Rat cerebral cortex neurons contain the five histone HI subtypes Hla-e and the subtype HIO present in other mammalian somatic tissues. The four subtypes Hla-d decay exponentially during postnatal development and are partially or totally replaced by Hle that becomes the major Bl subtype in adults. Hl0 accumulates in a period restricted to neuronal terminal differentiation. Here we study the synthesis of the Bl subtypes in cortical neurons and their neuroblasts by in vivo labeling with [14C]lysine. The subtype synthesis pattern of neuroblasts has been determined by labeling gravid rats during the period of proliferation of cortical neurons and synthesis in neurons has been studied by postnatal labeling. The subtype Hla is synthesized in neuroblasts but not in neurons and is therefore rapidly removed from neuronal chromatin. The synthesis of Hlb and Hld is much lower in neurons than in neuroblasts so that these subtypes are replaced to a large extent during postnatal development. Ole is synthesized at levels much higher than the other subtypes both in neurons and neuroblasts, but its very high turnover, about one order of magnitude faster than that of Hle in neurons, favors its partial replacement during postnatal development. Comparison of the synthesis rates of Hl0 in newborn and 30-day-old rats shows that the accumulation of HIO in differentiating neurons is due to an increased level of synthesis.
Histone H1 is necessary for the induction and stability of chromatin higher order structure. The H1 histone class is coded by a multigene family. The somatic H1 fraction from mammals has been resolved by two dimensional electrophoresis in five subtypes, Hla, b, c, d and e (Cole, 1984; Lennox et al., 1982; Lennox and Cohen, 1983; Lennox, 1984). In addition to these subtypes, some somatic tissues contain H1 ° (Panyim and Chalkley, 1969). The synthesis of Hl0 is greater in tissues with little or no cell division (Gerset et al., 1982). The H1 subtypes differ in extent of phosphorylation and in evolutionary and metabolic stability (Lennox and Cohen, 1983; Lennox, 1984). The subtypes also differ in their relative rates of synthesis and degradation in proliferating and quiescent cells (Lennox and Cohen, 1983; Pherson and Cole, 1982). In addition, it has been shown that some of the subtypes differ in their ability to condense DNA in vitro (Welsh and Cole, 1979, 1980; Liao and Cole, 1981). These properties have led to the suggestion that not all subtypes are equally efficient in promotmg chromatin higher order structures (Lennox, 1984; Huang and Cole, 1984).
In previous papers, we showed that in brain cortical neurons the proportions of the H1 subtypes change during postnatal development. The subtypes Hla-d decay exponentially and are replaced to different extents by Hle, which becomes the most abundant subtype in mature neurons. The changes affecting the composition in the subtypes Hla-e are already apparent at birth and the subtype composition is close to steady state by postnatal day 30 (Pina et al., 1987). H1 ° is differently regulated. It accumulates in a restricted period between days 8 and 18, coinciding with neuronal terminal differentiation, and its concentration remains stable thereafter (Pina et al., 1984). The replacement of the subtypes Hla-d by Hle during postnatal develop ment suggests that neurons and neuroblasts have distinct synthesis patterns. The purpose of this study has been to compare the H1 subtype synthesis patterns of neurons and neuroblasts in order to explain the changes m subtype composition in neurons during postnatal development.
Mammalian cortical neurons do not divide after birth (Jacobson, 1978). The incorporation of newly syn thesized histones into neuronal chromatin can thus be followed by means of postnatal injections of labeled amino acid precursors. The study of histone synthesis in neuroblasts is not so straightforward because these germinal cells cannot be isolated in enough quantities for analysis. We have devised a labeling format that allows access to histone synthesis in neuroblasts. For this purpose gravid rats were injected with [14C]lysine during the period of proliferation of the brain cortical neurons of the fetuses and the labeled histone incorpor ated in the dividing neuroblasts recovered from the neurons of the newborn. The method is reminiscent of that of Angevine and Sidman (1961) that permitted the establishment of the time of origin of neurons in the mammalian cerebral cortex. In this procedure, the gravid female was injected with tritiated thyrnidine, and the pattern of labeled neurons in the fetuses was determined postnatally at various intervals after injec tion. The relatively long half-life of DNA-bound H1 together with the very well-defined timing of prolifer ation of cortical neurons have made it possible to adapt this procedure to the study of histone synthesis in neuroblasts.
Materials and methods
In vivo labeling procedures
In order to study the synthesis of the H1 subtypes in neuroblasts, gravid Sprague-Dawley rats were mjected intra pentoneally once a day with 0.28 μCi of L-[14C]lysme per gram of body weight on days 16, 17, 18, 19, 20 and 21 of gestation The rats were born on day 22 and the protocol for h1stone isolation and histone extraction began two hours after birth.
For the study of the synthesIS of the H1 subtypes in neurons, either newborn or 30-day-old rats were injected intrapentoneally three times with doses of 2 μC1 of L [14C]lysme per gram of body weight at 4 hour intervals and killed 14 hours after the last injection.
