An age-related homeostasis mechanism is essential for spontaneous amelioration of hemophilia B Leyden

Abstract

Regulation of age-related changes in gene expression underlies many diseases. We previously discovered the first puberty-onset gene switch, the age-related stability element (ASE)/age-related increase element (AIE)-mediated genetic mechanism for age-related gene regulation. Here, we report that this mechanism underlies the mysterious puberty-onset amelioration of abnormal bleeding seen in hemophilia B Leyden. Transgenic mice robustly mimicking the Leyden phenotype were constructed. Analysis of these animals indicated that ASE plays a central role in the puberty-onset amelioration of the disease. Human factor IX expression in these animals was reproducibly nullified by hypophysectomy, but nearly fully restored by administration of growth hormone, being consistent with the observed sex-independent recovery of factor IX expression. Ets1 was identified as the specific liver nuclear protein binding only to the functional ASE, G/CAGGAAG, and not to other Ets consensus elements. This study demonstrates the clinical relevance of the first discovered puberty-onset gene switch, the ASE/AIE-mediated regulatory mechanism.

Fig. 1.

The hFIX gene and its minigene constructs with transient expression activities. (A) A schematic drawing of the hFIX gene and LSR. The hFIX gene is shown at the top with its 5′ end at left. Exons are shown by rectangles. Relative positions of ASE in the 5′ upstream, AIE in the 3′ UTR, and LSR in the 5′ UTR are shown. Right-angled arrow, asterisk with vertical thin line, and pA indicate the transcription start site, translation stop site, and polyadenylation site, respectively. The symbol sl (potential stem loop forming structure) in the last exon rectangle represents AIE. Solid line circles represent proteins binding to the wild-type LSR sequence while the dotted circle indicates the binding of UKP-26. Locations of representative mutations are shown with nucleotide (nt) numbering (12). (B) Human FIX minigene constructs and transient expression activities in vitro. Human FIX minigenes containing a representative Leyden phenotype mutation T-20A and their normal counterparts relevant to the present study are shown. These constructs extend 5′ end to nt -802 or nt -416, and have the middle portion of the 3′ UTR deleted (m1) or not deleted (m1/1.4). Transient expression activity levels relative to that of -416FIXm1 (approximately 50 ng/10⁶ HepG2 cells/48 h) are shown at the right with SDs (averages of 5 independent assays).

Fig. 2.

Age-related expression profiles of hFIX minigenes with a hemophilia B Leyden mutation in transgenic mice. (A) Age-related hFIX expression profiles of -416(-20A)FIXm1 in transgenic mice. Serum hFIX concentrations (vertical axis) are plotted against age (horizontal axis). Animals are identified as previously defined by the format [(F or P for founder or progeny, respectively)(identification number)(progeny generation)/(sex)] (1). For founder animals, no progeny generation number is given. Vertical thick green lines represent the relative puberty period. Age 0 represents the birth time point. (B) Age-related hFIX expression profiles of -802(-20A)FIXm1 in transgenic mice. All definitions are as described in A.

Fig. 3.

Identification of the ASE binding nuclear protein. (A) EMSAs with liver NEs prepared from mice at various ages. All lanes contain the ³²P-labeled ASE probe (approximately 40,000 cpm). Lane 1, without NEs; lanes 2–7, with NEs (80 μg) prepared from the liver tissues of 1-, 3-, 6-, 12-, 18-, and 21-month-old animals, respectively. Arrow: shifted band position. (B) Supershift EMSAs with antibodies specific to various Ets family proteins. All lanes contain the ³²P-labeled ASE probe (approximately 20,000 cpm). Lanes 1, 10, and 20, with no NEs; lanes 2–9, 12–19, and 21–26 with liver NEs (40 μg) prepared from 6-month-old mice. Lane 11, anti-Ets1 antibody with the ³²P-labeled probe with no NEs. Lanes 3–9, 13–19, and 22–26 are supershift EMSAs with various antibodies to specific proteins labeled at the top of lanes. Lanes without antibody are noted with (-) as shown at top. Arrow: shifted band position. (C) EMSAs and supershift EMSA with NEs prepared from 293T cells overexpressing mEts1. ³²P-labeled ASE probe and procedures are as described above differing only in NEs used. Lane 1, without NEs; lane 2, with 10 μg NEs from 6-month-old mouse liver; lane 3, with 10 μg NEs from wild-type 293T cells; lane 4, with 10 μg NEs from 293T cells overexpressing mEts1; lane 5, same as lane 4 with anti-Ets1 antibody. Arrow: shifted band position.

Fig. 4.

Effects of HP and hormone treatments on hFIX expression in transgenic mice carrying a hemophilia B Leyden phenotype minigene, -802(-20A) FIXm1. (A) Effects of HP and a course of administration of GH and DHT, followed by the second course of GH administration, on hFIX expression in a male mouse P53-m6(F2/m) carrying -802(-20A)FIXm1. HP was done at 6 months of age. Vertical arrows show time points for HP, initiation and termination of GH, DHT, and the 2nd GH administration. Vertical axis: serum hFIX concentration level; horizontal axis: age and duration of various treatments in month or day. (B) Effects of HP and subsequent administrations of PBS, sesame oil, and GH, on hFIX expression in a male animal P53-m10(F2/m) carrying -802(-20A) FIXm1. The rest are the same as in (A). (C) Northern blot analyses of mouse liver hFIX mRNA preparations. Total liver RNA samples (15 μg per lane) were used for analyses. Lane 1, wild-type animal; lane 2–4, Leyden phenotype animals carrying -802(-20A)FIXm1. Lane 2, animal P127(F1/f) without HP; lane 3, animal P126(F1/f) after HP; lane 4, P108(F1/f) animal sacrificed immediately after the course of GH injection at eighth day. (Upper) Filter probed with the SspI/BamHI fragment of hFIX cDNA; (Lower) filter re-hybridized with RNR18 probe (1). A horizontal arrow on the right indicates the hFIX mRNA band position with size information.

Fig. 5.

Molecular mechanisms underlying the puberty-onset amelioration of hemophilia B Leyden in relation to the ASE/AIE-mediated regulatory mechanism. Vertical and horizontal axes are for relative gene expression levels and age, respectively. Relative positions along the age axis for birth, weaning, and puberty are shown with vertical thin orange, green, and a fat light green lines, respectively. A thin gray dotted line represents hFIX expression from the perinatal stage through 1 month of age. Open gray arrows indicate directions of up or down changes in hFIX expression in the presence (+) or absence (−) of ASE and/or AIE. Four age-related patterns of hFIX expression generated by the ASE/AIE-mediated genetic regulation are shown by solid gray lines depicted with specific combinations of ASE and AIE shown with + or − on the right. Age-related patterns of hFIX expression from minigenes -802(-20A)FIXm1 and -416(-20A)FIXm1 carrying a representative Leyden mutation (T-20A) are shown by red and blue curved lines, respectively.