RESUMEN
The chromatin remodelers SWI/SNF and RSC function in evicting promoter nucleosomes at highly expressed yeast genes, particularly those activated by transcription factor Gcn4. Ino80 remodeling complex (Ino80C) can establish nucleosome-depleted regions (NDRs) in reconstituted chromatin, and was implicated in removing histone variant H2A.Z from the -1 and +1 nucleosomes flanking NDRs; however, Ino80C's function in transcriptional activation in vivo is not well understood. Analyzing the cohort of Gcn4-induced genes in ino80Δ mutants has uncovered a role for Ino80C on par with SWI/SNF in evicting promoter nucleosomes and transcriptional activation. Compared to SWI/SNF, Ino80C generally functions over a wider region, spanning the -1 and +1 nucleosomes, NDR and proximal genic nucleosomes, at genes highly dependent on its function. Defects in nucleosome eviction in ino80Δ cells are frequently accompanied by reduced promoter occupancies of TBP, and diminished transcription; and Ino80 is enriched at genes requiring its remodeler activity. Importantly, nuclear depletion of Ino80 impairs promoter nucleosome eviction even in a mutant lacking H2A.Z. Thus, Ino80C acts widely in the yeast genome together with RSC and SWI/SNF in evicting promoter nucleosomes and enhancing transcription, all in a manner at least partly independent of H2A.Z editing.
Asunto(s)
Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética , Activación Transcripcional/genética , Adenosina Trifosfatasas/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica/genética , Nucleosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/genéticaRESUMEN
Chromatin organization and epigenetic marks play a critical role in stem cell pluripotency and differentiation. Chromatin digestion by micrococcal nuclease (MNase) followed by high-throughput sequencing (MNase-seq) is the most widely used genome-wide method for studying nucleosome organization, that is, the first level of DNA packaging into chromatin. Combined with chromatin immunoprecipitation (ChIP), MNase-ChIP-seq represents a high-resolution method for investigating both chromatin organization and the distribution of epigenetic marks and histone variants. The plot2DO package presented here is a flexible tool for evaluating the quality of MNase-seq and MNase-ChIP-seq data, and for visualizing the distribution of nucleosomes near the functional regions of the genome. The plot2DO package is open-source software, and it is freely available from https://github.com/rchereji/plot2DO under the MIT license.
Asunto(s)
Biología Computacional/métodos , Nucleosomas/genética , Nucleosomas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Simulación por Computador , Epigénesis Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Análisis de Secuencia de ADN , Programas InformáticosRESUMEN
Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing â¼147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of â¼190 bp, corresponding to a â¼45 bp linker. However, cortical neurons have a shorter average spacing of â¼165 bp. The significance of this atypical global chromatin organization is unclear. We have compared the chromatin structures of purified mouse dorsal root ganglia (DRG) neurons, cortical oligodendrocyte precursor cells (OPCs) and cortical astrocytes. DRG neurons have short average spacing (â¼165 bp), whereas OPCs (â¼182 bp) and astrocytes (â¼183 bp) have longer spacing. We measured nucleosome positions by MNase-seq and gene expression by RNA-seq. Most genes in all three cell types have a promoter chromatin organization typical of active genes: a nucleosome-depleted region at the promoter flanked by regularly spaced nucleosomes phased relative to the transcription start site. In DRG neurons, the spacing of phased nucleosomes downstream of promoters (â¼182 bp) is longer than expected from the genomic average for DRG neurons, whereas phased nucleosome spacing in OPCs and astrocytes is similar to the global average for these cells (â¼183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.
Asunto(s)
Astrocitos/metabolismo , Cromatina/genética , Neuronas/metabolismo , Nucleosomas/genética , Células Precursoras de Oligodendrocitos/metabolismo , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Electroforesis en Gel de Agar , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Histonas , Ratones , Nucleasa Microcócica , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , RNA-Seq , Análisis de Secuencia de ADN , TranscriptomaRESUMEN
DNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at almost all genomic AluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, suggesting that nucleosome spacing is a major determinant of accessibility. DNA accessibility varies from cell to cell, such that essentially no sites are accessible or inaccessible in every cell. AluI sites in inactive mouse promoters are accessible in some cells, implying that transcription factors could bind without activating the gene. Euchromatin and heterochromatin have very similar accessibilities, suggesting that transcription factors can penetrate heterochromatin. Thus, DNA accessibility is not likely to be the primary determinant of gene regulation.
