Epidermal Differentiation Comple

5.2.3.1 Evolution

Speculation of the evolutionary origin of the three so far identified gene families within the EDC has generally been limited to comparisons of these genes within the human. The observation that loricrin, involucrin and SPRR proteins shared homology at the N and C termini, coupled with strikingly homologous exon structures suggested that these genes had common ancestry (Backendorf and Hohl, 1992). This notion was investigated further

after the identification of additional SPRR members and comparisons of the internal repeat structures (Gibbs et al, 1993), Loricrin, involucrin, and the SPRR proteins share homology at the N and C termini, separating gene specific internal repeats. It was noted that only three internal repeats existing in the seven SPRR2 genes suggested inter-genic duplication (creation of additional gene copies), while the 14 internal repeats of the single SPRR3 gene suggested intra-genic duplication (duplication within the single gene). In the SPRR 1A gene, 6 repeats were identified, coupled with SPRR IB this demonstrated a process of both inter- and intra-genic duplication (Gibbs et al, 1993). The promoter regions of the human SPRR genes have been examined in detail (Fischer et al, 1996; Sark

et al, 1998; Fischer et al, 1999). In all SPRRl and 2 genes studied, a common

transcriptional binding site was identified at the same position, 5 ’ of the transcriptional initiation site, whilst in the SPRR3 gene this binding site is some 180 nucleotides

downstream of the conserved region. This prompted the theory that the rearrangement of this binding site has led to the divergence of the SPRR3 gene (Fischer et al, 1999). More recently, a number o f the SPRR genes have been identified in mouse (Kartsova et al,

1996; Reddy et al, 1998; Song et al, 1999). These studies concentrate on the genomic structure and differential expression of the mouse SPRR genes, as opposed to

commenting on evolutionary relationships between mouse and man.

Expression of loricrin was demonstrated in a number of mammalian species by the use of monospecific polyclonal antibodies to the carboxy-terminal peptide of human loricrin (which is highly conserved in mouse, Hohl et al, 1993). Immunoblot analysis of total protein extracts from the skin of various species separated by SDS-PAGE demonstrated cross-reactivity with the antibodies to human loricrin in all mammalian species tested (human, mouse, hamster, rat, rabbit, lamb, and cow). Three non-mammalian vertebrate species were investigated in a similar manner and failed to show any significant cross­ reactivity with the human loricrin antibody {Xenopus laevis, garter snake and chicken). No cross-reactivity with human involucrin or SPRR proteins was noted, indicating that the presence of any possible ancestral loricrin/involucrin/SPRR proteins in the non­ mammalian vertebrate species cannot be discounted (an ancestral protein could possibly be more similar to involucrin or SPRR proteins and therefore would not show cross­ reactivity).

Early identification of multiple SlOO genes, coupled with the observation that they were homologous, led to the speculation of a gene family (Lagasse and Clerc, 1988). Later, when more SlOO genes and proteins had been identified, analysis of homology was performed with human SlOOAl-10, SIOOB, SI OOP, and CALB3 (Schaefer e ta l, 1995). The resulting dendrogram led to comments that two distinctive groups were present within the SlOO genes analyzed, but no speculation as to which could be the ancestral gene was made. Furthermore the authors comment on the fact that a number of SlOO genes were identified in other vertebrates but failed to analyze the relationship of interspecies SlOO genes. The more recent identification o f a similar clustering o f SlOO genes in mouse (see below) has led to the suggestion that the SlOO gene cluster on human

lq21 was present in a common ancestor (70 million years ago) and that two rearrangements have occurred since (Ridinger et al, 1999).

The genes profilaggrin, trichohyalin, and repetin, are all related structural proteins containing functional calcium binding domains (see section 1.3.1.2). It has been noted that the internal repeats of trichohyalin share homology with the internal repeats of involucrin (Lee et al, 1993), and that the internal repeats and C terminus of repetin share homology with involucrin (Kreig et al, 1997). All three genes share an identical exon structure as the majority of SlOO proteins (albeit larger with internal repeats). After the initial elucidation that profilaggrin can functionally bind calcium via two E-F hand domains it was proposed that this family (now including repetin which was unidentified at the time) arose during evolution by the fusion of a gene for a structural protein, containing internal repeats (of the loricrin, involucrin and SPRR family), with a calcium binding protein (SlOO) that were both co-localized to lq21 (Markova et al, 1993).

