May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
An insulator–like sequence acts as a negative regulatory element in between the mouse Shsp/B–crystallin enhancer and Mkbp/HspB2 promoter.
Author Affiliations & Notes
  • S.K. Swamynathan
    Laboratory of Molecular & Developmental Biology, National Eye Institute, Bethesda, MD
  • J. Piatigorsky
    Laboratory of Molecular & Developmental Biology, National Eye Institute, Bethesda, MD
  • Footnotes
    Commercial Relationships  S.K. Swamynathan, None; J. Piatigorsky, None.
  • Footnotes
    Support  none
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 2250. doi:
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      S.K. Swamynathan, J. Piatigorsky; An insulator–like sequence acts as a negative regulatory element in between the mouse Shsp/B–crystallin enhancer and Mkbp/HspB2 promoter. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2250.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Mouse small heat shock protein Shsp/αB–crystallin is expressed abundantly in the lens and moderately in the heart and skeletal muscle, while the related, closely linked, divergently transcribed Mkbp/HspB2 is expressed at a low level in the heart and the skeletal muscle. An intergenic enhancer (–436/–257 bp relative to transcription start site of Shsp/αB–crystallin) regulates the muscle and lens activity of the Shsp/αB–crystallin promoter, but not that of Mkbp/HspB2 promoter. Here, we have studied the molecular basis of this regulation. Methods: The –836/–628 bp sequence between the enhancer and the Mkbp/HspB2 promoter was aligned with the known CTCF (vertebrate insulator binding protein) binding insulator sequences. DNaseI footprint analysis and electrophoretic mobility shift assays (EMSAs) were used to study the interaction of nuclear factors with this fragment. The enhancer blocking ability of the –836/–628 bp fragment was tested in transient transfection assays by cloning it on either or both the sides of the SV40 enhancer in pGL3–Control plasmid, and in stable transformation assays using K–562 erythroleukemia cells by cloning it in between the globin HS2 enhancer and the γ–globin promoter driving NeoR gene. Results: Comparison of the –836/–628 bp sequence with the well characterized CTCF–binding insulator sequences revealed striking similarities with CTCF recognition sites. DNaseI footprint analysis showed extensive protection within these sites. In transient transfection assays using circular plasmids, the –836/–628 bp fragment suppressed the SV40 promoter activity by ∼50% only when present on both the sides of the enhancer. By contrast, in stably transformed K–562 cells, the –836/–628 bp fragment suppressed the globin promoter activity by about 4–5 fold when present on either side of the enhancer. EMSAs demonstrated that CTCF does not interact with any of the several oligonucleotides tested spanning the –836/–628 bp fragment while an unknown nuclear factor interacted in a sequence specific manner. Conclusions:The Mkbp/HspB2 promoter is shielded from the influence of the nearby Shsp/αB–crystallin enhancer by the newly identified negative regulatory element within the –836/–628 bp fragment. Coupled with prior observations on the tissue specificity of the Shsp/αB–crystallin promoter and orientation–specificity of the enhancer, the –836/–628 bp negative regulatory element largely explains the adaptive changes in gene regulation accompanying the functional diversification of these two related genes.

Keywords: crystallins • genetics • gene/expression 
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