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Basal core promoters control the equilibrium between negative cofactor 2 and preinitiation complexes in human cells.

Albert TK, Grote K, Boeing S, Meisterernst M - Genome Biol. (2010)

Bottom Line: We compare target genes of TFIIB and NC2 in human B cells and analyze associated core promoter architectures.TATA and TATA-like elements dictate TFIIB occupancy at a subset of genes.Biochemical data support a model in which preinitiation complex but not TBP-NC2 complex formation is regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Tumor Biology (IMTB), University of Muenster, Robert-Koch-Str, Muenster, Germany. albertt@uni-muenster.de

ABSTRACT

Background: The general transcription factor TFIIB and its antagonist negative cofactor 2 (NC2) are hallmarks of RNA polymerase II (RNAPII) transcription. Both factors bind TATA box-binding protein (TBP) at promoters in a mutually exclusive manner. Dissociation of NC2 is thought to be followed by TFIIB association and subsequent preinitiation complex formation. TFIIB dissociates upon RNAPII promoter clearance, thereby providing a specific measure for steady-state preinitiation complex levels. As yet, genome-scale promoter mapping of human TFIIB has not been reported. It thus remains elusive how human core promoters contribute to preinitiation complex formation in vivo.

Results: We compare target genes of TFIIB and NC2 in human B cells and analyze associated core promoter architectures. TFIIB occupancy is positively correlated with gene expression, with the vast majority of promoters being GC-rich and lacking defined core promoter elements. TATA elements, but not the previously in vitro defined TFIIB recognition elements, are enriched in some 4 to 5% of the genes. NC2 binds to a highly related target gene set. Nonetheless, subpopulations show strong variations in factor ratios: whereas high TFIIB/NC2 ratios select for promoters with focused start sites and conserved core elements, high NC2/TFIIB ratios correlate to multiple start-site promoters lacking defined core elements.

Conclusions: TFIIB and NC2 are global players that occupy active genes. Preinitiation complex formation is independent of core elements at the majority of genes. TATA and TATA-like elements dictate TFIIB occupancy at a subset of genes. Biochemical data support a model in which preinitiation complex but not TBP-NC2 complex formation is regulated.

