Infrared nanospectroscopic mapping of a single metaphase chromosome.

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.

Unreeling the chromatin thread: a genomic perspective on organization around the periphery of the Arabidopsis nucleus.

The first genome-wide examination of the chromatin landscape at the periphery of the plant cell nucleus reveals substantial enrichment of heterochromatin and Polycomb-based repressive chromatin.

Small chromosomal regions position themselves autonomously according to their chromatin class.

The spatial arrangement of chromatin is linked to the regulation of nuclear processes. One striking aspect of nuclear organization is the spatial segregation of heterochromatic and euchromatic domains. The mechanisms of this chromatin segregation are still poorly understood. In this work, we investigated the link between the primary genomic sequence and chromatin domains. We analyzed the spatial intranuclear arrangement of a human artificial chromosome (HAC) in a xenospecific mouse background in comparison to an orthologous region of native mouse chromosome. The two orthologous regions include segments that can be assigned to three major chromatin classes according to their gene abundance and repeat repertoire: (1) gene-rich and SINE-rich euchromatin; (2) gene-poor and LINE/LTR-rich heterochromatin; and (3) gene-depleted and satellite DNA-containing constitutive heterochromatin. We show, using fluorescence in situ hybridization (FISH) and 4C-seq technologies, that chromatin segments ranging from 0.6 to 3 Mb cluster with segments of the same chromatin class. As a consequence, the chromatin segments acquire corresponding positions in the nucleus irrespective of their chromosomal context, thereby strongly suggesting that this is their autonomous property. Interactions with the nuclear lamina, although largely retained in the HAC, reveal less autonomy. Taken together, our results suggest that building of a functional nucleus is largely a self-organizing process based on mutual recognition of chromosome segments belonging to the major chromatin classes.

Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast.

The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.

Survival in Quiescence Requires the Euchromatic Deployment of Clr4/SUV39H by Argonaute-Associated Small RNAs.

Quiescence (G0) is a ubiquitous stress response through which cells enter reversible dormancy, acquiring distinct properties including reduced metabolism, resistance to stress, and long life. G0 entry involves dramatic changes to chromatin and transcription of cells, but the mechanisms coordinating these processes remain poorly understood. Using the fission yeast, here, we track G0-associated chromatin and transcriptional changes temporally and show that as cells enter G0, their survival and global gene expression programs become increasingly dependent on Clr4/SUV39H, the sole histone H3 lysine 9 (H3K9) methyltransferase, and RNAi proteins. Notably, G0 entry results in RNAi-dependent H3K9 methylation of several euchromatic pockets, prior to which Argonaute1-associated small RNAs from these regions emerge. Overall, our data reveal another function for constitutive heterochromatin proteins (the establishment of the global G0 transcriptional program) and suggest that stress-induced alterations in Argonaute-associated sRNAs can target the deployment of transcriptional regulatory proteins to specific sequences.

Ccp1 Homodimer Mediates Chromatin Integrity by Antagonizing CENP-A Loading.

CENP-A is a centromere-specific histone 3 variant essential for centromere specification. CENP-A partially replaces canonical histone H3 at the centromeres. How the particular CENP-A/H3 ratio at centromeres is precisely maintained is unknown. It also remains unclear how CENP-A is excluded from non-centromeric chromatin. Here, we identify Ccp1, an uncharacterized NAP family protein in fission yeast that antagonizes CENP-A loading at both centromeric and non-centromeric regions. Like the CENP-A loading factor HJURP, Ccp1 interacts with CENP-A and is recruited to centromeres at the end of mitosis in a Mis16-dependent manner. These data indicate that factors with opposing CENP-A loading activities are recruited to centromeres. Furthermore, Ccp1 also cooperates with H2A.Z to evict CENP-A assembled in euchromatin. Structural analyses indicate that Ccp1 forms a homodimer that is required for its anti-CENP-A loading activity. Our study establishes mechanisms for maintenance of CENP-A homeostasis at centromeres and the prevention of ectopic assembly of centromeres.

[Cohesion inside eu- and heterochromatin in human cells].

