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PubMed2003

Embryonic stem cells: a perfect marriage between gene regulation and regenerative medicine.

The mechanism of mammalian gene regulation is highly complex, involving multiple layers of feedback control loops and dynamic chromatin remodeling. The current approach used to dissect the genetic circuitry of mammalian gene regulation utilizes somatic cells and protein fusion as a means to modulate protein interactions. This approach has several limitations that include (i) genome inaccessibility, (ii) high background interferences and, (iii) limited cellular phenotypes. Previously, the two broad fields of research "control of gene expression" and "stem cell biology" had been pursued separately by cell biologists; this review outlines evidence suggesting that integration of these two fields would provide a comprehensive platform for interdisciplinary research seeking to address mechanistic questions concerning gene regulation-that could have enormous implication for the development of therapeutic applications.

Animals↗
PubMedDate not supplied

[Endogenous DNases as a tool for isolation of nuclear matrix: critical parameters of nucleolysis].

Degree of nucleolysis has critical significance for isolation of nuclear matrix (NM) specifically enriched in transcribed DNA sequences as demonstrated at the example of inactive (c-fos, c-myc, and Ck) and active (p53, albumin, and 28S rRNA) genes in resting hepatocytes. Optimal degree of nucleolysis features degradation of loop domains of chromatin with preserved relatively uniform molecular weight distribution of DNA. Deviation from these parameters leads to nonspecific fragmentation of chromatin in various gene loci and isolation of NM samples nonspecifically enriched or depleted of transcribed DNA sequences. Under optimal hydrolytic conditions, the transcribed chromatin is more resistant to endogenous DNase attack, which allows selective conservation of its association with the nuclear matrix.

Albumins↗
PubMed2003

Visualization of chromatin domains created by the gypsy insulator of Drosophila.

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.

Animals↗
PubMed2006

Tpk3 and Snf1 protein kinases regulate Rgt1 association with Saccharomyces cerevisiae HXK2 promoter.

Hexokinase 2 is an essential factor for signalling repression through the Saccharomyces cerevisiae high-glucose sensing pathway. The main regulatory mechanism that controls the HXK2 gene expression in yeast is mediated by the Rgt1 and Med8 transcription factors, which repress HXK2 expression in low-glucose containing media. In this study, we show that the repression activity of Rgt1 is regulated by Snf1 and Tpk3 protein kinases. Binding of Rgt1 to the HXK2 promoter requires Rgt1 phosphorylation by Snf1 or by an Snf1-dependent protein kinase. Conversely, Rgt1 hyperphosphorylation by the Tpk3 or by a Tpk3-dependent protein kinase dissociates Rgt1 from the repressor complex. Two-hybrid and chromatin immunoprecipitation experiments indicate that an Snf1-dependent interaction between Rgt1 and Med8 in the repressor complex is also essential for Rgt1 repression. The repression of HXK2 transcription by Rgt1 likely occurs through the formation of a DNA loop in the HXK2 locus, spanning the promoter and coding regions. These results suggest that a novel silent-chromatin loop is responsible for Rgt1-dependent transcriptional regulation of the HXK2 gene.

Binding Sites↗
PubMed1989

The limits of the DNase I-sensitive domain of the human apolipoprotein B gene coincide with the locations of chromosomal anchorage loops and define the 5' and 3' boundaries of the gene.

In eukaryotic cells, chromatin is organized as domains or loops that are generated by periodic attachment of the chromatin fiber to protein components of a nuclear matrix, or scaffold. These chromosomal loops may have a function in gene regulation. The length of the chromatin domain encompassing the human apolipoprotein B gene was studied by determining the locations of nuclear matrix attachment sites as well as the boundaries of the DNase I-sensitive domain in cells that express the gene (such as HepG2 and CaCo-2 cells) and in those that do not (HeLa cells). Three nuclear matrix attachment regions (MARs) of the human apolipoprotein B gene have been localized: a 3' -proximal MAR, between nucleotides +43,186 and +43,850; a 5' -proximal MAR, between nucleotides -2,765 and -1,801; and a 5' -distal MAR, between nucleotides -5,262 and -4,048. Both the 3' -proximal and the 5' -distal MARS were present in cells that express the gene (HepG2 and CaCo-2 cells) as well as in cells that do not (HeLa cells), whereas the 5' -proximal MAR was detected only in HepG2 cells. These MARs were located at the bases of chromosomal loops in histone-extracted nuclei in all three cell lines. Various classes of A/T-rich sequences resembling the recognition site for topoisomerase II were present within the MAR-containing fragments. The boundaries of the DNase I-sensitive domain coincide with the positions of the 3' -proximal and 5' -distal matrix attachment sites. These results suggest the existence of a 47.5-kilobase domain that represents a topologically sequestered functional unit containing the coding region and all known cis-acting regulatory elements of the human apolipoprotein B gene.

