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Cancer cells contain significant alterations in their epigenomic landscape, which several enzyme families reversibly contribute to. One class of epigenetic modifying enzymes is that of histone deacetylases (HDAC), which are receiving considerable scrutiny clinically as a therapeutic target in many cancers. The underlying rationale is that inhibiting HDACs will reverse dysregulated target gene expression by modulating functional histone (or other) acetylation marks. This perspective will discuss a recent paper by Markozashvili and co-workers which appeared in Gene, which indicates that the mechanisms by which HDAC inhibitors (HDACis) alter the epigenetic landscape include widespread alternative effects beyond simply controlling regional epigenetic marks. HDACs are involved in many processes/diseases, and it is not surprising that HDACis have considerable off-target effects, and thus a major effort is being directed toward identification of inhibitors which are selective for HDAC isoforms often uniquely implicated in various cancers. This Perspective will also discuss some representative work with inhibitors targeting individual HDAC classes or isoforms. At present, it is not really clear that isoform-specific HDACis will avoid non-selective effects on other unrecognized activities of HDACs.

The pattern of genome evolution can be divided into two phases: the step-wise continuous phase (step-wise clonal evolution, stable dominant clonal chromosome aberrations (CCAs), and low frequency of non-CCAs, NCCAs) and punctuated phase (marked by elevated NCCAs and transitional CCAs). Depending on the phase, system stresses (the diverse CIN promoting factors) may lead to the very different phenotype responses. To address the contribution of chromosome instability (CIN) to phenotype changes of tumor cells, we characterized CCAs/NCCAs of HeLa and HEK293 cells, and their derivatives after genotoxic stresses (a stable plasmid transfection, ectopic expression of cancer-associated CHI3L1 gene or treatment with temozolomide) by conventional cytogenetics, copy number alterations (CNAs) by array comparative genome hybridization, and phenotype changes by cell viability and soft agar assays. Transfection of either the empty vector pcDNA3.1 or pcDNA3.1_CHI3L1 into 293 cells initiated the punctuated genome changes. In contrast, HeLa_CHI3L1 cells demonstrated the step-wise genome changes. Increased CIN correlated with lower viability of 293_pcDNA3.1 cells but higher colony formation efficiency (CFE). Artificial CHI3L1 production in 293_CHI3L1 cells increased viability and further contributed to CFE. The opposite growth characteristics of 293_CHI3L1 and HeLa_CHI3L1 cells were revealed. The effect and function of a (trans)gene can be opposite and versatile in cells with different genetic network, which is defined by genome context. Temozolomide treatment of 293_pcDNA3.1 cells intensified the stochastic punctuated genome changes and CNAs, and significantly reduced viability and CFE. In contrast, temozolomide treatment of HeLa_CHI3L1 cells promoted the step-wise genome changes, CNAs, and increased viability and CFE, which did not correlate with the ectopic CHI3L1 production. Thus, consistent coevolution of karyotypes and phenotypes was observed. CIN as a driving force of genome evolution significantly influences growth characteristics of tumor cells and should be always taken into consideration during the different experimental manipulations.

In interphase nuclei as in metaphase chromosomes, the genome is organized into topologically closed loop domains. Here, we have mapped the ends of the loop domain that contains the Ifng (interferon-gamma) gene in primary and cultured murine T-lymphocytes. To determine whether the ends of the loop are located in close proximity to each other in the nuclear space, the 3C (chromosome conformation capture) technique, which detects protein-mediated DNA-DNA interactions, was utilized. A strong interaction was demonstrated between the two ends of the loop, which were close enough to become cross-linked in vivo in the presence of paraformaldehyde. Chromatin immunoprecipitation combined with the 3C technique demonstrated that topoisomerase IIalpha and MeCP2, but not topoisomerase IIbeta, heterochromatin-associated protein HP1 or CTCF, were involved in this interaction. The present findings have important implications in terms of mechanisms of illegitimate recombination that can result in chromosomal translocations and deletions.

