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Nuclear thyroid hormone receptor binding to chromatin subunits: Implications from digestion studies with micrococcal nuclease
Molecular and CellularEndocrinology, 10 (1918) 217 -292 0 Elsevier/North-Holland Scientific ~blishers, Ltd.
NUCLEARTHYROIDHORMONERECEPTORBINDINGTOCHROMATIN SUBUNITS:IMPLICATIONSFROMDIGESTIONSTUDIESWITH MICROCOCCALNUCLEASE Robert S. GARDNER * Department ofInterna! Medicine, Universityof Iowa, Iowa city, Iowa 52242, U.S.A. Received 28 September 1977;a~~pted
12 January 1978
Nuclear thyroid hormone receptors are putative regulatory proteins which copurify with chromatin components and remain associated with chromatin following hydrodynamic shear. Rat liver chromatin containing bound receptors labeled in vivo with rssI-labeled L-triiodothyronine (Ts) is cleaved by micrococcal nuclease at 2°C into a series of nucleosomes or chromatin subunits. After velocity sedimentation of these subunits in glycerol gradients, receptors remain associated with oligomeric subunits but not with monomers. In studies with isolated nuclei, thyroid hormone receptors are concentrated in a particulate ahromatin fraction prepared by low temperature digestion of rat liver nuclei with micrococcal nuclease and isolation of the unsheared ‘core’ chromatin, On brief incubation at 37”C, core chromatin is digested by residual nuclease, yielding monom~ subunits. Thyroid hormone receptors dissociate from nucleoprote~ binding sites during this ~cubation. These observations with two different chromatin preparations suggest that receptor binding sites are preserved in nucleasegenerated chromatin oliiomers but not in monomer subunits. It is proposed that receptors bind to DNA segments linking monomer subunits, rather than to the subunits. Keywords: nuclear thyroid hormone receptor; rat liver chromatin; micrococcal nuclease; nucleosomes.
Nuclear thyroid hormone receptors are DNA-binding (MacLeod and Baxter, 1976) nor&stone proteins (Thomopoulos et al., 1974) with a high affkity for Ta and a lesser affmity for L-thyroxine (Oppe~eimer et al., 1972,1974; Samuels and Tsai, 1973). The high afftity receptor accounts for at least 90% of T3 binding components present in a soluble extract of rat liver nuclei (Latham et al., 1976). These receptors remain tightly bound to chromatin during extensive purification procedures (Surks et al., 1973; Charles et al., 1975; Gardner, 1975; Spindier et al., 1975) including hydrodynamic shear (Charles et al., 1975; Gardner, 1975). There is preliminary evidence supporting their role in mediating at least some of the cellular
* Present address: Department 92093, U.S.A.
of Psychiatry
MO03, University of California, La Jolla, CaJif.
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effects of thyroid hormones in mammalian organisms (Samuels and Tsai, 1973; Samuels et al., 1973; Oppenheimer et al., 1974; Koerner et al., 1975; Samuels et al., 1976). When rat liver nuclei labeled in vivo with [ ‘*‘I]Ta are incubated with excess micrococcal nuclease at 2”C, most of the chromatin is sheared into soluble particles, but only 17% of the [1251]Ta is released from nuclei with the soluble chromatin (Gardner, 1975). Core chromatin, representing 2% of the nuclear DNA, is prepared from the residual nuclear fraction remaining after nuclease treatment. Thyroid hormone receptors are strikingly associated with core chromatin. Almost 50% of the radioactive Ta which purifies with chromatin is isolated in the core chromatin fraction (Gardner, 1975). This represents a 14-fold enrichment of T3 receptor bound/mg DNA in core chromatin compared to total nuclear chromatin. A series of chromatin particles containing multiples of 200 base pairs of DNA is generated when rat liver nuclei are digested with micrococcal nuclease at 2°C or briefly at 37°C (No11 and Kornberg, 1977). The digestion products are resolvable into monomer, dimer and higher oligomer particle fractions on isokinetic sucrose gradient centrifugation (Finch et al., 1975). Monomer subunits, also known as mononucleosomes, consist of 200 base pairs of DNA and a histone octomer of histones H2A, H2B, H3 and H4 (Kornberg, 1974, 1975). Most, if not all, genomic sequences appear to be involved in the basic subunit structure of rat liver chromatin (Lacy and Axel, 1975). More prolonged digestion of chromatin with micrococcal nuclease at 37°C results in degradation of chromatin fragments from the ends, with release of histone Hl (No11 and Kornberg, 1977). In this paper, it is reported that thyroid hormone receptors are associated with nucleasegenerated oligonucleosomes prepared from chromatin. They appear not to be associated with mononucleosomes. This is compatible with the hypothesis that thyroid hormone receptors are associated with intemucleosomal regions of the chromosome, as suggested by Yoshizato et al. (1977). This is based on their observations that thyroid hormone receptors are localized to chromatin DNA regions sensitive to DNase. Our studies are performed using in vivo labeling of receptors with radioactive Ts to avoid potential artifacts associated with in vitro labeling techniques (Tata, 1975).
MATERIALS
AND METHODS
T3 and methemoglobin were obtained from Sigma (St. Louis, MO.). Metrizamide was obtained from Nyegaard and Co. A/S (Oslo, Norway). [12sI]Ta (spec. act. 70 and 500 mCi/mg) was purchased from Abbott Labs (Chicago, Ill.). [3H]RNA markers were obtained from Schwartz-Mann (Orangeburg, N.Y.). Micrococcal nuclease (spec. act. 16 000 units/mg) was obtained from Worthington Corp. (Freehold, N.J.). Male albino Sprague-Dawley rats, thyroidectomized at 100 g, were obtained
Thyroid hormorle receptor binding to chromatin
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from Hormone Assay Labs (Chicago, Ill.), and the animals were fed a low-iodine diet. After one week, residual thyroid was ablated with 0.5 mCi [1251]iodide. They were not used until at least four weeks post-radioiodine ablation. Preparation of rat liver chromatin
Nuclei prelabeled in vivo with 1 nmol [ 1251]T3 (spec. act. 70 mCi/mg) were purified from hypothyroid rat livers in 2.2 M sucrose Buffer A (100 mM NaCl, 10 mM Tricine, 8 mM MgCl*, 2 mM CaC12, pH 7.8), as previously described (Gardner, 1975). The nuclear pellet was homogenized in 0.25 M sucrose Buffer A containing 0.5% Triton X-100 and centrifuged at low speed. The purified nuclei contained 1.4 mg [1251]T3 bound/mg DNA. At least 90% of the radioactive T, represents T3 specifically bound to the nuclear receptor, based on studies of nuclear binding after injection of [ 1251]Ta with and without 10 nmol unlabeled Ta. Chromatin was purified from the Triton-washed nuclei, as previously described (Gardner, 1975). Z+-eparationof core chromutin and soluble core chromatin
Nuclei were purified from 30 g hypothyroid rat livers prelabeled in vivo with [12SI]Ta (spec. act. 70 mCi/mg), resuspended to an A260 of 10 and digested with 300 units/ml micrococcal nuclease with gentle stirring for 30 min at 2”C, under conditions as previously described (Gardner, 1975). After termination of the reaction with 4 mM EDTA, pH 7.5 (final concentration), the reaction mixture was centrifuged at 4000g for 10 min, and the residual chromatin pellet homogenized in 30 ml of 10 mM Tris, 5 mM EDTA, pH 7.5 and centrifuged at 8000g for 10 min. The chromatin pellet was then purified by repeating this procedure thrice, with 1 mM EDTA substituting for 5 mM EDTA in the last two washings. The final core chromatin pellet was resuspended by gentle homogenization to an AZe.,,of 15 in 10 mM Tris, pH 7.5, 1 mM CaC12and incubated for 60 s at 37’C. The reaction was then terminated with 4 mM EDTA (final concentration), and the particulate components removed by centrifugation at 18 000 g for 10 min. The chromatin component of the supematant fraction is called soluble core chromatin. Beparation of / ‘2SZJT3-labeled receptors
Rat liver nuclei labeled in vivo with [1251]Ta (spec. act., 500 mCi/mg) were isolated as previously described (Gardner, 1975), except that 3 mM MgC12was substituted for Buffer A in all of the sucrose solutions. The nuclei were further purified by centrifuging them in 0.25 M sucrose-3 mM MgC12solutions containing 0.5% Triton X-100. Purified nuclei were resuspended in 0.4 M KCl, 10 mM Tris, pH 8.5 and incubated for 20 min at 2’C. The nuclei were then centrifuged at 10 OOOgfor 10 min. The supernatant fraction, containing 75-85% of the nuclear-bound [12’I]T3 was collected and the T3 radioactivity specifically associated with the thyroid hormone receptors separated from unbound T3 by purification on a Biogel P-10 column and collection of the void volume fractions (Thomopoulos et al.,
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1974). The pooled fractions generally contained 100 000 dpm/ml represents 90% of the radioactivity applied to the column.
