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1.
Nat Methods ; 21(7): 1349-1363, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38849569

RESUMO

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.


Assuntos
Perfilação da Expressão Gênica , RNA-Seq , Humanos , Animais , Camundongos , RNA-Seq/métodos , Perfilação da Expressão Gênica/métodos , Transcriptoma , Análise de Sequência de RNA/métodos , Anotação de Sequência Molecular/métodos
2.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34857629

RESUMO

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.


Assuntos
Inativação Gênica/fisiologia , Histonas/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Acetilação , Cromatina/metabolismo , Heterocromatina/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/genética , Sirtuína 2/metabolismo , Telômero/metabolismo
3.
Epigenetics Chromatin ; 17(1): 28, 2024 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-39272151

RESUMO

Transcriptional silencing in Saccharomyces cerevisiae is a persistent and highly stable form of gene repression. It involves DNA silencers and repressor proteins that bind nucleosomes. The silenced state is influenced by numerous factors including the concentration of repressors, nature of activators, architecture of regulatory elements, modifying enzymes and the dynamics of chromatin.Silencers function to increase the residence time of repressor Sir proteins at silenced domains while clustering of silenced domains enables increased concentrations of repressors and helps facilitate long-range interactions. The presence of an accessible NDR at the regulatory regions of silenced genes, the cycling of chromatin configurations at regulatory sites, the mobility of Sir proteins, and the non-uniform distribution of the Sir proteins across the silenced domain, all result in silenced chromatin that only stably silences weak promoters and enhancers via changes in transcription burst duration and frequency.These data collectively suggest that silencing is probabilistic and the robustness of silencing is achieved through sub-optimization of many different nodes of action such that a stable expression state is generated and maintained even though individual constituents are in constant flux.


Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Saccharomyces cerevisiae , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Transcrição Gênica , Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética
4.
Cell Rep ; 43(4): 113983, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38517895

RESUMO

Transcriptional silencing in Saccharomyces cerevisiae involves the generation of a chromatin state that stably represses transcription. Using multiple reporter assays, a diverse set of upstream activating sequence enhancers and core promoters were investigated for their susceptibility to silencing. We show that heterochromatin stably silences only weak and stress-induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements, and the partial repression of these elements did not result in bistable expression states. Permutation analysis of enhancers and promoters indicates that both elements are targets of repression. Chromatin remodelers help specific regulatory elements to resist repression, most probably by altering nucleosome mobility and changing transcription burst duration. The strong enhancers/promoters can be repressed if silencer-bound Sir1 is increased. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating-type gene regulatory elements but not strong housekeeping gene regulatory sequences.


Assuntos
Regulação Fúngica da Expressão Gênica , Inativação Gênica , Heterocromatina , Regiões Promotoras Genéticas , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Heterocromatina/metabolismo , Heterocromatina/genética , Regiões Promotoras Genéticas/genética , Elementos Facilitadores Genéticos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sequências Reguladoras de Ácido Nucleico/genética , Nucleossomos/metabolismo , Nucleossomos/genética
5.
bioRxiv ; 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38562899

RESUMO

Genome-wide identification of chromatin organization and structure has been generally probed by measuring accessibility of the underlying DNA to nucleases or methyltransferases. These methods either only observe the positioning of a single nucleosome or rely on large enzymes to modify or cleave the DNA. We developed adduct sequencing (Add-seq), a method to probe chromatin accessibility by treating chromatin with the small molecule angelicin, which preferentially intercalates into DNA not bound to core nucleosomes. We show that Nanopore sequencing of the angelicin-modified DNA is possible and allows visualization and analysis of long single molecules with distinct chromatin structure. The angelicin modification can be detected from the Nanopore current signal data using a neural network model trained on unmodified and modified chromatin-free DNA. Applying Add-seq to Saccharomyces cerevisiae nuclei, we identified expected patterns of accessibility around annotated gene loci in yeast. We also identify individual clusters of single molecule reads displaying different chromatin structure at specific yeast loci, which demonstrates heterogeneity in the chromatin structure of the yeast population. Thus, using Add-seq, we are able to profile DNA accessibility in the yeast genome across long molecules.

