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Mitochondrial endonuclease G (EndoG) contributes to chromosomal degradation when it is released from mitochondria during apoptosis. It is presumed to also have a mitochondrial function because EndoG deficiency causes mitochondrial dysfunction. However, the mechanism by which EndoG regulates mitochondrial function is not known. Fat accumulation in metabolic dysfunction-associated steatotic liver disease (MASLD), which is more common in men, is caused in part by mitochondrial dysfunction. EndoG expression is reduced in MASLD liver, and EndoG deficiency causes MASLD in an obesity-independent manner but only in males. EndoG promotes mitochondrial respiration by resolving mitochondrial tRNA/DNA hybrids formed during mtDNA transcription by recruiting RNA helicase DHX30 to unwind them. EndoG also cleaves off the 3'-end of the H-strand transcript that can prevent mt-tRNA Thr precursor cloverleaf-folding, and processing, which increases mt-tRNA Thr production and mitochondrial translation. Using fluorescent lifetime imaging microscopy technology to visualize oxygen consumption at the individual mitochondrion level, we found that EndoG deficiency leads to the selective loss of a mitochondrial subpopulation with high-oxygen consumption. This defect was reversed with mt-tRNA Thr supplementation. Thus, EndoG promotes mitochondrial respiration by selectively regulating the production of mt-tRNA Thr in male mice., Competing Interests: Competing interests statement:The authors declare no competing interest.

R-loops, composed of DNA-RNA hybrids and displaced single-stranded DNA, are known to pose a severe threat to genome integrity. Therefore, extensive research has focused on identifying regulatory proteins involved in controlling R-loop levels. These proteins play critical roles in preventing R-loop accumulation and associated genome instability. Herein I summarize recent knowledge on R-loop regulators affecting R-loop homeostasis, involving a wide array of R-loop screening methods that have enabled their characterization, from forward genetic and siRNA-based screens to proximity labeling and machine learning. These approaches not only deepen our understanding on R-loop formation processes, but also hold promise to find new targets in R-loop dysregulation associated with human pathologies., (© 2024. The Author(s).)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the death of motor neurons, the aetiology of which is essentially unknown. Here, we present an integrative epigenomic study in blood samples from seven clinically characterised sporadic ALS patients to elucidate molecular factors associated with the disease. We used clinical exome sequencing (CES) to study DNA variants, DNA-RNA hybrid immunoprecipitation sequencing (DRIP-seq) to assess R-loop distribution, and reduced representation bisulfite sequencing (RRBS) to examine DNA methylation changes. The above datasets were combined to create a comprehensive repository of genetic and epigenetic changes associated with the ALS cases studied. This repository is well-suited to unveil new correlations within individual patients and across the entire patient cohort. The molecular attributes described here are expected to guide further mechanistic studies on ALS, shedding light on the underlying genetic causes and facilitating the development of new epigenetic therapies to combat this life-threatening disease., (© 2024. The Author(s).)

Arabidopsis NODULIN HOMEOBOX (NDX) is a plant-specific transcriptional regulator whose role in small RNA biogenesis and heterochromatin homeostasis has recently been described. Here we extend our previous transcriptomic analysis to the flowering stage of development. We performed mRNA-seq and small RNA-seq measurements on inflorescence samples of wild-type and ndx1-4 mutant (WiscDsLox344A04) Arabidopsis plants. We identified specific groups of differentially expressed genes and noncoding heterochromatic siRNA (hetsiRNA) loci/regions whose transcriptional activity was significantly changed in the absence of NDX. In addition, data obtained from inflorescence were compared with seedling transcriptomics data, which revealed development-specific changes in gene expression profiles. Overall, we provide a comprehensive data source on the coding and noncoding transcriptomes of NDX-deficient Arabidopsis flowers to serve as a basis for further research on NDX function., (© 2023. The Author(s).)

