Changes In Chromatin Condensation Research Articles

Zinc(II)-Sterol Hydrazone Complex as a Potent Anti-Leishmania Agent: Synthesis, Characterization, and Insight into Its Mechanism of Antiparasitic Action.

Searching for new alternatives for treating leishmaniasis, we present the synthesis, characterization, and biological evaluation against Leishmania amazonensis of the new ZnCl2(H3)2 complex. H3 is 22-hydrazone-imidazoline-2-yl-chol-5-ene-3β-ol, a well-known bioactive molecule functioning as a sterol Δ24-sterol methyl transferase (24-SMT) inhibitor. The ZnCl2(H3)2 complex was characterized by infrared, UV-vis, molar conductance measurements, elemental analysis, mass spectrometry, and NMR experiments. The biological results showed that the free ligand H3 and ZnCl2(H3)2 significantly inhibited the growth of promastigotes and intracellular amastigotes. The IC50 values found for H3 and ZnCl2(H3)2 were 5.2 µM and 2.5 µM for promastigotes, and 543 nM and 32 nM for intracellular amastigotes, respectively. Thus, the ZnCl2(H3)2 complex proved to be seventeen times more potent than the free ligand H3 against the intracellular amastigote, the clinically relevant stage. Furthermore, cytotoxicity assays and determination of selectivity index (SI) revealed that ZnCl2(H3)2 (CC50 = 5 μΜ, SI = 156) is more selective than H3 (CC50 = 10 μΜ, SI = 20). Furthermore, as H3 is a specific inhibitor of the 24-SMT, free sterol analysis was performed. The results showed that H3 was not only able to induce depletion of endogenous parasite sterols (episterol and 5-dehydroepisterol) and their replacement by 24-desalkyl sterols (cholesta-5,7,24-trien-3β-ol and cholesta-7,24-dien-3β-ol) but also its zinc derivative resulting in a loss of cell viability. Using electron microscopy, studies on the fine ultrastructure of the parasites showed significant differences between the control cells and parasites treated with H3 and ZnCl2(H3)2. The inhibitors induced membrane wrinkle, mitochondrial injury, and abnormal chromatin condensation changes that are more intense in the cells treated with ZnCl2(H3)2.

Chromatin condensation regulates endothelial cell adaptation to shear stress.

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS.

The Chromatin Remodeling Complex CHD1 Regulates the Primitive State of Mesenchymal Stromal Cells to Control Their Stem Cell Supporting Activity.

The primitive state (stemness) of mesenchymal stromal cells (MSCs) is responsible for supporting the function of tissue-specific stem cells to regenerate damaged tissues. However, molecular mechanisms regulating the stemness of MSCs remain unknown. In this study, we found that the primitive state of MSCs is hierarchically regulated by the expression levels of the chromatin remodeling complex, CHD1, with CHD1 expression levels higher in the undifferentiated state, and decreasing upon MSC differentiation. Consistently, CHD1 expression levels decrease during progressive loss of clonogenic progenitors (CFU-F) induced by passage cultures. Moreover, knockdown (KD) of CHD1 decreased CFU-F frequency, whereas CHD1 overexpression increased it. In addition, the expression of stem cell-specific genes was down- or upregulated upon KD or overexpression of CHD1, respectively, accompanied by associated changes in chromatin condensation. Importantly, altering CHD1 expression levels affected the ability of MSCs to support the self-renewing expansion of hematopoietic stem cells (HSCs). Furthermore, CHD1 levels were significantly decreased in MSCs from acute myeloid leukemia or aplastic anemia patients, where CFU-F and HSC-supporting activities are lost. Altogether, these findings show that chromatin remodeling by CHD1 is a molecular parameter that influences the primitive state of MSCs and their stem cell-supporting activity, which controls tissue regeneration.

