Definition of the SCAN Domain

Based on the analysis of a limited number of SCAN genes, the definition of the SCAN domain has varied, resulting in several forms of the domain in online databases.⁷ Following a more complete analysis of the SCAN domains found in the human genome, the definition of the SCAN domain has been refined.⁸ As will be discussed below, the human genome has at least 70 genes containing the SCAN domain.8 An alignment of a select number of these members is illustrated in Figure 1. A more extensive alignment of the highly homologous SCAN domains (as well as other associated domains) can be found online at the Project Website (http://www.scanfamily.org). Based on the sequence alignment, as well as the biochemical properties of the recombinant protein detailed below, the SCAN domain is defined as 84 residues, beginning with E-43 and ending with R-126 of ZNF174. In many sequences, there are proline residues both before and after the domain, helping to delineate the boundaries of the predicted secondary structural elements. Of the 84 residues that comprise the SCAN domain, a remarkable 62 are conserved in more than 50% of the family members.

Figure 1

SCAN domains in the human genome. To illustrate the homology of the SCAN domain, the amino acid sequences of a select number of human SCAN domains were aligned by consensus ClustalW sequence alignment using MacVector 7.1.1 (Genetics Computer Group) with (more...)

The SCAN domain has also been called the “leucine rich region” (LeR).⁹ This is an unfortunate designation because the similar term “leucine rich repeat” is used to describe a different domain found in a functionally distinct group of proteins involved in inflammation and cell death. For example, the caspase recruitment domain (CARD) containing protein CARD4 (also called NOD1) is a CED-4/Apaf-1 family member that activates NF-κB signaling and induces apoptosis.¹⁰ CARD4 contains several putative functional domains, including an N-terminal CARD domain and a C-terminal region consisting of leucine-rich repeats that likely function as a site for interactions with upstream signaling components. To complicate matters further, the leucine-rich repeat superfamily of proteins is quite diverse and includes the Toll-like receptors that are involved in transmembrane signaling.¹¹ Unfortunately, the use of the term “leucine rich region” to describe the SCAN domain, and the related term “leucine rich repeat,” found in mediators of immune responses and apoptosis, has resulted in confusion in the literature.

The SCAN Domain Is a Protein Interaction Motif

Unlike the KRAB and BTB/POZ domains, the SCAN domain does not have transcriptional activation or repression capabilities.^12,13 Findings using both mammalian and yeast two-hybrid systems demonstrate that the SCAN domain is an interaction motif.^12-14 For example, a mammalian two-hybrid assay was used to show that the SCAN domain mediates protein-protein interactions.¹² To demonstrate that the SCAN domain can self-associate, the ability of a ZNF174 SCAN-GAL4 construct to activate a reporter was tested in the presence of a ZNF174 SCAN-VP16 fusion construct. Coexpression of both fusion constructs markedly activated transcription of a GAL4-dependent reporter when compared with that of empty GAL4 or VP16 vectors, or with each of the SCAN domain fusion constructs alone. The SCAN domain did not interact with the leucine zipper motifs of either c-FOS or c-JUN, demonstrating that the SCAN domain did not interact nonspecifically with other transcription factors that contain amphipathic α-helices mediating oligomerization.¹² The mammalian two-hybrid system was also used to demonstrate that self-association of the ZNF174 SCAN domain requires the entire SCAN domain, in that smaller regions of the domain failed to interact in the assay.¹² Similarly, forms of the SCAN domain into which mutations were introduced that disrupted the predicted central helix of the domain failed to interact in the two-hybrid assay. Taken together, these findings suggest that the minimum length functional unit is the entire SCAN domain and that structural integrity of this domain is required for self-association with other SCAN domain partners.

The SCAN domain is capable of mediating protein-protein interactions in the context of an intact member of the family. Studies were done in which a tagged full-length form of ZNF174 was cotranslated with another SCAN protein, and associations between the two SCAN protein forms were then examined by immunoprecipitation with an antibody that recognized the tag.¹² These coimmunoprecipitation studies confirmed that the SCAN domain is responsible for self-association in the context of an intact SCAN family member and that the α-helical character of the domain is required for the interaction. Additionally, these in vitro studies demonstrate that intact ZNF174 can selectively bind other members of the SCAN family. Generalizing on these observations, members of the SCAN family can both self-associate and form heterodimers using the SCAN domain as an interaction motif.

