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Cell-cycle regulation of histone gene expression
Cell, Vol. 45, 471-472, May 23, 1986, Copyright
0 1986 by Ceil Press
Cell-Cycle Regulation of Histone Gene Expression Daniel Schtimperli lnstitut fur Molekutarbiologie der Universitat Zurich Honggerberg 8093 Zurich Switzerland
II
The histone proteins in higher eukaryotes can be grouped into three major classes: (i) the predominant, replicationdependent variants whose expression is tightly linked to the S phase of the cell cycle; (ii) replication-independent or replacement variants, which accumulate preferentially in nondividing, terminally differentiated tissues; and (iii) tissue-specificvariants, such as the erythroid cell-specific histone H5 found in birds and amphibia. In the chicken, and probably also in mammals, these histone gene classes differ considerably from each other in their genomic organization and mode of expression. The replication-independent and the tissue-specific histone variants seem to be encoded by single copy genes that are not linked to any other histone genes and are transcribed into polyadenylated mRNAs (Krieg et al., Nucl. Acids Res. 77,619-627, 1983; Brush et al., Mol. Cell. Biol. 5, 1307-1317, 1985). In contrast, the replication-dependent histone genes represent a family consisting of about 5-20 genes per type of histone protein, which are relatively well conserved even in the silent bases of the protein coding body. A total of 42 core and Hl histone genes-probably all or most of the replication-dependent histone genes of the chicken-have been mapped to four nonoverlapping chromosome regions @ ‘Andrea et al., Mol. Cell. Biol. 5, 3108-3115, 1985). These genes are not organized into the conserved repeats seen in the sea urchin and Drosophila histone genes. The replication-dependent histone genes differ from most other genes transcribed by RNA polymerase II in that they contain no introns and their mRNAs carry a conserved palindromic sequence instead of a poly(A) tail at the 3’ end (reviewed in Stein et al., Histone Genes: Structure, Organization and Regulation, John Wiley & Sons, New York, 1984). The periodic fluctuations in synthesis of these replication-dependent histones in proliferating cells are paralleled by similar fluctuations in the levels of the corresponding mRNAs. This regulation must involve both transcriptional and post-transcriptional factors, since the transcription rate of histone genes during S phase is only 2-5 times higher than that during Gl or G2; this variation alone cannot fully account for the usually much greater fluctuations in histone mRNA levels. Furthermore, although the genes are expressed with different efficiencies, the relative ratios of the transcripts from individual histone genes seem to remain constant throughout the cell cycle, indicating that all genes are controlled in parallel, perhaps by a common mechanism (Graves et al., J. Mol. E3iol. 783,179-194,1985). Thus, any explanation for the regulation of histone genes during the cell cycle must
inireview
account for the fact that more than forty different genes for all five histone types are controlled coordinately by an interplay of transcriptional and post-transcriptional processes. Transcriptional Regulation of ~ist~~~ Gene Expression during the Cell Cycle Early work on yeast histone genes suggested that the periodic transcription of an H2A-H25 gene pair during the cell cycle required an ARS (autonomous replicating sequence) downstream from the H gene (Osley and Hereford, PNAS 79, 7689-7693, 1 however, indicate that the ARS is dispensable for cell cycle regulation and that the regulatory sequences are in fact localized in the 5’ promoter region separating the divergently transcribed H2A-H2B genes (Osley et al., Cell, this issue). The human histone H4 gene is transcribed 3 to lo-fold more efficiently in nuclear extracts from S phase HeLa cells than in extracts from non-S phase cells (Heintz and Roeder, PNAS 87, 2713-2717, 1984). Mutational analyses indicate that promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site are required for maximal transcription and are recognized preferentially in extracts from S phase cells (Hanly et al., Mol. Cell. Biol. 5,380-389, 