Understanding why sexual reproduction-which involves syngamy (union of gametes) and meiosis-emerged and how it has subsisted for millions of years remains a fundamental problem in biology. Considered as the essence of sex, meiotic recombination is initiated by a DNA double-strand break (DSB) that forms on one of the pairing homologous chromosomes. This DNA lesion is subsequently repaired by gene conversion, the non-reciprocal transfer of genetic information from the intact homolog. A major issue is which of the pairing homologs undergoes DSB formation. Accumulating evidence shows that chromosomal sites where the pairing homologs locally differ in size, i.e., are heterozygous for an insertion or deletion, often display disparity in gene conversion. Biased conversion tends to duplicate insertions and lose deletions. This suggests that DSB is preferentially formed on the "shorter" homologous region, which thereby acts as the recipient for DNA transfer. Thus, sex primarily functions as a genome (re)loading mechanism. It ensures the restoration of formerly lost DNA sequences (deletions) and allows the efficient copying and, mainly in eukaryotes, subsequent spreading of newly emerged sequences (insertions) arising initially in an individual genome, even if they confer no advantage to the host. In this way, sex simultaneously repairs deletions and increases genetic variability underlying adaptation. The model explains a remarkable increase in DNA content during the evolution of eukaryotic genomes., (© 2022. The Author(s).)

Arabidopsis NODULIN HOMEOBOX (NDX) is a nuclear protein described as a regulator of specific euchromatic genes within transcriptionally active chromosome arms. Here we show that NDX is primarily a heterochromatin regulator that functions in pericentromeric regions to control siRNA production and non-CG methylation. Most NDX binding sites coincide with pericentromeric het-siRNA loci that mediate transposon silencing, and are antagonistic with R-loop structures that are prevalent in euchromatic chromosomal arms. Inactivation of NDX leads to differential siRNA accumulation and DNA methylation, of which CHH/CHG hypomethylation colocalizes with NDX binding sites. Hi-C analysis shows significant chromatin structural changes in the ndx mutant, with decreased intrachromosomal interactions at pericentromeres where NDX is enriched in wild-type plants, and increased interchromosomal contacts between KNOT-forming regions, similar to those observed in DNA methylation mutants. We conclude that NDX is a key regulator of heterochromatin that is functionally coupled to het-siRNA loci and non-CG DNA methylation pathways., (© 2022. The Author(s).)

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these "domesticated", transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a histone-lysine methyltransferase domain and an HsMar1 transposase. To elucidate the biological role of SETMAR, it is crucial to identify genomic targets to which SETMAR specifically binds and link these sites to the regulation of gene expression. Herein, we mapped the genomic landscape of SETMAR binding in a near-haploid human leukemia cell line (HAP1) in order to identify on-target and off-target binding sites at high resolution and to elucidate their role in terms of gene expression. Our analysis revealed a perfect correlation between SETMAR and inverted terminal repeats (ITRs) of HsMar1 transposon remnants, which are considered as natural target sites for SETMAR binding. However, we did not detect any untargeted events at non-ITR sequences, calling into question previously proposed off-target binding sites. We identified sequence fidelity of the ITR motif as a key factor for determining the binding affinity of SETMAR for chromosomes, as higher conservation of ITR sequences resulted in increased affinity for chromatin and stronger repression of SETMAR-bound gene loci. These associations highlight how SETMAR's chromatin binding fine-tune gene regulatory networks in human tumour cells., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

A serious adverse effect of cancer therapies is cardiovascular toxicity, which significantly limits the widespread use of antineoplastic agents. The promising new field of cardio-oncology offers the identification of potent anti-cancer therapeutics that effectively inhibit cancer cell proliferation without causing cardiotoxicity. Future introduction of recently identified cardio-safe compounds into clinical practice (including ERK dimerization inhibitors or BAX allosteric inhibitors) is expected to help oncologists avoid unwanted cardiological complications associated with therapeutic interventions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Szabó, Hornyák, Miskei and Székvölgyi.)

Uveal melanoma (UM) is the most common malignant tumor of the eye with extremely high metastatic potential. UM tumor cells can disseminate only hematogenously, thus, angiogenic signals have a particular role in the prognosis of the disease. Although the presence of cancer stem cells (CSCs) in densely vascularized UMs has been reported previously, their role in the process of hematogenous spread of UM has not been studied. In this study, we investigated the regulation of angiogenesis in UM in correlation with the presence of CSCs. Seventy UM samples were collected to analyze the expression of CSC markers and angiogenic factors. The expression of CSC markers was studied by RT-PCR, Western blotting techniques and IHC-TMA technique. RT-PCR showed high expression of CSC markers, particularly nestin, FZD6 and SOX10 and somewhat lower expression of NGFR. The protein expression of FZD6, HIF-1α and VEGFA was further evaluated in 52 UM samples by the IHC-TMA technique. We report here for the first time a significant correlation between FZD6 and VEGFA expression in UM samples. The observed correlation between FZD6 and VEGFA suggests the presence of CSCs in UM that are associated with the vascularization process.