The SCAN Domain Forms a Stable Dimer in Solution

In previous studies our group and others demonstrated that the SCAN domain functions as an interaction domain, mediating self-association or association with other proteins bearing SCAN domains. In part of those studies, a fragment of either the N-terminus of ZNF174 or ZNF202 that encompassed the SCAN domain behaved as an oligomeric species.^12,14 However, in those studies the definitive subunit stoichiometry of the oligomeric SCAN domain was not determined. Our group utilized the sequences of the SCAN domains in the human genome to more completely define the limits of the domain. The isolated ZNF174 SCAN domain was then overexpressed in bacteria, purified and characterized. Both size exclusion chromatography and equilibrium sedimentation analysis demonstrate that the isolated ZNF174 SCAN domain forms a homodimer.⁷ Additionally, thermal denaturation studies of the ZNF174 domain revealed a high melting temperature (74°C) and demonstrated the stability of the SCAN dimer. These studies resolve the issue of the oligomeric nature of the isolated SCAN domain in that the purified protein forms a stable dimer. However, study of the isolated SCAN domain does not address the important issue of the oligomerization state of the intact SCAN domain family member in cells.

Assembly of a Transcriptional Regulatory System Through SCAN-SCAN Interactions

Based on the ability of the SCAN domain to function as a dimerization domain, it is possible that the domain participates in the assembly of a family of regulatory proteins. To determine which SCAN domains have the ability to interact with one another, pairwise combinations of nine isolated SCAN motifs were tested in the mammalian two-hybrid system.¹² Several general features of SCAN domain interactions could be inferred from the findings in this system. First, not all SCAN domains are able to self-associate. Second, interactions between different SCAN domains are selective. For example, ZNF174 can interact with some, but not all, SCAN domains. Third, there is significant variation in the relative affinities of the SCAN domains. Although these studies with nine SCAN family members represent only a small fraction of the members of the family, they may provide some initial guidelines for the assembly of a SCAN protein network.

In addition to the findings from the mammalian two-hybrid system, various SCAN domains have been used as bait in the yeast two-hybrid system. These studies have identified SCAN containing proteins, revealing new interactions among family members. For example, a region of MZF1 (ZNF42) containing a SCAN domain interacted with RAZ1 (SCAN-related protein associated with MZF1B),¹³ which is also known as SDP1 (SCAN-domain containing protein 1) or SCAND1. Similarly, RAZ1 interacted with the SCAN domain of ZNF202 and inhibited its repressive function.^14,15 A summary of the existing interactions between SCAN family members is shown in the accompanying model (fig. 2). The majority of these observations outlined above were based on studying isolated SCAN-SCAN associations. Whether most of these interactions occur in the context of full-length proteins is uncertain. Additionally, understanding the nature of the partnerships occurring in vivo will be key to defining the family. Transcription factor dimerization can increase the selectivity of protein-DNA interactions and generate a large amount of diversity from a relatively small number of proteins.¹⁶ With a group of regulators the size of the SCAN family (see below), the range of potential dimers is tremendous. It will be important to determine the structural rules that regulate SCAN dimerization specificity and obtain experimental data both in vitro and in vivo that verifies some of the partners. Based on this information it may be possible to classify many of the SCAN family members based on dimerization properties, as has been done for the B-ZIP proteins.¹⁷

Figure 2

A model network of SCAN family members. A summary of the reported interactions between SCAN family members is shown. Each SCAN member is represented by a different color: ZNF174 (orange), ZNF192 (teal), ZNF24 (yellow), ZNF197 (red), MZF1 (blue), SCAND1 (more...)

Zinc Fingers

The majority of SCAN domains (91%) are associated with C₂H₂ zinc finger domains. These 58 members contain a variable number (2-22) of C₂H₂ zinc fingers, defined by the consensus sequence CX_2-4CX₁₂HX_2-6H (with X being any amino acid). All of the C₂H₂ zinc finger motifs are located at the C-terminal end of the SCAN proteins (fig. 3). Most of these C₂H₂ zinc fingers are Krüppel-type, as defined by the conserved link (TGEKP(Y/F)X) between the histidine of the preceding finger with the cysteine of the next finger (H-C link). Many members contain multiple-adjacent fingers that are arranged in clusters of 3 or more (e.g., ZNF197, ZFP95), while other members contain single or duplicate pairs of fingers (e.g., FLJ12895, ZNF215). Since Krüppel-type factors are frequently involved in DNA-binding, this variation in zinc finger number and spacing may have an effect on nucleotide base pair recognition.²¹ Application of the proposed DNA-zinc finger rules,^22,23 suggests that most of the C-terminal zinc fingers of the family members can participate in DNA binding. Notably, members of the SCAN family with multiple adjacent C₂H₂ zinc fingers may have more than one DNA binding activity, as is seen with other proteins with more than four zinc fingers.²⁴ Additionally, since C₂H₂ fingers have been implicated in protein-protein interactions,²⁵ it is possible that some of the zinc fingers in the SCAN family members may interact with other members of the family, or with other target proteins.