Apolipoproteins B↗
PubMed1987

Ribosomal DNA sequences attached to the nuclear matrix.

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.

Animals↗
PubMed2000

Organization of highly repeated sequences in surface-spread pachytene chromosomes of rye.

A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs) associated with fluorescence in situ hybridization (FISH) has been applied to analyze the location and organization of five different highly repeated DNA sequences in rye. Our observations indicate that, depending on the type of sequence, the chromatin displays different types of organization. Telomeric sequences were seen tightly associated with the SC while other repetitive DNA sequences were found to form loops that are associated with SCs only at their bases. On the contrary, the FISH signal of a centromeric satellite had a granular appearance, reflecting that the hybridization occurs only with parts of the chromatin loops.

Chromosomes↗
PubMed1989

Structure of transcriptionally active chromatin: radiological evidence for requirement of torsionally constrained DNA.

Synthesis of alpha- and beta-globin RNA in DMSO-induced Friend's erythroleukemia cells and synthesis of immunoglobulin gamma- and kappa-chain RNA, total RNA, 5S RNA, and tRNA in mouse myeloma cells (MPC-11) was inhibited by gamma-irradiation. For all RNA species, synthesis decreased nearly exponentially as a function of radiation dose, whereas RNA size distributions, turnover rates, and specific activities of radioactively labeled RNA were affected only insignificantly. D37 values for the loss of synthesis of various RNA species correspond to target sizes ranging from 21,000 to 53,000 kd, or 30-80 kbp of DNA. These target sizes are several-fold larger than the structural genes in question; however, they correspond well with the size of DNA loops, or "domains" constrained by the nuclear matrix. The data suggest that the eukaryotic transcription unit is the torsionally constrained chromatin loop, transcription of which may be inactivated, or significantly reduced by a DNA single-strand break.

Animals↗
PubMedDate not supplied

An endogenous nuclease in hamster, mouse, and human spermatozoa cleaves DNA into loop-sized fragments.

Recent work from our laboratory provided evidence for the existence of a nuclease in hamster spermatozoa. This endogenous nuclease cleaves sperm chromatin at the bases of DNA loop domains into large fragments with an average size of roughly 50 kb. Here, we demonstrate that this sperm nuclease is present in the sperm nucleus and that it is activated by the presence of both calcium and magnesium much more efficiently than with either ion alone, resulting in DNA degradation in 30 minutes. We also show that similar nucleases are present in mouse and human spermatozoa. The human nuclease can be activated by freeze-thawing spermatozoa in noncryoprotective media. The activity of the sperm nuclease in all 3 species resembles that of a group of somatic cell DNAses that also require both calcium and magnesium and that digest the chromatin into loop-sized fragments during apoptosis.

Animals↗
PubMed1980

Chromatin organization during meiotic prophase of Bombyx mori.

Chromatin organization during the early stages of male meiotic prophase in Bombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200-300 A in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5-25 mu of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.

Animals↗
PubMed2005

A new method to analyze boar sperm DNA fragmentation under bright-field or fluorescence microscopy.

We present a new, rapid and simple method to study DNA Fragmentation Index (DFI) in sperm samples from boar under bright-field and fluorescence microscopy. Discrimination of sperm cells containing fragmented DNA relies on the extreme peripheral diffusion of their chromatin fragments, whereas those sperm nuclei without DNA fragmentation do not disperse or show very restricted spreading of DNA loops close to the flagellum. The basic methodology provided in the commercial kit Sperm-Sus-Halomax allows, in addition to a direct estimation of DFI in a sperm sample under bright field microscopy, a direct visualization of DNA breaks by incorporation of labelled nucleotides using the DNA polymerase I following the in situ nick translation assay (ISNT methodology not provided in the kit). An external control using DBD-FISH (DNA breakage detection-fluorescence in situ hybridization) on human and boar sperm samples was used in this experiment. The results obtained show (i) low levels of background DNA fragmentation (from 0.7 to 10%), (ii) no significant differences for DFI after the application of Sperm-Sus-Halomax and ISNT, with a tendency to be underestimated after using DBD-FISH and (iii) a characteristic chromatin organization in boar sperm nucleus, with a particular response to chromatin loop relaxation and preferential DNA labelling by ISNT at the proximal nuclear area, close to the flagellum. This methodology allows the routine assessment of boar sperm samples for DFI, as well as basic and clinical research on this relevant topic in any laboratory of semen analysis.

Animals↗
PubMed1995

[Structure of chromatin. 2: levels of organization of DNA in the nucleus. Highly organized structures].

