Lenka Stixová

Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ± 50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.

Acetylation-dependent nuclear arrangement and recruitment of BMI1 protein to UV-damaged chromatin.

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. J. Cell. Physiol. 227: 1838–1850, 2012.

Recruitment of Oct4 Protein to UV-Damaged Chromatin in Embryonic Stem Cells

Background Oct4 is a specific marker of embryonic stem cell (ESC) pluripotency. However, little is known regarding how Oct4 responds to DNA damage. Here, we investigated whether Oct4 recognizes damaged chromatin in mouse ESCs stably expressing GFP-Oct4. These experiments should contribute to the knowledge of how ESC genomic integrity is maintained, which is crucial for potential application of human ESCs in regenerative medicine. Methodology/Principal Findings We used time-lapse confocal microscopy, microirradiation by UV laser (355 nm), induction of DNA lesions by specific agents, and GFP technology to study the Oct4 response to DNA damage. We found that Oct4 accumulates in UV-damaged regions immediately after irradiation in an adenosine triphosphate-dependent manner. Intriguingly, this event was not accompanied by pronounced Nanog and c-MYC recruitment to the UV-damaged sites. The accumulation of Oct4 to UV-damaged chromatin occurred simultaneously with H3K9 deacetylation and H2AX phosphorylation (γH2AX). Moreover, we observed an ESC-specific nuclear distribution of γH2AX after interference to cellular processes, including histone acetylation, transcription, and cell metabolism. Inhibition of histone deacetylases mostly prevented pronounced Oct4 accumulation at UV-irradiated chromatin. Conclusions/Significance Our studies demonstrate pluripotency-specific events that accompany DNA damage responses. Here, we discuss how ESCs might respond to DNA damage caused by genotoxic injury that might lead to unwanted genomic instability.

Trajectories and nuclear arrangement of PML bodies are influenced by A-type lamin deficiency

Promyelocytic leukaemia (PML) bodies are specific nuclear structures with functional significance for acute promyelocytic leukaemia. In this study, we analysed the trajectories of PML bodies using single‐particle tracking.

Heterogeneity in the kinetics of nuclear proteins and trajectories of substructures associated with heterochromatin.

BackgroundProtein 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.ResultsWe 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.ConclusionBased 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.

Differentiation-Independent Fluctuation of Pluripotency-Related Transcription Factors and Other Epigenetic Markers in Embryonic Stem Cell Colonies

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.

Lipid alterations in human colon epithelial cells induced to differentiation and/or apoptosis by butyrate and polyunsaturated fatty acids

The present study highlights the important association between lipid alterations and differentiation/apoptotic responses in human colon differentiating (FHC) and nondifferentiating (HCT-116) cell lines after their treatment with short-chain fatty acid sodium butyrate (NaBt), polyunsaturated fatty acids (PUFAs), and/or their combination. Our data from GC/MS and LC/MS/MS showed an effective incorporation and metabolization of the supplemented arachidonic acid (AA) or docosahexaenoic acid (DHA), resulting in an enhanced content of the respective PUFA in individual phospholipid (PL) classes and an altered composition of the whole cellular fatty acid spectrum in both FHC and HCT-116 cells. We provide novel evidence that NaBt combined with PUFAs additionally modulated AA and DHA cellular levels and caused their shift from triacylglycerol to PL fractions. NaBt increased, while AA, DHA and their combination with NaBt decreased endogenous fatty acid synthesis in FHC but not in HCT-116 cells. Fatty acid treatment also altered membrane lipid structure, augmented cytoplasmic lipid droplet accumulation, reactive oxygen species (ROS) production and dissipation of the mitochondrial membrane potential. All these parameters were significantly enhanced by combined NaBt/PUFA treatment, but only in FHC cells was this accompanied by highly increased apoptosis and suppressed differentiation. Moreover, the most significant changes of ROS production, differentiation and apoptosis among the parameters studied, the highest effects of combined NaBt/PUFA treatment and a lower sensitivity of HCT-116 cells were confirmed using two-way ANOVA. Our results demonstrate an important role of fatty acid-induced lipid alterations in the different apoptotic/differentiation response of colon cells with various carcinogenic potential.

