Matthias C. Huber

The Chicken Lysozyme Locus as a Paradigm for the Complex Developmental Regulation of Eukaryotic Gene Loci

Gene loci of higher organisms have complex structural features. In some cases their coding regions occupy many, even hundreds, of kilobases of DNA. Additionally, the sequences that contain the information for the correct spatial and temporal regulation of a particular gene locus during development often exceed the extensions of the coding region by severalfold. The question of what type of information is encoded in these vast amounts of DNA has puzzled researchers from the beginning. It is now clear that eukaryotic genes are regulated by a number of different cis-regulatory elements distributed over large distances. A convenient way to assay the number and the distribution of cis-regulatory elements has been the mapping of DHS in chromatin. Such local chromatin perturbations are in most cases caused by the binding of transcription factors to their cognate DNA sequences. The pattern of DHS can undergo dramatic developmental changes, indicating a change in the activity of cis-regulatory elements. In addition, the analysis of protein-DNA interactions at a single-nucleotide resolution level in vivo has demonstrated that, depending on the developmental stage, different combinations of transcription factors can occupy the same cis-regulatory element (1, 2). These experiments indicate that the transcriptional activation of a gene locus is achieved by the cooperation of several different cisregulatory elements, which, in turn, assemble transcription factors in a sequential, developmentally controlled fashion. However, the assembly of active transcription factor complexes on natural genes does not occur on naked DNA but in a chromatin context, where nucleosome-DNA interactions have to be counteracted. Hence, the activation of a gene locus requires at least the following steps: the perturbation of chromatin structure by the binding of transcription factors on cis-regulatory elements, the developmentally controlled reorganization of transcription factor complexes, the assembly of the basal transcription machinery and its interaction with upstream regulatory elements, the onset of mRNA synthesis, and, in many cases, the maintenance of an active transcriptional state during multiple rounds of DNA synthesis. How can the molecular basis of locus activation be experimentally studied? While the basal activities of individual cisregulatory elements of particular gene loci can be analyzed by transient and stable transfection experiments, the molecular mechanism of activation of a gene locus from the transcriptionally silent state can only be studied in a developing system, preferentially in transgenic animals. The ideal model locus should be small, thus facilitating the manipulation of individual cis-regulatory elements within the context of an entire genomic locus, and it should be extensively characterized on the molecular level. In addition, to dissect the role of different cis-regulatory elements in the developmental control of gene locus activation, it should be possible to follow cell differentiation experimentally, thus enabling the linkage of a stage-specific chromatin structure with the transcriptional activity of the gene. Here, we summarize recent studies on the molecular basis of the transcriptional activation of the chicken lysozyme locus, which may serve as a paradigm for other developmentally regulated eukaryotic gene loci.

Prerequisites for tissue specific and position independent expression of a gene locus in transgenic mice.

Abstract The elucidation of general parameters influencing the transcriptional activation of gene loci at distinct stages of development is an essential prerequisite for a reproducibly successful gene transfer in both gene therapy protocols and biotechnology. Up to now research has focused mostly on the identification and characterization of individual cis-regulatory elements by transient transfection and in vitro assays. However, the most relevant assay system to test gene constructs designed for gene therapy protocols is the transgenic animal. In such an experimental system exogenous genes are usually integrated randomly in the chromatin. For gene constructs not fulfilling the requirements for correct gene locus activation this can lead to genomic position effects on gene expression. The consequences are highly variable expression levels and a disturbance of temporal and spatial expression patterns. Hence it is important to examine how cis-elements function in a chromatin context, and how they cooperate during the developmentally controlled activation of an entire gene locus. One among a few gene loci which are sufficiently characterized to enable such investigations is the chicken lysozyme locus. This review summarizes recent results aimed at identifying the necessary prerequisites for a reproducibly correct expression of the lysozyme locus in transgenic mice and the implications of our findings for gene transfer. The complete lysozyme locus is expressed independent of the chromosomal position and at a high level in macrophages of transgenic mice. Correct transgene regulation requires the cooperation of all cis-regulatory elements. Chromatin of the lysozyme locus in both the active and the inactive state is highly structured. Each cis-regulatory element on the chicken lysozyme locus is organized in its own unique chromatin environment, with nucleosomes specifically placed on specific sequences. The transcriptional activation of the lysozyme locus is accompanied by extensive rearrangements of its chromatin structure, which are disturbed when the transgenes are subjects to genomic position effects. Based on these results, we propose that a complete locus is resistant to genomic position effects, and that a distinct chromatin architecture of a gene locus is required for its correct activation.