In the interphase nucleus, the 30 nm fiber, whose characteristics were discussed in the first part of this review, is condensed 10-15 fold to form loops or domains of chromatin with a DNA packing ratio of 600. In the metaphase chromosomes, the packing ratio is 12000. The existence of loops in the interphase nucleus has been suggested by experiments from electronic microscopy, sedimentation and nuclease digestion. These loops of 20-200Kb are attached at their bases to a non-histone protein network, that is the nuclear matrix or scaffold whose major component is the topoisomerase II. There is disagreement regarding the arrangement of the loops in metaphase chromosomes and several models have been proposed. Six of these models are presented. A number of data suggest that the sequences of attachment (SARs/MARs) define functional units containing related genes. Thus, the compartmentation of the genome in topographically independent domains might ensure the fidelity of epigenetic transmission of the chromatin structure, and it might help to control gene expression. In the interphase nucleus, the chromosomes coexist with other domains involved in major nuclear functions such as transcription, replication or post-transcriptional modifications and whose compartmentation is not always evident.

Centromere↗
PubMedDate not supplied

The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation.

Sperm DNA fragmentation is being increasingly recognized as an important cause of infertility. We herein describe the Sperm Chromatin Dispersion (SCD) test, a novel assay for sperm DNA fragmentation in semen. The SCD test is based on the principle that sperm with fragmented DNA fail to produce the characteristic halo of dispersed DNA loops that is observed in sperm with non-fragmented DNA, following acid denaturation and removal of nuclear proteins. This was confirmed by the analysis of DNA fragmentation using the specific DNA Breakage Detection-Fluorescence In Situ Hybridization (DBD-FISH) assay, which allows the detection of DNA breaks in lysed sperm nuclei. Sperm suspensions either prepared from semen or isolated from semen by gradient centrifugation were embedded in an agarose microgel on slides and treated with 0.08 N HCl and lysing solutions containing 0.8 M dithiothreitol (DTT), 1% sodium dodecyl sulfate (SDS), and 2 M NaCl. Then, the slides were sequentially stained with DAPI (4',6-diamidino-2-phenylindole) and/or the Diff-Quik reagent, and the percentages of sperm with nondispersed and dispersed chromatin loops were monitored by fluorescence and brightfield microscopy, respectively. The results indicate that all sperm with nondispersed chromatin displayed DNA fragmentation, as measured by DBD-FISH. Conversely, all sperm with dispersed chromatin had very low to undetectable DBD-FISH labeling. SCD test values were significantly higher in patients being screened for infertility than in normozoospermic sperm donors who had participated in a donor insemination program. The coefficient of variation obtained using 2 different observers, either by digital image analysis (DIA) or by brightfield microscopy scoring, was less than 3%. In conclusion, the SCD test is a simple, accurate, highly reproducible, and inexpensive method for the analysis of sperm DNA fragmentation in semen and processed sperm. Therefore, the SCD test could potentially be used as a routine test for the screening of sperm DNA fragmentation in the andrology laboratory.

Acids↗
PubMed2004

Highly specific interactions between bHLH transcription factors and chromatin during retina development.

Basic helix-loop-helix (bHLH) transcription factors such as atonal homolog 5 (ATH5) and neurogenin 2 (NGN2) determine crucial events in retinogenesis. Using chromatin immunoprecipitation, we demonstrate that their interactions with target promoters undergo dynamic changes as development proceeds in the chick embryo. Chick ATH5 associates with its own promoter and with the promoter of the beta3 nicotinic receptor specifically in retinal ganglion cells and their precursors. NGN2 binds to the ATH5 promoter in retina but not in optic tectum, suggesting that interactions between bHLH factors and chromatin are highly tissue specific. The transcriptional activations of both promoters correlate with dimethylation of lysine 4 on histone H3. Inactivation of the ATH5 promoter in differentiated neurons is accompanied by replication-independent chromatin de-methylation. This report is one of the first demonstrations of correlation between gene expression, binding of transcription factors and chromatin modification in a developing neural tissue.

Animals↗
PubMed1982

Isolation, properties and nucleolytic degradation of chromatin from Escherichia coli.

