Organization of the thyroid hormone receptor in the chromatin of C6 glial cells: Evidence that changes in receptor levels are not associated with changes in receptor distribution

The association of [¹²⁵I]T3-receptor complexes with C6 cell chromatin was analysed after a limited digestion with micrococcal nuclease (MN) or DNase I. Both nucleases solubilized up to 60–70% of receptor and 0.4 M KCl extracted 70%, of the non-digested receptor, thus showing that only a residual fraction of receptor is associated with the nuclear matrix. With DNase I the receptor was released 2–3-fold faster than the bulk of chromatin, whereas a preferential release of receptor over total chromatin was not observed with MN. The digestion of receptor with DNase I and MN occurred 14- and 6-fold faster, respectively, than the appearance of PCA-soluble chromatin. Preincubation for 48 h with 4 nM T3 of 2 mM butyrate significantly altered receptor levels but did not change sensitivity to the nucleases. These results suggest that the thyroid hormone receptor is associated with chromatin highly sensitive to nuclease digestion, and that changes in receptor number are not associated with changes in its distribution in chromatin.     

A phosphatase inhibitor enhances the DNase I sensitivity of active chromatin

Although it is well-known that active domains of chromatin have elevated DNase I sensitivity, it can be difficult to observe preferential sensitivity in many cell types. We show that the DNase I sensitivity of active chromatin is enhanced some 10-fold by treating nuclei with the phosphatase inhibitor p-(chloromercuri)benzenesulfonic acid (CMBS) whereas DNase I sensitivity in inactive domains is only 3-fold higher. We further show that CMBS-enhanced DNase I sensitivity is associated with at least two histone modifications. First, the negatively charged CMBS molecule becomes covalently attached to the thiol groups on histone H3. Second, histone H2A phosphorylation is significantly elevated in treated nuclei. The phosphorylation data along with other results point to the possibility that H2A phosphorylation plays a role in enhancing preferential DNase I sensitivity. Whatever the mechanism, CMBS treatment of nuclei followed by DNase I digestion provides a novel and reproducible assay for probing the chromatin structure of active domains.     

Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I

The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.     

The DNase I sensitive state of "active" globin gene chromatin resists trypsin treatments which disrupt chromatin higher order structure

Active genes in higher eukaryotes reside in chromosomal domains which are more sensitive to digestion by DNase I than the surrounding inactive chromatin. Although it is widely assumed that some modification of higher order structure is important to the preferential DNase I sensitivity of active chromatin, this has so far not been tested. Here we show that the structural distinction between DNase I sensitive and resistant chromatin is remarkably stable to digestion by trypsin. Chick embryonic red blood cell nuclei were subjected to increasing levels of trypsin digestion and then assayed in the following three ways: (1) by gel electrophoresis for histone cleavage, (2) by sedimentation and nuclease digestion for loss of higher order structure, and (3) by dot-blot hybridization to globin and ovalbumin probes for disappearance of preferential DNase I sensitivity. We have found that chromatin higher order structure is lost concomitantly with the cleavage of histones H1, H5, and H3. In contrast, the preferential sensitivity of the globin domain to DNase I persists until much higher concentrations of trypsin, and indeed is not completely abolished even by the highest levels of trypsin we have used. We therefore conclude that the structural distinction of active chromatin, recognized by DNase I, does not reside at the level of higher order structure.     

Comparison of non histone proteins selectively associated with nucleosomes with proteins released during limited DNase digestions.

Cultured mammary cells from GR mouse were used to analyse proteins associated with the mononucleosomes and released by a short micrococcal DNase treatment of nuclei. On metrizamide density gradients, mononucleosomes appear to be heterogeneous according to their content of associated non-histone proteins. Proteins associated with the denser fraction (1.22 - 1.24 g/ml) were analysed by two dimensional electrophoresis and compared to the proteins released by DNase I treatment. All the proteins associated with mononucleosomes were also released by DNase I treatment. It could then be assumed that these proteins are associated with the active part of the genome. Additional proteins were released by micrococcal DNase treatment of the nuclei. They could be involved in a higher order organization of chromatin.     

Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.     

DNase I sensitivity of immunoglobulin light chain genes in Abelson murine leukemia virus transformed pre-B cell lines.

We have used Abelson murine leukemia virus (A-MuLV) transformed pre-B cell lines to test the hypothesis that the rearrangement potential of a developing B-lymphocyte is dependent on an "opening" of the chromatin structure surrounding immunoglobulin (Ig) genes, thus allowing accessibility to an Ig gene recombinase. The chromatin structures surrounding heavy (H), kappa (kappa), and lambda (lambda) chain constant-region genes were assessed by DNase I sensitivity in A-MuLV transformed cell lines capable of H, kappa or lambda gene rearrangement. Our results indicate that DNase I-sensitive chromatin structures of these Ig constant-region genes correlate closely with the ability of the genes to undergo recombination. We also find that the chromatin structure of an Ig constant-region locus becomes DNase I sensitive before any DNA rearrangement events occur.     

