Identification of human epidermal differentiation complex (EDC)-encoded genes by subtractive hybridization of entire YACs to a gridded keratinocyte cDNA library

Abstract

The epidermal differentiation complex (EDC) comprises a large number of genes that are of crucial importance for the maturation of the human epidermis. So far, 27 genes of 3 related families encoding structural as well as regulatory proteins have been mapped within a 2-Mb region on chromosome 1q21. Here we report on the identification of 10 additional EDC genes by a powerful subtractive hybridization method using entire YACs (950_e_2 and 986_e_10) to screen a gridded human keratinocyte cDNA library. Localization of the detected cDNA clones has been established on a long-range restriction map covering more than 5 Mb of this genomic region. The genes encode cytoskeletal tropomyosin TM30nm (TPM3), HS1-binding protein Hax-1 (HAX1), RNA-specific adenosine deaminase (ADAR1), the 34/67-kD laminin receptor (LAMRL6), and the 26S proteasome subunit p31 (PSMD8L), as well as five hitherto uncharacterized proteins (NICE-1, NICE-2, NICE-3, NICE-4, and NICE-5). The nucleotide sequences and putative ORFs of the EDC genes identified here revealed no homology with any of the established EDC gene families. Whereas database searches revealed that NICE-3, NICE-4, and NICE-5 were expressed in many tissues, no EST or gene-specific sequence was found for NICE-2. Expression of NICE-1 was up-regulated in differentiated keratinocytes, pointing to its relevance for the terminal differentiation of the epidermis. The newly identified EDC genes are likely to provide further insights into epidermal differentiation and they are potential candidates to be involved in skin diseases and carcinogenesis that are associated with this region of chromosome 1. Moreover, the extended integrated map of the EDC, including the polymorphic sequence tag site (STS) markers D1S1664, D1S2346, and D1S305, will serve as a valuable tool for linkage analyses.

Figure 1

Hybridization of YACs 986_e_10 and 950_e_2#9 to a high-density filter of the gridded keratinocyte cDNA library. Autoradiographs show a quarter of a blot containing 18,432 double-spotted cDNA clones, which was successively hybridized with ³²P-labeled DNA derived from YACs 986_e_10 and 950_e_2#9, respectively. Positive clones are detected by two spots arranged in a specific pattern. Clones identified with both YACs were subtracted. Arrows indicate examples of a subtracted clone (black arrows) and of potentially EDC-specific cDNAs (white arrows).

Figure 2

Integrated map of the EDC. Genomic and YAC restriction mapping results yielded the order of genes (in bold), STSs (in italics), and other loci within the EDC as shown. STS markers coinciding with the newly assigned genes were not tested and are marked with an asterisk. Precise localization of 37m2/37m16, of the two SPRR1 genes and of S100A1/S100A13 was resolved previously (South et al. 1999). The order of loci underlined with a horizontal bar could not be resolved. (A) Genomic restriction map of the EDC. A continuous map spanning 3.5 Mb of region 1q21 with NotI (N), NruI (R), MluI (M), and BsiWI (B) restriction sites of the two haplotypes present in the H2LCL cell line (represented by the two lanes) is shown. White ovals indicate hybridization of the probe specified below to the corresponding restriction fragments that resulted from single and double restriction enzyme digests. Refined mapping results of S100A10, THH, FLG, IVL, SPRR3, SPRR1, SPRR2, LOR, S100A9, S100A8, and S100A6 obtained by digestion with additional restriction enzymes (not shown) were adopted from the established map (Mischke et al. 1996). (B) Refined YAC contig of the EDC. Sixteen YACs from the 6-Mb contig covering the EDC (Marenholz et al. 1996) are shown with their addresses. Eight overlapping YACs of 950_e_2 that were used to resolve the order in the distal region are included. Sizes of YACs as determined previously by rotating-field gel electrophoresis (ROFE) are represented by the sum of the length of the gray and black boxes (detected fragments) and of the black horizontal bars (unidentified DNA sequences). White ovals represent hybridization of the respective fragment with the corresponding probe, derived from the locus above. SalI restriction sites are indicated by vertical lines. A polymorphic restriction site in NICE-1 was detected for 907_e_6 and 874_d_5. Gray boxes correspond to continuously covered human DNA as deduced from marker content and from identical fragments in several independent YACs. Broken horizontal lines indicate deletions and black boxes indicate other rearranged fragments identified by aberrant size and/or marker content. Restriction fragments of distinct size that were only detected in a single YAC are potential end fragments or could be rearranged and lack the second SalI site marker. Arrangement of detected SalI restriction fragments with respect to the unidentified DNA sequences depended on YAC sizes and genomic mapping results. S100A3, S100A5, and S100A12 represented by gray ovals were mapped previously to YAC 100_f_3 (Schaefer et al. 1995; Wicki et al. 1996b). D1S1664, D1S2346, and D1S305 (vertical broken lines) were assigned to the corresponding YACs by PCR and therefore could not be positioned on single restriction fragments. The NICE-2, S100A7, and D1S3625 probes each hybridized to the same two SalI fragments, most likely indicating a duplication of sequences. Their order was resolved on the basis of signal intensities.

