Overview of Rodent Zic Genes
10.3 The Structure and Conservation of the Murine ZIC Proteins
The defining feature of all ZIC proteins is the inclusion of a zinc finger domain (ZFD) composed of five tandem Cys2His2-type zinc fingers (Fig. 10.1). This domain is most closely related to the ZFD of the GLI, GLIS and NKL families and is highly conserved between ZIC family members both within an individual species
Table 10.1 (continued)
Intron
number 5′UTR 3′UTR Introns
Coding DNA Protein Zic5
Mouse (NM_022987) 1 100.00 100.00 100.00 100.00 100.00
Human (NM_033132) 1 90.52 92.49 53.29 89.24 88.73
Rat (ENSRNOG00000014358) 1 97.07 95.38 80.49 96.36 97.27
Ground squirrel (ENSSTOG00000027399) 1 86.38 88.55 55.52 90.03 89.27 Chinese hamster
(ENSCGRG00001022535)
1 93.55 94.22 71.55 94.09 94.02 Naked mole rat (ENSHGLG00000019584) 1 87.02 90.70 53.39 87.71 85.81 Long-tailed chinchilla
(ENSCLAG00000003432)
1 86.47 89.53 54.41 79.91 88.96 Prairie vole (ENSMOCG00000003890) 1 94.13 91.33 70.92 93.88 94.70 Available DNA and protein sequences for the human (Homo sapiens), rat (Rattus norvegicus), Chinese hamster (Cricetulus griseus), guinea pig (Cavia porcellus), long-tailed chinchilla (Chinchilla lanigera), naked mole rat (Heterocephalus glaber), prairie vole (Microtus ochrogas- ter) and ground squirrel (Ictidomys tridecemlineatus) were compared to the laboratory mouse (Mus musculus). Blank cells indicate species where only a partial sequence was available. NA indicates sequences that could not be sufficiently aligned to the mouse
K. E. M. Diamand et al.
Fig. 10.1Structural features of the five murine ZIC proteins. All mouse ZIC proteins contain a zinc finger domain (ZFD) that consists of five tandem C2H2- type zinc fingers. This domain is highly conserved, with only the first zinc fingers of ZIC4 and ZIC5 showing some divergence. Additionally, all five proteins also contain a short (14–21 amino acids) highly conserved domain immediately upstream of the zinc fingers, called the ZF-NC domain, as well as low- complexity regions with the major amino acid found at each low-complexity region shown by the associated letter (A alanine, H histidine, P proline, S/G serine/ glycine). ZIC1, ZIC2 and ZIC3 share a small domain (9–10 amino acids) of homology towards the N-terminal of the protein, termed the ZOC motif. The ZIC proteins can be divided into two distinct structural subclasses on the basis of the presence or absence of the ZOC motif and the degree of conservation within the first zinc finger domain
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and across species. The ZIC ZFD is, however, distinguished by an atypical first zinc finger. Generally one to five amino acid residues separate the two cysteines of a C2H2 zinc finger, whereas, in the first zinc finger of ZIC proteins, this number is both increased and highly variable, ranging from 6 to 38 in species so far examined (Aruga et al. 2006; Layden et al. 2010). Additionally, structural analysis of the ZIC3 protein indicates that the first two zinc finger domains may not be canonical (DNA binding) C2H2-type zinc fingers. Instead these fingers may form a single structural unit called the tandem CWCH2 motif, the hallmark of which is a tryptophan residue located between the two canonical cysteines of each zinc finger (Hatayama et al.
2008). This motif is conserved across a wide range of metazoan species (Hatayama and Aruga 2010; Aruga et al. 2006), indicative of biological significance, as is the identification of a missense mutation of the tryptophan in the first zinc finger of human ZIC3 in association with congenital heart malformations. This mutation reduces protein stability and perturbs the nuclear localization of the protein (Chhin et al. 2007). The ZFD is intimately involved in ZIC protein function, mediating DNA binding and protein-protein interactions. Additionally, none of the ZIC pro- teins contain a canonical nuclear localization signal, but the human ZIC3 zinc finger domain (fingers 2–5) has been shown to be essential for this function and presum- ably harbours an interspersed nuclear localization signal (Bedard et al. 2007;
Hatayama et al. 2008).
The regions outside of the ZFD exhibit greater sequence variability between the five murine ZIC proteins, but evolutionary conserved domains are present (Fig. 10.1). The ZF-NC (zinc finger N-flanking conserved) domain, located directly prior to the ZFD, is a small (14–21 aa) conserved region that is SUMOylated in human ZIC3 (Aruga et al. 2006; Chen et al. 2013). Additionally, a small (9–10 aa), N-terminally located domain called the Zic-opa conserved (ZOC) domain is con- served in ZIC1-3. This domain is one of two shown to be involved in transcriptional activation (the other being the ZFD) and has also been shown to be involved in protein-protein interactions, such as binding the myogenic repressor protein, I-mfa (Mizugishi et al. 2004). As reviewed in Houtmeyers et al. (2013), phylogenetic comparison of the ZIC protein sequences has led to the classification of ZICs into two distinct structural subclasses based on the presence of the ZOC domain and the variation within the first zinc finger. The structural subclass A contains the more highly conserved ZIC1, 2 and 3 proteins, and subclass B contains the less conserved ZIC4 and ZIC5 proteins. Notably, the subclass division is reflected in the genome arrangement and evolution of the Zic genes, since each gene pair (i.e. Zic1/Zic4 and Zic2/Zic5) contains one subclass A and one subclass B protein.
Each murine ZIC protein contains several low-complexity regions (including poly-alanine, poly-histidine, poly-proline and poly-serine/poly-glycine tracts), with the number and type of sequence varying between the five ZIC proteins (Fig. 10.1). Low-complexity regions can undergo expansion and contraction muta- tions that alter protein function, and poly-alanine tract expansions in ZIC2 and ZIC3 are associated with human disease pathogenesis. Expansion of the alanine tract in ZIC2 is implicated in Holoprosencephaly, whilst expansion mutations of this domain in ZIC3 are associated with Heterotaxy (Brown et al. 2001; Wessels
K. E. M. Diamand et al.
et al. 2010). In vitro cell-based assays have shown that expansion of the ZIC2 ala- nine tract from 15As to 25As results in a near-complete loss of transactivation abil- ity, dependent on the promoter that is used (Brown et al. 2005), suggesting a role for the alanine tract in the modulation of ZIC transactivation. It is unclear, however, if poly-alanine tract expansions affect the functionality of other ZIC family mem- bers. Previous analysis of ZIC3 found no significant change in transactivation abil- ity upon modification of the alanine tract (Cowan et al. 2014), a result which may be attributed to the differences in promoters and experimental conditions in the two studies.