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• W2021097332 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. 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. Hemophilia B (MIM 306900) is an X-linked recessive blood-clotting disorder that affects approximately 1 in every 30,000 males.1Mannucci P.M. Tuddenham E.G. The hemophilias—From royal genes to gene therapy.N. Engl. J. Med. 2001; 344: 1773-1779Crossref PubMed Scopus (794) Google Scholar It is also infamously known as the “royal disease” because it afflicted European royal families descended from Queen Victoria.2Rogaev E.I. Grigorenko A.P. Faskhutdinova G. Kittler E.L. Moliaka Y.K. Genotype analysis identifies the cause of the “royal disease”.Science. 2009; 326: 817Crossref PubMed Scopus (75) Google Scholar It is caused by mutations that affect the normal expression or function of coagulation factor IX (F9 [MIM 300746]). Interestingly, mutations within the F9 proximal promoter are associated with a specific subtype, known as hemophilia B Leyden (MIM 306900), which was first reported in the Netherlands.3Veltkamp J.J. Meilof J. Remmelts H.G. van der Vlerk D. Loeliger E.A. Another genetic variant of haemophilia B: Haemophilia B Leyden.Scand. J. Haematol. 1970; 7: 82-90Crossref PubMed Scopus (46) Google Scholar Sufferers of hemophilia B Leyden express low levels of F9 up until puberty, at which point F9 levels rise and clinical symptoms improve; this phenomenon is associated with increasing androgen receptor and growth-factor activity.4Briët E. Bertina R.M. van Tilburg N.H. Veltkamp J.J. Hemophilia B Leyden: A sex-linked hereditary disorder that improves after puberty.N. Engl. J. Med. 1982; 306: 788-790Crossref PubMed Scopus (69) Google Scholar, 5Kurachi S. Huo J.S. Ameri A. Zhang K. Yoshizawa A.C. Kurachi K. An age-related homeostasis mechanism is essential for spontaneous amelioration of hemophilia B Leyden.Proc. Natl. Acad. Sci. USA. 2009; 106: 7921-7926Crossref PubMed Scopus (29) Google Scholar, 6Crossley M. Ludwig M. Stowell K.M. De Vos P. Olek K. Brownlee G.G. Recovery from hemophilia B Leyden: An androgen-responsive element in the factor IX promoter.Science. 1992; 257: 377-379Crossref PubMed Scopus (111) Google Scholar, 7Rimmer E.K. Seftel M.D. Israels S.J. Houston D.S. Unintended benefit of anabolic steroid use in hemophilia B leiden.Am. J. Hematol. 2012; 87: 122-123Crossref PubMed Scopus (4) Google Scholar, 8Brady J.N. Notley C. Cameron C. Lillicrap D. Androgen effects on factor IX expression: In-vitro and in-vivo studies in mice.Br. J. Haematol. 1998; 101: 273-279Crossref PubMed Scopus (9) Google Scholar To date, more than 80 families affected by hemophilia B Leyden have been analyzed worldwide and 21 distinct point mutations have been detected in the F9 promoter (Figure 1).12Giannelli F. Green P.M. Sommer S.S. Poon M. Ludwig M. Schwaab R. Reitsma P.H. Goossens M. Yoshioka A. Figueiredo M.S. Brownlee G.G. Haemophilia B: Database of point mutations and short additions and deletions—Eighth edition.Nucleic Acids Res. 1998; 26: 265-268Crossref PubMed Scopus (143) Google Scholar, 13Rallapalli, P.M., Kemball-Cook, G., Tuddenham, E.G., Gomez, K., and Perkins, S.J. (2013). Factor IX Mutation Database, http://www.factorix.org.Google Scholar The mutations fall into three clusters: one at nucleotide −20 relative to the transcription start site (TSS, designated +1), one at around nucleotide +10, and one at nucleotides –5 and –6 (−5/−6 site). The mutations at −20 prevent binding of the transcriptional activator HNF4α (hepatocyte nuclear factor 4α, encoded by HNF4A [MIM 600281]), and the +10 mutations disrupt a C/EBPα site (CCAAT/enhancer-binding protein α, encoded by CEBPA [MIM 116897]).6Crossley M. Ludwig M. Stowell K.M. De Vos P. Olek K. Brownlee G.G. Recovery from hemophilia B Leyden: An androgen-responsive element in the factor IX promoter.Science. 1992; 257: 377-379Crossref PubMed Scopus (111) Google Scholar, 14Reijnen M.J. Sladek F.M. Bertina R.M. Reitsma P.H. Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden.Proc. Natl. Acad. Sci. USA. 1992; 89: 6300-6303Crossref PubMed Scopus (89) Google Scholar, 15Crossley M. Brownlee G.G. Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B.Nature. 1990; 345: 444-446Crossref PubMed Scopus (103) Google Scholar However, around half of all known Leyden mutations lie in the third cluster, and their mechanism of action has remained unresolved.12Giannelli F. Green P.M. Sommer S.S. Poon M. Ludwig M. Schwaab R. Reitsma P.H. Goossens M. Yoshioka A. Figueiredo M.S. Brownlee G.G. Haemophilia B: Database of point mutations and short additions and deletions—Eighth edition.Nucleic Acids Res. 1998; 26: 265-268Crossref PubMed Scopus (143) Google Scholar, 13Rallapalli, P.M., Kemball-Cook, G., Tuddenham, E.G., Gomez, K., and Perkins, S.J. (2013). Factor IX Mutation Database, http://www.factorix.org.Google Scholar We noted that the −5/−6 site resembles the recently defined consensus site for the transcription factor ONECUT1 (encoded by ONECUT1 [MIM 604164]), also known as hepatocyte nuclear factor 6 (HNF6).10Laudadio I. Manfroid I. Achouri Y. Schmidt D. Wilson M.D. Cordi S. Thorrez L. Knoops L. Jacquemin P. Schuit F. et al.A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation.Gastroenterology. 2012; 142: 119-129Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar Importantly, ONECUT1 is abundant in the liver, the tissue in which F9 is produced.16Rausa F. Samadani U. Ye H. Lim L. Fletcher C.F. Jenkins N.A. Copeland N.G. Costa R.H. The cut-homeodomain transcriptional activator HNF-6 is coexpressed with its target gene HNF-3 beta in the developing murine liver and pancreas.Dev. Biol. 1997; 192: 228-246Crossref PubMed Scopus (157) Google Scholar We thus sought to determine whether ONECUT1 and the related protein ONECUT2 (encoded by ONECUT2 [MIM 604894])17Jacquemin P. Lannoy V.J. Rousseau G.G. Lemaigre F.P. OC-2, a novel mammalian member of the ONECUT class of homeodomain transcription factors whose function in liver partially overlaps with that of hepatocyte nuclear factor-6.J. Biol. Chem. 1999; 274: 2665-2671Crossref PubMed Scopus (86) Google Scholar, 18Dusing M.R. Maier E.A. Aronow B.J. Wiginton D.A. Onecut-2 knockout mice fail to thrive during early postnatal period and have altered patterns of gene expression in small intestine.Physiol. Genomics. 2010; 42: 115-125Crossref PubMed Scopus (16) Google Scholar constitute the missing Leyden regulators that operate through the −5/−6 site. To test whether ONECUT1 and ONECUT2 could recognize the −5/−6 site in the human F9 promoter, we first cloned the DNA-binding domains of ONECUT1 (residues 280–465) and ONECUT2 (residues 321–504) and fused them each individually to the C terminus of glutathione S-transferase (GST) contained in the vector pGEX-6P. Recombinant GST-fusion proteins were subsequently expressed in bacteria and were purified with glutathione beads as described previously.19Smith D.B. Johnson K.S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase.Gene. 1988; 67: 31-40Crossref PubMed Scopus (5046) Google Scholar The DNA-binding activities of the resulting extracts were assessed in electrophoretic mobility shift assays (EMSAs) with a double-stranded, radiolabeled DNA probe encompassing the −5/−6 region of the human F9 promoter (5′-AACTAATCGACCTTACCA-3′). As shown in Figure 2A, strong binding was observed for both ONECUT1 and ONECUT2. We also tested probes containing the hemophilia-B-Leyden-associated mutations (−6G>C, −6G>A, −5A>T, and −5A>G). The −6G>C mutation most significantly impaired binding, −6G>A had the mildest effects, and the two −5 mutations were of intermediate severity. This broadly reflects the clinical severity of the various mutations: the −6G>C mutation produces the most severe hemophilia, −6G>A produces the mildest form, and the −5 mutations are of intermediate severity.12Giannelli F. Green P.M. Sommer S.S. Poon M. Ludwig M. Schwaab R. Reitsma