Genome-wide organization of transcription machinery detected at single nucleotide resolution.

摘要

Gene expression plays an important role in control of cell growth and differentiation in eukaryotic cells. Many proteins control gene expression by interacting with each other or with specific DNA sequences on the genome. Thus, determining where these proteins bind to DNA in vivo is essential to understanding how they regulate gene expression in eukaryotes. Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) is the most widely used method to identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce erroneous binding locations and imprecision in mapping. Consequently, stringent data filtering produces missed binding locations.;To overcome these limitations, I have developed a ChIP-exo method by combining exonuclease digestion with ChIP. ChIP-exo allows us to map a vast majority of genomic protein-DNA interactions at near single nucleotide accuracy. In this dissertation, I present an unprecedented view into genome-wide binding of sequence-specific DNA-binding proteins from yeast to human using ChIP-exo. I find that binding sites become unambiguous and reveal diverse tendencies governing in vivo DNA binding specificity that include sequence variants, functionally distinct motifs, motif clustering, secondary interactions, and combinatorial modules within a compound motif.;Furthermore, I reveal the structural and positional organization of the eukaryotic transcription pre-initiation complex (PIC) across a genome. I use ChIP-exo to identify and probe the organization of ∼6,000 PICs at near single nucleotide resolution throughout the yeast genome. PICs, which include RNA polymerase (Pol) II and each general transcription factor (GTF), are precisely positioned around the transcription start site (TSS). Exonuclease patterns are entirely consistent with crystallographic models of the PIC, and are sufficiently precise to identify TATA-like elements at nearly all so-called TATA-less promoters. Their unambiguous detection may help distinguish bona-fide genes from transcriptional noise. Taken together, the work presented in this dissertation reveals a genome-wide organization of transcription machinery at near single nucleotide resolution and allows us a more complete assessment of regulatory networks in which transcription factors regulate gene expression.