Martin Falk

Chromatin structure influences the sensitivity of DNA to γ-radiation

For the first time, DNA double-strand breaks (DSBs) were directly visualized in functionally and structurally different chromatin domains of human cells. The results show that genetically inactive condensed chromatin is much less susceptible to DSB induction by gamma-rays than expressed, decondensed domains. Higher sensitivity of open chromatin for DNA damage was accompanied by more efficient DSB repair. These findings follow from comparing DSB induction and repair in two 11 Mbp-long chromatin regions, one with clusters of highly expressed genes and the other, gene-poor, containing mainly genes having only low transcriptional activity. The same conclusions result from experiments with whole chromosome territories, differing in gene density and consequently in chromatin condensation. It follows from our further results that this lower sensitivity of DNA to the damage by ionizing radiation in heterochromatin is not caused by the simple chromatin condensation but very probably by the presence of a higher amount of proteins compared to genetically active and decondensed chromatin. In addition, our results show that some agents potentially used for cell killing in cancer therapy (TSA, hypotonic and hypertonic) influence cell survival of irradiated cells via changes in chromatin structure and efficiency of DSB repair in different ways.

3D Structure of the human genome: Order in randomness

Abstract. A complex study of the spatial arrangement of different genetic elements (genes, centromeres and chromosomal domains) in the cell nucleus is presented and the principles of this arrangement are discussed. We show that the radial location of genetic elements in the three-dimensional (3D) space between the center of the nucleus and the nuclear membrane is element specific and dependent on the position of the element on the chromosome. In contrast, mutual angular positioning of both homologous and heterologous genetic elements is, in the majority of cases, random. In several cases, tethering of heterologous genetic elements was observed. This close proximity of specific loci may be responsible for their mutual rearrangement and the development of cancer. Comparison of our results with transcriptome maps shows that the nuclear location of chromosomal domains with highly expressed genes is more central when compared with chromosomes with low expression. The higher-order chromatin structure is strikingly similar in various human cell types, which correlates with the fact that the profiles of gene expression are also similar.

Higher-order chromatin structure in DSB induction, repair and misrepair.

Double-strand breaks (DSBs), continuously introduced into DNA by cell metabolism, ionizing radiation and some chemicals, are the biologically most deleterious type of genome damage, and must be accurately repaired to protect genomic integrity, ensure cell survival, and prevent carcinogenesis. Although a huge amount of information has been published on the molecular basis and biological significance of DSB repair, our understanding of DSB repair and its spatiotemporal arrangement is still incomplete. In particular, the role of higher-order chromatin structure in DSB induction and repair, movement of DSBs and the mechanism giving rise to chromatin exchanges, and many other currently disputed questions are discussed in this review. Finally, a model explaining the formation of chromosome translocations is proposed.

The 3D structure of human chromosomes in cell nuclei

The spatial arrangement of some genetic elements relative to chromosome territories and in parallel with the cell nucleus was investigated in human lymphocytes. The structure of the chromosome territories was studied in chromosomes containing regions (clusters) of highly expressed genes (HSA 9, 17) and those without such clusters (HSA 8, 13). In chromosomes containing highly expressed regions, the elements pertaining to these regions were found close to the centre of the nucleus on the inner sides of chromosome territories; those pertaining to regions with low expression were localized close to the nuclear membrane on the opposite sides of the territories. In chromosomes with generally low expression (HSA 8, 13), the elements investigated were found symmetrically distributed over the territories. Based on the investigations of the chromosome structure, the following conclusions are suggested: (1) Chromosome territories have a non-random internal 3D structure with defined average mutual positions between elements. For example, RARα, TP53 and Iso-q of HSA 17 are nearer to each other than they are to the HSA 17 centromere. (2) The structure of a chromosome territory reflects the number and chromosome location of clusters of highly expressed genes. (3) Chromosome territories behave to some extent as solid bodies: if the territory is found closer to the nuclear centre, the individual genetic elements of this chromosome are also found, on average, closer the centre of the nucleus. (4) The positions of centromeres are, on average, nearer to the fluorescence weight centre of the territory (FWCT) than to genes. (5) Active genes are not found near the centromeres of their own territory. A simple model of the structure of chromosome territory is proposed.

