Cloning, characterization and regulation of mouse H1 histone genes

Abstract

In this thesis I have characterized certain aspects of the structure and regulation of the H1 histone genes encoding the H1{dollar}\sp0{dollar}, H1a and H1c subtypes.;The promoter region of both mouse and human H1{dollar}\sp0{dollar} genes was found to contain an AC box (AAACACA) previously implicated in the cell cycle regulation of H1 gene expression. Sequence comparisons of the H1{dollar}\sp0{dollar} promoter regions and the related avian H5 gene with all sequenced replication-dependent H1 genes reveal that the latter promoter region also contains a CAAT box (CCAAT) that is not present in H1{dollar}\sp0{dollar}/H5 promoters.;Previous results in our laboratory showed that two H1{dollar}\sp0{dollar} mRNAs are rapidly induced during MEL cell terminal differentiation. To determine the mechanism of differential regulation of the two mRNAs, their half lives were measured by actinomycin D inhibition experiments at several times during MEL cell differentiation. The results indicate that both mRNAs are quite stable before and during the period of their rapid induction, and even during the period of decline of the short mRNA. The long half life of the short H1{dollar}\sp0{dollar} mRNA in presence of actinomycin D suggests that actinomycin D inhibits the synthesis of an unstable component which may be involved in the degradation of the short mRNA during the later stages of differentiation.;The mouse H1.3 histone gene, previously cloned and sequenced in our laboratory, was identified to encode the H1a subtype.;The H1c gene produces two mRNAs: a cell cycle regulated, 0.7 kb, poly A{dollar}\sp{lcub}-{rcub}{dollar} mRNA that ends in a conserved stem-loop sequence, and a non-cell cycle regulated, 1.7kb, poly A{dollar}\sp{lcub}+{rcub}{dollar} mRNA. Regulation of the poly A{dollar}\sp{lcub}+{rcub}{dollar} mRNA form was studied in cultured MEL cells, where it is rapidly induced and then subsequently down-regulated upon exposure to HMBA.;Nuclear run-on transcription experiments with set of probes lying along the H1c gene showed that in uninduced MEL cells, which produce mostly the short poly A{dollar}\sp{lcub}-{rcub}{dollar} mRNA, transcription of the H1c gene terminates/attenuates in a region (the T/A region) lying between the two mRNA 3{dollar}\sp\prime{dollar} end processing sites. In differentiating MEL cells, transcription termination appears to be largely inhibited, leading to a build-up of RNA polymerase molecules upstream of the T/A region. This may explain why the level of the H1c poly A{dollar}\sp{lcub}-{rcub}{dollar} mRNA declines. Surprisingly, only a two-fold increase in run-on transcription products downstream of the T/A region was observed in differentiating cells in which the longer poly A{dollar}\sp{lcub}+{rcub}{dollar} form is induced more than ten fold. RNase protection experiments showed that the level of a long nuclear H1c transcript ending beyond the polyadenylation site increases during induction of the poly A{dollar}\sp{lcub}+{rcub}{dollar} mRNA. This transcript is present only in the nucleus, is non-polyadenylated and presumably is a precursor for the poly A{dollar}\sp{lcub}+{rcub}{dollar} mRNA. Actinomycin D inhibition experiments were used to estimate possible changes in the half life of H1c poly A{dollar}\sp{lcub}+{rcub}{dollar} mRNA during the period of its rapid induction and decline in differentiating MEL cells. The half life of the H1c poly A{dollar}\sp{lcub}+{rcub}{dollar} mRNA was found to be longer than 12 hours in both uninduced MEL cells and even in differentiating MEL cells where the mRNA level rapidly declines. (Abstract shortened by UMI.).