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Validation of TFIIB target promoters. (a) Signal distribution of TFIIB enrichment. I, II and III denote groups of promoters from the upper 10th, 60th to 80th, or lower 10th percentile and correspond to high, mid-to-low, or no TFIIB occupancy. (b) Target gene validation. Selected genes from groups I to III were analyzed by ChIP-qPCR using TFIIB or IgG control antibody in a third chromatin sample from LCL721 cells in which two independent ChIP reactions were performed (upper panel). Genes are ordered from left to right according to TFIIB levels on the promoter arrays. The relative ChIP recovery is expressed as percentage of input (y-axis). The bars represent the mean, error bars the range of the two ChIP experiments. Corresponding gene expression levels in LCL721 B cells are shown in the lower panel. These were determined using Affymetrix U133 Plus 2.0 microarrays. They represent normalized hybridization signals of gene-specific microarray probesets. N.A., not analyzed. (c) Assembly of an active PIC at the GAPDH promoter. ChIP-qPCR was conducted with eight primer pairs spanning the human GAPDH locus (numbered boxes in top scheme). Results of ChIPs in LCL721 B cells with antibodies for TFIIB, TBP or the initiating form of RNAPII (CTD S5-P) are graphed as relative occupancy levels at the different amplicon locations (lower panel). (d) Scatter plot showing the genome-wide correlation of TFIIB binding to promoters (x-axis, log2 scale) and steady-state mRNA levels (y-axis, log2 scale) of the corresponding genes. The median of all expression array probesets with present calls is indicated by the dotted horizontal line. The red dots indicate the average expression in gene groups with increasing TFIIB occupancy. They were determined by moving a sliding window (step size 0.1) over the TFIIB data points and calculating the mean expression value for each increment. (e) Distribution of ranked gene expression quantiles (color-coding indicated to the right) in genes with increasing TFIIB occupancy levels. The difference in distributions was statistically evaluated using a Kolmogorov-Smirnov test (***P < 2e-16).
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Figure 2: Validation of TFIIB target promoters. (a) Signal distribution of TFIIB enrichment. I, II and III denote groups of promoters from the upper 10th, 60th to 80th, or lower 10th percentile and correspond to high, mid-to-low, or no TFIIB occupancy. (b) Target gene validation. Selected genes from groups I to III were analyzed by ChIP-qPCR using TFIIB or IgG control antibody in a third chromatin sample from LCL721 cells in which two independent ChIP reactions were performed (upper panel). Genes are ordered from left to right according to TFIIB levels on the promoter arrays. The relative ChIP recovery is expressed as percentage of input (y-axis). The bars represent the mean, error bars the range of the two ChIP experiments. Corresponding gene expression levels in LCL721 B cells are shown in the lower panel. These were determined using Affymetrix U133 Plus 2.0 microarrays. They represent normalized hybridization signals of gene-specific microarray probesets. N.A., not analyzed. (c) Assembly of an active PIC at the GAPDH promoter. ChIP-qPCR was conducted with eight primer pairs spanning the human GAPDH locus (numbered boxes in top scheme). Results of ChIPs in LCL721 B cells with antibodies for TFIIB, TBP or the initiating form of RNAPII (CTD S5-P) are graphed as relative occupancy levels at the different amplicon locations (lower panel). (d) Scatter plot showing the genome-wide correlation of TFIIB binding to promoters (x-axis, log2 scale) and steady-state mRNA levels (y-axis, log2 scale) of the corresponding genes. The median of all expression array probesets with present calls is indicated by the dotted horizontal line. The red dots indicate the average expression in gene groups with increasing TFIIB occupancy. They were determined by moving a sliding window (step size 0.1) over the TFIIB data points and calculating the mean expression value for each increment. (e) Distribution of ranked gene expression quantiles (color-coding indicated to the right) in genes with increasing TFIIB occupancy levels. The difference in distributions was statistically evaluated using a Kolmogorov-Smirnov test (***P < 2e-16).

Mentions: A subset of target genes was validated by quantitative ChIP-quantitative PCR (ChIP-qPCR) using a third independent B cell-derived chromatin sample. A total of 29 promoters were interrogated that represent high occupancy (group I, upper 10th percentile), mid-to-low occupancy (group II, 60th to 80th percentile), or no TFIIB occupancy (group III, lower 10th percentile) as determined by ChIP-chip (Figure 2a). Non-TSS regions were included as negative controls and a non-specific IgG ChIP served as background reference. With few exceptions, relative magnitudes of array signals were retained in the ChIP-qPCR analysis. Out of 25 promoters from groups I and II, 23 (92%) showed greater than 10-fold enrichment of TFIIB over control ChIP, proving them as true positives. Likewise, four of four group III promoters and four of four control regions were negative for TFIIB enrichment in ChIP-qPCR (Figure 2b). Based on the above confirmation rate, we estimate that approximately 6,000 (92% of 6,547) promoters - representing one-quarter of all 24,000 interrogated promoters - are bound by TFIIB. This is in line with previous estimates on the number of active promoters in human cells [25]. To corroborate specific promoter association of TFIIB, the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was scanned by ChIP-qPCR with eight primer pairs scattered throughout the locus (Figure 2c). Binding of TBP and the initiating form of RNAPII (phosphorylated at serine 5 in its carboxy-terminal domain) were monitored in parallel. All three factors showed pronounced binding to the GAPDH promoter, indicating assembly of an active PIC containing TFIIB. Similar results were obtained at other large gene loci (data not shown). TFIIB did not bind the 3' region of GAPDH or other genes [22] as was recently reported for yeast genes [27].