Meiotic recombination is initiated by Spo11-catalyzed DNA double-strand breaks (DSBs) that are promoted by histone modifications and histone modifying enzymes. Herein we investigated the role of histone H3 lysine 56 acetylation (H3K56ac) located near the entry/exit points of the DNA in the globular H3 domain. We generated a series of mutant cells ( asf1 Δ, rtt109 Δ, hst3/4 Δ, and H3K56A) in which the endogenous level of H3K56ac was manipulated and tracked during meiotic growth. We show that complete loss or increased abundance of H3K56ac in these mutants allows timely entry into meiosis and sporulation and does not impair S phase progression, first and second meiotic cell divisions, and spore viability. In the asf1 Δ, rtt109 Δ, hst3/4 Δ mutants, DSBs and crossovers form normal levels with a short (60-min) delay at the HIS4-LEU2 artificial recombination hotspot, however, DSB formation shows a ∼threefold decrease in the H3K56A mutant at the natural BUD23-ARE1 hotspot. The latter DSB phenotype, showing significant DSB reduction in the H3K56A mutant, was also observed at DSB sites using genome-wide mapping of Rfa1-coated single-stranded DNA flanking DSBs (RPA ChIP). Parallel mapping of H3K56-acetylated histones in wild type cells revealed strong depletion of the H3K56ac ChIP signal over Spo11-oligo DSBs, albeit most H3K56-acetylated histones were enriched adjacent to the identified RPA ChIP binding sites. Taken together, these associations demonstrate a prominent role of H3 lysine 56 acetylation in the formation of DNA breaks within recombination hotspot regions., (Copyright © 2020 Karányi, Hornyák and Székvölgyi.)

Unravelling the origin of genetic alterations from point mutations to chromosomal rearrangements was greatly enhanced by the discovery of RNA-DNA hybrids (R-loops) that behave as hotspots of genomic instability in a variety of organisms. Current models suggest that uncontrolled R-loops are a hazard to genome integrity, therefore, identifying proteins that are involved in recognising and signalling R-loop structures are of key importance. Herein we analysed key RNA-DNA hybrid binding proteins in humans taking advantage of large-scale gene expression, survival rate, and drug-sensitivity data from cancer genomics databases. We show that expression of RNA-DNA hybrid binding proteins in various cancer types is associated with survival and may have contrasting outcomes in responding to therapeutic treatments. Based on the revealed pharmacogenomic landscape of human RNA-DNA hybrid binding proteins, we propose that R-loops and R-loop binding proteins are potentially relevant new epigenetic markers and therapeutic targets in multiple cancers., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Molecular combing and gel electrophoretic studies revealed endogenous nicks with free 3'OH ends at ∼100 kb intervals in the genomic DNA (gDNA) of unperturbed and G1-synchronized Saccharomyces cerevisiae cells. Analysis of the distribution of endogenous nicks by Nick ChIP-chip indicated that these breaks accumulated at active RNA polymerase II (RNAP II) promoters, reminiscent of the promoter-proximal transient DNA breaks of higher eukaryotes. Similar periodicity of endogenous nicks was found within the ribosomal rDNA cluster, involving every ∼10th of the tandemly repeated 9.1 kb units of identical sequence. Nicks were mapped by Southern blotting to a few narrow regions within the affected units. Three of them were overlapping the RNAP II promoters, while the ARS-containing IGS2 region was spared of nicks. By using a highly sensitive reverse-Southwestern blot method to map free DNA ends with 3'OH, nicks were shown to be distinct from other known rDNA breaks and linked to the regulation of rDNA silencing. Nicks in rDNA and the rest of the genome were typically found at the ends of combed DNA molecules, occasionally together with R-loops, comprising a major pool of vulnerable sites that are connected with transcriptional regulation.

Spp1 is the H3K4me3 reader subunit of the Set1 complex (COMPASS/Set1C) that contributes to the mechanism by which meiotic DNA break sites are mechanistically selected. We previously proposed a model in which Spp1 interacts with H3K4me3 and the chromosome axis protein Mer2 that leads to DSB formation. Here we show that spatial interactions of Spp1 and Mer2 occur independently of Set1C. Spp1 exhibits dynamic chromatin binding features during meiosis, with many de novo appearing and disappearing binding sites. Spp1 chromatin binding dynamics depends on its PHD finger and Mer2-interacting domain and on modifiable histone residues (H3R2/K4). Remarkably, association of Spp1 with Mer2 axial sites reduces the effective turnover rate and diffusion coefficient of Spp1 upon chromatin binding, compared with other Set1C subunits. Our results indicate that "chromosomal turnover rate" is a major molecular determinant of Spp1 function in the framework of meiotic chromatin structure that prepares recombination initiation sites for break formation., (© 2018 Karányi et al.)