K.S. Gardner
[r*‘I]Ta
which
Isokinetic sucrose gradient centrifugation One-ml samples of [ 1251]TJabeled soluble core chromatin or partially purified [1251]Ts-labeled thyroid hormone receptors were layered onto 36.3 ml isokinetic sucrose gradients formed with an isokinetic gradient maker (Molecular Instruments Co., Evanston, Ill.) with C’, = 5%, Cr = 28.8% and V, = 33 ml (Finch et al., 1975), where PM = mixing volume of the flask, C, = concentration of sucrose at the top of the gradient, and Cr = concentration of sucrose at the bottom of the gradient (Nell, 1967). The sucrose gradient solutions also contain 0.1 mM EDTA, pH 7.5. The samples were centrifuged for 20 h at 27 000 rev./min in an SW 27 rotor, and 0.8 ml fractions were collected after passage through a turbulence-free flow cell (Molecular Instruments Co.) for measurement of the AZ60 using a Beckman model 24 spectrophotometer. Isopycnic centrifugation in metrizamide-50% D20 gradients Preformed linear gradients of metrizamide in 50% D20 were prepared using five solutions of metrizamide (containing 50% D20) ranging in concentration between 20 and 40% (w/w) (Htittermann and Guntermann, 1975). The 50% D20 is substituted for 99.5% D20 in the layering solutions to lower the density of the gradient. The metrizamide gradient solutions also contain 10 mM Tris, pH 7.5, 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride and 0.1 mM dithioerythritol. 0.5 ml samples of [rz51]Ta-labeled sonicated core chromatin or [1251]TJabeled sonicated nuclei were layered onto 3.5-ml gradients and centrifuged at 58 000 rev./ min for 16 to 20 h in an SW 60 rotor. 0.2 ml fractions were collected and DNA determined by a modified diphenylamine reaction after addition of 1 mg carrier bovine serum albumin to each fraction and extensive washing of the precipitates with 0.4 N perchloric acid (Burton, 1956). Gel electrophoresis of DNA fragments 1.2% agarose gels were prepared as previously described except that agarose was substituted for acrylamide (Peacock and Dingman, 1967). Chromatin samples were suspended in 1% sodium dodecyl sulfate, 1 M NaCl, 10 mM Tris and 2.5 mM EDTA, pH 8.0, and extracted with an equal volume of chloroform : isoamyl alcohol (25 : 1). The aqueous phase was precipitated with 3 volumes of ethanol and then dissolved in 0.1 ml electrophoresis buffer containing 15% sucrose and 0.1% bromphenol blue. Up to 50 $ samples were applied to agarose gels and electrophoresed at 175 V for 1 h at 20°C in a buffer system as described by Peacock and Dingman (1967). After electrophoresis, the gels were stained for 30 min with 0.4 &ml ethidium bromide in electrophoresis buffer and photographed with an orange filter under ultraviolet light.
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RESULTS
Purified hypothyroid rat liver chromatin labeled in vivo with [‘asI]T, was incubated at 2’C with micrococcal nuclease in the presence of 0.025 mM Ca2”. The reaction was stopped with excess EDTA, and the remaining unsheared chromatin pelleted. As i~ustrated in fig. 1, there was a progressive solub~zation of chromate during the 120.min incubation. After 30 min, only 35% of the A260 remained in the c~omati~ pellet, and after 60 min, this decreased to 5%. Virtually ail of the A 260 eventually became nonpelletable. After 60 min of incubation, only 10% of the AzeO was acid-soluble, ~dicating the extent of degradation of DNA under these conditions. Associated with the appearance of chromatin AzGO in the soluble fraction, a simiiar percentage of [ ‘251]T3 was solubilized. At 30min, when 65% of the AZ60 was soluble, 67% of the radioactive Ts was also soluble. However, later, when virtually all of the chromatin A 260 had been solubilized, there was a residual 1.5% of T3 radioactivity bound to the pellet. The nucle~e-digested chro-
TIME
fmin)
1, Kinetics of digestion of chromatin with micrococcal nuclease. Rat liver chromatin was resuspended to an AZ60 of 10 in 0.025 mM CaClz and 5 mM sodium phosphate buffer, pH 7.2, and incubated at 2°C with 300 units/ml micrococcal n&ease. At various times, aliquots were withdrawn and the reaction was stopped with 4 mM EDTA (final concentration). The aliquots were centrifuged at 28 OOOg for 25 min, and the pellet resuspended in incubation buffer containing 1 mM EDTA and sonicated for 30 s at 30 W. (Sonifier W185 with microtip). The A260 and radioactivity was determined on each supernataut and sonicated pellet fractions. -, d2ee; ---, cpm+ Fig,
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matin had a protein : DNA ratio of 1.8 and a histone : DNA ratio of 1.l . It had the usual complement of histones and non-histone proteins, as determined by SDSacrylamide gel electrophoresis (data not shown). Agarose-gel electrophoresis of DNA extracted from micrococcal nuclease digest of chromatin is shown in fig. 2. DNA bands corresponding to monomer, dimer and higher oligomer chromatin subunits were present and were comparable to DNA bands obtained from micrococcal nuclease digests of nuclei. Glycerol gradient ultracentrifugation of soluble chromatin prepared from 30-min and 60-min nuclease
123456 Fig. 2. Electrophoretic comparison of DNA fractions prepared from nuclei and chromatin DNA was extracted from nuclei and chromatin and electrophoresed at 175 V for 1 h through 1.2% agarose slab gels. Gels were stained with 0.4 ng/ml ethidium bromide and photographed under ultraviolet light using an orange filter. Wells l-4: Nuclei digested with five units of micrococcal nuclease/4aec for 30, 60, 90 and 120 s, respectively, at 20°C. Wells 5-6: Chromatin digested with five units of micrococcal nuclease/Aaeo for 45 and 90 s, respectively, at 20°C.
Thyroid hormone receptor binding to chromatin
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Fig. 3. Glycerol gradient centrifugation of 30-min nuciease-digested chromatiu. Rat liver chromatin was digested with micrococeal nuclease for 30 mm as described in the Iegeud of fii. 1 and 1.5 ml of the soluble chromatin was layered onto lo-30% w/w glycerol gradients containing 10 mM Tris, pH 7.5, and 0.1 mM dithioerythritoi and centrifuged for 16 h at 27 000 rev./min in an SW 27 rotor. 1.2 ml fractions were collected and A2eo and radioactivity determined. -,A260;---,cpm.
digests verified that [*251fT3 remained bound to chromate particles fo~o~g nuclease digestion (figs. 3 and 4). In each gradient, the A260 profile consisted of a slowsedimenting peak (fr. no. 10-12) identified as the 13 S monomer particle, using an 18 S ribosomal RNA marker to calibrate the gradient, and more rapidly
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gradient centrifugation of 6O-min nuclease-digested chromatin. Rat liver chromatin was digested with micrococcal nuclease for 60 mm and 1.5 ml samples applied to glycerol gradients as described in the legend of fii. 3. -,&eo; - - -, cpm.