Effects of combined fungicide in stingless bees Scaptotrigona bipunctata

Locker is a fungicide used in different crops for biological control, affecting both insect pests and pollinators, such as bees. This study aimed to evaluate toxicity and establish the lethal concentration (LC50) of the fungicide Locker in stingless bees Scaptotrigona bipunctata, investigating changes in brain cells chromatin structure and in midgut morphology. This agrochemical is a combined fungicide that contains three active ingredients: benzimidazole, triazole and strobilurin. Adult forage bees were orally exposed to the following concentrations: 1.7 mg a.i./L; 2.55 mg a.i./L; 3.4 mg a.i./L and 4.25 mg a.i./L of fungicide, and submitted to histochemical and morphological analysis 24, 48 and 72 hours after ingestion. The analysis of S. bipunctata brain cells showed changes in chromatin condensation in all treatments. There were also several morphological changes in the midgut at all concentrations and exposure times. Thus, the fungicide Locker significantly affected bee survival, inducing changes in their internal morphology, which can lead to alterations in their activities, directly interfering in their foraging behavior and survival.

Unravelling how in vitro capacitation alters ram sperm chromatin before and after cryopreservation.

Sperm chromatin structure provides valuable information for the prediction of male fertility and can be altered during different procedures. Previous studies have shown that sperm chromatin condensation decreased during in vitro capacitation. Moreover, cryopreservation can affect sperm DNA integrity and chromatin compaction. This study aimed to investigate dynamic modifications produced in the chromatin structure of ram spermatozoa during in vitro capacitation before and after cryopreservation. Chromatin decondensation (AB+), DNA methylation, DNA fragmentation index (%DFI) and high DNA stainability (HDS) were evaluated in fresh and frozen-thawed ram spermatozoa incubated under capacitating (CAP) conditions at 1, 5, 15, 30, 60, 120, 180 and 240minutes and under non-capacitating (NC) conditions at 0, 15 and 240minutes. Incubation in NC conditions did not induce significant changes in chromatin condensation (P>.05; AB+and HDS). However, incubation of fresh and cryopreserved ram spermatozoa under CAP conditions significantly increased chromatin decondensation (P<.05), reaching the highest percentage of AB+and HDS from 180 to 240minutes in fresh samples and from 5 to 30minutes in cryopreserved samples. Both variables (HDS and AB+) were positively correlated with tyrosine phosphorylation, total motility, progressive motility, curvilinear velocity and amplitude of lateral head displacement, as well as between them under CAP conditions in fresh and cryopreserved spermatozoa. DNA methylation significantly increased in cryopreserved spermatozoa (P<.05), but only after extended incubation under CAP conditions (60-240minutes), while the %DFI, albeit higher in cryopreserved samples, remained constant under CAP and NC conditions in both types of sample (P>.05). Our results suggest that sperm chromatin condensation decreased progressively during in vitro capacitation of ram spermatozoa, while sperm DNA integrity remained intact. Such changes in chromatin condensation appeared faster after sperm cryopreservation.

Advances in studies on histone deactylase inhibitors as anticancer drugs

Inhibitors of histone deacetylases (HDIs), by affecting the process of histone acetylation, lead to changes in chromatin condensation, and in consequence, to changes in the expression of numerous genes responsible for the cell cycle and differentiation. Therefore, they can be effective in the treatment of cancer. The antitumor activity for over 15 HDIs has been confirmed so far, and some of them have been approved in the USA and Europe, mainly in combination with cytostatics or radiotherapy. For several HDIs, large clinical trials are being carried out to estimate their effectiveness, in monotherapy and new combinations. Other synthetic and natural compounds with HDI activity are tested in preclinical studies. HDIs differ in terms of chemical structure, biological activity and specificity in relation to individual HDACs. The compounds active against classic HDACs (Zn2+-dependent metalloproteins) are usually characterized by the presence the Zn2+ binding group, the linker part and the capping group. Taking into account the type of Zn2+ binding group, classic HDIs are classified as short-chain fatty acids, hydroxamic acids, cyclic peptides and benzamides. In turn, the nicotinamide adenine dinucleotide-dependent SIRTs inhibitors are small molecules, mostly nicotinamide and β-naphthol derivatives. The presented paper summarizes the most important information regarding the use of HDIs as anticancer drugs, regarding their diversified chemical structure, activity against HDACs, additional therapeutic properties and side effects. The review was made taking into account the literature from the last five years (2013-2017).

