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2.
Nucleic Acids Res ; 48(15): 8408-8430, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32663283

RESUMO

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.


Assuntos
Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Ativação Transcricional/genética , Adenosina Trifosfatases/genética , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/genética
3.
Methods Mol Biol ; 2117: 93-108, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31960374

RESUMO

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.


Assuntos
Biologia Computacional/métodos , Nucleossomos/genética , Nucleossomos/metabolismo , Animais , Imunoprecipitação da Cromatina , Simulação por Computador , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA , Software
4.
Neurosci Lett ; 714: 134559, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639421

RESUMO

Eukaryotic chromosomes are composed of chromatin, in which regularly spaced nucleosomes containing ∼147 bp of DNA are separated by linker DNA. Most eukaryotic cells have a characteristic average nucleosome spacing of ∼190 bp, corresponding to a ∼45 bp linker. However, cortical neurons have a shorter average spacing of ∼165 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 (∼165 bp), whereas OPCs (∼182 bp) and astrocytes (∼183 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 (∼182 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 (∼183 bp). Thus, the atypical nucleosome spacing of neuronal chromatin does not extend to promoter-proximal regions.


Assuntos
Astrócitos/metabolismo , Cromatina/genética , Neurônios/metabolismo , Nucleossomos/genética , Células Precursoras de Oligodendrócitos/metabolismo , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Eletroforese em Gel de Ágar , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Histonas , Camundongos , Nuclease do Micrococo , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Análise de Sequência de DNA , Transcriptoma
5.
Genome Biol ; 20(1): 198, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519205

RESUMO

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.


Assuntos
Nuclease do Micrococo , Nucleossomos , Análise de Sequência de DNA , Animais , Sequência de Bases , Linhagem Celular , Cromatina , DNA/química , Drosophila melanogaster/genética , Sequenciamento de Nucleotídeos em Larga Escala , Cinética , Transcrição Gênica
6.
Genome Res ; 29(12): 1985-1995, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31511305

RESUMO

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.


Assuntos
Núcleo Celular , Cromatina , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Hepatócitos/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Camundongos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
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
8.
Genome Res ; 29(3): 407-417, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30683752

RESUMO

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.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Terminação da Transcrição Genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Nucleossomos/genética , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
9.
Sci Adv ; 4(11): eaav2131, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30417101

RESUMO

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).


Assuntos
Carbazóis/farmacologia , Montagem e Desmontagem da Cromatina/fisiologia , Fibrossarcoma/genética , Histonas/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Fibrossarcoma/tratamento farmacológico , Transferência Ressonante de Energia de Fluorescência , Histonas/genética , Humanos , Nucleossomos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Células Tumorais Cultivadas
10.
Mol Cell ; 72(5): 875-887.e9, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30318444

RESUMO

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.


Assuntos
Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Metalotioneína/genética , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Metalotioneína/metabolismo , Modelos Genéticos , Nucleossomos/química , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , Fatores de Transcrição/metabolismo , Transcrição Gênica
11.
Genes Dev ; 32(9-10): 695-710, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29785963

RESUMO

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.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética
12.
FEBS Lett ; 592(10): 1681-1692, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29683485

RESUMO

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.


Assuntos
Regulação da Expressão Gênica/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Interferon Tipo I/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Histona-Lisina N-Metiltransferase/metabolismo , Interferon Tipo I/metabolismo , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
13.
Biophys J ; 114(10): 2279-2289, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29628211

RESUMO

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.


Assuntos
Nucleossomos/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , DNA/genética , DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Transcrição Gênica
14.
Mol Cell ; 70(2): 297-311.e4, 2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29628310

RESUMO

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 ∼30% of Gcn4-binding motifs. Surprisingly, only ∼40% of the bound sites are in promoters, of which only ∼60% activate transcription, indicating extensive negative control over Gcn4 function. Most of the remaining ∼300 Gcn4-bound sites are within coding sequences (CDSs), with ∼75 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.


Assuntos
Região 5'-Flanqueadora , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ativação Transcricional , Fatores de Transcrição de Zíper de Leucina Básica/genética , Sítios de Ligação , DNA Fúngico/genética , Histonas/genética , Histonas/metabolismo , Mutação , Nucleossomos/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
Genome Biol ; 19(1): 19, 2018 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-29426353

RESUMO

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.


Assuntos
Genômica/métodos , Nucleossomos , Genes , Histonas , Modelos Biológicos , Transcrição Gênica
16.
Nucleic Acids Res ; 46(1): 203-214, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29126175

RESUMO

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.


Assuntos
Cromatina/metabolismo , Elementos Facilitadores Genéticos , Nucleossomos/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Cromatina/genética , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Dexametasona/metabolismo , Dexametasona/farmacologia , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Histonas/genética , Histonas/metabolismo , Camundongos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/efeitos dos fármacos , Nucleossomos/genética , Ligação Proteica/efeitos dos fármacos , Receptores de Glucocorticoides/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos
17.
Nucleic Acids Res ; 45(15): 8806-8821, 2017 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-28575439

RESUMO

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.


Assuntos
Actinas/metabolismo , Montagem e Desmontagem da Cromatina , DNA/química , DNA/metabolismo , Complexo Mediador/metabolismo , RNA/metabolismo , Elementos Reguladores de Transcrição , Sítios de Ligação/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Conformação de Ácido Nucleico , Organismos Geneticamente Modificados , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Mol Cell ; 65(3): 565-577.e3, 2017 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-28157509

RESUMO

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.


Assuntos
Nuclease do Micrococo/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Composição de Bases , Nucleossomos , Regiões Promotoras Genéticas
19.
Nucleic Acids Res ; 44(10): 4625-35, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26861626

RESUMO

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.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Montagem e Desmontagem da Cromatina , Genes Fúngicos , Histonas/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica
20.
Nucleic Acids Res ; 44(3): 1036-51, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26429969

RESUMO

Nucleosomal DNA is thought to be generally inaccessible to DNA-binding factors, such as micrococcal nuclease (MNase). Here, we digest Drosophila chromatin with high and low concentrations of MNase to reveal two distinct nucleosome types: MNase-sensitive and MNase-resistant. MNase-resistant nucleosomes assemble on sequences depleted of A/T and enriched in G/C-containing dinucleotides, whereas MNase-sensitive nucleosomes form on A/T-rich sequences found at transcription start and termination sites, enhancers and DNase I hypersensitive sites. Estimates of nucleosome formation energies indicate that MNase-sensitive nucleosomes tend to be less stable than MNase-resistant ones. Strikingly, a decrease in cell growth temperature of about 10°C makes MNase-sensitive nucleosomes less accessible, suggesting that observed variations in MNase sensitivity are related to either thermal fluctuations of chromatin fibers or the activity of enzymatic machinery. In the vicinity of active genes and DNase I hypersensitive sites nucleosomes are organized into periodic arrays, likely due to 'phasing' off potential barriers formed by DNA-bound factors or by nucleosomes anchored to their positions through external interactions. The latter idea is substantiated by our biophysical model of nucleosome positioning and energetics, which predicts that nucleosomes immediately downstream of transcription start sites are anchored and recapitulates nucleosome phasing at active genes significantly better than sequence-dependent models.


Assuntos
Cromatina/metabolismo , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Genoma , Nucleossomos/metabolismo , Animais , Imunoprecipitação da Cromatina , Drosophila melanogaster/embriologia
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