SUMMARY Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives (erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak-pairs by deep sequencing. Contaminating DNA is degraded or fails to form complementary peak-pairs. With the single bp accuracy provided by ChIP-exo, we show an unprecedented view into genome-wide binding of the yeast transcription factors Reb1, Gal4, Phd1, Rap1, and human CTCF. Each of these factors was chosen to address potential limitations of ChIP-exo. We found 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.

SUMMARYThe structural and positional organization of transcription pre-initiation complexes (PICs) across eukaryotic genomes is unknown. We employed ChIP-exo to precisely examine ~6,000 PICs in Saccharomyces. PICs, including RNA polymerase II and general factors TFIIA, -B, -D/TBP, -E, -F, -H, and -K were positioned within promoters and excluded from coding regions. Exonuclease patterns agreed with crystallographic models of the PIC, and were sufficiently precise to identify TATA-like elements at so-called TATA-less promoters. These PICs and their transcription start sites were positionally constrained at TFIID-engaged +1 nucleosomes. At TATA box-containing promoters, which are depleted of TFIID, a +1 nucleosome was positioned to be in competition with the PIC, which may afford greater latitude in start site selection. Our genomic localization of mRNA and noncoding RNA PICs reveal that two PICs, in inverted orientation, may occupy the flanking borders of nucleosome-free regions. Their unambiguous detection may help distinguish bona-fide genes from transcriptional noise.

SUMMARY Genes are packaged into nucleosomal arrays, each nucleosome typically having two copies of histones H2A, H2B, H3, and H4. Histones have distinct post-translational modifications, variant isoforms, and dynamics. Whether each histone copy within a nucleosome has distinct properties, particularly in relation to the direction of transcription, is unknown. Here we use ChlP-exo to resolve the organization of individual histones on a genomic scale. We detect widespread subnucleosomal structures in dynamic chromatin, including what appear to be half nucleosomes consisting of one copy of each histone. We also detect interactions of H3 tails with linker DNA between nucleosomes, which may be negatively regulated by methylation of H3K36. Histone variant H2A.Z is enriched on the promoter-distal half of the +1 nucleosome, whereas H2BK123 ubiquitylation and H3K9 acetylation are enriched on the promoter-proximal half in a transcription-linked manner. Subnucleosome asymmetries might serve as molecular beacons that guide transcription.

This unit describes ChIP-exo methodology, which involves chromatin immunoprecipitation (ChIP) combined with lambda exonuclease digestion followed by high-throughput sequencing. ChIP-exo allows us to identify a nearly complete set of binding locations of DNA-bound proteins at near single nucleotide resolution with almost no background. The process is initiated by crosslinking DNA and associated proteins. Chromatin is then isolated from nuclei and subjected to sonication. Subsequently, an antibody against a specific protein is used to immunoprecipitate specific DNA-protein complexes. ChIP DNA is purified, sequencing adaptors are ligated, and digested by lambda exonuclease. High-throughput sequencing generates 25 to 50 nucleotides sequences. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding locations. 5 ends of DNA fragments on the forward strand indicate the left border of DNA-protein boundaries and 5 ends of DNA fragments on the reverse strand indicate the right border of DNA-protein boundaries.

SUMMARY A canonical nucleosome architecture around promoters establishes the context in which proteins regulate gene expression. Whether gene regulatory proteins that interact with nucleosomes are selective for individual nucleosome positions across the genome is not known. Here we examine in Saccharomyces several protein-nucleosome interactions, including those that 1) bind histones (Bdf1/SWR1 and Srm1), 2) bind specific DNA sequences (Rap1 and Reb1), and 3) potentially collide with nucleosomes during transcription (RNA polymerase II). We find that the Bdf1/SWR1 complex forms a di-nucleosome complex that is selective for the +1 and +2 nucleosomes of active genes. Rap1 selectively binds to its cognate site on the rotationally exposed first and second helical turn of DNA inside either border of the −1 nucleosome, whereas Reb1 is selective for a single NFR-proximal border of the −1 nucleosome. We find that a transcribing RNA polymerase creates a delocalized state of resident nucleosomes. These findings suggest that nucleosomes around promoter regions have position-specific functions, and that some gene regulators have position-specific nucleosomal interactions.