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1.
Mol Cell ; 83(9): 1462-1473.e5, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-37116493

RESUMO

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.


Assuntos
DNA , Fatores de Transcrição , Sítios de Ligação , Fatores de Transcrição/metabolismo , Ligação Proteica , DNA/genética
2.
Mol Cell ; 79(3): 459-471.e4, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32553192

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/genética , Motivos de Nucleotídeos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deleção de Sequência , Fatores de Transcrição/genética , Sítios de Ligação , Biologia Computacional/métodos , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Estatísticos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
3.
Cell ; 150(5): 1016-28, 2012 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-22939625

RESUMO

Morphogen gradients pattern tissues and organs during development. When morphogen production is spatially restricted, diffusion and degradation are sufficient to generate sharp concentration gradients. It is less clear how sharp gradients can arise within the source of a broadly expressed morphogen. A recent solution relies on localized production of an inhibitor outside the domain of morphogen production, which effectively redistributes (shuttles) and concentrates the morphogen within its expression domain. Here, we study how a sharp gradient is established without a localized inhibitor, focusing on early dorsoventral patterning of the Drosophila embryo, where an active ligand and its inhibitor are concomitantly generated in a broad ventral domain. Using theory and experiments, we show that a sharp Toll activation gradient is produced through "self-organized shuttling," which dynamically relocalizes inhibitor production to lateral regions, followed by inhibitor-dependent ventral shuttling of the activating ligand Spätzle. Shuttling may represent a general paradigm for patterning early embryos.


Assuntos
Drosophila/embriologia , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Animais , Padronização Corporal , Proteínas de Drosophila , Mesoderma , Sulfotransferases
4.
RNA ; 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39353735

RESUMO

RNA binding proteins (RBPs) are composed of RNA binding domains (RBDs) often linked via intrinsically disordered regions (IDRs). Structural and biochemical analysis have shown that disordered linkers contribute to RNA binding by orienting the adjacent RBDs and also characterized certain disordered repeats that directly contact the RNA. However, the relative contribution of IDRs and predicted RBDs to the in-vivo binding pattern is poorly explored. Here, we upscaled the RNA tagging method to map the transcriptome-wide binding of sixteen RBPs in budding yeast. We then performed extensive sequence mutations to distinguish binding determinants within predicted RBDs and the surrounding IDRs in eight of these. The majority of the predicted RBDs tested were not individually essential for mRNA binding, while multiple IDRs that lacked predicted RNA binding potential appeared essential for binding affinity or specificity. Our results provide new insights into the function of poorly studied RBPs and emphasize the complex and distributed encoding of RBP-RNA interaction in-vivo.

5.
Mol Cell ; 70(6): 1121-1133.e9, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29910110

RESUMO

DNA replication introduces a dosage imbalance between early and late replicating genes. In budding yeast, buffering gene expression against this imbalance depends on marking replicated DNA by H3K56 acetylation (H3K56ac). Whether additional processes are required for suppressing transcription from H3K56ac-labeled DNA remains unknown. Here, using a database-guided candidate screen, we find that COMPASS, the H3K4 methyltransferase, and its upstream effector, PAF1C, act downstream of H3K56ac to buffer expression. Replicated genes show reduced abundance of the transcription activating mark H3K4me3 and accumulate the transcription inhibitory mark H3K4me2 near transcription start sites. Notably, in hydroxyurea-exposed cells, the S phase checkpoint stabilizes H3K56ac and becomes essential for buffering. We suggest that H3K56ac suppresses transcription of replicated genes by interfering with post-replication recovery of epigenetic marks and assign a new function for the S phase checkpoint in stabilizing this mechanism during persistent dosage imbalance.


Assuntos
Replicação do DNA/fisiologia , Histonas/metabolismo , Acetilação , Pontos de Checagem do Ciclo Celular/genética , Replicação do DNA/genética , Epigênese Genética/fisiologia , Epigenômica/métodos , Regulação Fúngica da Expressão Gênica/genética , Histona Acetiltransferases/metabolismo , Histona Metiltransferases/metabolismo , Histonas/fisiologia , Homeostase/genética , Lisina/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
Nucleic Acids Res ; 52(10): 5720-5731, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38597680

RESUMO

The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition.


Assuntos
Complexo de Reconhecimento de Origem , Origem de Replicação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Sítios de Ligação , Replicação do DNA , DNA Fúngico/metabolismo , DNA Fúngico/química , DNA Fúngico/genética , Mutação , Motivos de Nucleotídeos , Complexo de Reconhecimento de Origem/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/química , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química
7.
Nucleic Acids Res ; 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-39187372

RESUMO

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.