1985). Thus, ceil cycle regulation of human histone gene transcription appears to involve periodic fluctuations in specific transcription factors, which, in the case of the H4 gene, interact with upstream transcription signals in the promoter Several groups have introduced cloned histone genes into animal cells by DNA-mediated gene transfer and observed faithful cell cycle regulation of gene expression (Luscher et al., PNAS 82, 4389-4393, 1985; Alterman et al., Mol. Cell. Biol. 5, 2316-2324, 1985; Capasso and Heintz, PNAS 82, 5622-5626, 1985). Using a similar experimental approach, Artishevsky et al. (Science 230, 1061-1063, 1985) have shown that a DNA fragment derived from a hamster H3 gene, which contains about 1 kb of 5’flanking sequences as well as sequences encoding the first 20 amino acids of the gene product, confers cell cycle regulation to the coding sequence of the bacterial neomycin resistance gene. Post-transcriptional Regulation of xpression during the Cell Cycle Post-transcriptional regulation during the cell cycle or in response to changes in cell growth conditions is not a property unique to histone genes; such regulation is also observed for genes encoding ‘“housekeeping” functions, such as enzymes of nucleotide metabolism (Merrill et al., Mol. Cell. Biol. 4, 1777-1784, 1984; Leys et al., J. Cell Biol. 99, 180-187, 19849, and for cellular oncogenes (Blanchard et al., Nature 377,443-4451985; ‘i?eisman, Cell 42, 889-902, 1985). It is of interest to know whether this regulation acts at the level of histone m~NAstabil~ty or at some earlier step, possibly even prior to the appearance of newly synthesized mRNA in the cytoplasm. Preformed histone mRNAs are destabilized rapidly
Ceil 472
when cells are treated with inhibitors of DNA synthesis. The only exception is novobiocin, which is also the only inhibitor tested that does not affect deoxyribonucleotide metabolism (Graves and Marzluff, Mol. Cell. Biol. 4, 351-357, 1984). This destabilization of histone mRNA can be counteracted by treating cells with protein synthesis inhibitors. In fact, protein synthesis inhibitors can superinduce histone mRNAs by increasing histone mRNA stability both in the presence and absence of DNA synthesis (Stimac et al., Mol. Cell. Biol. 4,2082-2090, 1984; Sive et al., Mol. Cell. Biol. 4, 2723-2734, 1984). These results suggest that histone mRNA stability is sensitive to changes in DNA synthesis (or deoxyribonucleotide metabolism) and that a short-lived protein may be involved in histone mRNA degradation. However, it has not yet been shown that the regulatory mechanisms detected in these inhibitor experiments are the same ones controlling histone mRNA levels during a normal cell cycle. It has been suggested that histone biosynthesis is subject to some form of autoregulation (Butler and Mueller, Biochim. Biophys. Acta 294,481-496, 1973; Sariban et al., Mol. Cell. Biol. 5, 1279-1286, 1985); this also remains to be tested. Direct measurements of histone mRNA stability throughout an undisturbed cell cycle have been hampered by the small size of the mRNA pool outside of S phase. However, recent pulse-chase experiments with mouse erythroleukemia cells fractionated into cell cyclespecific stages show that the turnover of mouse H3 mRNAs follows a two-component decay curve in both S and G2 phase cells (Alterman et al., Mol. Cell. Biol. 4, 123-132, 1984). The half-lives of the two components are similar in both phases, but the less stable component appears to constitute a greater proportion of the H3 mRNA pool in G2 than in S. In contrast, the incorporation of 3Huridine into cytoplasmic H3 mRNA in 15 or 60 min pulses rises sharply during the transition from Gl to S and then begins to fall as cells approach G2. These results suggest that the changes in histone mRNA levels during the cell cycle are not due to differential stability of mature cytoplasmic mRNA (possibly the more stable component), but rather to some nuclear process that determines whether newly synthesized nuclear histone transcripts (possibly the less stable component) are exported to the
