroded are in red. Figure S Relationship of CpG island size and DNA methylation to chromatin state in human ES cells (A) Enrichment of was plotted against enrichment of HKme for all CpG islands in human ES cells (Ernst et al, 211). (-E) The relationship of CpG island size to chromatin state was examined for fully unmethylated (methylation <%) CpG islands in human ES cells. enrichment is plotted against HKme enrichment for CpG islands () <bp (C) -1bp (D) 1-2bp and (E) >2bp in size. Read density is displayed as reads per 1 million per bp window. To facilitate comparison, number of CGIs associated with low levels of both HKme and modifications (red box) are displayed on each plot in the following format: CGIs in red box / CGIs plotted = % in red box. Figure S Additional examples of PRC2 recruitment in ES cells and qpcr validation of selected peaks (A) Positive control region (Gata6 gene) () Negative control region (PTMA gene). Note the PTMA gene is expressed at a high level in pluripotent cells.

5 (C-D) Additional examples of de novo PRC2 recruitment at (C) Fat1 and (D) Ebf genes in cells. (E) ChIP qpcr validation of selected peaks. ChIP was performed with an antibody to in wild type and ES cells. Data are plotted as percentage of input material. Act and HS-26 are negative control points; Gata6 and Hox7 are positive controls. Figure S6 isulfite sequencing data at de novo sites of PRC2 recruitment in wild type and ES cell lines isulfite PCR amplicons were designed to overly de novo sites of PRC2 recruitment in ES cell lines. Locations are indicated in Figure 7. For each locus CpG methylation is compared in wild type (left) and mutant (right) cells. For each amplicon PCR products were cloned and multiple clones sequenced. Filled circles indicated methylated and open circles unmethylated CpG dinucleotides.

6 HA2 A HUMAN α globin locus MOUSE α globin locus HZ HM HA2 HA1 Theta Hbz Hba1 'theta Hba2 'theta Homology bloc 1 Homology bloc 2 Homology bloc 1 Homology bloc 2 Reads / 1M Reads / 1M Reads / 1M 1 HKme Reads / 1M Reads / 1M Reads / 1M 1 HKme C Reads / 1M Reads / 1M Reads / 1M 1 HKme D Reads / 1M Reads / 1M Reads / 1M 1 HKme Hba-a1 Hba-a2 kb kb FERDL (chr7: ) H (chr11: ) E Reads / 1M Reads / 1M Reads / 1M 1 HKme FERDL F Reads / 1M Reads / 1M Reads / 1M 1 H HKme.2kb 2.2kb %Input G H HA2 I H FERDL %Input %Input Fig S1

7 A HA2 HA2 Density CpG dinucleotides (%) Frag (i) Frag (ii) Frag (iii) Frag (iv) Content of G/C nucleotides (%) Frag (i) Frag (ii) Frag (iii) Frag (iv) kb Density of CpG dinucleotides in bp window G/C content in bp window kb C FERDL D FERDL Density CpG dinucleotides (%) Content of G/C nucleotides (%).2kb Density of CpG dinucleotides in bp window G/C content in bp window.2kb E H F H 8 Density CpG dinucleotides (%) Content of G/C nucleotides (%) kb Density of CpG dinucleotides in bp window G/C content in bp window 2.2kb Fig S2

8 A maximal CpG density maximal CpG density maximal density 1 Peak calling cutoff maximal density 2 CpG density > % CpG density <= % Fig S

9 A Methylation < %, any size 2 read density 1 1 HKme read density 1 Methylation < %, Size < bp C Methylation < %, Size -1bp 2 2 read density 1 6/267=16% 1/891=8% read density 1 1 HKme read density 1 1 HKme read density 1 D Methylation < %, Size 1-2bp E Methylation < %, Size >2bp read density /2918=% read density /729=1.% 1 HKme read density HKme read density Fig S

10 A Gata6 Gata6 CpG islands 2kb PTMA Ptma CpG islands 1kb Pde6d Reads / 1M Reads / 1M CpG / bp 7 CpG density CpG / bp 7 CpG density C CpG islands Fat1 1kb D Ebf Ebf Ebf Ebf CpG islands 2kb Reads / 1M Reads / 1M CpG / bp 7 CpG density CpG / bp 7 CpG density E chip validation peaks Ebf Zfp2 Fat1 KcnJ Olfr21 zscan2 Act hs-26 Gata6 Hvoxb7 Dnmtab-/- WT Fig S

11 A KcnJ Olfr21 C Sorbs1 D Kank1 E Stox2 F Zfp2 G DNMT a/b -/- Ebf Fig S6