Compartmentalization and compaction of DNA in the nucleus is the characteristic feature of eukaryotic cells. A fully extended DNA molecule has to be compacted 100,000 times to fit within the nucleus. At the same time it is critical that various DNA regions remain accessible for interaction with regulatory factors and transcription/replication factories. This puzzle is solved at the level of DNA packaging in chromatin that occurs in several steps: rolling of DNA onto nucleosomes, compaction of nucleosome fiber with formation of the so-called 30 nm fiber, and folding of the latter into the giant (50-200 kbp) loops, fixed onto the protein skeleton, the nuclear matrix. The general assumption is that DNA folding in the cell nucleus cannot be uniform. It has been known for a long time that a transcriptionally active chromatin fraction is more sensitive to nucleases; this was interpreted as evidence for the less tight compaction of this fraction. In this review we summarize the latest results on structure of transcriptionally active chromatin and the mechanisms of transcriptional regulation in the context of chromatin dynamics. In particular the significance of histone modifications and the mechanisms controlling dynamics of chromatin domains are discussed as well as the significance of spatial organization of the genome for functioning of distant regulatory elements.

SummaryAnimal cloning by nuclear-transfer experiments frequently fails due to the inability of transplanted nuclei to support normal embryonic development. We show here that the formation of mitotic chromosomes in the egg context is crucial for adapting differentiated nuclei for early development. Differentiated erythrocyte nuclei replicate inefficiently in Xenopus eggs but do so as rapidly as sperm nuclei if a prior single mitosis is permitted. This mitotic remodeling involves a topoisomerase II-dependent shortening of chromatin loop domains and an increased recruitment of replication initiation factors onto chromatin, leading to a short interorigin spacing characteristic of early developmental stages. It also occurs within each early embryonic cell cycle and dominantly regulates initiation of DNA replication for the subsequent S phase. These results indicate that mitotic conditioning is crucial to reset the chromatin structure of differentiated adult donor cells for embryonic DNA replication and suggest that it is an important step in nuclear cloning.

Most cells of higher eukaryotes may maintain several rounds of replication of circular DNA. Efficiency of replication is usually low, and depends on the length of the circular DNA rather than on the sequence context. We have isolated and characterized several short DNA fragments that form structural sites of attachment to the nuclear matrix (nmDNA) in chicken cells, and tested whether they would enhance autonomous replication of DNA in chicken cells as compared to the vector DNA. Indeed, a several-fold increase in a short-term replication efficiency was detected using a semi-conservative replication and a DpnI-resistance assay. Most of the cloned matrix-associated fragments were recovered in the nuclear matrix fraction when introduced into cultured chicken cells as a circular DNA. The data obtained suggest that the observed enhancement in the replication efficiency of the circular DNA may be due to their recruitment to the nuclear matrix by the nmDNA.

We have mapped nuclear matrix attachment regions (MARs), defined by their specific binding to nuclear matrices in vitro, and sites of topoisomerase II reaction, detected by DNA cleavage in vitro in the presence of the inhibitor VM-26, in the vicinity of the replication origin of the chicken alpha-globin gene domain. Two MARs are located close to the downstream end (in the direction of transcription) of a 3 kb fragment which includes the origin. These MARs contain sites for strong topoisomerase II binding and reaction. Our observations on this gene domain support two hypotheses concerning MARs in eukaryotic cells, namely that they are close to DNA replication origins and that they contain multiple topoisomerase II recognition sites.

The permanent sites of DNA attachment to the nuclear matrix in the domain of chicken alpha-globin genes originally found in erythrocyte nuclei have also been shown to exist in sperm and cultured fibroblast cells. A primary structure of a 1.7 kb fragment located in 5'-upstream region of chicken alpha-globin gene domain and containing both replication origin and permanent nuclear matrix attachment site has been determined. It was found to possess homologies with papovaviral replication origins and contain short internal repeats and GC-rich motifs.

A matrix-associated region (MAR)-containing fragment has been selected from the library of cloned chicken nuclear matrix-associated DNA fragments. Factors, which determine the specific binding of DNA fragments have been studied. Using topoisomerase II-specific inhibitor VM 26 we established that nuclear matrix-associated topoisomerase II interacted with the MAR-containing DNA fragment producing specific cleavage sites on DNA of the fragment.