The dynamic properties of neuronal chromatin are modulated by triiodothyronine

Neurochemical Research, VoL 17, No. 11, 1992, pp. 1049-1055

The Dynamic Properties of Neuronal Chromatin Are Modulated by Triiodothyronine

Alessandro Cestelli 1, Roberto Gristina 1, Daniele Castiglia l, Carlo Di Liegro 1, Giovanni Savettieri 2, Guiseppe Salemi 2, and Italia Di Liegro 1,3

(Accepted February 27, 1992)

The effect of triiodothyronine (T3) on the rate of synthesis of nuclear proteins was studied during terminal differentiation of rat cortical neurons cuitured in a serum-free medium. To this aim total and acid soluble nuclear proteins were analyzed by different electrophoretic techniques. Our results show that: 1) during maturation in vitro, neuronal nuclei undergo a dramatic change in the rate at which different classes of histones and high mobility group (HMG) proteins are synthesized; the synthetic activity, measured as incorporation of radioactive precursors into nuclear proteins, slows indeed down with age: especially evident is the decrease in core histones synthesis; at day 15, on the other hand, HMG 14 and 17 and ubiquitinated H2A (A24) are synthesized at a high rate, especially in T3-treated neurons; 2) neurons treated with T3 show, at any age tested, a higher level of lysine incorporation into nuclear proteins; 3) even if during the first days of culture neurons synthesize core histones more actively in the presence of T3, there is no accumulation of these proteins at later stages, as compared with untreated cells. Possible implications of these data and relationship with the chromatin rearrangement which accompanies neuronal terminal differentiation are discussed.

KEY WORDS: CNS development; chromatin; triiodothyronine; neurons.

INTRODUCTION

During brain maturation neuronal chromatin is rear- ranged so that the average length of nucleosomes scales down from 200 about 165 base pairs (4,22,33). This event has been reported to be accompanied in vivo by an increase in the molar ratio of core histones to DNA and by decrease in the H1 class of histones (18,30). Even if partially deprived of H1, neuronal chromatin has been shown to adopt a highly folded conformation (1). Al- though H1 class decreases as a whole, the variant H1 ~

1 Dipartimento di Biologia Cellulare e dello Sviluppo "Alberto Monroy". 2 Cliniea Neurologica; Universit~ degli Studi, Palermo, Italy. 3 To whom to address reprint requests at: Dipartimento di Biologia

Cellulare e dello Sviluppo "Alberto Monroy", via Archirafi 22, 90123 PaIerm•, RaCy.

1049

seems to accumulate during brain maturation (5): this finding together with H1 ~ localization in peripheral chromatin (2) suggests that this H1 variant can have some role in gene activation, probably by maintaining the special structural organization adopted by peripheral chromatin (12).

The modifications in chromatin composition which accompany neuronal terminal differentiation are probably responsible for the partial uncoiling of chromatin evidenced by the increase in the overall sensitivity to DNasel (17,21) which in turn may correlate with the high transcriptional activity of nuclei of mature neurons (17). This active state is likely to be triggered by the incorporation into chromatin of proteins like HMG 14 and 17 (14,34) and ubiquitinated H2A (13) which have been reported to be specifically localized in active chromatin of many ceils %,pes.

0364-3190/92/1100-1049506.50/0 �9 1992 Plenum Publishing Corporation

1050 Cestelli, et al.

The molecular basis and the functional conse- quences of the short repeat remain however largely un- known, also because of the cellular complexity of mammalian brain and the consequent difficulties in searching for specific biosynthetic activities in vivo.

We have previously reported that triiodothyronine (T3) is able to induce the shortening of the chromatin repeat in neurons cultured in a synthetic medium (6). This system, in which non-neuronal ceils as well as un- defined factors stemming from the serum are virtually absent (7), allows to study biosynthetic events concerning neurons more precisely: cells can be fed with radiolabeled precursors of macromolecules more easily if the corresponding unlabeled compounds are excluded at all in the formulation of the medium. By using this ap- proach we have studied the rate of incorporation of labeled lysine into nuclear proteins of neurons cultured for different periods of time either in the absence or in the presence of T3.

EXPERIMENTAL PROCEDURE

Celt cultures. Neurons were purified from fetal rat cortices at the 16th embryonal day (El6) and cultured for 5, 10, or 15 days in Maat medium, supplemented or not with T3, as described elsewhere (6,7). To investigate the synthesis of nuclear proteins, cells were cultured for the last 24 hrs in lysine-free Maat medium, supplemented with [3H]lysine monohydrochloride (Amersham, UK: specific activity > 70 Ci/mmol) at a final concentration of 5 ~Ci/ml.

