Functional analysis of a novel cis-acting regulatory region within the human ankyrin gene (ANK-1) promoter

Abstract

The characterization of atypical mutations in loci associated with diseases is a powerful tool to discover novel regulatory elements. We previously identified a dinucleotide deletion in the human ankyrin-1 gene (ANK-1) promoter that underlies ankyrin-deficient hereditary spherocytosis. The presence of the deletion was associated with a decrease in promoter function both in vitro and in vivo establishing it as a causative hereditary spherocytosis mutation. The dinucleotide deletion is located in the 5' untranslated region of the ANK-1 gene and disrupts the binding of TATA binding protein and TFIID, components of the preinitiation complex. We hypothesized that the nucleotides surrounding the mutation define an uncharacterized regulatory sequence. To test this hypothesis, we generated a library of more than 16,000 ANK-1 promoters with degenerate sequence around the mutation and cloned the functional promoter sequences after cell-free transcription. We identified the wild type and three additional sequences, from which we derived a consensus. The sequences were shown to be functional in cell-free transcription, transient-transfection, and transgenic mouse assays. One sequence increased ANK-1 promoter function 5-fold, while randomly chosen sequences decreased ANK-1 promoter function. Our results demonstrate a novel functional motif in the ANK-1 promoter.

FIG. 1.

(A) Erythroid ankyrin-1 (ANK-1) promoter. The erythroid ANK-1 TSSs at positions −106, −100, −84, −71, −63, and −56 relative to the ATG codon are indicated by arrows. These TSS correspond to positions −23, −16, +1, +16, +24, and +30 relative to the TSS listed in the database of transcriptional start sites (DBTSS) (37-39). The heavier arrow at position −84 represents the TSS from which the majority of the transcripts initiate. The location of the 9 nucleotides surrounding the TG dinucleotide deletion at positions −78 to −70 is indicated, along with a similar sequence between positions −158 and −150 (see Discussion). Sp1/CACCC sites are indicated as Sp1. (B) ChIPSeq analysis of a 10-kb region of the human erythroid ANK-1 promoter. The coordinates of the March 2006 human genome assemble (hg18) are shown along the y axis below the figure. The x axis shows the number of RNA Pol II-enriched sequence tags that map to 10-bp windows along the ANK-1 locus. Lines indicate the location of the peak windows in the region enlarged in panel A.

FIG. 2.

Experimental design. (A) The erythroid ANK-1 promoter was linked to a luciferase reporter gene. The region surrounding the TG dinucleotide deleted in the German HS patient was removed and replaced with a library of oligonucleotides with degenerate sequences that preserve the critical TG. (B) The promoter library was used as a template for cell-free transcription. Cell-free transcription products (∼290 bp) were separated on an agarose gel and cloned by RACE. Because the degenerate sequence is in the transcribed region, active promoter sequences can be determined by sequencing of RACE clones. M, DNA size markers (lambda/HindIII; φX174/HaeIII); the arrow indicates the position of the cell-free transcription products from a wild-type ANK-1 promoter template (WT; + control) and from the library of ANK-1 promoters with degenerate sequence in the −78 to −70 region (Lib.).

FIG. 3.

Comparison of the sequence of a 126-bp region of the human ANK-1 promoter extending from positions −179 to −53 in humans to the sequence of aligned regions in other mammals. The annotated erythroid ANK-1 mRNA (using the major TSS; −84) is indicated by the black line at the top of the figure. The consensus sequence between positions −78 and −70 in Old World primates is boxed, as is a similar sequence between positions −158 and −150 (see Discussion). The sequences for human and 20 other mammals (indicated at the right) are shown in the center. At the bottom of the figure, conserved sequences among primates (top) and all mammals (bottom) are indicated in green boxes.

FIG. 4.

