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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 binds active genes in Rin-m rat insulinoma cells. (A) RNA was isolated and cDNA was synthesized using reverse transcriptase. Transcript levels of active (c-Fos and Ins1) and repressed (Fcgr2b and Mycd) genes were measured by quantitative real-time PCR. Levels of cDNA detected (black bars) were determined using a standard curve generated with purified PCR amplicons. No reverse transcriptase controls (white bars) indicate that signal generated was RNA-dependent. The amount of Fcgr2b and Mycd was over 500-fold lower than c-Fos and therefore represents background amplification in the assay. (B) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg of TFIIB antibodies (black bars), 3 μg of RNAP II antibodies (gray bars), or 3 μg of Gal4 antibodies as a control IgGs (white bars). The level of immunoprecipitated Ins1, c-Fos, Fcgr2b, and Mycd promoters was measured by real-time PCR. The standard curve was derived from PCR reactions using serially diluted input chromatin DNA as the template. Error bars are SEM.
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Figure 1: TFIIB binds active genes in Rin-m rat insulinoma cells. (A) RNA was isolated and cDNA was synthesized using reverse transcriptase. Transcript levels of active (c-Fos and Ins1) and repressed (Fcgr2b and Mycd) genes were measured by quantitative real-time PCR. Levels of cDNA detected (black bars) were determined using a standard curve generated with purified PCR amplicons. No reverse transcriptase controls (white bars) indicate that signal generated was RNA-dependent. The amount of Fcgr2b and Mycd was over 500-fold lower than c-Fos and therefore represents background amplification in the assay. (B) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg of TFIIB antibodies (black bars), 3 μg of RNAP II antibodies (gray bars), or 3 μg of Gal4 antibodies as a control IgGs (white bars). The level of immunoprecipitated Ins1, c-Fos, Fcgr2b, and Mycd promoters was measured by real-time PCR. The standard curve was derived from PCR reactions using serially diluted input chromatin DNA as the template. Error bars are SEM.

Mentions: Initially, we tested whether TFIIB binds promoters of active genes in the rat insulinoma cell line, Rin-m. The Rin-m cell line was established from radiation-induced rat islet cell tumor maintained in athymic nude mice [18]. Using quantitative reverse transcriptase PCR (qRT-PCR), we found that transcript levels of two genes known to be expressed in Rin-m cells, insulin and cFos [18,19], were at least two-to-three orders of magnitude higher than repressed genes (the immune cell specific Fcgr2b and muscle specific, myocardin, Mycd) (Figure 1A). TFIIB binding was then tested in ChIP assays using promoter-specific primers. In addition, an antibody directed against the DNA binding domain of the yeast activator Gal4 was used in parallel ChIPs as a control for specificity. TFIIB bound to the insulin and cFos promoters, but not to the repressed Fcgr2b and Mycd promoters (Figure 1B). ChIP assays using antibodies specific for RNAP II confirmed that TFIIB associated with transcribed genes. We then constructed a SACO library with DNA isolated from TFIIB ChIPs.


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

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

TFIIB binds active genes in Rin-m rat insulinoma cells. (A) RNA was isolated and cDNA was synthesized using reverse transcriptase. Transcript levels of active (c-Fos and Ins1) and repressed (Fcgr2b and Mycd) genes were measured by quantitative real-time PCR. Levels of cDNA detected (black bars) were determined using a standard curve generated with purified PCR amplicons. No reverse transcriptase controls (white bars) indicate that signal generated was RNA-dependent. The amount of Fcgr2b and Mycd was over 500-fold lower than c-Fos and therefore represents background amplification in the assay. (B) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg of TFIIB antibodies (black bars), 3 μg of RNAP II antibodies (gray bars), or 3 μg of Gal4 antibodies as a control IgGs (white bars). The level of immunoprecipitated Ins1, c-Fos, Fcgr2b, and Mycd promoters was measured by real-time PCR. The standard curve was derived from PCR reactions using serially diluted input chromatin DNA as the template. Error bars are SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: TFIIB binds active genes in Rin-m rat insulinoma cells. (A) RNA was isolated and cDNA was synthesized using reverse transcriptase. Transcript levels of active (c-Fos and Ins1) and repressed (Fcgr2b and Mycd) genes were measured by quantitative real-time PCR. Levels of cDNA detected (black bars) were determined using a standard curve generated with purified PCR amplicons. No reverse transcriptase controls (white bars) indicate that signal generated was RNA-dependent. The amount of Fcgr2b and Mycd was over 500-fold lower than c-Fos and therefore represents background amplification in the assay. (B) Real-time PCR quantitation of DNA fragments precipitated in a ChIP assay using 3 μg of TFIIB antibodies (black bars), 3 μg of RNAP II antibodies (gray bars), or 3 μg of Gal4 antibodies as a control IgGs (white bars). The level of immunoprecipitated Ins1, c-Fos, Fcgr2b, and Mycd promoters was measured by real-time PCR. The standard curve was derived from PCR reactions using serially diluted input chromatin DNA as the template. Error bars are SEM.
Mentions: Initially, we tested whether TFIIB binds promoters of active genes in the rat insulinoma cell line, Rin-m. The Rin-m cell line was established from radiation-induced rat islet cell tumor maintained in athymic nude mice [18]. Using quantitative reverse transcriptase PCR (qRT-PCR), we found that transcript levels of two genes known to be expressed in Rin-m cells, insulin and cFos [18,19], were at least two-to-three orders of magnitude higher than repressed genes (the immune cell specific Fcgr2b and muscle specific, myocardin, Mycd) (Figure 1A). TFIIB binding was then tested in ChIP assays using promoter-specific primers. In addition, an antibody directed against the DNA binding domain of the yeast activator Gal4 was used in parallel ChIPs as a control for specificity. TFIIB bound to the insulin and cFos promoters, but not to the repressed Fcgr2b and Mycd promoters (Figure 1B). ChIP assays using antibodies specific for RNAP II confirmed that TFIIB associated with transcribed genes. We then constructed a SACO library with DNA isolated from TFIIB ChIPs.

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