Plant programmed cell death from a chromatin point of view.

Programmed cell death (PCD) is a ubiquitous genetically regulated process consisting of the activation of finely controlled signalling pathways that lead to cellular suicide. PCD can be part of a developmental programme (dPCD) or be triggered by environmental conditions (ePCD). In plant cells, as in animal cells, extensive chromatin condensation and degradation of the nuclear DNA are among the most conspicuous features of cells undergoing PCD. Changes in chromatin condensation could either reflect the structural changes required for internucleosomal fragmentation of nuclear DNA or relate to large-scale chromatin rearrangements associated with a major transcriptional switch occurring during cell death. The aim of this review is to give an update on plant PCD processes from a chromatin point of view. The first part will be dedicated to chromatin conformational changes associated with cell death observed in various developmental and physiological conditions, whereas the second part will be devoted to histone dynamics and DNA modifications associated with critical changes in genome expression during the cell death process.

Pluripotency factor opens chromatin

Pluripotency All cells in an organism share the same genome. Differences in form and function arise from cells varying the specific genes they express. Temporal and spatial cues trigger changes in chromatin condensation: An open arrangement supports, whereas a closed conformation blocks, gene expression. Lopes Novo et al. report a chromatin-opening role for the pluripotency transcription factor NANOG, even in typically condensed areas of the genome, such as repetitive sequences. To do this, NANOG works with the SALL1, a protein associated with condensed chromatin. When embryonic stem cells differentiate, they compact their chromatin and simultaneously reduce NANOG expression. This work highlights how pluripotent cells link their pluripotency network with chromatin organization. Genes Dev. 30 , 1101 (2016).

Response of exfoliated human buccal epithelium cells to combined gamma radiation, microwaves, and magnetic field exposure estimated by changes in chromatin condensation and cell membrane permeability

Modulation of the biological effects produced by ionizing radiation (IR) using microwave and magnetic fields has important theoretical and practical applications. Response of human buccal epithelium cells to different physical agents (single and combined exposure to 0.5–5 Gy γ-radiation (60Co); microwaves with the frequency of 36.64 GHz and power densities of 0.1 and 1 W/m2, and static magnetic field with the intensity of 25 mT) has been investigated. The stress response of the cells was evaluated by counting heterochromatin granules quantity (HGQ) in the cell nuclei stained with orcein. Membrane permeability was assessed by the percentage of cells stained with indigocarmine (cells with damaged membrane). The increase of heterochromatin granules quantity (HGQ), i.e. chromatin condensation was detected at the doses of 2 Gy and higher. Changes in the cell membrane permeability to indigocarmine expressed the threshold effect. Membrane permeability reached the threshold at the doses of 2–3 Gy for the cells of different donors and did not change with the increase of the dose of γ-radiation. Cells obtained from different donors revealed some individual peculiarities in their reaction to γ-radiation. The static magnetic field and microwaves applied before or after γ-radiation decreased its impact, as revealed by means of HGQ assessment.

The mystery of underground death: cell death in roots during ontogeny and in response to environmental factors.