cytoplasm. This is highly reminiscent of the post-transcriptional regulation of dihydrofolate reductase gene expression, which was found to operate through stabilization of transcripts in the nucleus (Leys et al., op. cit.). Gene transfer experiments have revealed that some, if not all, of the information for the post-transcriptional control of mouse H4 mRNA concentrations during the cell cycle is located at the 3’ end of the gene. lnsertion of a 463 bp fragment containing the 3’terminal half of a mouse W# gene, including 230 bp of spacer sequences, into a transcription unit controlled by the SV40 early promoter results in regulated expression of S/40-H4 fusion RNA in transformed cells of a temperature-sensitive mouse mastocytoma cell line (Lijscher et al., op. cit.). In contrast, longer transcripts that do not acquire histone-specific 3’ ends, but instead terminate downstream at sequences specified by a SV40 polyadenylation site, are not regulated. It is intriguing that although the same histone gene sequences are present in both the short and the long RNAs, only the short, specifically processed, RNA is regulated. The mature histone mRNA is generated from longer precursor transcripts by a post-transcriptional processing event involving the conserved 3’ terminal palindrome, some additional spacer sequences, and U7 snRNPs (Birchmeier et al., PNAS 87, 1057-1061, 1984; reviewed in Birnstiel et al., Cell 47, 349-359, 1985). It will be interesting to determine whether the target for cell cycle regulation is congruent with this highly conserved processing signal and whether the post-transcriptional regulation is effected at the level of mRNA processing. Of course, the conserved palindrome might also be important for other functions; e.g., mRNP formation, nucleocytoplasmic transport, or stabilization of the mature histone mRNA. Cell cycle regulation could operate at any of these levels. With the help of gene transfer experiments and in vitro systems, the analysis of the regulation of histone gene expression during the eukaryotic cell cycle has moved from phenomenology to reconstitutive science. Together with work on other growth- or cell cycle-regulated genes, recent research in this field has also helped to emphasize the importance of post-transcriptional gene regulation for normal cellular processes.
0 1986 by Ceil Press
Cell-Cycle Regulation of Histone Gene Expression Daniel Schtimperli lnstitut fur Molekutarbiologie der Universitat Zurich Honggerberg 8093 Zurich Switzerland
II
The histone proteins in higher eukaryotes can be grouped into three major classes: (i) the predominant, replicationdependent variants whose expression is tightly linked to the S phase of the cell cycle; (ii) replication-independent or replacement variants, which accumulate preferentially in nondividing, terminally differentiated tissues; and (iii) tissue-specificvariants, such as the erythroid cell-specific histone H5 found in birds and amphibia. In the chicken, and probably also in mammals, these histone gene classes differ considerably from each other in their genomic organization and mode of expression. The replication-independent and the tissue-specific histone variants seem to be encoded by single copy genes that are not linked to any other histone genes and are transcribed into polyadenylated mRNAs (Krieg et al., Nucl. Acids Res. 77,619-627, 1983; Brush et al., Mol. Cell. Biol. 5, 1307-1317, 1985). In contrast, the replication-dependent histone genes represent a family consisting of about 5-20 genes per type of histone protein, which are relatively well conserved even in the silent bases of the protein coding body. A total of 42 core and Hl histone genes-probably all or most of the replication-dependent histone genes of the chicken-have been mapped to four nonoverlapping chromosome regions @ ‘Andrea et al., Mol. Cell. Biol. 5, 3108-3115, 1985). These genes are not organized into the conserved repeats seen in the sea urchin and Drosophila histone genes. The replication-dependent histone genes differ from most other genes transcribed by RNA polymerase II in that they contain no introns and their mRNAs carry a conserved palindromic sequence instead of a poly(A) tail at the 3’ end (reviewed in Stein et al., Histone Genes: Structure, Organization and Regulation, John Wiley & Sons, New York, 1984). The periodic fluctuations in synthesis of these replication-dependent