Dissociation of single-stranded DNA from nucleosomes following modification with acetic anhydride

Modification with acetic anhydride of nucleosomes from chicken erythrocytes at low ionic strength (less than 0.1 M NaCl) is accompanied by the formation of residual particles and the release of free DNA. This DNA has been identified as single-stranded by thermal denaturation, digestion with nuclease S1, and elution from hydroxyapatite. In contrast, if modification takes place at 0.6 M NaCl, the liberated DNA is mainly double-stranded. The release of the free energy stored in folded nucleosomal DNA, triggered by the weakening of lysine-DNA interactions which takes place upon modification, might be responsible for the observed denaturation of DNA at low ionic stregth.This work was supported in part by the Fondo de Investigaciones Sanitarias and Comisión Asesora de Investigación Científica y Técnica (Spain).Peer Reviewe

Investigations into the role of histone H2A ubiquitination in chromatin

Bibliography: leaves 141-150.An in vitro system was used to determine the effect of histone H2A ubiquitination on linker histone binding to mononucleosomes. Hybrid octamers containing either H2A or ubiquitinated H2A (uH2A) were reconstituted onto random sequence 167 bp DNA. The affinity of the resultant nucleosome cores for linker histone H1 was determined from nucleoprotein gel shifts, protein analyses and thermal denaturation. Ubiquitinated H2A did not inhibit linker histone binding to nucleosome cores. The effect of uH2A on nucleosome and chromatosoine positioning on a 208 bp Lytechinus variegatus 5S rDNA fragment was investigated using a combination of micrococcal nuclease digestion and subsequent restriction enzyme digestion of the core particle or chromatosome DNA. Nucleosomes and chromatosomes containing uH2A were found to occupy the same positions on the template DNA as those containing H2A. Chromatin folding of nucleosomal arrays containing either H2A or uH2A was analysed using a quantitative agarose gel electrophoresis system developed by Hansen and co-workers. The extent of folding of nucleosomal arrays containing uH2A was comparable to that of control nucleosomal arrays. A differential centrifugation assay was used to monitor the extent of divalent cation induced oligomerisation of reconstituted nucleosomal arrays. Nucleosomal arrays containing uH2A were found to oligomerise at a lower magnesium concentration than control arrays. As a first step towards studying the effects of H2A ubiquitination in linker histone-bound nucleosomal arrays, a novel method for linker histone reconstitution onto long chromatin stripped of linker histones was developed. The fidelity of linker histone reconstitution was assayed by micrococcal nuclease digestion, thermal denaturation and determination of the orientation of neighbouring linker histone molecules in extended chromatin. In a separate study, the relationship between the observed repeat length of chromatin and the rate of micrococcal nuclease digestion was investigated. The repeat length of the same starting chromatin preparation at equivalent extents of digestion was found to vary according to the rate of digestion

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA

Dissociation of nucleosomal particles by chemical modification. Equivalence of the two binding sites for H2A.H2B dimers

3 páginas, 3 figuras.Treatment of nucleosomal particles with dimethylmaleic anhydride, a reagent for protein amino groups, is accompanied by a biphasic release of histones H2A plus H2B; one H2A.H2B dimer is more easily released than the other. This behavior allows the preparation of nucleosomal particles containing only one H2A.H2B dimer, which were complemented with 125I-labeled H2A.H2B. These reconstituted particles, which contain one labeled and one unlabeled H2A.H2B dimer, were treated with the amount of reagent needed to release one of the two H2A.H2B dimers. Radioactivity was equally distributed between residual particles and released proteins, which is consistent with equivalent binding sites in the nucleosomal particle for H2A.H2B dimers, rather than with intrinsically different sites. The asymmetric release of H2A.H2B dimers would be caused by a change in the binding site of one dimer following the release of the other. This behavior might be related to the structural dynamics of nucleosomes.This work was supported in part by the Fondo de Investigaciones Sanitarias and the Comisión Asesora de Investigacion Cientifica y Tecnica (Spain). The costs of publication of this article were defrayed in part by the payment of page charges.Peer reviewe

Contribution of histone N-terminal tails to the structure and stability of nucleosomes

AbstractHistones are the protein components of the nucleosome, which forms the basic architecture of eukaryotic chromatin. Histones H2A, H2B, H3, and H4 are composed of two common regions, the “histone fold” and the “histone tail”. Many efforts have been focused on the mechanisms by which the post-translational modifications of histone tails regulate the higher-order chromatin architecture. On the other hand, previous biochemical studies have suggested that histone tails also affect the structure and stability of the nucleosome core particle itself. However, the precise contributions of each histone tail are unclear. In the present study, we determined the crystal structures of four mutant nucleosomes, in which one of the four histones, H2A, H2B, H3, or H4, lacked the N-terminal tail. We found that the deletion of the H2B or H3 N-terminal tail affected histone–DNA interactions and substantially decreased nucleosome stability. These findings provide important information for understanding the complex roles of histone tails in regulating chromatin structure

