Andrea Harničarová

Histone Modifications and Nuclear Architecture: A Review

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.

Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases

The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

Epigenome and chromatin structure in human embryonic stem cells undergoing differentiation

Epigenetic histone (H3) modification patterns and the nuclear radial arrangement of select genetic elements were compared in human embryonic stem cells (hESCs) before and after differentiation. H3K9 acetylation, H3K9 trimethylation, and H3K79 monomethylation were reduced at the nuclear periphery of differentiated hESCs. Differentiation coincided with centromere redistribution, as evidenced by perinucleolar accumulation of the centromeric markers CENP‐A and H3K9me3, central repositioning of centromeres 1, 5, 19, and rearrangement of other centromeres at the nuclear periphery. The radial positions of PML, RARα genes, and human chromosomes 10, 12, 15, 17, and 19 remained relatively stable as hESCs differentiated. However, the female inactive H3K27‐trimethylated X chromosome occupied a more peripheral nuclear position in differentiated cells. Thus, pluripotent and differentiated hESCs have distinct nuclear patterns of heterochromatic structures (centromeres and inactive X chromosome) and epigenetic marks (H3K9me3, and H3K27me3), while relatively conserved gene density‐related radial chromatin distributions are already largely established in undifferentiated hES cells. Developmental Dynamics 237:3690–3702, 2008.

H3K9 acetylation and radial chromatin positioning

Histone variants and their epigenetic modifications determine genome function, particularly transcription. However, whether regulation of gene expression can be influenced by nuclear organization or vice versa is not completely clear. Here, we analyzed the effect of epigenetic changes induced by a histone deacetylase inhibitor (HDACi) on the nuclear radial rearrangement of select genomic regions and chromosomes. The HDACi, sodium butyrate (NaBt), induced differentiation of human adenocarcinoma HT29 cells as well as a genome‐wide increase in H3K9 acetylation. Three‐dimensional analysis of nuclear radial distributions revealed that this increase in H3K9 acetylation was often associated with a repositioning of select loci and chromosomes toward the nuclear center. On the other hand, many centromeres resided sites more toward the nuclear periphery, similar to sites occupied by chromosome X. In more than two‐thirds of events analyzed, central nuclear positioning correlated with a high level of H3K9 acetylation, while more peripheral positioning within interphase nuclei correlated with a lower level of acetylation. This was observed for the gene‐rich chromosomes 17 and 19, TP53, and CCND1 genes as well as for gene‐poor chromosome 18, APC gene, regions of low transcriptional activity (anti‐RIDGEs), and the relatively transcriptionally less active chromosome X. These results are consistent with a role for epigenetic histone modifications in governing the nuclear radial positioning of genomic regions during differentiation. J. Cell. Physiol. 220: 91–101, 2009.

Nuclear organization of PML bodies in leukaemic and multiple myeloma cells.

The nuclear arrangement of promyelocytic leukaemia nuclear bodies (PML NBs) was studied in vitro after the cell treatment by clinically used agents such as all-trans retinoic acid (RA) in human leukaemia and cytostatics or gamma radiation in multiple myeloma cells. In addition, the influence of phorbol ester (PMA) on PML NBs formation was analyzed. A reduced number of PML bodies, which led to relocation of PML NBs closer to the nuclear interior, mostly accompanied RA- and PMA-induced differentiation. Centrally located PML NBs were associated with transcriptional protein RNAP II and SC35 regions, which support importance of PML NBs in RNA processing that mostly proceeds within the nuclear interior. Conversely, the quantity of PML NBs was increased after cytostatic treatment, which caused re-distribution of PML NBs closer to the nuclear envelope. Here we showed correlations between the number of PML NBs and average Centre-to-PML distances. Moreover, a number of cells in S phase, especially during differentiation, influenced number of PML NBs. Studying the proteins involved in PML compartment, such as c-MYC, cell-type specific association of c-MYC and the PML NBs was observed in selected leukaemic cells undergoing differentiation, which was accompanied by c-MYC down-regulation.