S.2.3.2 Mouse EDC

M ouse genes corresponding to the majority of the human EDC genes have been described, in fact a number of the mouse EDC genes were characterized before their human equivalents (Mehrel et al, 1990; Ohta et al, 1991; Kreig et al, 1997). Initial linkage studies mapped mouse loricrin and profilaggrin to mouse chromosome 3 (Rothnagel et al, 1994) in a region of known synteny between human chromosome 1 (Moseley and Seldin, 1989; Hardas et al, 1994). Other human EDC equivalents to have

been localized to mouse chromosome 3 include S I00A4 and S100A6 (Debry and Seldin, 1996), SIOOAIO (Saris et al, 1987) and repetin (Kreig et al, 1997).

A recent study has defined the order of mouse SPRRs, loricrin, involucrin, and

profilaggrin on mouse chromosome 3 to be identical to the human EDC on chromosome lq21 (Song et al, 1999). The genetic distance between mouse profilaggrin and loricrin was calculated to be 1.4 cM, an estimated 2.8Mb. A mouse YAC clone has been

described that harbors a cluster of the mouse SlOO genes (equivalent human) A l, A3, A4, A5, A6, A8, A9 and A13 (Ridinger et al, 1999). The order of the mouse SlOO genes is slightly different to that in human, for which two inversion events have been suggested (see chapter 6). This YAC clone was localized to mouse chromosome 3 by fluorescent in situ hybridization (FISH) in agreement with other assignments o f individual SlOO genes to mouse chromosome 3 (Debry and Seldin, 1996). The degree of linkage between the identified mouse SlOO cluster and other mouse EDC-equivalent genes has been described using radiation hybrid mapping (Lueders et al, 1999). S100A6 is mapped closer to

loricrin than loricrin is to profilaggrin. Exact distances are not given as the measurements described are between profilaggrin and a non-EDC gene (97.5 cR away. Lenders et al,

1999). If the distance between profilaggrin and loricrin is estimated to be 2.8Mb from genetic mapping (Song et al, 1999) then a comparable distance between S100A6 and loricrin would be 1.7Mb. It remains to be seen whether these distances described using principally genetic mapping, will be confirmed by physical mapping.

5.2.3.3 Transcriptional orientation within the EDC located human SlOO gene family One of the ways to assess the evolutionary processes at work in the formation of a gene family is to look at the transcriptional orientation of the gene members. A duplication event generally results in a head to tail array of genes (Ohno, 1970), where an inversion (non-homologous recombination), or an insertion within a tandem array o f duplicated genes, can be indicated by opposing transcriptional orientation. Currently, the

transcriptional orientation of four SlOO genes is known (S100A 3,4, 5, and 6). This is based on the isolation of a YAC clone containing 9 SlOO genes (Schaefer et al, 1995) and long range physical mapping (Mischke et al, 1996), coupled with sequencing data

(Englekamp et al, 1993), see figure 3.1 for details. These four genes are arranged in a head to tail orientation (telomere to centromere).

5.2.3.4 Chicken EDC genes

In order to assess the possibility of an EDC in the chicken, a search of the available databases was performed using all known EDC gene names. The sequences o f Gallus gallus calcyclin (S100A6) mRNA (gb/U7635/GGU76365) and Gallus gallus calgizzarin (SlOOAl 1) mRNA (gb/U77733/GGU77733) were identified via an ENTREZ search (a search engine that identifies terms within the text of database entries, available at the NCBI web site - http://www.ncbi.nlm.nih.gov/Entrez). The database entries were submitted by: Allen, E.G., Sutherland, C., Andrea, I.E., Schonekess, B.C., and W alsh, M.P., 1996, unpublished (S100A6); and by Schonekess, B.O. and Walsh, M.P., 1996, unpublished (SlOOAl 1).