It is considered that sister chromatids are held together immediately after replication by special protein complex--cohesin that consists of Smc1--Smc3 core dimer and two additional subunits, Scc1 and Scc3. This process is called cohesion. We have characterized binding of cohesin complex to early- and late-replicated chromatin at different stages of the cell cycle in human cells HeLa and HT1080 using superresolution microscopy (based on Structural ilumination microscopy--SIM) and immunoelectron microscopy. It has been shown that cohesins do not play important role in cohesion of heterochromatic domains, but they provide cohesion and organization of subdomains in euchromatic regions.

Effects of ovariectomy on the secretory apparatus in the right atrial cardiomyocytes of middle-aged mice.

OBJECTIVE: The aim of the present study was to evaluate the effects of ovariectomy on the secretory apparatus of natriuretic peptides in right atrial cardiomyocytes. METHODS: Nine-month-old mice underwent bilateral ovariectomy or sham surgery. The blood exam of the ovariectomized mice showed results consistent with castrated females. Systolic blood pressure was measured after ovariectomy (9 mo of age) and at the moment of sacrifice (12 mo of age). Fragments of the right atrium were collected and prepared for electron microscopy examination. The following variables were quantified: the quantitative density and area of the natriuretic peptide granules, the relative volume of euchromatin in the nucleus, the number of pores per 10 µm of the nuclear membrane and the relative volumes of the mitochondria and Golgi complex. RESULTS: The cardiomyocytes obtained from ovariectomized mice indicated that the quantitative density and the area of secretory granules of natriuretic peptides were significantly lower compared with the sham-operated mice. Furthermore, there was a decrease in the relative volume of euchromatin, a lower density of nuclear pores, and lower relative volumes of the mitochondria and Golgi complex in the ovariectomized mice compared with the sham-operated mice. These findings suggest a pool with a low turnover rate, i.e., low synthesis and elimination of natriuretic peptides. CONCLUSION: A lack of estrogen caused hypotrophy of the secretory apparatus in right atrial cardiomyocytes that could explain the weak synthesis of natriuretic peptides in mice. Furthermore, one of the mechanisms of blood pressure control was lost, which may explain, in part, the elevated blood pressure in ovariectomized mice.

Effects of ovariectomy on the secretory apparatus in the right atrial cardiomyocytes of middle-aged mice

OBJECTIVE : The aim of the present study was to evaluate the effects of ovariectomy on the secretory apparatus of natriuretic peptides in right atrial cardiomyocytes. METHODS: Nine-month-old mice underwent bilateral ovariectomy or sham surgery. The blood exam of the ovariectomized mice showed results consistent with castrated females. Systolic blood pressure was measured after ovariectomy (9 mo of age) and at the moment of sacrifice (12 mo of age). Fragments of the right atrium were collected and prepared for electron microscopy examination. The following variables were quantified: the quantitative density and area of the natriuretic peptide granules, the relative volume of euchromatin in the nucleus, the number of pores per 10 μm of the nuclear membrane and the relative volumes of the mitochondria and Golgi complex. RESULTS: The cardiomyocytes obtained from ovariectomized mice indicated that the quantitative density and the area of secretory granules of natriuretic peptides were significantly lower compared with the sham-operated mice. Furthermore, there was a decrease in the relative volume of euchromatin, a lower density of nuclear pores, and lower relative volumes of the mitochondria and Golgi complex in the ovariectomized mice compared with the sham-operated mice. These findings suggest a pool with a low turnover rate, i.e., low synthesis and elimination of natriuretic peptides. CONCLUSION: A lack of estrogen caused hypotrophy of the secretory apparatus in right atrial cardiomyocytes that could explain the weak synthesis of natriuretic peptides in mice. Furthermore, one of the mechanisms of blood pressure control was lost, which may explain, in part, the elevated blood pressure in ovariectomized mice. .

Structural chromatin alterations in peripheral blood leukocytes of alcohol-dependent individuals during detoxification therapy.

