We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno ؊/؊ ) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno ؊/؊ fetal liver. Moreover, definitive erythropoiesis was affected in noncell autonomous manner in syno ؊/؊ embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno ؊/؊ mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno ؉/؉ mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

The human ␤-globin locus contains five developmentally regulated ␤-type globin genes. All five genes depend on the locus control region (LCR), located at the 5 end of the locus, for abundant globin gene transcription. The LCR is composed of five DNase I-hypersensitive sites (HSs), at least a subset of which appear to cooperate to form a holocomplex in activating genes within the locus. We previously tested the requirement for proper LCR polarity by inverting it in human ␤-globin yeast artificial chromosome transgenic mice and observed reduced expression of all the ␤-type globin genes regardless of developmental stage. This phenotype clearly demonstrated an orientation-dependent activity of the LCR, although the mechanistic basis for the observed activity was obscure. Here, we describe genetic evidence demonstrating that human HS5 includes enhancerblocking (insulator) activity that is both CTCF and developmental stage dependent. Curiously, we also observed an attenuating activity in HS5 that was specific to the -globin gene at the primitive stage and was independent of the HS5 CTCF binding site. These observations demonstrate that the phenotype observed in the LCR-inverted locus was in part attributable to placing the HS5 insulator between the LCR HS enhancers (HS1 to HS4) and the promoter of the ␤-globin gene.During the normal process of mammalian development, proper temporal and spatial expression of genetic information must be achieved. To this end, gene expression must be tightly regulated through cis-DNA elements: promoters, enhancers, and silencers. After the human genome had been completely sequenced, it became clear that the average size of intergenic regions is roughly 100 kbp. However, substantial evidence argues that enhancers can modulate promoter activity from very long distances, exceeding 100 kbp (19,20). In such a circumstance, one can envision an activity that protects a locus from neighboring gene regulatory elements to prevent improper gene regulation. Indeed, such an activity has been described: DNA insulators were first identified in Drosophila melanogaster, and similar activities were subsequently found in mammals.Insulators can protect a locus by two distinguishable means; one is a chromatin barrier function, while a second is referred to as enhancer-blocking activity. The most extensively characterized vertebrate insulator was originally identified in the chicken ␤-globin LCR, which consists of four DNase I-hypersensitive (HS) sites; the 5Ј-most is HS4. After stable transformation of K562 cells, a 1.2-kbp DNA fragment containing HS4 was found to interfere with enhancer/promoter interactions, but only when it was placed between them (10). Further analysis revealed two separable categories of activity within this 1.2-kbp DNA fragment, one of which protects stably integrated transgenes from silencing after long-term cell culture (28). This property of an insulator is referred to as its chromatin barrier activity.Chung et al. developed an enhancer-blocking assay to refine the position of a 250...

Synoviolin is an E3 ubiquitin ligase localized in the endoplasmic reticulum (ER) and serving as ERassociated degradation system. Analysis of transgenic mice suggested that synoviolin gene dosage is implicated in the pathogenesis of arthropathy. Complete deficiency of synoviolin is fatal embryonically. Thus, alternation of Synoviolin could cause breakdown of ER homeostasis and consequently lead to disturbance of cellular homeostasis. Hence, the expression level of Synoviolin appears to be important for its biological role in cellular homeostasis under physiological and pathological conditions. To examine the control of protein level, we performed promoter analysis to determine transcriptional regulation. Here we characterize the role of synoviolin transcription in cellular homeostasis. The Ets binding site (EBS), termed EBS-1, from position ؊76 to ؊69 of the proximal promoter, is responsible for synoviolin expression in vivo and in vitro. Interestingly, transfer of EBS-1 decoy into NIH 3T3 cells conferred not only the repression of synoviolin gene expression but also a decrease in cell number. Fluorescence-activated cell sorter analysis using annexin V staining confirmed the induction of apoptosis by EBS-1 decoy and demonstrated recovery of apoptosis by overexpression of Synoviolin. Our results suggest that transcriptional regulation of synoviolin via EBS-1 plays an important role in cellular homeostasis. Our study provides novel insight into the transcriptional regulation for cellular homeostasis.

