Cell cycle length governs heterochromatin reprogramming during early development in non-mammalian vertebrates.

Heterochromatin marks such as H3K9me3 undergo global erasure and re-establishment after fertilization, and the proper reprogramming of H3K9me3 is essential for early development. Despite the widely conserved dynamics of heterochromatin reprogramming in invertebrates and non-mammalian vertebrates, previous studies have shown that the underlying mechanisms may differ between species. Here, we investigate the molecular mechanism of H3K9me3 dynamics in medaka (Japanese killifish, Oryzias latipes) as a non-mammalian vertebrate model, and show that rapid cell cycle during cleavage stages causes DNA replication-dependent passive erasure of H3K9me3. We also find that cell cycle slowing, toward the mid-blastula transition, permits increasing nuclear accumulation of H3K9me3 histone methyltransferase Setdb1, leading to the onset of H3K9me3 re-accumulation. We further demonstrate that cell cycle length in early development also governs H3K9me3 reprogramming in zebrafish and Xenopus laevis. Together with the previous studies in invertebrates, we propose that a cell cycle length-dependent mechanism for both global erasure and re-accumulation of H3K9me3 is conserved among rapid-cleavage species of non-mammalian vertebrates and invertebrates such as Drosophila, C. elegans, Xenopus and teleost fish.

Incomplete erasure of histone marks during epigenetic reprogramming in medaka early development.

Epigenetic modifications undergo drastic erasure and reestablishment after fertilization. This reprogramming is required for proper embryonic development and cell differentiation. In mammals, some histone modifications are not completely reprogrammed and play critical roles in later development. In contrast, in nonmammalian vertebrates, most histone modifications are thought to be more intensively erased and reestablished by the stage of zygotic genome activation (ZGA). However, histone modifications that escape reprogramming in nonmammalian vertebrates and their potential functional roles remain unknown. Here, we quantitatively and comprehensively analyzed histone modification dynamics during epigenetic reprogramming in Japanese killifish, medaka (Oryzias latipes) embryos. Our data revealed that H3K27ac, H3K27me3, and H3K9me3 escape complete reprogramming, whereas H3K4 methylation is completely erased during cleavage stage. Furthermore, we experimentally showed the functional roles of such retained modifications at early stages: (i) H3K27ac premarks promoters during the cleavage stage, and inhibition of histone acetyltransferases disrupts proper patterning of H3K4 and H3K27 methylation at CpG-dense promoters, but does not affect chromatin accessibility after ZGA; (ii) H3K9me3 is globally erased but specifically retained at telomeric regions, which is required for maintenance of genomic stability during the cleavage stage. These results expand the understanding of diversity and conservation of reprogramming in vertebrates, and unveil previously uncharacterized functions of histone modifications retained during epigenetic reprogramming.

Targeted Manipulation of Histone Modification in Medaka Embryos.

Recent development of targeted manipulation of histone modification enables us to experimentally and directly test the functional relevance of histone modifications accumulated at specific genomic regions. In particular, dCas9 epigenome editing has been widely used for site-specific manipulation of epigenetic modification. Here, we describe how to apply dCas9 epigenome editing in fish (medaka, Oryzias latipes) embryos and how to analyze induced changes in histone modification.

Targeted in vivo epigenome editing of H3K27me3.

BACKGROUND: Epigenetic modifications have a central role in transcriptional regulation. While several studies using next-generation sequencing have revealed genome-wide associations between epigenetic modifications and transcriptional states, a direct causal relationship at specific genomic loci has not been fully demonstrated, due to a lack of technology for targeted manipulation of epigenetic modifications. Recently, epigenome editing techniques based on the CRISPR-Cas9 system have been reported to directly manipulate specific modifications at precise genomic regions. However, the number of editable modifications as well as studies applying these techniques in vivo is still limited. RESULTS: Here, we report direct modification of the epigenome in medaka (Japanese killifish, Oryzias latipes) embryos. Specifically, we developed a method to ectopically induce the repressive histone modification, H3K27me3 in a locus-specific manner, using a fusion construct of Oryzias latipes H3K27 methyltransferase Ezh2 (olEzh2) and dCas9 (dCas9-olEzh2). Co-injection of dCas9-olEzh2 mRNA with single guide RNAs (sgRNAs) into one-cell-stage embryos induced specific H3K27me3 accumulation at the targeted loci and induced downregulation of gene expression. CONCLUSION: In this study, we established the in vivo epigenome editing of H3K27me3 using medaka embryos. The locus-specific manipulation of the epigenome in living organisms will lead to a previously inaccessible understanding of the role of epigenetic modifications in development and disease.