Isolation of Nuclei. Nuclei were purified from labeled cells as described elsewhere (6), with minor modifications; briefly, neurons were scraped from the dish surfaces, rinsed twice in cold phosphate- buffered saline, resuspended in nuclei buffer (NB: 0.32M sucrose; 50 mM sodium phosphate buffer, pH 6.5; 50 mM KC1; 0.15 mM sperm- ine; 0.15 mM spermidine; 2 mM ethylenediaminetetraacetie acid (EDTA); 0.5 mM ethylene glycol-bis (13-aminoethyl ether) N,N,N',N'- Tetraacetic Acid (EGTA) and 1.0 mM phenylmethylsulfonyl fluoride, PMSF) and homogenized with several strokes of a tight-fitting pestle in a Dounce homogenizer. The homogenate was centrifuged at 1000 g for 10 rain and the nuclear pellet was resuspended in NB, stratified on top of a cushion of 1.0 M sucrose in NB and centrifuged at 64,000 g for 30 rain at 4~ The nuclear pellet was finally washed once in reticulocyte saline buffer (RSB: 10 mM NaCI; 10 mM Tris-HC1, pH 7.4; 1.5 mM MgCla).

Determination of [SH]Lysine Uptake and Incorporation Into Pro- teins. Samples (20 ~1) of each lysate were placed on two filter-paper discs; one of each pair of discs was directly counted in a Beckmann L-7500 scintillation counter (using a liquid scintillation mixture of toluene/2,5-Diphenyloxazole/Phenyloxazolylphenyloxazolylphenyl) to obtain the total radioactivity associated with ceils; the other was treated with trichloroacetic acid (TCA), followed by alcohol-ether and ether; the disc was finally counted in the liquid scintillation system mentioned, according to Greenberg and Rothstein (16). Synthesis of proteins was determined as TCA-precipitable counts. Both uptake and incorporation were normalized per Izg of DNA, according to absorbance at 260 nm of the last nuclear pellet.

The data obtained from different experiments were used for statistical analysis. Student's t test was used for comparing means; paired t test was used to evidence any significant difference between the two experimental conditions (C and T).

Isolation of HCl- and PCA-Soluble Proteins. Purified nuclei were resuspended in RSB, containing 1.0 mM PMSF and HC1 was added to a final concentration of 0.4M. After overnight incubation under continuous stirring at 4~ acid-insoluble material was pelleted at 10,000 g for 20 rain and HCI-soluble proteins were recovered from the supernatant by overnight precipitation with 10 vol of acetone at -20~ and centrifugation at 10,000 g for 20 min. Perchloric acid (PCA)-soluble proteins were obtained by overnight extraction of nuclear pellets with 5% PCA at 4~ in the presence of 1.0 mM PMSF. After centrifugation at 10,000 g for 20 min at 4~ the supernatants were dialyzed against distilled water (with 3 changes) and PCA-soluble proteins were recovered by overnight precipitation with 8 voI of acetone (0.4 M HCI) at -20~ Both HC1- and PCA-soluble proteins were finally dissolved in distilled water and protein concentration was determined according to Lowry et al. (25), using calf thymus histones (Sigma, St. Louis, MO) as a standard. Incorporation of [3H]lysine in these fractions was measured by counting small aliquots in a liquid scintillation system.

Electrophoretic Analysis of Total, HCl-Soluble, and PCA-Soluble Nuclear Proteins. When total nuclear proteins were to be analyzed, purified nuclei were resuspended in RSB, and DNA concentration was calculated from the absorbance at 260 nm; nuclear suspensions were then directly diluted in Laemmli protein sample buffer or in Triton X- 100 sample buffer. HC1- and PCA-soluble proteins were dissolved in distilled water, as described above.

Electrophoresis on Sodium dodecyl sulfate (SDS)-polyacrylamide slab gels (PAGE) was performed according to Laemmli (24). Electro- phoretic analysis of nuclear basic proteins on acetic acid/urea (AU) or acetic acid/urea/Triton X-100 (AUT) PAGE was carried out as described elsewhere (8).

Two-dimensional polyacrylamide gel electrophoresis was AUT in the first dimension and SDS in the second, according to Pina and Suau (31). Briefly, the track of interest was cut out from the AUT gel and soaked in the following solutions: 1) 0.46 M Tris(Hydroxyl- methyl)aminomethane (Tris)-HC1, pH 6.8; 0.4% SDS and 10% 13- mercaptoethanol for 1 hr; 2) twice in the same solution without 13- mercaptoethanol for 20 min and 3) SDS stacking gel solution without polymerisers for 10 rain. Finally, 0.5 ~Lllml of N,N,N',N'-Tetrame- thylethylenediamine (TEMED) and 15 I.L1/ml of 50 mg/ml ammonium persulfate were added and the polymerising solution, together with the equilibrated track, was introduced between the glass plates, over the resolving gel. After migration, gels were stained overnight to satura- tion with Coomassie Billiant blue R250 and photographed.