Renin, a major regulatory component of the renin-angiotensin system, plays a pivotal role in regulating blood pressure and electrolyte homeostasis and is predominantly expressed in the kidney. Several cAMP-responsive elements have been identified within renin gene promoters. Here, we study how 2 such elements, renin proximal promoter element-2 (RP-2) and overlapping cAMP and negative regulatory elements (CNRE), affect the transcriptional regulation of renin. We generated Tg mice (TgM) bearing BACs containing either WT or mutant RP-2 or CNRE, integrated at single chromosomal loci. Analysis of the TgM revealed that RP-2 was essential to basal promoter activity in the kidney, while renin mRNA levels did not significantly change in any tissues tested in the CNRE mutant TgM. To evaluate the physiological significance of these mutations, we used the BAC Tg to rescue hypotensive Renin-null mutant mice. As predicted, no renin expression was observed in the kidneys of RP-2 mutant/Renin-null compound mice, whereas renin expression in CNRE mutant compound mice was indistinguishable from that in control mice. Consistent with this, RP-2 mutant animals were hypotensive, while CNRE mutants had normal blood pressure. Thus, transcriptional regulation of renin expression via RP-2 but not CNRE is critical for blood pressure regulation by this gene. IntroductionRenin is an aspartyl protease synthesized mainly in the juxtaglomerular (JG) cells of the kidney. It catalyzes cleavage of a unique substrate, angiotensinogen, a plasma protein synthesized predominantly in the liver; cleavage yields the decapeptide angiotensin I (AI). AI is further processed by angiotensin-converting enzyme to produce the octapeptide angiotensin II (AII), which mediates vasoconstriction and aldosterone secretion through angiotensin receptors (ATs), leading to increased BP. Renin catalyzes the first, rate-limiting reaction of the renin-angiotensin system and thus plays a central role in regulating overall activity of the renin-angiotensin system. Our lab and others have shown by gene ablation in the mouse that renin is essential to BP homeostasis (1, 2).A number of physiological stimuli, such as renal sympathetic activity, sodium depletion, and low BP, induce renin transcription and secretion in order to maintain BP homeostasis. Several of these stimuli utilize cAMP as an intracellular signal (reviewed in ref.3). In JG cells, noradrenalin released from sympathetic nerve endings activates adenylate cyclase. Intracellular cAMP concentration in JG cells can also be modified directly or indirectly by low distal tubular salt load, increased NO and prostaglandins, and/or decreased adenosine release from the macula densa. Pharmacological blockade of AII synthesis or receptor function also increases

The function of putative regulatory sequences identified in cell transfection experiments can be elucidated only through in vivo experimentation. However, studies of gene regulation in transgenic mice (TgM) are often compromised by the position effects, in which independent transgene insertions differ in expression depending on their location in the genome. In order to overcome such a dilemma, a method called transgene coplacement has been developed in Drosophila melanogaster. In this method, any two sequences can be positioned at exactly the same genomic site by making use of Cre/loxP recombination. Here we applied this method to mouse genetics to characterize the function of direct repeat (DR) sequences in the promoter of the human angiotensinogen (hAGT) gene, the precursor of the vasoactive octapeptide angiotensin II. We modified a hAGT bacterial artificial chromosome to use Cre/loxP recombination in utero to generate TgM lines bearing a wildtype or a mutant promoter-driven hAGT locus integrated at a single chromosomal position. The expression analyses revealed that DR sequences contribute 50 or >95% to hAGT transcription in the liver and kidneys, respectively, whereas same sequences are not required in the heart and brain. This is the first in vivo dissection of DNA cis elements that are demonstrably indispensable for regulating both the level and cell type specificity of hAGT gene transcription.The renin-angiotensin system (RAS) plays a key role in the regulation of blood pressure (BP) and electrolyte homeostasis. Angiotensinogen (AGT), a plasma protein synthesized predominantly in the liver, is catalyzed by renin, an aspartyl protease synthesized mainly in the juxtaglomerular cells of the kidney, to produce decapeptide angiotensin I (AI). The latter is further processed by angiotensin-converting enzyme to an octapeptide, angiotensin II (AII), which mediates vasoconstriction and aldosterone secretion through specific cell surface receptors present throughout the cardiovascular system. Aldosterone then causes sodium reabsorption in the collecting ducts of the kidney to increase body fluid, leading to increased BP.Since the reaction catalyzed by renin is the rate-limiting step of this enzymatic cascade and AGT concentrations in plasma are close to the K m value for renin (12), the level of AGT is directly reflected by BP phenotype. In accord with this notion, transgenic overexpression (8, 18) or targeted gene inhibition of AGT in the mouse (39) has demonstrated that this gene is a key genetic determinant of BP. It has been also shown in experimental animal models that as little as a 20% change in AGT expression was reflected as a BP phenotype (18,19). Furthermore, in humans, genetic linkage between human AGT (hAGT) and essential hypertension has been reported (4, 16). Given these correlations, it may be of fundamental importance to elucidate the molecular mechanisms underlying hAGT transcriptional regulation in order to define the origins of BP physiology and pathology. To this end, we have identified a numbe...