KRAB Domain

Twenty-four members of the human SCAN domain family also contain a KRAB domain (fig. 3). As outlined elsewhere in this book, the KRAB domain is found at the N-terminus of approximately 200 C₂H₂ zinc finger proteins^19,20 and mediates repression of transcription.^26,27 The KRAB domain spans about 75 amino acids and has been divided into two subregions, designated A and B.³ It has been demonstrated that the KRAB-A domain contains transcriptional repression activity. This domain is predicted to form an amphipathic helix that interacts with KAP1 (KRAB-associated protein-1, also known as TIF-1β, transcription intermediary factor-1β).^28-34 KAP1 can enhance KRAB-A-mediated repression by recruitment of a histone deacetylase complex containing N-CoR (nuclear receptor corepressor),³⁵ or the Mi-2α subunit of the NuRD complex.³³ KAP1 can also associate with members of the heterochromatin protein 1 (HP1) family, a family of nonhistone heterochromatin-associated proteins with an established gene-silencing function.³¹

A sequence alignment of the KRAB domains from the SCAN family members is found on the project web site. As with other KRAB domain containing genes, the KRAB domain is located at the N-terminus of SCAN family members, usually C-terminal to the SCAN domain. KRAB-containing proteins can be classified into three types:³⁶ those that contain a KRAB-A domain alone, those with both —A and —B domains, or those that have an —A domain and a divergent —B domain. Twelve members of the human SCAN family contain both KRAB-A and -B domains, while another twelve members have a KRAB-A domain only. It is likely that the SCAN domain will influence the function of the KRAB domain, although this has not been extensively studied.

Novel Domain

Thirty-four members of the SCAN family contain a novel region of homology that was found at the very N-terminus of the predicted protein (fig. 3). A sequence alignment of a select number of the members is illustrated in Figure 4 and a full alignment of all the members is found on the project web site. This region is 13 residues in length, with the consensus EqEGLLiVKvEEe (where capital letters correspond to amino acids conserved over 51%, and lowercase letters to amino acids conserved over 35%), and may be similar to the previously described N-terminal acidic domain of the murine myeloid-specific zinc finger gene, MZF-2.³⁷ A search of the human genome with the consensus sequence indicates that this region may be found only in members of the SCAN family.

Figure 4

The conserved region N-terminal of the SCAN domain. The amino acid sequences of the N-terminal novel region from selected members of the human SCAN family were aligned by consensus ClustalW sequence alignment using MacVector 7.1.1 (Genetics Computer Group) (more...)

The core of this conserved N-terminal domain in the SCAN family members contains a sequence for conjugation to the small ubiquitin-related modifier, SUMO. Like ubiquitin, SUMO is a small polypeptide that is covalently attached to lysine residues in substrate proteins.³⁸ SUMO is attached to substrate proteins by a series of enzymatic reactions similar to those involved in ubiquitination. Following cleavage to expose a C-terminal glycine, SUMO is attached to a heterodimeric E1 SUMO activating enzyme (Aos1/Uba2), then transferred to an E2 SUMO conjugating enzyme (Ubc9), before being covalently attached to a lysine in the substrate protein. A consensus SUMO acceptor site has been identified consisting of the sequence ΨKXE, where Ψ is a large hydrophobic amino acid and K is the site of SUMO conjugation.³⁹ Contained within the core of the novel N-terminal region (EqEGLLiVKvEEe) is the sequence VKxE, which fits the consensus for a SUMO acceptor site. Additionally, a glycine residue is located five amino acids upstream from the acceptor lysine, consistent with the novel domain containing a sumoylation site (fig. 4).

