Persistence of chromosomal proteins HMG-14/-17 in myotubes following differentiation-dependent reduction of HMG mRNA.

The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both HMG-14 and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cycle-related event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein. These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis.

The expression of chromosomal proteins HMG-14 and HMG-17 during cellular differentiation was studied in cultured mouse myoblasts. During myogenesis the level of both  and HMG-17 mRNA decreased to less than 20% of that found in myoblasts. The down-regulation of HMG-14/-17 mRNA occurred simultaneously with activation of muscle-specific actin mRNA and was not linked to DNA synthesis, indicating that it is a differentiation-, rather than a cell cyclerelated event. Incorporation of radiolabeled lysine into HMG proteins was similar to that into the major histone fractions in that it was significant in myoblasts and undetectable in myotubes. The decrease in mRNA and protein synthesis did not affect the cellular levels of HMG protein.
These results indicate that the regulation of HMG-14/-17 mRNA levels is different from that of the histones and is linked to differentiation rather than to DNA synthesis.
Chromosomal proteins HMG-14 and HMG-1'7 are among the most abundant nonhistone proteins found in the nuclei of higher eukaryotes (1). The two proteins, which have higher affinity for nucleosomes than for DNA (2), bind to the inner side of the nucleosomal DNA (3) potentially altering the interaction between the DNA and the histone octamer. The limited amount of HMG proteins in the nucleus (4,5) confines their presence to a subset of the nucleosomes.
So far, the cellular role of these proteins is not understood.
The data obtained from nuclease digestion (6, 7), nucleosome reconstitution (2, 8), immunofluorescence tions of rapidly dividing myoblasts differentiate into myotubes (14). The process involves a switch in gene expression resulting in both activation and suppression of certain sets of genes (14). Therefore, this system is suited for studies on the cellular role and mode of action of putative regulatory proteins such as HMG-14/-17.
The present study on the levels of HMG-14 and HMG-17 mRNA and protein during various stages of myogenesis is the first examination of the pattern of HMG transcription in a differentiating system. The results indicate that during myogenesis both the mRNA levels and protein synthesis rate decrease without a significant alteration in the cellular protein concentration.
Interruption of DNA synthesis with hydroxyurea resulted in immediate down-regulation of histone mRNA levels without having any effect on HMG synthesis, indicating that the expression of these two classes of nucleosomal proteins are independently regulated. RNA was isolated from cells at various times after addition of differentiation media and analyzed by the Northern procedure. Duplicate blots were prepared, and one was sequentially probed with HMG-17 (0) and (oc-actin (a); fl-actin (V)), the other with HMG-14 (0) and glyceraldehyde-3'-phosphate dehydrogenase (GAPDH) (El) probe. The autoradiograms obtained with these probes were scanned in a Beckman DU8 spectrophotometer and the area under each peak quantified. The percentage of each mRNA during myogenesis is plotted. Note that for HMG-14, HMG-17 and p-actin the maximum level of RNA was observed after 15-39 h in culture. Nuclei were prepared from cells at various days after initiation of myotube formation (numbers at the bottom of the rows), and the HMG prepared by extraction of 1.25 N perchloric acid (PcA). A, HMG proteins resolved by electrophoresis in 20% polyacrylamide acid-urea gels. The left column contains marker proteins isolated from chicken erythrocytes (Eryth). The HMGs are identified by the numbers in the margins of the figures. Days 3 and 5, myoblasts; days 6 and 8, myotube stage. B, Synthesis of HMG-14/-17 and histones in myoblasts and myotubes. Proteins were extracted from nuclei labeled with ['4C]lysine and examined by sodium dodecyl sulfate-gel electrophoresis. The bands the HMG and histones are identified. Note that although the amount of myoblast and myotube protein applied is identical, only the proteins extracted from myoblasts incorporated radioactive lysine. Northern blot analysis of RNA isolated from control and hydroxyurea-treated cells. All tracks were loaded with 10 pg of total RNA and the blots sequentially probed with the various probes. A, myoblasts incubated with or without 10 mM hydroxyurea for the time indicated at the top of the figure. B, myoblasts incubated for 6 h with 10 mM hydroxyurea plus or minus 2 pM actinomycin D. Myoblasts were incubated with or without 10 mM hydroxyurea. At each time point cultures were radiolabeled with ["Cllysine for 1 h and cultures were harvested as described under "Materials and Methods." A, sequential staining with Coomassie Blue and silver. B, autoradiogram of gel following a l-week exposure to Kodak XAR film. -70 "C. removal of the growth factors and then decreased to 50% of maximum at 63 h and to 10% of maximum at 98 h after induction of differentiation. This decrease in HMG-14/-17 mRNA level paralleled the switch from synthesis of P-actin to a-actin and was not due to variation in amount of mRNA applied since the level of another marker, glyceraldehyde-3'phosphate dehydrogenase, exhibited a marked elevation at 98 h (Fig. 1).
