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First published online August 17, 2007
Journal of Experimental Biology 210, 3126-3132 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.004150

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Functional identification of an osmotic response element (ORE) in the promoter region of the killifish deiodinase 2 gene (FhDio2)

L. López-Bojórquez^*, P. Villalobos, C. García-G., A. Orozco and C. Valverde-R.

Departamento de Neurobiologia Celular y Molecular, Instituto de Neurobiología, Universidad Nacional Autónoma de México, Quéretaro, Qro. CP 76230, México

View larger version (10K): [in this window] [in a new window]   Fig. 1. Putative transcription factor binding sites (TFBS) in the FhDio2. (A) Schematic representation of the 1.3 kb promoter region of FhDio2 showing the localization of putative TFBS. Numbers refer to the position of the starting nucleotide of the element upstream of the transcription start sequence. Black arrows indicate the position of the two putative ORE-binding sites. (B) The sites and the corresponding oligonucleotide sequences of ORE1 and ORE2 are indicated.

View larger version (41K): [in this window] [in a new window]   Fig. 2. In vivo time course of nuclear recruitment of a putative osmotic response element binding protein (OREBP) after hypo-osmotic stress. (A) Translocation (EMSA, using ORE1 and ORE2 oligonucleotides) of putative OREBPs into the nuclei, and (B) their corresponding disappearance from the cytoplasmic compartment. Protein–DNA binding occurred in a biphasic mode: an initial protein translocation 2 h after hypo-osmotic stress, and a second and more intense wave of recruitment 8 h post-challenge. Panels show representative gels from 4–6 replicates. Below each gel is the corresponding quantification (relative intensity; arbitrary units) of the complexes. Notice the difference in the scales for nuclei (A) and cytoplasmic (B) graphs. Values are means ± s.e.m. of three separate quantifications. *P<0.05.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Specific binding of nuclear proteins to ORE. The specificity of nuclear extracts to radiolabeled ORE1 binding under different experimental conditions is shown. Control (not challenged; column 1) and 8 h post in vivo hypo-osmotic stress (column 2); competitive displacement in the presence of a 100-fold excess of unlabeled ORE-1 oligonucleotide (column 3); formation of a nonspecific complex using a random oligonuclotide (column 4), and control without nuclear extract (column 5). The arrow shows the putative OREBP–ORE complex.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Up-regulation of D2 mRNA and enzyme activity after hypo-osmotic stress. (A) D2 enzymatic activity from the in vivo time-course experiment. The increase in D2 activity became significant only 12 h after the osmotic challenge. (B) A separate set of experiments showing mRNA concentrations (filled bars) and D2 activity (open bars). The increase in mRNA precedes the corresponding rise in D2 activity, and attained maximum values 8 h after osmotic stress. For each panel, values are means ± s.e.m. (N=10) of two separate experiments, each in triplicate. *P<0.05 vs control (C).

View larger version (20K): [in this window] [in a new window]   Fig. 5. In vitro temporal course of putative OREBP nuclear binding and D2 activity after hypo-osmotic challenge. To assess whether liver cells could detect and respond directly to osmotic changes, liver explants from seawater-adapted killifish were pooled and randomly distributed into iso-osmotic (control, C) or hypo-osmotic L-15 medium. (A) Temporal course of recruitment of putative OREBP into the nuclear compartment. Significant recruitment can be observed as early as 2 h after challenge and peaks at 4 h post-stress. The lower portion of A shows the quantification (relative intensity) of this response. (B) The associated increment in hepatic D2 activity (filled bars). Values are means ± s.e.m. of four separate experiments, each in triplicate. *P<0.05 vs control (open bars). Normalized D2 activity is expressed in arbitrary units (see Materials and methods).

View larger version (18K): [in this window] [in a new window]   Fig. 6. (A) Genistein in vitro blocks nuclear translocation of putative OREBP. The drug prevents both the translocation of OREBP and the associated increase in D2 activity. Notice that even in those explants that were not challenged, genistein reduced both protein–DNA binding and enzyme activity. Lower portion of A shows the corresponding quantification (relatively intensity) of the complexes. (B) The associated D2 activity (means ± s.e.m. of three separate experiments, each in triplicate). Normalized D2 results are expressed in arbitrary units.