Saccharomyces cerevisiae BY4741
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA573518 | ENA |
REPOSITORIES: ENA
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Project description:Thousands of protein domains encoded in the human genome function by binding up to a million short linear motifs embedded in intrinsically disordered regions of other proteins. How affinity and specificity are encoded in these binding domains and the motifs themselves is not well understood. The evolvability of binding specificity - how rapidly and extensively it can change upon mutation - is also largely unexplored, as is the contribution of ‘fuzzy’ dynamic residues to affinity and specificity in protein-protein interactions. Here we produce the first global map of affinity and specificity encoding in a globular protein domain. Quantifying >200,000 energetic interactions between the domain and ligand allows us to identify 20 major energetically coupled pairs of sites. These are organised into six modules, with the vast majority of mutations in each module only reprogramming specificity for a single position in the ligand. Nine of the major energetic couplings encoding specificity are direct structural contacts and 11 have an allosteric mechanism of action. The dynamic tail of the ligand is more robust to mutation than the structured portion but contributes additively to binding affinity and communicates with structured residues to enable changes in specificity. Our results present how affinity and specificity are encoded in a globular protein domain interacting with a disordered peptide and a direct comparison of the encoding of affinity and specificity in structured and dynamic molecular recognition.
2024-04-25 | GSE265816 | GEO
Project description:Transcription factors harbour defined intrinsically disordered regulatory regions, which raises the question of how they mediate binding to structured co-regulators and how this regulates activity. Here, we present a detailed molecular regulatory mechanism of Forkhead box O4 (FOXO4) by the structured transcriptional co-regulator β-catenin. We find that the largely disordered FOXO4 C-terminal region, which contains its transactivation domain binds β-catenin through two defined interaction sites, and this is regulated by combined PKB/AKT- and CK1-mediated phosphorylation. Binding of β-catenin competes with the auto-inhibitory interaction of the FOXO4 disordered region with its DNA-binding forkhead domain, and thereby enhances FOXO4 transcriptional activity. Furthermore, we show that binding of the β-catenin inhibitor protein ICAT is compatible with FOXO4 binding to β-catenin, suggesting that ICAT acts as a molecular switch between anti-proliferative FOXO and pro-proliferative Wnt/TCF/LEF signalling. Together these data illustrate how the interplay of intrinsically disordered regions, post-translational modifications and co-factor binding contribute to transcription factor function. Highlights • The interaction network between FOXO4 and β-catenin was deciphered • FOXO4 auto-inhibition interferes with DNA binding and is counter-acted by β-catenin • FOXO4 exists in multiple conformations regulated by phosphorylation and co-factors • ICAT switches between FOXO4 and TCF/LEF transcription factors
2022-04-15 | PXD026263 | Pride
Project description:Disordered regions within RNA binding proteins are required to control mRNA decay and protein synthesis. To understand how these disordered regions modulate gene expression, we surveyed regulatory activity across the entire disordered proteome using a high-throughput functional assay. We identified hundreds of regulatory sequences within intrinsically disordered regions and demonstrate how these elements cooperate with core mRNA decay machinery to promote transcript turnover. Coupling high-throughput functional profiling with mutational scanning revealed diverse molecular features, ranging from defined motifs to overall sequence composition, underlying the regulatory effects of disordered peptides. Machine learning analysis implicated aromatic residues in particular contexts as critical determinants of repressor activity, consistent with their roles in forming protein-protein interactions with downstream effectors. Our results define the molecular principles and biochemical mechanisms that govern post-transcriptional gene regulation by disordered regions and exemplify the encoding of diverse yet specific functions in the absence of well-defined structure.
2024-02-02 | GSE254492 | GEO
Project description:Genome-wide nucleosome specificity and directionality of chromatin remodelers
| PRJNA189244 | ENA
Project description:Intrinsically disordered regions (IDRs) are abundant within eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as exemplified by the budding-yeast TF- Msn2. To examine how low-complexity IDRs encode multiple functions, we compared genomic binding preferences, gene induction, and coactivator recruitment amongst a large set of designed Mns2-IDR mutants. We show that multiple regions across the >500AA IDR contribute to both functions. Yet, transcription activity was readily disrupted by variants having no consequences on Msn2 binding. Our data attribute this differential sensitivity to the integration of relaxed, composition-based code directing binding preferences with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Interwoven sequence grammar may present a general paradigm through which low-complexity IDRs encode multiple functions.