Tumors are composed of abnormally transformed cell types and tissues that differ from normal tissues in their genetic and epigenetic makeup, metabolism, and immunology. Molecular compounds that modulate the immune response against neoplasms offer promising new strategies to combat cancer. Inhibitors targeting the indoleamine-2,3-dioxygenase 1 enzyme (IDO1) represent one of the most potent therapeutic opportunities to inhibit tumor growth. Herein, we assess the biochemical role of IDO1 in tumor metabolism and immune surveillance, and review current diagnostic and therapeutic approaches that are intended to increase the effectiveness of immunotherapies against highly aggressive and difficult-to-treat IDO-expressing cancers.

Lysine 27 to methionine (K27 M) mutation of the histone variant H3.3 drives the formation of an aggressive glioblastoma multiforme tumor in infants. Here we analyzed how the methionine substitution alters the stability of H3.3 nucleosomes in vitro and modifies its kinetic properties in live cells. We also determined whether the presence of mutant nucleosomes perturbed the mobility of the PRC2 subunit Ezh2 (enhancer-of-zeste homolog 2). We found that K27 M nucleosomes maintained the wild-type molecular architecture both at the level of bulk histones and single nucleosomes and followed similar diffusion kinetics to wild-type histones in live cells. Nevertheless, we observed a remarkable differential recovery of Ezh2 in response to transcriptional stress that was accompanied by a faster diffusion rate of the mobile fraction of Ezh2 and a significantly increased immobile fraction, suggesting tighter chromatin binding of Ezh2 upon transcription inhibition. The differential recovery of Ezh2 was dependent on transcription, however, it was independent from K27 M mutation status. These biophysical characteristics shed more light on the mechanism of histone H3.3 K27M in glioma genesis in relation to the kinetic properties of Ezh2., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The impact of R-loops on the physiology and pathology of chromosomes has been demonstrated extensively by chromatin biology research. The progress in this field has been driven by technological advancement of R-loop mapping methods that largely relied on a single approach, DNA-RNA immunoprecipitation (DRIP). Most of the DRIP protocols use the experimental design that was developed by a few laboratories, without paying attention to the potential caveats that might affect the outcome of RNA-DNA hybrid mapping. To assess the accuracy and utility of this technology, we pursued an analytical approach to estimate inherent biases and errors in the DRIP protocol. By performing DRIP-sequencing, qPCR, and receiver operator characteristic (ROC) analysis, we tested the effect of formaldehyde fixation, cell lysis temperature, mode of genome fragmentation, and removal of free RNA on the efficacy of RNA-DNA hybrid detection and implemented workflows that were able to distinguish complex and weak DRIP signals in a noisy background with high confidence. We also show that some of the workflows perform poorly and generate random answers. Furthermore, we found that the most commonly used genome fragmentation method (restriction enzyme digestion) led to the overrepresentation of lengthy DRIP fragments over coding ORFs, and this bias was enhanced at the first exons. Biased genome sampling severely compromised mapping resolution and prevented the assignment of precise biological function to a significant fraction of R-loops. The revised workflow presented herein is established and optimized using objective ROC analyses and provides reproducible and highly specific RNA-DNA hybrid detection., (© 2017 Halász et al.; Published by Cold Spring Harbor Laboratory Press.)

P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs) catalyzed by the evolutionary conserved Spo11 protein and accessory factors. DSBs are nonrandomly distributed along the chromosomes displaying a significant (~400-fold) variation of frequencies, which ultimately establishes local and long-range "hot" and "cold" domains for recombination initiation. This remarkable patterning is set up within the chromatin context, involving multiple layers of biochemical activity. Predisposed chromatin accessibility, but also a range of transcription factors, chromatin remodelers, and histone modifiers likely promote local recruitment of DSB proteins, as well as mobilization, sliding, and eviction of nucleosomes before and after the occurrence of meiotic DSBs. Here, we assess our understanding of meiotic DSB formation and methods to change its patterning. We also synthesize current heterogeneous knowledge on how histone modifications and chromatin remodeling may impact this decisive step in meiotic recombination., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