8.
Nucleic Acids Res ; 52(15): 8763-8777, 2024 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-38908024

RESUMO

Intrinsically disordered regions (IDRs) guide transcription factors (TFs) to their genomic binding sites, raising the question of how structure-lacking regions encode for complex binding patterns. We investigated this using the TF Gln3, revealing sets of IDR-embedded determinants that direct Gln3 binding to respective groups of functionally related promoters, and enable tuning binding preferences between environmental conditions, phospho-mimicking mutations, and orthologs. Through targeted mutations, we defined the role of short linear motifs (SLiMs) and co-binding TFs (Hap2) in stabilizing Gln3 at respiration-chain promoters, while providing evidence that Gln3 binding at nitrogen-associated promoters is encoded by the IDR amino-acid composition, independent of SLiMs or co-binding TFs. Therefore, despite their apparent simplicity, TF IDRs can direct and regulate complex genomic binding patterns through a combination of SLiM-mediated and composition-encoded interactions.


Assuntos
Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/química , Sítios de Ligação , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos
9.
Nucleic Acids Res ; 52(5): 2260-2272, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38109289

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA , Proteínas Intrinsicamente Desordenadas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Regulação da Expressão Gênica , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Mutação , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo
10.
Nucleic Acids Res ; 2024 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-39413205

RESUMO

DNA-binding domains (DBDs) within transcription factors (TFs) recognize short sequence motifs that are highly abundant in genomes. In vivo, TFs bind only a small subset of motif occurrences, which is often attributed to the cooperative binding of interacting TFs at proximal motifs. However, large-scale testing of this model is still lacking. Here, we describe a novel method allowing parallel measurement of TF binding to thousands of designed sequences within yeast cells and apply it to quantify the binding of dozens of TFs to libraries of regulatory regions containing clusters of binding motifs, systematically mutating all motif combinations. With few exceptions, TF occupancies were well explained by independent binding to individual motifs, with motif cooperation being of only limited effects. Our results challenge the general role of motif combinatorics in directing TF genomic binding and open new avenues for exploring the basis of protein-DNA interactions within cells.

11.
EMBO J ; 40(21): e108439, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34569643

RESUMO

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.


Assuntos
Replicação do DNA , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase III/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Galactoquinase/genética , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Hidroxiureia/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosfoproteínas , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Saccharomyces cerevisiae/genética , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Transcrição Gênica
12.
Genome Res ; 32(6): 1089-1098, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35609993

RESUMO

DNA replication perturbs chromatin by triggering the eviction, replacement, and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a genetically encoded sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing slow synchronous replication in nucleotide-limiting conditions. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S-phase-induced acetyltransferase, stabilizes nucleosomes behind the fork but promotes H3 replacement ahead of the fork. Increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56 and rather results from Vps75-dependent Rtt109 activity toward the H3 N terminus. Our results suggest that, at least under nucleotide-limiting conditions, selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, likely reflecting the distinct challenges for genome stability at these different regions.


Assuntos
Replicação do DNA , Histona Acetiltransferases , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Acetilação , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Nucleotídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Genome Res ; 32(6): 1099-1111, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35618416

RESUMO

Variations in noncoding regulatory sequences play a central role in evolution. Interpreting such variations, however, remains difficult even in the context of defined attributes such as transcription factor (TF) binding sites. Here, we systematically link variations in cis-regulatory sequences to TF binding by profiling the allele-specific binding of 27 TFs expressed in a yeast hybrid, in which two related genomes are present within the same nucleus. TFs localize preferentially to sites containing their known consensus motifs but occupy only a small fraction of the motif-containing sites available within the genomes. Differential binding of TFs to the orthologous alleles was well explained by variations that alter motif sequence, whereas differences in chromatin accessibility between alleles were of little apparent effect. Motif variations that abolished binding when present in only one allele were still bound when present in both alleles, suggesting evolutionary compensation, with a potential role for sequence conservation at the motif's vicinity. At the level of the full promoter, we identify cases of binding-site turnover, in which binding sites are reciprocally gained and lost, yet most interspecific differences remained uncompensated. Our results show the flexibility of TFs to bind imprecise motifs and the fast evolution of TF binding sites between related species.


Assuntos
Biologia Computacional , Fatores de Transcrição , Sítios de Ligação , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
14.
Nature ; 575(7782): 355-360, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31695196

RESUMO

Central to understanding cellular behaviour in multi-cellular organisms is the question of how a cell exits one transcriptional state to adopt and eventually become committed to another. Fibroblast growth factor-extracellular signal-regulated kinase (FGF -ERK) signalling drives differentiation of mouse embryonic stem cells (ES cells) and pre-implantation embryos towards primitive endoderm, and inhibiting ERK supports ES cell self-renewal1. Paracrine FGF-ERK signalling induces heterogeneity, whereby cells reversibly progress from pluripotency towards primitive endoderm while retaining their capacity to re-enter self-renewal2. Here we find that ERK reversibly regulates transcription in ES cells by directly affecting enhancer activity without requiring a change in transcription factor binding. ERK triggers the reversible association and disassociation of RNA polymerase II and associated co-factors from genes and enhancers with the mediator component MED24 having an essential role in ERK-dependent transcriptional regulation. Though the binding of mediator components responds directly to signalling, the persistent binding of pluripotency factors to both induced and repressed genes marks them for activation and/or reactivation in response to fluctuations in ERK activity. Among the repressed genes are several core components of the pluripotency network that act to drive their own expression and maintain the ES cell state; if their binding is lost, the ability to reactivate transcription is compromised. Thus, as long as transcription factor occupancy is maintained, so is plasticity, enabling cells to distinguish between transient and sustained signals. If ERK signalling persists, pluripotency transcription factor levels are reduced by protein turnover and irreversible gene silencing and commitment can occur.


