Project description:Cullin-RING ubiquitin ligases (CRLs) control the degradation of a wide landscape of human proteins in combination with ubiquitin-carrying enzymes (UCEs). CRL expansion during evolution is apparent, with a few dozen in yeast that function with a single UCE and as many as 300 in humans that function with at least 8 UCEs. A major unaddressed question is why human CRL buildup has been accompanied by additional UCEs that function with CRLs. Here we demonstrate that human CRLs and UCEs can display specificity, resulting in increased affinity for each other and enhanced rates of ubiquitin transfer to substrates. To uncover the structural basis for CRL-UCE specificity, cryo-EM was performed on a CRL2 subfamily member with substrate receptor subunit FEM1C (CRL2FEM1C) in complex with a proxy for catalytically active UCE. The structure elucidated an extensive CRL-UCE interface that promotes proximity between the UCE active site and CRL2FEM1C-bound substrate. Unanticipated selectivity was also observed between the CRL substrate Lys ubiquitylation sites and the identity of the UCE. CRL-UCE specificity also manifests during targeted protein degradation by affecting the activities of drugs that induce ubiquitylation of neosubstrates. An emerging CRL code is revealed that drives selective formation of CRL-UCE complexes to promote rapid substrate ubiquitylation.
Project description:We perform a self hybridisation comprative genomic hybridization (CGH) in order to validate the probe tiling design we done on Trichoderma reesei. This hybridization was done using QM6a wild type strain.
Project description:In this study we used mice lacking Evf2 (Evf2TS/TS) and mice expressing a truncated form of Evf2 (Evf1TS/TS) to determine UCE lncRNA epigenetic and chromosome toplogical control. We used 4Cseq to investigate how Evf2 regulates UCE interactions along chromosome 6 (where Evf2 is expressed). We used ChIpseq to compare histone methylation profiles from Evf2TS/TS and Evf1TS/TS. In addition, we used ChIPseq to determine Evf2-depedent regulation of cohesin subunit binding (SMC1 and SMC3) and histone H3K27acetylation. Together, these data support that Evf2 UCE lncRNA controls chromosome topology over multi-megabse distances, through cohesin binding and effects on histone methylation and acetylation. Also included is the ChIPseq profile of Dlx binding sites in SW (outbred strain of mice) from E13.5 GE.
Project description:Whole-transcriptome gene-expression analyses are commonly performed in species that have a sequenced genome and for which microarrays are commercially available. To do such analyses in species with no or limited genome data, i.e. non-model organisms, necessary transcriptomics resources, i.e. an annotated transcriptome and a validated gene-expression microarray, must first be developed. The aim of the present study was to establish an advanced approach for developing transcriptomics resources for non-model organisms by combining next-generation sequencing (NGS) and microarray technology. We applied our approach to the non-biting midge Chironomus riparius, an ecologically relevant species that is widely used in sediment ecotoxicity testing. We sampled extensively covering all C. riparius developmental stages as well as toxicant exposed larvae and obtained from a normalized cDNA library 1.5 M NGS reads totalling 501 Mbp. Using the NGS data we developed transcriptomics resources in several steps. First, we designed 844 k probes directly on the NGS reads, as well as 76 k probes targeting expressed sequence tags of related species. These probes were tested for their affinity to C. riparius DNA and mRNA, by performing two biological experiments with a 1 M probe-selection microarray that contained the entire probe-library. Subsequently, the 1.5 M NGS reads were assembled into 23,709 isotigs and 135,082 singletons, which were associated to ~55 k, respectively, ~61 k gene ontology terms and which corresponded together to 22,593 unique protein accessions. An algorithm was developed that took the assembly and the probe affinities to DNA and mRNA into account, what resulted in 59 k highly-reliable probes that targeted uniquely 95% of the isotigs and 18% of the singletons. Concluding, our approach allowed the development of high-quality transcriptomics resources for C. riparius, and is applicable to any non-model organism. It is expected, that these resources will advance ecotoxicity testing with C. riparius as whole-transcriptome gene-expression analysis are now possible with this species.
