A CpG mutational hotspot in a ONECUT binding site accounts for the prevalent variant of hemophilia B Leyden

Abstract

Hemophilia B, or the "royal disease," arises from mutations in coagulation factor IX (F9). Mutations within the F9 promoter are associated with a remarkable hemophilia B subtype, termed hemophilia B Leyden, in which symptoms ameliorate after puberty. Mutations at the -5/-6 site (nucleotides -5 and -6 relative to the transcription start site, designated +1) account for the majority of Leyden cases and have been postulated to disrupt the binding of a transcriptional activator, the identity of which has remained elusive for more than 20 years. Here, we show that ONECUT transcription factors (ONECUT1 and ONECUT2) bind to the -5/-6 site. The various hemophilia B Leyden mutations that have been reported in this site inhibit ONECUT binding to varying degrees, which correlate well with their associated clinical severities. In addition, expression of F9 is crucially dependent on ONECUT factors in vivo, and as such, mice deficient in ONECUT1, ONECUT2, or both exhibit depleted levels of F9. Taken together, our findings establish ONECUT transcription factors as the missing hemophilia B Leyden regulators that operate through the -5/-6 site.

Figure 1

The Proximal Promoter of Human F9 The major transcription start site (TSS) is indicated by an arrow and designated +1. Point mutations associated with hemophilia B Leyden are indicated above the sequence. The transcription factors known to bind this region are shown, and the consensus sequences that they recognize are aligned below the sequence. DNA-binding consensus sequences were determined from the ChIP-Seq data reported here (ArrayExpress accession number E-MTAB-890) and previously (ArrayExpress E-TABM-722) with the default settings of MEME and the following parameters: -revcomp -maxw 20 -minw 6 -nmotifs 5. We selected the top 500 peaks ordered by input-corrected read depth and used the 25 base pairs centered on the identified summit of the peak as our input for the motif-discovery analysis. The position weight matrices are consistent with previously determined DNA-binding consensuses for these transcription factors. The following protein abbreviations are used: HNF4α, hepatocyte nuclear factor 4 alpha; C/EBPα, CCAAT/enhancer-binding protein alpha; and OC1/2, ONECUT1/2.

Figure 2

ONECUT Proteins Recognize the −5/−6 Region of the Human F9 Promoter, and Binding Is Disrupted by Leyden Mutations (A–F) EMSAs using wild-type (WT) and mutant probes spanning the −5/−6 region. (A) ONECUT1 and ONECUT2 GST-fusion proteins purified from bacteria. Coomassie staining is shown on the right to indicate the level and purity of the recombinant proteins used. (B) Nuclear extracts from COS cells expressing full-length ONECUT1 and ONECUT2. (C) Murine liver nuclear extracts with antibodies against ONECUT1 and ONECUT2. (D) Murine liver nuclear extracts with WT and mutant probes. Nuclear extracts from COS cells transfected with ONECUT1 and ONECUT2 were included as positive controls. (E) ONECUT1 and ONECUT2 GST-fusion proteins purified from bacteria. Equivalent levels of protein were added as in (A). (F) A competition assay using radiolabeled WT probe (1.6 ng per lane) and cold competitor probes (0 ng, 16 ng, 160 ng, and 1.6 μg, that is, 0×, 10×, 100×, and 1,000× excesses, respectively) compares the ability of the different mutants to compete for binding by ONECUT1. Two microliters of nuclear extracts from COS cells expressing ONECUT1 was used per lane. Murine liver tissue was obtained under ethics approval number 09/128A (Animal Care and Ethics Committee, University of New South Wales). The following protein abbreviations are used: OC1, ONECUT1; and OC2, ONECUT2.

Figure 3

ONECUT1 Binds the F9 Promoter In Vivo along with HNF4α and C/EBPα (A) ONECUT1 ChIP using mouse liver tissue. Primer sets are specific to the TSS of F9 and to 1.0 kb downstream and 1.5 kb upstream. Negative-control primers specific to the promoter regions of F7 and F10 were included. Enrichment was normalized to input, and the lowest values for immunoglobulin G (IgG) and anti-ONECUT1 were set to 1.0. Error bars represent the SEM, and n = 3 for each data point. ^∗p < 0.05 (two-tailed t test) compared to the anti-ONECUT1 enrichment at each of the F9 distal regions and the F7 and F10 promoters. (B–C) Human-liver-tissue ChIP-Seq tracks for ONECUT1, C/EBPα, and HNF4α across the F9 locus (B) and around the TSS (C). (C) The HNF4α, ONECUT1, and C/EBPα binding sites at nucleotides −20, −5/−6, and +10, respectively, are denoted by colored boxes. Murine liver tissue was obtained under ethics approval number 09/128A (Animal Care and Ethics Committee, University of New South Wales). ChIP sequencing reads and input samples were aligned with MAQ with default parameters to NCBI Genome browser build 36. All sequence, genome-annotation, and comparative-genomics data were taken from Ensembl release 52. After alignment, binding events were discovered with a dynamic programming algorithm (SWEMBL) with the parameter “-R 0.005 -i –S” as previously described.

Figure 4

ONECUT Proteins Are Functionally Required for F9 Expression (A) A Luciferase reporter assay in HepG2 cells. Wild-type (WT) and mutant F9 promoter fragments were tested in the presence or absence of ONECUT1. Triplicate, independent DNA preparations for the promoter constructs were used. Firefly levels were normalized with the use of Renilla, and the value for the WT promoter in the absence of ONECUT1 was set to 1.0. ^∗p < 0.02 for −5/−6 mutant promoters relative to the WT promoter, and ^∗∗p < 0.05 for the WT and −7C>T promoters in the presence compared to the absence of ONECUT1 (two-tailed t tests). n = 3 for each data point or genotype, and error bars represent the SEM. (B and C) qRT-PCR analysis of the expression of F9 and other liver genes in embryonic day (E) 17.5 livers from WT and Onecut1^−/− and Onecut2^−/− single-knockout (KO) mice (B) and qRT-PCR analysis of E15.5 livers from WT and Onecut1^−/−/Onecut2^−/− double-knockout (DKO) mice (C). Expression was normalized to 18S rRNA for all genes, and WT levels were set to 1.0. Murine liver tissue was obtained under ethics approval number 09/128A (Animal Care and Ethics Committee, University of New South Wales). ^∗p < 0.002, ^∗∗p < 0.02 (two-tailed t tests). n = 3 for each data point or genotype, and error bars represent the SEM. The following protein abbreviations are used: OC1, ONECUT1; and OC2, ONECUT2. (D) Agarose gel showing ethidium-bromide-stained amplicons from (C).