P.H. Goossens M. Yoshioka A. Figueiredo M.S. Brownlee G.G. Haemophilia B: Database of point mutations and short additions and deletions—Eighth edition.Nucleic Acids Res. 1998; 26: 265-268Crossref PubMed Scopus (143) Google Scholar, 13Rallapalli, P.M., Kemball-Cook, G., Tuddenham, E.G., Gomez, K., and Perkins, S.J. (2013). Factor IX Mutation Database, http://www.factorix.org.Google Scholar, 20Vidaud D. Tartary M. Costa J.M. Bahnak B.R. Gispert-Sanchez S. Fressinaud E. Gazengel C. Meyer D. Goossens M. Lavergne J.M. et al.Nucleotide substitutions at the -6 position in the promoter region of the factor IX gene result in different severity of hemophilia B Leyden: Consequences for genetic counseling.Hum. Genet. 1993; 91: 241-244Crossref PubMed Scopus (10) Google Scholar We next assayed full-length ONECUT1 and ONECUT2 expressed in COS cells by using pCMV6-XL5 expression vectors (OriGene). COS cells were transfected and nuclear extracts were obtained as described previously.21Crossley M. Whitelaw E. Perkins A. Williams G. Fujiwara Y. Orkin S.H. Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells.Mol. Cell. Biol. 1996; 16: 1695-1705Crossref PubMed Scopus (209) Google Scholar, 22Funnell A.P. Maloney C.A. Thompson L.J. Keys J. Tallack M. Perkins A.C. Crossley M. Erythroid Krüppel-like factor directly activates the basic Krüppel-like factor gene in erythroid cells.Mol. Cell. Biol. 2007; 27: 2777-2790Crossref PubMed Scopus (77) Google Scholar No significant retarded bands were observed in untransfected cells, but strong binding was observed when ONECUT1 or ONECUT2 was overexpressed (Figure 2B). Again, all four Leyden mutations reduced the binding observed. ONECUT1- and ONECUT2-specific antibodies (sc-13050 X and sc-51177 X, respectively; Santa Cruz Biotechnology) supershifted the retarded species, confirming the identities of these protein-DNA complexes (Figure S1, available online). The reduced binding of full-length ONECUT1 and ONECUT2 to the −5/−6 Leyden mutants was further confirmed by competition assays using radiolabeled wild-type probe and 0×, 10×, 100×, and 1,000× excesses of cold competitor probe (Figure 2F and Figures S2 and S3). Next, we assessed nuclear extracts from murine liver (3 months of age). As shown in Figure 2C, we detected a single, distinct protein-DNA complex by using the wild-type probe. The addition of antibodies individually specific to ONECUT1 and ONECUT2 demonstrated that the retarded species contained both proteins but predominantly had ONECUT1 (Figure 2C). We then examined the binding of the complex to the Leyden mutant probes. Again, the intensity of this band was reduced when probes carrying the mutations were used (Figure 2D), and the degree of impairment correlated with the clinical severity of the mutation. Together, these data suggest that ONECUT1 and ONECUT2 (but particularly ONECUT1) constitute the majority of DNA-binding activity at the −5/−6 site in the liver and that the Leyden mutations disrupt binding of these endogenous factors. Extensive mapping of the F9 locus has illustrated the prevalence of CpG dinucleotides as hotspots for mutation in hemophilia B cases.23Sommer S.S. Assessing the underlying pattern of human germline mutations: Lessons from the factor IX gene.FASEB J. 1992; 6: 2767-2774PubMed Google Scholar In this regard, it is interesting that although both residues −6 and −7 are within a CpG dinucleotide, no C>T transition has been reported at −7 (Figure 1). In contrast, mutations at −6 have been reported at least 34 times and account for nearly half of all Leyden individuals.12Giannelli F. Green P.M. Sommer S.S. Poon M. Ludwig M. Schwaab R. Reitsma P.H. Goossens M. Yoshioka A. Figueiredo M.S. Brownlee G.G. Haemophilia B: Database of point mutations and short additions and deletions—Eighth edition.Nucleic Acids Res. 1998; 26: 265-268Crossref PubMed Scopus (143) Google Scholar, 13Rallapalli, P.M., Kemball-Cook, G., Tuddenham, E.G., Gomez, K., and Perkins, S.J. (2013). Factor IX Mutation Database, http://www.factorix.org.Google Scholar Importantly, the high incidence of this mutation is not simply due to a founder effect, because different haplotypes have been shown to be associated with the −6 mutation.24Morgan G.E. Figueiredo M.S. Winship P.R. Baker R. Bolton-Maggs P.H. Brownlee G.G. The high frequency of the -6G—>A factor IX promoter mutation is the result both of a founder effect and recurrent mutation at a CpG dinucleotide.Br. J. Haematol. 