Basal transcription activity of the dyskeratosis congenita gene is mediated by Sp1 and Sp3 and a patient mutation in a Sp1 binding site is associated with decreased promoter activity.

The multisystem disorder dyskeratosis congenita (DKC) is caused by mutations in the DKC1 gene. The protein dyskerin is a component of the box H+ACA small nucleolar RNAs (snoRNAs) and is also functionally associated with the RNA component of the human telomerase. The majority of mutations are missense mutations, although single examples of non-coding mutations have been described. One of these is a point mutation in a putative Sp1 binding site in the 5'-upstream region of the DKC1 gene which presumably represents the promoter region of the gene. In this report, we compare the promoter sequences of both the human and mouse genes and provide a first functional characterisation of the human DKC1 promoter. This includes a characterisation of the disease-associated implications caused by the mutation identified in one patient. By reporter gene analysis, functional regions of the DKC1 promoter were delineated. The core promoter region critical for basal level of transcription was found to lie at -10 to -180. Bandshift- and supershift experiments clearly demonstrated a mutual binding of transcription factors Sp1 and Sp3 to two of five putative GC-box/Sp1-binding sites located within the core promoter region. An additional GC-box interacts only with the Sp1 transcription factor. Further, we provide evidence that the DKC1 mutation in one of the Sp1 binding sites results in reduced promoter activity.

Physical and genetic characterization reveals a pseudogene, an evolutionary junction, and unstable loci in distal Xq28.

A large portion of human Xq28 has been completely characterized but the interval between G6PD and Xqter has remained poorly understood. Because of a lack of stable, high-density clone coverage in this region, we constructed a 1.6-Mb bacterial and P1 artificial chromosome (BAC and PAC, respectively) contig to expedite mapping, structural and evolutionary analysis, and sequencing. The contig helped to reposition previously mismapped genes and to characterize the XAP135 pseudogene near the int22h-2 repeat. BAC clones containing the distal int22h repeats also demonstrated spontaneous rearrangements and sparse coverage, which suggested that they were unstable. Because the int22h repeats are involved in genetic diseases, we examined them in great apes to see if they have always been unstable. Differences in copy number among the apes, due to duplications and deletions, indicated that they have been unstable throughout their evolution. Taking another approach toward understanding the genomic nature of distal Xq28, we examined the homologous mouse region and found an evolutionary junction near the distal int22h loci that separated the human distal Xq28 region into two segments on the mouse X chromosome. Finally, haplotype analysis showed that a segment within Xq28 has resisted excessive interchromosomal exchange through great ape evolution, potentially accounting for the linkage disequilibrium recently reported in this region. Collectively, these data highlight some interesting features of the genomic sequence in Xq28 and will be useful for positional cloning efforts, mouse mutagenesis studies, and further evolutionary analyses.