Rat growth hormone gene introns stimulate nucleosome alignment in vitro and in transgenic mice.

Abstract

Average hepatic expression (mRNA per cell per gene) of a metallothionein-rat growth hormone (rGH) gene with its natural introns was about 15-fold higher than an intronless version when tested in transgenic mice. We examined the idea that intron removal leads to an alteration in chromatin structure that might be responsible for this effect. Using an in vitro chromatin assembly system, we observed that nucleosomes were aligned in a characteristic ordered array over the gene and promoter when all introns were present. Linker histones were necessary for this alignment to occur. In contrast, nucleosome alignment was perturbed in constructs lacking some or all of the introns. A similar disruption of nucleosome alignment was observed when comparing chromatin from livers of transgenic mice carrying rGH transgenes with or without introns. In vitro, sequences at the 3' end of the rGH gene position nucleosomes and facilitate nucleosome alignment upstream; however, nucleosome alignment does not occur on the -3 kb of downstream flanking rat sequence. These observations suggest that signals present in genomic rGH DNA may serve to establish appropriate nucleosome alignment during development and, possibly, to restore nucleosome alignment to the transcribed region after disruption incurred by the passage of an RNA polymerase molecule, thereby facilitating subsequent rounds of transcription.The use of cDNA constructs or heterologous promoters in transgenic mice often leads to poor gene expression, even for constructs that permit efficient gene expression when transfected into cultured cells (1, 2). The rat growth hormone (rGH) gene fused to the mouse metallothionein (mMT) promoter has been studied in some detail. It was shown, in this case, that the effect of introns on expression was at the level of transcription. The transcriptional efficiency in transgenic mouse liver was increased by 10-to 100-fold when the natural rGH introns were included (2). Improvement in the average expression level and the number of mice that gave detectable expression was observed. The general lack of a marked stimulatory effect on transcription when introns were placed at various unnatural locations with respect to the promoter, along with the lack of a stimulatory effect when constructs were transfected into cultured cells, appears to rule out the existence of ordinary enhancers within introns. Moreover, although the first intron (intron A) alone, in its natural location, was able to rescue expression to a level of 50% in transgenic mice, the presence of introns A and B curiously led to lower average expression levels than when no introns were present (3).A possible explanation for these findings is that genomic DNA might contain sequence arrangements that facilitate the packaging of some genes into chromatin (2-6). Thus, unnat-ural sequence arrangements might lead to less well-defined chromatin structures that may be deleterious to either transcription initiation or elongation. To test this idea, we examined the ability of the same set of ...

Transgenic Complementation of Leptin-Receptor Deficiency

“…A rat growth hormone intron (generated by polymerase chain reaction [PCR]) was inserted into the AscI site between the NSE promoter/enhancer and the LEPR-B cDNA (Fig. 1) to enhance transcription of the LEPR-B transgene (28,29). A simian virus 40 (SV40) late polyadenylation signal, present in the pMET7 vector, was 3Ј of the LEPR-B cDNA.…”

Introns Regulate RNA and Protein Abundance in Yeast

“…The question arises: Are the introns directly responsible for increasing gene expression or does their removal act indirectly, by simply derailing the mRNA synthesis assembly line? Some examples in metazoans support a direct role in expression: introns containing transcriptional enhancers have been identified (Sleckman et al 1996) and one group showed that removing introns from a gene disrupts nucleosome binding (Liu et al 1995). There is, however, no consensus that introns serve to increase gene expression.…”

Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer

“…Transcriptional targeting by use of hepatocyte-specific expression cassettes may lead to immunological ignorance or tolerance for the transgene product. [1][2][3] Expression cassettes for hepatocyte-directed transfer have been improved by using new promoterenhancer combinations, [3][4][5][6][7][8][9] inclusion of introns 5,[10][11][12][13] and inclusion of additional transcriptional sequences like scaffold matrix-attached regions (SMAR) and hepatic control regions (HCR). 12,[14][15][16][17][18] However, because different gene transfer vectors, different doses and different transgenes were used in these studies, direct comparative data for different expression cassettes are often lacking and hamper the interpretation of existing literature.…”