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sedimenting components (fr. no. 15-30) representing dimer, trimer and higher oligomeric chromatin subunits. T3 radioactivity peaked with the oligomeric subunits, with no evidence of a radioactive peak coinciding with the slow-sedimenting Aa6e peak. This 11 S particle increased relative to the other AZ60 components as the nuclease digestion was prolonged from 30 to 60 mm. This is consistent with generation of monomer subunits from oligomers as the latter are cleaved by the nudease. The resolution of the gradients does not allow for a clear delineation between the indi~du~ ofigomeric subunits and the radioactive Ta associated with the subunits. Therefore, it is not possible to calculate the [f2sIjTa/A260 ratio as a function of the number of subunits in a chromatin oligomer, although this ratio appears to be reduced in the higher oligomers. Studies with core chromatin
Core chromatin containing bound [ ’ 25I] Ta-labeled thyroid hormone receptors was prepared from unsheared particulate components remaining after a 2’C microDENSITY (q/cm31 1””
BOTTOM
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Kg. 5. Isopycnic gradient centrifugation of sonicated [ t2sI]T~-labeled core chromatin in metrizamide-50% D20. Core chromatin containing bound Ts-labeled receptors was resuspended in S mM EDTA, 1 mM Tris, pH 7.5, and sonicated for 100 s at 30 W, using IS-s pulses. The sonicate was centrifuged for 10 min and the supernatant applied to preformed metrizamide-I&O gradients, and centrifuged as described in Methods. 0.2 ml fractions were coilected and DNA and radioactivity determined. .A260; - --, cpm.
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DENSITY (g/cm3)
FRACTION
No
Fig. 6. Isopycnic gradient centrifugation of sonicated [ rss~]Ts-labeled nuclei in metrizamide50% D20. Nuclei containing bound Ta-labeled receptors were resuspended in 5 mM EDTA, 1 mM Tris, pH 7.5, and sonicated for 100 s at 30 W. After centrifugation at 8000 g for 10 min, the supernatant was applied to preformed metrizamide-D20 gradients, as described in Methods and 0.2 ml fractions were collected and DNA and radioactivity determined. -, DNA; - - -, cpm.
coccal nuclease digest of hypothyroid rat liver nuclei. It was further characterized by isopycnic centrifugation in metrizamide-DzO gradients. Core chromatin, after sonication, banded at a density of 1.2 g/cm in these gradients (fig. 5). In contrast, total rat liver nuclear chromatin prepared from sonicated nuclei banded at a density of 1.18 g/cm (fig. 6). A major fraction of the radiolabeled T3 peaked at approximately the same density as the chromatin DNA band in both gradients, suggesting that the receptors remained associated with both core and chromatin DNA components following sonication and gradient centrifugation in metrizamide. Although there was a second, smaller, radioactive T3 peak near the top of the core chromatin gradient, presumably representing unbound T3, most of the radioactivity appeared to be chromatinassociated. Core chromatin was also sheared by brief incubation at 37°C in the presence of Ca2+. Approximately 65% of the A260 and 50% of the [12’I]T3 was solubilized on incubation of T3-labeled core chromatin for 30-60 s. Calcium was required for this
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Fig. 7. Electrophoretic comparison of chromatin DNA fractions. The agarose gel of extracted DNA was run as described in fig. 2. Well 1: Core chromatin; Well 2: Soluble core chromatin; Well 3: Core chromatin digested with 5 units of micrococcal nucleasq’A2eo for 3 min at 37°C; Well 4: Rat liver chromatin purifed using the same methods as for core chromatin (minus the nudease step) and digested by endogenous nuclease for 3 mm at 37°C; Well 5: Rat liver chromatin digested with 5 units of micrococcal nucIease/Azeo for 3 mm at 37°C.
reaction, with excess EDTA markedly inhibiting the release of both Asee and radioactivity into the supernatant fraction (data not shown). The chromatin present in this soluble fraction was called soluble core chromatin. DNA extracted from core chromatin and soluble core chromatin was further characterized by gel electrophoresis. Two DNA bands were identified in soluble core chromatin, with an electrophoretic mobility corresponding to chromatin monomer and dimer subunits (fig. 7).Themonomer component was clearly the predominant band. In contrast,
Thyroid hormone receptor binding to chromatin
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Fig. 8. Isokinetic sucrose gradient centrifugation of [ 125IlT34abeled soluble ‘core chromatin. Soluble core chromatin was prepared from core chromatin as described in Methods, and centrifuged in isokinetic sucrose gradients. 0.8 ml fractions were collected and A26o and radioaco- - -0, cpm. tivity determined. -,A2,e;
DNA extracted from core chromatin contained no discrete bands on gel electrophoresis. This suggested that core chromatin is composed of chromatin multimers which are cleaved to mainly monomer subunits on incubation at 37°C. Added micrococcal nuclease was not required for this reaction. However, in the presence of additional nuclease, the monomer DNA band had a greater mobility on gel electrophoresis, suggesting that it has been degraded to a subunit of lower molecular size (fig. 7). Soluble core chromatin was further characterized by centrifugation in an isokinetic sucrose gradient. As illustrated in fig. 8, two A260 peaks were identifiable. Peak 1 represents low molecular weight acid-soluble oligonucleotides, while peak 2 is acid-insoluble chromatin, corresponding to the monomer subunit. This is based on an estimated sedimentation coefficient of 11 S using 18 S and 28 S ribosomal RNA markers to calibrate the gradient. When peak 2 fractions were pooled and the extracted DNA electrophoresed on agarose gels, the DNA migrated as a single band with a mobility virtually the same as the DNA from monomer subunits generated by low-temperature digestion of rat liver nuclei with micrococcal nuclease (fig. 9). This suggests that monomer subunits are generated when core chromatin is incubated briefly at 37’C in the presence of Ca’+. The [t2’I]T3 peak in this gradient, representing at least 75% of the applied radioactivity, was clearly separable from the monomer DNA A260 peak (fig. 8). There was no evidence of significant radioactivity in the region of the gradient containing chromatin subunits, except in the multimer fraction near the bottom of the gradient. Saltextracted and partially purified [ ’ 25I] T3-labeled thyroid hormone receptors centrifuged in a similar isokinetic sucrose gradient sedimented as a single radioactive component with the same sedimentation coefficient as the radioactivity present in the soluble core chromatin gradient (data not shown). This suggests that
R.S. Gardner
Fig. 9. Electrophoretic comparison of pooled peak 2 fractions from an isokinetic sucrose gradient of soluble core chromatin with micrococcal nuciease digests of nuclei. Agarose gels of extracted DNA were run as described in fig. 2. Wells 1-2: Nuclei digested with 35 units micrococcal nuclease/ilzeo for 10 and 20 mm, respectively, at 2°C. Well 3: Pooled core chromatin fractions 23-27 from peak 2 of fig. 7.
the major radioactive component in soluble core chromatin is bound to thyroid hormone receptors or other macromolecules with similar sedimentation coefficients .
