Digestion Of Nuclei Research Articles

Discovering enhancers directly by activity

Discovering enhancers directly by activity

Genome‐Scale Mapping of DNase I Hypersensitivity

DNase I-seq is a global and high-resolution method that uses the nonspecific endonuclease DNase I to map chromatin accessibility. These accessible regions, designated as DNase I hypersensitive sites (DHSs), define the regulatory features, (e.g., promoters, enhancers, insulators, and locus control regions) of complex genomes. In this unit, methods are described for nuclei isolation, digestion of nuclei with limiting concentrations of DNase I, and the biochemical fractionation of DNase I hypersensitive sites in preparation for high-throughput sequencing. DNase I-seq is an unbiased and robust method that is not predicated on an a priori understanding of regulatory patterns or chromatin features.

Epigenome characterization at single base-pair resolution

We have combined standard micrococcal nuclease (MNase) digestion of nuclei with a modified protocol for constructing paired-end DNA sequencing libraries to map both nucleosomes and subnucleosome-sized particles at single base-pair resolution throughout the budding yeast genome. We found that partially unwrapped nucleosomes and subnucleosome-sized particles can occupy the same position within a cell population, suggesting dynamic behavior. By varying the time of MNase digestion, we have been able to observe changes that reflect differential sensitivity of particles, including the eviction of nucleosomes. To characterize DNA-binding features of transcription factors, we plotted the length of each fragment versus its position in the genome, which defined the minimal protected region of each factor. This process led to the precise mapping of protected and exposed regions at and around binding sites, and also determination of the degree to which they are flanked by phased nucleosomes and subnucleosome-sized particles. Our protocol and mapping method provide a general strategy for epigenome characterization, including nucleosome phasing and dynamics, ATP-dependent nucleosome remodelers, and transcription factors, from a single-sequenced sample.

Dexamethasone affected rabbit nucleus pulposus cell proliferation and expression of proliferating cell nuclear antigen

Objective To study the proliferative effects of Dexamethasone on rabbit nucleus pulposus cell, and the expression of proliferation cell nuclear antigen ( PCNA ) of nucleus pulposus cells.Methods The intervertebral disk was extracted by sterile process, after digestion of nucleus pulposus the cells were cultured in DMEM medium. Then 1-100 nmol/L of Dexamethasone was added into cultured nucleus pulposus cells. The expression of PCNA mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR) ;the expression of PCNA protein was detected by Western blotting. Results RT-PCR and Western blot results showed that 1-100 nmol/L of Dexamethasone has up-regulated the expression of PCNA,it has 0. 42 folder and 1.37 folder than control group. Conclusion Dexamethasone could promote nucleus pulposus cell proliferation and up-regulate the expression of PCNA. Key words: Dexamethasone; PCNA; nucleus pulposus cell; tissue engineering

Effects of dexamethasone on rabbit nucleus pulposus cell proliferation and expression of Aggrecan

Objective To study the proliferative effects of dexamethasone on rabbit nucleus pulpo-sus cell, and the expression of Aggreean in nucleus pulposus cells. Methods The intervertebral disk was extracted by sterile process, and after digestion of nucleus pulposus the cells were cultured in DMEM. 1-10 000 nmol/L of dexamethasone was added into cultured nucleus pulposus colls. The proliferative effects of dexamethasone on nucleus pulposus cells were measured by MTT assay, and the expression of Aggrecan mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Results MTT revealed that dexamethasone had the proliferative effects on cultured nucleus pulposus cells, and the optimal treated time was 48 h, the optimam concentration was 100 nmol/L. The RT-PCR results showed that dexamethasone could up-regulate the expression of Aggrecan mRNA as compared with control group. Conclusion Dexam-ethasone could promote nucleus pulposus cell proliferation and up-regulate the expression of Aggrecan. Key words: Dexamethasone; Aggrecan; Nucleus pulposus cell ; Tissue engineering

Chromatin condensation and differential sensitivity of mammalian and insect cells to DNA strand breaks induced by bleomycin