1995; 89: 672-674Crossref PubMed Scopus (13) Google Scholar Our assertion that ONECUT proteins operate in vivo predicts that the −7C>T mutation might occur naturally but that it would not have a functional effect on F9 expression, perhaps because it would not disrupt ONECUT binding. We used EMSAs with ONECUT1 and ONECUT2 GST-fusion proteins to test whether the anticipated mutation at −7 would impair binding of ONECUT factors (Figure 2E). We observed both ONECUT1 and ONECUT2 binding—slightly less than that observed with the wild-type sequence but significantly more than observed with the −6G>A mutation (compare with Figure 2A). Furthermore, competitor assays revealed that full-length ONECUT1 and ONECUT2 bind to probes containing the −7C>T mutation with considerably higher affinity than to any of the Leyden-associated mutations (Figure 2F and Figure S2). Thus, we conclude that −7C>T mutations would produce a minimal phenotype, and we propose that this is the reason that they have never been clinically encountered. Taken together, the investigations of the base substitutions at the −5, −6, and −7 sites concur well with the position weight matrix determined for ONECUT1 (Figure 1). The −5 and −6 sites, which are robustly conserved in the model, considerably disrupt binding when mutated, whereas the −7 site, which is more flexible and less conserved, is able to tolerate substitution. To confirm in vivo binding at the F9 promoter, we performed ONECUT1 chromatin immunoprecipitation (ChIP) on murine liver tissue as described previously.25Schmidt D. Wilson M.D. Spyrou C. Brown G.D. Hadfield J. Odom D.T. ChIP-seq: Using high-throughput sequencing to discover protein-DNA interactions.Methods. 2009; 48: 240-248Crossref PubMed Scopus (365) Google Scholar, 26Odom D.T. Dowell R.D. Jacobsen E.S. Gordon W. Danford T.W. MacIsaac K.D. Rolfe P.A. Conboy C.M. Gifford D.K. Fraenkel E. Tissue-specific transcriptional regulation has diverged significantly between human and mouse.Nat. Genet. 2007; 39: 730-732Crossref PubMed Scopus (417) Google Scholar Primers used are outlined in Table S1, and quantitative real-time PCR (qRT-PCR) runs were performed with Power SYBR Green PCR Master Mix and the 7500 Fast Real-Time PCR System (Applied Biosystems).22Funnell A.P. Maloney C.A. Thompson L.J. Keys J. Tallack M. Perkins A.C. Crossley M. Erythroid Krüppel-like factor directly activates the basic Krüppel-like factor gene in erythroid cells.Mol. Cell. Biol. 2007; 27: 2777-2790Crossref PubMed Scopus (77) Google Scholar Data analysis was conducted with 7500 Software v.2.0.4 (Applied Biosystems). As shown in Figure 3A, occupancy was detected immediately upstream of F9, but not at more distal locations (1.0 kb downstream and 1.5 kb upstream). Moreover, ONECUT1 was not detected at the proximal promoters of other liver-enriched genes, namely, coagulation factor VII (F7 [MIM 613878]) or factor X (F10 [MIM 613872]). We also carried out ONECUT1 ChIP sequencing (ChIP-seq) on human liver tissue. Human liver material (from two males of unknown age) was obtained from the Liver Tissue Distribution Program (National Institute of Diabetes and Digestive and Kidney Diseases contract N01-DK-9-2310) at the University of Pittsburgh and Addenbrooke’s Hospital, Cambridge under human tissue license 08/H0308/117. Immunoprecipitated material was end repaired, A tailed, ligated to single- or paired-end sequencing adapters, amplified by 18 cycles of PCR, and size selected (200–300 bp); this was followed by single-end sequencing on an Illumina Genome Analyzer II according to the manufacturer’s instructions. Data have been deposited in ArrayExpress and assigned the accession number E-MTAB-890. Analysis of the human F9 locus found a strong ONECUT1 peak over the −5/−6 site (Figures 3B and 3C). Similarly, human ChIP-seq data for HNF4α and C/EBPα, which we had previously reported (ArrayExpress E-TABM-722),9Schmidt D. Wilson M.D. Ballester B. Schwalie P.C. Brown G.D. Marshall A. Kutter C. Watt S. Martinez-Jimenez C.P. Mackay S. et al.Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding.Science. 2010; 328: 1036-1040Crossref PubMed Scopus (527) Google Scholar, 25Schmidt D. Wilson M.D. Spyrou C. Brown G.D. Hadfield J. Odom D.T. ChIP-seq: Using high-throughput sequencing to discover protein-DNA interactions.Methods. 2009; 48: 240-248Crossref PubMed Scopus (365) Google Scholar revealed at the F9 promoter a high level of occupancy corresponding to the sites that are mutated in hemophilia B Leyden and to which these factors have been reported to bind in vitro (Figures 3B and 3C). Thus, ONECUT1, HNF4α, and C/EBPα all bind to the F9 promoter in vivo and together encompass all 21 reported point mutations associated with hemophilia B Leyden (Figure 1). We next tested the functional effect of ONECUT1 and ONECUT2 on the F9 promoter by means of reporter assays in HepG2 cells as described previously.22Funnell A.P. Maloney C.A. Thompson L.J. Keys J. Tallack M. Perkins A.C. Crossley M. Erythroid Krüppel-like factor directly activates the basic Krüppel-like factor gene in erythroid cells.Mol. Cell. Biol. 2007; 27: 2777-2790Crossref PubMed Scopus (77) Google Scholar We used a fragment that encompassed a minimal region (−189 to +21) of the human F9 promoter and that had previously been shown to recapitulate key aspects of the Leyden phenotype in transfection studies and transgenic mice.15Crossley M. Brownlee G.G. Disruption of a C/EBP binding site in the factor IX promoter is associated with haemophilia B.Nature. 1990; 345: 444-446Crossref PubMed Scopus (103) Google Scholar, 28Boland E.J. Liu Y.C. Walter C.A. Herbert D.C. Weaker F.J. Odom M.W. Jagadeeswaran P. Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice.Blood. 1995; 86: 2198-2205PubMed Google Scholar This region was cloned into the firefly luciferase vector pGL4.10[luc2] (Promega). Cells in 6-well plates were transfected (FuGENE6, Roche Diagnostics) with 1 μg triplicate DNA preparations of pGL4.10[luc2] (wild-type or Leyden mutants of the –189 to +21 region) together with 100 ng of the Renilla luciferase vector pGL4.74[hRluc/TK] (Promega) as a transfection control. Expression of ONECUT1 and ONECUT2 was achieved with 1 μg pCMV6-XL5 (ONECUT1 or ONECUT2) (OriGene). We found that both ONECUT1 and ONECUT2 could potently transactivate the wild-type promoter but had less activity on the Leyden mutant promoters (Figure 4A and Figure S4). We also found that compared to the wild-type promoter, the −7C>T promoter did not appreciably reduce transactivation (Figure 4A), consistent with the view that such a mutation does not functionally impair ONECUT binding. Lastly, it should also be noted that C/EBPα and HNF4α are expressed in HepG2 cells.29Friedman A.D. Landschulz W.H. McKnight S.L. CCAAT/enhancer binding protein activates the promoter of the serum albumin gene in cultured hepatoma cells.Genes Dev. 1989; 3: 1314-1322Crossref PubMed Scopus (363) Google Scholar, 30Greenberg D. Miao C.H. Ho W.T. Chung D.W. Davie E.W. Liver-specific expression of the human factor VII gene.Proc. Natl. Acad. Sci. USA. 1995; 92: 12347-12351Crossref PubMed Scopus (39) Google Scholar This raises the possibility that in these cells, the ONECUT proteins might synergistically activate the F9 promoter constructs in combination with either or both of these endogenous factors. The results demonstrate that ONECUT proteins can bind to and activate the F9 promoter in experimental systems. To determine whether