Programmed cell death (PCD) is an essential part of the ontogeny of roots and their tolerance/resistance mechanisms, allowing adaptation and growth under adverse conditions. It occurs not only at the cellular and subcellular level, but also at the levels of tissues, organs and even whole plants. This process involves a wide spectrum of mechanisms, from signalling and the expression of specific genes to the degradation of cellular structures. The major goals of this review were to broaden current knowledge about PCD processes in roots, and to identify mechanisms associated with both developmental and stress-associated cell death in roots. Vacuolar cell death, when cell contents are removed by a combination of an autophagy-associated process and the release of hydrolases from a collapsed vacuole, is responsible for programming self-destruction. Regardless of the conditions and factors inducing PCD, its subcellular events usually include the accumulation of autophagosome-like structures, and the formation of massive lytic compartments. In some cases these are followed by the nuclear changes of chromatin condensation and DNA fragmentation. Tonoplast disruption and vacuole implosion occur very rapidly, are irreversible and constitute a definitive step toward cell death in roots. Active cell elimination plays an important role in various biological processes in the life history of plants, leading to controlled cellular death during adaptation to changing environmental conditions, and organ remodelling throughout development and senescence.

Quantification of chromatin condensation level by image processing

The level of chromatin condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors.This study describes a validated experimental technique to quantify chromatin condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of chromatin condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of chromatin condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting chromatin condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of chromatin condensation.

Biological Rationale for the Favorable Clinical Outcomes of Patients Carrying SF3B1 Mutations in Myelodysplastic Syndromes with Ring Sideroblasts

AbstractAbstract 922SF3B1, a RNA splicing factor is frequently mutated in refractory anemia with ring sideroblasts (RARS) and RARS with thrombocytosis (RARS-T). Aside from its phenotypic importance, SF3B1 has been associated with good outcomes in MDS. Consistent with others, we found favorable survival outcomes in SF3B1 mutants. To explain the clinical phenotypes (good outcomes, anemia, and less progression to acute myeloid leukemia (AML)), we delved into the consequences of SF3B1 dysfunction in MDS. We hypothesized that SF3B1 mutations result in changes in RNA splicing of key genes in erythropoiesis resulting in anemia. Moreover, we postulated that SF3B1 is a founder mutation in RS formation but insufficient as a sole abnormality to induce a deleterious phenotype. The acquisition of genetic/ epigenetic aberrations may trigger the final phenotype. We reported abundant iron deposits in the mitochondria of SF3B1 mutants. We hypothesized that SF3B1 mutations change the composition/ chemical valence of iron leading to free radicals and DNA damage. We performed Energy-dispersive X-ray/ Electron-Energy Loss Spectroscopy on WT and SF3B1 mutants (n=2) finding that Fe2O3 is the most prevalent iron form. Changes in iron might predispose to DNA damage. We used flow cytometry to measure gamma-H2AX, a marker of DNA damage finding higher gamma-H2AX levels in bone marrow of WT (n=4; 26.35±28.36) vs SF3B1 mutants (n=8; 4.19±4.34), healthy subjects (n=4; 6.15±3.74) and other WT low-risk MDS (n=5; 7.6±1.6). Since increased DNA damage induces chromatin remodeling, we found that WT have more condensed chromatin with granular cytoplasm vs SF3B1 mutants (n=2). Further, RNA-sequencing showed alterations in histone deacetylases (HDAC1/HDAC2) and modification (ASXL1) in SF3B1 mutants. This links splicing mutations to epigenetic events and may represent the 2nd hit to induce the disease phenotype. Other targets were interrogated. Since SF3B1 interacts with members of Class II polycomb group (PcG) in Sf3b1+/− mice and PcG genes, like ASXL1/ EZH2 are mutated in MDS, we performed sequencing on PcG Class II genes (RNF2/ PCGF2). No mutation was found in RARS/RARS-T pts (n=34; SF3B1 mutant=26; WT=8). mRNA levels might be changed in these pts. We also elucidated the role of SF3B1 in the phenotype of anemia. RNA-sequencing has been instructive. Ribosomal proteins (RPS14 and RPS17) are implicated in the pathogenesis of 5q- MDS and Diamond-Blackfan anemia. Both RPS14/ RPS17 expression was different in SF3B1 mutant vs WT (n=2). Further, cytokine analysis showed a trend to higher IL-6 levels in WT vs mutants (n=3). IL-6 is associated with anemia and poor response to therapy in some diseases. We assessed the response to erythropoietin (EPO) and found that none of the WT RARS/-T (n=4) responded to EPO while 15/19 (79%) of SF3B1 mutants responded (p=0.008). It’s suggested that SF3B1 mutants have better outcomes due to decreased AML transformation. To dissect this point, we looked at chromosomal defects in SF3B1 mutants (n=27) and WT (n=11), finding that 45% of WT carried complex karyotype (del(5q), del(17p) del(7), and i(17p); p=0.0009) while only 18% of mutants carried one additional abnormality (del(20q), inv3, and 16p). We also checked for concomitant mutations in genes of methylation (TET2, DNMT3A, IDH1/2), histone (ASXL1, UTX, EZH2), transcription (RUNX1, JAK2, TP53), signaling (CBL, NRAS, KRAS), and splicing (U2AF1, SRSF2). We analyzed the global mutational status in RARS/RARS-T (n=38; WT, n=11 and SF3B1 mutants, n=27) finding that 5/11 (45%) WT carried in total 6 other methylation (n=2), transcription (n=1), and splicing (n=3) gene mutations. Further, 15/27 (56%) of SF3B1 mutants carried a total of 16 mutations excluding SF3B1 in: methylation (n=9), transcription (n=4), splicing (n=1), histone (n=2). The most prevalent mutated genes (TET2, DNMT3A) predict for good treatment response. In-vitro treatment with decitabine (0.2 uM) showed changes in chromatin condensation, DNA damage, and response in SF3B1 mutants vs WT (n=2) suggesting that the pathogenetic effects of SF3B1 may be elicited through the methylation pathway. Lastly, SF3B1 predicts for good outcomes. SF3B1 may mediate effects on anemia through alterations in ribosomal function, cytokines, and epigenetic changes. Conversely, SF3B1 mutants have better survival and lower risk to AML evolution because of lower tendency to acquire poor prognostic defects. Disclosures:No relevant conflicts of interest to declare.