Bleomycin (BLM) induces DNA damage in living cells. In this report we analyzed the role of chromatin compactness in the differential response of mosquito (ATC-15) and mammalian (CHO) cells to DNA strand breaks induced by BLM. We used cells unexposed and exposed to sodium butyrate (NaB), which induces chromatin decondensation. By nucleoid sedimentation assay and digestions of nuclei with DNAse I, untreated mosquito cells (no BLM; no NaB) were shown to have more chromatin condensation than untreated CHO cells. By alkaline unwinding ATC-15 cells treated with NaB showed more BLM-induced DNA strand breaks than NaB-untreated CHO cells. The time-course of BLM-induced DNA damage to nuclear DNA was similar for NaB-untreated mammalian and insect cells, but with mosquito cells showing less DNA strand breaks, both at physiological temperatures and at 4 °C. However, when DNA repair was inhibited by low temperatures and chromatin was decondensed by NaB treatments, differences in BLM-induced DNA damage between these cells lines were no longer observed. In both cell lines, NaB did not affect BLM action on cell growth and viability. On the other hand, the low sensitivity of ATC-15 cells to BLM was reflected in their better growth efficiency. These cells exhibited a satisfactory growth at BLM doses that produced a permanent arrest of growth in CHO cells. The data suggest that mosquito cells might have linker DNAs shorter than those of mammalian cells, which would result in the observed both greater chromatin condensation and greater resistance to DNA damage induced by BLM as compared to CHO cells.

Ca2+-binding proteins in nuclei.

Nuclei isolated from skeletal muscle of 15-day-old chick embryos, adult chickens, rabbits and from rat liver contain on the average 8-18 nmol Ca2+/mg protein. Digestion of nuclei with DNAase I and RNAase at 37 degrees C for 8--12 h reduced the Ca2+ binding by more than 90%. After nuclease treatment, Ca2+-binding proteins were identified in the nonhistone chromosomal protein fractions and in the insoluble residue by equilibrium dialysis and centrifuge transport, in media of 0.1 M KCl and 1 mM MgCl2. The interaction of Ca2+-binding proteins with chromatin may be of importance in the regulation of the gene expression in response to changes in cytoplasmic and nucleoplasmic free-Ca2+ concentration.

Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococcal nuclease.

Nuclear matrix attachment regions (MARs) are thought to influence the expression of flanking genes. In this study, we investigated the activation of genes by tobacco MARs that had previously been identified in the 5' region of the basic class I chitinase gene, CHNS0. In transgenic tobacco cells, a construct consisted of the 35S promoter of cauliflower mosaic virus (CaMV) fused to a beta-glucuronidase gene (uidA) with 5' MAR elements was expressed at a 10-fold higher level than a similar construct without MAR sequences. However, expression of a similar construct with 3' MARs and of a construct with a truncated (-46) 35S minimal promoter and uidA with 5' MARs was not similarly enhanced, suggesting that MARs might act by increasing the activity of downstream enhancers. Deletion analysis of the MAR sequences revealed that the function of the MARs that increased the expression of the transgene was redundant. Moreover, assays of the transient expression of transgenes suggested that MAR elements might be involved in the structure and organization of chromatin. To examine the influence of MARs on chromatin structure, we investigated the effects of micrococcal nuclease (MNase) on the DNA in the reporter gene around the MARs. Analysis of the time-course of digestion of nuclei with MNase revealed that the 35S promoter region with 5' MARs was much more sensitive to MNase than the same region without MARs, suggesting that MARs might mediate the opening of chromatin in the region of a downstream promoter, with consequent enhancement of transcription.

A chromatin-associated protein from pea seeds preferentially binds histones H3 and H4.

Pisum sativum p16 is a protein present in the chromatin of ungerminated embryonic axes. The purification of p16 and the isolation of a cDNA clone of psp54, the gene encoding its precursor have been recently reported [Castillo, J., Rodrigo, M. I., Márquez, J. A., Zúñiga, A and Franco, L. (2000) Eur. J. Biochem.267, 2156-2165]. In the present paper, we present data showing that p16 is a nuclear protein. First, after subcellular fractionation, p16 was clearly found in nuclei, although the protein is also present in other organelles. Immunocytochemical methods also confirm the above results. p16 seems to be firmly anchored to chromatin, as only extensive DNase I digestion of nuclei allows its release. Far Western and pull-down experiments demonstrate a strong in vitro interaction between p16 and histones, especially H3 and H4, suggesting that p16 is tethered to chromatin through histones. Because the psp54 gene is specifically expressed during the late development of seed, the role of p16 might be related to the changes that occur in chromatin during the processes of seed maturation and germination.