histones in proliferating cells are paralleled by similar fluctuations in the levels of the corresponding mRNAs. This regulation must involve both transcriptional and post-transcriptional factors, since the transcription rate of histone genes during S phase is only 2-5 times higher than that during Gl or G2; this variation alone cannot fully account for the usually much greater fluctuations in histone mRNA levels. Furthermore, although the genes are expressed with different efficiencies, the relative ratios of the transcripts from individual histone genes seem to remain constant throughout the cell cycle, indicating that all genes are controlled in parallel, perhaps by a common mechanism (Graves et al., J. Mol. E3iol. 783,179-194,1985). Thus, any explanation for the regulation of histone genes during the cell cycle must
inireview
account for the fact that more than forty different genes for all five histone types are controlled coordinately by an interplay of transcriptional and post-transcriptional processes. Transcriptional Regulation of ~ist~~~ Gene Expression during the Cell Cycle Early work on yeast histone genes suggested that the periodic transcription of an H2A-H25 gene pair during the cell cycle required an ARS (autonomous replicating sequence) downstream from the H gene (Osley and Hereford, PNAS 79, 7689-7693, 1 however, indicate that the ARS is dispensable for cell cycle regulation and that the regulatory sequences are in fact localized in the 5’ promoter region separating the divergently transcribed H2A-H2B genes (Osley et al., Cell, this issue). The human histone H4 gene is transcribed 3 to lo-fold more efficiently in nuclear extracts from S phase HeLa cells than in extracts from non-S phase cells (Heintz and Roeder, PNAS 87, 2713-2717, 1984). Mutational analyses indicate that promoter elements between 70 and 110 nucleotides upstream from the transcription initiation site are required for maximal transcription and are recognized preferentially in extracts from S phase cells (Hanly et al., Mol. Cell. Biol. 5,380-389, 1985). Thus, ceil cycle regulation of human histone gene transcription appears to involve periodic fluctuations in specific transcription factors, which, in the case of the H4 gene, interact with upstream transcription signals in the promoter Several groups have introduced cloned histone genes into animal cells by DNA-mediated gene transfer and observed faithful cell cycle regulation of gene expression (Luscher et al., PNAS 82, 4389-4393, 1985; Alterman et al., Mol. Cell. Biol. 5, 2316-2324, 1985; Capasso and Heintz, PNAS 82, 5622-5626, 1985). Using a similar experimental approach, Artishevsky et al. (Science 230, 1061-1063, 1985) have shown that a DNA fragment derived from a hamster H3 gene, which contains about 1 kb of 5’flanking sequences as well as sequences encoding the first 20 amino acids of the gene product, confers cell cycle regulation to the coding sequence of the bacterial neomycin resistance gene. Post-transcriptional Regulation of xpression during the Cell Cycle Post-transcriptional regulation during the cell cycle or in response to changes in cell growth conditions is not a property unique to histone genes; such regulation is also observed for genes encoding ‘“housekeeping” functions, such as enzymes of nucleotide metabolism (Merrill et al., Mol. Cell. Biol. 4, 1777-1784, 1984; Leys et al., J. Cell Biol. 99, 180-187, 19849, and for cellular oncogenes (Blanchard et al., Nature 377,443-4451985; ‘i?eisman, Cell 42, 889-902, 1985). It is of interest to know whether this regulation acts at the level of histone m~NAstabil~ty or at some earlier step, possibly even prior to the appearance of newly synthesized mRNA in the cytoplasm. Preformed histone mRNAs are destabilized rapidly
Ceil 472
