Sumiko Kurachi

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

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.

Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene.

Background In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes. Principal Finding We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser359, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser359. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable. Conclusions We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.

Limitation in Use of Heterologous Reporter Genes for Gene Promoter Analysis SILENCER ACTIVITY ASSOCIATED WITH THE CHLORAMPHENICOL ACETYLTRANSFERASE REPORTER GENE

Various heterologous reporter genes have been widely used for the functional characterization of gene promoters. Many such studies often found weak to very strong silencer activities to be associated with specific parts of the basal promoter or further upstream regions. In this study, we carried out a systematic study on human blood coagulation factor IX (hFIX) and anti-coagulant protein C (hPC) genes, previously shown to have silencer activities associated with their 5′-flanking regions containing promoter sequences. With newly constructed chloramphenicol acetyltransferase (CAT) reporter vectors carrying hFIXor hPC gene promoter sequences, we confirmed the strong silencer activities associated with the regions nt −1895 through nt −416 of the hFIX gene or with the region nt −802 through nt −82 of the hPC gene. However, no such silencer activities associated with the specific regions were found when autologous hFIX cDNA, hFIX minigenes, orhPC minigenes were used as reporters in the expression vector system. Relative levels of CAT, hFIX, and hPC proteins produced in the transient assays correlated well with their mRNA levels. Human FIX minigene constructs containing a simian virus 40 (SV40) 3′-untranslated region (UTR) taken from the CAT reporter gene showed no silencer activity, indicating that SV40 3′-UTR sequence of the CAT reporter gene does not contribute to the silencer activity. Expression vectors constructed with the β-galactosidase gene under the control of hFIX gene promoter sequences also showed no silencer activity associated with the region nt −1895 through nt −416. These findings indicate that silencer activities associated with specific regions of promoter sequences as analyzed with CAT reporter genes may represent artifacts specific to the CAT reporter genes. Our findings strongly suggest a need for re-examination of promoter characterizations of many eukaryotic genes, which have been studied to date with CAT reporter genes.

Genetic and molecular mechanisms of age regulation (homeostasis) of blood coagulation.

Blood coagulation plays a critical role not only in hemostasis but also in many physiological and pathological conditions. Epidemiological studies have shown that blood coagulation capacity in humans increases with age. Towards understanding the underlying mechanisms, the age regulation of factor IX, a key blood coagulation factor, was extensively studied. A series of human factor IX minigenes, consisting of various components of the human factor IX gene, were constructed and subjected to systematic analyses with HepG2 cells in culture and over the entire life span of transgenic mice. These studies identified critical gene structures that are essential for the unique age‐dependent expression patterns of the human factor IX gene?one acting by stabilizing gene transcription and another increasing the amount of mRNA present, presumably by augmenting mRNA stability. These studies have set the stage for analyzing the overall age‐based regulatory mechanisms of blood coagulation.

Deficiencies in factors IX and VIII: what is now known.

Genetic defects both gross and subtle have recently been identified in about 900 patients with hemophilia A or B. The defects, which include deletions, insertions, and point mutations, reveal much about genetic structure-function relationships in hemophilia. These insights will lead to improved treatment of bleeding disorders and perhaps to their cure.

Sumo15A: a lambda phasmid that permits easy selection for and against cloned inserts

We report the construction of a phasmid vector, Sumo15A, designed for recombination-based screening of recombinant DNA libraries [Seed, Nucleic Acids Res. 11 (1983) 2427-2445]. This vector permits rapid selection in Escherichia coli for homology-mediated integration and excision between homologous DNA inserts cloned in a supF-carrying plasmid and in Sumo15A. The region available for recombination spans the homologous sequence shared by the plasmid and the phasmid. SupF is the selection tool that we used. Efficient selection for supF expression by Sumo15A requires recombination mediated by the lambda phage red gene, which promotes homologous recombination between phage and plasmid DNAs. Counterselection against supF expression by Sumo15A occurs because the presence of a pSC101-derived plasmid replicon in this phasmid permits the growth of Sumo15A as a plasmid in a specialized host, E. coli strain DK37. In strain DK37, Sumo15A cannot replicate as a phage, and the presence of a plasmid-carrying supF is lethal to cells plated on galactose plates. This scheme was developed to select for sequences that are transcribed from chromosomes of interest.

Age-Related Homeostasis and Hemostatic System

Aging is one of the inevitable aspects of life, affecting homeostatic states of every physiological system including that of hemostasis. Through systematic transgenic studies of age-related dynamic changes in expression of genes for hemostatic factors, we found the very first molecular mechanism of age-related homeostasis, the ASE/AIE-mediated genetic mechanism for age-related regulation of gene expression. In this mechanism, together with other essential elements for the promoter activity, two genetic elements—age-related stability element (ASE) and age-related increase element (AIE)—play critical roles, producing four age-related gene expression patterns. This mechanism is also found to be the first puberty-onset gene switch mechanism identified. This ASE/AIE-mediated regulatory mechanism has universal functionality across different genes and animal species. In addition to the age-related regulatory activity, ASE also has a unique tissue-specificity regulatory activity. Global analyses of lifetime age-related expression profiles of mouse liver genes and proteins were carried out, and the results support that there exists a small number of fundamental age-related regulatory mechanisms of genes and proteins that govern complex age-related homeostatic regulations. A large body of information obtained from the global analyses has been constructed into a versatile database, which is a platform resource for studying age-related homeostasis.

Age Dimension Homeostasis of Physiological Systems, a Slow Dynamics Model in Biology

Throughout the life span, complex physiological systems are regulated by specific age related mechanisms, thus maintaining the age axis homeostasis. Our efforts for understanding the underlying mechanisms began about a decade ago, successfully discovering the very fist such a mechanism, we called the ASE/AIE‐mediated age‐related regulatory mechanism of gene expression. This mechanism can explain the major feature of the age‐related increase profile of blood coagulation activity. Global analyses of the mouse liver genes and proteins revealed heir highly dynamic and complex age‐related regulation, suggesting the existence of several major phases over the life span. These studies facilitate a systematic investigation into the regulatory mechanisms of age‐axis homeostasis, a slow dynamics system of the complex biological systems.

Age-related regulation of genes: slow homeostatic changes and age-dimension technology

Through systematic studies of pro- and anti-blood coagulation factors, we have determined molecular mechanisms involving two genetic elements, age-related stability element (ASE), GAGGAAG and age-related increase element (AIE), a unique stretch of dinucleotide repeats (AIE). ASE and AIE are essential for age-related patterns of stable and increased gene expression patterns, respectively. Such age-related gene regulatory mechanisms are also critical for explaining homeostasis in various physiological reactions as well as slow homeostatic changes in them. The age-related increase expression of the human factor IX (hFIX) gene requires the presence of both ASE and AIE, which apparently function additively. The anti-coagulant factor protein C (hPC) gene uses an ASE (CAGGAG) to produce age-related stable expression. Both ASE sequences (G/CAGAAG) share consensus sequence of the transcriptional factor PEA-3 element. No other similar sequences, including another PEA-3 consensus sequence, GAGGATG, function in conferring age-related gene regulation. The age-regulatory mechanisms involving ASE and AIE apparently function universally with different genes and across different animal species. These findings have led us to develop a new field of research and applications, which we named “age-dimension technology (ADT)”. ADT has exciting potential for modifying age-related expression of genes as well as associated physiological processes, and developing novel, more effective prophylaxis or treatments for age-related diseases.