Regulated expression of a chimeric histone gene introduced into mouse fibroblasts.

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Introduction of the mouse histone H3.1 gene into tk- mouse L cells by cotransfection with the herpesvirus thymidine kinase gene resulted in the production of two mRNAs from the transfected gene, one with a normal 3' end and the other one with a longer 3'-untranslated region, ending at site X, which was poly(A)+. In contrast, the endogenous histone H3.1 gene only produced a single mRNA. The cryptic poly(A)+ site was only used when the histone H3.1 gene was transfected. To localize possible downstream cryptic processing sites, the hairpin loop at the end of the histone gene was deleted and the resulting deletions were introduced into L cells. Two major mRNAs were produced from this gene, one ending at site X and the major one ending at site Y, which was located 150 nucleotides before site X. Transcription extended downstream of site X efficiently in the endogenous gene, as judged by the extent of transcription of downstream sequences in isolated nuclei. Transcription extended downstream of site X in the transfected gene because the placement of a normal histone 3' end downstream of site X resulted in transcripts that ended at site X and longer transcripts that ended with the new histone 3' end. These results indicate that transcription may normally proceed a substantial distance past the hairpin loop (greater than 500 bases). The formation of the different 3' ends in these transfected genes was due to competition between different processing mechanisms.

“…1B and C) (2). We also obtained cell lines transfected with the H3.21(221) and H3.21(614) genes, in which these genes were expressed in much smaller amounts.…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Expression of mouse histone genes: transcription into 3' intergenic DNA and cryptic processing sites downstream from the 3' end of the H3 gene.

Chodchoy¹,

Levine²,

Sprecher³

et al. 1987

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“…The S-phase-specific histone gene transcription appears to be regulated by cisacting DNA elements present 5' of histone genes (3,23,31,40,42). Also, histone mRNAs appear to have a sequence element(s) for stabilization of the mRNAs during the S phase of the cell cycle (1,7,29,39,44). The 3' noncoding region of the histone mRNA has a highly conserved hairpin structure that appears to influence S-phase-specific stability of histone mRNAs (7, 39, 44).…”

mentioning

confidence: 99%

S-phase-specific transcription regulatory elements are present in a replication-independent testis-specific H2B histone gene.

Hwang¹,

Chae²

1989

The testis-specific H2B histone (TH2B) gene is expressed in pachytene spermatocytes during meiotic prophase I in the absence of any significant DNA synthesis. Unlike somatic histones, synthesis of testis-specific histones is not affected by inhibitors of DNA synthesis. A genomic rat TH2B gene was cloned by using a DNA fragment derived from TH2B cDNA as a probe. Expression of the cloned TH2B was investigated by gene transfer experiments. From these studies, we found that the 5' upstream region of the cloned TH2B gene contained S-phase-specific transcription elements which regulated expression of a reporter gene in an S-phase-specific manner. The S-phase-regulatory element was found to be located in two regions containing CCAAT elements between -153 and -110 base pairs (bp) and an octamer element (ATTTGCAT) between -109 and -84 bp. The two regions were required for a maximal stimulation of transcription of the cloned TH2B gene in S phase. On the other hand, only the octamer element was reported be important for the S-phase-specific transcription of human H2B gene. Since the synthesis of the TH2B histone is independent of DNA synthesis and specific for pachytene spermatocytes in vivo, the presence of the S-phase-specific transcription regulatory elements in the TH2B gene is surprising.During S phase in most eucaryotic cells, newly replicated DNA is assembled into chromatin by histone proteins (36). Therefore, there is a great demand for histones during S phase, when DNA replication occurs. Cells meet the demand for histone proteins by two different mechanisms: increased expression of histone genes and stabilization of histone mRNAs during the S phase (19,29,30,35,41,44). In this manner, cells achieve more than a 10-fold increase in histone mRNA during the S phase. The coupling of histone and DNA synthesis has been well established, but the mechanism of coupling is not clearly understood. Recently attention has been focused on the molecular mechanisms of the cell cycle-dependent expression of histone genes and the stabilization of mRNAs during S phase. The S-phase-specific histone gene transcription appears to be regulated by cisacting DNA elements present 5' of histone genes (3,23,31,40,42). Also, histone mRNAs appear to have a sequence element(s) for stabilization of the mRNAs during the S phase of the cell cycle (1,7,29,39,44). The 3' noncoding region of the histone mRNA has a highly conserved hairpin structure that appears to influence S-phase-specific stability of histone mRNAs (7, 39, 44).There are histone variants which are cell cycle-independent and cell type specific. One of the cell cycle-independent variants is the human H3.3, which has an intron and encodes polyadenylated mRNA (46). The best-known examples of cell type-specific histones are the avian erythrocyte-specific H5 (11, 45) and the mammalian testis-specific histones (4, 27). During spermatogenesis, testis-specific histones (TH1, TH2A, and TH2B) are synthesized in the mammalian testis in the absence of DNA replication, and these histones replace ...