when cells are treated with inhibitors of DNA synthesis. The only exception is novobiocin, which is also the only inhibitor tested that does not affect deoxyribonucleotide metabolism (Graves and Marzluff, Mol. Cell. Biol. 4, 351-357, 1984). This destabilization of histone mRNA can be counteracted by treating cells with protein synthesis inhibitors. In fact, protein synthesis inhibitors can superinduce histone mRNAs by increasing histone mRNA stability both in the presence and absence of DNA synthesis (Stimac et al., Mol. Cell. Biol. 4,2082-2090, 1984; Sive et al., Mol. Cell. Biol. 4, 2723-2734, 1984). These results suggest that histone mRNA stability is sensitive to changes in DNA synthesis (or deoxyribonucleotide metabolism) and that a short-lived protein may be involved in histone mRNA degradation. However, it has not yet been shown that the regulatory mechanisms detected in these inhibitor experiments are the same ones controlling histone mRNA levels during a normal cell cycle. It has been suggested that histone biosynthesis is subject to some form of autoregulation (Butler and Mueller, Biochim. Biophys. Acta 294,481-496, 1973; Sariban et al., Mol. Cell. Biol. 5, 1279-1286, 1985); this also remains to be tested. Direct measurements of histone mRNA stability throughout an undisturbed cell cycle have been hampered by the small size of the mRNA pool outside of S phase. However, recent pulse-chase experiments with mouse erythroleukemia cells fractionated into cell cyclespecific stages show that the turnover of mouse H3 mRNAs follows a two-component decay curve in both S and G2 phase cells (Alterman et al., Mol. Cell. Biol. 4, 123-132, 1984). The half-lives of the two components are similar in both phases, but the less stable component appears to constitute a greater proportion of the H3 mRNA pool in G2 than in S. In contrast, the incorporation of 3Huridine into cytoplasmic H3 mRNA in 15 or 60 min pulses rises sharply during the transition from Gl to S and then begins to fall as cells approach G2. These results suggest that the changes in histone mRNA levels during the cell cycle are not due to differential stability of mature cytoplasmic mRNA (possibly the more stable component), but rather to some nuclear process that determines whether newly synthesized nuclear histone transcripts (possibly the less stable component) are exported to the
cytoplasm. This is highly reminiscent of the post-transcriptional regulation of dihydrofolate reductase gene expression, which was found to operate through stabilization of transcripts in the nucleus (Leys et al., op. cit.). Gene transfer experiments have revealed that some, if not all, of the information for the post-transcriptional control of mouse H4 mRNA concentrations during the cell cycle is located at the 3’ end of the gene. lnsertion of a 463 bp fragment containing the 3’terminal half of a mouse W# gene, including 230 bp of spacer sequences, into a transcription unit controlled by the SV40 early promoter results in regulated expression of S/40-H4 fusion RNA in transformed cells of a temperature-sensitive mouse mastocytoma cell line (Lijscher et al., op. cit.). In contrast, longer transcripts that do not acquire histone-specific 3’ ends, but instead terminate downstream at sequences specified by a SV40 polyadenylation site, are not regulated. It is intriguing that although the same histone gene sequences are present in both the short and the long RNAs, only the short, specifically processed, RNA is regulated. The mature histone mRNA is generated from longer precursor transcripts by a post-transcriptional processing event involving the conserved 3’ terminal palindrome, some additional spacer sequences, and U7 snRNPs (Birchmeier et al., PNAS 87, 1057-1061, 1984; reviewed in Birnstiel et al., Cell 47, 349-359, 1985). It will be interesting to determine whether the target for cell cycle regulation is congruent with this highly conserved processing signal and whether the post-transcriptional regulation is effected at the level of mRNA processing. Of course, the conserved palindrome might also be important for other functions; e.g., mRNP formation, nucleocytoplasmic transport, or stabilization of the mature histone mRNA. Cell cycle regulation could operate at any of these levels. With the help of gene transfer experiments and in vitro systems, the analysis of the regulation of histone gene expression during the eukaryotic cell cycle has moved from phenomenology to reconstitutive science. Together with work on other growth- or cell cycle-regulated genes, recent research in this field has also helped to emphasize the importance of post-transcriptional gene regulation for normal cellular processes.