Supporting Information - PNAS...Supporting Information Yokochi et al. 10.1073/pnas.0906142106 Total...
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Supporting Information Yokochi et al. 10.1073/pnas.0906142106 Total genes = 24,210 Transcription Fold Change (OHT / Mock) Upregulated genes Downregulated genes Transcription (Mock) A Dub1 Magea1 Asz1 Gm397 Rhox2 AF067061 Pramel6 Actin Slc23a1 Mef2b Nr1h3 Grg13 Slc45a1 Atoh7 Dlx4 OHT - + OHT - + >4 15 >10 28 >8 10 >5 18 >5 5 4 27 4 10 0.6 0.5 0.9 1.1 0.7 0.6 1.0 1.0 B Fold Change PCR Array Fold Change PCR Array 0.3 0.28 0.28 0.27 0.26 0.24 0.24 0.87 Fig. S1. Transcriptional analysis after differentiation. (A) RNA preparations from the cells in Fig. 3A were analyzed by RT-PCR for the presence of several of the up-regulated genes (red) and all of the down-regulated genes (blue) that were in the group of NCBI best- annotated genes (RefSeq). PCR was performed for a number of cycles determined to be in the linear range and quantified using IMAGEJ, as described (1). (B) At 5 days after OHT or mock treatment, cells were differen- tiated to NPCs for 9 days, and microarray analysis was performed as in Fig. 2 A. After data normalization and consolidation of probe values into 1 value per gene, gene expression values for all 24,210 genes identified on the NimbleGen microarray were plotted on the x-axis for mock-treated cells vs. the fold change for each gene after OHT treatment relative to mock treatment. A 4-fold increase in transcription was chosen as a cut-off (red line) for up-regulated genes, and a3-fold decrease in tran- scription was chosen as a cut-off (blue line) for down- regulated genes, as in Fig. 2 A. 1. Hiratani I, Leskovar A, Gilbert DM (2004) Differentiation- induced replication-timing changes are restricted to AT-rich/ long interspersed nuclear element (LINE)-rich isochores. Proc Natl Acad Sci USA 101(48):16861–16866. Yokochi et al. www.pnas.org/cgi/content/short/0906142106 1 of 5
Transcript of Supporting Information - PNAS...Supporting Information Yokochi et al. 10.1073/pnas.0906142106 Total...
Supporting InformationYokochi et al. 10.1073/pnas.0906142106
Total genes = 24,210
Tran
scrip
tion
Fold
Cha
nge
(OH
T / M
ock)
Upregulated genes
Downregulated genes
Transcription (Mock)
A
Dub1 Magea1
Asz1 Gm397 Rhox2
AF067061 Pramel6
Actin
Slc23a1 Mef2b Nr1h3 Grg13
Slc45a1 Atoh7
Dlx4
OHT - +
OHT - +
>4 15 >10 28 >8 10 >5 18 >5 5 4 27 4 10
0.6 0.5 0.9 1.1 0.7 0.6 1.0 1.0
B
Fold Change PCR Array
Fold Change PCR Array
0.3 0.28 0.28 0.27 0.26 0.24 0.24 0.87
Fig. S1. Transcriptional analysis after differentiation.(A) RNA preparations from the cells in Fig. 3A wereanalyzed by RT-PCR for the presence of several of theup-regulated genes (red) and all of the down-regulatedgenes (blue) that were in the group of NCBI best-annotated genes (RefSeq). PCR was performed for anumber of cycles determined to be in the linear rangeand quantified using IMAGEJ, as described (1). (B) At 5days after OHT or mock treatment, cells were differen-tiated to NPCs for 9 days, and microarray analysis wasperformed as in Fig. 2A. After data normalization andconsolidation of probe values into 1 value per gene,gene expression values for all 24,210 genes identified onthe NimbleGen microarray were plotted on the x-axis formock-treatedcellsvs. thefoldchangeforeachgeneafterOHT treatment relative to mock treatment. A 4-foldincrease in transcription was chosen as a cut-off (red line)for up-regulated genes, and a3-fold decrease in tran-scription was chosen as a cut-off (blue line) for down-regulated genes, as in Fig. 2A.
1. Hiratani I, Leskovar A, Gilbert DM (2004) Differentiation-
induced replication-timing changes are restricted to AT-rich/
long interspersed nuclear element (LINE)-rich isochores. Proc
Natl Acad Sci USA 101(48):16861–16866.
Yokochi et al. www.pnas.org/cgi/content/short/0906142106 1 of 5
Fig. S2. Display of ChIP-chip data for H3K9me2 surrounding several G9a-repressed genes. Relative log2 ratio Cy5/Cy3 from Gene Expression Omnibus (GEO;accession number GSM336512) for H3K9me2 ChIP-chip experiments described in Wen et al. (1) were uploaded as in ref. 2, and regions immediately encompassingeach of the indicated genes (indicated by black lines and enlarged in the bottom panel for each gene) as well as a considerably larger region (shown in the toppanel for each gene) were displayed to assemble the figure. Red rectangles denote the start (indicated by an arrow) and end of each gene. Oct4 is provided asan example of a non-H3K9me2-enriched region. The horizontal dotted line at Cy5/Cy3 log2 ratio of 1.1 represents a 95% confidence threshold for significantH3K9me2 enrichment.
1. Wen B, Wu H, Shinkai Y, Irizarry RA, Feinberg AP (2009) Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells. Nat Genet41(2):246–250.
2. Weddington N, et al. (2008) ReplicationDomain: a visualization tool and comparative database for genome-wide replication timing data. BMC Bioinformatics 9(1):530.
Yokochi et al. www.pnas.org/cgi/content/short/0906142106 2 of 5
Fig. S3. G9a-repressed genes are enriched on the male X-chromosome. (Upper) Histogram of the percentage of genes up-regulated uniquely in ESCs (blue), uniquelyin NPCs (green), or in both cell types (red), as well as the total up-regulated genes in both cell types (black) that are found on each chromosome. (Lower) Percentageof genes on each chromosome that are found to be up-regulated in each of the categories of up-regulated genes, color-coded as in (Upper).
Yokochi et al. www.pnas.org/cgi/content/short/0906142106 3 of 5
Fig. S4. SKY of conditional G9a-knockout cells after knockout. SKY of cells 7 days after OHT treatment revealed that, even when aneuploidy (usually trisomy 7, 8,or 19) was found, no evidence of genomic rearrangement could be seen. Shown is a trisomy 8 karyotype. The top left and right panels show the original SKY imageand Giemsa staining, respectively. The middle panel shoes an alignment of homologous chromosomes, and the bottom panel shows an alignment of each chromosometo it’s Geimsa stained counterpart.
Yokochi et al. www.pnas.org/cgi/content/short/0906142106 4 of 5
Other Supporting Information Files
Table S1
Yokochi et al. www.pnas.org/cgi/content/short/0906142106 5 of 5