Unlinking the methylome pattern from nucleotide sequence, revealed by large-scale in vivo genome engineering and methylome editing in medaka fish.

The heavily methylated vertebrate genomes are punctuated by stretches of poorly methylated DNA sequences that usually mark gene regulatory regions. It is known that the methylation state of these regions confers transcriptional control over their associated genes. Given its governance on the transcriptome, cellular functions and identity, genome-wide DNA methylation pattern is tightly regulated and evidently predefined. However, how is the methylation pattern determined in vivo remains enigmatic. Based on in silico and in vitro evidence, recent studies proposed that the regional hypomethylated state is primarily determined by local DNA sequence, e.g., high CpG density and presence of specific transcription factor binding sites. Nonetheless, the dependency of DNA methylation on nucleotide sequence has not been carefully validated in vertebrates in vivo. Herein, with the use of medaka (Oryzias latipes) as a model, the sequence dependency of DNA methylation was intensively tested in vivo. Our statistical modeling confirmed the strong statistical association between nucleotide sequence pattern and methylation state in the medaka genome. However, by manipulating the methylation state of a number of genomic sequences and reintegrating them into medaka embryos, we demonstrated that artificially conferred DNA methylation states were predominantly and robustly maintained in vivo, regardless of their sequences and endogenous states. This feature was also observed in the medaka transgene that had passed across generations. Thus, despite the observed statistical association, nucleotide sequence was unable to autonomously determine its own methylation state in medaka in vivo. Our results apparently argue against the notion of the governance on the DNA methylation by nucleotide sequence, but instead suggest the involvement of other epigenetic factors in defining and maintaining the DNA methylation landscape. Further investigation in other vertebrate models in vivo will be needed for the generalization of our observations made in medaka.

Cough-challenge trial with a new angiotensin-converting enzyme inhibitor, imidapril.

This study was conducted to examine whether imidaprilat, an active diacid of the angiotensin-converting enzyme (ACE) inhibitor imidapril, preferentially inhibits angiotensin I degradation rather than bradykinin degradation, and whether imidapril is less active than other ACE inhibitors in inducing cough in patients with hypertension. The effect of imidaprilat on the inhibition of pressor response to angiotensin I and augmentation of depressor response to bradykinin was compared with that of enalaprilat and captopril in anesthetized rats. To determine the incidence of cough associated with imidapril, patients with a history of ACE inhibitor-induced dry cough were enrolled in a randomized, open-labeled, crossover trial with two 6-week periods to be treated with imidapril or amlodipine, a calcium-channel blocker. The recurrence of cough was assessed during both treatments. In the animal study, there were no significant differences in the ratio of inhibition of pressor response to angiotensin I and the augmentation of depressor response to bradykinin among the ACE inhibitors. In the cough-challenge trial, a total of 60 patients with hypertension were enrolled in the study. Cough and cough related symptoms recurred in 98.3% of the patients (59/ 60) during imidapril therapy. In contrast, only two patients reported cough during treatment with amlodipine. These results indicate that imidapril has no selectivity in inhibiting angiotensin I- and bradykinin-degradation in rats, and that clinically it is not different from other ACE inhibitors in inducing cough in patients with hypertension.

Fosinopril. Clinical pharmacokinetics and clinical potential.

