Regulation of beta-B1 crystallin expression

Crystallins are characterized as highly expressed soluble proteins of the lens. The expression of crystallins is generally regulated at the level of transcription, and in addition there are controls of RNA processing, translation, and protein stability. The lenses of different species contain distinct levels of both shared and unique crystallin members due to variations in the control of their expression.; In this work, I characterize two new beta-crystallin types found only in the lenses of chicken which result from alternative RNA splicing and translation. An extended version of exon four of betaB2-crystallin in chicken produces a protein form with 14 additional amino acids in its linker region. betaA2-crystallin is translated from a second initiation codon present only in chickens, in addition to the first initiation codon used in mammals, resulting in a protein with four fewer amino acids in a flexible N-terminal extension. These forms contribute to the unique diversity of crystallins in the chicken lens.; Translational controls of the level of crystallin protein production are evident. I created transgenic mice which expressed transgene mRNA for betaB2-crystallin in the lens, but this RNA was not translated into protein, indicating an absent translational control required for high-level protein expression of betaB2-crystallin, a predominant soluble protein of the adult rodent lens.; The high-level expression of chicken betaB1-crystallin is controlled at the transcriptional level. The -432/+30 promoter of chicken betaB1-crystallin was found to be capable of expressing transgene mRNA in lenses of transgenic mice at levels equivalent to endogenous betaB1-crystallin. Previous work demonstrated that a smaller promoter (-126/+30) drove expression levels 200-fold lower than -432/+30 in fiber cells. Here, I demonstrate that the -245/+30 promoter drives expression at an intermediate level, indicating the presence of regulatory elements throughout the -432/-126 region controlling high-level expression in lens fiber cells of transgenic mice. However, the transcription of the betaB1 promoter is regulated differently in epithelial cells, annular pad cells, and terminally differentiated fiber cells. In epithelial cells, a smaller promoter, -126/+30 is expressed at the highest levels, and in cultured chicken lens annular pad cells, the -245/+30 promoter expresses most highly. Thus, the -432/+30 promoter can function at full capacity only in lens fiber cells, and is repressed in other lens cell types.; Several transcription factors in the lens contribute to this high level of betaB1-crystallin expression specific to the fiber cells. Prox1, a transcription factor highly expressed in fiber cell nuclei, activates the betaB1-crystallin promoter at sites located in the region of the promoter controlling high-level expression of this gene. Pax-6, highly expressed in epithelium, represses the expression of endogenous betaB1-crystallin if exogenously expressed in fiber cells, and is a major contributor to fiber-cell specificity of betaB1. deltaEF1 is also expressed highly in lens epithelial cells, and also contributes to its lens fiber-cell specific expression by repressing betaB1-crystallin at two promoter elements, at -282 and -206.; While splicing and alternative translational initiation contribute to the diversity of crystallins, the control of high-level expression of the crystallin genes occurs at both the transcriptional and translational levels. Further work will determine the full spectrum of factors contributing to the high-level expression of the crystallins.