👤 Stanislav Kozubek

The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage. Show less

This review focuses on the function of heterochromatin protein HP1 in response to DNA damage. We specifically outline the regulatory mechanisms in which HP1 and its interacting partners are involved. HP1 protein subtypes (HP1α, HP1β, and HP1γ) are the main components of constitutive heterochromatin, and HP1α and HP1β in particular are responsible for heterochromatin maintenance. The recruitment of these proteins to DNA lesions is also important from the perspective of proper DNA repair mechanisms. For example, HP1α is necessary for the binding of the main DNA damage-related protein 53BP1 at DNA repair foci, which are positive not only for the HP1α protein but also for the RAD51 protein, a component of DNA repair machinery. The HP1β protein also appears in monomeric form in DNA lesions together with the evolutionarily well-conserved protein called proliferating cell nuclear antigen (PCNA). The role of HP1 in DNA lesions is also mediated via the Kap1 transcription repressor. Taken together, these results indicate that the function of HP1 after DNA injury depends strongly on the kinetics of other DNA repair-related factors and their post-translational modifications, such as the phosphorylation of Kap-1. Show less

We studied the histone signature of embryonic and adult brains to strengthen existing evidence of the importance of the histone code in mouse brain development. We analyzed the levels and distribution patterns of H3K9me1, H3K9me2, H3K9me3, and HP1β in both embryonic and adult brains. Western blotting showed that during mouse brain development, the levels of H3K9me1, H3K9me2, and HP1β exhibited almost identical trends, with the highest protein levels occurring at E15 stage. These trends differed from the relatively stable level of H3K9me3 at developmental stages E8, E13, E15, and E18. Compared with embryonic brains, adult brains were characterized by very low levels of H3K9me1/me2/me3 and HP1β. Manipulation of the embryonic epigenome through histone deacetylase inhibitor treatment did not affect the distribution patterns of the studied histone markers in embryonic ventricular ependyma. Similarly, Hdac3 depletion in adult animals had no effect on histone methylation in the adult hippocampus. Our results indicate that the distribution of HP1β in the embryonic mouse brain is related to that of H3K9me1/me2 but not to that of H3K9me3. The unique status of H3K9me3 in the brain was confirmed by its pronounced accumulation in the granular layer of the adult olfactory bulb. Moreover, among the studied proteins, H3K9me3 was the only posttranslational histone modification that was highly abundant at clusters of centromeric heterochromatin, called chromocenters. When we focused on the hippocampus, we found this region to be rich in H3K9me1 and H3K9me3, whereas H3K9me2 and HP1β were present at a very low level or even absent in the hippocampal blade. Taken together, these results revealed differences in the epigenome of the embryonic and adult mouse brain and showed that the adult hippocampus, the granular layer of the adult olfactory bulb, and the ventricular ependyma of the embryonic brain are colonized by specific epigenetic marks. Show less

The optimal repair of DNA lesions is fundamental for physiological processes. We asked whether the recruitment of HP1β, 53BP1 and BMI1 proteins to ultraviolet (UVA)-induced DNA lesions requires functional A-type lamins. We found that UVA irradiation of nuclear lamina abolished the fluorescence of mCherry-tagged A-type lamins and destroyed the nuclear lamina as also observed by electron microscopy studies. Similarly, an absence of endogenous A- and B-type lamins was found in irradiated regions by UVA. However, irradiation did not affect the recruitment of HP1β, 53BP1 and BMI1 to DNA lesions. The UVA-induced shrinkage of the nuclear lamina, which anchors chromatin, explains why UVA-micro-irradiated chromatin is relaxed. Conversely, additional experiments with γ-irradiation showed that the nuclear lamina remained intact and the genome-wide level of HP1β was stable. Fluorescence intensity of HP1β and BMI1 in UVA-induced DNA lesions and level of HP1β after γ-irradiation were unaffected by deficiency in A-type lamins, whereas those parameters of 53BP1 were changed. We conclude that only the 53BP1 status in DNA lesions, induced by UVA or γ-rays, is affected by A-type lamin deficiency, which was not observed for heterochromatin-related proteins HP1β and BMI1. Show less

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair. Show less

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. Show less

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

Heterochromatin protein 1 (HP1), which binds to sites of histone H3 lysine 9 (H3K9) methylation, is primarily responsible for gene silencing and the formation of heterochromatin. We observed that HP1 beta is located in both the chromocenters and fibrillarin-positive nucleoli interiors. However, HP1 alpha and HP1 gamma occupied fibrillarin-positive compartments to a lesser extent, corresponding to the distinct levels of HP1 subtypes at the promoter of rDNA genes. Deficiency of histone methyltransferases SUV39h and/or inhibition of histone deacetylases (HDACi) decreased HP1 beta and H3K9 trimethylation at chromocenters, but not in fibrillarin-positive regions that co-localized with RNA polymerase I. Similarly, SUV39h- and HDACi-dependent nucleolar rearrangement and inhibition of rDNA transcription did not affect the association between HP1 beta and fibrillarin. Moreover, the presence of HP1 beta in nucleoli is likely connected with transcription of ribosomal genes and with the role of fibrillarin in nucleolar processes. Show less

Recent studies have shown that histone code dictates the type and structure of chromatin. Bearing in mind the importance of A-type lamins for chromatin arrangement, we studied the effect of trichostatin A (TSA)-induced histone hyperacetylation in lamin A/C-deficient (LMNA-/-) fibroblasts. Lamin A/C deficiency caused condensation of chromosome territories and the nuclear reorganization of centromeric heterochromatin, which was accompanied by the appearance of a chain-like morphology of HP1beta foci. Conversely, histone deacetylase (HDAC) inhibition induced de-condensation of chromosome territories, which compensated the effect of lamin A/C deficiency on chromosome regions. The amount of heterochromatin in the area associated with the nuclear membrane was significantly reduced in LMNA-/- cells when compared with lamin A/C-positive (LMNA+/+) fibroblasts. TSA also decreased the amount of peripheral heterochromatin, similarly as lamin A/C deficiency. In both LMNA+/+ and LMNA-/- cells, physically larger chromosomes were positioned more peripherally as compared with the smaller ones, even after TSA treatment. Our observations indicate that lamin A/C deficiency causes not only reorganization of chromatin and some chromatin-associated domains, but also has an impact on the extent of chromosome condensation. As HDAC inhibition can compensate the lamin A/C-dependent chromatin changes, the interaction between lamins and specifically modified histones may play an important role in higher-order chromatin organization, which influences transcriptional activity. Show less