During meiosis, combinatorial associations of genetic traits arise from homologous recombination between parental chromosomes. Histone H3 lysine 4 trimethylation marks meiotic recombination hotspots in yeast and mammals, but how this ubiquitous chromatin modification relates to the initiation of double-strand breaks (DSBs) dependent on Spo11 remains unknown. Here, we show that the tethering of a PHD-containing protein, Spp1 (a component of the COMPASS complex), to recombinationally cold regions is sufficient to induce DSB formation. Furthermore, we found that Spp1 physically interacts with Mer2, a key protein of the differentiated chromosomal axis required for DSB formation. Thus, by interacting with H3K4me3 and Mer2, Spp1 promotes recruitment of potential meiotic DSB sites to the chromosomal axis, allowing Spo11 cleavage at nearby nucleosome-depleted regions.

Sexual reproduction depends on the success of faithful chromosome transmission during meiosis to yield viable gametes. Central to meiosis is the process of recombination between paternal and maternal chromosomes, which boosts the genetic diversity of progeny and ensures normal homologous chromosome segregation. Imperfections in meiotic recombination are the source of de novo germline mutations, abnormal gametes, and infertility. Thus, not surprisingly, cells have developed a variety of mechanisms and tight controls to ensure sufficient and well-distributed recombination events within their genomes, the details of which remain to be fully elucidated. Local and genome-wide studies of normal and genetically engineered cells have uncovered a remarkable stochasticity in the number and positioning of recombination events per chromosome and per cell, which reveals an impressive level of flexibility. In this minireview, we summarize our contemporary understanding of meiotic recombination and its control mechanisms, and address the seemingly paradoxical and poorly understood diversity of recombination sites. Flexibility in the distribution of meiotic recombination events within genomes may reside in regulation at the chromatin level, with histone modifications playing a recently recognized role.

Microcystin-LR (MC-LR) is a heptapeptide cyanotoxin, known to be a potent inhibitor of type 1 and 2A protein phosphatases in eukaryotes. Our aim was to investigate the effect of MC-LR on the organization of microtubules and mitotic chromatin in relation to its possible effects on cell and whole organ morphology in roots of common reed (Phragmites australis). P. australis is a widespread freshwater and brackish water aquatic macrophyte, frequently exposed to phytotoxins in eutrophic waters. Reed plantlets regenerated from embryogenic calli were treated with 0.001-40 microg ml(-1) (0.001-40.2 microM) MC-LR for 2-20 days. At 0.5 microg ml(-1) MC-LR and at higher cyanotoxin concentrations, the inhibition of protein phosphatase activity by MC-LR induced alterations in reed root growth and morphology, including abnormal lateral root development and the radial swelling of cells in the elongation zone of primary and lateral roots. Both short-term (2-5 days) and long-term (10-20 days) of cyanotoxin treatment induced microtubule disruption in meristems and in the elongation and differentiation zones. Microtubule disruption was accompanied by root cell shape alteration. At concentrations of 0.5-5 microg ml(-1), MC-LR increased mitotic index at long-term exposure and induced the increase of the percentage of meristematic cells in prophase as well as telophase and cytokinesis of late mitosis. High cyanotoxin concentrations (10-40 microg ml(-1)) inhibited mitosis at as short as 2 days of exposure. The alteration of microtubule organization was observed in mitotic cells at all exposure periods studied, at cyanotoxin concentrations of 0.5-40 microg ml(-1). MC-LR induced spindle anomalies at the metaphase-anaphase transition, the formation of asymmetric anaphase spindles and abnormal sister chromatid separation. This paper reports for the first time that MC-LR induces cytoskeletal changes that lead to alterations of root architecture and development in common reed and generally, in plant cells. The MC-LR induced alterations in cells of an ecologically important aquatic macrophyte can reveal the importance of the effects of a cyanobacterial toxin in aquatic ecosystems.