Inhibition of Histone Deacetylases Alters Allelic Chromatin Conformation at the Imprinted U2af1-rs1 Locus in Mouse Embryonic Stem Cells

Most loci that are regulated by genomic imprinting have differentially methylated regions (DMRs). Previously, we showed that the DMRs of the mouse Snrpn and U2af1-rs1 genes have paternal allele-specific patterns of acetylation on histones H3 and H4. To investigate the maintenance of acetylation at these DMRs, we performed chromatin immunoprecipitation on trichostatin-A (TSA)-treated and control cells. In embryonic stem (ES) cells and fibroblasts, brief (6-h) TSA treatment induces global hyperacetylation of H3 and H4. In ES cells only, TSA led to a selective increase in maternal acetylation at U2af1-rs1, at lysine 5 of H4 and at lysine 14 of H3. TSA treatment of ES cells did not affect DNA methylation or expression of U2af1-rs1, but was sufficient to increase DNase I sensitivity along the maternal allele to a level comparable with that of the paternal allele. In fibroblasts, TSA did not alter U2af1-rs1 acetylation, and the parental alleles retained their differential DNase I sensitivity. At Snrpn, no changes in acetylation were observed in the TSA-treated cells. Our data suggest that the mechanisms regulating histone acetylation at DMRs are locus and developmental stage-specific and are distinct from those effecting global levels of acetylation. Furthermore, it seems that the allelic U2af1-rs1 acetylation determines DNase I sensitivity/chromatin conformation.

Chromatin remodelling of the cardiac beta-myosin heavy chain gene.

To investigate the role of chromatin structure in cardiac gene expression, we used the DNase I and micrococcal nuclease to probe the chromatin structure of the hamster cardiac beta-MyHC gene. Two cardiac-specific DNase I hypersensitive sites (DHS) were identified, one of which was mapped to the -2.3 kb (beta-2.3 kb) region and the other to the proximal promoter region of the beta-MyHC gene. The two sites were readily detectable using nuclei from neonatal hamster heart; however, the proximal promoter site disappeared when adult hamster heart nuclei were used, and the -2.3 kb site decreased in intensity. We were able to demonstrate the gradual disappearance of this proximal promoter DHS by comparing heart nuclei isolated from animals at late-gestation and 1-day-old stages. Furthermore, injecting thyroid hormone caused the disappearance of the proximal promoter DHS in late gestational fetal ventricular nuclei. Digestion of nuclei from various tissues by micrococcal nuclease revealed that the beta-MyHC gene proximal promoter exists in an array of three specifically-positioned nucleosomes only in fetal heart chromatin. The beta-MyHC gene proximal promoter is DNase I hypersensitive within one of the nucleosomal particles. Our data suggest that chromatin structure may participate actively in cardiac gene expression.

An intron 1 regulatory region from the murine adenosine deaminase gene can activate heterologous promoters for ubiquitous expression in transgenic mice.

Ubiquitously expressed genes contain regulatory features which allow expression in virtually all cell types. In an effort to understand the molecular basis for this regulatory feature, the chromatin structure of the murine adenosine deaminase gene was examined by DNase I digestion in nuclei of several tissues. The promoter contained a strong hypersensitive site in all tissues examined, including those with very high and very low levels of ADA expression. Transgenic mouse studies revealed that a 3.3 kb EcoRI (3.3EE) fragment from intron I was required to generate a strong promoter DNase I hypersensitive site, and to produce ubiquitous expression. The 3.3EE fragment also contained a thymic enhancer activity which mapped to sequences conserved with the human ADA gene T-lymphocyte enhancer. Mutational analysis indicated that ubiquitous expression was not dependent on the presence of a functional thymic enhancer. Both the thymic enhancer and the ubiquitous activator within the 3.3EE fragment functioned with heterologous promoters in transgenic mice.

Androgen treatment protects mouse liver chromatin from cleavage by endogenous nucleases during aging. Androgen and nuclease activity.

We have examined the endogenous nuclease activity of the liver of intact, castrated and testosterone-treated mice of different ages. Both Mg(2+)- and Ca(2+)-dependent endogenous nuclease activities decline in old age. Withdrawal of the hormone increases nuclease activity in the immature and young. However, testosterone administration prevents the digestion of nuclei to different extents in all ages. These findings suggest a possible protective role of testosterone in the cleavage of liver chromatin by endogenous nucleases during the aging of mice.

Progressive Increases in the Methylation Status and Heterochromatinization of the myoD CpG Island during Oncogenic Transformation

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

Epigenetic mechanisms underlying the imprinting of the mouse H19 gene.