Epigenetics of multiple myeloma after treatment with cytostatics and gamma radiation

Genetic and epigenetic changes in multiple myeloma (MM) correlate with the stage of the disease. Therefore, we investigated how cytostatics and gamma radiation influence MM-associated histone modifications. ChIP-PCR and ChIP-on-chip technologies were used to quantify H3K9 acetylation and H3K9 dimethylation at select loci in MM patients, lymphoblastoid ARH-77, and myeloma MOLP-8 cells. Genome-wide analysis revealed that the cytostatic, melphalan, increased H3K9 acetylation at multiple gene promoters in ARH-77 cells. Melphalan and gamma radiation also influenced histone modification of prognostically important c-myc and CCND1 genes in ARH-77 and MOLP-8 cells. Moreover, H3K9 acetylation at c-myc and CCND1 promoters was increased in individual MM patients after melphalan treatment. Western blotting revealed that these effects were accompanied by changes in c-MYC and cyclin D1 protein levels. Taken together, we showed that cytostatics significantly alter histone modification of tumor-related genes which is indispensable for understanding cancer therapies.

Single-cell c-myc gene expression in relationship to nuclear domains.

Nuclear locations of the c-myc gene and its transcripts (c-mycT) have been investigated in relation to nuclear domains involved in RNA synthesis and processing. Transcription of the c-myc gene appears to be linked to the late G1- and preferentially to S-phases of the cell cycle. The c-myc gene and its transcripts were positioned non-randomly within the interphase nucleus; additionally, c-myc RNA signals accumulated at nucleoli. Using oligo-probes, designed to exon II and exon III of the c-myc gene, single c-mycT was preferentially observed in human carcinoma HT29 and A549 cells. Conversely, human embryonal teratocarcinoma NTERA cells were characterized by the presence of multiple c-myc RNA signals located in both the nucleoli and nucleoplasm. When accumulated at nucleoli, c-mycT occupied the periphery of this organelle, though not those associated with the cultivation surface. In HT29 cells, approximately 80% of c-mycT co-localized with the RNAP II positive regions, so-called transcription factories. However, in ∼20% of the cells with c-myc transcripts, the c-mycT was released from the site of synthesis, and was not associated with either transcription factories or SC35 domains. In ∼60% of nuclei with c-mycT, these signals were located in close proximity to the SC35 regions, but promyelocytic leukaemia bodies were associated with c-mycT only in ∼20% of the nuclei. Taken together, c-myc RNA signals were positioned in the most internal parts of the cell nuclei preferentially associated with the nucleoli. Specific nuclear and nucleolar positioning probably reflects the kinetics of c-myc RNA metabolism.

Chromatin Structure and Epigenetics of Tumour Cells: A Review

Tumour cells are characterized by karyotype instability, which is accompanied by specific events in the chromatin structure and epigenetic patterns. Epigenetics involves heritable changes in the physical and biochemical state of chromatin, which have no effect on DNA sequences; therefore, changes in the nuclear radial arrangement of chromosomes can also be considered epigenetic events. Nuclear radial distributions of select genomic regions have been studied in many tumour cells and are not influenced by aberrations in chromosome number. On the other hand, genes involved in translocations take up new positions midway between the original coding sequences. The differentiation of leukaemia cells with clinically used agents is often accompanied by nuclear repositioning of tumour-related genes. However, the nuclear rearrangement is cell-type specific and not always associated with changes in the transcriptional activity. Similarly, cell type-specific chromatin structure is observed in tumour cells treated with select cytostatics and inhibitors of epigenetic processes, which have significant influences on the histone code. Chromatin structure and histone modifications were also affected by gamma radiation in leukaemia, multiple myeloma, and solid tumour cells. Interestingly, gamma radiation induced loci proximity, which has been suggested to increase the probability of exchange aberrations typically associated with tumour progression.