Post-translational modification of the SCAN family member by SUMO may have a substantial effect on transcription factor activity. By analogy to other proteins, SUMO modification of the SCAN family member could prevent modification by ubiquitin at that position, potentially preventing degradation of the family member by the proteasome. SUMO may also compete for post-translational modification of lysines by acetylation. Additionally, SUMO modification of the SCAN family member may disrupt protein-protein interactions with some proteins, while promoting interaction with other proteins. In addition to playing a role in regulating transcription factor activity, SUMO modification also affects transcription factor subcellular localization. For example, SUMO-1 modified Sp3 accumulates at the nuclear periphery and in nuclear dots whereas the transcriptionally active form lacking SUMO-1 has a more diffuse nuclear localization.⁴⁰ Although experimental verification will be required, the potential sumoylation site N-terminal to the SCAN domain may have an important role in the function of some of the family members.

SCAN-(C₂H₂)_x

The first group of SCAN domain proteins consists of family members with just a SCAN domain and a variable number of C-terminal C₂H₂ zinc fingers (fig. 3). There are at least 40 of these genes in the SCAN family, but only a few have been characterized. Some of the better known representatives from this class of genes are indicated in Table 1.

The myeloid zinc finger gene (MZF1, also known as ZNF42) is expressed preferentially in hematopoietic progenitor cells of the myeloid lineage.⁴² Inactivation of the murine MZF1 gene results in a striking increase in hematopoetic progenitors, with the eventual development of lethal myeloid neoplasias,⁴³ suggesting that MZF1 is involved in growth, differentiation and tumorigenesis of myeloid progenitors. In both human and mouse cells, several isoforms of MZF proteins are produced from a single gene by the use of alternative promoters and alternative splicing.^37,44 Of the isoforms, MZF1B, and its murine counterpart MZF-2A, seems to represent the major product of the gene. MZF1B shares identity to the carboxy-terminus of MZF1, a C-terminal DNA binding domain consisting of 13 C₂H₂ zinc finger modules, but MZF1B encodes an additional amino terminal extension that contains a novel N-terminal domain, a SCAN domain and a transactivation domain.

To further characterize this member of the SCAN family, proteins that interact with several of the domains of MZF1B (MZF-2A) have been defined. The SCAN domain of MZF1B interacts with RAZ1,¹³ a member of the SCAN family that lacks zinc fingers, as discussed below. The transactivation domain of MZF-2A works specifically in myeloid cells and can function as an autonomous transactivation motif when fused to a heterologous DNA binding domain.⁴⁵ In studies exploring the regulation of the activity of the transactivation domain, three serine residues were found to be phosphorylated by ERK and p38 MAP kinases.⁴⁶ This suggests that the activity of the transactivation region of MZF-2A is negatively regulated through phosphorylation by MAP kinases. To understand the mechanism of MZF-2A dependent transcriptional activation, a yeast two-hybrid Ras recruitment screen was done to look for interacting proteins.⁴⁷ A novel SWI2/SNF2-related protein, termed mammalian Domino, was identified as a candidate MZF-2A interacting partner. Notably, mDomino contains a SWI2/SNF2-type ATPase/helicase domain, a SANT domain, and a glutamine rich domain. The C-terminal Q-rich domain physically associated with the transactivation domain of MZF-2A and overexpression of mDomino enhanced MZF-2A mediated activation of a reporter gene.⁴⁷ Collectively, these findings suggest that an ATF-dependent chromatin-remodeling complex interacts with MZF-2A to regulate gene expression in myeloid cells.

To address the key issue of defining genes regulated by MZF1, a DNA binding site was determined for the zinc fingers of MZF1 and consensus elements were found in the promoters of several hematopoietic cell-specific genes, such as CD34, c-myb, lactoferin and myeloperoxidase.^48,49 The MZF1 binding motif is also found in the promoter of the telomerase reverse transcriptase gene,⁵⁰ and in an intron in the gene for the high affinity IgE receptor,⁵¹ where MZF1 is likely to function as a negative regulator for gene expression. Interestingly, MZF1 activates gene expression in cells of the hematopoietic lineage, while in nonhematopoietic cells it functions as a transcriptional repressor.⁴² It is possible that other SCAN family members may be both positive and negative regulators of target gene expression, depending upon the cell type.

SCAN-KRAB-(C₂H₂)_x

A second class of SCAN family members consists of a SCAN-KRAB-(C₂H₂)_x modular architecture (fig. 3). There are at least 24 genes in this class of SCAN family members. Some of the better known members of this class of SCAN family members are listed in Table 1.

