In vivo activity of the hepatitis B virus core promoter: tissue specificity and temporal regulation.

The contribution of the hepatitis B virus enhancers I and II in the regulation of the activity of the core and the X promoters was assessed in transgenic mice. Surprisingly, despite the presence of heterologous promoters linked 5' of the X gene, the transgene expression is mostly due to core promoter (Cp) activity present in the X coding sequence. Moreover, the restriction of Cp activity to hepatic tissue required the combined action of both enhancers I and II, whereas the proximity of these two enhancers was insufficient to confer tissue specificity on Xp activity. Furthermore, the liver-specific activity of the Cp was developmentally regulated in an enhancer I-independent manner.     

Cutting edge: distal regulatory elements are required to achieve selective expression of IFN-gamma in Th1/Tc1 effector cells

Using a transgenic approach, we analyzed the contribution of introns located within the IFN-gamma gene and distal regulatory regions to IFN-gamma gene expression. Intron 1 and 3 from the IFN-gamma gene displayed strong enhancer activity. This activity appeared to be dependent upon integration into the genome but resulted in a loss of Th1 selectivity. We also found that distal regulatory elements are not required for high level expression of the human IFN-gamma gene, but rather for cell lineage-specific expression. An 8.6-kb human IFN-gamma transgene was sufficient to yield high level expression but a 191-kb IFN-gamma transgene with approximately 90 kb of flanking 5' and 3' sequence was necessary to achieve both high level and Th1 selective expression of human IFN-gamma.     

Random activation of a transgene under the control of a hybrid hCD2 locus control region/Ig enhancer regulatory element.

Locus control regions such as those of human CD2 and beta-globin differ from classical enhancers in that, whereas the former confer high level, copy-dependent, position-independent expression to linked genes in transgenic mice, the latter do not, expression levels being dependent on the site of integration. We report that the position independence of the CD2 locus control region is modified by coupling it to the immunoglobulin heavy chain enhancer. Whilst in the majority of transgenic lines the Ig heavy chain enhancer has little or no effect on T cell expression of the hCD2 transgene, in others transgene expression is non-specifically extinguished in a proportion of lymphoid cells. The transgenic locus chromatin appears inaccessible to DNase I in these cells, which do not express the gene. Furthermore, mice homozygous for the hybrid hCD2-Ig heavy chain enhancer construct contain T cells with both an active and an inactive transgene. The 'decision' to express or repress the gene appears to be a random process which involves each chromosome separately, occurs at early stages in differentiation and is heritable by daughter cells. These data suggest the possibility that stochastic decisions might control a number of biological processes.     

Molecular analysis of the distal enhancer of the mouse alpha-fetoprotein gene.

The mouse alpha-fetoprotein (AFP) gene is transcribed at high levels in the visceral endoderm of the yolk sac and fetal liver and at much lower rates in the endoderm of the fetal gut. Expression of the gene in vivo requires the presence of at least one of three enhancers which lie in its 5' flanking region. In this report, we establish that the most distal AFP enhancer directed consistent expression of a linked AFP minigene in all three endodermal tissues in transgenic mice. The enhancer is composed of three domains, each of which is essential for full enhancer function by transient transfection assays. DNase I footprinting identified three regions of the enhancer which are protected by human hepatoma nuclear extracts, one of which corresponded to a consensus site for HNF-3 binding. Site-directed mutations in this site caused a 10-fold reduction in enhancer function by transient transfection. In transgenic mice, however, the mutation resulted in sporadic expression of the transgene, dependent on the site of integration. A similar acquisition of position-dependent sporadic expression of the transgene was observed with a mutation in a second protein binding site, despite the fact that this mutation had very little effect on enhancer function as assessed by transient transfection. These studies underscore the value of examining the functions of specific protein binding sites in vivo.     

Homologous and heterologous enhancers modulate spatial expression but not cell-type specificity of the murine gamma F-crystallin promoter

Previous studies have shown that mouse gamma F-crystallin sequences -759 to +45, which include the core promoter and two upstream enhancer elements, contain sufficient information for directing gene expression to terminally differentiated fiber cells of the ocular lens. To investigate the role that proximal sequences of the mouse gamma F-crystallin promoter play in the developmental regulation of gene expression, we generated transgenic mice containing the lacZ gene driven by either mouse gamma F-crystallin sequences -171 to +45, which lack functional enhancers, or a hybrid hamster alpha A-/mouse gamma F-crystallin promoter, which contains the hamster alpha A-crystallin enhancer instead of operational gamma F-crystallin enhancers. In situ analysis of lacZ expression in these mice revealed that the mouse gamma F-crystallin promoter segment -171 to +45, which shows low activity in vitro, is able to direct gene expression to the fiber cells in the nucleus of the lens. However, animals expressing gamma 171-lacZ show both a lower level of expression of the lacZ gene and a narrower pattern of staining in the lens nucleus than mice expressing gamma 759-lacZ, which contains the two enhancer elements located between -392 and -278 and -226 to -123.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sheltering of gamma-globin expression from position effects requires both an upstream locus control region and a regulatory element 3' to the A gamma-globin gene.

Integration position-independent expression of human globin transgenes in transgenic mice requires the presence of regulatory elements from the beta-globin locus control region (LCR) in the transgene construct. However, several recent studies have suggested that, while clearly necessary, such elements are not by themselves sufficient to realize this effect. In the case of the human fetal gamma-globin genes, previous results have indicated that additional regulatory information required for sheltering of gamma-globin transgene expression from position effects may reside downstream from the A gamma gene. To investigate this possibility, we established 17 lines of transgenic mice carrying constructs comprising a micro-LCR (microLCR) element, an A gamma-globin gene fragment, and a variable length of 3' sequence information beyond the A gamma 3' HindIII site. gamma-Globin expression during development was studied in 170 individual F2 progeny from these lines. We find that gamma-globin expression becomes sheltered from position effects when the normally position-sensitive microLCR-A gamma construct is extended by 600 bp beyond the 3' HindIII site to include a previously identified regulatory sequence (the A gamma-globin enhancer), the functional significance of which in vivo had heretofore been unclear. The results suggest that the mechanism whereby an upstream LCR achieves sheltering of globin gene expression from position effects involves cooperation with a gene-proximal regulatory element distinct from the promoter region.