Histone mRNAs Do Not Accumulate during S Phase of either Mitotic or Endoreduplicative Cycles in the ChordateOikopleura dioica

Abstract

Metazoan histones are generally classified as replication-dependent or replacement variants. Replicationdependent histone genes contain cell cycle-responsive promoter elements, their transcripts terminate in an unpolyadenylated conserved stem-loop, and their mRNAs accumulate sharply during S phase. Replacement variant genes lack cell cycle-responsive promoter elements, their polyadenylated transcripts lack the stem-loop, and they are expressed at low levels throughout the cell cycle. During early development of some organisms with rapid cleavage cycles, replication-dependent mRNAs are not fully S phase restricted until complete cell cycle regulation is achieved. The accumulation of polyadenylated transcripts during this period has been considered incompatible with metazoan development. We show here that histone metabolism in the urochordate Oikopleura dioica does not accord with some key tenets of the replication-dependent/replacement variant paradigm. During the premetamorphic mitotic phase of development, expressed variants shared characteristics of replication-dependent histones, including the 3 stem-loop, but, in contrast, were extensively polyadenylated. After metamorphosis, when cells in many tissues enter endocycles, there was a global downregulation of histone transcript levels, with most variant transcripts processed at the stem-loop. Contrary to the 30-fold S-phase upregulation of histone transcripts described in common metazoan model organisms, we observed essentially constant histone transcript levels throughout both mitotic and endoreduplicative cell cycles.

Plasticity of histone modifications across the invertebrate to vertebrate transition: histone H3 lysine 4 trimethylation in heterochromatin

“…Animal and cell culture Oikopleura dioica were obtained and cultured as described (Chioda et al 2004). NIH3T3 cells were cultured in DMEM supplemented with 5% FCS (Sigma), 10 000 IU/ml of penicillin, 10 000 mg/ml of streptomycin and 2 mmol/L L-glutamine in a 5% CO 2 humidified atmosphere at 37 C. Where indicated, cells were cultured in the presence of 20 mg/ml of a-amanitin (Sigma) for 24 h. Drosophila embryonic Kc cells were cultured in Insect-Xpress medium (BioWhittaker) supplemented with penicillin and streptomycin as above and 1.2 mmol/L L-glutamine at 25 C. Cells were seeded on glass coverslips at least 24 h before fixation or in situ run on transcription.…”

“…Immunofluorescent labelling of whole mount O. dioica samples was performed as described (Chioda et al 2004). Cultured cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT), permeabilized with 0.5% Triton X-100 in PBS containing 0.1 mol/L glycine and blocked with 3% BSA in PBS containing 0.1% Tween 20 (PBT).…”

Histone H4 post‐translational modifications in chordate mitotic and endoreduplicative cell cycles

“…dioica were obtained and cultured as described [Chioda et al, 2004]. NIH3T3 and HeLa S3 cells were cultured in DMEM containing 5 and 10% FCS (Sigma), respectively, in a 5% CO 2 humidified atmosphere at 378C.…”

Phosphorylation of the histone H3.3 variant in mitosis and meiosis of the urochordate Oikopleura dioica

“…Both genes contain introns, are found outside histone clusters and their transcripts are polyadenylated, i.e. they lack the stem loop sequence in the 3 0 UTR that couples the translation of replication-dependent histone mRNAs to S-phase (Chioda et al 2004). Many studies examining histone modifications have concluded that the combination of modifications on nucleosomes may be the key to determining chromatin structure and function in a variety of organisms (reviewed in Margueron et al 2005).…”

“…There is evidence, however, that some histone modifications and their dynamics may have different interpretations and roles across divergent evolutionary groups of organisms (Manzanero et al 2000, Spada et al 2005a). Our previous finding, that H3.3 becomes a major H3 variant in the postmetamorphic stages of O. dioica (Chioda et al 2004), led us to investigate its state of phosphorylation at different developmental stages. To determine whether phosphorylation of H3.3 is a conserved temporal and spatial marker of specific mitotic stages at the invertebrate/vertebrate transition we used an H3.3S31P-specific antibody .…”