DNase I site mapping and micrococcal nuclease digestion of pachytene chromatin reveal novel structural features

A comparison of the DNase I digestion products of the 32P-5'-end-labeled pachytene nucleosome core particles (containing histones H2A, TH2A, X2, H2B, TH2B, H3, and H4) and liver nucleosome core particles (containing somatic histones H2A, H2B, H3, and H4) revealed that the cleavage sites that are 30, 40, and 110 nucleotides away from the 5'-end are significantly more accessible in the pachytene core particles than in the liver core particles. These cleavage sites correspond to the region wherein H2B interacts with the nucleosome core DNA. These results, therefore, suggest that the histone-DNA interaction at these sites in the pachytene core particles is weaker, possibly because of the presence of the histone variant TH2B interacting at similar topological positions in the nucleosome core as that of its somatic counterpart H2B. Such a loosened structure may also be maintained even in the native pachytene chromatin since micrococcal nuclease digestion of pachytene nuclei resulted in a higher ratio of subnucleosomes (SN4 + SN7) to mononucleosomes than that observed in liver chromatin

Interaction of spermatid-specific protein TP2 with nucleic acids, in vitro. A comparative study with TP1

TP2 was purified from rat testes employing a gentle method involving differential salt extraction of the sonication-resistant spermatid nuclei. The nucleic acid binding properties of TP2 were studied by fluorescence quenching, thermal denaturation, circular dichroism techniques and compared with those of TP1 (Singh, J., and Rao, M. R. S. (1987) J. Biol. Chem. 262, 734-740). The tyrosine fluorescence of TP2 was quenched upon binding to double-stranded and denatured DNA and poly(rA). The apparent association constants for binding of TP2 to these nucleic acids were calculated from the fluorescence quenching data, obtained at 50 mM NaCl, and found to be 1.63 × 105 M−1, 6.5 × 105 M−1, and 7.3 × 105 M−1, respectively. Thermal denaturation studies of calf thymus DNA and its complexes with TP2 showed that at 1 mM NaCl, TP2 shifted the Tm from 53°C to 62-67°C, while at 50 mM NaCl, the Tm was shifted from 72 to 78°C suggesting that TP2 is a DNA stabilizing protein. Circular dichroism studies of TP1·DNA and TP2·DNA complexes have revealed that TP2 has a better DNA condensing property than TP1. Furthermore, in contrast to TP1, TP2 does not destabilize in vitro the compactness of liver nucleosome core particles. The DNA binding properties of TP1 and TP2 have been discussed in relation to the significance of their transient appearance during mammalian spermiogenesis

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Influence of histone octamer core distortion on nucleosome thermal mobility