Table 1

Summary of representative SCAN family members from the three largest classes.

An interesting example of this subgroup of SCAN family members is the candidate hypoalphalipoproteinemia susceptibility gene, ZNF202. A low HDL cholesterol locus on chromosome 11q23 was identified that is distinct from the apoA-I/C-III/A-IV/AV cluster of genes.⁵² This new familial susceptibility locus for hypoalphalipoproteinemia contains the SCAN family member, ZNF202. The gene for ZNF202 encodes a protein predicted to contain a SCAN domain, an intact KRAB domain and eight C₂H₂ zinc-finger motifs. Two splice forms have been identified for ZNF202, the m1 form encoding the full-length protein, and the m3 form encoding a smaller version containing only the SCAN domain and lacking the zinc fingers.⁵³ The ZNF202 gene product is a transcriptional repressor that binds to elements found predominantly in genes that participate in lipid metabolism, such as the group of genes that comprise the apoA-I/C-III/A-IV/A-V gene cluster on chromosome 11, the apoE/C-I/C-IV/C-II gene cluster on chromosome 19, phospholipid transfer protein, and a series of enzymes involved in lipid processing, including lipoprotein lipase, hepatic triglyceride lipase and lecithin cholesteryl ester transferase.⁵³ Additional targets for repression by ZNF202 include the ATP-binding cassette A1 (ABCA1) and ABCG1.¹⁵ ABCA1 is a key regulator of plasma HDL levels, while ABCG1 supports lipid efflux in human macrophages. ZNF202, which acts as a transcriptional repressor of both genes, is able to reduce phospholipid and cholesterol efflux in transiently transfected macrophages, demonstrating the functional relevance of the SCAN family member.¹⁵ Additionally, ZNF202 expression is inversely regulated with respect to the expression of its target genes ABCA1 and apoE during macrophage differentiation and foam cell formation.⁵⁴ Collectively, these findings demonstrate that ZNF202 regulates reverse-cholesterol transport and are consistent with the proposal that ZNF202 controls the balanced expression of genes involved in lipid metabolism.

Isolated SCAN Domain Proteins

For six members of the human SCAN family there is no current evidence for associated C₂H₂ zinc finger motifs (fig. 3). These SCAN domain-only sequences might represent novel genes without zinc fingers, or splice forms of larger transcripts that contain zinc finger motifs. Indeed, there is cDNA evidence that some SCAN-containing genes are alternatively spliced generating transcripts that lack the SCAN, KRAB, or zinc finger domains, as discussed below.

A murine SCAN domain containing protein was identified as an adipogenic cofactor bound by the differentiation domain of the nuclear receptor peroxisome proliferator-activated receptor γ(PPARγ).⁵⁵ This mouse protein, termed PPARγ coactivator-2 (PGC-2), or its human homolog, SCAND1 (also known as SDP1, SCAN-domain containing protein 1; or RAZ1, SCAN-related protein associated with MZF1B), interacts with the ligand-independent activation function region (AF-1) of PPARγ and can potentiate PPARγ-dependent gene expression. This effect is presumably mediated by facilitating the assembly of a coactivation complex and enhancing fat cell formation. PGC-2 does not contain zinc fingers, but does contain a partial SCAN domain that consists of the N-terminal 60 residues of the “authentic” SCAN domain. Interestingly, SCAND1 and PGC-2 may interact with PPARγ through their SCAN domains, even though PPARγ does not contain a SCAN domain.^14,56 Although the SCAN domain was required for interaction with PPARγ, it was not sufficient for coactivator function. Interestingly, SCAND1 has been shown to interact with ZNF202 via its SCAN domain, thereby preventing recruitment of KAP1 and transcriptional repression.¹⁵ Thus by enhancing PPARγ-dependent transcription, and blocking KAP binding to ZNF202, SCAND1 may be an important coregulator of genes controlling the cellular lipid machinery.^54,56