To calculate the relative proportion of different histone species, the gels of interest were immediately dried and scanned at 560 nm using a Beckman DU-8 spectrophotometer. The planimetry of the re- suiting densitometer scans was used for calculations, as described elsewhere (5). Tritium-labeled proteins were fluorographed by treating stained gels with Amplify TM (Amersham UK) for 30 min under shak- ing, after which procedure the gels were dried and exposed to Kodak XS film at - 8 & C for appropriate intervals. The films were scanned as described above and the planimetry of the scans was used to calculate the percent of de novo synthesis with respect to the total amount of protein in a given band; this latter value was obtained by scanning the negative of the photograph of the stained gel. The values were corrected by taking into account the number of lysine residues in each class of proteins: in particular, lysine represents about 30% of ami- noacid residues in the HI class of histones, whereas it accounts for an

Dynamic Properties of Neuronal Chromatin are Modulated by Triiodothyronine 1051

average of 10% in core histones (3,20). Because of the large variability due to different conditions of film exposures, for the calculation of the average values reported in Table III, it was also necessary to refer the values obtained for each gel to internal labeled standards of known concentration.

RESULTS

Figure 1 shows an SDS-PAGE on which total nuclear proteins from neurons cultured for the indicated intervals were run. According to absorbance at 260 nm, equal amounts of nuclear lysates (and hence of DNA) were loaded on each lane. The densitometric analysis of the stained gel (Figure 1A) indicated that roughly similar quantities of proteins had been analyzed for each age and condition. Within the limits of this assay, it seems, therefore, that there is no change in the total amount of core histone/nucleus during neuronal differentiation in culture, either in the presence or in the absence of T3. This finding agrees with our previous results concerning neuronal maturation in vivo (5).

In spite of a constant core histone/DNA ratio, differences are evident in the corresponding fluorography (Figure 1B and 1C): the rate of lysine incorporation decreases in fact with time (longer exposures are necessary to visualize bands at day 15: figure 1C); moreover, neurons treated with T3 show, at any age tested, a higher level of labeling. These phenomena do not depend on differences in the lysine uptake since ceils are labeled at the same level independent of the treatment with hormone (Table I, 1st column); a difference in the general

rate of protein synthesis in the two conditions can be also ruled out according to Table I (2nd column). Nuclei appear therefore the primary targets of thyroid hormone action; the most significant difference regards the higher rate of labeling of non-histone proteins in T3-treated neurons, as compared with controls. Although differences could be also observed in specific bands corresponding to histone proteins, the electrophoretic analysis of total nuclear lysates is not adequate to infer more precise informations.

Moreover, the starting point of our study was the finding that T3 is able to induce in vitro a shortening of the chromatin repeat length similar to that observed in vivo (6); if this rearrangement requires a higher amount of histones to organize new nucleosomes, a higher level of synthesis of histones in T3-treated neurons could be expected. For these reasons, in the next analyses we have especially focused on HCI- and PCA-soluble nuclear proteins. Figure 2 shows an SDS-PAGE on which equal amounts of HC1- and PCA-soluble nuclear proteins from treated and untreated cells, cultured for 15 days, were applied.

The analysis of nuclear basic proteins confirms the already noticed difference in the rate of lysine incorporation between treated and untreated neurons (expressed as total cpm/~g of protein loaded onto each lane). The densitometrie analysis of the gels shown in Figures 2 and 3 reveals differences in specific bands; in particular, in T3-treated ceils Hla is more labeled than Hlb, whereas in control neurons the two main H1 subtypes are equally labeled (Figure 2); H1 ~ on the other hand, shows a slightly higher specific activity relative to total H1, in

Fig. 1. Electrophoretic analysis on an 18% SDS-polyacrylamide slab gel of total nuclear proteins from rat cortical neurons cultured for 5, 10 or 15 days in Maat medium with (T) or without (C) T3. Equal amounts of nuclear suspension (about 25 l~g of DNA, according to absorbance at 260 nm) were loaded on each lane. A" Coomassie blue staining; B: fluorography (7 days of exposure); C: fluorography of the last two samples presented in B, after a longer exposure (30 days). Marker sizes (in KDa) are indicated on the right margin.


