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Localization of TFIIB binding regions using serial analysis of chromatin occupancy.

Yochum GS, Rajaraman V, Cleland R, McWeeney S - BMC Mol. Biol. (2007)

Bottom Line: The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay.The 5' TSSs for internal transcripts were confirmed by primer extension.Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA.

ABSTRACT

Background: RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome.

Results: A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20-22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of the 1783 protein-coding genes in this sample of the SACO library, TFIIB binding mapped near the characterized 5' promoter that is upstream of the transcription start site (TSS). However, internal TFIIB binding positions were identified in 57% of the 1783 protein-coding genes. Internal positions are defined as those within an inclusive region greater than 2.5 kb downstream from the 5' TSS and 2.5 kb upstream from the transcription stop. We demonstrate that both TFIIB and TFIID (an additional component of PICs) bound to internal regions using chromatin immunoprecipitation (ChIP). The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay. The 5' TSSs for internal transcripts were confirmed by primer extension. Additionally, an analysis of the functional annotation of mouse 3 (FANTOM3) databases indicates that internally initiated transcripts identified by TFIIB SACO in rat are conserved in mouse.

Conclusion: Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated.

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Internal transcripts associated with TFIIB GSTs in rat are conserved in mouse. Diagram depicting the spatial relationship between TFIIB GSTs and the 5' end of sense and antisense transcripts determined by cap-trapping. The 3' portion of rat CBP is shown in black with exons as rectangles and introns as a horizontal line. Black vertical lines indicate the position of the feature listed. Black vertical lines with an arrow conferring direction of the transcript indicate the 5' nucleotides of cap-trapped transcripts. Rat sense and antisense CAP are the positions of the experimentally capped transcripts. Mouse-TSS are the positions of the 5' nucleotides of mouse CBP transcripts identified by cap analysis of gene expression (CAGE). The mouse TSS data was obtained from the FANTOM3 consortium [11].
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Figure 5: Internal transcripts associated with TFIIB GSTs in rat are conserved in mouse. Diagram depicting the spatial relationship between TFIIB GSTs and the 5' end of sense and antisense transcripts determined by cap-trapping. The 3' portion of rat CBP is shown in black with exons as rectangles and introns as a horizontal line. Black vertical lines indicate the position of the feature listed. Black vertical lines with an arrow conferring direction of the transcript indicate the 5' nucleotides of cap-trapped transcripts. Rat sense and antisense CAP are the positions of the experimentally capped transcripts. Mouse-TSS are the positions of the 5' nucleotides of mouse CBP transcripts identified by cap analysis of gene expression (CAGE). The mouse TSS data was obtained from the FANTOM3 consortium [11].

Mentions: We then utilized data from the FANTOM3 consortium as an independent source to validate our findings and to determine whether the internal transcripts we identified in rat are conserved in mouse. The FANTOM3 consortium recently determined the 5' and 3' boundaries for over 180,000 unique RNAs comprising the mouse transcriptome [11]. Using NCBI's Homologene, we identified 1462 RefSeq mouse homologs of the rat genes in our TFIIB SACO library. Of the mouse homologs, 1284 (88%) had cap analysis of gene expression (CAGE) evidence for internal TSSs. We then positionally co-aligned the rat TFIIB GSTs identified in our TFIIB SACO screen with the mouse TSSs reported by the FANTOM3 consortium. Overall, with respect to distance, 84% of the GSTs were within 2.5 kb of a TSS (80% of these sites were within 750 bp). To illustrate this co-alignment, we considered CBP as an example (Figure 5). Rat CBP had the most TFIIB GSTs and the highest number of experimentally confirmed TSS by our modified cap-trapping method. The rat TFIIB GSTs and the rat TSS that we identified in CBP were mapped to mouse CBP. We then overlaid transcript start positions within mouse CBP identified by FANTOM3. The positions of internal TSS in mouse CBP co-align with TFIIB GSTs and TSS in rat CBP, suggesting that the internal promoters are conserved across the two species. Together, this analysis suggests that full-length protein-coding transcripts may comprise only a fraction of the transcripts derived from a gene.


Localization of TFIIB binding regions using serial analysis of chromatin occupancy.