A new procedure has been developed for the isolation of the chromosome complex, termed chromatin, from Escherichia coli. The bacteria were subjected to low ionic strength and T4 lysozyme, followed by detergent treatment analogous to that employed for the isolation of eukaryotic chromosomes. The chromatin was an insoluble viscous material which contained approximately equal amounts of DNA and RNA. The protein content of the chromatin was almost three times greater than the nucleic acid content. Electron microscopy revealed that the chromatin was highly condensed, having multiple loops and beaded structures with various diameters. The chromatin could be completely solubilized by both micrococcal nuclease and DNAase I, whereas RNAase had no effect. The initial degradation by micrococcal nuclease resulted in the production of a DNA-protein particle, sedimentation coefficient 10S, and an RNA-protein complex of 24S. Further degradation led to a decrease in sedimentation coefficient of the DNA-protein complex, but not of the RNA-protein particle. The peak size of the DNA of the initial DNA-protein particle was approximately 2400 bp. The action of micrococcal nuclease also resulted in the production of several discrete RNA species of various sizes. Several low molecular weight proteins (12000-27000) were found in the DNA-protein complex. The DNA-binding protein HU was present in the undigested chromatin; varying amounts of HU were, however, detected in the DNA-protein and RNA-protein particles.

Bacterial Proteins↗
PubMed2025

Setdb2 Regulates Inflammatory Trigger-Induced Trained Immunity of Macrophages Through Two Different Epigenetic Mechanisms.

"Trained immunity" of innate immune cells occurs through a sequential two-step process where an initial pathogenic or sterile inflammatory trigger is followed by an amplified response to a later un-related secondary pathogen challenge. The memory effect is mediated at least in part through epigenetic modifications of the chromatin landscape. Here, we investigated the role of the epigenetic modifier Setdb2 in microbial (β-glucan) or sterile trigger (Western-diet-WD/oxidized-LDL-oxLDL)-induced trained immunity of macrophages. Using genetic mouse models and genomic analysis, we uncovered a critical role of Setdb2 in regulating proinflammatory and metabolic pathway reprogramming. We further show that Setdb2 regulates trained immunity through two different complementary mechanisms: one where it positively regulates glycolytic and inflammatory pathway genes via enhancer-promoter looping, and is independent of its enzymatic activity; while the second mechanism is associated with both increased promoter associated H3K9 methylation and repression of interferon response pathway genes. Interestingly, while both mechanisms occur in response to pathogenic training, only the chromatin-looping mechanism operates in response to the sterile inflammatory stimulus. These results reveal a previously unknown bifurcation in the downstream pathways that distinguishes between pathogenic and sterile inflammatory signaling responses associated with the innate immune memory response and may provide potential therapeutic opportunities to target cytokine vs. interferon pathways to limit complications of chronic inflammation.

Setdb2↗
PubMed1989

The ultrastructure of upstream and downstream regions of an active Balbiani ring gene.

When active, the 37 kb Balbiani ring genes are known to form transcription loops with an almost fully extended chromatin axis. Here we examine the upstream and downstream regions of such transcription loops by electron microscopy. We demonstrate that a loop starts and ends in tightly packed chromatin; the two anchoring sites are clearly separated from each other in space. The upstream, nontranscribed region consists of a thin, extended, apparently flexible and nucleosome-free fiber corresponding to about 0.5 kb DNA. The downstream, nontranscribed region appears as a 200 nm long nucleofilament loosely coiled into a short, thick chromatin fiber and estimated to contain about 3 kb DNA.

Animals↗
PubMed1998

Scaffold-associated regions in the human type I interferon gene cluster on the short arm of chromosome 9.

Scaffold-associated regions (SARs) function at the level of modeling or shaping the chromatin of DNA into loop domains. We have mapped 36 SARs in the human type I interferon (IFN) gene complex on chromosome 9, band p21-22, to examine the overall structure of this gene complex. A total of 29 strong SARs and 7 weak SARs were mapped to the flanking regions of the different interferon genes. Twenty-two strong SARs mapped to the flanking regions of 13 interferon (IFNA) alpha genes; 2 strong SARs mapped to one interferon omega (IFNW) gene; 2 strong SARs mapped to one interferon alpha pseudogene (IFNAP); and 3 strong SARs mapped to two interferon omega pseudogenes (IFNWP). One weak SAR mapped to the flanking region of one IFNA gene, whereas 6 weak SARs flanked four IFN pseudogenes (P11, P12 P20, P23). The IFN SAR structure was comparable between the BV173 leukemia cell line and the U373 glioma cell line. Analysis of two glioma deletion breakpoint junctions, where breaks occur within and outside the IFN gene cluster, revealed an association with SARs. IFN SARs showed evidence for cooperativity among the SARs, while DNA sequence analysis revealed a series of clustered A-tracts within strong SARs. These data suggest that the IFN genes may be organized into a series of small (2-10 kb) DNA loop domains, with each loop containing a coding region flanked by SARs. In our model, the SAR enrichment and the clustering of A-tracts observed at the SARs within the IFN gene complex represent a higher level of chromatin organization, which may predispose this region to breakage.

Chromatin↗