Levels of the tissue-specific linker histone H5 are elevated in mgture erythroid cells as compared with levels in dividing cells of the same lineage. We examined levels of H5 mRNA in relation to the cell cycle in early erythroid cells transformed by avian erythroblastosis virus to determine whether the gene for this unusual histone is S-phase regulated. Northern blotting analyses revealed that during the cell cycle steady-state levels of H5 mRNA remained relatively constant in contrast to levels of the major core and Hl mRNAs which increased approximately 15-fold during S phase. In vitro pulse-labeling experiments involving nuclei isolated from synchronized cells at various stages of the cell cycle revealed that transcription of the H5 gene was not initiated at any particular stage of the cell cycle but was constitutive. In contrast, transcription of the H2A gene(s) initiated in early S phase, was present throughout the DNA replicative phase, and was essentially absent in Gl and G2 phases.The H5 histone gene is unusual in several ways. It is a single-copy gene which is not closely linked to the clustered core and Hi genes in the chicken genome (23). It is expressed only in cells of the erythroid lineage (27,29), and its mRNA is polyadenylated (22). H5 proteins gradually replace Hi histones during maturation of erythroid cells, although fully mature erythrocytes still contain significant levels of Hi proteins. In this work we investigated the regulation of H5 gene transcription in relation to other chicken histone genes during the cell cycle.A large body of evidence indicates that synthesis of core and Hi histones is coincident with DNA synthesis during the cell cycle (11, 35), although Groppi and Coffino (17) showed that histone synthesis in S49 mouse lymphoma cells can also occur in Gi. Nevertheless, the general consensus is that histone protein synthesis is temporally coupled to DNA replication to facilitate the coordinated assembly of nucleosomes.The controlling factors governing histone protein levels in eucaryotic cells are not clearly understood. The molecular cloning of histone genes from a range of eucaryotes has made experiments investigating the content and metabolism of the major histone (Hi, H2A, H2B, H3, and H4) mRNA species possible. Studies with synchronous populations of tissue culture cells obtained by treatment with aphidicolin (19) or hydroxyurea (38), serum deprivation (9), use of cell-cycle mutants (2), and centrifugal elutriation (1) have shown that levels of the major histone mRNA species increase by approximately 10-to 20-fold during S phase. Furthermore, treatment of synchronized cells in S phase with inhibitors of DNA synthesis rapidly reduces levels of the major histone mRNAs (3, 16, 34), indicating a coupling between histone gene expression and DNA replication. Further data indicate that elevated histone mRNA levels in S phase result from an increased rate of transcription and increased mRNA stability (1,19,38).We show in this study that these parameters are not valid for the histone...
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