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.

• Klíčová slova
• FLIM-FRET, HP1, epigenetics, irradiation, mass spectrometry, nucleolus, phosphorylation,
• MeSH
• buněčné jadérko metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• fosforylace MeSH
• HeLa buňky MeSH
• homolog proteinu s chromoboxem 5 MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• optické zobrazování MeSH
• poškození DNA MeSH
• rezonanční přenos fluorescenční energie MeSH
• serin metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CBX1 protein, human MeSH Prohlížeč
• CBX5 protein, human MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• homolog proteinu s chromoboxem 5 MeSH
• serin MeSH

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.

• Klíčová slova
• Brain sections, Epigenetics, Hippocampus, Histone methylation, Histones, Olfactory bulb,
• MeSH
• chromozomální proteiny, nehistonové analýza   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• histonlysin-N-methyltransferasa metabolismus   MeSH
• imunohistochemie MeSH
• mozek embryologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Cbx1 protein, mouse MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• histonlysin-N-methyltransferasa MeSH

Embryonic stem cells (ESCs) maintain their pluripotency through high expression of pluripotency-related genes. Here, we show that differing levels of Oct4, Nanog, and c-myc proteins among the individual cells of mouse ESC (mESC) colonies and fluctuations in these levels do not disturb mESC pluripotency. Cells with strong expression of Oct4 had low levels of Nanog and c-myc proteins and vice versa. In addition, cells with high levels of Nanog tended to occupy interior regions of mESC colonies. In contrast, peripherally positioned cells within colonies had dense H3K27-trimethylation, especially at the nuclear periphery. We also observed distinct levels of endogenous and exogenous Oct4 in particular cell cycle phases. The highest levels of Oct4 occurred in G2 phase, which correlated with the pKi-67 nuclear pattern. Moreover, the Oct4 protein resided on mitotic chromosomes. We suggest that there must be an endogenous mechanism that prevents the induction of spontaneous differentiation, despite fluctuations in protein levels within an mESC colony. Based on the results presented here, it is likely that cells within a colony support each other in the maintenance of pluripotency.

• MeSH
• antigen Ki-67 metabolismus   MeSH
• buněčná diferenciace * MeSH
• buněčné jádro genetika   metabolismus   MeSH
• embryonální kmenové buňky cytologie   metabolismus   MeSH
• epigeneze genetická MeSH
• FRAP MeSH
• G2 fáze MeSH
• histony metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• konfokální mikroskopie MeSH
• kultivované buňky MeSH
• lysin metabolismus   MeSH
• metylace MeSH
• myši MeSH
• nanog MeSH
• nika kmenových buněk MeSH
• oktamerní transkripční faktor 3 genetika   metabolismus   MeSH
• pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• protoonkogenní proteiny c-myc genetika   metabolismus   MeSH
• western blotting MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigen Ki-67 MeSH
• histony MeSH
• homeodoménové proteiny MeSH
• lysin MeSH
• Myc protein, mouse MeSH Prohlížeč
• Nanog protein, mouse MeSH Prohlížeč
• nanog MeSH
• oktamerní transkripční faktor 3 MeSH
• protoonkogenní proteiny c-myc MeSH
• zelené fluorescenční proteiny MeSH

BACKGROUND: Protein exchange kinetics correlate with the level of chromatin condensation and, in many cases, with the level of transcription. We used fluorescence recovery after photobleaching (FRAP) to analyse the kinetics of 18 proteins and determine the relationships between nuclear arrangement, protein molecular weight, global transcription level, and recovery kinetics. In particular, we studied heterochromatin-specific heterochromatin protein 1β (HP1β) B lymphoma Mo-MLV insertion region 1 (BMI1), and telomeric-repeat binding factor 1 (TRF1) proteins, and nucleolus-related proteins, upstream binding factor (UBF) and RNA polymerase I large subunit (RPA194). We considered whether the trajectories and kinetics of particular proteins change in response to histone hyperacetylation by histone deacetylase (HDAC) inhibitors or after suppression of transcription by actinomycin D. RESULTS: We show that protein dynamics are influenced by many factors and events, including nuclear pattern and transcription activity. A slower recovery after photobleaching was found when proteins, such as HP1β, BMI1, TRF1, and others accumulated at specific foci. In identical cells, proteins that were evenly dispersed throughout the nucleoplasm recovered more rapidly. Distinct trajectories for HP1β, BMI1, and TRF1 were observed after hyperacetylation or suppression of transcription. The relationship between protein trajectory and transcription level was confirmed for telomeric protein TRF1, but not for HP1β or BMI1 proteins. Moreover, heterogeneity of foci movement was especially observed when we made distinctions between centrally and peripherally positioned foci. CONCLUSION: Based on our results, we propose that protein kinetics are likely influenced by several factors, including chromatin condensation, differentiation, local protein density, protein binding efficiency, and nuclear pattern. These factors and events likely cooperate to dictate the mobility of particular proteins.