DISCUSSION This paper describes investigations in which thyroid hormone receptor binding to chromatin structures is probed, using micrococcal nuclease to shear chromatin
Thyroid hormone receptor binding to chromatin
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into subunits or nucleosomes. Two different chromatin fractions labeled in vivo with [ 1251]T3 were employed to define receptor interactions with subunits under conditions where at least 90% of the nuclear radioactivity is specifically bound to the receptor. The first, purified hypothyroid rat liver chromatin, was digested with micrococcal nuclease at 2°C generating soluble chromatin particles which were subsequently purified. During the digestion, radioactive T3 was also solubilized. Purified chromatin used in these studies was prepared by lysis of nuclei in low ionic strength buffers. The u&eared chromatin preparation was then digested with micrococcal nuclease, which shears chromatin into a series of subunits which are resolvable on glycerol velocity gradients and gel electrophoresis of the extracted DNA. Liew and Chan (1976) prepared chromatin by conventional low ionic strength methods and found that the characteristic nucleosomal structure was preserved. Woodhead and Johns (1976) prepared chromatin in 0.14 M NaCl and sheared the chromatin in a blender, again with preservation of the subunit structure. In contrast, Noll et al. (1975) found that the nucleosomal structure was destroyed by mechanical shear of chromatin, and this has been confirmed by Axe1 (1975). It appears that under low ionic strength conditions in the absence of vigorous mechanical shear, the native structure of the chromatin is preserved, and this was confirmed in the present study, The second chromatin fraction used, known as core chromatin, was prepared from hypothyroid rat liver nuclei which have been digested with excess micrococcal nuclease at 2”C, and the residual unsheared chromatin enriched in thyroid hormone receptors was purified. Core chromatin was subsequently sheared to soluble chromatin particles by brief incubation at 37’C in the presence of Ca”, but without additional nuclease. Micrococcal nuclease bound to core chromatin from the prior digestion of nuclei with this nuclease is the probable explanation for the observed nuclease activity in core chromatin. This is consistent with a recent report that micrococcal nuclease copurifies with chromatin prepared from micrococcal nuclease digests of rat liver nuclei (Carter and Levinger, 1977). During nuclease shear of chromatin, 85% of the radioactive T; was solubilized. The residual 15% of the radioactivity was not associated with DNA, since it remained with the pellet after all of the chromatin DNA was solubilized. In contrast, when nuclei were incubated with micrococcal nuclease, most of the radioactive T3 remained with the nuclear pellet and copurified with the residual (core) chromatin. It is unclear whether core chromatin DNA sequences are also present in bulk chromatin. Clarification of this question will require the identification of specific DNA sequences in core chromatin. It is our working assumption that core chromatin is associated with a specific nuclear structure which is relatively protected from micrococcal nuclease in the intact nucleus, but becomes susceptible to this nuclease in isolated chromatin. The identity of this structure is unknown, although the nucleolus has been postulated to be the intranuclear location of core chromatin (Gardner, 1975). Nuclear thyroid hormone receptors bind tightly to both rat liver chromatin
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preparations. This is based on velocity sedimentation of hydrodynamically sheared total rat liver chromatin (Charles et al., 1975) and core chromatin (Gardner, 1975). Also, as shown in this paper, total nuclear chromatin as well as core chromatin both band with associated thyroid hormone receptors in metrizamide isopycnic gradients. The banding pattern observed with rat liver chromatin in metrizamide gradients is consistent with previous studies of the behavior of this chromatin in isopycnic gradients (Monahan and Hall, 1974). In their studies, the major chromatin components have a buoyant density of 1 .I 8 g/cm, the same as found in our studies. Core chromatin has a slightly higher density in these gradients, suggesting a slightly higher protein-to-DNA ratio. Following nuclease shear of rat liver chromatin, thyroid hormone receptors sediment with chromatin oligomers in glycerol velocity gradients. Of particular interest is the small percent of binding of Ta to monomer subunits in this preparation. It is unclear whether receptors bind to chromatin dimers as well as longer oligomers following nuclease cleavage. Better resolution of chromatin oligomers will require multiple gradient centrifugations to further purify the dimer and trimer subunits. Core chromatin is cleaved to predominantly monomer subunits on brief incubation at 37°C. Compared to monomer subunits prepared from 2°C nuclease digestion of nuclei which have been reported to consist of 198 * 6 base pairs of DNA, the monomer subunit prepared from core chromatin has similar mobility on agarose gels (Noll and Kornberg, 1977) suggesting that core chromatin monomers are similar in size to bulk monomer subunits. Digestion of chromatin with micrococcal nuclease at 2°C rather than at 37” generates subunits with minimal degradation from the ends, therefore making mononucleosomes a reference marker for 200 base-pair fragments (Noll and Kornberg, 1977). During the incubation, Ts is released as receptor-bound T3. This is further evidence that receptors are not bound to monomer subunits, although it is not clear whether the release of receptor is always linked with formation of monomer subunits. The binding of thyroid hormone receptors to oligomeric chromatin subunits but not to monomeric subunits would be consistent with receptor binding to linker regions between subunits. There is convincing evidence that DNA regions bridging monomer subunits are more sensitive to nuclease cleavage than monomer subunits (Shaw et al., 1976; Whitlock and Simpson, 1976; No11 and Kornberg, 1977). Thyroid hormone receptors may bind to these linker regions with receptor released during nuclease digestion of these regions. This is similar to the behavior of histone Hl which apparently is associated with these linker regions and is released during their digestion by micrococcal nuclease (Shaw et al., 1976; Whitlock and Simpson, 1976; No11 and Kornberg, 1977). Although receptor localization on linker regions is consistent with these studies, there are clearly other explanations. Receptors may bind to monomer subunit DNA sites that are digested when dimers are cleaved to monomers. Alternatively, they may bind to monomer DNA sites that undergo conformational changes during nuclease digestion of adjacent linker regions leading to release of receptor. Regardless of the mechanism, thyroid hormone receptor bind-
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ing sites appear to be preserved in nuclease-generated chromatin oligomers provided the oligomer consists of at least two (or possibly three) linked subunits. Further studies are planned to determine whether the minimum chromatin structure supporting receptor binding is the dimer or trimer subunit, and this has clear implications for the hypothesis discussed in this paper.
ACKNOWLEDGEMENTS The author wishes to thank Duane Rossman and Ellyn Murphy for their dedication in the performance of skillful technical assistance. I also wish to thank Dr. Vaughn Jackson for technical help with gel electrophoresis of DNA fragments. This work was supported in part by grants from the NIH and Veterans Administration.
REFERENCES Axel, R. (1975) Biochemistry 14,2921-2925. Burton, K. (1956) Biochem. J. 62, 315-323. Carter, Jr., C.W. and Levinger, L.F. (1977) Biochem. Biophys. Res. Commun. 74,955-960. Charles, M.A., Ryffel, G.U., Obinata, M., McCarthy, B.J. and Baxter, J.D. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 1787-1791. Finch, J.T., Nell, M. and Kornberg, R.D. (1975) Proc. Natl. Acad. Sci. U.S.A. 72,3320-3322. Gardner, R.S. (1975) Biochem. Biophys. Res. Commun. 67,625-633. Hiittermann, A. and Guntermann, U. (1975) Anal. Biochem. 64,360-366. Koerner, D., Schwartz, H.L., Surks, M.I., Oppenheimer, J.H. and Jorgensen, E.C. (1975) J. Biol. Chem. 250,6417-6423. Kornberg, R.D. (1974) Science 184,868-871. Kornberg, R.D. (1975) In: The Eucaryotic Chromosome, Eds.: W.J. Peacock and R.D. Brock (Australian National University Press, Canberra) pp. 245-253. Lacy, E. and Axel, R. (1975) Proc. Natl. Acad. Sci. U.S.A. 72,3978-3982. Latham, K.R., Ring, J.C. and Baxter, J.D. (1976) J. Biol. Chem. 251,7388-7397. Liew, CC. and Chan, P.K. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3458-3462. MacLeod, K.M. and Baxter, J.D. (1976) J. Biol. Chem. 251,7380-7387. Monahan, J.J. and Hall, R.H. (1974) Nucleic Acids Res. 1,1359-1370. Noll, M., Thomas, SO. and Kornberg, R.D. (1975) Science 187,1203-1206. Nell, M. and Kornberg, R.D. (1977) J. Mol. Biol. 109,393-404. Oppenheimer, J.H., Schwartz, H.L., Koerner, D. and Surks, M.I. (1974) J. Clin. Invest. 53, 768-777. Oppenheimer, J.H., Koerner, D., Schwartz, H.L. and Surks, M.I. (1972) J. Clin. Endocrinol. Metab. 35, 330-333. Peacock, A. and Dingman, C. (1967) Biochemistry 6,1818-1827. Samuels, H.H. and Tsai, J.S. (1973) Proc. Natl. Acad. Sci. U.S.A. 70,3488-3492. Samuels, H.H., Stanley, F. and Shapiro, L.E. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 38773881. Samuels, H.H., Tsai, J.S. and Cintron, R. (1973) Science 181,1253-1256.