Basal core promoters control the equilibrium between negative cofactor 2 and preinitiation complexes in human cells.

Albert TK, Grote K, Boeing S, Meisterernst M - Genome Biol. (2010)

Validation of TFIIB target promoters. (a) Signal distribution of TFIIB enrichment. I, II and III denote groups of promoters from the upper 10th, 60th to 80th, or lower 10th percentile and correspond to high, mid-to-low, or no TFIIB occupancy. (b) Target gene validation. Selected genes from groups I to III were analyzed by ChIP-qPCR using TFIIB or IgG control antibody in a third chromatin sample from LCL721 cells in which two independent ChIP reactions were performed (upper panel). Genes are ordered from left to right according to TFIIB levels on the promoter arrays. The relative ChIP recovery is expressed as percentage of input (y-axis). The bars represent the mean, error bars the range of the two ChIP experiments. Corresponding gene expression levels in LCL721 B cells are shown in the lower panel. These were determined using Affymetrix U133 Plus 2.0 microarrays. They represent normalized hybridization signals of gene-specific microarray probesets. N.A., not analyzed. (c) Assembly of an active PIC at the GAPDH promoter. ChIP-qPCR was conducted with eight primer pairs spanning the human GAPDH locus (numbered boxes in top scheme). Results of ChIPs in LCL721 B cells with antibodies for TFIIB, TBP or the initiating form of RNAPII (CTD S5-P) are graphed as relative occupancy levels at the different amplicon locations (lower panel). (d) Scatter plot showing the genome-wide correlation of TFIIB binding to promoters (x-axis, log2 scale) and steady-state mRNA levels (y-axis, log2 scale) of the corresponding genes. The median of all expression array probesets with present calls is indicated by the dotted horizontal line. The red dots indicate the average expression in gene groups with increasing TFIIB occupancy. They were determined by moving a sliding window (step size 0.1) over the TFIIB data points and calculating the mean expression value for each increment. (e) Distribution of ranked gene expression quantiles (color-coding indicated to the right) in genes with increasing TFIIB occupancy levels. The difference in distributions was statistically evaluated using a Kolmogorov-Smirnov test (***P < 2e-16).
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Figure 2: Validation of TFIIB target promoters. (a) Signal distribution of TFIIB enrichment. I, II and III denote groups of promoters from the upper 10th, 60th to 80th, or lower 10th percentile and correspond to high, mid-to-low, or no TFIIB occupancy. (b) Target gene validation. Selected genes from groups I to III were analyzed by ChIP-qPCR using TFIIB or IgG control antibody in a third chromatin sample from LCL721 cells in which two independent ChIP reactions were performed (upper panel). Genes are ordered from left to right according to TFIIB levels on the promoter arrays. The relative ChIP recovery is expressed as percentage of input (y-axis). The bars represent the mean, error bars the range of the two ChIP experiments. Corresponding gene expression levels in LCL721 B cells are shown in the lower panel. These were determined using Affymetrix U133 Plus 2.0 microarrays. They represent normalized hybridization signals of gene-specific microarray probesets. N.A., not analyzed. (c) Assembly of an active PIC at the GAPDH promoter. ChIP-qPCR was conducted with eight primer pairs spanning the human GAPDH locus (numbered boxes in top scheme). Results of ChIPs in LCL721 B cells with antibodies for TFIIB, TBP or the initiating form of RNAPII (CTD S5-P) are graphed as relative occupancy levels at the different amplicon locations (lower panel). (d) Scatter plot showing the genome-wide correlation of TFIIB binding to promoters (x-axis, log2 scale) and steady-state mRNA levels (y-axis, log2 scale) of the corresponding genes. The median of all expression array probesets with present calls is indicated by the dotted horizontal line. The red dots indicate the average expression in gene groups with increasing TFIIB occupancy. They were determined by moving a sliding window (step size 0.1) over the TFIIB data points and calculating the mean expression value for each increment. (e) Distribution of ranked gene expression quantiles (color-coding indicated to the right) in genes with increasing TFIIB occupancy levels. The difference in distributions was statistically evaluated using a Kolmogorov-Smirnov test (***P < 2e-16).
Mentions: A subset of target genes was validated by quantitative ChIP-quantitative PCR (ChIP-qPCR) using a third independent B cell-derived chromatin sample. A total of 29 promoters were interrogated that represent high occupancy (group I, upper 10th percentile), mid-to-low occupancy (group II, 60th to 80th percentile), or no TFIIB occupancy (group III, lower 10th percentile) as determined by ChIP-chip (Figure 2a). Non-TSS regions were included as negative controls and a non-specific IgG ChIP served as background reference. With few exceptions, relative magnitudes of array signals were retained in the ChIP-qPCR analysis. Out of 25 promoters from groups I and II, 23 (92%) showed greater than 10-fold enrichment of TFIIB over control ChIP, proving them as true positives. Likewise, four of four group III promoters and four of four control regions were negative for TFIIB enrichment in ChIP-qPCR (Figure 2b). Based on the above confirmation rate, we estimate that approximately 6,000 (92% of 6,547) promoters - representing one-quarter of all 24,000 interrogated promoters - are bound by TFIIB. This is in line with previous estimates on the number of active promoters in human cells [25]. To corroborate specific promoter association of TFIIB, the glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) was scanned by ChIP-qPCR with eight primer pairs scattered throughout the locus (Figure 2c). Binding of TBP and the initiating form of RNAPII (phosphorylated at serine 5 in its carboxy-terminal domain) were monitored in parallel. All three factors showed pronounced binding to the GAPDH promoter, indicating assembly of an active PIC containing TFIIB. Similar results were obtained at other large gene loci (data not shown). TFIIB did not bind the 3' region of GAPDH or other genes [22] as was recently reported for yeast genes [27].

