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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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TFIIB and TFIID bind internal positions in RefSeq genes and are associated with internally initiated transcripts. (A) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg TFIIB antibody (black bars), 10 μl of TFIID antibody (gray bars) or 3 μg Gal4 antibody as a control IgG (white bars). Regions interrogated correspond to a subset of genes with internal sites that were chosen at random. The levels of Ins1 and CBP 5' promoters precipitated are included as controls. Negative regions included the repressed HBB and IL2 genes. Data is presented as percent input and error is SEM. 5', internal, and 3' refer to the positions of the TFIIB GST relative to RefSeq gene boundaries. (B) An agarose gel of the amplified transcript from an internally initiated CBP transcript identified by a modified cap trapping procedure (see methods). Two independent PCR clones (lanes 1 and 2) mapped the start site to nucleotide 11687697 on chromosome 10.
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Figure 3: TFIIB and TFIID bind internal positions in RefSeq genes and are associated with internally initiated transcripts. (A) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg TFIIB antibody (black bars), 10 μl of TFIID antibody (gray bars) or 3 μg Gal4 antibody as a control IgG (white bars). Regions interrogated correspond to a subset of genes with internal sites that were chosen at random. The levels of Ins1 and CBP 5' promoters precipitated are included as controls. Negative regions included the repressed HBB and IL2 genes. Data is presented as percent input and error is SEM. 5', internal, and 3' refer to the positions of the TFIIB GST relative to RefSeq gene boundaries. (B) An agarose gel of the amplified transcript from an internally initiated CBP transcript identified by a modified cap trapping procedure (see methods). Two independent PCR clones (lanes 1 and 2) mapped the start site to nucleotide 11687697 on chromosome 10.

Mentions: The high percentage of internal TFIIB GSTs prompted us to further validate whether these TFIIB sites identified promoter regions; i.e. whether they were associated with transcriptional start sites. First, we performed ChIP assays to confirm TFIIB binding to 18 internal positions of 9 genes demarcated by TFIIB GSTs. TFIIB bound each of the 18 internal positions examined (Figure 3A). TFIIB also bound a region at the 3' end of CBP. To test whether the PIC was recruited to the internal TFIIB positions, we assayed for TFIID binding by ChIP. TFIID plays a role in promoter recognition within the PIC [3]. Like TFIIB, TFIID bound each internal position tested (Figure 3A). The levels of TFIIB and TFIID binding to the internal and 3' positions were similar to that at the 5' promoter of Ins1 and CBP. No significant binding for TFIIB or TFIID was detected at the repressed HBB and IL2 genes.


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

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

TFIIB and TFIID bind internal positions in RefSeq genes and are associated with internally initiated transcripts. (A) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg TFIIB antibody (black bars), 10 μl of TFIID antibody (gray bars) or 3 μg Gal4 antibody as a control IgG (white bars). Regions interrogated correspond to a subset of genes with internal sites that were chosen at random. The levels of Ins1 and CBP 5' promoters precipitated are included as controls. Negative regions included the repressed HBB and IL2 genes. Data is presented as percent input and error is SEM. 5', internal, and 3' refer to the positions of the TFIIB GST relative to RefSeq gene boundaries. (B) An agarose gel of the amplified transcript from an internally initiated CBP transcript identified by a modified cap trapping procedure (see methods). Two independent PCR clones (lanes 1 and 2) mapped the start site to nucleotide 11687697 on chromosome 10.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: TFIIB and TFIID bind internal positions in RefSeq genes and are associated with internally initiated transcripts. (A) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg TFIIB antibody (black bars), 10 μl of TFIID antibody (gray bars) or 3 μg Gal4 antibody as a control IgG (white bars). Regions interrogated correspond to a subset of genes with internal sites that were chosen at random. The levels of Ins1 and CBP 5' promoters precipitated are included as controls. Negative regions included the repressed HBB and IL2 genes. Data is presented as percent input and error is SEM. 5', internal, and 3' refer to the positions of the TFIIB GST relative to RefSeq gene boundaries. (B) An agarose gel of the amplified transcript from an internally initiated CBP transcript identified by a modified cap trapping procedure (see methods). Two independent PCR clones (lanes 1 and 2) mapped the start site to nucleotide 11687697 on chromosome 10.
Mentions: The high percentage of internal TFIIB GSTs prompted us to further validate whether these TFIIB sites identified promoter regions; i.e. whether they were associated with transcriptional start sites. First, we performed ChIP assays to confirm TFIIB binding to 18 internal positions of 9 genes demarcated by TFIIB GSTs. TFIIB bound each of the 18 internal positions examined (Figure 3A). TFIIB also bound a region at the 3' end of CBP. To test whether the PIC was recruited to the internal TFIIB positions, we assayed for TFIID binding by ChIP. TFIID plays a role in promoter recognition within the PIC [3]. Like TFIIB, TFIID bound each internal position tested (Figure 3A). The levels of TFIIB and TFIID binding to the internal and 3' positions were similar to that at the 5' promoter of Ins1 and CBP. No significant binding for TFIIB or TFIID was detected at the repressed HBB and IL2 genes.

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