Fosinopril is a phosphorus-containing ester prodrug of an angiotensin-converting enzyme (ACE) inhibitor. It is hydrolysed mainly in the gastrointestinal mucosa and liver to the active diacid, fosinoprilat, which has unique pharmacological properties. The majority of the active moieties of other ACE inhibitors are excreted in the urine. This means that an adjustment in either the dosage and/or the administration interval is needed in patients with moderate to severe renal dysfunction, in order to reduce drug accumulation and the possibility of an excessive decrease in blood pressure or other adverse effects. On the other hand, fosinoprilat is excreted both in urine and bile (as with temocaprilat, zofenoprilat and spiraprilat), and thus an adjustment of dosage and/or administration interval may be unnecessary in patients with moderate to severe renal dysfunction, as impaired renal function influences little of the pharmacokinetics of fosinoprilat. Furthermore, the available evidence suggests that the pharmacokinetic variables of fosinoprilat in patients receiving haemodialysis were similar to those in patients with moderate to severe renal dysfunction. Dosage modifications or supplemental dose administration following dialysis may be unnecessary. The hypotensive effect of the combination of fosinopril and a diuretic is synergistic. Pharmacokinetic interactions with fosinopril are unlikely in patients receiving thiazide or loop diuretics. Fosinopril has beneficial effects for patients with hypertension and left ventricular hypertrophy because it produces an adequate reduction in blood pressure and reversal of left ventricular hypertrophy. There are a large number of studies of the pharmacokinetics of fosinopril. However studies of its pharmacokinetic drug interactions with other drugs are far fewer. Further investigations are needed in several clinical settings.

Effect of doxazosin therapy on glucose tolerance and lipid metabolism in hypertensive patients with impaired glucose tolerance.

The effects of long-term monotherapy with doxazosin, an alpha 1-blocker, or placebo on blood pressure (BP), glucose tolerance, and serum lipid levels were investigated prospectively in 43 hypertensive patients with impaired glucose tolerance. The levels of plasma glucose, serum lipids, fructosamine, and glycated hemoglobin A1c (Hb A1c) were determined before and during long-term (mean treatment period, 6.7 months) therapy with doxazosin (n = 23) or placebo (n = 20). A 75-g oral glucose tolerance test was performed before and during therapy. Significant decreases in both systolic and diastolic BP were maintained during doxazosin therapy; BP did not change in the placebo group. Neither fasting nor post-glucose-load venous plasma glucose levels were altered, and there was no significant change in the insulinogenic index in either group. Glucose intolerance was slightly improved with significant reductions in Hb A1c and fructosamine levels during doxazosin therapy. Serum total cholesterol (TC) and low-density lipoprotein (LDL) cholesterol levels were significantly decreased, and high-density lipoprotein cholesterol levels were significantly increased in patients treated with doxazosin. Moreover, TC, LDL cholesterol, and apolipoprotein B levels were significantly decreased in patients with hypercholesterolemia (TC > or = 5.69 mmol/L). In contrast, there were no significant changes in Hb A1c, fructosamine, and lipid levels in the placebo group. These results suggest that long-term doxazosin therapy may improve glucose and lipid metabolism in hypertensive patients. Doxazosin appears useful as an antihypertensive agent for hypertensive patients with either impaired glucose metabolism or dyslipidemia.

Long-term captopril therapy with no effect on glucose metabolism in patients with essential hypertension.

The long-term antihypertensive response to captopril (25-50 mg/day) and 75g oral glucose tolerance (75g oGTT) following a 6-10 month period of captopril administration was evaluated in 20 patients with essential hypertension without persistent proteinuria. Eleven of these 20 patients exhibited impaired glucose tolerance (IGT), while the remaining nine patients had normal glucose tolerance (NGT). All patients tolerated long-term captopril therapy with no untoward effects. Six months' administration of captopril significantly decreased blood pressure in patients with NGT from 171 +/- 12/105 +/- 5 mm Hg (mean +/- SE) to 146 +/- 7/88 +/- 4 mm Hg. Also in patients with IGT, long-term captopril therapy decreased blood pressure from 165 +/- 3/97 +/- 2 to 143 +/- 5/86 +/- 2 mm Hg. No patient with NGT developed diabetes mellitus. Neither fasting nor post-glucose-load venous blood glucose deteriorated in any of the patients during the therapy. There were no significant changes in the insulinogenic index (delta IRI/delta BG at 30 minutes after glucose load) in both the patients with NGT and IGT. In patients with IGT, the concentration of glycosylated hemoglobin (Hb A1 and Hb A1c slightly but significantly decreased from 8.1 +/- 0.3 to 7.7 +/- 0.4% (P less than 0.05) and from 5.9 +/- 0.3 to 5.5 +/- 0.3% (P less than 0.01) after 6.2 +/- 1.4 months' captopril therapy. These results suggest that in addition to its antihypertensive effects, long-term captopril therapy does not compromise glucose metabolism in hypertensive patients.