Bottom Line: We compare target genes of TFIIB and NC2 in human B cells and analyze associated core promoter architectures.TATA and TATA-like elements dictate TFIIB occupancy at a subset of genes.Biochemical data support a model in which preinitiation complex but not TBP-NC2 complex formation is regulated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular Tumor Biology (IMTB), University of Muenster, Robert-Koch-Str, Muenster, Germany. albertt@uni-muenster.de

ABSTRACT

Background: The general transcription factor TFIIB and its antagonist negative cofactor 2 (NC2) are hallmarks of RNA polymerase II (RNAPII) transcription. Both factors bind TATA box-binding protein (TBP) at promoters in a mutually exclusive manner. Dissociation of NC2 is thought to be followed by TFIIB association and subsequent preinitiation complex formation. TFIIB dissociates upon RNAPII promoter clearance, thereby providing a specific measure for steady-state preinitiation complex levels. As yet, genome-scale promoter mapping of human TFIIB has not been reported. It thus remains elusive how human core promoters contribute to preinitiation complex formation in vivo.

Results: We compare target genes of TFIIB and NC2 in human B cells and analyze associated core promoter architectures. TFIIB occupancy is positively correlated with gene expression, with the vast majority of promoters being GC-rich and lacking defined core promoter elements. TATA elements, but not the previously in vitro defined TFIIB recognition elements, are enriched in some 4 to 5% of the genes. NC2 binds to a highly related target gene set. Nonetheless, subpopulations show strong variations in factor ratios: whereas high TFIIB/NC2 ratios select for promoters with focused start sites and conserved core elements, high NC2/TFIIB ratios correlate to multiple start-site promoters lacking defined core elements.

Conclusions: TFIIB and NC2 are global players that occupy active genes. Preinitiation complex formation is independent of core elements at the majority of genes. TATA and TATA-like elements dictate TFIIB occupancy at a subset of genes. Biochemical data support a model in which preinitiation complex but not TBP-NC2 complex formation is regulated.

Show MeSH
Related in: MedlinePlus