Revisiting the model for coactivator recruitment: Med15 can select its target sites independent of promoter-bound transcription factors.

Activation domains (ADs) within transcription factors (TFs) induce gene expression by recruiting coactivators such as the Mediator complex. Coactivators lack DNA binding domains (DBDs) and are assumed to passively follow their recruiting TFs. This is supported by direct AD-coactivator interactions seen in vitro but has not yet been tested in living cells. To examine that, we targeted two Med15-recruiting ADs to a range of budding yeast promoters through fusion with different DBDs. The DBD-AD fusions localized to hundreds of genomic sites but recruited Med15 and induced transcription in only a subset of bound promoters, characterized by a fuzzy-nucleosome architecture. Direct DBD-Med15 fusions shifted DBD localization towards fuzzy-nucleosome promoters, including promoters devoid of the endogenous Mediator. We propose that Med15, and perhaps other coactivators, possess inherent promoter preference and thus actively contribute to the selection of TF-induced genes.

Coactivators localize to specific regulatory regions to activate gene expression. As coactivators lack known DNA binding domains (DBDs), they are assumed to passively follow the binding location of their recruiting transcription factors but not to influence the identity of their activated genes. In this work, we revisited this model in the context of Med15, a component of the mediator tail subunit that is recruited by multiple TFs within budding yeast. Using genomic measurements of Med15 recruitment, genomic localization, and transcriptional changes, applied to engineered minimal TFs and direct Med15-DBD fusions, we provide evidence that Med15 actively directs the selection of induced genes by its inherent preference to localize at specific target promoters independent of promoter-localizing TFs.

Intrinsically disordered regions of the Msn2 transcription factor encode multiple functions using interwoven sequence grammars.

Intrinsically disordered regions (IDRs) are abundant in eukaryotic proteins, but their sequence-function relationship remains poorly understood. IDRs of transcription factors (TFs) can direct promoter selection and recruit coactivators, as shown for the budding yeast TF Msn2. To examine how IDRs encode both these functions, we compared genomic binding specificity, coactivator recruitment, and gene induction amongst a large set of designed Msn2-IDR mutants. We find that both functions depend on multiple regions across the > 600AA IDR. Yet, transcription activity was readily disrupted by mutations that showed no effect on the Msn2 binding specificity. Our data attribute this differential sensitivity to the integration of a relaxed, composition-based code directing binding specificity with a more stringent, motif-based code controlling the recruitment of coactivators and transcription activity. Therefore, Msn2 utilizes interwoven sequence grammars for encoding multiple functions, suggesting a new IDR design paradigm of potentially general use.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

Complementary strategies for directing in vivo transcription factor binding through DNA binding domains and intrinsically disordered regions.

DNA binding domains (DBDs) of transcription factors (TFs) recognize DNA sequence motifs that are highly abundant in genomes. Within cells, TFs bind a subset of motif-containing sites as directed by either their DBDs or DBD-external (nonDBD) sequences. To define the relative roles of DBDs and nonDBDs in directing binding preferences, we compared the genome-wide binding of 48 (∼30%) budding yeast TFs with their DBD-only, nonDBD-truncated, and nonDBD-only mutants. With a few exceptions, binding locations differed between DBDs and TFs, resulting from the cumulative action of multiple determinants mapped mostly to disordered nonDBD regions. Furthermore, TFs' preferences for promoters of the fuzzy nucleosome architecture were lost in DBD-only mutants, whose binding spread across promoters, implicating nonDBDs' preferences in this hallmark of budding yeast regulatory design. We conclude that DBDs and nonDBDs employ complementary DNA-targeting strategies, whose balance defines TF binding specificity along genomes.

The molecular grammar of protein disorder guiding genome-binding locations.

Intrinsically disordered regions (IDRs) direct transcription factors (TFs) towards selected genomic occurrences of their binding motif, as exemplified by budding yeast's Msn2. However, the sequence basis of IDR-directed TF binding selectivity remains unknown. To reveal this sequence grammar, we analyze the genomic localizations of >100 designed IDR mutants, each carrying up to 122 mutations within this 567-AA region. Our data points at multivalent interactions, carried by hydrophobic-mostly aliphatic-residues dispersed within a disordered environment and independent of linear sequence motifs, as the key determinants of Msn2 genomic localization. The implications of our results for the mechanistic basis of IDR-based TF binding preferences are discussed.

Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis.

Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.

Order through disorder: The role of intrinsically disordered regions in transcription factor binding specificity.

Transcription factors (TFs) must bind at specific genomic locations to accurately regulate gene expression. The ability of TFs to recognize specific DNA sequence motifs arises from the inherent preferences of their globular DNA-binding domains (DBDs). Yet, these preferences are insufficient to explain the in vivo TF binding site selection. TFs are enriched with intrinsically disordered regions (IDRs), most of which are poorly characterized. While not generally considered as determinants of TF binding specificity, IDRs guide protein-protein interactions within transcriptional condensates, and multiple examples exist in which short IDRs flanking the DBD contribute to binding specificity via direct contact with the DNA. We recently reported that long IDRs, present away from the DBD, act as major specificity determinants at the genomic scale. Here, we discuss mechanisms through which IDRs contribute to DNA binding specificity, highlighting the role of long IDRs in dictating the in vivo binding site selection.

