RESUMO
The chromatin-regulatory principles of histone post-translational modifications (PTMs) are discussed with a focus on the potential alterations in chromatin functional state due to steric and mechanical constraints imposed by bulky histone modifications such as ubiquitin and SUMO. In the classical view, PTMs operate as recruitment platforms for histone "readers," and as determinants of chromatin array compaction. Alterations of histone charges by "small" chemical modifications (e.g., acetylation, phosphorylation) could regulate nucleosome spontaneous dynamics without globally affecting nucleosome structure. These fluctuations in nucleosome wrapping can be exploited by chromatin-processing machinery. In contrast, ubiquitin and SUMO are comparable in size to histones, and it seems logical that these PTMs could conflict with canonical nucleosome organization. An experimentally testable hypothesis that by adding sterical bulk these PTMs can robustly alter nucleosome primary structure is proposed. The model presented here stresses the diversity of mechanisms by which histone PTMs regulate chromatin dynamics, primary structure and, hence, functionality.
Assuntos
Histonas/química , Histonas/metabolismo , Nucleossomos/metabolismo , Acetilação , Montagem e Desmontagem da Cromatina , Modelos Biológicos , Nucleossomos/química , Nucleossomos/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Sumoilação , Transcrição Gênica , UbiquitinaçãoRESUMO
DNA in nucleosomes has restricted nucleosome dynamics and is refractory to DNA-templated processes. Histone post-translational modifications play important roles in regulating DNA accessibility in nucleosomes. Whereas most histone modifications function either by mitigating the electrostatic shielding of histone tails or by recruiting 'reader' proteins, we show that ubiquitylation of H2B K34, which is located in a tight space protected by two coils of DNA superhelix, is able to directly influence the canonical nucleosome conformation via steric hindrances by ubiquitin groups. H2B K34 ubiquitylation significantly enhances nucleosome dynamics and promotes generation of hexasomes both with symmetrically or asymmetrically modified nucleosomes. Our results indicate a direct mechanism by which a histone modification regulates the chromatin structural states.
Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Cromatina/genética , Cromatina/metabolismo , Histonas/genética , Lisina/metabolismo , Nucleossomos/genética , Processamento de Proteína Pós-Traducional , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Histone posttranslational modifications (PTM) control gene activity by targeting chromatin-regulatory proteins. By altering histone charges PTMs could also modulate inter- and intra-nucleosomal interactions, and thus affect chromatin high-order compaction and nucleosome stochastic folding, respectively. However, recently it has been shown that histone H2BK34- ubiquitylation (which is deposited in vivo by MOF-MSL) can destabilize one of the nucleosomal H2A-H2B dimers in symmetrically and (albeit to a lesser extend) asymmetrically modified nucleosomes, and thus promote formation of a hexasome particle. Here we have studied ubiquitylation patterns by purified MSL1/MSL2 using nucleosomes and different histone substrates. We have shown that H2B-ubiquitylation by MSL1/2 depends on substrate configuration. In addition, MSL1/2 efficiently ubiquitylate histone substrates but very poorly modify nucleosomes, which implies a requirement for nucleosome structural alteration for efficient ubiquitylation of H2BK34. Nucleosome modification by MSL1/MSL2 in vitro was analyzed directly using nucleosome gel-mobility shift assay, which suggested that MSL1/2 can deposit two ubiquitin moieties in one nucleosome.
Assuntos
Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Técnicas In Vitro , Nucleossomos/metabolismo , Especificidade por Substrato , UbiquitinaçãoRESUMO
Catalytic activity of ISWI chromatin remodelers, which regulate nucleosome positioning on the DNA, depends on interactions of the putative acidic patch in ISWI helicase domain with the N-termini of nucleosomal H4--such, that removal of H4 termini abolishes ISWI remodeling. Acetylation of H4 termini is also known to disrupt H4 interactions with acidic protein surfaces, and thus, histone acetylation could potentially impede ISWI functions. Since active chromatin in vivo is hyperacetylated, it is important to clarify if ISWI activities can function on the in vivo hyperacetylated nucleosomes. We evaluated if purified yeast ISW2 can act on mononucleosomes in which all four core histones are highly acetylated. Mononucleosomes were assembled using purified histones from mammalian CV1 cells grown in the presence of deacetylase inhibitor Trichostatin A (TSA). The CV1 cell line is characterized by fast kinetic of accumulation of highly acetylated histone isoforms in response to TSA treatment. However, such 'native' histone hyperacetylation had no apparent effects on the nucleosome remodeling propensities, suggesting that histone hyperacetylation does not necessarily block ISWI functions and that ISWI enzymes can function on active chromatin as well.