Nephron deficit is not required for progressive proteinuria development in the Munich Wistar Fromter rat

The Munich Wistar Frömter (MWF) rat represents a genetic model with an inherited nephron deficit and exhibits mild hypertension and progressive albuminuria, which is more pronounced in males than females. Previously, we demonstrated in a consomic strain that replacement of a quantitative trait locus on chromosome 6 normalized the nephron deficit and suppressed albuminuria development, suggesting a link between the two findings. Here we tested the role of a second major locus linked to albuminuria in MWF on chromosome 8 and generated the consomic strain MWF-8(SHR) by transfer of chromosome 8 from spontaneously hypertensive rats (SHR) into MWF. The early onset of albuminuria at 8 wk of age in MWF (>50-fold increase compared with SHR) was significantly suppressed in consomic animals, and the development of marked proteinuria at 32 wk significantly diminished. Total nephron number in consomic rats (23,771 +/- 1,352) and MWF (27,028 +/- 1,322) were similar and significantly lower (-36%) compared with SHR (36,979 +/- 1,352, P < 0.0001). The development of mild albuminuria in female MWF was also significantly diminished in MWF-8(SHR). Thus, the development of overt and mild albuminuria in male and female MWF rats is not a mandatory consequence of the inherited nephron deficit. The locus on chromosome 8 appears of interest, because its exchange between MWF and SHR protects against the development of albuminuria in MWF-8(SHR) animals despite their inherited nephron deficit and higher systolic blood pressure.

Genetic locus on MWF rat chromosome 6 affects kidney damage in response to l-NAME treatment in spontaneously hypertensive rats

A major quantitative trait locus (QTL) on rat chromosome (RNO)6 was linked to albuminuria in Munich Wistar Frömter rats (MWF). We tested whether transfer of MWF RNO6 into the background of albuminuria-resistant spontaneously hypertensive rats (SHR) induces albuminuria in consomic SHR-6(MWF) animals. Male MWF, SHR, and SHR-6(MWF) were sham operated and treated between 6 and 24 wk of age with normal water (Sham) or with water containing 20 mg/l N(G)-nitro-L-arginine methyl ester (L-NAME) or unilaterally nephrectomized (Nx). Compared with SHR albuminuria was not increased in SHR-6(MWF) in both Sham and Nx groups. All animals survived the observation period in Sham and Nx groups, while premature mortality occurred from 12-14 wk on in L-NAME-treated SHR and SHR-6(MWF) compared with MWF L-NAME animals, in which survival was not affected (P < 0.005, respectively). Subsequent further analysis of L-NAME-treated animals at 12 wk of age showed significantly increased arterial blood pressures in both SHR and SHR-6(MWF) compared with control (P < 0.05), with higher levels in SHR compared with consomics (P < 0.05). However, L-NAME-treated consomic animals demonstrated increased albuminuria compared with SHR (12.7 +/- 3.5 vs. 0.8 +/- 0.2 mg/24 h; P < 0.05) and an induction of tubulointerstitial structural injury and expression of neutrophil gelatinase-associated lipocalin mRNA (P < 0.05 vs. other strains). Our study demonstrates that isolation of the RNO6 albuminuria QTL from the MWF background and transfer into SHR fails to induce an albuminuria phenotype during normal conditions or after nephron reduction. Moreover, our data indicate that genes on RNO6 contribute to the development of L-NAME-induced renal damage in the SHR strain.

Role of the angiotensin II type 2 receptor gene (+1675G/A) polymorphism on left ventricular hypertrophy and geometry in treated hypertensive patients.

Objective A genetic polymorphism in the angiotensin II type 2 receptor (AGTR2 +1675G/A) has been associated with left ventricular hypertrophy (LVH). We tested whether this polymorphism affects LVH and left ventricular geometry parameters in patients with essential hypertension and cardiovascular disease who are treated according to guidelines. Methods We analyzed a cohort of 208 women and 1030 men with essential hypertension, associated cardiovascular disease and left ventricular ejection fractions 40% or more. Previous cardiac diseases included coronary heart disease (81%) and myocardial infarction (MI; 52%). Ten parameters of left ventricular mass, geometry and function were determined by echocardiography. Genotyping was performed by PCR. Due to the X chromosomal location of AGTR2, genotype–phenotype analysis was separated for women and men. Statistical analysis was performed by univariate and multivariate analysis accounting for confounding factors. Results The mean age was 58.4 ± 10 years. In the overall cohort, mean left ventricular mass index was 54 ± 23.6 g/h2.7 without significant differences between patients with and without MI. The frequency of LVH (49% overall) was also similar in patients with or without MI. In men, AGTR2 +1675G/A had no influence on echocardiographic parameters. Similar findings were obtained in women, with the exception that the thickness of the interventricular septum was significantly lower in A allele carriers (−11%) in both crude (P = 0.002) and multivariate analysis (P = 0.044). Conclusion In treated patients with arterial hypertension, cardiac disease and preserved left ventricular systolic function AGTR2 (+1675G/A) exhibits only a minor effect on left ventricular geometry in women and none in men.