This isolated SCAN domain protein may play a role in differentiation of stem cells in the bone marrow. Pluripotent mesenchymal stem cells in bone marrow differentiate into adipocytes and other cells. Although balanced cytodifferentiation of stem cells is essential for the formation and maintenance of bone marrow, the mechanisms that control this balance remain largely unknown. Whereas PPARγ is a key inducer of adipogenesis, inflammatory cytokines, such as interleukin-1 and tumor necrosis factor-α, inhibit adipogenesis. Recent results suggest that the ligand induced transactivation function of PPARγ is suppressed by the inflammatory cytokines and that this suppression is mediated through a signaling cascade that results in NF-κB activation. NF-κB blocks PPARγ binding to DNA by forming a complex with PPARγ and its coactivator PGC-2.⁵⁷ These findings suggest that expression of the cytokines in bone marrow may alter the fate of stems cells by suppressing PPARγ function, directing cellular differentiation towards osteoblasts rather than adipocytes. Since SCAND1 also interacts with MZF1B,^13,58 a factor that plays a role in the differentiation of myeloid progenitors, it is possible that similar SCAND1-dependent mechanisms may regulate the differentiation of other types of stem cells in the bone marrow.

This brief overview of just one representative gene from each of the three largest classes of SCAN family members demonstrates that the SCAN domain containing transcription factors perform a wide range of functions important in cell development or differentiation.

Isoforms Lacking the SCAN Domain

As mentioned above, the MZF1 gene generates a transcript that encodes a SCAN domain-containing zinc finger protein, MZF1B.⁴⁴ MZF1B shares identity to the carboxy-terminus of MZF1, including the 13 C₂H₂ zinc finger modules, but MZF1B encodes an additional amino terminal extension that contains a SCAN domain (fig. 5). Thus while MZF1 and MZF1B may bind to the same DNA target, it is likely that they exert distinct regulatory effects because of their distinct amino termini. Additionally, it is possible that when MZF1 is bound to a target gene it may act as a dominant-negative inhibitor of MZF1B function.

Figure 5

Structure and function of some of SCAN family member isoforms. Representative isoforms from selected SCAN family members discussed in the text. The predicted structure of the isoforms from three representative human SCAN-containing family members is shown. (more...)

Isoforms Lacking Zinc Fingers

Other SCAN family member genes also generate forms that lack the C-terminal zinc fingers. Thus in a general context, a SCAN domain-only protein may dimerize with another member of the family and alter nucleotide binding, since the resulting heterodimer would lack the pair of DNA binding motifs (fig. 6). The expression of these alternate transcripts from SCAN containing genes would, therefore, increase the available number of protein combinations within the family. Such forms may help regulate the function of the SCAN family members.

Figure 6

The corresponding model illustrates that selective SCAN dimerization may be a potential mechanism for functional diversity in the SCAN family. In one example, a SCAN-containing gene generates two isoforms; a SCAN domain-containing zinc finger protein (more...)

One interesting example of this type of isoform is derived from ZNF197 because it interacts with the von Hipple Lindau tumor suppressor (pVHL). pVHL is a component of an E3 ubiquitin ligase and targets hypoxia-inducible factor-1α (HIF-1α) for ubiquitination and degradation under normoxic conditions.⁵⁹ pVHL also directly inhibits HIF-1α transactivation by recruiting histone deacetylases. In a yeast two-hybrid screen for proteins interacting with pVHL, an isoform of ZNF197 was identified which was given the name pVHL-associated KRAB-A domain-containing protein (VHLaK).⁶⁰ It contains a SCAN domain and a KRAB A-domain, but lacks the 22 zinc fingers present in ZNF197 (fig. 5). The KRAB A domain in VHLaK mediates pVHL binding and functions as a transcriptional repression module,⁶⁰ consistent with previous reports that the KRAB domain of ZNF197 could repress transcription of a reporter gene.⁹ The SCAN domain in VHLaK mediates homo-oligomerization and enhances VHLaK repressive activity. pVHL can recruit VHLaK to repress HIF-1α transcriptional activity and HIF-1α-induced VEGF expression.⁶⁰ Additionally, the ZNF197 isoform can recruit both KAP1 and pVHL simultaneously, indicating that KAP1 may participate in pVHL mediated transcriptional repression of HIF-1α.⁶⁰

In summary, SCAN family members can generate isoforms lacking the SCAN domain as well as isoforms lacking the zinc fingers. Expression of these isoforms may be static, but more likely, the production of these isoforms may be biologically relevant. For example, it is possible that in tumors, relative to the corresponding normal tissues, the levels of the various isoforms of the SCAN family members may change.⁶¹