Assuntos
Adenosina Trifosfatases/farmacologia , Montagem e Desmontagem da Cromatina/fisiologia , Histonas/metabolismo , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Fatores de Transcrição/farmacologia , Acetilação , Catálise , Montagem e Desmontagem da Cromatina/efeitos dos fármacosRESUMO
The solid-phase protein assays using blotting membranes as hard support do not allow achieving the low background and sensitivity of protein staining in clear gels. The membrane opacity complicates imaging of results on standard lab documentation systems. We describe a low-cost transparent matrix that can be used as an alternative to polymeric membranes for solid-phase assays. Protein samples are spotted onto a dry film of composite agarose-polyacrylamide matrix covering standard glass microscopic slides. After rehydration in protein-fixing solution, matrix with protein samples can be detached from glass support and stained as conventional protein polyacrylamide gels.
Assuntos
Resinas Acrílicas/química , Proteínas/análise , Sefarose/química , Animais , Bovinos , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional , Soroalbumina Bovina/análise , Coloração e RotulagemRESUMO
A new solid-phase protein nano-assay is suggested for simple and sensitive estimation of protein content in sample buffers (a 1-µl sample is sufficient for analysis). The assay is different from conventional "on-filter" assays in that it uses inexpensive fully transparent polyacrylamide gel (PAAG)-coated glass plates as solid support and, thus, combines the convenience of "on-membrane" staining with the sensitivity and ease of documentation of "in-gel" staining (and, therefore, is especially suited for standard lab gel documentation systems). The PAAG plates assay is compatible with all dyes for in-gel protein staining. Depending on the sensitivity of the staining protocol, the assay can be used in macro-, micro-, and nano-assay formats. We also describe a low-cost two-component colloidal Coomassie brilliant blue G-250 (CBB G-250) staining protocol for fast quantitative visualization of proteins spotted on a PAAG plate (the detection limit is up to 2 ng of proteins even when using a Nikon CoolPix digital camera and white light transilluminator instead of a gel scanner). The suggested colloidal CBB G-250 protocol could also be used for visualizing nano-amounts of proteins in polyacrylamide gels. The PAAG plate assay could be useful for proteomic applications and, in general, for all cases where a fast, sensitive, and easily documentable cost-effective solid-phase protein assay is required.
Assuntos
Resinas Acrílicas/química , Vidro/química , Nanotecnologia/economia , Nanotecnologia/métodos , Proteínas/análise , Animais , Soluções Tampão , Bovinos , Análise Custo-Benefício , Limite de Detecção , Soroalbumina Bovina/análise , Fatores de TempoRESUMO
Nucleosome remodeling studies in vitro have primarily focused on the use of mononucleosome templates, which, however, can provide only limited information on how nucleosome mobilization occurs in the context of chromatin, in which internucleosome interactions might influence remodeling. We tried to evaluate whether nucleosome mobilization by yeast Isw1a, Isw1b and Isw2 could be affected by neighboring nucleosomes. We compared mono- and dinucleosomes positioned by the '601' sequence, the studied constructs contain variation in linker length between nucleosomes and variation in the length of flanking sequences. The data characterizing the remodeling were based on gel retardation of the mono and dinucleosomes, keeping in mind the observation that the relative position of the nucleosome will change the mobility of the complex in well defined ways. We found that Isw1a, Isw1b and Isw2 process nucleosomes differently whether they exist as mononucleosomes or dinucleosomes, such as, the Isw1a and Isw1b nucleosome repositioning patterns, which were very similar for mononucleosomes, appeared to be profoundly different in case of dinucleosome templates. We also examined the DNase I protection patterns of remodeled mono- and dinucleosomes. The data suggest that nucleosome mobilizing activity of Isw1a, Isw1b and Isw2 complexes could be significantly influenced by neighboring nucleosomes.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Montagem e Desmontagem da Cromatina , DNA Fúngico/metabolismo , Histonas/metabolismo , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The three Saccharomyces cerevisiae ISWI chromatin remodeling complexes, Isw1a, Isw1b, and Isw2, are implicated in the regularization of arrayed nucleosomes and regulation of gene activity. Although Isw1a and Isw1b are based on the same catalytic unit, in general, their functions in vivo do not overlap. To better understand the structural consequences of these complexes, we compared the putative nucleosome disrupting activities of the purified Isw1a, Isw1b, and Isw2. To account for the putative effects of nucleosomal environment, we employed reconstituted dinucleosomes in which the histone octamers were specifically positioned by the 146 base pair high-affinity nucleosome sequence "601". We have compared the MNase and deoxyribonuclease I protection patterns of remodeled nucleosome templates and evaluated the nucleosome destabilizing abilities of the Isw1a/b and Isw2 using restriction endonucleases. Although the Isw2 showed little evidence of nucleosome disassembly, the Isw1b remodeled dinucleosomes exhibited some common features with the ySwi-Snf remodeling products. The nuclease digestion data suggest that Isw1a can also promote ATP-dependent distortion of nucleosome structure, although less efficiently than the Isw1b complex.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Montagem e Desmontagem da Cromatina , Desoxirribonuclease I/metabolismo , Histonas/metabolismo , Nuclease do Micrococo/metabolismo , Nucleossomos/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/fisiologiaRESUMO
Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semi-synthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3 K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA Polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.