TFIID binding to variant ANK-1 promoters. (A) In vitro DNase I footprint analysis of purified TFIID incubated with ³²P-labeled 175-bp fragments containing the wild type (WT, TGCGGTGAG) and variant ANK-1 promoter sequences (GC, GCGGGTGAG; GG, GGCGGTGAG; 3G, GGGGGTGAG). T, T lane from sequencing ladder. (B) In vitro DNase I footprint analysis of purified TFIID incubated with a ³²P-labeled 173-bp fragment containing the ANK-1^−TG (TGCGG__AG) sequence. The sequence between −78 and −70 and for each promoter is shown to the right. The amount of TFIID added is indicated below each lane. The ANK-1^−TG footprint has been described previously (20). (C) Mg agarose EMSA analysis of TFIID binding to wild-type and variant ANK-1 promoters. The probes were 281-bp fragments of the ANK-1 promoter extending from positions −296 to −15. (D) Ratios of bound and unbound probe from three independent experiments normalized to the wild-type ratio. The ANK-1^rTG Mg agarose EMSA has been described previously (20). *, P < 0.05.

FIG. 5.

Cell Free Transcription of wild type (WT, TGCGGTGAG) and variant (GC, GCGGGTGAG; GG, GGCGGTGAG; 3G, GGGGGTGAG) ANK-1 promoters in HeLa and K562 cell nuclear extracts (indicated above the panels). (A) Each ANK-1 template is linked to an internal control CMV promoter fragment to ensure equimolar amounts of template. The CMV transcript gives a single band (lane C). Multiple transcripts are transcribed by the ANK templates (arrowheads show the position of the −106, −100, −84, −71, −63, and −56 transcripts). (B) Cell-free transcription of wild type and ANK-1^−TG (TGCGG__AG) ANK-1 promoters in HeLa and K562 cell nuclear extracts (indicated below panels). (C) Relative amounts of ANK-1 transcripts from individual templates. The data are the ratios of ANK-1 transcripts to the control CMV transcript from three independent experiments normalized to the wild-type ratio. The ANK-1^−TG cell-free transcription has been described previously (20). *, P < 0.05.

FIG. 6.

Transient-transfection analysis of the variant ANK-1 promoters in K562 cells. Each promoter was linked to a luciferase reporter gene and transfected into K562 cells. The data are the ratio of luciferase expression normalized to the wild type (WT, TGCGGTGAG). (A) Comparison of the activity of the ANK-1^WT promoter to ANK-1 promoters with the TG dinucleotide deletion at −72/73 (−TG, TGCGG__AG) or nonconsensus sequences flanking the TG dinucleotide (1, AATTGTGTT; 2, GGTTCTGTA; 3, TGTGTTGTT; 4, TACATTGCT; 5, ATGGTTGGG). (B) Comparison of the activity of the ANK-1^WT promoter to that of the variant ANK-1 promoters. (GC, GCGGGTGAG; GG, GGCGGTGAG; 3G, GGGGGTGAG). *, P < 0.01.

FIG. 7.

Analysis of the variant ANK-1 promoters in transgenic mice. Each promoter was linked to a human ^Aγ-globin reporter gene and injected into fertilized mouse eggs. Founder animals were bred to generate F₁ animals for analysis of the copy number and γ-globin mRNA level. (A) DNA, Southern blot analysis of wild type (+C) and low-copy-number animals with the variant ANK-1 promoters (GC, GCGGGTGAG; GG, GGCGGTGAG; 3G, GGGGGTGAG). The estimated copy number is indicated above the image of the Southern blot. RNA, RNase protection analysis of γ-globin mRNA levels in wild type (+C) and low-copy-number animals with the variant ANK-1 promoters (GC, GG, and GGG). (B) The relative levels of human γ-globin and endogenous mouse α-globin mRNA per transgene copy. Wild type (WT): n = 16; GC, n = 5; GG, n = 6; and GGG, n = 5). *, P < 0.01 compared to the wild type. (C) Fluorescence activated cell sorting analysis of human γ-globin protein in erythrocytes from wild type (+C) and low-copy-number animals with the variant ANK-1 promoters. −C, erythrocytes from a transgene negative littermate control. +C, erythrocytes from a mouse carrying >20 copies of the wild-type ANK-1 promoter/γ-globin transgene. GC, GG, and GGG, erythrocytes from mice carrying one to six copies of the indicated variant ANK-1 promoter/γ-globin transgene. Analysis results for all of the animals are presented in Table 2.