Mapping E. coli RNA polymerase and associated transcription factors and identifying promoters genome-wide.

The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions vary depending upon the organism and the nature of the DNA-binding proteins under study. Here, we describe a customized ChIP protocol to examine the genome-wide distribution of a mobile DNA-binding enzyme, Escherichia coli RNA Polymerase (RNAP) as well as the factors that dynamically associate with RNAP during different stages of transcription. We describe new data analysis methods for determining the association of a broadly distributed DNA-binding complex. Further, we describe our approach of combining small molecules and antibiotics that perturb specific cellular events with ChIP and genomic platforms to dissect mechanisms of gene regulation in vivo. The chemical genomic methods can be leveraged to map natural and cryptic promoters and transcription units, annotate genomes, and reveal coupling between different processes in regulation of genes. This approach provides the framework for engineering gene networks and controlling biological output in a desired manner.

Use of an electronic medical record to characterize cases of intermediate statin-induced muscle toxicity.

Statin use can be accompanied by a variety of musculoskeletal complaints. The authors describe the clinical characteristics of case patients who experienced adverse statin-induced musculoskeletal symptoms within a large population-based cohort in Central Wisconsin. Case status was determined based on elevated serum creatine kinase (CK) levels and the presence of at least 1 physician note reflecting an increased index of suspicion for statin intolerance. From the medical records of nearly 2 million unique patients, the authors identified more than 20,000 potential study patients ( approximately 1%) having CK data and at least 1 exposure to a statin drug. Manual screening was completed on 2227 patients with CK levels in the upper 10th percentile. Of those screened, 267 met inclusion criteria (12.0% eligibility) and 218 agreed to participate in a retrospective study characterizing the risk determinants of statin-induced muscle toxicity. Three categoric pain variables were graded retrospectively (distribution, location, and severity of pain). The presenting complaints of the case patients were extremely heterogeneous. The number of patients with a compelling pain syndrome (diffuse, proximal muscle pain of high intensity) increased at higher serum CK levels; the number of patients with indeterminate pain variables decreased at higher serum CK levels. The lines reflecting these relationships cross at a CK level of approximately 1175 U/L, approximately half the threshold level needed to make a clinical diagnosis of "myopathy" (ie, CK >10-fold the upper limit of normal).

Chemical inhibition of the TFIIH-associated kinase Cdk7/Kin28 does not impair global mRNA synthesis.

The process of gene transcription requires the recruitment of a hypophosphorylated form of RNA polymerase II (Pol II) to a gene promoter. The TFIIH-associated kinase Cdk7/Kin28 hyperphosphorylates the promoter-bound polymerase; this event is thought to play a crucial role in transcription initiation and promoter clearance. Studies using temperature-sensitive mutants of Kin28 have provided the most compelling evidence for an essential role of its kinase activity in global mRNA synthesis. In contrast, using a small molecule inhibitor that specifically inhibits Kin28 in vivo, we find that the kinase activity is not essential for global transcription. Unlike the temperature-sensitive alleles, the small-molecule inhibitor does not perturb protein-protein interactions nor does it provoke the disassociation of TFIIH from gene promoters. These results lead us to conclude that other functions of TFIIH, rather than the kinase activity, are critical for global gene transcription.

Holoenzyme switching and stochastic release of sigma factors from RNA polymerase in vivo.

We investigated the binding of E. coli RNA polymerase holoenzymes bearing sigma70, sigma(S), sigma32, or sigma54 to the ribosomal RNA operons (rrn) in vivo. At the rrn promoter, we observed "holoenzyme switching" from Esigma70 to Esigma(S) or Esigma32 in response to environmental cues. We also examined if sigma factors are retained by core polymerase during transcript elongation. At the rrn operons, sigma70 translocates briefly with the elongating polymerase and is released stochastically from the core polymerase with an estimated half-life of approximately 4-7 s. Similarly, at gadA and htpG, operons that are targeted by Esigma(S) and Esigma32, respectively, we find that sigma(S) and sigma32 also dissociate stochastically, albeit more rapidly than sigma70, from the elongating core polymerase. Up to approximately 70% of Esigma70 (the major vegetative holoenzyme) in rapidly growing cells is engaged in transcribing the rrn operons. Thus, our results suggest that at least approximately 70% of cellular holoenzymes release sigma70 during transcript elongation. Release of sigma factors during each round of transcription provides a simple mechanism for rapidly reprogramming polymerase with the relevant sigma factor and is consistent with the occurrence of a "sigma cycle" in vivo.

Immobilization of Escherichia coli RNA polymerase and location of binding sites by use of chromatin immunoprecipitation and microarrays.

The genome-wide location of RNA polymerase binding sites was determined in Escherichia coli using chromatin immunoprecipitation and microarrays (chIP-chip). Cross-linked chromatin was isolated in triplicate from rifampin-treated cells, and DNA bound to RNA polymerase was precipitated with an antibody specific for the beta' subunit. The DNA was amplified and hybridized to "tiled" oligonucleotide microarrays representing the whole genome at 25-bp resolution. A total of 1,139 binding sites were detected and evaluated by comparison to gene expression data from identical conditions and to 961 promoters previously identified by established methods. Of the detected binding sites, 418 were located within 1,000 bp of a known promoter, leaving 721 previously unknown RNA polymerase binding sites. Within 200 bp, we were able to detect 51% (189/368) of the known sigma70-specific promoters occurring upstream of an expressed open reading frame and 74% (273/368) within 1,000 bp. Conversely, many known promoters were not detected by chIP-chip, leading to an estimated 26% negative-detection rate. Most of the detected binding sites could be associated with expressed transcription units, but 299 binding sites occurred near inactive transcription units. This map of RNA polymerase binding sites represents a foundation for studies of transcription factors in E. coli and an important evaluation of the chIP-chip technique.