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On Human Promoters

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On Human Promoters Global distribution of negative cofactor 2 subunit-␣ on human promoters Thomas K. Albert*, Korbinian Grote†, Stefan Boeing*, Gertraud Stelzer*, Aloys Schepers‡, and Michael Meisterernst*§ Departments of *Gene Expression and ‡Gene Vectors, GSF–National Research Center for Environment and Health, Marchioninistrasse 25, 81377 Munich, Germany; and †Genomatix Software GmbH, Bayerstrasse 85a, 80335 Munich, Germany Communicated by Robert G. Roeder, The Rockefeller University, New York, NY, April 30, 2007 (received for review November 16, 2006) Negative cofactor 2 (NC2) forms a stable complex with TATA- of multiple sequence-specific transcription factors in mammalian binding protein (TBP) on promoters in vitro. Its association with TBP genomes. However, surprisingly little is known about the genome- prevents the binding of TFIIB and leads to inhibition of preinitiation wide location of GTFs and general cofactors in mammalian cells. complex formation. Here, we investigate the association of NC2 One exception is the investigation of the genome-wide location of subunit-␣ with human RNA polymerase II promoter regions by TAF1 conducted along with RNAPII (16). using gene-specific ChIP and genome-wide promoter ChIPchip In our analysis, the human cofactor NC2␣ occupied Ͼ20% of analyses. We find NC2␣ associated with a large number of human all human gene promoters. We observed a positive correlation promoters, where it peaks close to the core regions. NC2 occupancy of NC2 gene occupancy with mRNA levels. On the other hand, in vivo positively correlates with mRNA levels, which perhaps NC2 occupancy negatively correlated with the presence of BRE, reflects its capacity to stabilize TBP on promoter regions. In single the TFIIB core promoter recognition element. Collectively, the gene analyses, we confirm core promoter binding and in addition data support the notion of a global role for NC2 in gene control. map the NC2 complex to enhancer proximal regions. High- At the single gene level the NC2/TFIIB ratio is controlled in cis. occupancy histone genes display a stable NC2/TFIIB ratio during the cell cycle, which otherwise varies markedly from one gene to Results another. The latter is at least in part explained by an observed Genome-Wide Promoter Association of NC2␣. ChIPchip analysis was negative correlation of NC2 occupancy with the presence of the conducted with high-density promoter tiling arrays that cover TFIIB recognition element in core promoter regions. Our data Ϸ24,000 human genes. NC2-DNA complexes were precipitated establish the genome-wide basis for general and gene-specific from a human Epstein–Barr virus (EBV)-transformed lympho- functions of NC2 in mammalian cells. blastoid B cell line, LCL721, by using a rat monoclonal antibody directed against the ␣-subunit of the NC2 complex (17). Pro- genome-wide ͉ occupancy moters were subsequently ranked according to the median of the fold enrichments (in comparison with input DNA) on 15 probes ranscription control of gene expression in eukaryotes in- for each individual promoter. Of the 24,099 promoter regions ␣ volves a dynamic interplay of positively and negatively acting analyzed, 2,759 (11.4%) displayed high NC2 levels with a fold T Ͼ factors. The central nucleation factor for the assembly of RNA enrichment of 1.4, and another 3,517 promoters (14.6%) ␣ Ͼ polymerase II (RNAPII) initiation complexes at promoters is showed medium NC2 enrichment of 1.2 (Fig. 1b). The TBP, the TATA-binding protein (1). Binding of TBP to the remaining 17,823 genes displayed fold enrichments below the ␣ TATA box is a rate-limiting step during preinitiation complex threshold of 1.2 and represent genes with low or no NC2 (PIC) formation. Multiple factors target TBP or contribute to promoter occupancy [supporting information (SI) Data Set 1]. the formation of TBP–DNA complexes by using distinct mech- As a control, ChIPchip was conducted in parallel with a anisms. Specific examples include general transcription factors nonspecific IgG monoclonal antibody (SI Fig. 7). The alignment ␣ (GTFs) such as TFIIA and TFIIB, TBP-associated factors of both data sets indicated high specificity of the NC2 ChIP. (TAFs), and a group of proteins termed cofactors, among them Only a small fraction of the promoters (269/6,089, or 4.4%) with Ͼ ␣ BTAF1/Mot1p and negative cofactor NC2 (2). a median fold enrichment 1.2 in the NC2 ChIP showed a NC2 was originally defined as a TBP-binding activity in human comparable enrichment in the control ChIP (SI Fig. 7). nuclear extracts that repressed RNAPII transcription (3). The Although we clearly identified peaks on numerous genes with Ͻ cofactor is composed of two subunits, NC2␣ and NC2␤, that median fold enrichment 1.2 (for selected examples see SI