Krtap16, Characterization of a New Hair Keratin-associated Protein (KAP) Gene Complex on Mouse Chromosome 16 and Evidence for Regulation by Hoxc13

Abstract

Intermediate filament (IF) keratins and keratin-associated proteins (KAPs) are principal structural components of hair and encoded by members of multiple gene families. The severe hair growth defects observed upon aberrant expression of certain keratin and KAP genes in both mouse and man suggest that proper hair growth requires their spatio-temporally coordinated activation. An essential prerequisite for studying these cis-regulatory mechanisms is to define corresponding gene families, their genomic organization, and expression patterns. This work characterizes eight recently identified high glycine/tyrosine (HGT)-type KAP genes collectively designated Krtap16-n. These genes are shown to be integrated into a larger KAP gene domain on mouse chromosome 16 (MMU16) that is orthologous to a recently described HGT-and high sulfur (HS)-type KAP gene complex on human chromosome 21q22.11. All Krtap16 genes exhibit strong expression in a narrowly defined pattern restricted to the lower and middle cortical region of the hair shaft in both developing and cycling hair. During hair follicle regression (catagen), expression levels decrease until expression is no longer detectable in follicles at resting stage (telogen). Since isolation of the Krtap16 genes was based on their differential expression in transgenic mice overexpressing the Hoxc13 transcriptional regulator in hair, we examined whether bona fide Hoxc13 binding sites associated with these genes might be functionally relevant by performing electrophoretic mobility shift assays (EMSAs). The data provide evidence for sequence-specific interaction between Hoxc13 and Krtap16 genes, thus supporting the concept of a regulatory relationship between Hoxc13 and these KAP genes.

Evidence That the Satin Hair Mutant Gene Foxq1 Is among Multiple and Functionally Diverse Regulatory Targets for Hoxc13 during Hair Follicle Differentiation

“…cDNA suppression subtraction hybridization (18). Both KAP genes were identified as members of a novel KAP gene domain on MMU16 in a region of conserved linkage to HSA21q22.11 (18,22), and most of the cDNAs isolated by suppression subtraction hybridization in the previous study corresponded to this domain (18,22), a bias that was not observed in the DNA microarray data set presented here. The up-regulation of Krt2-1 in the epidermis (Table 1) was also noted in the previous screen (18), and although this finding is consistent with the epidermal hyperproliferation and ichthyosiform condition observed in GC13 mice, a potential mechanistic link to the overexpression of Hoxc13 in hair follicles is currently obscured since epidermal Hoxc13 expression was not detectable by ISH in both normal and GC13 mice (data not shown).…”

“…7, A and B). Hoxc13-oligo complex formation was inhibited by HOXC13-specific antiserum (21), which reacts specifically with mouse Hoxc13 in both supershift and immunohistochemical assays (22,36), whereas anti-tyrosinase-related protein 1 antiserum (see "Materials and Methods") used as a control did not affect complex formation (Fig. 7B).…”

“…PCR products were cloned into pCRII-TOPO vector (Invitrogen) for the generation of digoxigenin (Roche Applied Science)-labeled antisense and sense (control) RNA probes as described (22). Hybridization with 10-m cryosections and colorimetric signal detection using alkaline phosphatase-conjugated anti-digoxigenin antibodies (Roche Applied Science) was performed as previously described (22,31).…”

“…Hybridization with 10-m cryosections and colorimetric signal detection using alkaline phosphatase-conjugated anti-digoxigenin antibodies (Roche Applied Science) was performed as previously described (22,31).…”

Comparative analysis of genes downstream of the Hoxd cluster in developing digits and external genitalia

“…With such a technique, Hedlund et al (Hedlund et al, 2004) identified candidate target genes by comparing embryonic spinal cord tissue from wild-type and Hoxd10-null mice. In a related approach, Pruett et al (Pruett et al, 2004) used transgenic mice overexpressing the Hoxc13 gene to identify keratin genes of the Krtap16 family as likely direct targets of HOXC13. The use of such approaches is nevertheless complicated by the severity of some Hox mutations and the functional redundancy of these genes.…”