Glucose tolerance during chronic captopril therapy in patients with essential hypertension.

We conducted a prospective evaluation of the effects of chronic captopril therapy on glucose tolerance in 8 nondiabetic, hypertensive patients and 6 hypertensive patients with impaired glucose tolerance, including 3 diabetic patients. Captopril was well tolerated by all patients, and no untoward effects were observed. Chronic captopril therapy produced a significant decrease in blood pressure in all patients. No patients with normal glucose tolerance developed diabetes mellitus. Neither fasting nor post-glucose-load venous plasma glucose deteriorated in any patients during chronic captopril therapy. There were no significant changes in the insulinogenic index (delta IRI/delta BS at 30 min post-glucose load) in patients with either normal or impaired glucose tolerance. These results suggest that, in addition to its antihypertensive effect, chronic captopril therapy does not compromise glucose metabolism in hypertensive patients. Thus, captopril may have a clinical advantage in that it apparently can be given safely to hypertensive patients with either normal or impaired glucose metabolism.

Bilateral pheochromocytoma associated with papillary adenocarcinoma of the thyroid gland; report of an unusual case.

A 31-year old woman was admitted to our clinic complaining of high blood pressure, dizziness, constipation, mental irritability and weight loss. The physical examination revealed goiter in her neck. The plasma levels of norepinephrine and epinephrine were 3.45 and 0.76 ng/ml, respectively. Urinary excretion of norepinephrine was 1 mg and epinephrine was 32.2 micrograms/24-hours. The examination by radiography and radioactive isotope revealed a tumor in the left adrenal region and another in the left lower lobe of the thyroid. After the operations, pheochromocytoma and papillary adenocarcinoma of the thyroid gland were recognized pathologically. However, 17 months later, the recurrence of pheochromocytoma in the contralateral adrenal region was discovered and removed. Although the co-existence of bilateral pheochromocytoma and papillary adenocarcinoma of the thyroid gland is not one of multiple endocrine neoplasia, to the best of our knowledge, only 7 such cases have been reported in the published literature.

Involvement of alpha-adrenergic mechanisms in growth hormone release induced by opioid peptides in conscious rats.

The effects of synthetic enkephalin analog (KF 33-824) and beta-endorphin on growth hormone (GH) secretion and their interaction with brain monoamines were investigated in unanesthetized male rats. Blood samples (0.4 ml each) were withdrawn every 10-20 min for 6 h from a catheter chronically implanted in the right atrium. In all control rats, immunoreactive GH secretion was pulsatile in nature and two major GH bursts were found to occur around 12.00 and 15.30. The opioid peptides were injected between bursts at 14.00. Following an intravenous administration of FK 33-824 (10 microgram/100 g b.w.), there was an abrupt increase in plasma GH, which was significantly suppressed by naloxone (125 microgram/100 g b.w., i.v.), a specific opiate antagonist. Pretreatment with reserpine (1 mg/100 g b.w., i.p.) abolished not only the natural GH burst but also the GH response to FK 33-824. Pretreatment with dopamine-beta-hydroxylase inhibitors, diethyldithiocarbamate (DDC, 100 mg/100 g b.w., i.v.) and fusarate (10 mg/100 g b.w., i.v.) also inhibited the natural GH burst and GH rise induced by FK 33-824. Intravenous injection of clonidine (15 microgram/100 g b.w.), an alpha-adrenergic stimulant, resulted in an increase in plasma GH in the rats pretreated with reserpine, DDC or fusarate. Phenoxybenzamine (1 mg/100 g b.w., i.v.), an alpha-adrenergic blocking agent, inhibited the GH response to KF 33-824. On the other hand, GH release induced by FK 33-824 was not influenced by propranolol (1 mg/100 g b.w., i.v.), a beta-adrenergic blocking agent, nor pimozide (0.1 mg/100 g b.w., i.v.), a dopamine antagonist. Intraventricular administration of beta-endorphin (5 microgram/rat) also increased the plasma GH levels which were lowered by phenoxybenzamine. These findings suggest that alpha-adrenergic mechanisms are involved in GH release induced by opioid peptides in the rat.