Table I. pH]Lysine Uptake and Incorporation in Neurons Cultured in Maat Medium

[3H]Lysine Uptake [3H]Lysine Incorporation

Means • SD DIV (cpm x 10-3/l~g DNA)

C 165.2 --- 39.9 (5) 100.5 • 17.2 (5) 5 T 145.4 _ 49.9 (5) 91.0 • 5.4 (5)

C 139.4 • 75.2 (7) 100.7 • 54.5 (7) 10 T 146.6 • 80.5 (7) 94.5 • 48.5 (7)

C 130.1 • 60.0 (8) 90.4 • 58.1 (8) 15 T 136.7 • 63.1 (8) 91.6 • 50.5 (8)

Numbers in parentheses indicate independent experiments; in each case C and T samples were cultured and processed in parallel; at any age tested, the differences between C and T samples are not significant.

Fig. 3. Analysis by AUT-PAGE of HCl-soluble proteins from rat cortical neurons cultured for 15 days in Maat medium supplemented (T) or not (C) with T3. A: Coomassie blue staining; B: fluorography.

Fig. 2. Analysis by SDS-PAGE of HCl-(a,b) and PCA-soluble (c,d) nuclear proteins from rat cortical neurons cultured for 15 days in Maat medium supplemented (b and d) or not (a and c) with T3. A: Coomassie blue staining; B: fluorography.

Table II. Percent of Lysine Incorporation Into Different H1 Subtypes Relative to Total Incorporation into H1, in T3-Treated (T)

and Untreated (C) Neurons, at the 15th Day of Culture

Means - SD

H1 subtype C T

Hla 46 • 2.5 57 • 2.1 Hlb 50 - 2.5 39 --- 2.1 H1 ~ 4.5 + 0.5 3.9 --- 0.4

The data reported represent the average of 3 different gels. When C and T samples are compared, differences concerning Hla and Hlb subtypes are significant (p<0.05), while the percent of lysine incorporation into H1 ~ does not differ significantly (p>0.05).

control neurons, as compared with treated cells (see Ta- ble II).

Interestingly, there is a T3-dependence in the labeling of HMG proteins 14 and 17. In this case, however, it was not possible to calculate specific activity because these proteins are present at very low concen- trations and the densitometric analyses of the stained bands are not reliable. In addition the electrophoretic pattern of PCA-soluble proteins (Figure 2, lanes c and d) shows some bands whose identity is not known. Some of them (indicated as B,C, and D in Figure 2B) are labeled at a higher level in treated neurons; the protein indicated as " A " is instead labeled more strongly in control neurons. None of these proteins was detected in

the staining pattern: also in this case it is therefore im- possible to calculate absolute variations in specific activity.

As far as core histones are concerned, as shown in Table 3, de novo synthesis after 15 days of culture is very low, independent of hormone treatment. Similar analyses performed at 5 and 10 days of culture revealed that at day 5, neurons actively synthesize new histones; the rate of lysine incorporation is higher in T3-treated neurons; at day 10, there is still a significant synthesis of core histones; as shown in Figure 4, however, at this age the main differences between the two conditions concem non-histone proteins.

Among core histones, the only species which shows a relative high rate of labeling up to 15 days is the ubi-


Table III. Lysine Incorporation (Arbitrary Units) into H1 and Core Histones During Neuronal Differentiation in Culture, in the Presence

(T) or Absence (C) of T3

DIV H1 Core Histones

Means • SD

C 46 + 11 35 - 7 5 T 158 _+ 21 74 • 20

C 63 ---_ 23 26 ... 6 10 T 98 -2_ 26 39 ... 11

C 11 --- 1 3 .4-+ 0.9 15 T 14 -. 3 4.0 -_ 1.5

The data reported represent the average of 3 different gels. Lysine incorporation was normalized for total protein present in each band and for the percent of lysine residues present in the corresponding proteins, as described in Materials and Methods. Lysine incorporation into H1 and core histones is significantly higher (p < 0.001 and p <0.05 respectively) in T3-treated neurons than in con- trois, at day 5. Student's t test also reveals a significant difference (p<0.001) in the total rate of incorporation of lysine into both classes of proteins, either in the presence or in the absence of T3, when days 5 and 15 are compared.

quitinated form of H2A (uH2A or A24) that is labeled at slightly higher level in hormone-treated nuclei (Figure 2). It is worth noting that, even if during the first few days in culture core histones are synthesized more actively in the presence of T3, there is no difference in the total concentration of these proteins (expressed as ~g of core histones per equal amounts of neuronal nuclei) at later stages: i.e., as mentioned above, no accumulation of new histones could be evidenced. Moreover, the densitometric analysis of the stained gels shown in figures 2 and 3 (among others) has also demonstrated that both treated and untreated cells have a ratio of total H1 to core histones (calculated as molecules of H1/core histone octamere) of about 0.5 since the 5th day in culture. This ratio is equal to that found in mature neurons (5).

