François Robert

Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity.

Set1, the yeast histone H3-lysine 4 (H3-K4) methylase, is recruited by the Pol II elongation machinery to a highly localized domain at the 5 portion of active mRNA coding regions. Set1 association depends upon the TFIIH-associated kinase that phosphorylates the Pol II C-terminal domain (CTD) and mediates the transition between initiation and elongation, and Set1 interacts with the form of Pol II whose CTD is phosphorylated at serine 5 but not serine 2. The Rtf1 and Paf1 components of the Pol II-associated Paf1 complex are also important for Set1 recruitment. Although the level of dimethylated H3-K4 is fairly uniform throughout the genome, the pattern of trimethylated H3-K4 strongly correlates with Set1 occupancy. Hypermethylated H3-K4 within the mRNA coding region persists for considerable time after transcriptional inactivation and Set1 dissociation from the chromatin, indicating that H3-K4 hypermethylation provides a molecular memory of recent transcriptional activity.

Computational discovery of gene modules and regulatory networks

We describe an algorithm for discovering regulatory networks of gene modules, GRAM (Genetic Regulatory Modules), that combines information from genome-wide location and expression data sets. A gene module is defined as a set of coexpressed genes to which the same set of transcription factors binds. Unlike previous approaches that relied primarily on functional information from expression data, the GRAM algorithm explicitly links genes to the factors that regulate them by incorporating DNA binding data, which provide direct physical evidence of regulatory interactions. We use the GRAM algorithm to describe a genome-wide regulatory network in Saccharomyces cerevisiae using binding information for 106 transcription factors profiled in rich medium conditions data* from over 500 expression experiments. We also present a genome-wide location analysis data set for regulators in yeast cells treated with rapamycin, and use the GRAM algorithm to provide biological insights into this regulatory network.*Note: In the version of this article initially published online, the word and was omitted from the fourth sentence of the abstract, altering the meaning. The sentence should read: We use the GRAM algorithm to describe a genome-wide regulatory network in Saccharomyces cerevisiae using binding information for 106 transcription factors profiled in rich medium conditions and data from over 500 expression experiments. This mistake has been corrected for the HTML and print versions of the article.

Use of Site-Specific Protein-DNA Photocrosslinking to Analyze the Molecular Organization of the RNA Polymerase II Initiation Complex

Site-specific protein–DNA photocrosslinking has proved to be the method of choice for analysis of the formation of nucleoprotein complexes such as those involved in transcription by mammalian RNA polymerase II (RNA Pol II). The method has two principal advantages. First, it yields structural information on large, multisubunit complexes that in general cannot be analyzed using standard high-resolution techniques such as X-ray crystallography or nuclear magnetic resonance (NMR). For example, site-specific protein–DNA photocrosslinking, in conjunction with complementary methods such as protein-affinity chromatography and electron microscopy, has produced information on both the molecular organization and the composition of the RNA Pol II pre-initiation complex on promoter DNA (1–4). This complex contains RNA Pol II and the general transcription factors TBP, TFIIA, TFIIB, TFIIE, TFIIF (RAP74 and RAP30), and TFIIH, and is composed of more than 25 polypeptides ranging in Mr from 10 to 220 kDa (5). Neither X-ray crystallography nor NMR, which can only resolve the structure of complexes containing short protein fragments bound to small pieces of promoter DNA, could provide any detailed structural information on this complex. Second, the method has sufficient technical flexibility so as to allow the rapid analysis of complexes assembled under various conditions. Over the past few years, we have analyzed a large collection of complexes assembled in the presence of various combinations of the general transcription factors (wild-type or different deletion mutants) and RNA Pol II (1–4). These experiments have enabled us to draw conclusions on the dynamics of RNA Pol II pre-initiation complex assembly and has led to the notion that isomerization of the RNA Pol II pre-initiation complex proceeds through wrapping of the promoter DNA around the enzyme (4). n nSite-specific protein–DNA photocrosslinking is a method composed of two successive steps. First, a number of photoprobes that place one (or a few) photoreactive nucleotide(s) into juxtaposition with one (or a few) radiolabeled nucleotide(s) at various specific positions along the promoter DNA are prepared. Second, transcription complexes are assembled onto the various photoprobes, irradiated with ultraviolet (UV) light so as to induce protein–DNA crosslinking, and the processed in order to identify the crosslinked polypeptides. Because the crosslinking of protein to DNA is site-specific, the use of a series of photoprobes that place the photonucleotide derivative at various positions along the promoter DNA provides information on the relative position of the various factors within the complex.