One-Hybrid Approach

Perhaps most compelling are results from groups that have functionally characterized a specific regulatory element in a gene of interest and then used that site in a “one-hybrid” screen to identify the cognate regulatory protein. The yeast one-hybrid system is based on the principle of the yeast two-hybrid system and is used for isolating novel genes encoding proteins that bind to a target, cis-acting regulatory element.⁶² Several members of the SCAN family have been identified by this approach. EZF-2 (endothelial zinc finger protein-2 or ZNF444) is a member of the family with four zinc fingers and specifically targets the scavenger receptor expressed by endothelial cells.⁶³ ZNF263 has nine zinc fingers and a KRAB domain and interacts with an element in the promoter of the α2(XI) collagen gene.⁶⁴ ZNF24 (ZNF191) interacts with an intronic polymorphic TCAT repeat in the tyrosine hydroxylase gene, the rate-limiting enzyme in the synthesis of catecholamines.⁶⁵

Binding Site Selection

A second experimental approach to determine the genes regulated by a SCAN family member is to determine its DNA binding site and then utilize this information to identify candidate target genes. As outlined above, based on the binding site determination, ZNF202 has been shown to interact with and control the expression of a series of genes involved in lipid metabolism, including the ATP binding cassette transporter A1.¹⁵ Binding sites for MZF1 (ZNF42) were found in CD34, c-myb and myeloperoxidase, genes that are expressed in hematopoietic cells.

Overexpression

In these studies, members of the SCAN family are overexpressed with promoter-reporter constructs of candidate target genes. In this type of approach, ZNF174 selectively repressed the expression of PDGF-B chain and TGF-β1 promoter-reporters.⁶ Establishing the biological relevance of the findings is always the challenge with this type of experimental approach.

To date, chromatin immunoprecipitation (ChIP) has not been used to verify the interaction of SCAN family members with DNA binding sites in an authentic gene or to clone SCAN family member target promoters.⁶⁶ Additionally, it should be possible to identify physiologic targets of SCAN family members by looking at altered gene expression profiles using microarrays following overexpression of a SCAN family member.

Human SCAN Family Genes

The genes for the human SCAN domain family have been mapped to specific human chromosome locations.⁸ Of the 71 SCAN-containing genes, 14 are isolated single genes. The majority of genes (80%) are found in clusters on human chromosomes 3p21, 6p21.3, 7q22, 15q25, 16p13.3, 17p11.2, 18q12, 19q13.4 and Xq26, and some of these genes are arrayed in tandem. Some of these locations are sites frequently disrupted and associated with cytogenetic abnormalities. For example, ZNF197 is located at 3p21, a region frequently involved in cytogenetic abnormalities associated with epithelial malignancies of the kidney, lung, thyroid and breast, as well as other tumors.⁶⁷ The gene for MZF1 is located at the extreme end of the long arm of chromosome 19. Since telomeres are known to shorten as cells age, the location of MZF1 at the telomere may make it vulnerable to dysregulation or disruption during aging.⁶⁸

When the amino acid sequences of human SCAN domains were analyzed using a cladistics program, the majority of SCAN domains within these clustered regions grouped into distinct sets sharing sequence similarities.⁸ For example, several SCAN domains from the clustered regions on chromosomes 3, 6, 18 and 19 are grouped into subsets, whereas the isolated SCAN domains on chromosomes 1 and 11 are assorted elsewhere due to lesser sequence similarity. SCAN-containing genes that are tandemly linked present a target for mispairing and unequal crossover, which could result in duplication and divergence of the genes. Over time, these tandemly duplicated SCAN genes could become physically separated through chromosomal rearrangements and translocations. These local duplications may account for the high degree of sequence similarity shared by neighboring genes. Such SCAN gene duplication events taking place in the clustered region on chromosome 19q13.4, for example, would account for the striking similarity of the neighboring genes MGC4161, LOC126209, LOC1262110 and LOC126211. These four genes encode putative SCAN-containing C₂H₂ zinc finger transcription factors that are 80% identical at the nucleotide level. The SCAN domains are highly conserved (94% identity), whereas differences at the nucleotide level are most predominant in the zinc finger regions (68% identity). Within the five predicted zinc fingers found in each gene, most of the amino acids that differ are found in portions that determine the recognition specificities of the fingers. This suggests that after a possible duplication event, the genes may have acquired changes that permit functional divergence.