Asunto(s)
Núcleo Celular , Cromatina , ADN de Hongos , Regulación Fúngica de la Expresión Génica , Hepatocitos/metabolismo , Regiones Promotoras Genéticas , Saccharomyces cerevisiae , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Ratones , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.
Asunto(s)
Nucleasa Microcócica , Nucleosomas , Análisis de Secuencia de ADN , Animales , Secuencia de Bases , Línea Celular , Cromatina , ADN/química , Drosophila melanogaster/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Cinética , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Cromatina/metabolismo , Cromatina/ultraestructura , ARN de Transferencia/genética , Sitios de Unión , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Ensamble y Desensamble de Cromatina , Cromosomas/genética , Cromosomas/metabolismo , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción TFIII/metabolismoRESUMEN
Most yeast genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. We have examined the interplay between RSC (a conserved essential SWI/SNF-type complex that determines NDR size) and the ISW1, CHD1, and ISW2 nucleosome spacing enzymes in chromatin organization and transcription, using isogenic strains lacking all combinations of these enzymes. The contributions of these remodelers to chromatin organization are largely combinatorial, distinct, and nonredundant, supporting a model in which the +1 nucleosome is positioned by RSC and then used as a reference nucleosome by the spacing enzymes. Defective chromatin organization correlates with altered RNA polymerase II (Pol II) distribution. RSC-depleted cells exhibit low levels of elongating Pol II and high levels of terminating Pol II, consistent with defects in both termination and initiation, suggesting that RSC facilitates both. Cells lacking both ISW1 and CHD1 show the opposite Pol II distribution, suggesting elongation and termination defects. These cells have extremely disrupted chromatin, with high levels of closely packed dinucleosomes involving the second (+2) nucleosome. We propose that ISW1 and CHD1 facilitate Pol II elongation by separating closely packed nucleosomes.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Elongación de la Transcripción Genética , Factores de Transcripción/genética , Terminación de la Transcripción Genética , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Nucleosomas/genética , Nucleosomas/metabolismo , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Human FACT (facilitates chromatin transcription) is a multifunctional protein complex that has histone chaperone activity and facilitates nucleosome survival and transcription through chromatin. Anticancer drugs curaxins induce FACT trapping on chromatin of cancer cells (c-trapping), but the mechanism of c-trapping is not fully understood. Here, we show that in cancer cells, FACT is highly enriched within the bodies of actively transcribed genes. Curaxin-dependent c-trapping results in redistribution of FACT from the transcribed chromatin regions to other genomic loci. Using a combination of biochemical and biophysical approaches, we have demonstrated that FACT is bound to and unfolds nucleosomes in the presence of curaxins. This tight binding to the nucleosome results in inhibition of FACT-dependent transcription in vitro in the presence of both curaxins and competitor chromatin, suggesting a mechanism of FACT trapping on bulk nucleosomes (n-trapping).
