Cloning and functional characterization of Roaz, a zinc finger protein that interacts with O/E-1 to regulate gene expression: implications for olfactory neuronal development

Abstract

We have identified a protein, Rat O/E-1-associated zinc finger protein (Roaz), that plays a role in regulating the temporal and spatial pattern of olfactory neuronal-specific gene expression. This protein functions by interacting with the olfactory factor O/E-1 and modulating its transcriptional activity. Roaz, isolated via a yeast two-hybrid screen, encoded a protein containing 29 C2H2 zinc fingers of the TFIIIA type. The Roaz mRNA was found in brain, eye, olfactory epithelium, spleen, and heart. In situ hybridization data indicated that Roaz was expressed in the basal layer, consisting of neural precursor cells and immature sensory neurons of the olfactory epithelium, but not in the mature receptor cells. We showed that the Roaz protein bound specifically to O/E-1 by using the yeast two-hybrid system. The two proteins formed a stable complex in coimmunoprecipitation and in vitro binding assays. Introduction of Roaz and O/E-1 into cells containing an olfactory promoter-driven luciferase reporter demonstrated that Roaz abolished O/E-1-mediated transcriptional activation. We propose that the function of Roaz is to modulate negatively the transactivational activity of O/E-1 and to act as a switch protein in the coordination of olfactory sensory neuron differentiation.

Fig. 1.

Structure and sequence of Roaz cDNA clones.A, Schematic representation of Roaz and various cDNA clones isolated from cDNA library screens and RACE-PCR. Eachshaded box represents one zinc finger structure.RoazD86 is the original clone isolated from the yeast two-hybrid screen. The other clones represent isolates from subsequent cDNA library screens and RACE-PCR, as indicated in the text. The 3′-UTR of Roaz is not shown in this diagram. B, cDNA sequence and predicted open reading frame of Roaz. The amino acid sequences of zinc finger structures areunderlined. Figure continues.

Fig. 1.

Structure and sequence of Roaz cDNA clones.A, Schematic representation of Roaz and various cDNA clones isolated from cDNA library screens and RACE-PCR. Eachshaded box represents one zinc finger structure.RoazD86 is the original clone isolated from the yeast two-hybrid screen. The other clones represent isolates from subsequent cDNA library screens and RACE-PCR, as indicated in the text. The 3′-UTR of Roaz is not shown in this diagram. B, cDNA sequence and predicted open reading frame of Roaz. The amino acid sequences of zinc finger structures areunderlined. Figure continues.

Fig. 2.

Expression pattern of Roaz and O/E-1.A, RT-PCR of Roaz expression in tissues with the use of a gene-specific oligonucleotide. M, 1 kb marker;B, forebrain; E, eye; H, heart; I, intestine; K, kidney;Lg, lung; Lv, liver; O, olfactory epithelium; S, spleen; T, testis; P, 5 ng of Roaz-containing plasmid DNA as a positive control; G, 100 ng of genomic DNA.B, RT-PCR of O/E-1 and RNA Polymerase II.

Fig. 3.

In Situ hybridization. Digoxigeninin situ hybridization of Roaz, O/E-1, and OMP in adult olfactory epithelium. The pseudostratified epithelium is composed of the sustentacular cell layer (SL), the basal/immature cells (B/IN) and mature olfactory receptor neurons (ORN). Sections were hybridized with Roaz antisense probe (A, E), Roaz sense probe (B, F), OMP antisense probe (C), and O/E-1 antisense probe (D). Images in A and B are low-magnification coronal images of the mouse epithelium. Scale bar, 200 μm. Images inC–F are from the same sections at high magnification.

Fig. 4.