A Single Nucleotide Polymorphism near the CYP17A1 Gene Is Associated with Left Ventricular Mass in Hypertensive Patients under Pharmacotherapy

Cytochrome P450 17A1 (CYP17A1) catalyses the formation and metabolism of steroid hormones. They are involved in blood pressure (BP) regulation and in the pathogenesis of left ventricular hypertrophy. Therefore, altered function of CYP17A1 due to genetic variants may influence BP and left ventricular mass. Notably, genome wide association studies supported the role of this enzyme in BP control. Against this background, we investigated associations between single nucleotide polymorphisms (SNPs) in or nearby the CYP17A1 gene with BP and left ventricular mass in patients with arterial hypertension and associated cardiovascular organ damage treated according to guidelines. Patients (n = 1007, mean age 58.0 ± 9.8 years, 83% men) with arterial hypertension and cardiac left ventricular ejection fraction (LVEF) ≥40% were enrolled in the study. Cardiac parameters of left ventricular mass, geometry and function were determined by echocardiography. The cohort comprised patients with coronary heart disease (n = 823; 81.7%) and myocardial infarction (n = 545; 54.1%) with a mean LVEF of 59.9% ± 9.3%. The mean left ventricular mass index (LVMI) was 52.1 ± 21.2 g/m2.7 and 485 (48.2%) patients had left ventricular hypertrophy. There was no significant association of any investigated SNP (rs619824, rs743572, rs1004467, rs11191548, rs17115100) with mean 24 h systolic or diastolic BP. However, carriers of the rs11191548 C allele demonstrated a 7% increase in LVMI (95% CI: 1%–12%, p = 0.017) compared to non-carriers. The CYP17A1 polymorphism rs11191548 demonstrated a significant association with LVMI in patients with arterial hypertension and preserved LVEF. Thus, CYP17A1 may contribute to cardiac hypertrophy in this clinical condition.

Role of CYP2C9 genetic variants for salt sensitivity and the regulation of the renin-angiotensin-aldosterone system in normotensive men.

Objective Cytochrome P450 (CYP) 2C9 gene polymorphisms have been implicated in regulation of the renin–angiotensin–aldosterone system (RAAS) and salt sensitivity in hypertensive patients. We tested the relevance of CYP2C9 genotypes for regulation of plasma renin activity (PRA), plasma aldosterone concentrations and blood pressure (BP) in response to changes in salt intake in normotensive individuals. Methods Three hundred and ten normotensive men (mean age 24.9 ± 0.1) were studied after a standardized low = 20 mmol/day or high = 220 mmol/day sodium intake for 7 days. Individuals were classified as salt sensitive when the mean arterial BP was more than 3 mmHg higher after high compared with low-salt exposure. Results CYP2C9*2 and CYP2C9*3 alleles were not associated with salt-sensitivity status or BP phenotypes; CYP2C9*2 had no effect on PRA or plasma aldosterone. CYP2C9*3 carriers showed a significantly lower PRA compared with CYP2C9*1/*1 individuals in the overall study cohort (high salt: 0.39 ± 0.05 vs. 0.62 ± 0.04 ng/ml per h, P = 0.009; low salt: 2.19 ± 0.27 vs. 2.87 ± 0.13 ng/ml per h, P = 0.013). Salt-sensitive CYP2C9*3 carriers exhibited the lowest PRA values and significantly lower 24 h sodium excretion rates during high-salt intake (P = 0.005 vs. CYP2C9*1/*1). Lower plasma aldosterone concentrations were only observed in salt-resistant CYP2C9*3 carriers under low salt (P = 0.039). Conclusion The present study confirms an association between CYP2C9 polymorphism and activity of the RAAS. Specifically, we detected an overall effect of CYP2C9*3 on lower PRA, but not on salt-sensitive BP regulation in normotensive men. Further studies are needed to analyze the long-term effects of CYP2C9*3 for salt sensitivity and hypertensive diseases.

