In vivo competition of delta-crystallin gene expression by DNA fragments containing a GC box.

Abstract

Expression of the chicken 8-crystallin gene 1 injected into the nuclei of mouse cells is lens specific. Coinjection of GC box-containing DNA fragments from 8-crystallin, simian virus 40 early, and herpes simplex virus type 1 tk promoters effectively suppressed 8-crystallin expression in the lens, but coinjection with DNA fragments not containing the GC box did not. This suppression was likely due to the competition of an Spl-like transcription factor(s) and indicates involvement of the apparently ubiquitous factor(s) in the tissue-specific expression of the 8-crystallin gene.B-Crystallin is the most abundantly produced lens-specific protein of avians. The cloned 8-crystallin gene 1 (17,18), the active entity of two nonallelic genes of chickens (2,16,23), is expressed with a high lens specificity after nuclear injection into mouse cells (9,11,13). By introducing a series of deletions and substitutions in the gene and its flanking sequences ( Fig. 1), we located the DNA region responsible for the lens-specific gene regulation within 136 base pairs (bp) encompassing the transcription initiation site (from nucleotide positions -81 to + 55) (9). Most remarkably, deletion of the segment from -81 to -65 reduced the level of expression 20-fold. This segment contains the GC box sequence ( Fig. 1) which is often part of viral and animal gene promoters as an essential cis-acting element for transcription initiation (5-7, 10, 15). To characterize regulatory interactions of putative trans-acting cellular factors with the cisacting DNA element of the 5-crystallin gene, we analyzed competition between the nuclear-injected gene and various coinjected DNA fragments.First, a 72-bp fragment of the promoter region (from positions -107 to -36) of the B-crystallin gene (fragment A, Fig. 2B) was tested for its effect on b-crystallin expression upon coinjection with the gene. Two test genes were used: one was the 5-crystallin gene on plasmid pSClB (12) and the other was a chimeric gene (Mov-8 gene) on p5Kp-Mov (11) in which the Moloney murine leukemia virus (MoMuLV) long terminal repeat (LTR) (22) was linked to the 5-crystallin gene at the second intron. The former directs the synthesis of a 50-kilodalton polypeptide (Fig. 3A, lane 1), and the latter directs the synthesis of 48 and 51-kilodalton polypeptides (11) (Fig. 3A, lane 3); therefore, their products could be separately analyzed on an immunoblot (Fig. 3A, lane 2). A mixture of both test genes (125 pLg of each per ml) and fragment A (500 ,ug/ml) was injected into the nuclei of mouse lens epithelial cells in primary culture (13). Assuming an average injection volume of 10 fl (1,3,8), we estimated the numbers of injected competitor fragments and each of the test genes to be 6 x 104 and 60 per nucleus, respectively. The injected cells were harvested 48 h later, and expression of the test genes was examined (Fig. 3A, lane 4) (Fig. 3B). It is difficult to estimate the actual number of regulatory-factor molecules which may bind to the fragment A region of the 5-crystallin promoter, but...

Functional cooperation of lens-specific and nonspecific elements in the delta 1-crystallin enhancer.

“…If B3 or core multimers act as enhancers through interaction with trans-acting nuclear components, there may be competition from cointroduced, excess enhancer segments. We set up conditions for the competition by using microinjection, which allowed us to introduce up to 1,000 copies of an enhancer segment into a nucleus (6). For tkCAT genes without enhancers, injection of 100 cells with an average of 60 gene copies per nucleus resulted in reasonable CAT expression after 24 h (Fig.…”

“…These conditions are easily satisfied by microinjection of DNAs into the nucleus. In fact, we were able to demonstrate competition for an Spl-like factor between the GC boxdependent 81-crystallin promoter and a large number of competitor DNA fragments which contained GC boxes which were coinjected (6).…”

Lens-specific enhancer in the third intron regulates expression of the chicken delta 1-crystallin gene.

“…2 into the BamHI site (B) to test for an enhancer activity. (B) Promoter and enhancer-promoter complexes to construct the plasmids, p~CAT carries the region -459 bp to + 57 bp of the al-crystallin gene Ohno et al 1985;Hayashi and Kondoh 1986), shown to be sufficient for full promoter activity (Borr~is et al 1985;Hayashi et al 1985;Das and Piatigorsky 1986;Hayashi et al 1985;K. Goto, S. Hayashi, Y. Ueda, and H. Kondoh, in prep.).…”

Chicken beta B1-crystallin gene expression: presence of conserved functional polyomavirus enhancer-like and octamer binding-like promoter elements found in non-lens genes.

“…This observation agrees with the weak involvement of the Spl sites in the ,B1 promoter as observed in the site-specific mutagenesis experiments. Thus, the transcription of the ,B1 gene differs from that of the 81-crystallin gene in a HeLa extract, in which the same competitor essentially eliminated transcription (13), consistent with the involvement of Spl in expression of the 81-crystallin gene (26).…”