Figure 3

Alignment of the cDNA clones 31131 8 and 3162o12 representing two different alleles of the SPRR3 gene. Black boxes indicate divergences in the nucleotide sequences, open boxes indicate divergences in the deduced amino acid sequences that contain 14 (clone 31131 8) and 13 (clone 3162o12) octapeptide repeats (shaded gray), respectively. Among the four mutations within the coding region, only one results in a substituted amino acid residue, with higher homology to the consensus repeat sequence TKVPEPGC of clone 3162o12.

Figure 4

Alternatively spliced gene products of NICE-3. The nucleotide sequences of the three cDNA clones were identical except for the lacking exons (broken lines) of clones 3038m19 (exon 279–380) and 1023j12 (exons 279–380 and 499–552). Incomplete 5′-termini of clones 3038m19 and 3038j13 were filled up with overlapping sequences of clone 1023j12 (a) and EST T27537 (b). Sequence information derived from the respective clones is represented by open boxes; ORFs are shaded gray. The first nucleotide of the putative initiation methionine codon, exon boundaries, and the last nucleotide of the coding sequence, as well as the size of the predicted protein are indicated for each transcript. Additional spliced exons and combinations of them were identified in ESTs by similarity search: exon 202–278 in AA354455 from Jurkat T-cells, exons 202–278 and 279–380 in AA463392 from total fetus, exon 381–498 in AA057488 from colon, exons 279–380 and 381–498 in Z42265 from infant brain.

Figure 5

Alternative 3′-termini of the NICE-4 gene products. Sequence information derived from cDNA clones 1056f 5, 3114f17, and cDNA KIAA0144 is represented by open boxes. Clone 3114f17 only contains the 3′-terminal sequence of the transcript and was filled up with overlapping sequences of clone 1056f 5 and cDNA KIAA0144. Nonhomologous regions are cross-hatched, ORFs are shaded gray. The first and last nucleotide of the putative coding sequence and of identical regions are indicated above, the size of the predicted protein below the corresponding transcripts.

Figure 6

Sequence analysis of NICE-1 cDNA. (A) Nucleotide sequence of NICE-1 and the amino acid sequence deduced from its largest ORF. The putative initiation methionine codon ATG and the termination codon TGA are shaded gray. The polyadenylation signal AATAAA is underlined. (B) Comparison of the protein sequences of NICE-1 and xp5. Black boxes indicate identical, gray boxes indicate similar amino acids. Dashes indicate missing amino acids. Pairwise sequence alignment was performed at http://vega.crbm.cnrs-mop.fr/bin/align-guess.cgi.

Figure 7

Sequence analysis of the NICE-5 and NICE-2 gene products. cDNA clones 11591 8 (NICE-5) (top) and 1192j18 (NICE-2) (bottom) contain the 3′-termini of the corresponding transcripts. Overlapping ESTs AA287174 and AA641342 were used to extend the NICE-5 sequence. Sequence information derived from the respective clones is represented by open boxes; broken line boxes indicate lacking sequences as deduced from the approximate size of the entire mRNA, which was determined by Northern hybridization. The ORF of NICE-5 (shaded gray) is likely to be incomplete. No significant ORF was detected within the NICE-2 sequence. Numbering is relative to the start of the available sequence (+1). In the case of NICE-5, the last nucleotide of the putative coding sequence and the minimum protein size are indicated. The size of the protein resulting from the first initiation methionine codon within the available sequence and the position of the initial adenine (▵) are in parenthesis.

Figure 8

Northern blot analysis of NICE-1. A Northern blot containing RNA from different cell populations was hybridized with a specific probe for NICE-1. Total RNA (20 μg) in each lane was isolated from the following tissues or cultured cells: (1) human skin; (2) stripped human keratinocytes maintained in medium without calcium (Fischer et al. 1999); (3) stripped human keratinocytes maintained in a proliferative state in medium with 1.8 mM strontium (Praeger et al. 1987); (4) stripped human keratinocytes induced to differentiate in medium with 1.8 mM calcium; (5) HaCaT cells; (6) HeLa cells; (7) SCC4 cells; (8) primary human fibroblasts; (9) primary human melanocytes. The top band is due to cross-reaction with 28S ribosomal RNA.