Wladyslaw A. Krajewski

Alterations in the internucleosomal DNA helical twist in chromatin of human erythroleukemia cells in vivo influences the chromatin higher-order folding

In the present study chloroquine diphosphate, a DNA intercalating drug, was used to alter the internucleosomal DNA helical twist in chromatin of living mammalian cells. The intercalative binding of chloroquine effectively unwinds the DNA double helix and its binding is restricted to nucleosomal linker regions without noticeable disruption of nucleosomes. The results presented here imply that the alterations in the rotation angle between the adjacent nucleosomes in chromatin of eukaryotic cells in vivo significantly influences the way the chain of nucleosomes folds in higher‐order chromatin structures, as evidenced by specific alterations in nuclease susceptibility of chromatin.

Organization of specific DNA sequence elements in the region of the replication origin and matrix attachment site in the chicken α-globin gene domain

SummaryThe distribution of specific DNA sequence elements in a 2.9 kb HindIII fragment of chicken DNA containing the replication origin and the upstream matrix attachment site (MAR) of the α-globin gene domain was investigated. The fragment was shown to contain a CR1-type repetitive element and two stably bent DNA sequences. One of them colocalizes with the previously described MAR element and with the recognition site for a proliferating-cell-specific, DNA-binding protein. The melting pattern of a set of subfragments of the region proved to be non random. No correlation between the distribution of readily melting sequences and bent DNA was found. The possible importance of curved, low-melting and repetitive DNA sequences for the organization of the upstream boundary of the α-globin gene domain and the function of the replication origin is discussed.

Histone hyperacetylation facilitates chromatin remodelling in a Drosophila embryo cell-free system.

Abstract We found that Drosophila embryo extract contains a protein activity (or activities) that can destabilize nucleosomes, resulting in increased sensitivity to DNase I, release of nucleosomal supercoiling, high levels of conformational flexibility of DNA and more diffuse micrococcal nuclease digestion patterns. Incorporation of histone H1 did not significantly affect this nucleosome remodelling. Remodelling occurs more efficiently in hyperacetylated chromatin. It was shown previously that hyperacetylated chromatin, reconstituted in a Drosophila embryo cell-free system, exhibits increased DNase I sensitivity and a high degree of conformational flexibility of DNA. The present data suggest that the more diffuse structure of acetylated chromatin is a result of chromatin remodelling by protein activities in the Drosophila embryo extract.

The Saccharomyces cerevisiae Swi/Snf complex can catalyze formation of dimeric nucleosome structures in vitro.

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.

ENHANCEMENT OF TRANSCRIPTION BY SHORT ALTERNATING C . G TRACTS INCORPORATED WITHIN A ROUS SARCOMA VIRUS-BASED CHIMERIC PROMOTER : IN VIVO STUDIES

In view of the wide chromosomal distribution of short alternating purine-pyrimidine sequences capable of adopting a number of superhelical stress-dependent structural configurations (left-handed helices and cruciforms), the question has been posed whether such sequences exert any functional effects in vivo. A series of eukaryotic expression vectors were constructed which contained C·G tracts of various lengths in the promoter region. It was shown that insertion of C·G tracts of 12–16 bp significantly increased the level of expression of the chloramphenicol acetyltransferase reporter gene. It was also demonstrated that the formation of additional activation complexes and the use of a preferred “face” or side of the DNA molecule is not responsible for the increased transcription which was observed upon insertion of the C·G tracts. Comparative assays of chromatin structure at the chimeric promoters indicate that the alternating C·G tracts adopt a structure which is incapable of binding histone proteins. These results strongly suggest that control of access to chromatin is involved in regulating the transcriptional activity of the chimeric promoters. Possible molecular bases for this phenomena are discussed.

Chromatin structural transitions in Drosophila embryo cell-free extract result in a high conformational flexibility of nucleosomal DNA

DNA within chromatin has considerably more restricted flexibility in comparison with naked DNA. This raises the main question of how the functioning multi‐enzyme complexes overcome the nucleosomal level of DNA packaging. We studied the DNA conformational flexibility of reconstituted chromatin in a cell‐free system derived from Drosophila embryo extracts. Using this system, we have found evidence for a energy‐ independent chromatin remodelling process that efficiently destabilizes the nucleosome structure resulting in a high conformational flexibility of nucleosomal DNA. The described chromatin remodelling process may lay on the basis of defined molecular principles governing the molecular heterogeneity of chromatin structures in vivo.

Alternating purine-pyrimidine tract activates transcription from the Rouse sarcoma virus LTR lacking promoter and enhancer elements

The transcriptional control region of the Rouse sarcoma virus long terminal repeats (LTR) was shown to contain enhancer and promoter elements located within 200 base pairs upstream from the transcription initiation site [Cullen et al. (1985) Mol. Cell. Biol. 5, 438–447]. Deletion of these elements results in significant loss of LTR transcriptional activity. In the present paper it is shown that a short alternating purine‐pyrimidine sequence can restore the constitutive activity of the Rouse sarcoma virus LTR in the absence of upstream elements when inserted in close proximity to the transcription initiator site. The possible molecular bases of this phenomena are discussed.

Flexibility of DNA Within Transcriptionally Active Nucleosomes: Analysis by Circular Dichroism Measurements

The conformational flexibility of DNA in transcriptionally active chromatin fractions has been estimated by circular dichroism spectroscopy analysis and was found to be restricted in the same fashion as in bulk chromatin. The observation is discussed in the context of different models of active chromatin organization.

Isw1a does not have strict limitations on the length of extranucleosomal DNAs for mobilization of nucleosomes assembled with HeLa cell histones

The Saccharomyces cerevisiae Isw1a and Isw2 ATP-dependent chromatin-remodeling complexes have important roles in vivo in the regulation of nucleosome positioning and modulation of gene activity. We studied the ability of the Isw1a- and Isw2-remodeling enzymes to reposition nucleosomes in mono- and dinucleosomes templates with variably positioned histone octamers (in the center or at the ends of the DNA fragment). To compare the Isw1a and Isw2 nucleosome-mobilizing activities, we utilized mono- and dinucleosome templates reconstituted with purified HeLa cell histones and DNA containing one or two copies of the “601” nucleosome high-affinity sequence used to specifically position nucleosomes on the DNA. The obtained data suggest that Isw1a is able to mobilize HeLa cell histone-assembled mononucleosomes with long (more than 30 bp) extranucleosomal DNAs protruding from both sides, which contrasts to the previously reported inability of Isw1 to mobilize similar nucleosomes assembled with recombinant yeast histones. The results also suggest that Isw1a and Isw2 can mobilize nucleosomes with unfavorably short linker DNA lengths, and the presence of internucleosomal interactions promotes mobilization of nucleosomes even when the linkers are short.

On the role of inter-nucleosomal interactions and intrinsic nucleosome dynamics in chromatin function

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.