Biochemical characterization of Roaz/O/E-1 interaction. A, Left, Demonstration of Roaz interaction with O/E-1 by affinity chromatography. Purified proteins (GST or GST fusions) were mixed with whole-cell protein isolated from HEK293 cells transfected with pCIS-O/E-1 or pCIS vector. Bound protein was extracted with 30 μl of sample buffer, and 20 μl was fractionated on a 10% SDS-PAGE and detected by Western blotting with the use of anti-O/E-1 antibody (JH1132); one-twentieth of the input was loaded for comparison. Right, Interaction of O/E-1 with full-length Roaz protein. Proteins were purified as described above except that pCIS-GST-Roaz was transfected. Protein yields for the full-length construct were consistently lower than those observed for the pCISRoazD86 vector. B, Demonstration of Roaz interaction with O/E family proteins by affinity chromatography. Purified proteins (GST or GST fusions, 9 μg) were mixed with whole-cell proteins (400 μg) isolated from HEK293 cells transfected with pCIS-O/E-1, pCIS-O/E-2, or pCIS-O/E-3. Bound proteins were extracted with 60 μl of sample buffer. One-fifth (12 μl) was fractionated on a 10% SDS-PAGE and detected by Western blotting with the use of the anti-O/E-1 antibody (JH1132); one-twentieth of the input was loaded for comparison. C, Coimmunoprecipitation of Roaz and O/E-1 with anti-O/E-1 antibody (JH865). Whole-cell extract from HEK293 cells transfected with different constructs was mixed with anti-O/E-1 antibody (JH865) or preimmune serum and protein A-Sepharose. After being washed, resins were extracted with 50 μl of sample buffer; 20 μl was loaded on a 10% SDS-PAGE and detected after Western blotting with the use of anti-XPress antibody. One-fortieth of the input was loaded as a reference.

Fig. 5.

Localization of interaction domain of O/E-1 with Roaz. Yeast strain Y190 expressing GAL4(TA)-RoazD86 was transformed with constructs encoding C-terminal deletions (cnd), N-terminal deletions (nnd) of O/E-1, orOED5 (a partial sequence of O/E-1 cDNA isolated from original yeast two-hybrid screen) as GAL4(DB) fusions. Double transformants were grown in Leu/Trp drop-out medium before assaying for β-galactosidase activity. The strength of interaction for an individual mutant is expressed as a percentage relative to that of intact proteins, which was set at 100% and corresponded to 43 ± 7 U. All measurements were determined from at least four independent colonies. Numbers in parentheses indicate the corresponding starting and ending amino acids of the O/E-1 sequence. Previously characterized protein motifs in O/E-1 are indicated. Zf, Zinc finger; nls, nuclear localization signal; rhlh, repeat helix-loop-helix motif.

Fig. 6.

Transcriptional activation of O/E binding site-containing reporter constructs. A, Schematic representation of reporter constructs containing the type III adenylyl cyclase promoter (pGL-AC3R/B) and the OMP promoter (pGL-OMP) is shown. The filled boxes represent O/E binding sites, and thearrows represent the transcriptional start sites.Luc, Luciferase; AAAA, polyadenylation sequence. B, Luciferase activity assay of the O/E family proteins on ACIII and OMP promoters. Left panel shows inhibitory effect of Roaz on O/E-1-mediated transactivation.Lane 1, pCIS transfection; lane 2, pCIS-O/E-1 transfection; lane 3, pCIS-GST-Roaz transfection; lane 4, pCIS-O/E-1 and pCIS-GST-Roaz cotransfection. Right panel shows a similar effect of Roaz on O/E-2 and O/E-3-mediated activation. The data are derived from six independent transfection experiments. Relative luciferase activity was calculated by dividing the relative light units (RLU) of each transfection with that of the pCIS expression vector control. The absolute luciferase activity for each reporter construct cotransfected with pCIS vector was pGL-AC3R/B, 126,542 RLU; pGL-OMP, 156,119 RLU.C, Roaz interaction with O/E-1 protein/DNA complex. The pCIS-XPRoazD86 or pCIS-transfected whole-cell extracts (120 ng) were mixed with 312.5 ng of pCIS-O/E-1-transfected whole-cell lysate in an EMSA assay, using synthetic O/E binding site as a probe, and fractionated on a 6% SDS-PAGE. Quantitative assessment of binding revealed a 76% reduction of complex formation in the presence of XPRoazD86 (lane 1).

Fig. 7.

O/E-1 and Roaz interactions with the SV40 early promoter. A, Top, The pGL-P plasmid contains a 200 bp SV40 early promoter lacking the enhancer sequence.SV-P, SV40 early promoter. Bottom, Transcriptional activation of the pGL-P reporter construct was assessed after cotransfection with pCIS-GST-Roaz and/or the pCIS-O/E expression vectors. The absolute luciferase activity for each reporter construct cotransfected with pCIS vector waspGL-B, 22,975 RLU; pGL-P, 304,316 RLU. B, DNA binding ability of Roaz and O/E-1 on SV40 early promoter. GST-purified O/E-1 or Roaz (200 ng) was mixed with the indicated probes (p1–p5) in an EMSA, as shown in this figure. Shifted complexes were fractionated on a 1.5% agarose gel in 0.25× TBE running buffer.

Fig. 8.

A model for the transregulatory function of Roaz.