scholarly journals BZLF1 interacts with chromatin remodelers promoting escape from latent infections with EBV

2019 ◽  
Vol 2 (2) ◽  
pp. e201800108 ◽  
Author(s):  
Marisa Schaeffner ◽  
Paulina Mrozek-Gorska ◽  
Alexander Buschle ◽  
Anne Woellmer ◽  
Takanobu Tagawa ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Viral Transcription ◽  
Sequence Motifs ◽  
Viral Dna ◽  
Accessible Chromatin

A hallmark of EBV infections is its latent phase, when all viral lytic genes are repressed. Repression results from a high nucleosome occupancy and epigenetic silencing by cellular factors such as the Polycomb repressive complex 2 (PRC2) and DNA methyltransferases that, respectively, introduce repressive histone marks and DNA methylation. The viral transcription factor BZLF1 acts as a molecular switch to induce transition from the latent to the lytic or productive phase of EBV’s life cycle. It is unknown how BZLF1 can bind to the epigenetically silenced viral DNA and whether it directly reactivates the viral genome through chromatin remodeling. We addressed these fundamental questions and found that BZLF1 binds to nucleosomal DNA motifs both in vivo and in vitro. BZLF1 co-precipitates with cellular chromatin remodeler ATPases, and the knock-down of one of them, INO80, impaired lytic reactivation and virus synthesis. In Assay for Transposase-Accessible Chromatin-seq experiments, non-accessible chromatin opens up locally when BZLF1 binds to its cognate sequence motifs in viral DNA. We conclude that BZLF1 reactivates the EBV genome by directly binding to silenced chromatin and recruiting cellular chromatin-remodeling enzymes, which implement a permissive state for lytic viral transcription. BZLF1 shares this mode of action with a limited number of cellular pioneer factors, which are instrumental in transcriptional activation, differentiation, and reprogramming in all eukaryotic cells.

scholarly journals BZLF1 interacts with the chromatin remodeler INO80 promoting escape from latent infections with Epstein-Barr virus

2018 ◽  
Author(s):  
Marisa Schaeffner ◽  
Paulina Mrozek-Gorska ◽  
Anne Woellmer ◽  
Takanobu Tagawa ◽  
Alexander Buschle ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Epstein Barr Virus ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Viral Transcription ◽  
Chromatin Remodeler ◽  
Barr Virus ◽  
Pioneer Factors ◽  
Epstein Barr

ABSTRACTA hallmark of Epstein-Barr virus (EBV) infections is its latent phase, when all viral lytic genes are repressed. Repression results from a high nucleosome occupancy and epigenetic silencing by cellular factors such as the Polycomb repressive complex 2 (PRC2) and DNA methyltransferases that respectively introduce repressive histone marks and DNA methylation. The viral transcription factor BZLF1 acts as molecular switch to induce the transition from the latent to the lytic or productive phase of EBV’s life cycle. It is unknown how BZLF1 can bind to the epigenetically silenced viral DNA and whether it directly reactivates the viral genome through chromatin remodeling. We addressed these fundamental questions and found that BZLF1 binds to nucleosomal DNA motifs bothin vivoandin vitro, a property characteristic ofbona fidepioneer factors. BZLF1 co-precipitates with cellular chromatin remodeler ATPases, and the knock-down of one of them, INO80, impaired lytic reactivation and virus synthesis. We conclude that BZLF1 reactivates the EBV genome by directly binding to silenced chromatin and recruiting cellular chromatin remodeling enzymes, which implement a permissive state for viral transcription. BZLF1 shares this mode of action with a limited number of cellular pioneer factors, which are instrumental in transcriptional activation, differentiation, and reprogramming in all eukaryotic cells.

scholarly journals Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Adrian L Sanborn ◽  
Benjamin T Yeh ◽  
Jordan T Feigerle ◽  
Cynthia V Hao ◽  
Raphael J L Townshend ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Structural Constraints ◽  
Sequence Motifs ◽  
Activation Domains ◽  
Activator Proteins ◽  
Transcriptional Activation Domain ◽  
Shape Complementarity ◽  
And Function

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

scholarly journals Gal4p-Mediated Chromatin Remodeling Depends on Binding Site Position in Nucleosomes but Does Not Require DNA Replication

1998 ◽  
Vol 18 (3) ◽  
pp. 1201-1212 ◽  
Author(s):  
Mai Xu ◽  
Robert T. Simpson ◽  
Michael P. Kladde
Keyword(s):  
Dna Replication ◽  
Binding Site ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Chromatin Modification ◽  
Factor Binding ◽  
Affect Factor ◽  
Nucleosome Binding