BACKGROUND/AIM: The aim of this study was to investigate the state of chromatin condensation in peripheral blood leukocytes of alcoholics, during the early detoxification period, in order to highlight structural modifications, indicating epigenetic mechanisms regulated by alcohol. MATERIALS AND METHODS: Blood samples were obtained from alcoholic patients, who were admitted for detoxification on an inpatient basis, and from healthy controls. The level of condensed heterochromatin and de-condensed euchromatin were detected through the ratio of lysine to arginine residues, by the application of the ammoniacal silver reaction (ASR) staining on leukocyte pellets, and through immunohistochemical localization of histone H1 on peripheral blood smears. RESULTS: Lymphocytes and neutrophils with relaxed de-condensed chromatin were found, indicating a more reactive genome in alcoholics, even at the stage of detoxification. CONCLUSION: The results underline the importance of chromatin structure of leukocytes as a sensitive, peripheral, biological marker for epigenetic studies in living chronic alcoholics.

Loss of neuronal 3D chromatin organization causes transcriptional and behavioural deficits related to serotonergic dysfunction.

The interior of the neuronal cell nucleus is a highly organized three-dimensional (3D) structure where regions of the genome that are linearly millions of bases apart establish sub-structures with specialized functions. To investigate neuronal chromatin organization and dynamics in vivo, we generated bitransgenic mice expressing GFP-tagged histone H2B in principal neurons of the forebrain. Surprisingly, the expression of this chimeric histone in mature neurons caused chromocenter declustering and disrupted the association of heterochromatin with the nuclear lamina. The loss of these structures did not affect neuronal viability but was associated with specific transcriptional and behavioural deficits related to serotonergic dysfunction. Overall, our results demonstrate that the 3D organization of chromatin within neuronal cells provides an additional level of epigenetic regulation of gene expression that critically impacts neuronal function. This in turn suggests that some loci associated with neuropsychiatric disorders may be particularly sensitive to changes in chromatin architecture.

[Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions].

A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.

Epigenetics of eu- and heterochromatin in inverted and conventional nuclei from mouse retina.

To improve light propagation through the retina, the rod nuclei of nocturnal mammals are uniquely changed compared to the nuclei of other cells. In particular, the main classes of chromatin are segregated in them and form regular concentric shells in order; inverted in comparison to conventional nuclei. A broad study of the epigenetic landscape of the inverted and conventional mouse retinal nuclei indicated several differences between them and several features of general interest for the organization of the mammalian nuclei. In difference to nuclei with conventional architecture, the packing density of pericentromeric satellites and LINE-rich chromatin is similar in inverted rod nuclei; euchromatin has a lower packing density in both cases. A high global chromatin condensation in rod nuclei minimizes the structural difference between active and inactive X chromosome homologues. DNA methylation is observed primarily in the chromocenter, Dnmt1 is primarily associated with the euchromatic shell. Heterochromatin proteins HP1-alpha and HP1-beta localize in heterochromatic shells, whereas HP1-gamma is associated with euchromatin. For most of the 25 studied histone modifications, we observed predominant colocalization with a certain main chromatin class. Both inversions in rod nuclei and maintenance of peripheral heterochromatin in conventional nuclei are not affected by a loss or depletion of the major silencing core histone modifications in respective knock-out mice, but for different reasons. Maintenance of peripheral heterochromatin appears to be ensured by redundancy both at the level of enzymes setting the epigenetic code (writers) and the code itself, whereas inversion in rods rely on the absence of the peripheral heterochromatin tethers (absence of code readers).

Beyond repair foci: DNA double-strand break repair in euchromatic and heterochromatic compartments analyzed by transmission electron microscopy.