F9 expression is dependent on ONECUT proteins in vivo, we analyzed tissue from Onecut−/− mice.31Clotman F. Jacquemin P. Plumb-Rudewiez N. Pierreux C.E. Van der Smissen P. Dietz H.C. Courtoy P.J. Rousseau G.G. Lemaigre F.P. Control of liver cell fate decision by a gradient of TGF beta signaling modulated by Onecut transcription factors.Genes Dev. 2005; 19: 1849-1854Crossref PubMed Scopus (266) Google Scholar, 32Jacquemin P. Durviaux S.M. Jensen J. Godfraind C. Gradwohl G. Guillemot F. Madsen O.D. Carmeliet P. Dewerchin M. Collen D. et al.Transcription factor hepatocyte nuclear factor 6 regulates pancreatic endocrine cell differentiation and controls expression of the proendocrine gene ngn3.Mol. Cell. Biol. 2000; 20: 4445-4454Crossref PubMed Scopus (277) Google Scholar Because ONECUT1-deficient mice typically die shortly after birth, we assessed F9 expression in fetal liver. Total RNA was extracted from fetal livers and used as a template for cDNA synthesis by standard methods. qRT-PCR was performed as described above and with primer sequences shown in Table S2. We observed clear reductions in F9 expression in both Onecut1−/− and Onecut2−/− single knockouts (6-fold and 3-fold, respectively) at day 17.5 of gestation (Figure 4B). Given that both ONECUT1 and ONECUT2 can bind the −5/−6 site and could thus potentially compensate in each other’s absence, we next examined tissues from Onecut1−/− Onecut2−/− double-knockout animals. These animals die in utero, so we analyzed the liver at embryonic day 15.5, when F9 was already expressed and the embryos were still viable. We observed a dramatic reduction (>100-fold) in F9 levels in Onecut1−/− Onecut2−/− double-knockout samples (Figures 4C and 4D). Importantly, we found near-normal expression of a panel of other liver-specific genes, including F7, F10, and coagulation factor XI (F11 [MIM 264900]), and of the other transcription factors (HNF4α6Crossley M. Ludwig M. Stowell K.M. De Vos P. Olek K. Brownlee G.G. Recovery from hemophilia B Leyden: An androgen-responsive element in the factor IX promoter.Science. 1992; 257: 377-379Crossref PubMed Scopus (111) Google Scholar, 14Reijnen M.J. Sladek F.M. Bertina R.M. Reitsma P.H. Disruption of a binding site for hepatocyte nuclear factor 4 results in hemophilia B Leyden.Proc. Natl. Acad. Sci. USA. 1992; 89: 6300-6303Crossref PubMed Scopus (89) Google Scholar and C/EBPα12) known to drive F9, indicating that the deficit in F9 is specific and not due to a general failure in liver development at this embryonic stage (Figures 4C and 4D). In conclusion, we have established that the missing regulators in hemophilia B Leyden are the ONECUT transcription factors ONECUT1 and ONECUT2. These proteins bind to the proximal promoter and drive expression of F9. Their binding, and hence functional activity, is disrupted by −5/−6 base substitutions, which are found in more than half of all individuals with hemophilia B Leyden. We have shown here that the different Leyden mutations at −5/−6 disrupt ONECUT binding to varying degrees in a manner that correlates well with their clinical severities. It should be noted, however, that other factors might also influence the severity of hemophilia in individual cases, and we thus caution against solely using the mutations to predict clinical outcome. The comprehensive compilation of hemophilia B mutations worldwide provides what might be the most complete mapping of a human gene promoter.12Giannelli F. Green P.M. Sommer S.S. Poon M. Ludwig M. Schwaab R. Reitsma P.H. Goossens M. Yoshioka A. Figueiredo M.S. Brownlee G.G. Haemophilia B: Database of point mutations and short additions and deletions—Eighth edition.Nucleic Acids Res. 1998; 26: 265-268Crossref PubMed Scopus (143) Google Scholar, 13Rallapalli, P.M., Kemball-Cook, G., Tuddenham, E.G., Gomez, K., and Perkins, S.J. (2013). Factor IX Mutation Database, http://www.factorix.org.Google Scholar Detailed consensus sequences have now been determined for HNF4α, ONECUT1, and C/EBPα9Schmidt D. Wilson M.D. Ballester B. Schwalie P.C. Brown G.D. Marshall A. Kutter C. Watt S. Martinez-Jimenez C.P. Mackay S. et al.Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding.Science. 2010; 328: 1036-1040Crossref PubMed Scopus (527) Google Scholar, 10Laudadio I. Manfroid I. Achouri Y. Schmidt D. Wilson M.D. Cordi S. Thorrez L. Knoops L. Jacquemin P. Schuit F. et al.A feedback loop between the liver-enriched transcription factor network and miR-122 controls hepatocyte differentiation.Gastroenterology. 2012; 142: 119-129Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 11Bolotin E. Liao H. Ta T.C. Yang C. Hwang-Verslues W. Evans J.R. Jiang T. Sladek F.M. Integrated approach for the identification of human hepatocyte nuclear factor 4alpha target genes using protein binding microarrays.Hepatology. 2010; 51: 642-653Crossref PubMed Scopus (136) Google Scholar (data presented here), and the mutations associated with hemophilia B Leyden fit remarkably well with predictions from these consensus sequences (Figure 1). In the position weight matrices for these transcription factors, the most robustly conserved bases are, by and large, those that are mutated in cases of hemophilia. These proteins are also relevant in terms of understanding the liver-restricted expression of F9. HNF4α, ONECUT1, and C/EBPα are well known as major regulators of hepatocyte gene expression,33Lemaigre F.P. Mechanisms of liver development: concepts for understanding liver disorders and design of novel therapies.Gastroenterology. 2009; 137: 62-79Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar and this regulatory module satisfyingly explains the liver-restricted expression pattern of F9. Their cognate DNA elements are very tightly clustered within the F9 promoter, and the fact that disruption of any single site severely disrupts promoter activity suggests that these transcription factors must cooperate to ensure proper F9 expression. Taken together, these findings contribute to a thorough characterization of the human F9 promoter and its transacting factors and provide an illustrative example of how disrupted local promoter architecture can result in human disease. This research was supported by the Australian Research Council and National Health and Medical Research Council (M.C., A.P.W.F., K.S.M., J.B., and R.C.M.P.), the European Research Council and EMBO Young Investigator Program (D.T.O.), Cancer Research UK and University of Cambridge (M.D.W. and D.T.O.), the Wellcome Trust (D.T.O., B.B., and P.F.), the D.G. Higher Education and Scientific Research of the French Community of Belgium (F.P.L.), the Fund for Scientific Medical Research Belgium (F.P.L.), and the European Molecular Biology Laboratory (P.F.). Download .pdf (.33 MB) Help with pdf files Document S1. Figures S1–S4 and Tables S1 and S2 The URLs for data presented herein are as follows:Archive Ensemble release 52, http://Dec2008.archive.ensembl.org/ArrayExpress, http://www.ebi.ac.uk/arrayexpress/Factor IX Mutation Database, http://www.factorix.orgMEME Suite, http://meme.nbcr.net/memeOnline Mendelian Inheritance in Man (OMIM), http://www.omim.org/SWEMBL, http://www.ebi.ac.uk/∼swilder/SWEMBL/ The ArrayExpress accession number for the ONECUT1 ChIP-Seq data reported in this paper is E-MTAB-890. ChIP-Seq experiments for C/EBPα and HNF4α have previously been reported under ArrayExpress accession number E-TABM-722.9Schmidt D. Wilson M.D. Ballester B. Schwalie P.C. Brown G.D. Marshall A. Kutter C. Watt S. Martinez-Jimenez C.P. Mackay S. et al.Five-vertebrate ChIP-seq reveals the evolutionary dynamics of transcription factor binding.Science. 2010; 328: 1036-1040Crossref PubMed Scopus (527) Google Scholar" @default.
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• W2021097332 cites W2081631093 @default.
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• W2021097332 cites W2089793634 @default.
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