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

Abstract

We have previously shown that the tissue specificity determinant of the chicken ~l-crystallin gene lies 3' of position -100 (Hayashi et al. 1985). Since the promoter of the gene (81-crystallin promoter) did not show any tissue specificity, we examined various segments of the ~l-crystallin gene for a tissue-specific enhancer activity by placing each segment downstream of a heterologous transcriptional unit coding for chloramphenicol acetyltransferase (CAT) and by transfecting chicken tissues in primary culture. We found that a segment spanning the third intron bears a strong lens-specific enhancer activity. This "~l-crystallin enhancer" activates transcription from the 81-crystallin promoter 20-to 40-fold in lens cells and to various degrees with other promoters. Deletion analysis of the enhancer region indicated that it covered nearly 1 kb but did not indicate clear-cut boundaries. For its enhancer effect the core region of 120 bp and associations with certain adjoining regions were required. Removal of the enhancer from the gene totally abolished ~l-crystallin expression, and reinsertion of the enhancer in either upstream, internal, or downstream positions restored expression. We conclude that the ~l-crystallin enhancer is an essential and major determinant for lens-specificity of 81-crystallin expression.

Genetic and epigenetic mechanisms of gene regulation during lens development

“…The entire δ1-crystallin locus was found to be methylated in non-lens cells while the locus was hypomethylated in lens cells expressing δ1-crystallin (Sullivan et al, 1989). The 5' flanking sequence of the δ1-crystallin gene is only weakly active in transfections and lens specific expression was found to depend on an enhancer located in the third intron (Hayashi et al, 1987). The intron 3 enhancer was dissected into three fragments, B3, B4 and B5 which do not have enhancer activity alone but synergistically interact to form the functional enhancer.…”

Two mechanisms in the action of repressor δEF1: binding site competition with an activator and active repression

“…Chicken embryo lens cells (Hayashi et al 1987) were transfected with 0.5 mg of a reporter plasmid (0.1 pmole) and 0.1 pmole of an effector pCMVX plasmid carrying a form of dEF1 cDNA, and total DNA was adjusted to 1 mg with pUC19 DNA. Luciferase activity was measured 24 h after transfection, and normalized to the value obtained with insert-less effector plasmid.…”

Puzzle of crystallin diversity in eye lenses

“…Transfection (Hayashi et al, 1987;Thomas et al, 1990;Goto et al, 1990) and transgenic mouse (Kondoh et al, 1987) experiments have established the importance of transcriptional events for controlling 6-crystallin gene expression in lens and non-lens tissues. For example, transgenic mice expressed a fusion gene composed of a 62-crystallin promoter fragmenthacterial chloramphenicol acetyltransferase reporter genel62-crystallin enhancer (from intron 3) in the lens, neuroretina and brain (Purkinje cells of the cerebellum), reminescent of the expression pattern of the ASLl62-crystallin gene in the chicken embryo (Li et al, 1993a(Li et al, , 1993b; see cover immunofluorescence photomicrograph of this issue).…”