Multi-omic analysis of the effects of low frequency ventilation during cardiopulmonary bypass surgery.

BACKGROUND: Heart surgery with cardio-pulmonary bypass (CPB) is associated with lung ischemia leading to injury and inflammation. It has been suggested this is a result of the lungs being kept deflated throughout the duration of CPB. Low frequency ventilation (LFV) during CPB has been proposed to reduce lung dysfunction. METHODS: We used a semi-biased multi-omic approach to analyse lung biopsies taken before and after CPB from 37 patients undergoing coronary artery bypass surgery randomised to both lungs left collapsed or using LFV for the duration of CPB. We also examined inflammatory and oxidative stress markers from blood samples from the same patients. RESULTS: 30 genes were induced when the lungs were left collapsed and 80 by LFV. Post-surgery 26 genes were significantly higher in the LFV vs. lungs left collapsed, including genes associated with inflammation (e.g. IL6 and IL8) and hypoxia/ischemia (e.g. HIF1A, IER3 and FOS). Relatively few changes in protein levels were detected, perhaps reflecting the early time point or the importance of post-translational modifications. However, pathway analysis of proteomic data indicated that LFV was associated with increased "cellular component morphogenesis" and a decrease in "blood circulation". Lipidomic analysis did not identify any lipids significantly altered by either intervention. DISCUSSION: Taken together these data indicate the keeping both lungs collapsed during CPB significantly induces lung damage, oxidative stress and inflammation. LFV during CPB increases these deleterious effects, potentially through prolonged surgery time, further decreasing blood flow to the lungs and enhancing hypoxia/ischemia.

CRISPR-based reagents to study the influence of the epigenome on gene expression.

The use of epigenome editing is set to expand our knowledge of how epigenetic landscapes facilitate gene expression capacity within a given cell. As epigenetic landscape profiling in health and disease becomes more commonplace, so does the requirement to assess the functional impact that particular regulatory domains and DNA methylation profiles have upon gene expression capacity. That functional assessment is particularly pertinent when analysing epigenomes in disease states where the reversible nature of histone and DNA modification might yield plausible therapeutic targets. In this review we discuss first the nature of the epigenetic landscape, secondly the types of factors that deposit and erase the various modifications, consider how modifications transduce their signals, and lastly address current tools for experimental epigenome editing with particular emphasis on the immune system.

The nasal mucosal late allergic reaction to grass pollen involves type 2 inflammation (IL-5 and IL-13), the inflammasome (IL-1ß), and complement.

Non-invasive mucosal sampling (nasosorption and nasal curettage) was used following nasal allergen challenge with grass pollen in subjects with allergic rhinitis, in order to define the molecular basis of the late allergic reaction (LAR). It was found that the nasal LAR to grass pollen involves parallel changes in pathways of type 2 inflammation (IL-4, IL-5 and IL-13), inflammasome-related (IL-1ß), and complement and circadian-associated genes. A grass pollen nasal spray was given to subjects with hay fever followed by serial sampling, in which cytokines and chemokines were measured in absorbed nasal mucosal lining fluid, and global gene expression (transcriptomics) assessed in nasal mucosal curettage samples. Twelve of 19 subjects responded with elevations in interleukin (IL)-5, IL-13, IL-1ß and MIP-1ß/CCL4 protein levels in the late phase. In addition, in these individuals whole-genome expression profiling showed upregulation of type 2 inflammation involving eosinophils and IL-4, IL-5 and IL-13; neutrophil recruitment with IL-1α and IL-1ß; the alternative pathway of complement (factor P and C5aR); and prominent effects on circadian-associated transcription regulators. Baseline IL-33 mRNA strongly correlated with these late-phase responses, whereas a single oral dose of prednisone dose-dependently reversed most nasal allergen challenge-induced cytokine and transcript responses. This study shows that the LAR to grass pollen involves a range of inflammatory pathways and suggests potential new biomarkers and therapeutic targets. Furthermore, the marked variation in mucosal inflammatory events between different patients suggests that in the future precision mucosal sampling may enable rational specific therapy.

Control of cytokine gene transcription in Th1 and Th2 cells.

Summary Analysis of T-helper cell differentiation to T-helper type 1 (Th1) and Th2 lineages has begun to reveal a complex mechanism whereby transcription factors, enzymes that either deposit or remove covalent modifications from histone tails and DNA methylating enzymes are recruited to cytokine genes. Each resultant cell lineage subsequently displays a programme of transcriptional restrictions that firstly, facilitates expression of a particular subset of signature cytokines and secondly, silences expression of the cytokines normally recognized as being markers of the opposite differentiation limb. Some essential proteins in this differentiative paradigm, such as the transcription factors GATA3 and T-bet, are well studied; however, the types of enzymatic activities that these proteins recruit in order to implement differentiation are more obscure. Recent genome-wide studies of histone modifications have begun to clarify how specific modifications of histones impact upon both transcriptional regulation and chromatin organization. Here we review how this information has enlightened our knowledge of how Th1/Th2 differentiation is orchestrated.