Isolation of neuronal nuclei
The cerebral cortices were homogenized by hand With 30 up and-down strokes of a Dounce homogenizer in 1 M sucrose, 1 mM sodium cacodylate, 5 mM MgCl2, 1 mM dith1othre1tol, 1% thiod1glycol, pH 6.5. Cortex nuclei were fractioned according to Thomson (1973). All operations were performed at 2°C and phenylmethylsulfonyl fluoride (0.1 mM) was used throughout to inhibit proteolytic activity. The degree of contamination of the neuronal fraction with glial nuclei was of the order of 10%, as judged by phase-contrast microscopy.
The pellets of neuronal nuclei were resuspended in 10 mM EDTA, 1% thiodiglycol, pH 7.4 and sonicated. Perchloric acid-msoluble material was precipitated by addition of concentrated HCIO4. The combined supernatants containing H1 were acrdified by addition of concentrated HCI up to 0.25 Mand proteins precipitated overnight at -20°C by addition of 6 vol. of cold acetone. Proteins were collected by centnfu gation. The pellet was washed with acetone/thiod1glycol/HCl (97:2:1) and acetone/thiodiglycol (98:2) and vacuum dried.
Two-dimensional gel electrophoresis was performed basically as previously described (Pina et al., 1987). The firstdimensionwas a 30-cm-long urea/acetic acid gel, and the second a modified SDS gel according to Lennox and Cohen (1984), in which the separating gel contained 15% acrylarnide and 0.56% bisacrylamide, and the stacking gel contained 6% acrylamide with the pH reduced to 6.5 Gels were stamed for 24 hours with Coomassie brillant blue R-250 m methanoV water/acetic acid (5:5.1) and destained in methanoVethanoV water/acetic acid (7:1:1:1). Fluorograms were prepared according to the method of Laskey and Mills (1975).
Measurement of the spot intensities
Fluorograrns were measured by densitometry using a VINIX system for the digital processmg of the images.
In the rat brain, cortical neurons are generated continuously by mitosis of germmal cells in a penod that goes from intrauterine day 16 to shortly before birth on gestational day 22 (Berry et al., 1964). In order to establish the H1 subtype synthesis pattern in dividing neuroblasts, gravid rats were injected intraperitoneally with [14C]lysine during the proliferation period of the brain cortical neurons of the fetuses, that is on days 16 through 21 of gestation. The labeled histone syn thesized in dividing neuroblasts was recovered from the neuronal fraction of the newborn. Although histones are synthesized outside the S-phase of the cell cycle, most histone synthesis is tightly linked to S-phase (Wu and Bonner, 1981; D’Incalci et al., 1986). Therefore, the H1 prenatally labeled should essentially correspond to that synthesized and incorporated onto the DNA during the S-phase of dividing neuroblasts.
The synthesis pattern of neurons was studied by postnatal labeling. The absence of DNA replication in neurons makes the incorporation of newly synthesized histone molecules onto neuronal DNA rate limited by the turnover of the DNA-bound histone.
Synthesis of the H1 subtypes in neuroblasts
The synthesis pattern of dividing neuroblasts was obtained by prenatal labeling and the H1 fraction was purified from the neuronal nuclei of the newborn. The fluorography shows that all subtypes are incorporated in dividing neuroblasts (Fig. 1A). The intensities increase in the order Hl0<Hla<Hlb≈Hld<Hle< Hlc, according to the proportions shown in the histogram of Fig. 3. The most striking feature of this pattern is the high incorporation of Hlc, which is equivalent to that of all other subtypes taken together.
The subtype composition of the H1 newly incorpor ated onto the DNA during S-phase is basically determined by the relative synthesis rates of the subtypes. A steady-state for the H1 subtype compo sition of newly replicated chromatin cannot, however, be defined because as soon the chromatin assembly process is completed the representation of the different subtypes will either increase or decrease according to the balance of synthesis and turnover rates. In the case of the Hl subtypes, the balance favors the accumulation of Hle and the replacement to different extents of all the other subtypes during postnatal development.
Considering that the half-life of brain H1 has an average value of 13 days (Duerre and Lee, 1974), the fluorographic pattern obtained with prenatal labeling can be regarded as a good approximation of the subtype composition of the H1 newly incorporated into chroma tin. The case of Hlc deserves special consideration as this subtype has a half-life shorter than the average. A value as short as 2 1/2 days can be calculated from data of Pherson and Cole (1982) on mouse neuroblastoma. With such a half-life, about 45% of the Hlc incorpor ated during S-phase should be lost by exchange. However, these exchange losses are replaced to a large extent because Hlc is also actively synthesized in neurons as described in the next section.
Synthesis of the H1 subtypes in neurons
Neuron proliferation in the rat cortex stops shortly before birth. Neuronal synthesis patterns can therefore be studied by postnatal labeling without interference from cell proliferation. The synthesis pattern obtained by postnatal labeling of newborn rats is clearly distinct from that of neuroblasts. Hla is not synthesized in neurons, the synthesis of Hlb decreases to very low levels and that of Hld also decreases, although to a lesser extend. In contrast, Hlc and Hle are incorpor ated at levels eqmvalent to those observed in neuroblasts. Hlc dominates the pattern with about 50% of the total incorporation.
