Limits...
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.

Show MeSH

Related in: MedlinePlus

Distribution of TFIIB GSTs relative to RefSeq gene annotations. (A) A segment of chromosome 10 depicting internal and 3' localization relative to four RefSeq genes from the Gamma-aminobutyric acid A receptor (Gabrs) subfamily. Rectangles are exons and horizontal lines introns with hatch marks indicating the direction of transcription. (B) Locations of TFIIB GSTs relative to each of the 1783 RefSeq genes identified in the SACO library. 5' UTR; 5' untranslated region, 3' UTR; 3' untranslated region, Internal; Internal region, Multiple; the RefSeq gene contains more than one TFIIB GST. RefSeq genes comprising the "multiple" category contain TFIIB GSTs that localize to 5' and internal regions, 3' and internal regions, 5' and 3' regions, or 5', 3' and internal regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2211499&req=5

Figure 2: Distribution of TFIIB GSTs relative to RefSeq gene annotations. (A) A segment of chromosome 10 depicting internal and 3' localization relative to four RefSeq genes from the Gamma-aminobutyric acid A receptor (Gabrs) subfamily. Rectangles are exons and horizontal lines introns with hatch marks indicating the direction of transcription. (B) Locations of TFIIB GSTs relative to each of the 1783 RefSeq genes identified in the SACO library. 5' UTR; 5' untranslated region, 3' UTR; 3' untranslated region, Internal; Internal region, Multiple; the RefSeq gene contains more than one TFIIB GST. RefSeq genes comprising the "multiple" category contain TFIIB GSTs that localize to 5' and internal regions, 3' and internal regions, 5' and 3' regions, or 5', 3' and internal regions.

Mentions: A key question of interest was to identify the position of TFIIB GSTs relative to RefSeq gene boundaries. We found that TFIIB occupied internal and 3' positions in addition to the more traditional 5' promoter region. One example of the distribution of putative TFIIB binding sites is shown in Figure 2A for genes of the gamma-aminobutyric acid A receptor subfamily (Gabrs). We then localized all TFIIB GSTs in the library relative to RefSeq gene boundaries. TFIIB GSTs were classified as 5' promoter associated if they were found in an inclusive region 2.5 kb outside and 2.5 kb inside the transcript start site (TSS), 3' promoter associated if they were within a region 2.5 kb outside and 2.5 kb inside the 3' end, and internal if they were greater than 2.5 kb inside either the 5' or 3' ends of gene boundaries. The 2.5 kb limit was chosen based upon agarose gel determination of the uppermost size of the chromatin fragments included in library construction (see Additional file 3). There is a total of 1783 RefSeq genes with uniquely assigned GSTs in the TFIIB SACO library. 21% of the genes localized TFIIB binding to the 5' UTR relative to the characterized TSS of the RefSeq gene (Figure 2B). 14% had 3' UTR localizations thereby identifying potential promoters that drive antisense transcripts. Surprisingly, most genes (57%) contained internal TFIIB binding positions. Moreover, 8% of the RefSeq genes contained multiple TFIIB binding regions; most with an internal position. Therefore, internal TFIIB binding is a common characteristic of protein-coding genes.


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

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

Distribution of TFIIB GSTs relative to RefSeq gene annotations. (A) A segment of chromosome 10 depicting internal and 3' localization relative to four RefSeq genes from the Gamma-aminobutyric acid A receptor (Gabrs) subfamily. Rectangles are exons and horizontal lines introns with hatch marks indicating the direction of transcription. (B) Locations of TFIIB GSTs relative to each of the 1783 RefSeq genes identified in the SACO library. 5' UTR; 5' untranslated region, 3' UTR; 3' untranslated region, Internal; Internal region, Multiple; the RefSeq gene contains more than one TFIIB GST. RefSeq genes comprising the "multiple" category contain TFIIB GSTs that localize to 5' and internal regions, 3' and internal regions, 5' and 3' regions, or 5', 3' and internal regions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Distribution of TFIIB GSTs relative to RefSeq gene annotations. (A) A segment of chromosome 10 depicting internal and 3' localization relative to four RefSeq genes from the Gamma-aminobutyric acid A receptor (Gabrs) subfamily. Rectangles are exons and horizontal lines introns with hatch marks indicating the direction of transcription. (B) Locations of TFIIB GSTs relative to each of the 1783 RefSeq genes identified in the SACO library. 5' UTR; 5' untranslated region, 3' UTR; 3' untranslated region, Internal; Internal region, Multiple; the RefSeq gene contains more than one TFIIB GST. RefSeq genes comprising the "multiple" category contain TFIIB GSTs that localize to 5' and internal regions, 3' and internal regions, 5' and 3' regions, or 5', 3' and internal regions.
Mentions: A key question of interest was to identify the position of TFIIB GSTs relative to RefSeq gene boundaries. We found that TFIIB occupied internal and 3' positions in addition to the more traditional 5' promoter region. One example of the distribution of putative TFIIB binding sites is shown in Figure 2A for genes of the gamma-aminobutyric acid A receptor subfamily (Gabrs). We then localized all TFIIB GSTs in the library relative to RefSeq gene boundaries. TFIIB GSTs were classified as 5' promoter associated if they were found in an inclusive region 2.5 kb outside and 2.5 kb inside the transcript start site (TSS), 3' promoter associated if they were within a region 2.5 kb outside and 2.5 kb inside the 3' end, and internal if they were greater than 2.5 kb inside either the 5' or 3' ends of gene boundaries. The 2.5 kb limit was chosen based upon agarose gel determination of the uppermost size of the chromatin fragments included in library construction (see Additional file 3). There is a total of 1783 RefSeq genes with uniquely assigned GSTs in the TFIIB SACO library. 21% of the genes localized TFIIB binding to the 5' UTR relative to the characterized TSS of the RefSeq gene (Figure 2B). 14% had 3' UTR localizations thereby identifying potential promoters that drive antisense transcripts. Surprisingly, most genes (57%) contained internal TFIIB binding positions. Moreover, 8% of the RefSeq genes contained multiple TFIIB binding regions; most with an internal position. Therefore, internal TFIIB binding is a common characteristic of protein-coding 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