36 research outputs found
Targeted protein degradation via intramolecular bivalent glues
Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)âbifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target 1â4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change âgluesâ BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic targetâligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4âIBG1âDCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.</p
Targeted protein degradation via intramolecular bivalent glues
Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)âbifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target 1â4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change âgluesâ BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic targetâligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4âIBG1âDCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.</p
iRegulon and i-cisTarget: Reconstructing Regulatory Networks Using Motif and Track Enrichment
Gene expression profiling is often used to identify genes that are co-expressed in a biological process or disease. Downstream analyses of co-expressed gene sets using bioinformatics methods can reveal candidate transcription factors (TF) that co-regulate these genes, based on the presence of shared TF binding sites. Drawing gene regulatory networks that connect TFs to their predicted target genes can uncover gene modules that implement a particular function. Here, we describe several protocols to analyze any set of co-expressed genes using iRegulon and i-cisTarget. These tools perform regulatory sequence analysis (motif discovery) and integrate and mine large collections of existing regulatory data, such as ChIP-Seq, DHS-seq, and FAIRE-seq (track discovery). While iRegulon focuses on sets of co-expressed genes, i-cisTarget also analyses genomic regions as input. The following protocols describe how to install and use these tools, how to interpret the obtained results, and will thus help to create meaningful regulatory networks. © 2015 by John Wiley & Sons, Inc.status: publishe
Integrative analysis of regulatory tracks for the identification of direct TF-target interactions
status: accepte
EXPLORING THE P53 TRANSCRIPTIONAL NETWORK THROUGH INTEGRATIVE GENOMICS REVEALS NEW CANDIDATE TARGET GENES AND CO-FACTORS
As tumor suppressor many roles have been ascribed to p53 like cell cycle arrest and apoptosis but also metabolism and developmental processes. p53 functions as a transcription factor (TF) by interacting with a variety of target genes of which many have been reported but p53âs full targetome is likely incomplete. In addition, many other aspects of p53âs activity require further investigation. To address these questions we performed RNA-seq on MCF7-cells, revealing a list of differentially expressed genes. On this set we applied an in-house developed motif discovery tool called iRegulon, generating subsets of direct and indirect target genes. It also enabled us to retrieve possible master regulators like p53 itself but also possible new co-factors like AP-1. We observed E2F as regulator of the downregulated targets with a pronounced absence of the p53 motifs amongst these genes supporting the possibility of a p21-Rb-E2F approach for p53-repression. Next, we performed both ChIP-seq and FAIRE-seq in order to get a comprehensive view on the genomic landscape of p53 binding. While the p53 ChIP peaks improved our predicted set of p53 targets, the FAIRE profile established a correlation between open chromatin regions and upregulated genes. Finally we selected four enhancers from our direct targets for in vitro validation. Three of four enhancers showed the ability to functionally drive gene expression. In conclusion, by using NGS experiments, motif discovery and experimental validation we were able to address key questions about p53âs transcriptional mechanism and identify several new candidate target genes.status: accepte
Identification of High-Impact cis-Regulatory Mutations Using Transcription Factor Specific Random Forest Models
Cancer genomes contain vast amounts of somatic mutations, many of which are passenger mutations not involved in oncogenesis. Whereas driver mutations in protein-coding genes can be distinguished from passenger mutations based on their recurrence, non-coding mutations are usually not recurrent at the same position. Therefore, it is still unclear how to identify cis-regulatory driver mutations, particularly when chromatin data from the same patient is not available, thus relying only on sequence and expression information. Here we use machine-learning methods to predict functional regulatory regions using sequence information alone, and compare the predicted activity of the mutated region with the reference sequence. This way we define the Predicted Regulatory Impact of a Mutation in an Enhancer (PRIME). We find that the recently identified driver mutation in the TAL1 enhancer has a high PRIME score, representing a "gain-of-target" for MYB, whereas the highly recurrent TERT promoter mutation has a surprisingly low PRIME score. We trained Random Forest models for 45 cancer-related transcription factors, and used these to score variations in the HeLa genome and somatic mutations across more than five hundred cancer genomes. Each model predicts only a small fraction of non-coding mutations with a potential impact on the function of the encompassing regulatory region. Nevertheless, as these few candidate driver mutations are often linked to gains in chromatin activity and gene expression, they may contribute to the oncogenic program by altering the expression levels of specific oncogenes and tumor suppressor genes.status: publishe
Identification of High-Impact cis-Regulatory Mutations Using Transcription Factor Specific Random Forest Models.
Cancer genomes contain vast amounts of somatic mutations, many of which are passenger mutations not involved in oncogenesis. Whereas driver mutations in protein-coding genes can be distinguished from passenger mutations based on their recurrence, non-coding mutations are usually not recurrent at the same position. Therefore, it is still unclear how to identify cis-regulatory driver mutations, particularly when chromatin data from the same patient is not available, thus relying only on sequence and expression information. Here we use machine-learning methods to predict functional regulatory regions using sequence information alone, and compare the predicted activity of the mutated region with the reference sequence. This way we define the Predicted Regulatory Impact of a Mutation in an Enhancer (PRIME). We find that the recently identified driver mutation in the TAL1 enhancer has a high PRIME score, representing a "gain-of-target" for MYB, whereas the highly recurrent TERT promoter mutation has a surprisingly low PRIME score. We trained Random Forest models for 45 cancer-related transcription factors, and used these to score variations in the HeLa genome and somatic mutations across more than five hundred cancer genomes. Each model predicts only a small fraction of non-coding mutations with a potential impact on the function of the encompassing regulatory region. Nevertheless, as these few candidate driver mutations are often linked to gains in chromatin activity and gene expression, they may contribute to the oncogenic program by altering the expression levels of specific oncogenes and tumor suppressor genes
Identification of cis-regulatory mutations generating de novo edges in personalized cancer gene regulatory networks
Abstract The identification of functional non-coding mutations is a key challenge in the field of genomics. Here we introduce ÎŒ-cisTarget to filter, annotate and prioritize cis-regulatory mutations based on their putative effect on the underlying âpersonalâ gene regulatory network. We validated ÎŒ-cisTarget by re-analyzing the TAL1 and LMO1 enhancer mutations in T-ALL, and the TERT promoter mutation in melanoma. Next, we re-sequenced the full genomes of ten cancer cell lines and used matched transcriptome data and motif discovery to identify master regulators with de novo binding sites that result in the up-regulation of nearby oncogenic drivers. ÎŒ-cisTarget is available from http://mucistarget.aertslab.org