The SCAN domain is a protein-protein interaction domain. (A) Schematic diagram of the mammalian two-hybrid assay. Specific interactions between SCAN domains from various zinc finger genes were tested by using a mammalian two-hybrid assay system. Plasmids encoding a SCAN domain fused with the yeast GAL4 DNA-binding domain (GAL4 BD) and a SCAN domain fused with the herpes simplex virus VP16 transactivating region (VP16 AD) were transfected into COS-7 cells, along with a reporter plasmid (pG5CAT) containing five consensus GAL4 binding sites and an E1b minimal promoter upstream of the CAT gene. Measured levels of CAT activity can be correlated with the relative affinity of an interaction between hybrid proteins. (B) The SCAN domain of ZNF174 can interact with itself in a mammalian two-hybrid assay system. COS cells were transfected with 2 μg of reporter construct and 10 μg each of the indicated GAL4 BD and VP16 AD expression plasmids. Reporter construct pE1b-CAT (lane 5) is identical to pG5-CAT except that it lacks GAL4 binding sites. ZNF174 in either the GAL4 BD or VP16 AD vector refers to the SCAN domain region (aa 41 to 128) of this gene, and JUN-LZ and FOS-LZ refer to two-hybrid constructs which contain the leucine zipper regions of these genes. CAT activity is shown in counts per minute. (C) Specificity of SCAN-SCAN protein interactions. The table indicates the relative amounts of CAT activity seen when specific pairwise combinations of SCAN domains are tested for interaction in the mammalian two-hybrid assay, normalized to self-association of the ZNF174 SCAN domain. The results observed with different pairs of SCAN domains are presented relative to this amount. Results from ZNF191-GAL4 are not included here due to the endogenous transcriptional activating activity of ZNF191 when fused to the GAL4 DNA-binding domain. Data are representative of at least three independent experiments. Asterisks indicate that the pooled data from these particular experiments were analyzed by the nonparametric Wilcoxon signed rank test and found to be significantly different (P < 0.05) from the level of activity seen with ZNF174-GAL4 and ZNF174-VP16.

Selective oligomerization via the SCAN domain. Schematic diagrams of the proteins generated by in vitro translation are shown on the left side of the figure. Volumes shown in the input lane are half the amounts used for the immunoprecipitation assays. (A) The SCAN domain mediates self-association. Analysis of [³⁵S]methionine-labeled AU1 epitope-tagged full-length ZNF174[1-408] cotranslated with [³⁵S]methionine-labeled nontagged shorter forms of ZNF174. Lanes 1 to 6, ZNF174[1-172]; lanes 7 to 12, ZNF174[136-408]. (B) α-Helical character of the SCAN domain is required for oligomerization. [³⁵S]methionine-labeled nontagged wild-type SCAN domain and a mutant form of the SCAN domain in which leucines at positions 44 and 45 were changed to prolines (mutant L>P). Lanes 1 to 6, ZNF174[1-250, wild type]; lanes 7 to 12, ZNF174[1-250, mutant L>P]. (C) Selective interaction of SCAN domains. [³⁵S]methionine-labeled nontagged heterologous SCAN domains. Lanes 1 to 6, ZnF20[1-284]; lanes 7 to 12, ZNF191[1-140]; lanes 13 to 18, FPM315[1-129].

Amino acid alignment of SCAN domains. (A) The amino acid sequences of the SCAN domains from the following genes are aligned: ZNF174 (GenBank accession number U31248), ZNF165 (X84801), ZNF191 (U68536, also known as ZNF24 and KOX 17 [P17028]), ZNF192 (U57796, also known as LD5-1), ZNF193 (U62392), ZNF202 (AF027219), ZNF213 (AF017433, also known as CR53), (ZnF20 (AF011573, also known as p18 [Z21707]), ZnFPH (taken from cosmid Q25 sequence, Z68344), CTfin51 (D10630, also known as Zfp-38 [X63747] and RU49 [U41671]), FPM315 (D88827), mMZF-2 (AB007407), KIAA0427 (AB007887), TRFA (L32162, also known as 3c3 and p20), Zfp94 (U62906), Zfp95 (U62907), Zfp96 (U62908), Zfp-29 (X55126), and SRE-ZBP (Z11773). A consensus sequence for the SCAN domain is presented underneath the alignment. Residues that are invariant among all the sequences are indicated within the consensus sequence in boldface type. Identical residues fitting the SCAN consensus have been boxed and are shaded in dark gray, conserved amino acid differences are indicated by light-gray shading. Putative α-helical regions are indicated (H) below the sequence. The helical predictions were made using the Predict Protein program (33a). (B) Unrooted phylogenetic tree relating SCAN domain sequences. All genes shown in the alignment (Fig. 1A) were used to calculate this tree. Amino acid sequences were aligned by using MacVector. Alignments were loaded into CLUSTALX, which calculated an unrooted tree and all branch lengths by using the neighbor-joining method of Saitou and Nei, which calculates distances (percent divergence) between all pairs of sequence from a multiple alignment and then applies the neighbor-joining method to the distance matrix. The resultant tree was produced in Phylip format. TreeViewPPC, version 1.5.3, was used to convert the tree into graphical format. The species of each SCAN domain is indicated to the right of the gene name as an “h” for human or “m” for mouse. (C) α-Helical character of the SCAN domain. CD spectra of the intact SCAN domain (aa 3 to 128) of ZNF174 (▴) and a specifically mutated form of the intact domain in which two conserved leucines at positions 44 and 45 are mutated to prolines (▵). (D) Gel filtration chromatography of the ZNF174 SCAN domain. A Bio-Rad SE-100/17 high-performance liquid chromatography column was used to determine the oligomeric state of the SCAN domain in 67 mM phosphate, 150 mM NaCl, and 1 mM DTT at pH 7.0. Chromatograms are plotted as the A₂₈₀ versus the elution time. The elution profile for the protein standards is indicated by a dotted line, and the molecular masses shown are in daltons. The elution profile for the SCAN domain is indicated by a solid line. The domain elutes as a single species with an estimated molecular weight of 41,980, consistent with the formation of elongated dimers or globular trimers.

Structural features of the SCAN domain required for self-association. A schematic representation of ZNF174 is shown at the top. The regions of the SCAN domain that were cloned into GAL4 BD and VP16 AD expression plasmids are indicated below. The first construct contains the full-length wild-type SCAN domain. The next two constructs represent smaller regions of the SCAN domain, with each one expressing individual predicted α-helices. The last construct contains the full-length SCAN domain in which two conserved leucines have been mutated to prolines. α-helical regions within the SCAN domain are indicated (H) below each construct. Relative levels of CAT activity between pairs of the expression constructs when tested by two-hybrid analysis are shown on the right.