Sub-nuclear fractionation. I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of ${}^3\text{H-actinomycin D}$, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].     

Rat liver chromatin. Distribution of histone and nonhistone proteins in eu- and heterochromatin.

Rat liver chromatin was sheared and separated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by glycerol gradient centrifugation. Chromosomal proteins associated with the chromatin fractions were analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. Histone composition did not vary qualitatively, but more histone protein was consistently found associated with the euchromatin fractions. Nonhistone protein banding patterns for these chromatin fractions exhibited marked heterogeneity, with a number of bands unique to either eu- or heterochromatin.     

Dauer der DNS-Replikation von Eu- und Heterochromatin bei Microtus agrestis

The duration of DNA replication of eu- and heterochromatin in kidney epithelial cell cultures of female Microtus agrestis was determined with combined H³-thymidine pulse labelling and cytophotometric determination of Feulgen DNA. The average duration of the total cell cycle was 23.3 hrs, with a G1 period of 14.6 hrs, S period of 5 hrs, G2 period of 2.7 hrs, and mitosis of 1 hr. The replication time of eu- and heterochromatin was determined by the frequency of the different labelling patterns after pulse labelling. The time sequence of the labelling patterns was ascertained by DNA measurements. During the S period, euchromatin replicates at first alone for 3 hrs (60% of the length of S) and 1 hr (19.3%) together with heterochromatin. During the last hour (20.7%), only heterochromatic regions replicate. The sex chromatin part of the one X chromosome starts synthesis 20 minutes (7.3% of S) before the remainder of the heterochromatic X material and ends 30 minutes (9.7% of S) prior to the termination of the S period. Replication of euchromatin takes about 80% of the duration of the total S period, whereas that of heterochromatin takes only 40%.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungs-Gemein-schaft.     

Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro.

Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of mercury in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of copper in both fractions. Fractionation of nuclei following in vivo challenge with copper showed enrichment of copper in heterochromatin, relative to euchromatin, while in vivo exposure to mercury resulted in a 20-fold accumulation of mercury in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward mercury and copper similar to that observed in the in vivo metal challenge experiments. The level of mercury which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 mug of Hg/mg of DNA) to that of in vivo euchromatin-bound mercury after 1 month of challenge with dietary metal. In contrast, copper showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both mercury and copper appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of copper.     

Rat liver chromatin. Fractionation into eu- and heterochromatin with localization of ribosomal genes.

Purified, normal rat liver chromatin was sheared under controlled conditions in a Virtis “60” homogenizer and then separated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by glycerol gradient centrifugation. The euchromatin portions of the glycerol gradients possessed greater than 90% of the in vitro template activity when estimated with Escherichia coli RNA polymerase and contained more than 90% of the nascent RNA formed in vivo. Inhibitor studies performed in vivo indicated that the leading edge of the heterochromatin portion of the glycerol gradients contains the genes coding for ribosomal RNA. Recentrifugation of putative eu- and heterochromatin fractions demonstrated that the isolation procedure produces fractions which are distinct and are characterized by little or no cross contamination. The data suggest that controlled shearing in conjunction with gradient centrifugation provides a means of fractionating with great fidelity the transcribable portions of the rat liver genome.     

URIDINE-5-H3 RADIOAUTOGRAPHY OF THE HUMAN SEX CHROMATIN BODY

To obtain an estimate of the rate of RNA synthesis by the heterochromatic sex chromatin body, human female fibroblasts were labeled with uridine-5-H³ and radioautographed. The number of grains over the sex chromatin body was compared with the number of grains over a comparable area of euchromatin. The ratio was 0.37. When corrected for the higher content of DNA per unit area in heterochromatin, the maximum rate of RNA synthesis by the DNA of the sex chromatin body was approximately 18% of the rate of RNA synthesis by a comparable amount of euchromatin DNA. The rate of RNA synthesis by the sex chromatin body did not increase significantly with partial despiralization of this chromatin at prophase.     

Nuclease accessibility of chromatin from a heterotic hybrid and from parental inbreds

The DNAase II, Mg²⁺ procedure was used to fractionate the seedling chromatin of a heterotic maize hybrid and its parental inbreds FRM017 and FRN28. The hybrid and the more vigorous maize inbred FRN28 have 14 and 11 percent soluble chromatin (euchromatin) respectively, while the less vigorous FRM017 contains 30 percent. The unfractionated chromatin of the hybrid contains less protein than either inbred. The RNA contents of the unfractionated chromatin of the hybrid and of FRN28 are similar and are one-half that of less vigorous FRM017. Hybrid euchromatin contains relatively more protein and RNA than DNA as well as higher proportions of protein and RNA than heterochromatin, unfractionated chromatin, or inbred euchromatin; this suggests a more “efficient” type of activity, as reflected by the low amount of euchromatin and a high proportion of RNA and chromosomal proteins.     

Persistent and heritable structural damage induced in heterochromatic DNA from rat liver by N-nitrosodimethylamine

Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.     

A comparative study of the nonhistone proteins of rat liver euchromatin and heterochromatin

Rat liver was fractionated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by controlled shearing and glycerol gradient centrifugation. The histone and nonhistone proteins associated with each fraction were compared. No qualitative differences in histone content were observed, but heterochromatin contained 1.5 times more histone protein than did euchromatin. The nonhistone proteins of each chromatin fraction were fractionated on the basis of salt solubility into loosely bound (those extracted by 0.35 m NaCl), tightly bound (those extracted by 2.0 m NaCl), and residual nonhistone proteins (those not extracted by 2.0 m NaCl). Euchromatin contained 3.7 times more loosely bound nonhistone proteins than did heterochromatin, while the latter contained twice as much residual nonhistone protein. Euchromatin was devoid of tightly bound nonhistone protein, a component of heterochromatin. Electrophoretic analysis of these nonhistone protein fractions revealed marked heterogeneity, with a number of bands unique to either eu- or heterochromatin.     