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018O nucleossoma é a subunidade fundamental da cromatina eucariótica. As histonas, proteínas características do nucleossoma, são pequenas proteínas básicas subdivididas em duas superfamílias: linker (H1) e core (H2A, H2B, H3 e H4). As histonas core oligomerizam para formar o octamero de histonas que, conjuntamente com ADN de cadeia dupla, estabelecem a estrutura complexa do nucleossoma. A estrutura do nucleossoma foi inicialmente identificada em 1975 recorrendo à digestão da cromatina por uma nuclease micrococal, sendo que apenas em 1997 foi divulgada a estrutura do nucleossoma obtida por difração de raios-X com uma resolução de 2.8 Å, e mais tarde, em 2002, com uma resolução de 1.9 Å, o que revela ser mais do que suficiente para as cadeias laterais e a cadeia principal serem observadas com elevado grau de confiança. As histonas partilham um domínio entre elas: o histone fold, constituído por cerca de 70 resíduos de aminoácidos, capaz de facilitar a heterodimerização das histonas e que se encontra localizado na região C-terminal e consiste em três hélices-α (α1, α2 e α3) ligadas entre si por dois loops (L1 e L2) flanqueando a hélice-α 2. As histonas são constituídas por um domínio globular e uma região N-terminal flexível rica em resíduos de lisina e arginina. Estas proteínas heterodimerizam com o auxílio de interações hidrofóbicas, formando o handshake motif. No contexto celular, o nucleossoma permite o empacotamento do genoma no espaço relativamente limitado disponível dentro do núcleo, através do folding numa série de estruturas moleculares de ordem gradualmente superior. Mais ainda, permite a regulação temporal e espacial de vários processos modelados pelo ADN, como por exemplo a transcrição, a replicação e o reparo de ADN, necessários para que as células prosperem e exerçam as suas funções. Desde a sua descoberta na década de 1960, as modificações pós-tradicionais de histonas (e.g. metilação, fosforilação e acetilação) foram associadas à regulação da expressão génica, com algumas modificações sendo associadas à ativação de genes, enquanto outras foram associadas ao seu silenciamento. Vários estudos demonstraram a existência de um “código de histonas” que estipula que diferentes modificações pós-tradicionais nas histonas afetam as afinidades de ligação de proteínas capazes de estabelecerem uma ligação à cromatina, levando a estados transcricionais alterados do ADN subjacente. A informação epigenética codificada por estas modificações que ocorrem nas histonas pode ser passada para a próxima geração de células, agindo como uma camada adicional de informação que pode ser armazenada dentro da célula, aumentando ainda mais a complexidade e a variabilidade do material genético. Foi previamente demonstrado que o nucleossoma exibe um deslocamento, quando incubado a altas temperaturas, para os terminais da molécula de ADN que está enrolada em volta do nucleossoma. Neste contexto, o presente projeto visa observar a influência da distorção do octamero de histonas na mobilidade térmica do nucleossoma. Deste modo, vários mutantes de histonas foram preparados, embora apenas um nucleossoma mutante tenha sido gerado, além do nucleossoma wild-type. Para a obtenção de histonas recombinantes com elevado grau de pureza, recorreu-se a técnicas cromatográficas. Em primeiro lugar, foram expressas histonas recombinantes (wild-type e mutantes) em células Escherichia coli BL21 (DE3) Rosetta competentes a partir de plasmídeos in house. As histonas core H2A e H2B, bem como H3 e H4, foram coexpressas, uma vez que a coexpressão destes dois pares de proteínas reduz o tempo necessário para a sua síntese, para além de produzir complexos solúveis passíveis de serem purificados por sucessivas técnicas experimentais. Posteriormente, executou-se a purificação das mesmas através do uso de uma cromatografia de afinidade seguida duma cromatografia de troca iónica. Após todas as histonas terem sido geradas em grandes quantidades e elevado grau de pureza, efetuou-se a montagem do octamero. Paralelamente, foi sintetizada a sequência Widom 601, previamente descrita na literatura como sendo uma sequência de muito alta afinidade para o nucleossoma, via PCR usando primers apropriados. Neste trabalho foram geradas duas variantes da sequência supramencionada: curta (147 pb) e longa (227 pb). Uma vez obtidos todos os componentes necessários para a montagem do nucleossoma, recorreu-se à reconstituição do mesmo através de diálises (com gradiente de salinidade). Por fim, os nucleossomas gerados foram usados para serem efetuados thermal shift assays e native gel shift assays. Os thermal shift assays foram usados para se observar o deslocamento do nucleossoma ao longo do ADN (baseado na sequência Widom 601) e o efeito da distorção do octamero na mobilidade do nucleossoma induzida pela temperatura. Quanto aos native gel shift assays, um ensaio experimental rápido e sensível para a deteção de interações proteína-ácido nucleico, foram usados para observar a afinidade de ligação de diversas proteínas com ligação putativa à cromatina. Este trabalho corroborou o padrão observado de deslocamento de nucleossomas wild-type para as extremidades do ADN associado ao mesmo. O nucleossoma mutante H4_V43C H3_F104C concebido neste projeto demonstrou estar associado ao aumento da integridade estrutural e rigidez do nucleossoma, possivelmente através do estabelecimento de uma ponte dissulfeto entre L1 (loop 1) da histona H4 e 2 (hélice- 2) da histona H3. O estudo do impacto da plasticidade do octamero de histonas, consequência das diversas modificações que podem ocorrer nos seus resíduos de aminoácidos e que levam à distorção do core de histonas, é