Methylation of histones in myeloid leukemias as a potential marker of granulocyte abnormalities

We show that common heterochromatin antigenic protein markers [HP1α, ‐β, ‐γ and mono‐, di‐, and trimethylated histone H3 lysine 9 (H3K9)], although present in human blood progenitor CD34+ cells, differentiated lymphocytes, and monocytes, are absent in neutrophil granulocytes and to large extent, in eosinophils. Monomethylated and in particular, dimethylated H3K9 are present to variable degrees in the granulocytes of chronic myeloid leukemia (CML) patients, without being accompanied by HP1 proteins. In patients with an acute phase of CML and in acute myeloid leukemia patients, strong methylation of H3K9 and all isoforms of HP1 are detected. In chronic forms of CML, no strong correlations among the level of histone methylation, disease progression, and modality of treatment were observed. Histone methylation was found even in “cured” patients without Philadelphia chromosome (Ph) resulting from +(9;22)(q34;q11) BCR/ABL translocation, suggesting an incomplete process of developmentally regulated chromatin remodeling in the granulocytes of these patients. Similarly, reprogramming of leukemia HL‐60 cells to terminal differentiation by retinoic acid does not eliminate H3K9 methylation and the presence of HP1 isoforms from differentiated granulocytes. Thus, our study shows for the first time that histone H3 methylation may be changed dramatically during normal cell differentiation. The residual histone H3 methylation in myeloid leukemia cells suggests an incomplete chromatin condensation that may be linked to the leukemia cell proliferation and may be important for the prognosis of disease treatment and relapse.

Two New Faces of Amifostine: Protector from DNA Damage in Normal Cells and Inhibitor of DNA Repair in Cancer Cells

Amifostine protects normal cells from DNA damage induction by ionizing radiation or chemotherapeutics, whereas cancer cells typically remain uninfluenced. While confirming this phenomenon, we have revealed by comet assay and currently the most sensitive method of DNA double strand break (DSB) quantification (based on γH2AX/53BP1 high-resolution immunofluorescence microscopy) that amifostine treatment supports DSB repair in γ-irradiated normal NHDF fibroblasts but alters it in MCF7 carcinoma cells. These effects follow from the significantly lower activity of alkaline phosphatase measured in MCF7 cells and their supernatants as compared with NHDF fibroblasts. Liquid chromatography-mass spectrometry confirmed that the amifostine conversion to WR-1065 was significantly more intensive in normal NHDF cells than in tumor MCF cells. In conclusion, due to common differences between normal and cancer cells in their abilities to convert amifostine to its active metabolite WR-1065, amifostine may not only protect in multiple ways normal cells from radiation-induced DNA damage but also make cancer cells suffer from DSB repair alteration.

Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure

Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction.

Function of chromatin structure and dynamics in DNA damage, repair and misrepair: γ-rays and protons in action.

According to their physical characteristics, protons and ion beams promise a revolution in cancer radiotherapy. Curing protocols however reflect rather the empirical knowledge than experimental data on DNA repair. This especially holds for the spatio-temporal organization of repair processes in the context of higher-order chromatin structure-the problematics addressed in this work. The consequences for the mechanism of chromosomal translocations are compared for gamma rays and proton beams.

Granulocyte maturation determines ability to release chromatin NETs and loss of DNA damage response; these properties are absent in immature AML granulocytes.

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL.

Topography of genetic elements of X-chromosome relative to the cell nucleus and to the chromosome X territory determined for human lymphocytes

Topography of three genetic elements--dystrophin (dmd) exons 5-7 (E(1)), 46-47 (E(2)), and centromere of chromosome X (N(X)) were studied relative to cell nuclei and to chromosome X territories of spatially fixed human lymphocytes. Repeated three-dimensional (3D) dual color fluorescence in situ hybridization combined with high-resolution cytometry was used. In addition, the nuclear location of fluorescence weight centers (FWC), spatial volume, and maximal area per one section of chromosome-X territories were investigated. The larger (X(L)) and smaller (X(S)) homologous X-chromosomes were distinguished for each nucleus according to the 3D volume of their territories. The distributions of the [center of nucleus]-to-[genetic element] distances (radial distributions) of dmd exons E(1), E(2), centromere N(X) and FWC were very similar for both homologous X-chromosomes of female lymphocytes as well as for the chromosome X of the human male. On the other hand, larger average mutual distances between all pairs of signals (E(1), E(2), N(X), FWC) and larger average maximal area were observed for the larger chromosome (X(L)) in comparison with the smaller one (X(S)). The territory of the larger homologue showed also more irregular surface. The most significant differences between homologous X-chromosomes were found for N(X)-E(1), N(X)-E(2) and E(1)-E(2) distances that were in average about twice longer for X(L) as compared with X(S). These parameters correlate to each other and can be used for the reliable determination of more (de)condensed X-chromosome territory. The longer E(1)-E(2) distances for X(L) indicate more open chromatin structure of the dystrophin gene on this chromosome in contrary to closed structure on X(S). Substantially shorter distances of the dystrophin exons from the centromeric heterochromatin in X(S) as compared to X(L) can be explained by silencing effect of centromeres as described in Nature 1 (2000) 137.