The expression of the H19 gene is governed by parental imprinting in mammals. H19, an unusual gene encoding an RNA with no known function, is exclusively expressed from the maternal chromosome. In mouse, it lies 90 kb downstream from the Igf2 gene, which encodes a fetal-specific growth factor, insulin-like growth factor II, and is expressed primarily from the paternally inherited chromosome. In this report we have utilized interspecific hybrid mice to identify male-specific DNA methylation of a 7- to 9-kb domain surrounding the H19 gene and its promoter. This allele-specific methylation could function as a mark to suppress transcription of the H19 paternal allele. Consistent with this proposal, the H19 promoter displayed an open chromatin conformation only on the relatively unmethylated active maternal allele. In contrast, a cell type-specific enhancer that lies outside the methylation domain is hypersensitive to restriction enzyme digestion in nuclei on both maternal and paternal chromosomes. That the allele-specific methylation domain, coupled to the two H19 enhancers, contains all the information necessary for its imprinting was tested by examining two transgenic lines containing an internally deleted H19 transgene. Both displayed paternal-specific methylation of the transgene and maternal-specific expression. Although neither line has been tested in an inbred genetic background, and therefore the action of complex modifiers cannot be formally excluded, the result suggests that the sequences necessary for the imprinting of H19 have been identified.

Dissection of the mouse N-ras gene upstream regulatory sequences and identification of the promoter and a negative regulatory element.

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

Dissection of the Mouse N-ras Gene Upstream Regulatory Sequences and Identification of the Promoter and a Negative Regulatory Element

The 5' flanking region of the mouse N-ras gene was investigated to determine the elements governing transcriptional activity of the gene. The promoter did not contain typical TATA or CCAAT boxes, and according to primer extension and RNase protection analyses, transcription started at several sites. These assays also confirmed the short nucleotide distance interposed between the N-ras transcription unit and the previously described upstream unr gene. Chromatin studies performed by digestion of nuclei with DNase I revealed the presence of four hypersensitive sites: a, b, c, and d. Deletion mutagenesis of the 5' flanking region revealed sequences responsible for both promotion and inhibition of transcription. These sequences resided within 230 bp upstream of the transcription initiation site. Hypersensitive site b colocalized with the 76-bp segment with promoter activity. The negative regulatory element at position -180 colocalized with hypersensitive site a, was active on the N-ras promoter in stable as well as transient assays, and down-regulated the heterologous herpes simplex virus thymidine kinase promoter. Footprint analysis and in vivo transfection-competition experiments indicated that a trans-acting factor is responsible for the negative effect on transcription. The interaction between the cis-acting negative regulatory element and the promoter region may play a role in the tissue- and developmental-stage-specific patterns of expression of the N-ras gene.

Conformation and expression of the albumin gene of young and old rats

The conformation and expression of the albumin gene in the liver of young (21-) and old (85-week) rats were studied. Digestion of nuclei with MNase shows no differences in the nucleosomal organization in the coding region of the gene in the two ages. The gene has a DNase I hypersensitive site which is distinctly less sensitive in old rats. Its 5'-CCGG-3' sequences are more methylated in the old in which its rate of transcription is also lower.

Intranuclear location of the adenovirus type 5 E1B 55-kilodalton protein

The intracellular location of the adenovirus type 5 E1B 55-kilodalton (kDa) protein, particularly the question of whether it is associated with nuclear pore complexes, was examined. Fractionation of adenovirus type 5-infected HeLa cell nuclei by an established procedure (N. Dwyer and G. Blobel, J. Cell. Biol. 70:581-591, 1976) yielded one population of E1B 55-kDa protein molecules released by digestion of nuclei with RNase A and a second population recovered in the pore complex-lamina fraction. Free and E1B 55-kDa protein-bound forms of the E4 34-kDa protein (P. Sarnow, C. A. Sullivan, and A. J. Levine, Virology 120:387-394, 1982) were largely recovered in the pore complex-lamina fraction. Nevertheless, the association of E1B 55-kDa protein molecules with this nuclear envelope fraction did not depend on interaction of the E1B 55-kDa protein with the E4 34-kDa protein. Comparison of the immunofluorescence patterns observed with antibodies recognizing the E1B 55-kDa protein or cellular pore complex proteins and of the behavior of these viral and cellular proteins during in situ fractionation suggests that the E1B 55-kDa protein does not become intimately or stably associated with pore complexes in adenovirus-infected cells.