• Publikační typ
• časopisecké články MeSH

Determining averaged effective diffusion constants from experimental measurements of fluorescent proteins in an inhomogeneous medium in the presence of ligand-receptor interactions poses problems of analytical tractability. Here, we introduced a nonfitting method to evaluate the averaged effective diffusion coefficient of a region of interest (which may include a whole nucleus) by mathematical processing of the entire cellular two-dimensional spatial pattern of recovered fluorescence. Spatially and temporally resolved measurements of protein transport inside cells were obtained using the fluorescence recovery after photobleaching technique. Two-dimensional images of fluorescence patterns were collected by laser-scanning confocal microscopy. The method was demonstrated by applying it to an estimation of the mobility of green fluorescent protein-tagged heterochromatin protein 1 in the nuclei of living mouse embryonic fibroblasts. This approach does not require the mathematical solution of a corresponding system of diffusion-reaction equations that is typical of conventional fluorescence recovery after photobleaching data processing, and is most useful for investigating highly inhomogeneous areas, such as cell nuclei, which contain many protein foci and chromatin domains.

• MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• chromozomální proteiny, nehistonové chemie   genetika   metabolismus   MeSH
• difuze MeSH
• fluorescence MeSH
• fotovybělování MeSH
• FRAP metody   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• konfokální mikroskopie metody   MeSH
• lidé MeSH
• matematika MeSH
• molekulární modely * MeSH
• myši MeSH
• reprodukovatelnost výsledků MeSH
• roztoky MeSH
• zelené fluorescenční proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• homolog proteinu s chromoboxem 5 MeSH
• roztoky MeSH
• zelené fluorescenční proteiny MeSH

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.

• MeSH
• buněčné jadérko metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• fibroblasty metabolismus   MeSH
• kultivované buňky MeSH
• methyltransferasy metabolismus   MeSH
• myši MeSH
• represorové proteiny metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Cbx1 protein, mouse MeSH Prohlížeč
• chromozomální proteiny, nehistonové MeSH
• fibrillarin MeSH Prohlížeč
• methyltransferasy MeSH
• represorové proteiny MeSH
• Suv39h1 protein, mouse MeSH Prohlížeč

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.

• MeSH
• acetylace MeSH
• buněčné jádro metabolismus   ultrastruktura   MeSH
• chromatin ultrastruktura   MeSH
• chromozomální proteiny, nehistonové fyziologie   MeSH
• epigeneze genetická MeSH
• exprese genu MeSH
• histony genetika   metabolismus   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• inhibitory histondeacetylas MeSH
• interfáze MeSH
• lidé MeSH
• lidské chromozomy X metabolismus   MeSH
• metylace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• chromatin MeSH
• chromozomální proteiny, nehistonové MeSH
• histony MeSH
• homolog proteinu s chromoboxem 5 MeSH
• inhibitory histondeacetylas MeSH

Nuclear locations of the c-myc gene and its transcripts (c-myc (T)) 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 G(1)- 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-myc (T) 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-myc (T) occupied the periphery of this organelle, though not those associated with the cultivation surface. In HT29 cells, approximately 80% of c-myc (T) co-localized with the RNAP II positive regions, so-called transcription factories. However, in approximately 20% of the cells with c-myc transcripts, the c-myc (T) was released from the site of synthesis, and was not associated with either transcription factories or SC35 domains. In approximately 60% of nuclei with c-myc (T), these signals were located in close proximity to the SC35 regions, but promyelocytic leukaemia bodies were associated with c-myc (T) only in approximately 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.

• MeSH
• buněčné jádro genetika   metabolismus   ultrastruktura   MeSH
• buňky HT-29 MeSH
• exprese genu MeSH
• genetická transkripce MeSH
• geny myc * MeSH
• lidé MeSH
• lidské chromozomy, pár 8 MeSH
• messenger RNA metabolismus   MeSH
• nádorové buňky kultivované MeSH
• protoonkogenní proteiny c-myc metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• tkáňová distribuce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• MYC protein, human MeSH Prohlížeč
• protoonkogenní proteiny c-myc MeSH
• RNA-polymerasa II MeSH