We explore the possibilities offered by flow cytometric microbead analysis to develop high throughput methods for the detection of deletions/insertions and single-strand DNA lesions. The products of PCR reactions derived from reference and test samples are denatured and reannealed, then exposed to enzymatic or chemical treatments distinguishing homoduplices from heteroduplices. The biotin- and dye labeled reaction products are immobilized on microbeads and the homo- and heteroduplices are assessed in separate fluorescence channels, by flow cytometry. Using a model system based on the mixed lineage leukemia gene breakpoint cluster region, we demonstrate that deletions and insertions in genomic DNA can be detected, using S1 nuclease and chemical cleavage to distinguish hetero- from homoduplices, or a restriction enzyme cleaving only the homoduplices. Single-strand discontinuities can also be detected, by combining nick-translation, using labeled nucleotide, and flow cytometric microbead analysis. The methodical approaches demonstrated are applicable in a versatile manner in basic cell and molecular biological research and also promise direct application for high throughput screening of genetic diseases and lesions, including insertions or deletions of short sequence elements and single-strand lesions formed at hypersensitive sites in response to apoptotic stimuli., (Copyright 2008 International Society for Analytical Cytology.)

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at approximately 50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining approximately 50-kbp domains.

Background: Chromatin immunoprecipitation (ChIP) is a widely used technique for the detection of in vivo DNA-protein interactions underlying epigenetic regulation. The standard readout of ChIP is based on semi-quantitative or quantitative PCR measurements; however, the development of alternative platforms with high throughput potentialities is expected to facilitate the introduction of this method into routine diagnostics., Methods: We have established a flow-cytometry-based alternative for the evaluation of ChIP data. The method is based on the capture of the products of a conventional PCR run to low cycle numbers, on microbeads., Results: In vivo histone H4 acetylation and H3 lysine 4 methylation was detected at the promoter of the tissue transglutaminase type 2 gene. These results were confirmed by QPCR measurements. The levels of modifications decreased significantly upon apoptosis and were accompanied by the down-regulation of TGM2 mRNA expression., Conclusions: This method that we named ChIP-on-beads, a combination of flow cytometry and conventional PCR, is a reliable and efficient alternative in the quantitative analysis of ChIP results, especially promising when high throughput monitoring of epigenetic markers of diagnostic importance is required. The method is simple enough to be easily implemented in a routine flow-cytometric laboratory.

Phenomena involving the disassembly of chromosomes to approximately 50 kbp double-stranded fragments upon protein denaturing treatments of normal and apoptotic mammalian nuclei as well as yeast protoplasts may be an indication of special, hypersensitive regions positioned regularly at loop-size intervals in the eukaryotic chromatin. Here we show evidence in yeast cell systems that loop-size fragmentation can occur in any phase of the cell cycle and that the plating efficiency of these cells is approximately 100%. The possibility of sequence specificity was investigated within the breakpoint cluster region (bcr) of the human MLL gene, frequently rearranged in certain leukemias. Our data suggest that DNA isolated from yeast cultures or mammalian cell lines carry nicks or secondary structures predisposing DNA for a specific nicking activity, at non-random positions. Furthermore, exposure of MLL bcr-carrying plasmid DNA to S1 nuclease or nuclear extracts or purified topoisomerase II elicited cleavages at the nucleotide positions of nick formation on human genomic DNA. These data support the possibility that certain sequence elements are preferentially involved in the cleavage processes responsible for the en masse disassembly of chromatin to loop-size fragments upon isolation of DNA from live eukaryotic cells.

Background: Introduction of microbeads into flow-cytometry has created a new scenario, making quantitative measurement of molecules dispersed in a homogeneous phase, with an extremely wide realm of already realized and potential applications possible. Development of this field has lead to specialized instrumentation and microbead arrays, dedicated to certain applications., Methods: Formaldehyde-fixed yeast and bacterial cells were conjugated with avidin and applied as microbeads, to establish a simple, convenient, flexible, and inexpensive flow-cytometric platform for various immunological and biochemical assays., Results: We have tested these "biological microbeads" for the simultaneous titration of human alpha-fetoprotein (AFP) and human Chorionic Gonadotropin (betahCG) hormone levels, for the titration of proteolytic and nucleolytic (restriction) enzymes, and for quantitative PCR, using biotinylated and fluorescent primers., Conclusions: The use of biological microbeads for various immunological and biochemical assays has been demonstrated. The flow-cytometric methods proved to be at least as sensitive as the standard biochemical or immunological tests. For proteinase K activity measurements, a single enzyme molecule in the sample could be detected. The sensitivity, versatility, and low cost of the assays may advance flow-cytometry to become a central methodological platform in most laboratories. The biological microbeads offer virtually unlimited possibilities for fluorescent labeling (addressing), conjugation of ligand binding molecules, and they are easy to handle and perform well in a multiplex format.

Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements., (Copyright 2003 Wiley-Liss, Inc.)