Assuntos
Linhagem da Célula , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , Complexo Mediador/deficiência , Complexo Mediador/metabolismo , Camundongos , Ligação Proteica , Transcrição Gênica
15.
Nucleic Acids Res ; 51(10): 4831-4844, 2023 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-36938874

RESUMO

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.


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Genômica , Proteínas Intrinsicamente Desordenadas/química , Mutação , Ligação Proteica , Fatores de Transcrição/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo
16.
Nucleic Acids Res ; 51(16): 8496-8513, 2023 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-37493599

RESUMO

DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with overlapping in-vitro activities. Defining specific in-vivo activities requires monitoring histone dynamics during regulator depletion, which has been technically challenging. We have recently generated histone-exchange sensors in Saccharomyces cerevisiae, which we now use to define the contributions of 15 regulators to histone dynamics genome-wide. While replication-independent exchange in unperturbed cells maps to promoters, regulator depletions primarily affected gene bodies. Depletion of Spt6, Spt16 or Chd1 sharply increased nucleosome replacement sequentially at the beginning, middle or end of highly expressed gene bodies. They further triggered re-localization of chaperones to affected gene body regions, which compensated for nucleosome loss during transcription complex passage, but concurred with extensive TF binding in gene bodies. We provide a unified quantitative screen highlighting regulator roles in retaining nucleosome binding during transcription and preserving genomic packaging.


Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Trends Genet ; 37(5): 421-432, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33414013

RESUMO

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.


Assuntos
Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma , Ligação Proteica
18.
Genome Res ; 31(3): 426-435, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33563717

RESUMO

The wrapping of DNA around histone octamers challenges processes that use DNA as their template. In vitro, DNA replication through chromatin depends on histone modifiers, raising the possibility that cells modify histones to optimize fork progression. Rtt109 is an acetyl transferase that acetylates histone H3 before its DNA incorporation on the K56 and N-terminal residues. We previously reported that, in budding yeast, a wave of histone H3 K9 acetylation progresses ∼3-5 kb ahead of the replication fork. Whether this wave contributes to replication dynamics remained unknown. Here, we show that the replication fork velocity increases following deletion of RTT109, the gene encoding the enzyme required for the prereplication H3 acetylation wave. By using histone H3 mutants, we find that Rtt109-dependent N-terminal acetylation regulates fork velocity, whereas K56 acetylation contributes to replication dynamics only when N-terminal acetylation is compromised. We propose that acetylation of newly synthesized histones slows replication by promoting replacement of nucleosomes evicted by the incoming fork, thereby protecting genome integrity.


Assuntos
Replicação do DNA , Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Histonas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética
19.
Development ; 148(24)2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34918740

RESUMO

Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.


Assuntos
Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Morfogênese/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , RNA Mensageiro/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Núcleo Celular/genética , Drosophila melanogaster/genética , Embrião não Mamífero , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Íntrons/genética , RNA Polimerase II/genética
20.
Genome Res ; 30(7): 1000-1011, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32699020

RESUMO

Changes in gene expression drive novel phenotypes, raising interest in how gene expression evolves. In contrast to the static genome, cells modulate gene expression in response to changing environments. Previous comparative studies focused on specific conditions, describing interspecies variation in expression levels, but providing limited information about variation across different conditions. To close this gap, we profiled mRNA levels of two related yeast species in hundreds of conditions and used coexpression analysis to distinguish variation in the dynamic pattern of gene expression from variation in expression levels. The majority of genes whose expression varied between the species maintained a conserved dynamic pattern. Cases of diverged dynamic pattern correspond to genes that were induced under distinct subsets of conditions in the two species. Profiling the interspecific hybrid allowed us to distinguish between genes with predominantly cis- or trans-regulatory variation. We find that trans-varying alleles are dominantly inherited, and that cis-variations are often complemented by variations in trans Based on these results, we suggest that gene expression diverges primarily through changes in expression levels, but does not alter the pattern by which these levels are dynamically regulated.


Assuntos
Evolução Molecular , Regulação Fúngica da Expressão Gênica , RNA Mensageiro/metabolismo , Amônia-Liases/genética , Amônia-Liases/metabolismo , Perfilação da Expressão Gênica , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Transcriptoma
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