Open Access Highly Accessed Research article

Identification of Pou5f1, Sox2, and Nanog downstream target genes with statistical confidence by applying a novel algorithm to time course microarray and genome-wide chromatin immunoprecipitation data

Alexei A Sharov1, Shinji Masui2, Lioudmila V Sharova1, Yulan Piao1, Kazuhiro Aiba1, Ryo Matoba1, Li Xin1, Hitoshi Niwa2 and Minoru SH Ko1*

Author Affiliations

1 Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute on Aging, NIH, Baltimore, MD 21224, USA

2 Laboratory of Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan

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BMC Genomics 2008, 9:269  doi:10.1186/1471-2164-9-269

Published: 3 June 2008

Additional files

Additional file 1:

Summary of data used in this study

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Additional file 2:

Log-transformed gene expression data for non-redundant genes in ZHBTc4 cell line after suppression of Pou5f1. The time series was combined from 2 data sets: (1) data for 0, 3, 6, 12, and 24 hr after adding tetracycline analyzed with NIA 44 k array (probe names in the 1st column), and (2) data for 0, 24, 48, 72, 96, and 120 hr are from the experiment of [2] analyzed with NIA 22 k Dev-2 array (probe names are in column 24).

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Additional file 3:

Suppression of tet-inducible transgene Pou5f1 in ZHBTc4 ES cells in a time course after adding tetracycline to the media. (A) Log expression change from microarray data (log10), oligo is in ORF. (B) Expression change in real-time PCR data normalized by expression in parental EB5 cell line, primers are in transgene-specific region. (C) Western blot showing decrease in POU5F1 protein amount, UBTF is used as a control.

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Additional file 4:

Genes that responded to Pou5f1 suppression in ZHBTc4 ES cells

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Additional file 5:

Comparison of genes that responded to Pou5f1 suppression in various studies. (A) Venn diagram of genes that responded to Pou5f1 suppression by > 2 fold in our experiment with tet-inducible ES cell line ZHBTc4 and in published studies of [5,1], which used shRNA as a method of Pou5f1 suppression. (B) Scatter-plot of gene expression change after Pou5f1 suppression in this study and in [1] based on 1225 common genes.

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Additional file 6:

Genes that responded to Sox2 suppression in 2TS22C ES cells (data from [16])

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Additional file 7:

Genes that responded to Nanog suppression with shRNA in ES cells (analysis of data from [5])

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Additional file 8:

Genes that responded to Nanog suppression with shRNA in ES cells (analysis of data from [1])

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Additional file 9:

Genes that responded to Nanog overexpression in ES cells

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Additional file 10:

Co-localization of POU5F1 and NANOG binding sites in the mouse genome based on ChIP-PET data from [1]. (A) Proportion of ChIP-POU5F1 regions with different number of ditags that were co-localized (withn 1 Kb) with ChIP-NANOG regions (number of ditags indicates binding strength); (B) Density distribution of OCT-SOX composite binding motifs at various distances from the binding site identified with ChIP-PET [1] estimated for groups of genes with ≥ 4 POU5F1 ditags, with ≥ 4 NANOG ditags, and with ≥ 4 NANOG ditags and no POU5F1 ditags. Search for binding motifs was done using components of CisView software [17], which combines pattern-matching and position-weight matrix (PWM) approaches. PWM for OCT-SOX composite binding site was taken from [1].

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Additional file 11:

Sets of training genes that responded to Pou5f1 suppression and control genes used for optimizing the score of potential functionality of binding sites

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Additional file 12:

Parameters of equation for the score of potential functionality (SPF)

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Additional file 13:

POU5F1 binding sites with the highest score of potential functionality (SPF) for each gene

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Additional file 14:

ChIP-PET regions (from [1]) listed in Additional file 8

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Additional file 15:

Regression of the cumulative proportion of control genes (p) versus the score of potential function (SPF) of their binding regions identified with ChIP-PET [1]. Cumulative proportion of genes was estimated after sorting them by decreasing SPF. Magenta line shows the regression estimated using top 300 genes with highest SPF. (A) SPF parameters estimated by optimization against down-regulated genes; (B) SPF parameters estimated by optimization against up-regulated genes.

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Additional file 16:

Additional tentative target genes for POU5F1.

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Additional file 17:

Comparison of tentative target genes of POU5F1 identified in this paper with previously published lists of genes

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Additional file 18:

Frequency distribution of a score [SPF·abs(logratio)] among tentative target genes TTGs of POU5F1 identified in this paper and TTGs (non-matching with ours) from [1,2]

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Additional file 19:

Gene Ontology (GO) annotations of POU5F1 tentative target genes

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Additional file 20:

Venn diagram of tentative target genes (TTGs) of POU5F1, SOX2, and NANOG

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Additional file 21:

Combined tentative target genes (TTGs) of POU5F1, SOX2, and NANOG

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Additional file 22:

Additional tentative target genes for SOX2 identified using ChIP data from human ES cells [6] and > 2 fold expression change in mouse ES cells [16]

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Additional file 23:

Expression change of Cdx2 after suppression of Pou5f1 and Sox2 analyzed using quantitative RT-PCR (SE estimated with ANOVA)

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