PURPOSE: DNA double-strand breaks (DSBs) generated by ionizing radiation pose a serious threat to the preservation of genetic and epigenetic information. The known importance of local chromatin configuration in DSB repair raises the question of whether breaks in different chromatin environments are recognized and repaired by the same repair machinery and with similar efficiency. An essential step in DSB processing by non-homologous end joining is the high-affinity binding of Ku70-Ku80 and DNA-PKcs to double-stranded DNA ends that holds the ends in physical proximity for subsequent repair. METHODS AND MATERIALS: Using transmission electron microscopy to localize gold-labeled pKu70 and pDNA-PKcs within nuclear ultrastructure, we monitored the formation and repair of actual DSBs within euchromatin (electron-lucent) and heterochromatin (electron-dense) in cortical neurons of irradiated mouse brain. RESULTS: While DNA lesions in euchromatin (characterized by two pKu70-gold beads, reflecting the Ku70-Ku80 heterodimer) are promptly sensed and rejoined, DNA packaging in heterochromatin appears to retard DSB processing, due to the time needed to unravel higher-order chromatin structures. Complex pKu70-clusters formed in heterochromatin (consisting of 4 or ≥ 6 gold beads) may represent multiple breaks in close proximity caused by ionizing radiation of highly-compacted DNA. All pKu70-clusters disappeared within 72 hours post-irradiation, indicating efficient DSB rejoining. However, persistent 53BP1 clusters in heterochromatin (comprising ≥ 10 gold beads), occasionally co-localizing with γH2AX, but not pKu70 or pDNA-PKcs, may reflect incomplete or incorrect restoration of chromatin structure rather than persistently unrepaired DNA damage. DISCUSSION: Higher-order organization of chromatin determines the accessibility of DNA lesions to repair complexes, defining how readily DSBs are detected and processed. DNA lesions in heterochromatin appear to be more complex, with multiple breaks in spatial vicinity inducing severe chromatin disruptions. Imperfect restoration of chromatin configurations may leave DSB-induced epigenetic memory of damage with potentially pathological repercussions.

Peroxisome proliferator-activated receptor gamma activation restores islet function in diabetic mice through reduction of endoplasmic reticulum stress and maintenance of euchromatin structure.

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR-gamma) is an important target in diabetes therapy, but its direct role, if any, in the restoration of islet function has remained controversial. To identify potential molecular mechanisms of PPAR-gamma in the islet, we treated diabetic or glucose-intolerant mice with the PPAR-gamma agonist pioglitazone or with a control. Treated mice exhibited significantly improved glycemic control, corresponding to increased serum insulin and enhanced glucose-stimulated insulin release and Ca(2+) responses from isolated islets in vitro. This improved islet function was at least partially attributed to significant upregulation of the islet genes Irs1, SERCA, Ins1/2, and Glut2 in treated animals. The restoration of the Ins1/2 and Glut2 genes corresponded to a two- to threefold increase in the euchromatin marker histone H3 dimethyl-Lys4 at their respective promoters and was coincident with increased nuclear occupancy of the islet methyltransferase Set7/9. Analysis of diabetic islets in vitro suggested that these effects resulting from the presence of the PPAR-gamma agonist may be secondary to improvements in endoplasmic reticulum stress. Consistent with this possibility, incubation of thapsigargin-treated INS-1 beta cells with the PPAR-gamma agonist resulted in the reduction of endoplasmic reticulum stress and restoration of Pdx1 protein levels and Set7/9 nuclear occupancy. We conclude that PPAR-gamma agonists exert a direct effect in diabetic islets to reduce endoplasmic reticulum stress and enhance Pdx1 levels, leading to favorable alterations of the islet gene chromatin architecture.

A multi-instrumental approach to identify and purify very small embryonic like stem cells (VSELs) from adult tissues.

We employed several complementary cell image analytical methods including ImageStream system (ISS analysis) and molecular approaches to identify and purify from adult murine organs a population of very small embryonic like stem cells (VSELs). These cells are (i) small in size, (ii) possess high cytoplasmic/nuclear ratio, (iii) contain primitive unorganized euchromatin, (iv) in mice are found among Sca-1(+) Lin(-) CD45(-) cells and in humans among CD133(+) CXCR4(+) CD34(+) Lin(-) CD45(-) cells and (v) express embryonic markers such as Oct-4 protein in nuclei and SSEA antigens on the surface. In mice the highest number of these cells resides in brain, kidney, pancreas and bone marrow. Data from our laboratory indicate that VSELs are most likely a population of germ line/epiblast-derived pluripotent stem cells, that is deposited during organogenesis in developing tissues as a source of tissue committed stem cells and that the number of these cells decreases with the age. We believe that VSELs could be harnessed as a source of pluripotent stem cells for regenerative medicine.