ABSTRACT Biochemical studies have demonstrated decreased binding of various proteins to DNA in nucleosome cores as their cognate sites are moved from the edge of the nucleosome to the pseudodyad (center). However, to date no study has addressed whether this structural characteristic of nucleosomes modulates the function of a transcription factor in living cells, where processes of DNA replication and chromatin modification or remodeling could significantly affect factor binding. Using a sensitive, high-resolution methyltransferase assay, we have monitored the ability of Gal4p in vivo to interact with a nucleosome at positions that are known to be inaccessible in nucleosome cores in vitro. Gal4p efficiently bound a single cognate site (UASG) centered at 41 bp from the edge of a positioned nucleosome, perturbing chromatin structure and inducing transcription. DNA binding and chromatin perturbation accompanying this interaction also occurred in the presence of hydroxyurea, indicating that DNA replication is not necessary for Gal4p-mediated nucleosome disruption. These data extend previous studies, which demonstrated DNA replication-independent chromatin remodeling, by showing that a single dimer of Gal4p, without the benefit of cooperative interactions that occur at complex wild-type promoters, is competent for invasion of a preestablished nucleosome. When the UASG was localized at the nucleosomal pseudodyad, relative occupancy by Gal4p, nucleosome disruption, and transcriptional activation were substantially compromised. Therefore, despite the increased nucleosome binding capability of Gal4p in cells, the precise translational position of a factor binding site in one nucleosome in an array can affect the ability of a transcriptional regulator to overcome the repressive influence of chromatin.

Dynamic changes of global DNA methylation and hypermethylation of cell adhesion-related genes in rat kidneys in response to ochratoxin A

2015 ◽  
Vol 8 (4) ◽  
pp. 465-476 ◽  
Author(s):  
X. Li ◽  
J. Gao ◽  
K. Huang ◽  
X. Qi ◽  
Q. Dai ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Adhesion ◽  
Ochratoxin A ◽  
Transcriptional Activation ◽  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Global Dna Methylation ◽  
Specific Cell

Ochratoxin A (OTA), which is found in a variety of food products, is associated with the development of nephrotoxicity and carcinogenicity in rats and has raised public health concerns. A previous study in our laboratory indicated that OTA exposure induced cytotoxicity by decreasing global DNA methylation in vitro. However, the relationship between OTA-induced nephrotoxicity and DNA methylation changes in vivo remains unclear. The object of this study was to investigate whether OTA can change global DNA methylation or alter the expression of several critical tumour-related genes by inducing methylation modifications before carcinogenesis. We focused on the mechanism of action of OTA in regard to DNA methylation, including the expression of DNA methyltransferases and the regulation of specific cell signalling pathways. Dynamic and dose-dependent changes of global DNA methylation were observed during OTA-induced nephrotoxicity and probably associated with the expression of DNA methyltransferase 1. 13-week exposure of OTA caused hypermethylation in the promoters of critical cell adhesion-related genes, E-cadherin and N-cadherin, leading to reduction of the corresponding mRNA expression, accompanied by transcriptional activation of the Wnt and PI3K/AKT pathways. These findings suggested that long-term OTA exposure could disrupt DNA methylation profile, which might be one of the possible mechanisms of OTA-induced nephrotoxicity.

scholarly journals Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

2018 ◽  
Author(s):  
Leslie Y. Beh ◽  
Galia T. Debelouchina ◽  
Derek M. Clay ◽  
Robert E. Thompson ◽  
Kelsi A. Lindblad ◽  
...  
Keyword(s):  
De Novo ◽  
Nucleosome Occupancy ◽  
Dna Methyltransferases ◽  
Chromatin Organization ◽  
Sexual Cycle ◽  
List Type ◽  
Adenine Methylation ◽  
The Impact

SummaryDNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1in vivoleads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organizationin vitroby reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomesin vitroin a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.HighlightsThe MT-A70 protein MTA1 mediates DNA N6-adenine methylation inOxytrichaMTA1 mutants exhibit subtle changes in nucleosome organization and transcriptionin vivo6mA directly disfavors nucleosome occupancy in natural and synthetic chromosomesin vitroDe novosynthesis of complete, epigenetically definedOxytrichachromosomes

scholarly journals The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling

2021 ◽  
Vol 22 (4) ◽  
pp. 1793
Author(s):  
Wenbo Dong ◽  
Punit Prasad ◽  
Andreas Lennartsson ◽  
Karl Ekwall
Keyword(s):  
Chromatin Remodeling ◽  
Nucleosome Occupancy ◽  
Strong Association ◽  
Atpase Domain ◽  
Coupling Region ◽  
Nucleosome Remodeling ◽  
Catalytic Domains

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.

scholarly journals Distinct Domains of Erythroid Krüppel-Like Factor Modulate Chromatin Remodeling and Transactivation at the Endogenous β-Globin Gene Promoter