DISCUSSION

Thyroid hormones, like steroids, act on target tis- sues mostly by interacting with DNA-binding receptors which belong to the c erb A superfamily of proteins (11,15,28,29). This interaction plays an important role in regulating tissue-specific gene expression and is accompanied by changes in the structural organization of chromatin including 5'-flanking regulatory sequences of the target genes (for review, see: 10,11). In some in- stances the specific response (i.e. activation or repres- sion of a given gene) immediately follows hormonal administration (19,23,27), while in others many hours

are required (26). It is likely, however, that in most cases the hormone acts by triggering a cascade of biosynthetic events.

Mammalian brain development and maturation are deeply influenced by thyroid hormones, the sensitive period covering in the rat the few weeks during which post-mitotic neurons undertake extensive outgrowth of processes and stabilize synaptic contacts (for review, see: 32). This critical period is also characterized by a dramatic structural rearrangement of neuronal chromatin as a whole: namely the shortening of the average spacing of nucleosomes (4,22,33). Moreover, a correlation between this latter event and the hormone-sensitivity has been suggested by us because of finding that neurons, removed from rat cerebral hemispheres and cultured in a synthetic medium, do not show the shortening of the chromatin repeat, unless T3 is present in the medium (6).

The dramatic changes which alter the structural organization of whole chromatin probably require, as a prerequisite, activation of specific genes as well as a general preparation of the cell to manage with a large biosynthetic effort. In this study this working hypothesis was tested by using a cell culture system which allows selective maintenance of cortical neurons and accurate analysis of radiolabeled precursor incorporation into macromolecules. Statistical analysis of lysine uptake and incorporation has shown that, in spite of obvious experimental variability, there is no significant T3-dependent difference in the whole rate of cell metabolism. On the other hand, a generally higher synthetic rate of nuclear proteins in T3-treated neurons was noticed, which is in good agreement with the hypothesis that this hormone is involved in chromatin remodelling: the active synthesis of nuclear enzymes and regulatory factors, as well as of structural proteins could be in fact the necessary preparatory condition. The finding that these proteins, though synthesized at a higher rate, do not accumulate is however difficult to interpret, unless one supposes that the turnover as a whole and not only net synthesis of nuclear proteins is the key event that permits (or is caused by) the large functional rearrangement of neuronal nuclei; indeed, the shift in the nucleosomal length is likely to require sliding of preexisting cores; this latter event might cause loss and substitution of chromatin proteins.

The fact that, although at low rate, also untreated neurons synthesize histones up to 10 days of culture and both HMG 14 and 17 and uH2A at day 15, suggests that at least some of the basic processes which cause acquir- ing differentiated neuronal phenotype are anyway pro- grammed in postmitotic neurons; we have found for instance that the developmental curves of neuron spe-


Fig. 4. Two-dimensional PAGE of HCl-soluble proteins from rat cortical neurons cultured for 10 days irt Maat medium supplemented (T) or not (C) with T3. The first dimension was an AUT gel (right to left for C; left to right for T) and the second was a 18% polyacrylamide-SDS slab gel (top to bottom); both Coomassie blue staining and fluorography are shown; the staining of the 1st dimension gel has been included for reference. HCl-soluble proteins from C neurons, cultured for 5 days, were loaded as internal markers on the 2rid dimension gel in between the lanes cut from the AUT gel.

cific- (NSE) and nonneuronal-enolase (NNE) mRNAs expression are almost the same in vivo as well as in hormone-treated and untreated cells: T3 has indeed only minor effects, namely an earlier onset in NSE mRNA expression and a precocious drop in NNE mRNA level (9). The chromatin remodelling, on the other hand, seems to depend on T3 and, although its functional significance remains still uncertain, it is likely to have a profound effect on nuclear activity. It is worth noting in addition that the chromatin rearrangement which accompanies neuronal terminal differentiation is not in accord with a generally accepted view according to which major structural modifications and gene reprogramming require at least one cell cycle.

We conclude that the described events are related

to a fine tuning of neuronal activity rather than a general differentiative process.

ACKNOWLEDGMENTS

We wish to thank Mr. Domenico Cascino and Mr. Salvatore Buccoleri for preparation of figure photographs. This work was partially supported by the CNR Target Project "Biotechnology and Bioin- strumentation". D.C. is supported by a fellowship from CNR.

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The dynamic properties of neuronal chromatin are modulated by triiodothyronine

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