6.
EMBO J ; 28(17): 2583-600, 2009 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-19629037

RESUMO

Insulators bind transcription factors and use chromatin remodellers and modifiers to mediate insulation. In this report, we identified proteins required for the efficient formation and maintenance of a specialized chromatin structure at the yeast tRNA insulator. The histone acetylases, SAS-I and NuA4, functioned in insulation, independently of tRNA and did not participate in the formation of the hypersensitive site at the tRNA. In contrast, DNA polymerase epsilon, functioned with the chromatin remodeller, Rsc, and the histone acetylase, Rtt109, to generate a histone-depleted region at the tRNA insulator. Rsc and Rtt109 were required for efficient binding of TFIIIB to the tRNA insulator, and the bound transcription factor and Rtt109 in turn were required for the binding of Rsc to tRNA. Robust insulation during growth and cell division involves the formation of a hypersensitive site at the insulator during chromatin maturation together with competition between acetylases and deacetylases.


Assuntos
Cromatina/química , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histona Acetiltransferases/metabolismo , Elementos Isolantes , RNA de Transferência/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , Histonas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
7.
bioRxiv ; 2023 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-37873261

RESUMO

The interplay between nucleosomes and transcription factors leads to programs of gene expression. Transcriptional silencing involves the generation of a chromatin state that represses transcription and is faithfully propagated through DNA replication and cell division. Using multiple reporter assays, including directly visualizing transcription in single cells, we investigated a diverse set of UAS enhancers and core promoters for their susceptibility to heterochromatic gene silencing. These results show that heterochromatin only stably silences weak and stress induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements and the partial repression did not result in bistable expression states. Permutation analysis of different UAS enhancers and core promoters indicate that both elements function together to determine the susceptibility of regulatory sequences to repression. Specific histone modifiers and chromatin remodellers function in an enhancer specific manner to aid these elements to resist repression suggesting that Sir proteins likely function in part by reducing nucleosome mobility. We also show that the strong housekeeping regulatory elements can be repressed if silencer bound Sir1 is increased, suggesting that Sir1 is a limiting component in silencing. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating type gene regulatory elements but not strong housekeeping gene regulatory sequences which could help explain why these genes are often found at the boundaries of silenced domains.

8.
bioRxiv ; 2023 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-37546854

RESUMO

The Long-read RNA-Seq Genome Annotation Assessment Project (LRGASP) Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. The consortium generated over 427 million long-read sequences from cDNA and direct RNA datasets, encompassing human, mouse, and manatee species, using different protocols and sequencing platforms. These data were utilized by developers to address challenges in transcript isoform detection and quantification, as well as de novo transcript isoform identification. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. When aiming to detect rare and novel transcripts or when using reference-free approaches, incorporating additional orthogonal data and replicate samples are advised. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

9.
bioRxiv ; 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33851162

RESUMO

We report a SARS-CoV-2 lineage that shares N501Y, P681H, and other mutations with known variants of concern, such as B.1.1.7. This lineage, which we refer to as B.1.x (COG-UK sometimes references similar samples as B.1.324.1), is present in at least 20 states across the USA and in at least six countries. However, a large deletion causes the sequence to be automatically rejected from repositories, suggesting that the frequency of this new lineage is underestimated using public data. Recent dynamics based on 339 samples obtained in Santa Cruz County, CA, USA suggest that B.1.x may be increasing in frequency at a rate similar to that of B.1.1.7 in Southern California. At present the functional differences between this variant B.1.x and other circulating SARS-CoV-2 variants are unknown, and further studies on secondary attack rates, viral loads, immune evasion and/or disease severity are needed to determine if it poses a public health concern. Nonetheless, given what is known from well-studied circulating variants of concern, it seems unlikely that the lineage could pose larger concerns for human health than many already globally distributed lineages. Our work highlights a need for rapid turnaround time from sequence generation to submission and improved sequence quality control that removes submission bias. We identify promising paths toward this goal.

