👤 Lenka Stixová

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (∼40%) recovery of GFP-HP1α and GFP-HP1β after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic. Show less

Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture. Show less

Polycomb group (PcG) proteins, organized into Polycomb bodies, are important regulatory components of epigenetic processes involved in the heritable transcriptional repression of target genes. Here, we asked whether acetylation can influence the nuclear arrangement and function of the BMI1 protein, a core component of the Polycomb group complex, PRC1. We used time-lapse confocal microscopy, micro-irradiation by UV laser (355 nm) and GFP technology to study the dynamics and function of the BMI1 protein. We observed that BMI1 was recruited to UV-damaged chromatin simultaneously with decreased lysine acetylation, followed by the recruitment of heterochromatin protein HP1β to micro-irradiated regions. Pronounced recruitment of BMI1 was rapid, with half-time τ = 15 sec; thus, BMI1 is likely involved in the initiation step leading to the recognition of UV-damaged sites. Histone hyperacetylation, stimulated by HDAC inhibitor TSA, suppression of transcription by actinomycin D, and ATP-depletion prevented increased accumulation of BMI1 to γH2AX-positive irradiated chromatin. Moreover, BMI1 had slight ability to recognize spontaneously occurring DNA breaks caused by other pathophysiological processes. Taken together, our data indicate that the dynamics of recognition of UV-damaged chromatin, and the nuclear arrangement of BMI1 protein can be influenced by acetylation and occur as an early event prior to the recruitment of HPβ to UV-irradiated chromatin. Show less