Asunto(s)
Carbazoles/farmacología , Ensamble y Desensamble de Cromatina/fisiología , Fibrosarcoma/genética , Histonas/metabolismo , Nucleosomas/metabolismo , Transcripción Genética/efectos de los fármacos , Antineoplásicos/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Fibrosarcoma/tratamiento farmacológico , Transferencia Resonante de Energía de Fluorescencia , Histonas/genética , Humanos , Nucleosomas/genética , Regiones Promotoras Genéticas , Unión Proteica , Células Tumorales CultivadasRESUMEN
It is unknown how the dynamic binding of transcription factors (TFs) is molecularly linked to chromatin remodeling and transcription. Using single-molecule tracking (SMT), we show that the chromatin remodeler RSC speeds up the search process of the TF Ace1p for its response elements (REs) at the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates transcription bursts of CUP1 only by modulating TF occupancy but does not affect initiation and elongation rates. We show by SMT that RSC binds to activated promoters transiently, and based on MNase-seq data, that RSC does not affect the nucleosomal occupancy at CUP1. Therefore, transient binding of Ace1p and rapid bursts of transcription at CUP1 may be dependent on short repetitive cycles of nucleosome mobilization. This type of regulation reduces the transcriptional noise and ensures a homogeneous response of the cell population to heavy metal stress.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Metalotioneína/genética , ARN Mensajero/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/metabolismo , Metalotioneína/metabolismo , Modelos Genéticos , Nucleosomas/química , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagen Individual de Molécula/métodos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The nucleosome remodeling complex RSC functions throughout the yeast genome to set the positions of -1 and +1 nucleosomes and thereby determines the widths of nucleosome-depleted regions (NDRs). The related complex SWI/SNF participates in nucleosome remodeling/eviction and promoter activation at certain yeast genes, including those activated by transcription factor Gcn4, but did not appear to function broadly in establishing NDRs. By analyzing the large cohort of Gcn4-induced genes in mutants lacking the catalytic subunits of SWI/SNF or RSC, we uncovered cooperation between these remodelers in evicting nucleosomes from different locations in the promoter and repositioning the +1 nucleosome downstream to produce wider NDRs-highly depleted of nucleosomes-during transcriptional activation. SWI/SNF also functions on a par with RSC at the most highly transcribed constitutively expressed genes, suggesting general cooperation by these remodelers for maximal transcription. SWI/SNF and RSC occupancies are greatest at the most highly expressed genes, consistent with their cooperative functions in nucleosome remodeling and transcriptional activation. Thus, SWI/SNF acts comparably with RSC in forming wide nucleosome-free NDRs to achieve high-level transcription but only at the most highly expressed genes exhibiting the greatest SWI/SNF occupancies.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The compact structure of the nucleosome limits DNA accessibility and inhibits the binding of most sequence-specific proteins. Nucleosomes are not randomly located on the DNA but positioned with respect to the DNA sequence, suggesting models in which critical binding sites are either exposed in the linker, resulting in activation, or buried inside a nucleosome, resulting in repression. The mechanisms determining nucleosome positioning are therefore of paramount importance for understanding gene regulation and other events that occur in chromatin, such as transcription, replication, and repair. Here, we review our current understanding of the major determinants of nucleosome positioning: DNA sequence, nonhistone DNA-binding proteins, chromatin-remodeling enzymes, and transcription. We outline the major challenges for the future: elucidating the precise mechanisms of chromatin opening and promoter activation, identifying the complexes that occupy promoters, and understanding the multiscale problem of chromatin fiber organization.
Asunto(s)
Nucleosomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , ADN/genética , ADN/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Transcripción GenéticaRESUMEN
Gcn4 is a yeast transcriptional activator induced by amino acid starvation. ChIP-seq analysis revealed 546 genomic sites occupied by Gcn4 in starved cells, representing â¼30% of Gcn4-binding motifs. Surprisingly, only â¼40% of the bound sites are in promoters, of which only â¼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining â¼300 Gcn4-bound sites are within coding sequences (CDSs), with â¼75 representing the only bound sites near Gcn4-induced genes. Many such unconventional sites map between divergent antisense and sub-genic sense transcripts induced within CDSs adjacent to induced TBP peaks, consistent with Gcn4 activation of cryptic bidirectional internal promoters. Mutational analysis confirms that Gcn4 sites within CDSs can activate sub-genic and full-length transcripts from the same or adjacent genes, showing that functional Gcn4 binding is not confined to promoters. Our results show that internal promoters can be regulated by an activator that functions at conventional 5'-positioned promoters.
