Interaction of a lens cell transcription factor with the proximal domain of the mouse gamma F-crystallin promoter.

Abstract

The elements regulating lens-specific expression of the mouse yF-crystallin gene were examined. Here we show that mouse yF-crystallin sequences -67 to +45 contain a low basal level of lens-specific promoter activity and that sequences -67 to -25, which are highly conserved among different y-crystallin genes, are able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also show that nuclear factors from lens and nonlens cells are able to form different complexes with sequences centered at -46 to -36 and demonstrate that binding of the factor from lens cells correlates with lens-specific promoter activity of the mouse -yF-crystallin gene.The vertebrate lens is a transparent spheroidal structure consisting of essentially two cell types: terminally differentiated fiber cells, which are longitudinally arrayed and make up the main body of the lens, and epithelial cells, which are mitotically active and form a monolayer at the anterior surface. In mammals, terminal lens fiber cell differentiation is characterized by expression of the y-crystallins, a group of closely related polypeptides of =20 kDa (3-5, 7, 14, 15). The exact function of these proteins is unknown, although it is generally believed that they play a critical role in determining the optical properties of the transparent lens.In mice, the -y-crystallins are encoded by six closely linked genes located on chromosome 1 (18, 21). The different members of this gene family are coordinately activated at the onset of lens fiber cell differentiation but are temporally and spatially regulated during development, resulting in differences in the relative abundance of individual transcripts in different regions of the lens (2, 17). Insight into the mechanisms regulating expression of the mouse y-crystallins has come primarily from investigations of the mouse yF-crystal- or no activity in vitro, contain sufficient information for imparting lens fiber cell specificity, while homologous and heterologous enhancers are able to act in concert with these sequences to modulate the spatial pattern of gene expression within the lens.In the present study, we have examined the proximal sequences of the mouse -yF-crystallin promoter for determinants of lens specificity. We show that 5'-flanking sequences -67 to +45 contain lens-specific promoter activity and that the interval -67 to -25, which defines the proximal domain, is able to function as a strong transcriptional activator when duplicated and placed upstream of the TATA box. We also demonstrate that lens-specific promoter activity correlates with the ability of sequences centered at -46 to -36 to bind a lens cell nuclear factor and report that this factor, or its binding activity, is not detectable in various cell types of nonlens origin. MATERIALS AND METHODSPlasmid constructions. All recombinant plasmids containing the bacterial chloramphenicol acetyltransferase (CAT) gene (cat) were based on the parent vector pSVoATCAT (12, 16). Plasmid -y226-CAT, which carries the cat...

Developmental regulation and cell type‐specific expression of the murine γF‐crystallin gene is mediated through a lens‐specific element containing the γF‐1 binding site

“…5C,D). Three of the strains also showed distinct patterns of lacZ staining in variable subregions of the brain (for yF-1, respectively (Liu et al, 1991). Mutated nucleotides within the DR segments of constructs y226DRMI-CAT and y226DRM2-CAT are shaded.…”

“…1). The rationale behind this study stemmed from our previous observations that sequences -67 to -25 are required for lens-specific promoter activity and that tandem duplication of these sequences renders the Dy67 promoter segment about 100-fold more active than 767 in cultured chicken lens cells, but not in other non-lens cell types (Liu et al, 1991).…”

“…Within this interval, we have previously shown that sequences centered at -46 to -36 bind a chicken lens cell nuclear factor, yF-1, and that promoter mutations that block yF-1 binding also drastically reduce promoter activity as measured transiently in cultured chicken lens cells (Liu et al, 1991). To investigate whether the sequence conservation extending upstream of the yF-1 binding site is also of functional significance, we generated and assayed promoter-constructs bearing mutations within this region.…”

“…Transient expression studies in cultured chicken lens cells showed that these 5' flanking sequences contain two upstream enhancers located between -392 and -278 and -226 and -121, as well as a proximal promoter region between -67 and +45 (Lok et al, 1989;Liu et al, 1991). Basal activity of the proximal promoter depends upon binding of a lens cell nuclear factor, yF-1, to sequences centered between -46 and -36 (Liu et al, 1991).…”

“…Transient expression studies in cultured chicken lens cells showed that these 5' flanking sequences contain two upstream enhancers located between -392 and -278 and -226 and -121, as well as a proximal promoter region between -67 and +45 (Lok et al, 1989;Liu et al, 1991). Basal activity of the proximal promoter depends upon binding of a lens cell nuclear factor, yF-1, to sequences centered between -46 and -36 (Liu et al, 1991). The proximal enhancer is necessary for maximum lens specific promoter activity in vitro (Lok et al, 1989); however, sequences -171 to + 45 are adequate to direct gene expression to central nuclear fiber cells of the adult lens, but to a temporally more restricted subpopulation of cells than that obtained upon inclusion of the native yF-crystallin enhancers or a heterologous enhancer derived from the aA-crystallin gene (Yu et al, 1990).…”