2023-11-19 | GSE239882 | GEO
Project description:Intrinsically disordered regions (IDRs) are abundant within eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as exemplified by the budding-yeast TF- Msn2. To examine how low-complexity IDRs encode multiple functions, we compared genomic binding preferences, gene induction, and coactivator recruitment amongst a large set of designed Mns2-IDR mutants. We show that multiple regions across the >500AA IDR contribute to both functions. Yet, transcription activity was readily disrupted by variants having no consequences on Msn2 binding. Our data attribute this differential sensitivity to the integration of relaxed, composition-based code directing binding preferences with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Interwoven sequence grammar may present a general paradigm through which low-complexity IDRs encode multiple functions.
2023-11-19 | GSE239883 | GEO
Project description:H3 ChIP and input DNA were hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R Array Genome-wide mapping of nucleosomes generated by micrococcal nuclease (MNase) suggests that yeast promoter and terminator regions are very depleted of nucleosomes, predominantly because their DNA sequences intrinsically disfavor nucleosome formation. However, MNase has strong DNA sequence specificity that favors cleavage at promoters and terminators and accounts for some of the correlation between occupancy patterns of nucleosomes assembled in vivo and in vitro. Using an improved method for measuring nucleosome occupancy in vivo that does not involve MNase, we confirm that promoter regions are strongly depleted of nucleosomes, but find that terminator regions are much less depleted than expected. Unlike at promoter regions, nucleosome occupancy at terminators is strongly correlated with the orientation of and distance to adjacent genes. In addition, nucleosome occupancy at terminators is strongly affected by growth conditions, indicating that it is not primarily determined by intrinsic histone-DNA interactions. Rapid removal of RNA polymerase II (Pol II) causes increased nucleosome occupancy at terminators, strongly suggesting a transcription-based mechanism of nucleosome depletion. However, the distinct behavior of terminator regions and their corresponding coding regions suggests that nucleosome depletion at terminators is not simply associated with passage of Pol II, but rather involves a distinct mechanism linked to 3’ end formation.
2010-09-21 | GSE23778 | GEO
Project description:Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro – the hypoxia response element (HRE) – but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is dictated by the AD-containing intrinsically disordered regions (IDR). Using orthogonal genomic approaches such as Cut-and-Run and RNA-seq, we also confirmed IDR-dependent binding and activation for a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search process that may play a role in selective functional target site binding on chromatin.
2022-07-08 | GSE207574 | GEO
Project description:Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1α and HIF-2α subunit isoforms recognize the same DNA motif in vitro – the hypoxia response element (HRE) – but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF-α subunits and their dimerization partner HIF-1β exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2α specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is dictated by the AD-containing intrinsically disordered regions (IDR). Using orthogonal genomic approaches such as Cut-and-Run and RNA-seq, we also confirmed IDR-dependent binding and activation for a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search process that may play a role in selective functional target site binding on chromatin.
2022-07-08 | GSE207573 | GEO
Project description:Protein-protein interactions (PPIs) mediated by intrinsically disordered regions (IDRs) are often based on short linear motifs (SLiM). SLiMs are implicated in signal transduction and gene regulation, yet remain technically laborious and notoriously challenging to study. Here, we present an optimized method for a PRotein Interaction Screen on a peptide MAtrix (PRISMA) in combination with quantitative mass spectrometry. The protocol was benchmarked with previously described SLiM based PPIs using peptides derived from EGFR, SOS1, GLUT1 and CEBPB and extended to map binding partners of kinase activation loops. The detailed protocol provides practical considerations for setting up a PRISMA screen and subsequently implementing PRISMA on a liquid handling robotic platform as a cost effective high-throughput method. Optimized PRISMA can be universally applied to systematically study SLiM based interactions and associated post translational modifications (PTMs) or mutations to advance our understanding of the largely uncharacterized interactomes of intrinsically disordered protein regions.
2021-08-11 | PXD027265 | Pride