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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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Related in: MedlinePlus

Primer extension analysis of transcripts derived from the rat CBP locus. RNA was isolated from Rin-m cells and primer extension analysis was performed based on TSSs determined by cap-trapping. Products were resolved by denaturing polyacrylamide gel electrophoresis. Marker (M): End labeled Φ X174 Hinf 1 digested DNA. 5', internal, and 3' refer to the position of the TFIIB GST and TSS relative to CBP gene boundaries.
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Figure 4: Primer extension analysis of transcripts derived from the rat CBP locus. RNA was isolated from Rin-m cells and primer extension analysis was performed based on TSSs determined by cap-trapping. Products were resolved by denaturing polyacrylamide gel electrophoresis. Marker (M): End labeled Φ X174 Hinf 1 digested DNA. 5', internal, and 3' refer to the position of the TFIIB GST and TSS relative to CBP gene boundaries.

Mentions: The cap-trapping experiments confirmed that transcripts were initiated at internal positions within the protein-coding genes identified by TFIIB SACO. We then used primer extension to confirm the cap-trapping results with an alternative method. Primer extension requires an oligonucleotide that anneals downstream of a TSS to prime a cDNA synthesis reaction. The reverse transcriptase extends the cDNA until it reaches the 5' end of the transcript. The resulting products are ultimately run on a polyacrylamide gel. We isolated five capped transcripts associated with TFIIB GSTs for CBP. Four of the GSTs were associated with internal-sense transcripts and one with an antisense transcript. For the primer extension assay, the characterized 5' promoter was included as a control. We detected transcripts that initiated from the internal and antisense promoters, in addition to the known 5' promoter (Figure 4). Moreover, CBP int-1 and the CBP 3' antisense transcript appeared to be more abundant that the CBP transcript derived from the 5' promoter.


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

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

Primer extension analysis of transcripts derived from the rat CBP locus. RNA was isolated from Rin-m cells and primer extension analysis was performed based on TSSs determined by cap-trapping. Products were resolved by denaturing polyacrylamide gel electrophoresis. Marker (M): End labeled Φ X174 Hinf 1 digested DNA. 5', internal, and 3' refer to the position of the TFIIB GST and TSS relative to CBP gene boundaries.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Primer extension analysis of transcripts derived from the rat CBP locus. RNA was isolated from Rin-m cells and primer extension analysis was performed based on TSSs determined by cap-trapping. Products were resolved by denaturing polyacrylamide gel electrophoresis. Marker (M): End labeled Φ X174 Hinf 1 digested DNA. 5', internal, and 3' refer to the position of the TFIIB GST and TSS relative to CBP gene boundaries.
Mentions: The cap-trapping experiments confirmed that transcripts were initiated at internal positions within the protein-coding genes identified by TFIIB SACO. We then used primer extension to confirm the cap-trapping results with an alternative method. Primer extension requires an oligonucleotide that anneals downstream of a TSS to prime a cDNA synthesis reaction. The reverse transcriptase extends the cDNA until it reaches the 5' end of the transcript. The resulting products are ultimately run on a polyacrylamide gel. We isolated five capped transcripts associated with TFIIB GSTs for CBP. Four of the GSTs were associated with internal-sense transcripts and one with an antisense transcript. For the primer extension assay, the characterized 5' promoter was included as a control. We detected transcripts that initiated from the internal and antisense promoters, in addition to the known 5' promoter (Figure 4). Moreover, CBP int-1 and the CBP 3' antisense transcript appeared to be more abundant that the CBP transcript derived from the 5' promoter.

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