Fig. 2 shows the pattern of synthesis obtained when rats were labeled at postnatal day 30, when terminal differentiation of cortical neurons is completed and the composition in subtypes, including H1 °, is already close to steady-state (Piña et al., 1984, 1987). The pattern of mcorporatJon of the subtypes Hlb-e is identical within the statistical error to that of the newborn. In contrast, the level of incorporation of Hl0 is much higher, indicating that the accumulation of H1 ° in differen tiating neurons is a simple consequence of a higher level of synthesis. This result also shows that H1 ° turns over in mature neurons.
The mass pattern of the H1 sample isolated from newborn animals was obtained with Coomassie blue staining (Fig. lC). It shows that all H1 subtypes are present in the chromatin of the newborn. Hlb, Hlc, Hld and Hle are present in similar amounts, varying between 21% and 26%. Hla represents less than 5%. A very small amount of H1 ° is also present. This subtype composition is still far from equilibrium and thus further modified during postnatal development. Be yond postnatal day 60, when Hlb, Hlc, Hld and Hle represent, respectively, 6%, 16%, 9% and 69% of total Hl, the subtype composition remains stable as reported by Piña et al. (1987).
The relationship between the relative levels of incorporation of the subtypes in mature chromatin (Fig. 3) and the steady-state proportions reported by Piña et al. (1987) gives an estimation of the relative turnover rates of the whole set of subtypes present in neurons. It can be seen in Table 1 that the fastest turnover corresponds to Hlc, which turns over one order of magnitude faster that Hle, whereas H1b, H1d and H1° occupy intermediate positions.
The proportions of the H1 subtypes have been shown to change during postnatal development m brain cortical neurons (Piña et al., 1984, 1987). The changes in subtype composition can be explained by comparing the synthesis patterns of neurons and neuroblasts. Hla 1s synthesized in neuroblasts but not in neurons and is thus rapidly removed from neuronal chromatin. The de crease of Hlb and Hld is due to a lower synthesis level in neurons. The high level of synthesis of Hlc together with its low concentration in chromatm indicates that this subtype has a high turnover rate. Considering the steady-state concentrations and the relative synthesis rates of Hlc and Hle in mature neurons, we can estimate that Hlc turns over about one order of magnitude faster than Hle, which is the subtype with the slowest turnover. The accumulation of H1° during neuronal terminal differentiation is due to an increased level of synthesis as shown by the comparison of the synthesis patterns from inmature and terminally differ entiated neurons. Differential synthesis of H1 subfrac tions has previously been reported by Sizemore and Cole (1981) and D’Anna et al. (1982; 1985). D’Incalci et al. (1986) also observed that in quiescent human fibroblasts the synthesis of three H1 subtypes was greatly decreased. These subtypes presumably corre spond to Hla, Hlb and Hld. However, a definitive assignment has not been possible because these authors used a gel system different from that of this study.
The subtype composition of the H1 newly incorpor ated onto the DNA in S-phase is determined by the relative concentrations of the different subtypes avail able for binding to DNA, which in turn should be determined by the corresponding synthesis rates. The latter are established with good approximation by neuroblast labeling. Subsequently, differential turnover also contributes to the definition of the subtype composition of chromatin together with the changes in synthesis pattern associated to neuronal commitment. It is important to emphasize that even if the synthesis patterns of neurons and neuroblasts were identical, differential turnover would cause by itself the replace ment of some subtypes by others during postnatal development. The role of differential turnover in the control of H1 subtype proportions was given consider able emphasis in studies on butyrate treated neuroblas toma cells (Hall and Cole, 1985). It is apparent from the results that there is no single cause explaining the changes in H1 subtype proportions in neuronal chroma tin during postnatal development. Hla is removed from neuronal chomatin as a consequence of the strict dependence of its synthesis on DNA replication. Hlb and Hld are less dependent on DNA replication because, although their synthesis rates are greatly decreased in neurons, they remain present as minor components in mature chromatin. Hlc and Hle are not dependent on DNA replication and their relative synthesis rates are similar in neurons and neuroblasts. However, in this case, the much higher turnover of Hlc favors the accumulation of Hle and a slight decrease of Hlc during postnatal development.
Several lines of evidence indicate that the H1 subtypes differ in their capacity to condense DNA invitro. Results of Huang and Cole (1984) show that these differences are likely to be relevant to chromatin condensation in nuclei. On the basis of metabolic and developmental considerations, Lennox (1984) sugges ted that Hlc would be much less efficient than the other subtypes m promoting chromatin higher order struc ture. If it was so, a clearer expression of the properties of Hlc should be found in newly replicated chromatin before its concentration has been leveled down as a result of its high turnover. In contrast, the subtype composition of mature chromatin is dominated by Hle, the subtype with the slowest turnover, which in neuronal chromatin reaches nearly 70% of total Hl.
This work has been supported by Grants PB85-0096 and PB88-0233 from the Comisión Interministerial de C1encia y Tecnologfa