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Related Experiment Videos

The murine Hoxb1 3' RAIDR5 enhancer contains multiple regulatory elements

J R Thompson1, D Y Huang, L J Gudas

  • 1Department of Pharmacology, Cornell University Medical College, New York, New York 10021, USA.

Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research
|December 30, 1998
PubMed
Summary

Retinoic acid (RA) signaling regulates homeobox gene expression. This study identifies conserved elements CE1 and CE2 in the Hoxb1 enhancer that act as negative regulators, with their function dependent on RA receptor gamma and specific protein binding during embryonic development.

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Area of Science:

  • Developmental Biology
  • Molecular Biology
  • Genetics

Background:

  • Homeobox genes are crucial for vertebrate embryonic development, specifying body part identity.
  • Retinoids, particularly retinoic acid (RA), are key signaling molecules that regulate homeobox gene expression.
  • A previously identified RA-inducible enhancer (RAIDR5) 3' to the murine Hoxb1 gene contains conserved regulatory elements.

Purpose of the Study:

  • To investigate the function of two conserved elements, CE1 and CE2, within the Hoxb1 3' enhancer.
  • To determine the protein binding interactions and regulatory roles of CE1 and CE2 in Hoxb1 gene expression.
  • To elucidate the involvement of RA receptor gamma (RARgamma) in the function of these regulatory elements.

Main Methods:

  • Reporter gene assays using beta-galactosidase activity in F9 cells to assess enhancer function.

Related Experiment Videos

  • Gel shift assays to detect DNA element:protein binding complexes in nuclear extracts.
  • UV cross-linking to determine the molecular weight of binding proteins.
  • Analysis using a variant F9 cell line with disrupted RARgamma genes.

    • Main Results:

    • CE1 and CE2 function as negative regulatory elements in cultured F9 cells; mutation of either element increases reporter gene activity.
    • A specific protein complex binds to the Hoxb1 CE2 element, and this binding is dependent on RARgamma.
    • A distinct protein complex binds to the Hoxb1 CE1 element, with binding sensitive to sequence variations conserved across species.
    • Identical CE1 and CE2 binding complexes are found in nuclear extracts from both F9 cells and day 9.0 mouse embryos.

    Conclusions:

    • The CE1 and CE2 elements within the Hoxb1 3' enhancer play significant roles in repressing Hoxb1 gene expression.
    • The function of CE2 as a repressor is mediated by a RARgamma-dependent protein complex.
    • The conserved nature and specific protein interactions of CE1 and CE2 suggest their critical involvement in regulating Hoxb1 gene expression during embryonic development.