DNA replication stress induces deregulation of the cell cycle events in root meristems of Allium cepa

Prolonged treatment of Allium cepa root meristems with changing concentrations of hydroxyurea (HU) results in either premature chromosome condensation or cell nuclei with an uncommon form of biphasic chromatin organization. The aim of the current study was to assess conditions that compromise cell cycle checkpoints and convert DNA replication stress into an abnormal course of mitosis. Interphase-mitotic (IM) cells showing gradual changes of chromatin condensation were obtained following continuous 72 h treatment of seedlings with 0·75 mm HU (without renewal of the medium). HU-treated root meristems were analysed using histochemical stainings (DNA-DAPI/Feulgen; starch-iodide and DAB staining for H(2)O(2) production), Western blotting [cyclin B-like (CBL) proteins] and immunochemistry (BrdU incorporation, detection of γ-H2AX and H3S10 phosphorylation). Continuous treatment of onion seedlings with a low concentration of HU results in shorter root meristems, enhanced production of H(2)O(2), γ-phosphorylation of H2AX histones and accumulation of CBL proteins. HU-induced replication stress gives rise to axially elongated cells with half interphase/half mitotic structures (IM-cells) having both decondensed and condensed domains of chromatin. Long-term HU treatment results in cell nuclei resuming S phase with gradients of BrdU labelling. This suggests a polarized distribution of factors needed to re-initiate stalled replication forks. Furthermore, prolonged HU treatment extends both the relative time span and the spatial scale of H3S10 phosphorylation known in plants. The minimum cell length and a threshold level of accumulated CBL proteins are both determining factors by which the nucleus attains commitment to induce an asynchronous course of chromosome condensation. Replication stress-induced alterations in an orderly route of the cell cycle events probably reflect a considerable reprogramming of metabolic functions of chromatin combined with gradients of morphological changes spread along the nucleus.

