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Identification of highly conserved putative developmental enhancers bound by SOX3 in neural progenitors using ChIP-Seq.

McAninch D, Thomas P - PLoS ONE (2014)

Bottom Line: SOX3 binding was identified at over 8,000 sites, most of which were intronic or intergeneic and were significantly associated with neurodevelopmental genes.In addition, we identified a subset of highly conserved putative enhancers for CNS development genes common to SOXB1 members in NP cells, all of which contained the SOX consensus motif (ACAAWR).Together these data implicate SOX3 in the direct regulation of hundreds of NP genes and provide molecular insight into the overlapping roles of SOXB1 proteins in CNS development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Molecular & Biomedical Science and Robinson Research Institute, The University of Adelaide, Adelaide, Australia.

ABSTRACT
The transcription factor SOX3 is expressed within most neural progenitor (NP) cells of the vertebrate central nervous system (CNS) and is essential for normal brain development in mice and humans. However, despite the widespread expression of Sox3, CNS defects in mice are relatively mild due to functional redundancy with the other SOXB1 sub-group members Sox1 and Sox2. To further understand the molecular function of SOX3, we investigated the genome-wide binding profile of endogenous SOX3 in NP cells using ChIP-seq. SOX3 binding was identified at over 8,000 sites, most of which were intronic or intergeneic and were significantly associated with neurodevelopmental genes. The majority of binding sites were moderately or highly conserved (phastCons scores >0.1 and 0.5, respectively) and included the previously characterised, SOXB1-binding Nestin NP cell enhancer. Comparison of SOX3 and published ChIP-Seq data for the co-activator P300 in embryonic brain identified hundreds of highly conserved putative enhancer elements. In addition, we identified a subset of highly conserved putative enhancers for CNS development genes common to SOXB1 members in NP cells, all of which contained the SOX consensus motif (ACAAWR). Together these data implicate SOX3 in the direct regulation of hundreds of NP genes and provide molecular insight into the overlapping roles of SOXB1 proteins in CNS development.

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Overview of SOX3 ChIP-Seq data from mouse neural progenitor cells.(A) Genomic classification of SOX3 binding sites relative to nearest transcriptional start sites. (B) Validation of SOX3 ChIP by qPCR. Fold change is relative to both input DNA and IgG control values for the same genomic location. Error bars correspond to standard deviation of three independent sample replicates, P-values indicated as <0.05 (ns), >0.05 (*) and >0.001 (**). (C) Highest enriched DNA motifs identified by MEME-ChIP as i. a SOX motif and ii. a SOX-POU motif. (D) Enriched Gene Ontology terms associated with subsets of SOX3 ChIP peaks.
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pone-0113361-g001: Overview of SOX3 ChIP-Seq data from mouse neural progenitor cells.(A) Genomic classification of SOX3 binding sites relative to nearest transcriptional start sites. (B) Validation of SOX3 ChIP by qPCR. Fold change is relative to both input DNA and IgG control values for the same genomic location. Error bars correspond to standard deviation of three independent sample replicates, P-values indicated as <0.05 (ns), >0.05 (*) and >0.001 (**). (C) Highest enriched DNA motifs identified by MEME-ChIP as i. a SOX motif and ii. a SOX-POU motif. (D) Enriched Gene Ontology terms associated with subsets of SOX3 ChIP peaks.

Mentions: To identify genomic binding sites of endogenous SOX3 protein, we performed ChIP-Seq analysis of NP cells generated from embryonic stem cells by N2B27 neuroinduction [20]. We have shown previously that these NP cells exhibit robust SOX3 expression [21] and that the SOX3 antibody used for ChIP has specific activity in immunohistochemistry [6] and Western blot analyses [22]. A total of 8067 common binding sites were identified across three independent samples (Figure 1A; Table S1). ChIP-Seq data was validated using ChIP-qPCR on independently generated samples, with all but one of the SOX3 binding sites (SBS) tested showing enrichment (Figure 1B). A de novo analysis of the full set of ChIP peaks was performed to identify enriched DNA motifs. Comparison with the JASPAR database [23] confirmed that the most common motif was a SOX binding motif (Figure 1C) (with at least one occurrence within >70% of peaks, p-value less than 10−6, and an expected background occurrence of 39%), which was similar to the motif identified in a recently published SOX3 ChIP dataset [2]. The second most common motif features paired SOX/POU binding sites separated by a single nucleotide (Figure 1C) (with at least one occurrence within >40% of peaks). Motifs for other neural TF classes, such as the Zic, Klf and Engrailed families, were also enriched within 1215, 979 and 647 peaks respectively (all with p-values less than 0.0001). Together, these data indicate successful immunoprecipitation of SOX3-associated chromatin.