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Shaw, B.R., Herman, T.M., Kovacic, R.T., Beaudreau, G.S. and Van Holde. K.E. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 505-509. Spindler, B.J., MacLeod, K.M., Ring, J. and Baxter, J.D. (1975) J. Biol. Chem. 250, 41134119. Surks, M.I., Koerner, D., Dillman, W. and Oppenheimer, J.H. (1973) J. Biol. Chem. 248, 70667072. Tata, J.R. (1975) Nature 257, 18-23. Thomopoulos, P., Dastugue, B. and Defer, N. (1974) Biochem. Biophys. Res. Commun. 58. 499-506. Whitlock, Jr., J.P. and Simpson, R.T. (1976) Biochemistry 15, 3307-3314. Woodhead, L. and Johns, E.W. (1976) FEBS Lett. 62,115-117. Yoshizato, K., Nakajima, Y., Kawakami, M. and Shioya, N. (1977) Mol. Cell. Endocrinol. 7. 89-100.
NUCLEARTHYROIDHORMONERECEPTORBINDINGTOCHROMATIN SUBUNITS:IMPLICATIONSFROMDIGESTIONSTUDIESWITH MICROCOCCALNUCLEASE Robert S. GARDNER * Department ofInterna! Medicine, Universityof Iowa, Iowa city, Iowa 52242, U.S.A. Received 28 September 1977;a~~pted
12 January 1978
Nuclear thyroid hormone receptors are putative regulatory proteins which copurify with chromatin components and remain associated with chromatin following hydrodynamic shear. Rat liver chromatin containing bound receptors labeled in vivo with rssI-labeled L-triiodothyronine (Ts) is cleaved by micrococcal nuclease at 2°C into a series of nucleosomes or chromatin subunits. After velocity sedimentation of these subunits in glycerol gradients, receptors remain associated with oligomeric subunits but not with monomers. In studies with isolated nuclei, thyroid hormone receptors are concentrated in a particulate ahromatin fraction prepared by low temperature digestion of rat liver nuclei with micrococcal nuclease and isolation of the unsheared ‘core’ chromatin, On brief incubation at 37”C, core chromatin is digested by residual nuclease, yielding monom~ subunits. Thyroid hormone receptors dissociate from nucleoprote~ binding sites during this ~cubation. These observations with two different chromatin preparations suggest that receptor binding sites are preserved in nucleasegenerated chromatin oliiomers but not in monomer subunits. It is proposed that receptors bind to DNA segments linking monomer subunits, rather than to the subunits. Keywords: nuclear thyroid hormone receptor; rat liver chromatin; micrococcal nuclease; nucleosomes.
Nuclear thyroid hormone receptors are DNA-binding (MacLeod and Baxter, 1976) nor&stone proteins (Thomopoulos et al., 1974) with a high affkity for Ta and a lesser affmity for L-thyroxine (Oppe~eimer et al., 1972,1974; Samuels and Tsai, 1973). The high afftity receptor accounts for at least 90% of T3 binding components present in a soluble extract of rat liver nuclei (Latham et al., 1976). These receptors remain tightly bound to chromatin during extensive purification procedures (Surks et al., 1973; Charles et al., 1975; Gardner, 1975; Spindier et al., 1975) including hydrodynamic shear (Charles et al., 1975; Gardner, 1975). There is preliminary evidence supporting their role in mediating at least some of the cellular
* Present address: Department 92093, U.S.A.
of Psychiatry
MO03, University of California, La Jolla, CaJif.
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effects of thyroid hormones in mammalian organisms (Samuels and Tsai, 1973; Samuels et al., 1973; Oppenheimer et al., 1974; Koerner et al., 1975; Samuels et al., 1976). When rat liver nuclei labeled in vivo with [ ‘*‘I]Ta are incubated with excess micrococcal nuclease at 2”C, most of the chromatin is sheared into soluble particles, but only 17% of the [1251]Ta is released from nuclei with the soluble chromatin (Gardner, 1975). Core chromatin, representing 2% of the nuclear DNA, is prepared from the residual nuclear fraction remaining after nuclease treatment. Thyroid hormone receptors are strikingly associated with core chromatin. Almost 50% of the radioactive Ta which purifies with chromatin is isolated in the core chromatin fraction (Gardner, 1975). This represents a 14-fold enrichment of T3 receptor bound/mg DNA in core chromatin compared to total nuclear chromatin. A series of chromatin particles containing multiples of 200 base pairs of DNA is generated when rat liver nuclei are digested with micrococcal nuclease at 2°C or briefly at 37°C (No11 and Kornberg, 1977). The digestion products are resolvable into monomer, dimer and higher oligomer particle fractions on isokinetic sucrose gradient centrifugation (Finch et al., 1975). Monomer subunits, also known as mononucleosomes, consist of 200 base pairs of DNA and a histone octomer of histones H2A, H2B, H3 and H4 (Kornberg, 1974, 1975). Most, if not all, genomic sequences appear to be involved in the basic subunit structure of rat liver chromatin (Lacy and Axel, 1975). More prolonged digestion of chromatin with micrococcal nuclease at 37°C results in degradation of chromatin fragments from the ends, with release of histone Hl (No11 and Kornberg, 1977). In this paper, it is reported that thyroid hormone receptors are associated with nucleasegenerated oligonucleosomes prepared from chromatin. They appear not to be associated with mononucleosomes. This is compatible with the hypothesis that thyroid hormone receptors are associated with intemucleosomal regions of the chromosome, as suggested by Yoshizato et al. (1977). This is based on their observations that thyroid hormone receptors are localized to chromatin DNA regions sensitive to DNase. Our studies are performed using in vivo labeling of receptors with radioactive Ts to avoid potential artifacts associated with in vitro labeling techniques (Tata, 1975).