“…Here we show that the effect of the 3' sequences is to confer a higher efficiency of processing on the histone transcripts from chromosome 3, suggesting that a substantial portion of the transcripts from the genes on chromosome 13 must be degraded in the nucleus and never reach the cytoplasm. and the histone gene constructs, using calcium phosphate precipitation as described previously (1,6). Chinese hamster ovary (CHO) cells were cotransfected with pSV2neo (30) and the histone gene constructs, using the Polybrene procedure (5) as described previously (17).…”

mentioning

confidence: 99%

The efficiency of 3'-end formation contributes to the relative levels of different histone mRNAs.

Liu¹,

Levine²,

Skoultchi³

et al. 1989

Self Cite

Sequences at both the 5' and 3' ends of mouse histone genes contribute to the expression of individual genes. The 3' sequences required for high expression of the mouse H2a-614 gene are the same as the sequences required for 3'-end formation. When these sequences were substituted for the 3' end of the poorly expressed H2a-291 gene, expression of the H2a-291 gene was increased fivefold. A 65-nucleotide fragment containing the H2a-614 3' processing signal increased expression of the H2a-291 gene when it was placed in the proper orientation downstream of the H2a-291 3' end. The only mRNAs that accumulated from this gene ended at the H2a-291 3' end, which suggests that the transcript is sequentially processed. In an in vitro processing system, the different histone 3' ends showed different processing efficiencies, which correlated with their expression in cells. These results suggest that the efficiency of processing is important in determining the steady-state levels of individual mouse histone mRNAs.The steady-state concentrations of an mRNA are determined by three factors: (i) rate of transcription, (ii) efficiency of processing and transport to the cytoplasm, and (iii) half-life in the cytoplasm. There are numerous examples of the importance of the rate of transcription and half-life to the steady-state concentrations of an mRNA. Much less is known about the efficiency of RNA processing and transport of RNA to the cytoplasm.The replication-dependent histone genes are unique among eucaryotic genes in that they lack intervening sequences and code for mRNAs that are not polyadenylated. Instead, histone mRNAs end in a stem-loop structure, consisting of a 6-base stem and 4-base loop, which has been highly conserved in evolution (4, 15). The 3' end of histone mRNA is formed in a processing event that requires a U7 small nuclear ribonucleoprotein (snRNP) and at least one other component (10,19,22,23). The levels of histone mRNA are regulated during the cell cycle by multiple mechanisms, including alterations in transcription rate, efficiency of 3'-end formation, and changes in mRNA stability (reviewed in references 21 and 26).In mice, the histone genes are present in two clusters on chromosomes 3 and 13 (20). The individual genes on chromosome 3 account for a much higher proportion of the histone mRNA than do the histone genes on chromosome 13 (13), although both sets of genes are regulated coordinately with DNA synthesis during the cell cycle (8, 13). We have recently shown that sequences both 5' and 3' to the genes on chromosome 3 contribute independently to the high level of expression (17). Here we show that the effect of the 3' sequences is to confer a higher efficiency of processing on the histone transcripts from chromosome 3, suggesting that a substantial portion of the transcripts from the genes on chromosome 13 must be degraded in the nucleus and never reach the cytoplasm. and the histone gene constructs, using calcium phosphate precipitation as described previously (1,6). Chinese hamster ovary (CHO) cells we...