A high-resolution map of active promoters in the human genome

Abstract

In eukaryotic cells, transcription of every protein-coding gene begins with the assembly of an RNA Polymerase II preinitiation complex (PIC) on the promoter 1 . The promoters, in conjunction with enhancers, silencers and insulators, define the combinatorial codes that specify gene expression patterns 2 . Our ability to analyze the control logic encoded in the human genome is currently limited by a lack of accurate information of the promoters for most genes 3 . Here, we describe a genomewide map of active promoters in human fibroblast cells, determined by experimentally locating the sites of PIC binding throughout the human genome. This map defines 10,571 active promoters corresponding to 6,763 known genes and at least 1,199 un-annotated transcriptional units. Features of the map suggest extensive usage of multiple promoters by the human genes and widespread clustering of active promoters in the genome. In addition, examination of the genome-wide expression profile reveals four general classes of promoters that define the transcriptome of the cell. These results provide a global view of the functional relationship among the transcriptional machinery, chromatin structure and gene expression in human cells.The PIC consists of the RNA Polymerase II (RNAP), the transcription factor IID (TFIID) and other general transcription factors 4 . Our strategy to map the PIC binding sites involves a chromatin immunoprecipitation coupled DNA microarray analysis (ChIP-on-chip), which combines the immunoprecipitation of PIC-bound chromatin from formaldehyde crosslinked cells with parallel identification of the resulting bound DNA sequences using DNA microarrays 5,6 . Previously, we have demonstrated the feasibility of this strategy by successfully mapping active promoters in 1% of the human genome that correspond to the 44 genomic loci known as the ENCODE regions 6,7 . To apply this strategy to the entire human genome, we fabricated a series of DNA microarrays 8 containing roughly 14.5 million 50-mer oligonucleotides, designed to represent all the non-repeat DNA throughout the human genome at 100 basepairs (bp) resolution. We immunoprecipitated TFIID-bound DNA from the primary fibroblast IMR90 cells with a monoclonal antibody that specifically recognizes the TAF1 subunit of this complex (TBP associated factor 1, formerly TAF II 250 9, Fig 1a). We then amplified and fluorescently labeled the resulting DNA, and hybridized it to the above microarrays along with a differentially labeled control DNA (Fig. 1a). We determined 9,966 potential TFIID-binding regions using a simple algorithm requiring a stretch of four neighboring probes to have a hybridization signal significantly above the background. To 6 To whom correspondence should be addressed. Email: biren@ucsd.edu. Phone: 858 822 5766; Fax: 858 534 7750. 5 These two authors contributed equally to this work. Author to which correspondence and material request should be addressed: Bing Ren, biren@ucsddu. The microarray datasets are available from GEO (accession numbers to be ...

TRF2: TRansForming the view of general transcription factors

“…10 Although the TATA box is wellknown and extensively studied, it is only present in 10%-15% of mammalian core promoters, and in about 20% of Drosophila genes. 7,[11][12][13][14][15] The BRE motifs function together with the TATA box. The BRE u is located immediately upstream of the TATA box (with a SSRCGCC consensus sequence), and the BRE d is located immediately downstream of the TATA box (with a RTDKKKK consensus sequence).…”

“…[16][17][18] The initiator (Inr) is the most commonly occurring motif among core promoter elements. [12][13][14][15]19 The Inr encompasses the TSS 20 and its consensus is YYANWYY in humans and TCAKTY in Drosophila. Focused transcription generally starts at the A nucleotide of the Inr consensus that is referred to as "C1" of the TSS (whether transcription starts at this nucleotide or nearby).…”

Promoter architectures and developmental gene regulation

“…Various features of active promoters such as the presence of the PIC, promoter-associated histone marks and accessible and open chromatin have been used to localise promoters [16]. These approaches can only identify loci that serve as promoters, but cannot map precise transcription start sites or quantify the level of transcription from the detected promoters.…”

Identifying regulatory elements in eukaryotic genomes

“…For instance, ChIP experiments profiling the insulator protein CTCF have identified locations of putative CTCF-binding insulators in multiple organisms and cell types [56][57][58]. Similar experiments with different proteins have been used to identify promoters, enhancers and silencers [59][60][61][62].…”