RESUMO
Post-translational modifications of histone proteins often mediate gene regulation by altering the global and local stability of the nucleosome, the basic gene-packing unit of eukaryotes. We employed semisynthetic approaches to introduce histone H2B ubiquitylations at K34 (H2BK34ub) and K120 (H2BK120ub) and H3K79 trimethylation (H3K79me3). With these modified histones, we investigated their effects on the kinetics of transcription elongation by RNA polymerase II (Pol II) using single-molecule FRET. Pol II pauses at several locations within the nucleosome for a few seconds to minutes, which governs the overall transcription efficiency. We found that H2B ubiquitylations suppress pauses and shorten the pause durations near the nucleosome entry while H3K79me3 shortens the pause durations and increases the rate of RNA elongation near the center of the nucleosome. We also found that H2BK34ub facilitates partial rewrapping of the nucleosome upon Pol II passage. These observations suggest that H2B ubiquitylations promote transcription elongation and help maintain the chromatin structure by inducing and stabilizing nucleosome intermediates and that H3K79me3 facilitates Pol II progression possibly by destabilizing the local structure of the nucleosome. Our results provide the mechanisms of how these modifications coupled by a network of regulatory proteins facilitate transcription in two different regions of the nucleosome and help maintain the chromatin structure during active transcription.
Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Transcrição Gênica , RNA Polimerase II/química , UbiquitinaçãoRESUMO
Functioning of histone lysine methyltransferases (HKMTs) involves interactions of their catalytic domain "SET" with the N-termini of histone H3. However, these interactions are restricted in canonical nucleosomes due to the limited accessibility of H3 termini. Here we investigated whether nucleosome remodeling with the yeast Isw2 affects nucleosome affinity to the SET domain of ALL-1 HKMT. Reconstitution of mononucleosomes by salt dilutions also produces some nucleosome-dimer particles (self-associated mononucleosomes, described by: Tatchell and van Holde (1977) Biochemistry, 16, 5295-5303). The GST-tagged SET-domain polypeptide of ALL-1 was assayed for binding to assembled mononucleosomes and nucleosome-dimer particles, either intact or remodeled with purified yeast Isw2. Remodeling of mononucleosomes does not noticeably affect their affinity to SET domain; however, yIsw2 remodeling of nucleosome-dimer particles facilitated their association with GST-SET polypeptide. Therefore, it is conceivable that nucleosome interactions in trans could be implicated in the maintenance of chromatin methylation patterns in vivo.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina , Dimerização , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Humanos , Técnicas In Vitro , Proteína de Leucina Linfoide-Mieloide/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Tight packaging of DNA in chromatin severely constrains DNA accessibility and dynamics. In contrast, nucleosomes in active chromatin state are highly flexible, can exchange their histones, and are virtually "transparent" to RNA polymerases, which transcribe through gene bodies at rates comparable to that of naked DNA. Defining mechanisms that revert nucleosome repression, in addition to their value for basic science, is of key importance for the diagnosis and treatment of genetic diseases. Chromatin activity is largely regulated by histone posttranslational modifications, ranging from small chemical groups up to the yet understudied "bulky" ubiquitylation and sumoylation. However, it is to be revealed how histone marks are "translated" to permissive or repressive changes in nucleosomes: it is a general opinion that histone modifications act primarily as "signals" for recruiting the regulatory proteins or as a "neutralizer" of electrostatic shielding of histone tails. Here, we would like to discuss recent evidence suggesting that histone ubiquitylation, in a DNA stress-dependent manner, can directly regulate the dynamics of the nucleosome and their primary structure and can promote nucleosome decomposition to hexasome particles or additionally stabilize nucleosomes against unwrapping. In addition, nucleosome repression/ derepression studies are usually performed with single mononucleosomes as a model. We would like to review and discuss recent findings showing that internucleosomal interactions could strongly modulate the dynamics and rearrangements of nucleosomes. Our hypothesis is that bulky histone modifications, nucleosome inherent dynamics, internucleosome interactions, and DNA torsions could act in cooperation to orchestrate the formation of different dynamic states of arrayed nucleosomes and thus promote chromatin functionality and diversify epigenetic programming methods.