10.
PLoS One ; 16(12): e0261230, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34919584

RESUMO

The systematic screening of asymptomatic and pre-symptomatic individuals is a powerful tool for controlling community transmission of infectious disease on college campuses. Faced with a paucity of testing in the beginning of the COVID-19 pandemic, many universities developed molecular diagnostic laboratories focused on SARS-CoV-2 diagnostic testing on campus and in their broader communities. We established the UC Santa Cruz Molecular Diagnostic Lab in early April 2020 and began testing clinical samples just five weeks later. Using a clinically-validated laboratory developed test (LDT) that avoided supply chain constraints, an automated sample pooling and processing workflow, and a custom laboratory information management system (LIMS), we expanded testing from a handful of clinical samples per day to thousands per day with the testing capacity to screen our entire campus population twice per week. In this report we describe the technical, logistical, and regulatory processes that enabled our pop-up lab to scale testing and reporting capacity to thousands of tests per day.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Programas de Rastreamento/métodos , Pandemias/prevenção & controle , Programas de Triagem Diagnóstica , Humanos , Universidades
11.
Synth Biol (Oxf) ; 5(1): ysaa007, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32775697

RESUMO

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements-enhancers (upstream activating sequences), core promoters, 5' untranslated regions (5' UTRs) and transcription terminators/3' untranslated regions (3' UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5' UTRs and transcription terminators/3' UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicates that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.

12.
Mol Cell Biol ; 26(2): 489-501, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16382141

RESUMO

Histone H2A variants are highly conserved proteins found ubiquitously in nature and thought to perform specialized functions in the cell. Studies in yeast on the histone H2A variant H2A.Z have shown a role for this protein in transcription as well as chromosome segregation. Our studies have focused on understanding the role of H2A.Z during cell cycle progression. We found that htz1delta cells were delayed in DNA replication and progression through the cell cycle. Furthermore, cells lacking H2A.Z required the S-phase checkpoint pathway for survival. We also found that H2A.Z localized to the promoters of cyclin genes, and cells lacking H2A.Z were delayed in the induction of these cyclin genes. Several different models are proposed to explain these observations.


Assuntos
Ciclo Celular/fisiologia , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Replicação do DNA , Histonas/genética , Mutação , Regiões Promotoras Genéticas , Origem de Replicação , Fase S/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Mol Cell Biol ; 39(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30718362

RESUMO

The genome is packaged and organized in an ordered, nonrandom manner, and specific chromatin segments contact nuclear substructures to mediate this organization. tRNA genes (tDNAs) are binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the roles of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacked any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromatin architecture or chromosome tethering and mobility. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long-range HML-HMR heterochromatin clustering with concomitant effects on gene silencing. We propose that the tDNAs primarily affect local chromatin structure, which results in effects on long-range chromosome architecture.


Assuntos
Cromatina/metabolismo , Cromatina/ultraestrutura , RNA de Transferência/genética , Sítios de Ligação , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Montagem e Desmontagem da Cromatina , Cromossomos/genética , Cromossomos/metabolismo , Heterocromatina/metabolismo , Heterocromatina/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFIII/metabolismo
14.
Curr Opin Genet Dev ; 12(2): 188-92, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11893492

RESUMO

The establishment and restriction of transcriptionally inactive regions in the nucleus is mediated by silencer and barrier elements. Silencer-bound proteins recruit additional factors to establish the silenced domain during the S-phase of the cell cycle but, contrary to previous models, DNA replication is not a pre-requisite for the establishment. Characteristically, silenced domains contain hypoacetylated histones and recent data have identified residue-specific methylation of histone H3 as an additional signature that distinguishes active regions from inactive ones. Peaks of acetylated histones demarcate the boundaries between these regions and recruitment of HAT activities provides a mechanism to restrict the spread of heterochromatin.