Asunto(s)
Región de Flanqueo 5' , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Activación Transcripcional , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Sitios de Unión , ADN de Hongos/genética , Histonas/genética , Histonas/metabolismo , Mutación , Nucleosomas/genética , Unión Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The role of the histone chaperone SPT6 in mammalian cells is not fully understood. Here, we investigated the involvement of SPT6 in type I interferon (IFN)-induced transcription in murine fibroblasts. In RNA-seq analysis, Spt6 siRNA attenuates about half of ~ 200 IFN-stimulated genes (ISGs), while not affecting housekeeping genes. ISGs with high mRNA induction are more susceptible to Spt6 siRNA than those with lower levels of induction. ChIP analysis shows that SPT6 is recruited to highly inducible, Spt6 siRNA-sensitive ISGs, but not to other siRNA-insensitive ISGs. Furthermore, SPT6 recruitment is abrogated in cells lacking the histone methyltransferase NSD2. In co-IP experiments, SPT6 interacts with NSD2. In summary, SPT6 facilitates IFN-induced transcription, highlighting its critical role in gene activation.
Asunto(s)
Regulación de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Interferón Tipo I/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Animales , Células Cultivadas , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina/metabolismo , Interferón Tipo I/metabolismo , Ratones , Unión Proteica , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
We developed a chemical cleavage method that releases single nucleosome dyad-containing fragments, allowing us to precisely map both single nucleosomes and linkers with high accuracy genome-wide in yeast. Our single nucleosome positioning data reveal that nucleosomes occupy preferred positions that differ by integral multiples of the DNA helical repeat. By comparing nucleosome dyad positioning maps to existing genomic and transcriptomic data, we evaluated the contributions of sequence, transcription, and histones H1 and H2A.Z in defining the chromatin landscape. We present a biophysical model that neglects DNA sequence and shows that steric occlusion suffices to explain the salient features of nucleosome positioning.
Asunto(s)
Genómica/métodos , Nucleosomas , Genes , Histonas , Modelos Biológicos , Transcripción GenéticaRESUMEN
Glucocorticoid hormone plays a major role in metabolism and disease. The hormone-bound glucocorticoid receptor (GR) binds to a specific set of enhancers in different cell types, resulting in unique patterns of gene expression. We have addressed the role of chromatin structure in GR binding by mapping nucleosome positions in mouse adenocarcinoma cells. Before hormone treatment, GR-enhancers exist in one of three chromatin states: (i) Nucleosome-depleted enhancers that are DNase I-hypersensitive, associated with the Brg1 chromatin remodeler and flanked by nucleosomes incorporating histone H2A.Z. (ii) Nucleosomal enhancers that are DNase I-hypersensitive, marked by H2A.Z and associated with Brg1. (iii) Nucleosomal enhancers that are inaccessible to DNase I, incorporate little or no H2A.Z and lack Brg1. Hormone-induced GR binding results in nucleosome shifts at all types of GR-enhancer, coinciding with increased recruitment of Brg1. We propose that nucleosome-depleted GR-enhancers are formed and maintained by other transcription factors which recruit Brg1 whereas, at nucleosomal enhancers, GR behaves like a pioneer factor, interacting with nucleosomal sites and recruiting Brg1 to remodel the chromatin.
Asunto(s)
Cromatina/metabolismo , Elementos de Facilitación Genéticos , Nucleosomas/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Cromatina/efectos de los fármacos , Cromatina/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo , Dexametasona/metabolismo , Dexametasona/farmacología , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Histonas/genética , Histonas/metabolismo , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleosomas/efectos de los fármacos , Nucleosomas/genética , Unión Proteica/efectos de los fármacos , Receptores de Glucocorticoides/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.