Identification of highly conserved putative developmental enhancers bound by SOX3 in neural progenitors using ChIP-Seq.

McAninch D, Thomas P - PLoS ONE (2014)

Overview of SOX3 ChIP-Seq data from mouse neural progenitor cells.(A) Genomic classification of SOX3 binding sites relative to nearest transcriptional start sites. (B) Validation of SOX3 ChIP by qPCR. Fold change is relative to both input DNA and IgG control values for the same genomic location. Error bars correspond to standard deviation of three independent sample replicates, P-values indicated as <0.05 (ns), >0.05 (*) and >0.001 (**). (C) Highest enriched DNA motifs identified by MEME-ChIP as i. a SOX motif and ii. a SOX-POU motif. (D) Enriched Gene Ontology terms associated with subsets of SOX3 ChIP peaks.
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pone-0113361-g001: Overview of SOX3 ChIP-Seq data from mouse neural progenitor cells.(A) Genomic classification of SOX3 binding sites relative to nearest transcriptional start sites. (B) Validation of SOX3 ChIP by qPCR. Fold change is relative to both input DNA and IgG control values for the same genomic location. Error bars correspond to standard deviation of three independent sample replicates, P-values indicated as <0.05 (ns), >0.05 (*) and >0.001 (**). (C) Highest enriched DNA motifs identified by MEME-ChIP as i. a SOX motif and ii. a SOX-POU motif. (D) Enriched Gene Ontology terms associated with subsets of SOX3 ChIP peaks.
Mentions: To identify genomic binding sites of endogenous SOX3 protein, we performed ChIP-Seq analysis of NP cells generated from embryonic stem cells by N2B27 neuroinduction [20]. We have shown previously that these NP cells exhibit robust SOX3 expression [21] and that the SOX3 antibody used for ChIP has specific activity in immunohistochemistry [6] and Western blot analyses [22]. A total of 8067 common binding sites were identified across three independent samples (Figure 1A; Table S1). ChIP-Seq data was validated using ChIP-qPCR on independently generated samples, with all but one of the SOX3 binding sites (SBS) tested showing enrichment (Figure 1B). A de novo analysis of the full set of ChIP peaks was performed to identify enriched DNA motifs. Comparison with the JASPAR database [23] confirmed that the most common motif was a SOX binding motif (Figure 1C) (with at least one occurrence within >70% of peaks, p-value less than 10−6, and an expected background occurrence of 39%), which was similar to the motif identified in a recently published SOX3 ChIP dataset [2]. The second most common motif features paired SOX/POU binding sites separated by a single nucleotide (Figure 1C) (with at least one occurrence within >40% of peaks). Motifs for other neural TF classes, such as the Zic, Klf and Engrailed families, were also enriched within 1215, 979 and 647 peaks respectively (all with p-values less than 0.0001). Together, these data indicate successful immunoprecipitation of SOX3-associated chromatin.

Bottom Line: SOX3 binding was identified at over 8,000 sites, most of which were intronic or intergeneic and were significantly associated with neurodevelopmental genes.In addition, we identified a subset of highly conserved putative enhancers for CNS development genes common to SOXB1 members in NP cells, all of which contained the SOX consensus motif (ACAAWR).Together these data implicate SOX3 in the direct regulation of hundreds of NP genes and provide molecular insight into the overlapping roles of SOXB1 proteins in CNS development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Molecular & Biomedical Science and Robinson Research Institute, The University of Adelaide, Adelaide, Australia.

ABSTRACT
The transcription factor SOX3 is expressed within most neural progenitor (NP) cells of the vertebrate central nervous system (CNS) and is essential for normal brain development in mice and humans. However, despite the widespread expression of Sox3, CNS defects in mice are relatively mild due to functional redundancy with the other SOXB1 sub-group members Sox1 and Sox2. To further understand the molecular function of SOX3, we investigated the genome-wide binding profile of endogenous SOX3 in NP cells using ChIP-seq. SOX3 binding was identified at over 8,000 sites, most of which were intronic or intergeneic and were significantly associated with neurodevelopmental genes. The majority of binding sites were moderately or highly conserved (phastCons scores >0.1 and 0.5, respectively) and included the previously characterised, SOXB1-binding Nestin NP cell enhancer. Comparison of SOX3 and published ChIP-Seq data for the co-activator P300 in embryonic brain identified hundreds of highly conserved putative enhancer elements. In addition, we identified a subset of highly conserved putative enhancers for CNS development genes common to SOXB1 members in NP cells, all of which contained the SOX consensus motif (ACAAWR). Together these data implicate SOX3 in the direct regulation of hundreds of NP genes and provide molecular insight into the overlapping roles of SOXB1 proteins in CNS development.

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