Fig. dimerize by means of histone-fold domains. The NC2 complex 8), the choice of the thresholds remains arbitrary. To substan- binds to TBP–DNA from the underside, thereby preventing the tiate their relevance, we plotted the complete set of data against binding of TFIIB (Fig. 1a). Biochemical and genetic data the annotated main start sites of the genes. The group of suggested that NC2 further interferes with recruitment of promoters representing median fold enrichment of 1.2 to 1.4 TFIIA, thereby counteracting PIC assembly (3–7). showed maximum probe signals near the transcription start site The genes encoding NC2 are absent in prokaryotes including (Fig. 1c). Peak formation is even more pronounced in the group of promoters with median fold enrichment Ͼ1.4. In contrast, Archea but are conserved in all eukaryotes. In yeast, the corre- ␣ sponding genes, BUR6 and YDR1, emerged as transcriptional low-occupancy NC2 target genes and control sets showed a cofactors in independent genetic screens (8–10). Yeast gene expression profiling confirmed repressive functions but also Author contributions: T.K.A. and M.M. designed research; T.K.A., S.B. and G.S. performed revealed stimulatory effects of NC2 on gene transcription (11– research; A.S. contributed analytical tools; T.K.A. and K.G. analyzed data; and T.K.A. and 13). The cofactor associates with yeast and human promoters in M.M. wrote the paper. vivo. Mammalian NC2 displayed little specificity for TATA. The authors declare no conflict of interest. Consistently, both TATA and TATA-less core promoter were Abbreviations: ChIPchip, ChIP on DNA microarrays; NC2, negative cofactor 2; RNAPII, RNA recognized by NC2 in vivo (11, 14). polymerase II; TBP, TATA-binding protein; TCR␤, T cell receptor ␤. Here, we initiated chromatin immunoprecipitation on microar- §To whom correspondence should be addressed. E-mail: [email protected]. rays (ChIPchip) investigations with human NC2 in B cells. First This article contains supporting information online at www.pnas.org/cgi/content/full/ applied to low-complexity genomes such as those of yeast (15), 0703490104/DC1. ChIPchip has now been successfully used to determine the location © 2007 by The National Academy of Sciences of the USA 10000–10005 ͉ PNAS ͉ June 12, 2007 ͉ vol. 104 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703490104 Downloaded by guest on October 2, 2021 a TBP TBP TATA TATA NC2α NC2β TFIIB b 3% 4% 9% 16% 28% 15% NC2α Control 46% 80% Median fold N Occupancy enrichment Fig. 2. NC2␣ ChIPchip profile on individual promoters. Shown are NC2␣ occu- 616 Very high > 2.0 pancies at the selected ribosomal protein gene RPL7A (Left) and at the RNA 2,143 High 2.0 - 1.4 processing factor gene CSTF3 (Right) in LCL721 cells. Log2 ratios of the NC2␣ ChIP 3,517 Medium 1.4 - 1.2 versus input DNA on individual array probes are plotted. The approximate width 11,008 Low 1.2 - 1.0 6,815 No < 1.0 of the peak area is outlined. The broken arrows mark transcription start sites. 80 c )%( the mean of all signals 7,154 peaks were detected (low- s 60 confidence set). In the corresponding set of data set 2, 72.5% of e b o the peaks identified under stringent conditions in data set 1 are r p f p 40 NC2α cut-off >1.4 present . If the top 5% or 10% of median fold enrichments in o o n NC2α cut-off >1.2 data set 2 were compared with the top 25% of data set 1 o it 20 α Ͼ c NC2 no cut-off (referring to all genes with a median fold 1.2), 92% and 83%, arF Control respectively, of the genes were recovered. Similar numbers were 0 0 004 0 0021- 0 obtained with alternative algorithms (18). As documented below 0 0 4 8 - - (Fig. 3) the fraction of NC2-positive genes further increases if Distance from TSS (bp) only active genes are taken into account. Furthermore, our own unpublished observation show that occupancy numbers of NC2␣ ␣ Fig. 1. Genome-wide promoter association of NC2 .(a) Structure of the are close to the one observed for the general factor TFIIB NC2–TBP–TATA complex (31) and of the TFIIB–TBP–TATA complex (38). (b) Charts showing the distribution of enrichment classes of 24,099 human pro- (T.K.A. and M.M., unpublished observation). Altogether, these moters in a NC2␣ ChIP (Left) and an isotype IgG control ChIP (Right) performed data support the argument for global gene occupancy and for a in LCL721 B cells. Five enrichment classes were ranked according to the general role of NC2 in gene transcription. indicated median fold enrichments on 15 probes per individual promoter. (c) Distribution of the distance between NC2␣-bound probes and the closest NC2␣ Occupancy Positively Correlates with mRNA Levels. Affymetrix transcription start site (TSS). The fraction of bound probes that map to 100-bp microarrays (SI Data Set 2) were used to correlate NC2␣ BIOCHEMISTRY intervals from the TSS at the indicated cut-offs is shown.
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