Abstract
Crystallins are a diverse group of abundant soluble proteins that are responsible for the refractive properties of the transparent eye lens. We showed previously that Pax-6 can activate the alphaB-crystallin/small heat shock protein promoter via the lens-specific regulatory regions LSR1 (-147/-118) and LSR2 (-78/-46). Here we demonstrate that retinoic acid can induce the accumulation of alphaB-crystallin in N/N1003A lens cells and that retinoic acid receptor heterodimers (retinoic acid receptor/retinoid X receptor; RAR/RXR) can transactivate LSR1 and LSR2 in cotransfection experiments. DNase I footprinting experiments demonstrated that purified RAR/RXR heterodimers will occupy sequences resembling retinoic acid response elements within LSR1 and LSR2. Electrophoretic mobility shift assays using antibodies indicated that LSR1 and LSR2 can interact with endogenous RAR/RXR complexes in extracts of cultured lens cells. Pax-6 and RAR/RXR together had an additive effect on the activation of alphaB-promoter in the transfected lens cells. Thus, the alphaB-crystallin gene is activated by Pax-6 and retinoic acid receptors, making these transcription factors examples of proteins that have critical roles in early development as well as in the expression of proteins characterizing terminal differentiation.
Highlights
The molecular basis for the specialized expression of crystallin genes has been investigated for some time [7]
We show by DNase I footprinting, antibody/electrophoretic mobility shift assay (EMSA), site-directed mutagenesis, and transient-cotransfection experiments that RAR/RXR heterodimers can interact at retinoic acid-responsive elements (RAREs) within LSR1 and LSR2 and can activate the ␣B-crystallin promoter in lens cells either alone or collaboratively with Pax-6
Northern Blot Hybridization of ␣B-crystallin mRNA—In order to test whether retinoid signaling can induce endogenous ␣B-crystallin gene expression in lens cells, Northern blot hybridizations were performed with total RNA isolated from N/N1003A cells treated with increasing concentrations of RA (Fig. 1)
Summary
Vol 273, No 28, Issue of July 10, pp. 17954 –17961, 1998 Printed in U.S.A. Involvement of Retinoic Acid/Retinoid Receptors in the Regulation of Murine ␣B-crystallin/Small Heat Shock Protein Gene Expression in the Lens*. The ␣B-crystallin gene is activated by Pax-6 and retinoic acid receptors, making these transcription factors examples of proteins that have critical roles in early development as well as in the expression of proteins characterizing terminal differentiation. The refractive properties of the transparent eye lens depend on a diverse group of globular proteins called crystallins that comprise approximately 90% of the water-soluble proteins of this tissue [1, 2]. We show by DNase I footprinting, antibody/electrophoretic mobility shift assay (EMSA), site-directed mutagenesis, and transient-cotransfection experiments that RAR/RXR heterodimers can interact at retinoic acid-responsive elements (RAREs) within LSR1 and LSR2 and can activate the ␣B-crystallin promoter in lens cells either alone or collaboratively with Pax-6
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