Yochum GS, Rajaraman V, Cleland R, McWeeney S - BMC Mol. Biol. (2007)

Internal transcripts associated with TFIIB GSTs in rat are conserved in mouse. Diagram depicting the spatial relationship between TFIIB GSTs and the 5' end of sense and antisense transcripts determined by cap-trapping. The 3' portion of rat CBP is shown in black with exons as rectangles and introns as a horizontal line. Black vertical lines indicate the position of the feature listed. Black vertical lines with an arrow conferring direction of the transcript indicate the 5' nucleotides of cap-trapped transcripts. Rat sense and antisense CAP are the positions of the experimentally capped transcripts. Mouse-TSS are the positions of the 5' nucleotides of mouse CBP transcripts identified by cap analysis of gene expression (CAGE). The mouse TSS data was obtained from the FANTOM3 consortium [11].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211499&req=5

Figure 5: Internal transcripts associated with TFIIB GSTs in rat are conserved in mouse. Diagram depicting the spatial relationship between TFIIB GSTs and the 5' end of sense and antisense transcripts determined by cap-trapping. The 3' portion of rat CBP is shown in black with exons as rectangles and introns as a horizontal line. Black vertical lines indicate the position of the feature listed. Black vertical lines with an arrow conferring direction of the transcript indicate the 5' nucleotides of cap-trapped transcripts. Rat sense and antisense CAP are the positions of the experimentally capped transcripts. Mouse-TSS are the positions of the 5' nucleotides of mouse CBP transcripts identified by cap analysis of gene expression (CAGE). The mouse TSS data was obtained from the FANTOM3 consortium [11].
Mentions: We then utilized data from the FANTOM3 consortium as an independent source to validate our findings and to determine whether the internal transcripts we identified in rat are conserved in mouse. The FANTOM3 consortium recently determined the 5' and 3' boundaries for over 180,000 unique RNAs comprising the mouse transcriptome [11]. Using NCBI's Homologene, we identified 1462 RefSeq mouse homologs of the rat genes in our TFIIB SACO library. Of the mouse homologs, 1284 (88%) had cap analysis of gene expression (CAGE) evidence for internal TSSs. We then positionally co-aligned the rat TFIIB GSTs identified in our TFIIB SACO screen with the mouse TSSs reported by the FANTOM3 consortium. Overall, with respect to distance, 84% of the GSTs were within 2.5 kb of a TSS (80% of these sites were within 750 bp). To illustrate this co-alignment, we considered CBP as an example (Figure 5). Rat CBP had the most TFIIB GSTs and the highest number of experimentally confirmed TSS by our modified cap-trapping method. The rat TFIIB GSTs and the rat TSS that we identified in CBP were mapped to mouse CBP. We then overlaid transcript start positions within mouse CBP identified by FANTOM3. The positions of internal TSS in mouse CBP co-align with TFIIB GSTs and TSS in rat CBP, suggesting that the internal promoters are conserved across the two species. Together, this analysis suggests that full-length protein-coding transcripts may comprise only a fraction of the transcripts derived from a gene.

Bottom Line: The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay.The 5' TSSs for internal transcripts were confirmed by primer extension.Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vollum Institute, Oregon Health and Science University, Portland, OR 97239, USA.

ABSTRACT

Background: RNA Polymerase II (RNAP II) is recruited to core promoters by the pre-initiation complex (PIC) of general transcription factors. Within the PIC, transcription factor for RNA polymerase IIB (TFIIB) determines the start site of transcription. TFIIB binding has not been localized, genome-wide, in metazoans. Serial analysis of chromatin occupancy (SACO) is an unbiased methodology used to empirically identify transcription factor binding regions. In this report, we use TFIIB and SACO to localize TFIIB binding regions across the rat genome.

Results: A sample of the TFIIB SACO library was sequenced and 12,968 TFIIB genomic signature tags (GSTs) were assigned to the rat genome. GSTs are 20-22 base pair fragments that are derived from TFIIB bound chromatin. TFIIB localized to both non-protein coding and protein-coding loci. For 21% of the 1783 protein-coding genes in this sample of the SACO library, TFIIB binding mapped near the characterized 5' promoter that is upstream of the transcription start site (TSS). However, internal TFIIB binding positions were identified in 57% of the 1783 protein-coding genes. Internal positions are defined as those within an inclusive region greater than 2.5 kb downstream from the 5' TSS and 2.5 kb upstream from the transcription stop. We demonstrate that both TFIIB and TFIID (an additional component of PICs) bound to internal regions using chromatin immunoprecipitation (ChIP). The 5' cap of transcripts associated with internal TFIIB binding positions were identified using a cap-trapping assay. The 5' TSSs for internal transcripts were confirmed by primer extension. Additionally, an analysis of the functional annotation of mouse 3 (FANTOM3) databases indicates that internally initiated transcripts identified by TFIIB SACO in rat are conserved in mouse.

Conclusion: Our findings that TFIIB binding is not restricted to the 5' upstream region indicates that the propensity for PIC to contribute to transcript diversity is far greater than previously appreciated.

Show MeSH
Related in: MedlinePlus