Assuntos
Inativação Gênica/fisiologia , Genes Reguladores/fisiologia , Heterocromatina/fisiologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologia
15.
Mol Biol Cell ; 26(7): 1395-410, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25631822

RESUMO

Heterochromatin formation and nuclear organization are important in gene regulation and genome fidelity. Proteins involved in gene silencing localize to sites of damage and some DNA repair proteins localize to heterochromatin, but the biological importance of these correlations remains unclear. In this study, we examined the role of double-strand-break repair proteins in gene silencing and nuclear organization. We find that the ATM kinase Tel1 and the proteins Mre11 and Esc2 can silence a reporter gene dependent on the Sir, as well as on other repair proteins. Furthermore, these proteins aid in the localization of silenced domains to specific compartments in the nucleus. We identify two distinct mechanisms for repair protein-mediated silencing-via direct and indirect interactions with Sir proteins, as well as by tethering loci to the nuclear periphery. This study reveals previously unknown interactions between repair proteins and silencing proteins and suggests insights into the mechanism underlying genome integrity.


Assuntos
Montagem e Desmontagem da Cromatina , Cromossomos Fúngicos/metabolismo , Reparo do DNA , Heterocromatina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
Genetics ; 183(1): 131-48, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19596900

RESUMO

Chromatin insulators separate active from repressed chromatin domains. In yeast the RNA pol III transcription machinery bound to tRNA genes function with histone acetylases and chromatin remodelers to restrict the spread of heterochromatin. Our results collectively demonstrate that binding of TFIIIC is necessary for insulation but binding of TFIIIB along with TFIIIC likely improves the probability of complex formation at an insulator. Insulation by this transcription factor occurs in the absence of RNA polymerase III or polymerase II but requires specific histone acetylases and chromatin remodelers. This analysis identifies a minimal set of factors required for insulation.


Assuntos
Elementos Isolantes/fisiologia , RNA de Transferência/genética , Fatores de Transcrição TFIII/fisiologia , Transcrição Gênica/genética , Montagem e Desmontagem da Cromatina/genética , Mapeamento Cromossômico , Histona Acetiltransferases/metabolismo , Organismos Geneticamente Modificados , Multimerização Proteica , RNA de Transferência/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Fatores de Transcrição TFIII/metabolismo , Transcrição Gênica/fisiologia , Leveduras/genética
17.
Mol Cell Biol ; 28(6): 1924-35, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18195043

RESUMO

Gene regulation involves long-range communication between silencers, enhancers, and promoters. In Saccharomyces cerevisiae, silencers flank transcriptionally repressed genes to mediate regional silencing. Silencers recruit the Sir proteins, which then spread along chromatin to encompass the entire silenced domain. In this report we have employed a boundary trap assay, an enhancer activity assay, chromatin immunoprecipitations, and chromosome conformation capture analyses to demonstrate that the two HMR silencer elements are in close proximity and functionally communicate with one another in vivo. We further show that silencing is necessary for these long-range interactions, and we present models for Sir-mediated silencing based upon these results.


Assuntos
Cromossomos Fúngicos/genética , DNA Fúngico/genética , Elementos Facilitadores Genéticos/genética , Regulação Fúngica da Expressão Gênica , Inativação Gênica/fisiologia , Genes Fúngicos Tipo Acasalamento/genética , Região de Controle de Locus Gênico/genética , Modelos Genéticos , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/fisiologia , Imunoprecipitação da Cromatina , Cromossomos Fúngicos/ultraestrutura , DNA Fúngico/ultraestrutura , Proteínas de Saccharomyces cerevisiae/fisiologia , Complexo Shelterina , Proteínas de Ligação a Telômeros/fisiologia , Fatores de Transcrição/fisiologia
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