Ranbp2 haploinsufficiency mediates distinct cellular and biochemical phenotypes in brain and retinal dopaminergic and glia cells elicited by the Parkinsonian neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Many components and pathways transducing multifaceted and deleterious effects of stress stimuli remain ill-defined. The Ran-binding protein 2 (RanBP2) interactome modulates the expression of a range of clinical and cell-context-dependent manifestations upon a variety of stressors. We examined the role of Ranbp2 haploinsufficiency on cellular and metabolic manifestations linked to tyrosine-hydroxylase (TH+) dopaminergic neurons and glial cells of the brain and retina upon acute challenge to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonian neurotoxin, which models facets of Parkinson disease. MPTP led to stronger akinetic parkinsonism and slower recovery in Ranbp2 +/− than wild-type mice without viability changes of brain TH+-neurons of either genotype, with the exception of transient nuclear atypia via changes in chromatin condensation of Ranbp2 +/− TH+-neurons. Conversely, the number of wild-type retinal TH+-amacrine neurons compared to Ranbp2 +/− underwent milder declines without apoptosis followed by stronger recoveries without neurogenesis. These phenotypes were accompanied by a stronger rise of EdU+-proliferative cells and non-proliferative gliosis of GFAP+-Müller cells in wild-type than Ranbp2 +/− that outlasted the MPTP-insult. Finally, MPTP-treated wild-type and Ranbp2 +/− mice present distinct metabolic footprints in the brain or selective regions thereof, such as striatum, that are supportive of RanBP2-mediated regulation of interdependent metabolic pathways of lysine, cholesterol, free-fatty acids, or their β-oxidation. These studies demonstrate contrasting gene-environment phenodeviances and roles of Ranbp2 between dopaminergic and glial cells of the brain and retina upon oxidative stress-elicited signaling and factors triggering a continuum of metabolic and cellular manifestations and proxies linked to oxidative stress, and chorioretinal and neurological disorders such as Parkinson.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1071-9) contains supplementary material, which is available to authorized users.

Characterization of monomeric DNA-binding protein Histone H1 inLeishmania braziliensis

Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features. The localization and the aggregation state of L. braziliensis H1 has been determined by immunolocalization, mass spectrometry, cross-linking and electrophoretic mobility shift assays. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. braziliensis. An antibody raised against recombinant L. braziliensis H1 recognized specifically that protein by immunoblot in L. braziliensis extracts, but not in other Leishmania species, a consequence of the sequence divergences observed among Leishmania species. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. braziliensis H1 is monomeric in solution, but oligomerizes upon binding to DNA. Finally, despite the lack of a globular domain, L. braziliensis H1 is able to form complexes with DNA in vitro, with higher affinity for supercoiled compared to linear DNA.

Genistein Induced Apotosis in Human Gastric Carcinoma Cells

The apoptosis in gastric cancer cells induced by genistein and the relationship between this apoptosis and expression of NF-κB and Caspase-3 were studied. In vitro experiments, MTT assay was used to determine the cell growth inhibitory rate. Transmission fluorescence microscope, transmission electron microscopy, DNA-fragment assay were used to qualitively detect the apoptosis status of gastric cancer line SGC-7901 before and after the genistein treatment. Immunohistochemical staining and Western Blotting were performed to detect the expression of gene NF-κB and Caspase-3. The results showed that genistein inhibited the growth of gastric carcinoma cell line SGC-7901 in a dose-and time-dependent manner. Genistein induced SGC-7901 cells to undergo apoptosis with typically apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation and apoptotic body formation. Genistein could reduce the expression of gene NF-κB, and improve the expression of gene Caspase-3.