Nuclear architecture and gene regulation.

The spatial organization of eukaryotic genomes in the cell nucleus is linked to their transcriptional regulation. In mammals, on which this review will focus, transcription-related chromatin positioning is regulated at the level of chromosomal sub-domains and individual genes. Most of the chromatin remains stably positioned during interphase. However, some loci display dynamic relocalizations upon transcriptional activation, which are dependent on nuclear actin and myosin. Transcription factors in association with chromatin modifying complexes seem to play a central role in regulating chromatin dynamics and positioning. Recent results obtained in this regard also give insight into the question how the different levels of transcriptional regulation are integrated and coordinated with other processes involved in gene expression. Corresponding findings will be discussed.

Interchromatidal central ridge and transversal symmetry in early metaphasic human chromosome one.

The topographic structure of Giemsa-banded (G-banded) early metaphase human chromosomes adsorbed on glass was analyzed by atomic force microscope using amplitude modulation mode (AM-AFM). Longitudinal height measurements for early metaphasic human chromosomes showed a central ridge that was further characterized by transversal height measurements. The heterochromatic regions displayed a high level of transversal symmetry, while the euchromatic ones presented several peaks across the transversal height measurements. We suggest that this central ridge and symmetry patterns point out a transitional arrangement of the early metaphase chromosome and support evidence for interchromatidal interactions prior to disjunction.

[Ultrastructural characteristics of endotheliocytes of skeletal muscle hemomicrocirculatory bed during tibial lengthening using G. A. Ilizarov technique].

The method of transmission electron microscopy was used to study the ultrastructure of endotheliocytes in the hemomicrocirculatory bed of the anterior tibial muscle of adult dogs during tibial lengthening with Ilizarov method. The capacity of endotheliocytes to reduction and renewal was established. The tension stress induced in the biological tissues of the lengthened limb stimulates and supports high activity of endotheliocyte nuclear apparatus, which is characterized by the prevalence of euchromatin. Presence of capillaries with closed lumen at all time points studied is indicative of a prolonged angiogenesis process in the terminal parts of the hemomicrocirculatory bed, which is accompanied by regeneration and growth of the muscle fibers. After the cessation of distraction, while the muscle tissue continued to grow and differentiate, the endotheliocyte growth and the activation of their synthetic apparatus was noted.

Non-telomeric sites as evidence of chromosomal rearrangement and repetitive (TTAGGG)n arrays in heterochromatic and euchromatic regions in four species of Akodon (Rodentia, Muridae).

Comparative studies among four species--Akodonazarae (2n = 38), A. lindberghi (2n = 42), A. paranaensis (2n = 44) and A. serrensis (2n = 46)--employing classic cytogenetics (C- and G-bands) and fluorescence in situ hybridization with telomeric (TTAGGG)n sequencesare reported here. Non-telomeric signals in addition to the regular telomeric sites were detected in three species:A. azarae, A. lindberghi and A. serrensis. One interstitial telomeric site (ITS) was observed proximally at the long arm of chromosome 1 of A. azarae. The comparison of G-banding patterns among the species indicated that the ITS was due to a tandem fusion/fission rearrangement. Non-telomeric signals of A. lindberghi and A. serrensis were not related to chromosomal rearrangements; instead, the sequences co-localized with (i) heterochromatic regions of all chromosomes in A. serrensis; (ii) some heterochromatic regions in A. lindberghi, and (iii) both euchromatic and heterochromatic regions in the metacentric pair of A. lindberghi. These exceptional findings revealed that ITS in Akodon can be related to chromosomal rearrangements and repetitive sequences in the constitutive heterochromatin and that the richness of TTAGGG-like sequences in the euchromatin could be hypothesized to be a result of amplification of the referred sequence along the chromosome arms.