Post-Translational Modifications of Histones in Human Sperm

We examined the levels and distribution of post‐translationally modified histones and protamines in human sperm. Using western blot immunoassay, immunofluorescence, mass spectrometry (MS), and FLIM‐FRET approaches, we analyzed the status of histone modifications and the protamine P2. Among individual samples, we observed variability in the levels of H3K9me1, H3K9me2, H3K27me3, H3K36me3, and H3K79me1, but the level of acetylated (ac) histones H4 was relatively stable in the sperm head fractions, as demonstrated by western blot analysis. Sperm heads with lower levels of P2 exhibited lower levels of H3K9ac, H3K9me1, H3K27me3, H3K36me3, and H3K79me1. A very strong correlation was observed between the levels of P2 and H3K9me2. FLIM‐FRET analysis additionally revealed that acetylated histones H4 are not only parts of sperm chromatin but also appear in a non‐integrated form. Intriguingly, H4ac and H3K27me3 were detected in sperm tail fractions via western blot analysis. An appearance of specific histone H3 and H4 acetylation and H3 methylation in sperm tail fractions was also confirmed by both LC‐MS/MS and MALDI‐TOF MS analysis. Taken together, these data indicate that particular post‐translational modifications of histones are uniquely distributed in human sperm, and this distribution varies among individuals and among the sperm of a single individual. J. Cell. Biochem. 116: 2195–2209, 2015.

Basic nuclear processes affected by histone acetyltransferases and histone deacetylase inhibitors

AIM The optimal balance between histone acetylation and deacetylation is important for proper gene function. Therefore, we addressed how inhibitors of histone-modifying enzymes can modulate nuclear events, including replication, transcription, splicing and DNA repair. MATERIALS & METHODS Changes in cell signaling pathways upon treatment with histone acetyltransferases and/or histone deacetylase inhibitors were studied by cDNA microarrays and western blots. RESULTS We analyzed the effects of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) and the histone acetylase inhibitor MG149. SAHA altered the expression of factors involved in DNA replication complexes, basal transcription and the spliceosome pathway. DNA repair-related genes, including Rad51, Rad54 and BRCA2, were significantly downregulated by SAHA. However, MG149 had no effect on the investigated nuclear processes, with the exception of the spliceosome network and Sestrins, involved in DNA repair. CONCLUSION Based on our results, we propose that the studied epigenetic drugs have the distinct potential to affect specific cell signaling pathways depending on their respective molecular targets.

Nuclear Structures Surrounding Internal Lamin Invaginations

A‐ and C‐type lamins are intermediate filament proteins responsible for the maintenance of nuclear shape and most likely nuclear architecture. Here, we propose that pronounced invaginations of A/C‐type lamins into the nuclear interior represent channels for the transport of regulatory molecules to and from nuclear and nucleolar regions. Using fluorescent protein technology and immunofluorescence, we show that A‐type lamin channels interact with several nuclear components, including fibrillarin‐ and UBF‐positive regions of nucleoli, foci of heterochromatin protein 1 β, polycomb group bodies, and genomic regions associated with DNA repair. Similar associations were observed between A/C‐type lamin channels and nuclear pores, lamin‐associated protein LAP2α, and promyelocytic leukemia nuclear bodies. Interestingly, regions with high levels of A/C‐type lamins had low levels of B‐type lamins, and vice versa. These characteristics were observed in primary and immortalized mouse embryonic fibroblasts as well as human and mouse embryonic stem cell colonies exhibiting stem cell‐specific lamin positivity. Our findings indicate that internal channels formed by nuclear lamins likely contribute to normal cellular processes through association with various nuclear and nucleolar structures. J. Cell. Biochem. 115: 476–487, 2014.