2002 ◽  
Vol 22 (1) ◽  
pp. 161-170 ◽  
Author(s):  
R. Clark Brown ◽  
Scott Pattison ◽  
Janine van Ree ◽  
Elise Coghill ◽  
Andrew Perkins ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Gene Transcription ◽  
Transcriptional Activation ◽  
Globin Gene ◽  
Regulatory Elements ◽  
Binding Studies ◽  
Amino Terminal ◽  
Cellular Models

ABSTRACT Characterization of the mechanism(s) of action of trans-acting factors in higher eukaryotes requires the establishment of cellular models that test their function at endogenous target gene regulatory elements. Erythroid Krüppel-like factor (EKLF) is essential for β-globin gene transcription. To elucidate the in vivo determinants leading to transcription of the adult β-globin gene, functional domains of EKLF were examined in the context of chromatin remodeling and transcriptional activation at the endogenous locus. Human EKLF (hEKLF) sequences, linked to an estrogen-responsive domain, were studied with an erythroblast cell line lacking endogenous EKLF expression (J2eΔeklf). J2eΔeklf cells transduced with hEKLF demonstrated a dose-dependent rescue of β-globin transcription in the presence of inducing ligand. Further analysis using a series of amino-terminal truncation mutants of hEKLF identified a distinct internal domain, which is sufficient for transactivation. Interestingly, studies of the chromatin structure of the β-promoter revealed that a smaller carboxy-terminal domain generated an open promoter configuration. In vitro and in vivo binding studies demonstrated that this region interacted with BRG1, a component of the SWI/SNF chromatin remodeling complex. However, further study revealed that BRG1 interacted with an even smaller domain of EKLF, suggesting that additional protein interactions are required for chromatin remodeling at the endogenous β-promoter. Taken together, our findings support a stepwise process of chromatin remodeling and coactivator recruitment to the β-globin promoter in vivo. The J2eΔeklf inducible hEKLF system will be a valuable tool for further characterizing the temporal series of events required for endogenous β-globin gene transcription.

scholarly journals Plasmodium falciparum Histone Acetyltransferase, a Yeast GCN5 Homologue Involved in Chromatin Remodeling

2004 ◽  
Vol 3 (2) ◽  
pp. 264-276 ◽  
Author(s):  
Qi Fan ◽  
Lijia An ◽  
Liwang Cui
Keyword(s):  
Plasmodium Falciparum ◽  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Histone Acetyltransferase ◽  
Northern Analysis ◽  
Binding Assays ◽  
Core Histones ◽  
Chromatin Remodeling Complexes

ABSTRACT The yeast transcriptional coactivator GCN5 (yGCN5), a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcriptional activation. Like other eukaryotes, the malaria parasite DNA is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Here we show that GCN5 is conserved in Plasmodium species and that the most homologous regions are within the HAT domain and the bromodomain. The Plasmodium falciparum GCN5 homologue (PfGCN5) is spliced with three introns, encoding a protein of 1,464 residues. Mapping of the ends of the PfGCN5 transcript suggests that the mRNA is 5.2 to 5.4 kb, consistent with the result from Northern analysis. Using free core histones, we determined that recombinant PfGCN5 proteins have conserved HAT activity with a substrate preference for histone H3. Using substrate-specific antibodies, we determined that both Lys-8 and -14 of H3 were acetylated by the recombinant PfGCN5. In eukaryotes, GCN5 homologues interact with yeast ADA2 homologues and form large multiprotein HAT complexes. We have identified an ADA2 homologue in P. falciparum, PfADA2. Yeast two-hybrid and in vitro binding assays verified the interactions between PfGCN5 and PfADA2, suggesting that they may be associated with each other in vivo. The conserved function of the HAT domain in PfGCN5 was further illustrated with yeast complementation experiments, which showed that the PfGCN5 region corresponding to the full-length yGCN5 could partially complement the yGCN5 deletion mutation. Furthermore, a chimera comprising the PfGCN5 HAT domain fused to the remainder of yeast GCN5 (yGCN5) fully rescued the yGCN5 deletion mutant. These data demonstrate that PfGCN5 is an authentic GCN5 family member and may exist in chromatin-remodeling complexes to regulate gene expression in P. falciparum.

scholarly journals Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

2020 ◽  
Author(s):  
Adrian L. Sanborn ◽  
Benjamin T. Yeh ◽  
Jordan T. Feigerle ◽  
Cynthia V. Hao ◽  
Raphael J. L. Townshend ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Structural Constraints ◽  
Sequence Motifs ◽  
Activation Domains ◽  
Activator Proteins ◽  
Transcriptional Activation Domain ◽  
Shape Complementarity ◽  
And Function

SUMMARYGene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

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