Activator-dependent p300 Acetylation of Chromatin in Vitro: ENHANCEMENT OF TRANSCRIPTION BY DISRUPTION OF REPRESSIVE NUCLEOSOME-NUCLEOSOME INTERACTIONS

Condensation of chromatin into higher order structures is mediated by intra- and interfiber nucleosome-nucleosome interactions. Our goals in this study were to determine the impact specific activator-dependent histone acetylation had on chromatin condensation and to ascertain whether acetylation-induced changes in chromatin condensation were related to changes in RNA polymerase II (RNAPII) activity. To accomplish this, an in vitro model system was constructed in which the purified transcriptional activators, Tax and phosphorylated CREB (cAMP-response element-binding protein), recruited the p300 histone acetyltransferase to nucleosomal templates containing the human T-cell leukemia virus type-1 promoter sequences. We find that activator-dependent p300 histone acetylation disrupted both inter- and intrafiber nucleosome-nucleosome interactions and simultaneously led to enhanced RNAPII transcription from the decondensed model chromatin. p300 histone acetyltransferase activity had two distinct components: non-targeted, ubiquitous activity in the absence of activators and activator-dependent activity targeted primarily to promoter-proximal nucleosomes. Mass spectrometry identified several unique p300 acetylation sites on nucleosomal histone H3 (H3K9, H3K27, H3K36, and H3K37). Collectively, our data have important implications for understanding both the mechanism of RNAPII transcriptional regulation by chromatin and the molecular determinants of higher order chromatin structure.

Potassium deprivation is sufficient to induce a cell death program in Saccharomyces cerevisiae

Cell culture in low potassium (K(+)) media has been associated to programmed cell death (PCD) in metazoans. In this study, deprivation of K(+) led Saccharomyces cerevisiae cells to a death process that involved phosphatidylserine externalization, changes in chromatin condensation, DNA and vacuole fragmentation as well as enhanced accumulation of reactive oxygen species. During the course of K(+) starvation, plasma membrane hyperpolarization and increased accumulation of calcium (Ca(2+)) took place. The presence of rubidium (Rb(+)), a K(+)-analogue element, in the K(+)-deprived medium was accompanied by Rb(+) accumulation but did not fully prevent the appearance of PCD markers. This argues for a specific effect of K(+) on the course of cell death. While the absence of the YCA1 metacaspase did not have a major effect, the absence of TRK (transport of K(+)) K(+)-transporters led to changes in the pattern of annexin V/propidium iodide labeling. This change paralleled a fast accumulation of Ca(2+). Addition of ethylene glycol tetraacetic acid improved growth and reduced cell death in trk1Deltatrk2Delta cells. These findings reveal that K(+) deprivation is sufficient to induce PCD in a cell-walled eukaryotic organism and suggest that the phenotype attributed to the lack of TRK genes is partially due to the effect of the encoded transporters on Ca(2+) homeostasis.

UTX, a Histone Demethylase, Is Inactivated through Homozygous Deletion, Mutation, and DNA Methylation in Multiple Myeloma.