Microangiopathy and visual deficits characterize the retinopathy of a spontaneously hypertensive rat model with type 2 diabetes and metabolic syndrome.

Retinopathy has been increasing in prevalence as a consequence of type 2 diabetes and a cluster of coexisting risk factors characterized as the metabolic syndrome. However, the combined effects of these conditions on the retina are poorly understood. Therefore, we focused on the spontaneously hypertensive corpulent rat (SHR/N-cp), a model with type 2 diabetes, obesity and features of the metabolic syndrome to characterize retinal changes at a structural and functional level. SHR/N-cp males at 4 and 8 months of age were used in this study. Metabolic parameters and blood pressure were measured by standard methods. Morphology was investigated by histological techniques supplemented by nicotinamide adenine dinucleotide phosphate-diaphorase staining of whole mounts and fluorescein angiography to analyze the retinal vasculature. The in vivo function of the retina was examined by electroretinography (ERG). Obese SHR/N-cp rats were hypertensive and showed significant increases in body weight, serum levels of glucose, triglycerides, total cholesterol and urinary glucose excretion compared with lean controls (P<0.01 for each). Histology indicated an overall intact integrity of the retina and aspects of microangiopathy in obese SHR/N-cp rats. ERG revealed intact processing of light signals but significantly decreased amplitudes of b-waves for all (P<0.01) and of a-waves for some examined light intensities (P<0.05). Oscillatory potentials were significantly protracted (P<0.01), whereas amplitudes were not reduced. Microangiopathy and electroretinographic deficits combine to produce an early non-proliferative retinopathy phenotype in the obese SHR/N-cp rats. Thus, this model represents a valuable experimental tool to obtain further insights into the mechanisms of retinopathy in the context of obesity, type 2 diabetes and metabolic syndrome.

Automated detection of protein unfolding events in atomic force microscopy force curves.

Atomic force microscopy is not only a high‐resolution imaging device but also a mechanical machine, which can be used either to indent or stretch (soft) biomaterials. Due to the statistical nature of such materials (i.e., hydrogels or polymers) hundreds of force‐distance curves are required to describe their mechanical properties. In this manuscript, we present an automated system for polymer unfolding detection based on continuous wavelet analysis. We have tested the automated program on elastin, which is an important protein that provides elasticity to tissues and organs. Our results show that elastin changes its mechanical behavior in the presence of electrolytes. In particular, we show that NaCl has a different effect on the contour length than CaCl2 for similar unfolding forces. In addition, we provide the program in the supporting information for the researches facing such kind of problem.

Dialysis-associated hypertension treated with Telmisartan--DiaTel: a pilot, placebo-controlled, cross-over, randomized trial.

Treatment of hypertension in hemodialysis (HD) patients is characterised by lack of evidence for both the blood pressure (BP) target goal and the recommended drug class to use. Telmisartan, an Angiotensin receptor blocker (ARB) that is metabolised in the liver and not excreted via HD extracorporeal circuit might be particularly suitable for HD patients. We designed and conducted a randomised, placebo-controlled, double-blind and cross-over trial for treatment of dialysis–associated hypertension with telmisartan 80 mg once daily or placebo on top of standard antihypertensive treatment excluding other Renin-Angiotensin-System (RAS) blockers. In 29 patients after randomization we analysed BP after a treatment period of 8 weeks, while 13 started with telmisartan and 16 with placebo; after 8 weeks 11 continued with telmisartan and 12 with placebo after cross-over, respectively. Patients exhibited a significant reduction of systolic pre-HD BP from 141.9±21.8 before to 131.3±17.3 mmHg after the first treatment period with telmisartan or placebo. However, no average significant influence of telmisartan was observed compared to placebo. The latter may be due to a large inter-individual variability of BP responses reaching from a 40 mmHg decrease under placebo to 40 mmHg increase under telmisartan. Antihypertensive co-medication was changed for clinical reasons in 7 out of 21 patients with no significant difference between telmisartan and placebo groups. Our starting hypothesis, that telmisartan on top of standard therapy lowers systolic office BP in HD patients could not be confirmed. In conclusion, this small trial indicates that testing antihypertensive drug efficacy in HD patients is challenging due to complicated standardization of concomitant medication and other confounding factors, e.g. volume status, salt load and neurohormonal activation, that influence BP control in HD patients. Trial Registration Clinicaltrialsregister.eu 2005-005021-60