Phosphorylation of Serine 114 of the transcription factor ABSCISIC ACID INSENSITIVE 4 is essential for activity.

The transcription factor ABA-INSENSITIVE(ABI)4 has diverse roles in regulating plant growth, including inhibiting germination and reserve mobilization in response to ABA and high salinity, inhibiting seedling growth in response to high sugars, inhibiting lateral root growth, and repressing light-induced gene expression. ABI4 activity is regulated at multiple levels, including gene expression, protein stability, and activation by phosphorylation. Although ABI4 can be phosphorylated at multiple residues by MAPKs, we found that S114 is the preferred site of MPK3. To examine the possible biological role of S114 phosphorylation, we transformed abi4-1 mutant plants with ABI4pro::ABI4 constructs encoding wild type (114S), phosphorylation-null (S114A) or phosphomimetic (S114E) forms of ABI4. Phosphorylation of S114 is necessary for the response to ABA, glucose, salt stress, and lateral root development, where the abi4 phenotype could be complemented by expressing ABI4 (114S) or ABI4 (S114E) but not ABI4 (S114A). Comparison of root transcriptomes in ABA-treated roots of abi4-1 mutant plants transformed with constructs encoding the different phosphorylation-forms of S114 of ABI4 revealed that 85 % of the ABI4-regulated genes whose expression pattern could be restored by expressing ABI4 (114S) are down-regulated by ABI4. Phosphorylation of S114 was required for regulation of 35 % of repressed genes, but only 17 % of induced genes. The genes whose repression requires the phosphorylation of S114 are mainly involved in embryo and seedling development, growth and differentiation, and regulation of gene expression.

Speed-Specificity Trade-Offs in the Transcription Factors Search for Their Genomic Binding Sites.

Transcription factors (TFs) regulate gene expression by binding DNA sequences recognized by their DNA-binding domains (DBDs). DBD-recognized motifs are short and highly abundant in genomes. The ability of TFs to bind a specific subset of motif-containing sites, and to do so rapidly upon activation, is fundamental for gene expression in all eukaryotes. Despite extensive interest, our understanding of the TF-target search process is fragmented; although binding specificity and detection speed are two facets of this same process, trade-offs between them are rarely addressed. In this opinion article, we discuss potential speed-specificity trade-offs in the context of existing models. We further discuss the recently described 'distributed specificity' paradigm, suggesting that intrinsically disordered regions (IDRs) promote specificity while reducing the TF-target search time.

Intrinsically Disordered Regions Direct Transcription Factor In Vivo Binding Specificity.

Transcription factors (TFs) that bind common DNA motifs in vitro occupy distinct sets of promoters in vivo, raising the question of how binding specificity is achieved. TFs are enriched with intrinsically disordered regions (IDRs). Such regions commonly form promiscuous interactions, yet their unique properties might also benefit specific binding-site selection. We examine this using Msn2 and Yap1, TFs of distinct families that contain long IDRs outside their DNA-binding domains. We find that these IDRs are both necessary and sufficient for localizing to the majority of target promoters. This IDR-directed binding does not depend on any localized domain but results from a multitude of weak determinants distributed throughout the entire IDR sequence. Furthermore, IDR specificity is conserved between distant orthologs, suggesting direct interaction with multiple promoters. We propose that distribution of sensing determinants along extended IDRs accelerates binding-site detection by rapidly localizing TFs to broad DNA regions surrounding these sites.

Transcription Factor Binding to Replicated DNA.

Genome replication perturbs the DNA regulatory environment by displacing DNA-bound proteins, replacing nucleosomes, and introducing dosage imbalance between regions replicating at different S-phase stages. Recently, we showed that these effects are integrated to maintain transcription homeostasis: replicated genes increase in dosage, but their expression remains stable due to replication-dependent epigenetic changes that suppress transcription. Here, we examine whether reduced transcription from replicated DNA results from limited accessibility to regulatory factors by measuring the time-resolved binding of RNA polymerase II (Pol II) and specific transcription factors (TFs) to DNA during S phase in budding yeast. We show that the Pol II binding pattern is largely insensitive to DNA dosage, indicating limited binding to replicated DNA. In contrast, binding of three TFs (Reb1, Abf1, and Rap1) to DNA increases with the increasing DNA dosage. We conclude that the replication-specific chromatin environment remains accessible to regulatory factors but suppresses RNA polymerase recruitment.

Resolving noise-control conflict by gene duplication.

Gene duplication promotes adaptive evolution in two main ways: allowing one duplicate to evolve a new function and splitting ancestral functions between the duplicates. The second scenario may resolve adaptive conflicts that can rise when one gene performs different functions. In an apparent departure from both scenarios, low-expressing transcription factor (TF) duplicates commonly bind to the same DNA motifs and act in overlapping conditions. To examine for possible benefits of this apparent redundancy, we examined the Msn2 and Msn4 duplicates in budding yeast. We show that Msn2,4 function as one unit by inducing the same set of target genes in overlapping conditions. Yet, the two-factor composition allows this unit's expression to be both environmentally responsive and with low noise, resolving an adaptive conflict that limits expression of single genes. We propose that duplication can provide adaptive benefit through cooperation rather than functional divergence, allowing two-factor dynamics with beneficial properties that cannot be achieved by a single gene.