The promoter of a lysosomal membrane transporter gene, CTNS, binds Sp-1, shares sequences with the promoter of an adjacent gene, CARKL, and causes cystinosis if mutated in a critical region

Abstract

Although >55 CTNS mutations occur in patients with the lysosomal storage disorder cystinosis, no regulatory mutations have been reported, because the promoter has not been defined. Using CAT reporter constructs of sequences 5' to the CTNS coding sequence, we identified the CTNS promoter as the region encompassing nucleotides -316 to +1 with respect to the transcription start site. This region contains an Sp-1 regulatory element (GGCGGCG) at positions -299 to -293, which binds authentic Sp-1, as shown by electrophoretic-mobility-shift assays. Three patients exhibited mutations in the CTNS promoter. One patient with nephropathic cystinosis carried a -295 G-->C substitution disrupting the Sp-1 motif, whereas two patients with ocular cystinosis displayed a -303 G-->T substitution in one case and a -303 T insertion in the other case. Each mutation drastically reduced CAT activity when inserted into a reporter construct. Moreover, each failed either to cause a mobility shift when exposed to nuclear extract or to compete with the normal oligonucleotide's mobility shift. The CTNS promoter region shares 41 nucleotides with the promoter region of an adjacent gene of unknown function, CARKL, whose start site is 501 bp from the CTNS start site. However, the patients' CTNS promoter mutations have no effect on CARKL promoter activity. These findings suggest that the CTNS promoter region should be examined in patients with cystinosis who have fewer than two coding-sequence mutations.

Figure 1

Promoter region and its activity. a, Nucleotides within the 501-bp intergene region separating the start sites of CTNS and CARKL. The number on the left shows the distance from the CTNS start site. The Sp-1, AP-4, and CAAT-binding protein (CBP) motifs are underlined and in boldface. The mutations in patients 1 and 2 are boxed. The 41 nucleotides in uppercase font may contribute to both expression of CTNS and expression of CARKL. b, CAT activities of CTNS promoter constructs. Nucleotide +1 represents the transcription start site. The constructs portrayed on the left were inserted upstream of pbCAT. The pSV40—that is, the promoterless CAT control—plasmid, which contains both an SV40 enhancer and an SV40 promoter, served as a positive control, and pbCAT served as a negative control. CAT activity was determined for each construct after transient transfection into HeLa cells. A vector carrying the gene for β-galactosidase was cotransfected as a control, for transfection efficiency. The bars represent means ± SD for three determinations of CAT activity. c, CAT activities of CARKL promoter constructs. Nucleotide +1 represents the transcription start site of CARKL.

Figure 2

CTNS promoter mutations. The normal sequence is given for nucleotides −311 to −292. Arrows indicate the mutated bases—that is, a G→C change at position −295 in patient 1, a G→T change at position −303 in patient 2, and a T insertion after position −303 in patient 3.

Figure 3

EMSAs of CTNS promoter region bearing −295 G→C mutation in patient 1. a, Results of double-stranded–DNA probe consisting of nucleotides −308 to −279, radiolabeled with α[³²P]-dATP. Lane 1, No addition. Lane 2, NE added. Lane 3, NE and 100-fold excess nonradioactive probe added. Lane 4, NE and 30-fold-molar excess nonspecific (calf thymus) DNA added. b, Results of double-stranded–DNA probe consisting of nucleotides −308 to −279, containing the −295 G→C mutation in patient 1, radiolabeled with α[³²P]-dATP. Lane 1, No addition. Lane 2, NE added. c, Results of Sp-1 oligonucleotide probe radiolabeled with γ[³²P]-ATP. Lane 1, No addition. Lane 2, NE added. Lane 3, NE and 100-fold excess nonradioactive Sp-1 oligonucleotide added. Lane 4, NE and 100-fold excess nonradioactive probe consisting of nucleotides −308 to −279 added. Lane 5, NE and 100-fold excess nonradioactive probe consisting of nucleotides −308 to −279, with the −295 G→C mutation in patient 1, added. d, Results of double-stranded–DNA probe consisting of nucleotides −308 to −279, radiolabeled with α[³²P]-dATP. Lane 1, No addition. Lane 2, Authentic Sp-1 peptide added. Lane 3, Sp-1 peptide and nonradioactive probe consisting of nucleotides −308 to −279 added. Lane 4, Sp-1 peptide and 30-fold-molar excess nonspecific (calf thymus) DNA added.

Figure 4

EMSAs of CTNS promoter region bearing mutations in patients 2 and 3. a, Results of double-stranded DNA probe consisting of nucleotides −327 to −300, radiolabeled with α[³²P]-dATP. Lane 1, No addition. Lane 2, NE added. Lane 3, NE and 100-fold excess nonradioactive probe added. Lane 4, NE and 300-fold excess nonradioactive probe added. Lane 5, NE and 600-fold excess nonradioactive probe added. Lane 6, NE and 600-fold excess nonradioactive probe consisting of nucleotides −327 to −300, containing the −303 G→T mutation in patient 2, added. Lane 7, NE and 600-fold excess nonradioactive probe consisting of nucleotides −327 to −300, containing the −303 T insertion in patient 3, added. Arrows indicate the locations of DNA-protein complexes that can be competed against by the normal but not by the mutant oligonucleotides. b, Results of double-stranded DNA probe consisting of nucleotides −327 to −300, containing the −303 G→T mutation in patient 2, radiolabeled with α[³²P]-dATP. Lane 1, No addition. Lane 2, NE added. Lane 3, NE and 600-fold excess nonradioactive mutant probe added. c, Results of double-stranded DNA probe consisting of nucleotides −327 to −300, containing the T insertion after position −303 in patient 3, radiolabeled with α[³²P]-dATP. Lane 1, No addition. Lane 2, NE added. Lane 3, NE and 600-fold excess nonradioactive mutant probe added.