Characterization of a 190-Kilobase Pair Domain of Human Type I Hair Keratin Genes

Abstract

Polymerase chain reaction-based screening of an arrayed human P1 artificial chromosome (PAC) library using primer pairs specific for the human type I hair keratins hHa3-II or hHa6, led to the isolation of two PAC clones, which covered 190 kilobase pairs (kbp) of genomic DNA and contained nine human type I hair keratin genes, one transcribed hair keratin pseudogene, as well as one orphan exon. The hair keratin genes are 4 -7 kbp in size, exhibit intergenic distances of 5-8 kbp, and display the same direction of transcription. With one exception, all hair keratin genes are organized into 7 exons and 6 positionally conserved introns. On the basis of sequence homologies, the genes can be grouped into three subclusters of tandemly arranged genes. One subcluster harbors the highly related genes hHa1, hHa3-I, hHa3-II, and hHa4. A second subcluster of highly related genes comprises the novel genes hHa7 and hHa8, as well as pseudogene ⌿hHaA, while the structurally less related genes hHa6, hHa5, and hHa2 are constituents of the third subcluster. As shown by reverse transcription-polymerase chain reaction, all hair keratin genes, including the pseudogene, are expressed in the human hair follicle. The transcribed pseudogene ⌿hHaA contains a premature stop codon in exon 4 and exhibits aberrant pre-mRNA splicing. Evolutionary tree construction reveals an early divergence of hair keratin genes from cytokeratin genes, followed by the segregation of the genes into the three subclusters. We suspect that the 190-kbp domain contains the entire complement of human type I hair keratin genes.The keratin multigene family comprises the cytokeratins or soft ␣-keratins, which are expressed in the various types of epithelia, and the hair keratins or hard ␣-keratins, involved in the formation of hard keratinized structures. Both can be divided into type I (acidic) and type II (basic-neutral) proteins that form the 10-nm intermediate filament network of epithelial cells by obligatory association of equimolar amounts of type I and type II keratins (1, 2). Disturbances of intermediate filament formation through deleterious mutations in keratins can lead to a weakening of the structural integrity of the respective epithelial cells, resulting in hereditary disorders of skin, mucosa, nail, or hair (3-7). Although initial studies of hair keratin proteins of several species indicated the existence of eight major type hair keratins, four type I members, termed Ha1-Ha4, and four type II members, termed Hb1-Hb4, as well as of one minor hair keratin pair, Hax/Hbx (8 -11), it has recently been shown that the hair keratin family is distinctly more complex. In man, sequences of seven type I hair keratins, hHa1, hHa2, hHa3-I, hHa3-II, hHa4, 1 hHa5, hHa6 (previously designated hHRa1) 1 and four type II hair keratins, hHb1, hHb3, hHb5, and hHb6, have been elucidated by molecular cloning, and their differential expression in the hair matrix, cortex, and cuticle of the hair follicle has been shown (12-17). To date, complete sequences for one human type I and t...

Transcription Regulation and Protein Subcellular Localization of the Truncated Basic Hair Keratin hHb1-ΔN in Human Breast Cancer Cells

“…However, in the hHb1-⌬N promoter, neither Sp1 expression nor its binding activity were notably altered in response to 5-Aza-2ЈdC. 2 We thus exclude the possibility that the effect of 5-Aza-2ЈdC occurs at the Sp1 gene locus or at a gene locus whose product modifies Sp1 activity. hHb1-⌬N induction through demethylation is thus dependent on the synthesis and/or activation of an unknown protein.…”

“…genomic region encompassing the hHb1-⌬N coding region and the 5Ј-and 3Ј-flanking sequences. 2 Interestingly, transcription from the TATA-less promoter of the leukosialin gene is also mediated by a single GTbox element 40 bp upstream of the transcription start site, but no other cis-acting regulatory activity responsible for the cell type-specific expression could be identified (31). However, DNA methylation around the 5Ј-flanking region of the leukosialin gene is required to shut off the high level of expression, showing that tissue-specific expression is achieved by alteration of DNA methylation (32).…”

HOXC13 Is Involved in the Regulation of Human Hair Keratin Gene Expression

“…and promoter deletion constructs of type I hair keratin genes hHa5, hHa2, hHaA, and hHa7 (GenBank TM accession numbers Y16791, X90761, Y16795, and Y16793) were generated by PCR using the previously described PAC3 clone (12) as template DNA as well as forward primers containing an EcoRI-site and reverse primers containing a XhoI-site. After EcoRI/XhoI digestion of the PCR products, the gelpurified fragments were cloned into the EcoRI/XhoI-digested ␤-galactosidase reporter vector pNass␤ (CLONTECH, Heidelberg, Germany).…”

“…In the past years, our laboratory has elucidated the organization of the human type I and type II hair keratin gene loci and characterized the sequential expression of their members during trichocyte differentiation (12)(13)(14)(15) Based on this knowledge, we selected three human type I hair keratin genes hHa5, hHa2, and hHa7 as well as the transcribed pseudogene hHaA, whose mRNA expression patterns in the lower hair forming compartment of the human hair follicle (13,16) corresponded to that described for Hoxc13 in mouse hair follicles (6), and investigated whether they are target genes for HOXC13. In the present study, we provide strong evidence that these hair keratin genes are transcriptionally up-regulated by HOXC13 primarily via binding of the transcription factor to distinct core recognition motifs, TAAT and TTAT, in the respective proximal promoters.…”

Characterization of a First Domain of Human High Glycine-Tyrosine and High Sulfur Keratin-associated Protein (KAP) Genes on Chromosome 21q22.1

“…The main structural proteins of the hair fiber are the hair keratins and the hair keratin-associated proteins, KAPs, 1 the latter being encoded by a large number of multigene families (2). Hair keratins, a subset of the large keratin family whose members are found in all cells of epithelial origin (3,4), represent two multigene families, the type I (acidic) and type II (basic) families that comprise 15 members in humans (5,6). They form the 8 -10-nm intermediate filaments of trichocytes by co-polymerization of type I and type II members, which are differentially expressed during hair fiber development (7,8).…”