RESUMO
The Swi/Snf chromatin-remodeling complexes, human BAF/PBAF and yeast RSC, can catalyze formation of stably altered dimeric forms of nucleosomes. However, the ability to create remodeled dimers has not yet been reported for the Saccharomyces cerevisiae Swi/Snf complex. Despite its similarity with the other Swi/Snf proteins, the yeast Swi/Snf complex features certain structural and functional differences. This raises the question of whether ySwi/Snf can in fact catalyze formation of dimeric nucleosomes. Dimer formation was proposed to have a specific impact on chromatin regulatory effects. Thus, the answer to the above question may be helpful in clarifying the ySwi/Snf functions in vivo and generalizing the notions of the regulatory principles of Swi/Snf family proteins. Here we describe ySwi/Snf-catalyzed formation of nucleosome dimers using mono- and dinucleosome templates assembled from purified histones and DNA of the high-affinity (601) nucleosome positioning sequence. We evaluated effects of nucleosome template geometry on the formation of altered dimers and assayed formation of altered nucleosome pairs on reconstituted dinucleosomes.
Assuntos
Proteínas Cromossômicas não Histona/biossíntese , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/biossíntese , Catálise , DNA Fúngico/metabolismo , Dimerização , Histonas/metabolismo , Proteínas Nucleares/metabolismoRESUMO
The inheritable methylation pattern of gene activity, created upon cell differentiation, is further maintained by the "SET" (methyltransferase)-domain proteins. However, it is still not clear how SET-proteins can decide on the required gene activity state and the way their chromatin association is maintained. Here we have found that high levels of histone acetylation--the hallmarks of active chromosome regions in vivo--can increase the affinity of reconstituted nucleosomes to the SET domain of ALL-1 histone methyltransferase in a defined system in vitro.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nucleossomos/enzimologia , Acetilação , Células HeLa , Humanos , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Apart the gene-regulatory functions as docking sites for histone 'readers', some histone modifications could directly affect nucleosome structure. The H2BK34-ubiquitylation deposited by MOF-MSL complex, increases nucleosome dynamics in vitro and promotes donation of one H2A/H2B dimer to histone acceptors. METHODS: We evaluated temperature-depended stability of H2BK34-ubiquitylated nucleosomes under 'physiological' ionic conditions in the presence or absence of histone acceptor, and examined assembly and disassembly of ubiquitylated nucleosomes in vitro by recombinant mouse NAP1. RESULTS: H2BK34ub modification is sufficient to promote selective eviction of only one H2A/H2B dimer independently of histone-binding agents. Despite the robust H2A/H2B dimer-displacement effect of mNAP1 with the H2BK34ub (but not unmodified) nucleosomes, NAP1 could assemble symmetrically- or asymmetrically ubiquitylated nucleosomes under 'physiological' conditions in vitro. CONCLUSIONS AND GENERAL SIGNIFICANCE: The increased mobility of one nucleosomal H2A/H2B dimer is an intrinsic nucleosome destabilizing property of H2BK34 ubiquitylation that has the intranucleosome bases. The ability of NAP to reasonably efficiently assemble H2BK34-ubiquitylated nucleosomes supposes a potential mechanism for deposition/distribution of H2BK34ub mark in the MOF-MSL independent manner (for example, during histone dimer exchange upon transcription elongation).