relevante no sentido de tentar entender o seu papel na regulação da expressão génica. Uma dada mutação irá, hipoteticamente, reforçar a estrutura do nucleossoma, potencialmente impossibilitando o acesso do complexo basal de transcrição, devido à alteração da capacidade de mobilidade do nucleossoma em relação ao ADN, e silenciando o(s) gene(s) codificados no ADN subjacente que se encontra “blindado” pelo nucleossoma, e vice-versa. Também será possível que alguns complexos capazes de remodelar a cromatina possam tirar partido da alteração da integridade estrutural conferida pelas diversas mutações ou pela presença de variantes de histonas. Numa visão geral, os resultados obtidos neste projeto demonstraram que é necessária uma certa plasticidade do octamero de histonas para se observar o movimento não-catalisado do nucleossoma. Adicionalmente, foi tentada a análise da Fkbp39, uma peptidilprolil isomerase com um domínio NPL (nucleoplasmin-like) conservado na região N-terminal, que se pensa ser uma chaperona de histonas em S. pombe e que é responsável pelo aumento da taxa de isomerização cis-trans das prolinas na cauda N-terminal (região N-terminal flexível) da histona H3. A análise foi feita por criomicroscopia eletrónica, tanto com a Fkbp39 não-complexada, como em complexo com (H2A-H2B) e com (H3-H4)2. Nesse sentido, expressou-se a proteína Fkbp39 em células E. coli BL21 (DE3) Rosetta competentes e foi executada a sua purificação com elevado grau de pureza através do uso de diferentes técnicas cromatográficas (cromatografias de afinidade, de troca iónica, e de exclusão molecular). Embora as purificações tenham sido bem-sucedidas, nem a proteína Fkbp39 isolada, nem em complexo com o dímero (H2A-H2B) ou com o tetramero (H3-H4)2 foram passíveis de serem observadas por criomicroscopia electrónica. É possível que, devido à presença de uma região central dinâmica intrinsecamente desordenada, estratégias sucessivas de otimização sejam necessárias para estabilizar os complexos putativos. Por último, várias proteínas putativas de ligação à cromatina foram rastreadas quanto à ligação aos nucleossomas sintetizados neste projeto recorrendo a native gel shift assays. Hpf1 (Histone PARylation factor 1), Parp2 (Poly [ADP-ribose] polymerase 2) e Alf (TFIIA-alpha and beta-like factor), provenientes de Homo sapiens, demonstraram ser os melhores candidatos para estudos estruturais posteriores mais aprofundados para averiguar com maior confiança as suas afinidades de ligação à cromatina.The nucleosome is the fundamental repeating subunit of eukaryotic chromatin. The histones, hallmarks of the nucleosome, are small basic proteins subdivided into two super families: linker (H1) and core histones (H2A, H2B, H3 and H4). Core histones assemble together to form the histone octamer which then, together with double-stranded DNA, embodies the complex structure of the nucleosome. Most importantly, the nucleosome allows for the tight packaging of the large genome into the relatively constricted space available inside the nucleus by folding into a series of higher-order molecular structures. Furthermore, it allows for the fine-tuned temporal and spatial regulation of several DNA-templated processes, such as DNA transcription, replication and repair, unequivocally required for the cells to thrive and exert their functions. Since their discovery in the 1960s, histone post-translational modifications (e.g. methylation, phosphorylation and acetylation) have been shown to affect gene expression regulation, with some modifications being associated with gene activation, whilst others have been associated with gene silencing. Interestingly, several studies have demonstrated the existence of a “histone code” which states that the presence of post-translational modifications on histones affect binding affinities of chromatin-binding proteins leading to altered transcriptional states of the underlying DNA. The epigenetic information encoded via these modifications occurring on histones can be passed on to the next generation of cells, acting as an additional layer of information that can be stored inside the cell, further increasing the complexity and versatility of the genetic material. It has been shown that the nucleosome exhibits shifting when incubated at high temperatures towards the DNA ends in regard to the nucleosomal DNA. In this context, the current project aims to observe the influence of histone octamer core distortion on nucleosome thermal mobility. To do so, several histone mutants were prepared, albeit only one mutant nucleosome was assembled, apart from the wild-type nucleosome. This work corroborates the observed thermally-driven shifting pattern of wild-type nucleosomes. The H4_V43C H3_F104C mutant nucleosome assembled in this project has shown itself to increase nucleosome structural integrity, possibly through the establishment of a disulphide bridge between L1 (loop 1) of core histone H4 and 2 (-helix 2) of core histone H3, conversely, hindering nucleosome mobility. This suggests that for nucleosome to slide along DNA in a noncatalyzed fashion, there must be a requirement of histone octamer plasticity. Moreover, structural analysis of Fkbp39, a putative histone chaperone in Saccharomyces pombe, both alone and in complex with (H2A-H2B) and with (H3-H4)2 was attempted with electron cryomicroscopy, though unsuccessful. Further optimization strategies are likely required to stabilize the putative complexes. Lastly, several chromatin-binding proteins were screened for binding to the nucleosome, as a complementary experiment. Hpf1, Parp2 and Alf from Homo sapiens seem like the best candidates for more in-depth studies to fully disclose their binding affinities to chromatin