This apparent clustered expansion of SCAN genes at chromosome 19q13.4 appears similar to the human-specific expansion of KRAB-containing zinc finger genes clustered at this same locus.⁶⁹ In addition to chromosome 19, chromosome 6 contains highly similar SCAN-C₂H₂ genes (P1 p373c6 and ZNF306 at 6p21.3) that may represent a duplication event, suggesting that the expansion of SCAN genes at these two clustered sites is similar.

Mouse SCAN Family Genes

An analysis of conserved segments between human and mouse chromosomes becomes a useful approach in the identification of likely gene orthologs. To establish potential SCAN-containing gene orthologs, two approaches were taken. First, BLAST searches were performed against available mouse genome databases using representative mouse SCAN domains (Zfp38 or Skz1-pending). Second, conserved reference sets of genes that flank the genomic segment of the human SCAN gene(s) were located in the mouse genome by Mouse Genome Informatics (MGI) and Ensembl. The mouse DNA region between the markers was then searched; significant matches to DNA sequences corresponding to SCAN domains were identified.⁸

A reciprocal comparison of the human and mouse SCAN-containing genes within regions of conserved synteny identified likely orthologs. Based on the SCAN-containing genes represented in the Ensembl database, at least 23 of these members are represented by putative orthologs on conserved segments of the human chromosomes.⁸ In several cases, homologous SCAN family members within the human clusters were indistinguishable from each other when compared to the mouse and, as a result, an ortholog assignment was not possible. Interestingly, we found human SCAN clusters that are represented by a smaller number of SCAN genes in the conserved syntenic regions of the mouse. For example, the six clustered SCAN genes on human chromosome 16p13.3 are represented by two clusters of only four SCAN genes in the conserved segment of mouse chromosomes 16 and 17 (fig. 7). These findings provide evidence for human-specific cluster expansions of SCAN family members. This argues that some genes within the SCAN family are lineage-specific and may have been selected independently since the divergence of primate and rodent lineages.

Figure 7

Comparison of selected human and mouse SCAN family members. The SCAN genes on human 16p13.3 and the corresponding regions in the mouse genome are indicated. The cluster of six SCAN genes on human chromosome 16p13.3 is represented by only four SCAN genes (more...)

Similar to the human SCAN family, a large percentage (>75%) of the mouse SCAN domain-containing genes are predicted to encode one SCAN domain and a variable number of C₂H₂ zinc fingers. Where orthologous relationships could be established, predicted structures of the human and mouse family members were highly similar in that they had comparable amino acid lengths and the same number of conserved domains (KRAB and zinc fingers). For example, the human SCAN gene, ZNF287, and its mouse ortholog, Zfp287/Skat-2, encode predicted proteins of 754 and 758 amino acids, respectively, containing one SCAN domain, one KRAB domain, and 14 zinc fingers. Interestingly, in some pairs there is substantial conservation of the amino acids in the region of the C₂H₂ zinc fingers that determine the recognition specificity, suggesting that the contact sites in target genes may be conserved.

Possible Recent Retroviral Origin of SCAN Domains

Remarkably, the SCAN domains found in lower vertebrates are not associated with C₂H₂ zinc finger genes, but are contained in large retrovirus-like polyproteins that are reminiscent of those found in the retrovirus-like polyproteins.⁸

The absence of the SCAN domain in invertebrates, and the nonexistence of SCAN-ZFP in lower vertebrates, suggests that these genes originated and rapidly expanded during recent evolution. The rapid and lineage-specific expansion of the SCAN family may contribute to the diversity that is seen in higher vertebrates (fig. 8). As a result of the ability of the SCAN domain to mediate dimerization, a diverse network of transcription factor dimers could be generated that may play a key role in the uniqueness of higher vertebrates.

Figure 8

SCAN C₂H₂ proteins are vertebrate specific. The apparent number of SCAN domains found in databases of the worm, fly, zebrafish, frog, chicken, mouse, and human are indicated in the first column. The apparent number of SCAN-containing retrovirus-like polyproteins, (more...)

A number of interesting questions remain about the SCAN domain, including a detailed understanding of its structure, mechanism of partner choice and the nature of interacting proteins. Additionally, identifying the target genes regulated by this family will be useful in determining the function of this group of zinc finger transcription factors. Like the POZ/BTB and KRAB domains, the presence of a SCAN domain may provide some mechanistic insights into the large number of C₂H₂ type zinc finger proteins.