MATERIALS
AND METHODS
T3 and methemoglobin were obtained from Sigma (St. Louis, MO.). Metrizamide was obtained from Nyegaard and Co. A/S (Oslo, Norway). [12sI]Ta (spec. act. 70 and 500 mCi/mg) was purchased from Abbott Labs (Chicago, Ill.). [3H]RNA markers were obtained from Schwartz-Mann (Orangeburg, N.Y.). Micrococcal nuclease (spec. act. 16 000 units/mg) was obtained from Worthington Corp. (Freehold, N.J.). Male albino Sprague-Dawley rats, thyroidectomized at 100 g, were obtained
Thyroid hormorle receptor binding to chromatin
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from Hormone Assay Labs (Chicago, Ill.), and the animals were fed a low-iodine diet. After one week, residual thyroid was ablated with 0.5 mCi [1251]iodide. They were not used until at least four weeks post-radioiodine ablation. Preparation of rat liver chromatin
Nuclei prelabeled in vivo with 1 nmol [ 1251]T3 (spec. act. 70 mCi/mg) were purified from hypothyroid rat livers in 2.2 M sucrose Buffer A (100 mM NaCl, 10 mM Tricine, 8 mM MgCl*, 2 mM CaC12, pH 7.8), as previously described (Gardner, 1975). The nuclear pellet was homogenized in 0.25 M sucrose Buffer A containing 0.5% Triton X-100 and centrifuged at low speed. The purified nuclei contained 1.4 mg [1251]T3 bound/mg DNA. At least 90% of the radioactive T, represents T3 specifically bound to the nuclear receptor, based on studies of nuclear binding after injection of [ 1251]Ta with and without 10 nmol unlabeled Ta. Chromatin was purified from the Triton-washed nuclei, as previously described (Gardner, 1975). Z+-eparationof core chromutin and soluble core chromatin
Nuclei were purified from 30 g hypothyroid rat livers prelabeled in vivo with [12SI]Ta (spec. act. 70 mCi/mg), resuspended to an A260 of 10 and digested with 300 units/ml micrococcal nuclease with gentle stirring for 30 min at 2”C, under conditions as previously described (Gardner, 1975). After termination of the reaction with 4 mM EDTA, pH 7.5 (final concentration), the reaction mixture was centrifuged at 4000g for 10 min, and the residual chromatin pellet homogenized in 30 ml of 10 mM Tris, 5 mM EDTA, pH 7.5 and centrifuged at 8000g for 10 min. The chromatin pellet was then purified by repeating this procedure thrice, with 1 mM EDTA substituting for 5 mM EDTA in the last two washings. The final core chromatin pellet was resuspended by gentle homogenization to an AZe.,,of 15 in 10 mM Tris, pH 7.5, 1 mM CaC12and incubated for 60 s at 37’C. The reaction was then terminated with 4 mM EDTA (final concentration), and the particulate components removed by centrifugation at 18 000 g for 10 min. The chromatin component of the supematant fraction is called soluble core chromatin. Beparation of / ‘2SZJT3-labeled receptors
Rat liver nuclei labeled in vivo with [1251]Ta (spec. act., 500 mCi/mg) were isolated as previously described (Gardner, 1975), except that 3 mM MgC12was substituted for Buffer A in all of the sucrose solutions. The nuclei were further purified by centrifuging them in 0.25 M sucrose-3 mM MgC12solutions containing 0.5% Triton X-100. Purified nuclei were resuspended in 0.4 M KCl, 10 mM Tris, pH 8.5 and incubated for 20 min at 2’C. The nuclei were then centrifuged at 10 OOOgfor 10 min. The supernatant fraction, containing 75-85% of the nuclear-bound [12’I]T3 was collected and the T3 radioactivity specifically associated with the thyroid hormone receptors separated from unbound T3 by purification on a Biogel P-10 column and collection of the void volume fractions (Thomopoulos et al.,
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K.S. Gardner
[r*‘I]Ta
which
Isokinetic sucrose gradient centrifugation One-ml samples of [ 1251]TJabeled soluble core chromatin or partially purified [1251]Ts-labeled thyroid hormone receptors were layered onto 36.3 ml isokinetic sucrose gradients formed with an isokinetic gradient maker (Molecular Instruments Co., Evanston, Ill.) with C’, = 5%, Cr = 28.8% and V, = 33 ml (Finch et al., 1975), where PM = mixing volume of the flask, C, = concentration of sucrose at the top of the gradient, and Cr = concentration of sucrose at the bottom of the gradient (Nell, 1967). The sucrose gradient solutions also contain 0.1 mM EDTA, pH 7.5. The samples were centrifuged for 20 h at 27 000 rev./min in an SW 27 rotor, and 0.8 ml fractions were collected after passage through a turbulence-free flow cell (Molecular Instruments Co.) for measurement of the AZ60 using a Beckman model 24 spectrophotometer. Isopycnic centrifugation in metrizamide-50% D20 gradients Preformed linear gradients of metrizamide in 50% D20 were prepared using five solutions of metrizamide (containing 50% D20) ranging in concentration between 20 and 40% (w/w) (Htittermann and Guntermann, 1975). The 50% D20 is substituted for 99.5% D20 in the layering solutions to lower the density of the gradient. The metrizamide gradient solutions also contain 10 mM Tris, pH 7.5, 1 mM EDTA, 0.1 mM phenylmethylsulfonylfluoride and 0.1 mM dithioerythritol. 0.5 ml samples of [rz51]Ta-labeled sonicated core chromatin or [1251]TJabeled sonicated nuclei were layered onto 3.5-ml gradients and centrifuged at 58 000 rev./ min for 16 to 20 h in an SW 60 rotor. 0.2 ml fractions were collected and DNA determined by a modified diphenylamine reaction after addition of 1 mg carrier bovine serum albumin to each fraction and extensive washing of the precipitates with 0.4 N perchloric acid (Burton, 1956). Gel electrophoresis of DNA fragments 1.2% agarose gels were prepared as previously described except that agarose was substituted for acrylamide (Peacock and Dingman, 1967). Chromatin samples were suspended in 1% sodium dodecyl sulfate, 1 M NaCl, 10 mM Tris and 2.5 mM EDTA, pH 8.0, and extracted with an equal volume of chloroform : isoamyl alcohol (25 : 1). The aqueous phase was precipitated with 3 volumes of ethanol and then dissolved in 0.1 ml electrophoresis buffer containing 15% sucrose and 0.1% bromphenol blue. Up to 50 $ samples were applied to agarose gels and electrophoresed at 175 V for 1 h at 20°C in a buffer system as described by Peacock and Dingman (1967). After electrophoresis, the gels were stained for 30 min with 0.4 &ml ethidium bromide in electrophoresis buffer and photographed with an orange filter under ultraviolet light.
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RESULTS
Purified hypothyroid rat liver chromatin labeled in vivo with [‘asI]T, was incubated at 2’C with micrococcal nuclease in the presence of 0.025 mM Ca2”. The reaction was stopped with excess EDTA, and the remaining unsheared chromatin pelleted. As i~ustrated in fig. 1, there was a progressive solub~zation of chromate during the 120.min incubation. After 30 min, only 35% of the A260 remained in the c~omati~ pellet, and after 60 min, this decreased to 5%. Virtually ail of the A 260 eventually became nonpelletable. After 60 min of incubation, only 10% of the AzeO was acid-soluble, ~dicating the extent of degradation of DNA under these conditions. Associated with the appearance of chromatin AzGO in the soluble fraction, a simiiar percentage of [ ‘251]T3 was solubilized. At 30min, when 65% of the AZ60 was soluble, 67% of the radioactive Ts was also soluble. However, later, when virtually all of the chromatin A 260 had been solubilized, there was a residual 1.5% of T3 radioactivity bound to the pellet. The nucle~e-digested chro-
TIME
fmin)
1, Kinetics of digestion of chromatin with micrococcal nuclease. Rat liver chromatin was resuspended to an AZ60 of 10 in 0.025 mM CaClz and 5 mM sodium phosphate buffer, pH 7.2, and incubated at 2°C with 300 units/ml micrococcal n&ease. At various times, aliquots were withdrawn and the reaction was stopped with 4 mM EDTA (final concentration). The aliquots were centrifuged at 28 OOOg for 25 min, and the pellet resuspended in incubation buffer containing 1 mM EDTA and sonicated for 30 s at 30 W. (Sonifier W185 with microtip). The A260 and radioactivity was determined on each supernataut and sonicated pellet fractions. -, d2ee; ---, cpm+ Fig,
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matin had a protein : DNA ratio of 1.8 and a histone : DNA ratio of 1.l . It had the usual complement of histones and non-histone proteins, as determined by SDSacrylamide gel electrophoresis (data not shown). Agarose-gel electrophoresis of DNA extracted from micrococcal nuclease digest of chromatin is shown in fig. 2. DNA bands corresponding to monomer, dimer and higher oligomer chromatin subunits were present and were comparable to DNA bands obtained from micrococcal nuclease digests of nuclei. Glycerol gradient ultracentrifugation of soluble chromatin prepared from 30-min and 60-min nuclease
123456 Fig. 2. Electrophoretic comparison of DNA fractions prepared from nuclei and chromatin DNA was extracted from nuclei and chromatin and electrophoresed at 175 V for 1 h through 1.2% agarose slab gels. Gels were stained with 0.4 ng/ml ethidium bromide and photographed under ultraviolet light using an orange filter. Wells l-4: Nuclei digested with five units of micrococcal nuclease/4aec for 30, 60, 90 and 120 s, respectively, at 20°C. Wells 5-6: Chromatin digested with five units of micrococcal nuclease/Aaeo for 45 and 90 s, respectively, at 20°C.