The contradictory definitions of heterochromatin: transcription and silencing

Eukaryotic genomes are packaged in two general varieties of chromatin: gene-rich euchromatin and gene-poor heterochromatin. Each type of chromatin has been defined by the presence of distinct chromosomal proteins and posttranslational histone modifications. This review addresses recent findings that appear to blur the definitions of euchromatin and heterochromatin by pointing to the presence of typically heterochromatic modifications (including H3K9me) in euchromatin and typically euchromatic enzymes (including RNA polymerases) in heterochromatin. We discuss the implications of these new findings for the current definition of heterochromatin.     

Gadd45a is a heterochromatin relaxer that enhances iPS cell generation

Reprogramming of somatic cells to induced pluripotent stem cells rewrites the code of cell fate at the chromatin level. Yet, little is known about this process physically. Here, we describe a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and show significant heterochromatin loosening at the initial stage of reprogramming. We identify growth arrest and DNA damage‐inducible protein a (Gadd45a) as a chromatin relaxer in mouse embryonic fibroblasts, which also enhances somatic cell reprogramming efficiency. We show that residue glycine 39 (G39) in Gadd45a is essential for interacting with core histones, opening chromatin and enhancing reprogramming. We further demonstrate that Gadd45a destabilizes histone–DNA interactions and facilitates the binding of Yamanaka factors to their targets for activation. Our study provides a method to screen factors that impact on chromatin structure in live cells, and identifies Gadd45a as a chromatin relaxer.     

Sub-nuclear fractionation. II. Intranuclear compartmentation of transcription in vivo and in vitro

DNA-dependent RNA polymerase activities were measured in subnuclear fractions obtained from rat liver by the procedure described in the preceding paper [14]. Most of the total nuclear enzyme was recovered in a form bound to chromatin with only small amounts as free enzyme in the nucleoplasm. The multiple eukaryotic RNA polymerases were resolved according to the endogenous template to which they were bound and which they continue to transcribe in vitro. The A and B forms of the enzyme were distinguished from each other by their differential sensitivities to α-amanitin, exogenous native and denatured DNA, thermal denaturation at 45 °, Mg²⁺ and Mn² ions, high ionic strength and by the binding of ${}^{14}\text{C-methyl}\text{-γ-}\text{amanitin}$. RNA polymerase B (α-amanitin-sensitive) was exclusively recovered in the nucleoplasmic and euchromatin fractions. RNA polymerase A was recovered in the dispersed nucleolar as well as in heterochromatin. By assaying in the presence of α-amanitin subnuclear fractions that had been pre-incubated at 45 °C a third enzyme (form C) was located exclusively in heterochromatin fractions. Only the euchromatin associated RNA polymerase B was capable of initiating the synthesis of new RNA chains in vitro on endogenous template at low ionic strength. Raising the ionic strength abolished initiation but accelerated chain elongation by this form of enzyme.When nuclear RNA was labelled in vivo, newly made RNA turned over rapidly in the nucleoplasm but accumulated in the euchromatin + membrane fraction. RNA in the nucleolar fraction accumulated gradually after a lag period, whereas a significant amount of rapidly-labelled nuclear RNA was recovered in the heterochromatin fractions. The distribution of RNA labelled in vivo compared with that of RNA polymerase activities suggested that RNA synthesized in vivo is rapidly translocated from its site of synthesis to some other sites within the nucleus.     

Interaction of benzo[alpha]pyrene diol-epoxide with nuclei and isolated chromatin.

Chicken erythrocyte chromatin and nuclei were labeled with benzo[alpha]-pyrene (B[alpha]P) diol-epoxide (anti) and digested with micrococcal nuclease to mono- and dinucleosomes. Analysis of the distribution of the carcinogen showed that the internucleosomal region bound 3-4 times more carcinogen per unit DNA than did nucleosomes. The enhanced binding of the 'ultimate' carcinogen to the internucleosomal region was similar when isolated chromatin or nuclei were used for in vitro labeling. Furthermore, isolation of the histone core proteins, H2A, H2B, H3 and H4, revealed that only 15% of the carcinogen was associated with the histones and that the majority of the carcinogen was bound to chromosomal DNA. Fluorography of purified nucleosomal histones showed that the covalent association of the carcinogen was mainly with histones H3 and H2B.     

Critical electrolyte concentration of the heterochromatin and euchromatin of Triatoma infestans

The binding of toluidine blue molecules under Mg2+ competitive staining conditions was investigated in chromocentres and the euchromatin of single- and multi-chromocentred nuclei of Triatoma infestans Malpighian tubule cells. It was demonstrated that the chromocentre of single-chromocentred nuclei exhibited the largest critical electrolyte concentration (CEC) value (0.4 M), followed by the chromocentres of multi-chromocentred nuclei (0.3 M) and the euchromatin (0.2 M). The differences in CEC values were assumed to be due to differences in availability of free DNA phosphates and in packing states of the DNA-protein complexes of these chromatin types. Differences in chromatin supra-organization were evident for the chromocentral heterochromatin of single vs multi-chromocentred nuclei. This was also valid for the chromocentral heterochromatin in some multi-chromocentred nuclei, when one of the heterochromatic bodies was especially larger than the others.