Asunto(s)
Actinas/metabolismo , Ensamble y Desensamble de Cromatina , ADN/química , ADN/metabolismo , Complejo Mediador/metabolismo , ARN/metabolismo , Elementos Reguladores de la Transcripción , Sitios de Unión/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Conformación de Ácido Nucleico , Organismos Modificados Genéticamente , Unión Proteica , Multimerización de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Micrococcal nuclease (MNase) is commonly used to map nucleosomes genome-wide, but nucleosome maps are affected by the degree of digestion. It has been proposed that many yeast promoters are not nucleosome-free but instead occupied by easily digested, unstable, "fragile" nucleosomes. We analyzed the histone content of all MNase-sensitive complexes by MNase-ChIP-seq and sonication-ChIP-seq. We find that yeast promoters are predominantly bound by non-histone protein complexes, with little evidence for fragile nucleosomes. We do detect MNase-sensitive nucleosomes elsewhere in the genome, including at transcription termination sites. However, they have high A/T content, suggesting that MNase sensitivity does not indicate instability, but rather the preference of MNase for A/T-rich DNA, such that A/T-rich nucleosomes are digested faster than G/C-rich nucleosomes. We confirm our observations by analyzing ChIP-exo, chemical mapping, and ATAC-seq data from other laboratories. Thus, histone ChIP-seq experiments are essential to distinguish nucleosomes from other DNA-binding proteins that protect against MNase.
Asunto(s)
Nucleasa Microcócica/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Composición de Base , Nucleosomas , Regiones Promotoras GenéticasRESUMEN
Adenosine triphosphate-dependent chromatin remodeling machines play a central role in gene regulation by manipulating chromatin structure. Most genes have a nucleosome-depleted region at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. In vitro, the three known yeast nucleosome spacing enzymes (CHD1, ISW1 and ISW2) form arrays with different spacing. We used genome-wide nucleosome sequencing to determine whether these enzymes space nucleosomes differently in vivo We find that CHD1 and ISW1 compete to set the spacing on most genes, such that CHD1 dominates genes with shorter spacing and ISW1 dominates genes with longer spacing. In contrast, ISW2 plays a minor role, limited to transcriptionally inactive genes. Heavily transcribed genes show weak phasing and extreme spacing, either very short or very long, and are depleted of linker histone (H1). Genes with longer spacing are enriched in H1, which directs chromatin folding. We propose that CHD1 directs short spacing, resulting in eviction of H1 and chromatin unfolding, whereas ISW1 directs longer spacing, allowing H1 to bind and condense the chromatin. Thus, competition between the two remodelers to set the spacing on each gene may result in a highly dynamic chromatin structure.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina , Genes Fúngicos , Histonas/metabolismo , Nucleosomas/metabolismo , Transcripción GenéticaRESUMEN
Chaperones, nucleosome remodeling complexes, and histone acetyltransferases have been implicated in nucleosome disassembly at promoters of particular yeast genes, but whether these cofactors function ubiquitously, as well as the impact of nucleosome eviction on transcription genome-wide, is poorly understood. We used chromatin immunoprecipitation of histone H3 and RNA polymerase II (Pol II) in mutants lacking single or multiple cofactors to address these issues for about 200 genes belonging to the Gcn4 transcriptome, of which about 70 exhibit marked reductions in H3 promoter occupancy on induction by amino acid starvation. Examining four target genes in a panel of mutants indicated that SWI/SNF, Gcn5, the Hsp70 cochaperone Ydj1, and chromatin-associated factor Yta7 are required downstream from Gcn4 binding, whereas Asf1/Rtt109, Nap1, RSC, and H2AZ are dispensable for robust H3 eviction in otherwise wild-type cells. Using ChIP-seq to interrogate all 70 exemplar genes in single, double, and triple mutants implicated Gcn5, Snf2, and Ydj1 in H3 eviction at most, but not all, Gcn4 target promoters, with Gcn5 generally playing the greatest role and Ydj1 the least. Remarkably, these three cofactors cooperate similarly in H3 eviction at virtually all yeast promoters. Defective H3 eviction in cofactor mutants was coupled with reduced Pol II occupancies for the Gcn4 transcriptome and the most highly expressed uninduced genes, but the relative Pol II levels at most genes were unaffected or even elevated. These findings indicate that nucleosome eviction is crucial for robust transcription of highly expressed genes but that other steps in gene activation are more rate-limiting for most other yeast genes.