Thyroid hormone receptor binding to chromatin
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Fig. 3. Glycerol gradient centrifugation of 30-min nuciease-digested chromatiu. Rat liver chromatin was digested with micrococeal nuclease for 30 mm as described in the Iegeud of fii. 1 and 1.5 ml of the soluble chromatin was layered onto lo-30% w/w glycerol gradients containing 10 mM Tris, pH 7.5, and 0.1 mM dithioerythritoi and centrifuged for 16 h at 27 000 rev./min in an SW 27 rotor. 1.2 ml fractions were collected and A2eo and radioactivity determined. -,A260;---,cpm.
digests verified that [*251fT3 remained bound to chromate particles fo~o~g nuclease digestion (figs. 3 and 4). In each gradient, the A260 profile consisted of a slowsedimenting peak (fr. no. 10-12) identified as the 13 S monomer particle, using an 18 S ribosomal RNA marker to calibrate the gradient, and more rapidly
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sedimenting components (fr. no. 15-30) representing dimer, trimer and higher oligomeric chromatin subunits. T3 radioactivity peaked with the oligomeric subunits, with no evidence of a radioactive peak coinciding with the slow-sedimenting Aa6e peak. This 11 S particle increased relative to the other AZ60 components as the nuclease digestion was prolonged from 30 to 60 mm. This is consistent with generation of monomer subunits from oligomers as the latter are cleaved by the nudease. The resolution of the gradients does not allow for a clear delineation between the indi~du~ ofigomeric subunits and the radioactive Ta associated with the subunits. Therefore, it is not possible to calculate the [f2sIjTa/A260 ratio as a function of the number of subunits in a chromatin oligomer, although this ratio appears to be reduced in the higher oligomers. Studies with core chromatin
Core chromatin containing bound [ ’ 25I] Ta-labeled thyroid hormone receptors was prepared from unsheared particulate components remaining after a 2’C microDENSITY (q/cm31 1””
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Kg. 5. Isopycnic gradient centrifugation of sonicated [ t2sI]T~-labeled core chromatin in metrizamide-50% D20. Core chromatin containing bound Ts-labeled receptors was resuspended in S mM EDTA, 1 mM Tris, pH 7.5, and sonicated for 100 s at 30 W, using IS-s pulses. The sonicate was centrifuged for 10 min and the supernatant applied to preformed metrizamide-I&O gradients, and centrifuged as described in Methods. 0.2 ml fractions were coilected and DNA and radioactivity determined. .A260; - --, cpm.
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DENSITY (g/cm3)
FRACTION
No
Fig. 6. Isopycnic gradient centrifugation of sonicated [ rss~]Ts-labeled nuclei in metrizamide50% D20. Nuclei containing bound Ta-labeled receptors were resuspended in 5 mM EDTA, 1 mM Tris, pH 7.5, and sonicated for 100 s at 30 W. After centrifugation at 8000 g for 10 min, the supernatant was applied to preformed metrizamide-D20 gradients, as described in Methods and 0.2 ml fractions were collected and DNA and radioactivity determined. -, DNA; - - -, cpm.
coccal nuclease digest of hypothyroid rat liver nuclei. It was further characterized by isopycnic centrifugation in metrizamide-DzO gradients. Core chromatin, after sonication, banded at a density of 1.2 g/cm in these gradients (fig. 5). In contrast, total rat liver nuclear chromatin prepared from sonicated nuclei banded at a density of 1.18 g/cm (fig. 6). A major fraction of the radiolabeled T3 peaked at approximately the same density as the chromatin DNA band in both gradients, suggesting that the receptors remained associated with both core and chromatin DNA components following sonication and gradient centrifugation in metrizamide. Although there was a second, smaller, radioactive T3 peak near the top of the core chromatin gradient, presumably representing unbound T3, most of the radioactivity appeared to be chromatinassociated. Core chromatin was also sheared by brief incubation at 37°C in the presence of Ca2+. Approximately 65% of the A260 and 50% of the [12’I]T3 was solubilized on incubation of T3-labeled core chromatin for 30-60 s. Calcium was required for this
286
Fig. 7. Electrophoretic comparison of chromatin DNA fractions. The agarose gel of extracted DNA was run as described in fig. 2. Well 1: Core chromatin; Well 2: Soluble core chromatin; Well 3: Core chromatin digested with 5 units of micrococcal nucleasq’A2eo for 3 min at 37°C; Well 4: Rat liver chromatin purifed using the same methods as for core chromatin (minus the nudease step) and digested by endogenous nuclease for 3 mm at 37°C; Well 5: Rat liver chromatin digested with 5 units of micrococcal nucIease/Azeo for 3 mm at 37°C.
reaction, with excess EDTA markedly inhibiting the release of both Asee and radioactivity into the supernatant fraction (data not shown). The chromatin present in this soluble fraction was called soluble core chromatin. DNA extracted from core chromatin and soluble core chromatin was further characterized by gel electrophoresis. Two DNA bands were identified in soluble core chromatin, with an electrophoretic mobility corresponding to chromatin monomer and dimer subunits (fig. 7).Themonomer component was clearly the predominant band. In contrast,
Thyroid hormone receptor binding to chromatin
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Fig. 8. Isokinetic sucrose gradient centrifugation of [ 125IlT34abeled soluble ‘core chromatin. Soluble core chromatin was prepared from core chromatin as described in Methods, and centrifuged in isokinetic sucrose gradients. 0.8 ml fractions were collected and A26o and radioaco- - -0, cpm. tivity determined. -,A2,e;
DNA extracted from core chromatin contained no discrete bands on gel electrophoresis. This suggested that core chromatin is composed of chromatin multimers which are cleaved to mainly monomer subunits on incubation at 37°C. Added micrococcal nuclease was not required for this reaction. However, in the presence of additional nuclease, the monomer DNA band had a greater mobility on gel electrophoresis, suggesting that it has been degraded to a subunit of lower molecular size (fig. 7). Soluble core chromatin was further characterized by centrifugation in an isokinetic sucrose gradient. As illustrated in fig. 8, two A260 peaks were identifiable. Peak 1 represents low molecular weight acid-soluble oligonucleotides, while peak 2 is acid-insoluble chromatin, corresponding to the monomer subunit. This is based on an estimated sedimentation coefficient of 11 S using 18 S and 28 S ribosomal RNA markers to calibrate the gradient. When peak 2 fractions were pooled and the extracted DNA electrophoresed on agarose gels, the DNA migrated as a single band with a mobility virtually the same as the DNA from monomer subunits generated by low-temperature digestion of rat liver nuclei with micrococcal nuclease (fig. 9). This suggests that monomer subunits are generated when core chromatin is incubated briefly at 37’C in the presence of Ca’+. The [t2’I]T3 peak in this gradient, representing at least 75% of the applied radioactivity, was clearly separable from the monomer DNA A260 peak (fig. 8). There was no evidence of significant radioactivity in the region of the gradient containing chromatin subunits, except in the multimer fraction near the bottom of the gradient. Saltextracted and partially purified [ ’ 25I] T3-labeled thyroid hormone receptors centrifuged in a similar isokinetic sucrose gradient sedimented as a single radioactive component with the same sedimentation coefficient as the radioactivity present in the soluble core chromatin gradient (data not shown). This suggests that
R.S. Gardner
Fig. 9. Electrophoretic comparison of pooled peak 2 fractions from an isokinetic sucrose gradient of soluble core chromatin with micrococcal nuciease digests of nuclei. Agarose gels of extracted DNA were run as described in fig. 2. Wells 1-2: Nuclei digested with 35 units micrococcal nuclease/ilzeo for 10 and 20 mm, respectively, at 2°C. Well 3: Pooled core chromatin fractions 23-27 from peak 2 of fig. 7.
the major radioactive component in soluble core chromatin is bound to thyroid hormone receptors or other macromolecules with similar sedimentation coefficients .