HMG and histone Hl protein levels were analyzed during myogenesis by acid urea-gel electrophoresis of 1.25 N perchloric acid cell extracts (Fig. 2A). The location and relative amount of the protein bands were confirmed by Western analysis (data not shown). Densitometric analysis of the proteins resolved on either urea or SDS-containing gels, indicated that for each time point the HMG protein level varied less than 5% when compared to histone Hl (data not shown). Previous studies have found that, although the relative proportion-of the various Hl subfractions changes during myogenesis, the total amount of Hl does not (22,23). Therefore, the constant ratio of Hl to HMG indicates that during myoblast differentiation the levels of HMG protein remain relatively constant.
Next we examined the relative synthesis of histone and HMG proteins in myoblasts and myotubes. Nuclei from l-h pulse-labeled cell cultures were extracted with either 1.25 N perchloric acid to obtain a histone Hl and HMG fraction or 0.4 N H2S04 to obtain a fraction containing all histones and HMGs. Protein extracts were then electrophoresed in an 18% polyacrylamide gel containing sodium dodecyl sulfate. Autoradiograms of the gels (Fig. 2B) show that in fused myotubes the synthesis of both histone and HMG protein was negligible as compared to that seen in myoblasts. These results suggest that the HMG-14 and -17 chromosomal proteins are synthesized in the myoblast stage of differentiation, and they support previous results that indicated the proteins have relatively long half-lives (4, 24). However, it is also possible that the rate of protein degradation changes during differentiation.
During myogenesis the synthesis of the nucleosomal histones is tightly coupled to DNA replication (22,23). To test whether the synthesis of HMG-14 and -17 in myoblasts is similarly linked to DNA synthesis growing cultures were treated with 10 pM hydroxyurea. Under these conditions incorporation of radiolabeled thymidine into trichloroacetic acid-precipitated material was less than 20% of that of untreated myoblasts indicating that synthesis of DNA was blocked. Whereas a 3-h incubation with hydroxyurea decreased the amount of histone H4 mRNA by more than 90%, the HMG-14 and HMG-17 mRNAs were not affected even after a 9-h incubation with hydroxyurea (Fig. 3A). However, inhibition of RNA synthesis by addition of actinomycin D to the hydroxyurea-treated cells drastically reduced the amount of HMG-14/-17 mRNAs (Fig. 3B). These and other' results indicate that HMG mRNAs are not unusually stable and their persistence during the hydroxyurea treatment reflects continued synthesis. HMG and histone protein synthesis under these conditions followed a similar pattern similar to that detected for RNA synthesis (Fig. 4) and -17 synthesis was unaffected during the entire 9-h incubation. Fig. 4A shows that equal amount of protein was applied to each lane. The data indicate that hydroxyurea treatment drastically reduced the synthesis of histone Hl without affecting the synthesis of the HMG proteins.
We conclude that the down-regulation of HMG-14/-17 synthesis observed during myogenesis is a consequence of differentiation and is distinct from that seen with histone during suppression of DNA synthesis and cell cycle inhibition.