Abstract 1798Poster Board I-824Histone modifications are known to mediate transcriptional regulation through changes in chromatin condensation and as such can lead to aberrant transcriptional patterns resulting in malignant transformation. Modulation of chromatin structure via histone modification is becoming recognised as an important pathogenic mechanism in myeloma and has been suggested by the over-expression of MMSET, a histone methyltransferase, by the t(4;14) chromosomal rearrangement. More recently inactivation of UTX, a histone demethylase, has also been suggested to have a role in myeloma pathogenesis and both UTX and MMSET are mediators of transcriptional repression. UTX is inactivated in a number of different cancer cell lines but importantly, mutations and deletions have been detected in myeloma cell lines and we wished to follow up on this observation in uniformly treated clinical cases. UTX is a large gene found on the X chromosome covering 240 kb of genomic DNA and consists of 29 exons encoding a protein with both JmjC-domains and tricopeptide repeats responsible for histone demethylation and polycomb protein interactions. Inactivation of UTX occurs through deletions of individual exons through to large whole gene deletions as well as by mutations scattered throughout the 29 exons. A further mechanism of UTX inactivation which has not been looked for to date is via DNA methylation of the CpG island upstream of the transcriptional start site. We set out to determine the status of UTX in our dataset which includes expression, mapping, and methylation array data from presenting myeloma samples entered into the MRC Myeloma IX clinical trial. The gene expression of UTX was measured on 272 samples using Affymetrix U133 Plus 2.0 arrays and showed that 80% of samples do not express UTX transcripts but using expression quartile analysis we could not detect an effect on overall survival. The mechanism underlying the abrogation of expression was investigated further using the Affymetrix 500K SNP mapping array on a subset of 114 samples to detect copy number alterations. UTX was hemizygously deleted in 21 (42%) female samples and was completely deleted in 1 male sample, at the resolution of the arrays. In order to determine if individual exons were deleted, at a resolution below that detectable by mapping arrays, we performed quantitative PCR coupled with high resolution melting (HRM) analysis using the Rotor-gene Q real-time cycler (Qiagen). Exons were amplified, over 40 cycles, to obtain products of ∼200 bp using LC Green Plus mastermix (Idaho Technologies) in a 10 μl reaction on the Rotor-gene Q with a final HRM step from 72-95 °C with increments of 0.1 °C. Amplification plots combined with the HRM step allows us to identify both homozygous deletions and mutations within the exons. We screened all 114 samples for micro-deletions and mutations and found homozygous deletions in ∼7% of samples and identified a significant proportion of mutations using the HRM method which accounted for a total of ∼10% of gene inactivation. In order to determine if methylation could be responsible for inactivation of the remaining allele we used the Illumina Infinium humanmethylation27 array to study the methylation status at the UTX locus. This array interrogates 27,578 highly informative CpG sites per sample at the single-nucleotide resolution using bisulfite converted DNA. The results of this analysis are presented as an average beta-score where 1.0 is fully methylated and 0 is fully unmethylated. Samples were analyzed using Illumina GenomeStudio and the custom differential methylation algorithm. In samples with a diploid copy number of UTX the methylation signals covered 2 ranges: hemi-methylated (0.35-0.55, n=7) and hyper-methylated (0.73-0.89, n=14). In samples with 1 copy of UTX, which includes all males, there were 3 ranges: hypomethylated (0.08-0.21, n=5), hemi-methylated (0.35-0.51, n=3), and hypermethylated (0.66-0.88, n=48). All of the hypomethylated samples with a single copy of UTX were male, and at least 1 of these samples contained an inactivating exonic deletion resulting in complete loss of function. These data indicate that methylation of the residual allele contributes significantly to the inactivation of UTX along with interstitial deletions and mutations. We will go on to present data on the interaction of UTX with variation at the UTY locus and how this modulates behaviour of the myeloma clone. DisclosuresNo relevant conflicts of interest to declare.

The Gcn5 Bromodomain of the SAGA Complex Facilitates Cooperative and Cross-tail Acetylation of Nucleosomes

Bromodomains are acetyl lysine binding modules found in many complexes that regulate gene transcription. In budding yeast, the coactivator complex SAGA (Spt-Ada-Gcn5-acetyl-transferase) predominantly facilitates transcription of stress-activated genes and requires the bromodomain of the Gcn5 subunit for full activation of a number of these genes. This bromodomain has previously been shown to promote retention of the complex to H3 and H4 acetylated nucleosomes. Because the SAGA complex mediates histone H3 acetylation, we sought to determine to what extent the Gcn5 bromodomain directly modulates histone acetylation activity. Kinetic analysis of SAGA-mediated acetylation of nucleosomal substrates reveals that this bromodomain: 1) is required for the cooperative acetylation of nucleosomes, 2) enhances acetylation of an H3 histone tail when the other H3 tail within a nucleosome is already acetylated, and 3) augments the acetylation turnover of nucleosomes previously acetylated at lysine 16 of the histone H4 tails. These results indicate that the Gcn5 bromodomain promotes the establishment of nucleosome acetylation through multiple mechanisms and more generally show how chromatin recognition domains can modulate the enzymatic activity of chromatin modifying complexes.