During pituitary development, the homeo domain protein GHF-1 is required for generation of somatotropes and lactotropes and for growth hormone (GH) and prolactin (PRL) gene expression. GHF-1 mRNA is detectable several days before the emergence of GH-or PRL-expressing cells, suggesting the existence of a somatotropic progenitor cell in which GHF-1 transcription is first activated. We have immortalized this cell type by using the GHF-1 regulatory region to target SV40 T-antigen (Tag) tumorigenesis in transgenic mice. The GHF-Tag transgene caused developmental entrapment of somatotropic progenitor cells that express GHF-I but not GH or PRL, resulting in dwarfism. Immortalized cell lines derived from a transgenic pituitary tumor maintain the characteristics of the somato/lactotropic progenitor in that they express GHF-1 mRNA and protein yet fail to activate GH or PRL transcription. Using these cells, we identified an enhancer that activates GHF-1 transcription at this early stage of development yet is inactive in cells representing later developmental stages of the somatotropic lineage or in other cell types. These experiments not only demonstrate the potential for immortalization of developmental progenitor cells using the regulatory regions from cell type-specific transcription factor genes but illustrate the power of such model systems in the study of developmental control.

We have analyzed the expression of endogenous histone H4 genes and of a newly introduced H4 gene in 21-Tb cells, a mouse mastocytoma cell-cycle mutant. Endogenous H4 mRNAs were less abundant by a factor of 120-180 in Gl-arrested than in exponentially multiplying cells. However, H4 transcription rates were only decreased by a factor of 3 under these conditions, as determined by in vitro elongation of nascent transcripts. This indicates that post-transcriptional control of histone mRNA levels is important, in accord with published data. We introduced a mouse H4 gene, modified by a 12-base-pair (bp) insertion in its coding sequence, into 21-Tb cells by DNA-mediated gene transfer. The levels of transcripts from this gene were regulated in parallel with those of the endogenous genes. Moreover, fusion of the simian virus 40 (SV40) early promoter to a 463-bp fragment containing the 3'-terminal half of the mouse H4 gene, including 230 bp of spacer sequences, led to the regulated expression of SV40/H4 fusion RNA. However, a small proportion of SV40-initiated transcripts were not processed to histone-specific 3' ends, but extended farther through the downstream Escherichia coli galactokinase gene to a SV40 polyadenylylation site. In contrast to the short SV40/H4 RNA, the levels of these longer transcripts were not reduced in Gl-arrested cells. These resultsshow that sequences in the 3'-terminal part of the H4 gene can regulate gene expression in the cell cycle, presumably at the post-transcriptional level, as long as they are not positioned much more distant from the terminus than normal.Histone biosynthesis in most eukaryotic cells is tightly coupled to nuclear DNA synthesis such that translatable histone mRNAs are present in significant amounts only during the S phase ofthe cell cycle (1-3). Recent studies using synchronized murine or human cells and homologous histone genes as hybridization probes have shown that histone mRNA steady-state levels vary over a 10-to 50-fold range, and similar variations were also observed in cells treated with inhibitors of DNA synthesis (4-9). Where analyzed, histone gene transcription varied only 2-to 5-fold (6-9), suggesting a major contribution of some post-transcriptional mechanism(s) to regulation of histone gene expression during the cell cycle.We are interested in defining the genetic information responsible for cell-cycle regulation of histone gene expression. To this end, we have transformed a temperature-sensitive mouse mastocytoma cell-cycle mutant with a recombinant mouse H4 gene. We describe the faithful regulation of this gene and present evidence indicating that sequences in the 3' part of the H4 gene can effectively control RNA metabolism during the cell cycle. MATERIALS AND METHODSCell Growth and Transformation. The K21 line of P-815-X2 mouse mastocytoma and its heat-sensitive cell-cycle variant 21-Tb (10, 11) were cultured in suspension in medium 1 (12) supplemented with 10% horse serum (Amimed, Basel, Switzerland). Before transfection, =106 cells were immobiliz...

A short histone-like fusion RNA, generated when the RNA 3' processing signal from a mouse histone H4 gene is inserted into a heterologous transcription unit, becomes correctly down-regulated in G1-arrested cells of a temperature-sensitive mouse mastocytoma cell cycle mutant (21-Tb; Stauber et al., EMBO J. 5, 3297-3303 [1986]), due to a specific deficiency in histone RNA processing (Lüscher and Schümperli, EMBO J. 6, 1721-1726 [1987]). In contrast, inhibitors of DNA synthesis, known to stimulate histone mRNA degradation, have little or no effect on the fusion RNA. This RNA can therefore be used to discriminate between regulation by RNA 3' processing and RNA stability, respectively. The fusion RNA is also faithfully regulated in 21-Tb cells arrested in G1 phase by the drug indomethacin or in C127 mouse fibroblasts during a serum starvation experiment. Moreover, nuclear extracts from serum-starved C127 cells show a specific deficiency in a heat-labile component of the histone RNA processing apparatus, similar to that previously observed for temperature-arrested 21-Tb cells. These results suggest that RNA 3' processing is a major determinant for the response of histone mRNA levels to changes in cell proliferation.

Fragments from the 3′ end of a mouse histone H4 gene, when introduced into transcription units controlled by the SV40 early promoter, yield correctly processed RNA with histone‐specific 3′ ends, both in monkey and mouse cell lines. The processed RNA is regulated in parallel with endogenous H4 mRNAs in 21‐Tb cells, a temperature‐sensitive mouse mastocytoma cell cycle mutant that is specifically blocked in G1 phase at the non‐permissive temperature. Mutational analyses of the H4 gene fragment indicate that the minimal sequences for this regulation and for RNA 3′ processing are both contained within the same 80 bp. This fragment contains two histone‐specific, highly conserved sequence elements that are located at the 3′ end of histone mRNA and in the adjacent spacer region, respectively. Our data suggest that the observed cell cycle regulation is achieved either at RNA 3′ processing or at some later step involving the conserved 3′‐terminal sequence element of mature histone mRNA.

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