To identify DNA sequence elements from the human apolipoprotein B (apoB) gene required for high-level, correct tissue-specific expression in transgenic mice, we made several constructs that included one or more of the key regulatory elements that were previously characterized with cultured liver-derived and intestine-derived cell lines. Our data show that the apoB promoter alone (-898 to +121) is not sufficient to direct transcription in transgenic mice. An enhancer located in the second intron is absolutely required to specify transcription by the homologous apoB promoter in the livers of transgenic mice; this enhancer does not direct transcription in the small intestines. Thus, the elements controlling transcriptional activation of the apoB gene in the liver and the intestine in vivo are distinct and separable. Analysis of the DNase I hypersensitivity of the integrated human transgenes in various lines of expressing and nonexpressing mice suggests that the formation of DH4, a strong hypersensitive site in intron 2, may be a prerequisite for hepatic expression of the apoB gene. Nuclear matrix association regions (MARs) of the apoB gene may play a role in transgene expression. Constructs including MAR sequences displayed higher levels of expression than those lacking them. However, these MARs did not completely insulate the associated transgenes from position effects.

Transcription

Nuclear matrix

10.1128/mcb.14.4.2243-2256.1994

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Thyroid hormone receptor and albumin gene expression in Xenopus laevis ovary and liver

Alan R. Brooks

I have isolated a full length cDNA clone encoding a thyroid hormone receptor from a Xenopus laevis mature oocyte cDNA library. This receptor binds thyroid hormone with high affinity when expressed in COS cells. Expression from the thymidine kinase promoter containing an artificial thyroid hormone response element could be induced by thyroid hormone in the presence (but not in the absence) of this receptor. Thyroid hormone receptors isolated from other organisms are known to act directly upon gene expression at the level of transcription by binding to regulatory sequences within thyroid hormone responsive genes. My results with the Xenopus thyroid hormone receptor are consistent with this mode of action. There appear to be several rare transcripts in X. laevis ovary which hybridised to the thyroid hormone receptor cDNA clone. These transcripts could not be detected in eggs, suggesting that they are located in the follicle cells rather than the oocyte itself. Several rare transcripts which hybridised to the cDNA clone are also present in early embryonic stages. These differently sized but clearly related transcripts are suggestive of differential splicing. Synthetic RNA prepared from the entire cDNA translates very poorly in vitro and this is almost certainly due to the presence of several AUG codons upstream of the long open reading frame. Thyroid hormone is known to play a vital role in inducing the many biochemical and physiological changes that occur during amphibian metamorphosis. It is almost certain that some of these changes are the result of the activation and repression of specific genes at a transcriptional level. I have demonstrated that transcription of the Xenopus albumin genes is activated at the onset of metamorphosis. The albumin genes are therefore candidates for genes whose expression is activated by thyroid hormone. I have tested the response of a cloned X. laevis 68Kda albumin gene to thyroid hormone in a transient transfection assay. From this experiment I tentatively conclude that thyroid hormone does increase expression of the 68Kda albumin gene, at the level of the messenger RNA, and that this increase requires the presence of the co-transfected (Xenopus) thyroid hormone receptor. The liver-specific expression of albumin genes from Xenopus and other organisms is known to be controlled at the level of transcription by liver-specific trans-acting factors which interact with regulatory sequences upstream of these genes. In vivo, in the developing liver, transcription of the Xenopus albumin genes might require the presence of both tissue-specific trans-acting factors and the thyroid hormone receptor complex.

Thyroid hormone receptor

Growth-hormone-releasing hormone receptor

PAX8

Hormone response element

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Journal of Biological Chemistry (1996)

Miguel Abal Abd Alla Aberle Hermann Magnus Abrahamson Abu-Elneel Kawther

10.1016/s0021-9258(17)46099-6

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