Assuntos
Histonas/metabolismo , Naftalenos/metabolismo , Oligopeptídeos/metabolismo , Animais , Cromatina/metabolismo , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/fisiologia , Histonas/fisiologia , Camundongos , Proteína 1 de Modelagem do Nucleossomo/química , Proteína 1 de Modelagem do Nucleossomo/genética , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Nucleossomos/metabolismo , Oligopeptídeos/fisiologia , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ubiquitinação/fisiologiaRESUMO
Steric hindrances by bulky histone modifications (such as ubiquitylation) could destabilize and remodel canonical nucleosome structure. This highlights a novel mechanism by which bulky modifications directly regulate chromatin, distinct from the more generally accepted roles of histone modifications in the recruitment of downstream effectors and histone charge shielding.
Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Ubiquitinação , Cromatina/metabolismo , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Using cell-free system derived from Drosophila embryos, we found evidence for a regulated nucleosome disruption process, which depends on the phosphorylation status of 120 kDa protein (complex). Dephosphorylation enables the remodeling activity to destabilize nucleosomes, which assume a more accessible structure, possessing increased DNase I sensitivity and high conformational flexibility of DNA; remodeling was more efficient on highly acetylated chromatin templates. This phosphorylation-regulated nucleosome destabilization, acting synergistically with histone acetylation, is discussed as a possible mechanism to provide regulated disrupt of histone-DNA interaction.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Nucleossomos/metabolismo , Acetilação , Animais , Sistema Livre de Células , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Histonas/metabolismo , FosforilaçãoRESUMO
The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trxZ11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.
Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Super-Helicoidal/metabolismo , Nucleossomos/metabolismo , Transcrição Gênica/fisiologia , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Animais , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Humanos , Mutação/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , RNA/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
On the nucleosome level, histone posttranslational modifications function mainly as the regulatory signals; in addition, some posttranslational modifications can enhance nucleosome stochastic folding, which is restricted in "canonic" nucleosomes. Recently, it has been shown in vitro that symmetric or asymmetric nucleosome ubiquitylation at H2BK34 (and H2BK120, to a lesser extent) can destabilize one of the nucleosomal H2A-H2B dimers and promote nucleosome conversion to a hexasome particle [Krajewski et al. (2018). Nucleic Acids Res., 46, 7631-7642]. Such lability of H2Bub nucleosomes raises a question of whether they could accommodate transient changes in DNA torsional tensions, which are generated by virtually any process that manipulates DNA strands. Using positively or negatively supercoiled DNA minicircles and homogeneously-modified H2Bub histones, we have found that DNA topology could strongly and selectively affect nucleosome stability depending on its ubiquitylation state (here the term "nucleosome stability" means the nucleosome property to maintain its structural integrity and dynamics characteristic to "canonic" nucleosomes). The results point to a role for H2B ubiquitylation in amplifying or mitigating the effects of a DNA torque on the nucleosome stability and dynamics.
Assuntos
DNA Super-Helicoidal/metabolismo , Histonas/metabolismo , Nucleossomos/química , Modelos Moleculares , Conformação Molecular , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
Evidence is emerging that many diseases result from defects in gene functions, which, in turn, depend on the local chromatin environment of a gene. However, it still remains not fully clear how chromatin activity code is 'translated' to the particular 'activating' or 'repressing' chromatin structural transition. Commonly, chromatin remodeling in vitro was studied using mononucleosomes as a model. However, recent data suggest that structural reorganization of a single mononucleosome is not equal to remodeling of a nucleosome particle under multinucleosomal content - such as, interaction of nucleosomes via flexible histone termini could significantly alter the mode (and the resulting products) of nucleosome structural transitions. It is becoming evident that a nucleosome array does not constitute just a 'polymer' of individual 'canonical' nucleosomes due to multiple inter-nucleosomal interactions which affect nucleosome dynamics and structure. It could be hypothesized, that inter-nucleosomal interactions could act in cooperation with nucleosome inherent dynamics to orchestrate DNA-based processes and promote formation and stabilization of highly-dynamic, accessible structure of a nucleosome array. In the proposed paper we would like to discuss the nucleosome dynamics within the chromatin fiber mainly as it pertains to the roles of the structural changes mediated by inter-nucleosomal interactions.