DISCUSSION This paper describes investigations in which thyroid hormone receptor binding to chromatin structures is probed, using micrococcal nuclease to shear chromatin
Thyroid hormone receptor binding to chromatin
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into subunits or nucleosomes. Two different chromatin fractions labeled in vivo with [ 1251]T3 were employed to define receptor interactions with subunits under conditions where at least 90% of the nuclear radioactivity is specifically bound to the receptor. The first, purified hypothyroid rat liver chromatin, was digested with micrococcal nuclease at 2°C generating soluble chromatin particles which were subsequently purified. During the digestion, radioactive T3 was also solubilized. Purified chromatin used in these studies was prepared by lysis of nuclei in low ionic strength buffers. The u&eared chromatin preparation was then digested with micrococcal nuclease, which shears chromatin into a series of subunits which are resolvable on glycerol velocity gradients and gel electrophoresis of the extracted DNA. Liew and Chan (1976) prepared chromatin by conventional low ionic strength methods and found that the characteristic nucleosomal structure was preserved. Woodhead and Johns (1976) prepared chromatin in 0.14 M NaCl and sheared the chromatin in a blender, again with preservation of the subunit structure. In contrast, Noll et al. (1975) found that the nucleosomal structure was destroyed by mechanical shear of chromatin, and this has been confirmed by Axe1 (1975). It appears that under low ionic strength conditions in the absence of vigorous mechanical shear, the native structure of the chromatin is preserved, and this was confirmed in the present study, The second chromatin fraction used, known as core chromatin, was prepared from hypothyroid rat liver nuclei which have been digested with excess micrococcal nuclease at 2”C, and the residual unsheared chromatin enriched in thyroid hormone receptors was purified. Core chromatin was subsequently sheared to soluble chromatin particles by brief incubation at 37’C in the presence of Ca”, but without additional nuclease. Micrococcal nuclease bound to core chromatin from the prior digestion of nuclei with this nuclease is the probable explanation for the observed nuclease activity in core chromatin. This is consistent with a recent report that micrococcal nuclease copurifies with chromatin prepared from micrococcal nuclease digests of rat liver nuclei (Carter and Levinger, 1977). During nuclease shear of chromatin, 85% of the radioactive T; was solubilized. The residual 15% of the radioactivity was not associated with DNA, since it remained with the pellet after all of the chromatin DNA was solubilized. In contrast, when nuclei were incubated with micrococcal nuclease, most of the radioactive T3 remained with the nuclear pellet and copurified with the residual (core) chromatin. It is unclear whether core chromatin DNA sequences are also present in bulk chromatin. Clarification of this question will require the identification of specific DNA sequences in core chromatin. It is our working assumption that core chromatin is associated with a specific nuclear structure which is relatively protected from micrococcal nuclease in the intact nucleus, but becomes susceptible to this nuclease in isolated chromatin. The identity of this structure is unknown, although the nucleolus has been postulated to be the intranuclear location of core chromatin (Gardner, 1975). Nuclear thyroid hormone receptors bind tightly to both rat liver chromatin
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preparations. This is based on velocity sedimentation of hydrodynamically sheared total rat liver chromatin (Charles et al., 1975) and core chromatin (Gardner, 1975). Also, as shown in this paper, total nuclear chromatin as well as core chromatin both band with associated thyroid hormone receptors in metrizamide isopycnic gradients. The banding pattern observed with rat liver chromatin in metrizamide gradients is consistent with previous studies of the behavior of this chromatin in isopycnic gradients (Monahan and Hall, 1974). In their studies, the major chromatin components have a buoyant density of 1 .I 8 g/cm, the same as found in our studies. Core chromatin has a slightly higher density in these gradients, suggesting a slightly higher protein-to-DNA ratio. Following nuclease shear of rat liver chromatin, thyroid hormone receptors sediment with chromatin oligomers in glycerol velocity gradients. Of particular interest is the small percent of binding of Ta to monomer subunits in this preparation. It is unclear whether receptors bind to chromatin dimers as well as longer oligomers following nuclease cleavage. Better resolution of chromatin oligomers will require multiple gradient centrifugations to further purify the dimer and trimer subunits. Core chromatin is cleaved to predominantly monomer subunits on brief incubation at 37°C. Compared to monomer subunits prepared from 2°C nuclease digestion of nuclei which have been reported to consist of 198 * 6 base pairs of DNA, the monomer subunit prepared from core chromatin has similar mobility on agarose gels (Noll and Kornberg, 1977) suggesting that core chromatin monomers are similar in size to bulk monomer subunits. Digestion of chromatin with micrococcal nuclease at 2°C rather than at 37” generates subunits with minimal degradation from the ends, therefore making mononucleosomes a reference marker for 200 base-pair fragments (Noll and Kornberg, 1977). During the incubation, Ts is released as receptor-bound T3. This is further evidence that receptors are not bound to monomer subunits, although it is not clear whether the release of receptor is always linked with formation of monomer subunits. The binding of thyroid hormone receptors to oligomeric chromatin subunits but not to monomeric subunits would be consistent with receptor binding to linker regions between subunits. There is convincing evidence that DNA regions bridging monomer subunits are more sensitive to nuclease cleavage than monomer subunits (Shaw et al., 1976; Whitlock and Simpson, 1976; No11 and Kornberg, 1977). Thyroid hormone receptors may bind to these linker regions with receptor released during nuclease digestion of these regions. This is similar to the behavior of histone Hl which apparently is associated with these linker regions and is released during their digestion by micrococcal nuclease (Shaw et al., 1976; Whitlock and Simpson, 1976; No11 and Kornberg, 1977). Although receptor localization on linker regions is consistent with these studies, there are clearly other explanations. Receptors may bind to monomer subunit DNA sites that are digested when dimers are cleaved to monomers. Alternatively, they may bind to monomer DNA sites that undergo conformational changes during nuclease digestion of adjacent linker regions leading to release of receptor. Regardless of the mechanism, thyroid hormone receptor bind-
Thyroid hormone receptor binding to chromatin
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ing sites appear to be preserved in nuclease-generated chromatin oligomers provided the oligomer consists of at least two (or possibly three) linked subunits. Further studies are planned to determine whether the minimum chromatin structure supporting receptor binding is the dimer or trimer subunit, and this has clear implications for the hypothesis discussed in this paper.
ACKNOWLEDGEMENTS The author wishes to thank Duane Rossman and Ellyn Murphy for their dedication in the performance of skillful technical assistance. I also wish to thank Dr. Vaughn Jackson for technical help with gel electrophoresis of DNA fragments. This work was supported in part by grants from the NIH and Veterans Administration.
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