Genome-wide analysis of ETS-family DNA-binding in vitro and in vivo

Peer reviewe

Objective sequence-based subfamily classifications of mouse homeodomains reflect their in vitro DNA-binding preferences

Classifying proteins into subgroups with similar molecular function on the basis of sequence is an important step in deriving reliable functional annotations computationally. So far, however, available classification procedures have been evaluated against protein subgroups that are defined by experts using mainly qualitative descriptions of molecular function. Recently, in vitro DNA-binding preferences to all possible 8-nt DNA sequences have been measured for 178 mouse homeodomains using protein-binding microarrays, offering the unprecedented opportunity of evaluating the classification methods against quantitative measures of molecular function. To this end, we automatically derive homeodomain subtypes from the DNA-binding data and independently group the same domains using sequence information alone. We test five sequence-based methods, which use different sequence-similarity measures and algorithms to group sequences. Results show that methods that optimize the classification robustness reflect well the detailed functional specificity revealed by the experimental data. In some of these classifications, 73–83% of the subfamilies exactly correspond to, or are completely contained in, the function-based subtypes. Our findings demonstrate that certain sequence-based classifications are capable of yielding very specific molecular function annotations. The availability of quantitative descriptions of molecular function, such as DNA-binding data, will be a key factor in exploiting this potential in the future.Canadian Institutes of Health Research (MOP#82940)Sickkids FoundationOntario Research FundNational Science Foundation (U.S.)National Human Genome Research Institute (U.S.) (R01 HG003985

A Library of Yeast Transcription Factor Motifs Reveals a Widespread Function for Rsc3 in Targeting Nucleosome Exclusion at Promoters

The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes, one-third of which have not been previously reported. Several newly discovered binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially ~100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters

A snoRNA that guides the two most conserved pseudouridine modifications within rRNA confers a growth advantage in yeast

International audienceRibosomal RNAs contain a number of modified nucleotides. The most abundant nucleotide modifications found within rRNAs fall into two types: 2′-O-ribose methylations and pseudouridylations. In eukaryotes, small nucleolar guide RNAs, the snoRNAs that are the RNA components of the snoRNPs, specify the position of these modifications. The 2′-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs, respectively. The role of these modifications in rRNA remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2′-O-ribose methylations or pseudouridylations. Only very recently, a slight translation defect and perturbation of polysome profiles was reported in yeast for the absence of the Ψ at position 2919 within the LSU rRNA. Here we report the identification and characterization in yeast of a novel intronic H/ACA snoRNA that we called snR191 and that guides pseudouridylation at positions 2258 and 2260 in the LSU rRNA. Most interestingly, these two modified bases are the most conserved pseudouridines from bacteria to human in rRNA. The corresponding human snoRNA is hU19. We show here that, in yeast, the presence of this snoRNA, and hence, most likely, of the conserved pseudouridines it specifies, is not essential for viability but provides a growth advantage to the cell

TFCat: the curated catalog of mouse and human transcription factors

Unravelling regulatory programs governed by transcription factors (TFs) is fundamental to understanding biological systems. TFCat is a catalog of mouse and human TFs based on a reliable core collection of annotations obtained by expert review of the scientific literature. The collection, including proven and homology-based candidate TFs, is annotated within a function-based taxonomy and DNA-binding proteins are organized within a classification system. All data and user-feedback mechanisms are available at the TFCat portal http://www.tfcat.ca .Medical Genetics, Department ofMedicine, Faculty ofMolecular Medicine and Therapeutics, Centre forReviewedFacult

Novel Widespread Marine Oomycetes Parasitising Diatoms, Including the Toxic Genus Pseudo-nitzschia: Genetic, Morphological, and Ecological Characterisation

Parasites are key drivers of phytoplankton bloom dynamics and related aquatic ecosystem processes. Yet, the dearth of morphological and molecular information hinders the assessment of their diversity and ecological role. Using single-cell techniques, we characterise morphologically and molecularly, intracellular parasitoids infecting four potentially toxin-producing Pseudo-nitzschia and one Melosira species on the North Atlantic coast. These sequences define two, morphologically indistinguishable clades within the phylum Oomycota, related to the genera of algal parasites Anisolpidium and Olpidiopsis and the diatom parasitoid species Miracula helgolandica. Our morphological data are insufficient to attribute either clade to the still unsequenced genus Ectrogella; hence it is proposed to name the clades OOM_1 and OOM_2. A screening of global databases of the barcode regions V4 and V9 of the 18S rDNA demonstrate the presence of these parasitoids beyond the North Atlantic coastal region. During a biweekly metabarcoding survey (Concarneau Bay, France), reads associated with one sequenced parasitoid coincided with the decline of Cerataulina pelagica bloom, whilst the other parasitoids co-occurred at low abundance with Pseudo-nitzschia. Our data highlight a complex and unexplored diversity of the oomycete parasitoids of diatoms and calls for the investigation of their phenology, evolution, and potential contribution in controlling their host spatial-temporal dynamics

Xrn1 depletion and eIF2A deletion are synthetic lethal.

Wild-type and mutant strains were serially diluted and spotted on YPGlu rich medium supplemented or not with CFW in the presence or not of IAA (100 μM Auxin) to deplete the cells of Xrn1. uth1Δ and hsp150Δ mutants were used as a control.</p

Decrease of Sun4 protein level upon <i>eIF2A</i> overexpression requires the presence of Ssd1.

(A) Wild-type and the ssd1Δ mutant cells harboring pCM190 or pCM190: eIF2A plasmids and expressing Sun4-TAP protein were grown in -URA medium and harvested after 5 hours of eIF2A overexpression (+) or not (-). Protein extracts were separated on a polyacrylamide gel and Sun4-TAP was revealed by Western Blot using PAP antibodies. G6PDH was used as a loading control. (B) Quantification of Western Blot analyzes was performed as described in Fig 3B. Error bars indicate the standard deviations of averages from at least three independent experiments. Statistical analysis was performed using a t-test, p = 0.0054. The dots correspond to the value obtained for each individual replicate. Asterisks indicate statistical significances (*: p-value ≤0.05, **: p-value ≤ 0.01).</p

The <i>SUN4</i>, <i>CTS1</i>, <i>SRL1</i> mRNAs are enriched by eIF2A-TAP only in the presence of Ssd1.

Wild-type and the ssd1Δ mutant strains expressing eIF2A-TAP protein were cultivated to exponential-growth phase. RIP experiment was performed as described in Materials and Methods. 8 μg of total RNA (input) and 1 μg of immunoprecipitated RNA (RIP eIF2A-TAP) were respectively separated by an agarose gel. SUN4, CTS1, SRL1 and RPL28 mRNAs were revealed by Northern Blot with appropriate DIG-labeled probes and anti-DIG antibody.</p