Structural study of the porcine NA+/H+ exchanger NHE1 gene and its 5′-flanking region

The poly(ADP-ribosyl)ation system, associated with different nuclear fractions of rat testis, has been analyzed for both pADPR and pADPR acceptor proteins. The DNase I sensitive and resistant chromatin contain 35% and 40%, respectively, of the total pADPR synthesized in intact nuclei incubated with [³²P]NAD. Moreover, the residual 25% were estimated to be associated with the nuclear matrix.Three different classes of pADPR are present in the nuclei. The longest and branched ADPribose polymers modify proteins present in the DNase I resistant (2 M NaCl extractable) chromatin and in the nuclear matrix, whereas polymers of > 20 residues interact with the components of the DNase I sensitive chromatin and oligomers of 6 ADPribose residues are bound specifically to the acid-soluble chromosomal proteins, present in isolated nuclear matrix. The main pADPR acceptor protein in all the nuclear fractions is represented by the PARP itself (auto-modification reaction). The hetero-modification reaction occurs mostly on histone H1 and core histones, that have been found associated to DNase I sensitive and resistant chromatin, respectively. Moreover, an oligo(ADP-ribosyl)ation occurs on core histones tightly-bound to the matrix associated regions (MARs) of chromatin loops.     

Distribution of DNase I-sensitive sites in simian virus 40 nucleoprotein complexes from disrupted virus particles.

Nucleoprotein complexes (core particles) released from simian virus 40 (SV40) virions were compared with similar complexes (SV40 chromatin) extracted from nuclei of infected cells. Core particles were sensitive to cleavage by DNase I at about the same enzyme concentration required to cleave SV40 chromatin. DNase I preferentially cleaved SV40 chromatin adjacent to the viral origin of replication; however, cleavage of core particles occurred with much less selectivity. The difference between these nucleoproteins was not due to a structural alteration induced by the virion disruption procedure, since SV40 chromatin retained its pattern of DNase I-sensitive sites when subjected top this treatment. On the other hand, core particles did not acquire the nuclease-sensitive feature typical of SV40 chromatin when they were exposed to infected cell nuclei and the Triton X-100-EDTA extraction procedure. Hence, the nuclease-sensitive feature was lost or altered during the normal process of virion assembly and maturation.     

The Higher Structure of Chromatin in the LCR of the β-Globin Locus Changes during Development

The β-globin locus control region (LCR) is able to enhance the expression of all globin genes throughout the course of development. However, the chromatin structure of the LCR at the different developmental stages is not well defined. We report DNase I and micrococcal nuclease hypersensitivity, chromatin immunoprecipitation analyses for histones H2A, H2B, H3, and H4, and 3C (chromatin conformation capture) assays of the normal and mutant β-globin loci, which demonstrate that nucleosomes at the DNase I hypersensitive sites of the LCR could be either depleted or retained depending on the stages of development. Furthermore, MNase sensitivity and 3C assays suggest that the LCR chromatin is more open in embryonic erythroblasts than in definitive erythroblasts at the primary- and secondary-structure levels; however, the LCR chromatin is packaged more tightly in embryonic erythroblasts than in definitive erythroblasts at the tertiary chromatin level. Our study provides the first evidence that the occupancy of nucleosomes at a DNase I hypersensitive site is a developmental stage-related event and that embryonic and adult cells possess distinct chromatin structures of the LCR.     

Cell cycle variations in chromatin structure detected by DNase I

We have recently developed a reproducible method for the use of DNase I as a sensitive probe of chromatin structure (Prentice, D A & Gurley, L R, Biochim biophys acta 740 (1983) 134) [12] and have used this probe to investigate chromatin structure during the interphase of the cell cycle. Chinese hamster cells (line CHO) were synchronized by: (1) mitotic detachment, to obtain M-phase cells; (2) isoleucine deprivation, to obtain G1-phase cells; and (3) sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea, to obtain cells blocked at the start of S phase. The cells were released from the various blocking schemes and nuclei were isolated and digested with DNase I at various times. The digestion kinetics were monitored to detect possible changes in chromatin condensation through the cell cycle. The chromatin was much more accessible to DNase I in G1 phase than in S or G2 phase, with only small variations in structure detected in late G1 and very early S phase. From early S phase up to mitosis, the chromatin became increasingly condensed and inaccessible to DNase I action. These results support the concept of a chromatin condensation cycle during interphase as well as during mitosis.     

Accessibility of histone H4 gene of Physarum polycephalum to DNase I during the cell cycle

DNase I was used as a probe to detect conformational changes of the H4 histone gene of Physarum polycephalum during the cell cycle. The degradation of histone genes was followed by gel electrophoresis and hybridization with a probe for the H4 histone gene. It was found that even during mitosis when chromatin is condensed into chromosomes, the histone genes are preferentially degraded by DNase I. The histone genes retain a characteristic structure which is recognized by DNase I during all stages of the cell cycle and thus independently of the biosynthesis of histones.