Suppression of granulocyte-macrophage colony-stimulating factor expression by glucocorticoids involves inhibition of enhancer function by the glucocorticoid receptor binding to composite NF-AT/activator protein-1 elements.

Increased expression of a number of cytokines including GM-CSF is associated with chronic inflammatory conditions such as bronchial asthma. Glucocorticoid therapy results in suppression of cytokine levels by a mechanism(s) not yet fully understood. We have examined regulation of GM-CSF expression by the synthetic glucocorticoid dexamethasone in human T cells. Transient transfection assays with reporter constructs revealed that dexamethasone inhibited the function of the GM-CSF enhancer, but had no effect on regulation of GM-CSF expression occurring through the proximal promoter. Activation of the GM-CSF enhancer involves cooperative interaction between the transcription factors NF-AT and AP-1. We demonstrate here that glucocorticoid-mediated inhibition of enhancer function involves glucocorticoid receptor (GR) binding to the NF-AT/AP-1 sites. These elements, which do not constitute recognizable glucocorticoid response elements, support binding of the GR, primarily as a dimer. This binding correlates with the ability of dexamethasone to inhibit enhancer activity of the NF-AT/AP-1 elements, suggesting a competition between NF-AT/AP-1 proteins and GR.

Regulation of Th2 cytokine gene transcription.

Because of the different functions of IL-4, IL-13 and IL-5, it would perhaps be surprising if common transcriptional mechanisms occur. However, because of the physical proximity of their genes within the human 5q locus, chromatin remodelling during T-cell differentiation may make transcription of all the genes permissible. If co-ordinate regulation were to take place, it might be argued that similar factors might be involved in expression of all of the cytokines. Emerging data on c-Maf and GATA3 regulation of IL-4 and IL-5 respectively argues that diverse proteins may be required for transcriptional activation. Alternatively, these factors may be responsible for regulating transcriptional competence, allowing fine control over generation of particular cytokines depending upon recognition of physiological cues. If competent for transcription, common factors, such as members of the NFAT and/or AP-1 families, may operate to regulate cytokine levels. To support this, we have recently identified a conserved palindrome located within the promoters of the different Th2-type cytokines, which acts as an enhancer of transcription. Central to the capacity to express Th2 cytokines is likely to be the ability to remodel chromatin at the locus. It remains to be determined whether a single factor, or combination of factors acts to regulate this event. It is also unclear what the boundaries of remodelling within the locus are, i.e. whether IL-4 and IL-13 may be within open chromatin and IL-5 in a closed environment, and whether there is a hierarchy which determines whether particular cytokines are preferentially expressed irrespective of competence.

Regulation of Wnt signaling by Sox proteins: XSox17 alpha/beta and XSox3 physically interact with beta-catenin.

Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17 beta. Ectopic expression of XSox17 beta ventralizes embryos by inhibiting the Wnt pathway downstream of beta-catenin but upstream of the Wnt-responsive gene Siamois. XSox17 beta also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and beta-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses beta-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17 beta maps to a region C-terminal to the HMG box; this region of XSox17 beta physically interacts with the Armadillo repeats of beta-catenin. Two additional Sox proteins, XSox17 alpha and XSox3, likewise bind to beta-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways.

The maize retinoblastoma protein homologue ZmRb-1 is regulated during leaf development and displays conserved interactions with G1/S regulators and plant cyclin D (CycD) proteins.

Recent discoveries of plant retinoblastoma (Rb) protein homologues and D-type cyclins suggest that control of the onset of cell division in plants may have stronger parallels with mammalian G1/S controls than with yeasts. In mammals, the Rb protein interacts specifically with D-type cyclins and regulates cell proliferation by binding and inhibiting E2F transcription factors. However, the developmental role of Rb in plants and its potential interaction with cell cycle regulators is unknown. We show that the maize Rb homologue ZmRb-1 is temporally and spatially regulated during maize leaf development. ZmRb-1 is highly expressed in differentiating cells, but almost undetectable in proliferating cells. In vitro, both ZmRb-1 and human Rb bind all classes of plant D-type cyclins with the involvement of a conserved N-terminal Leu-x-Cys-x-Glu (LxCxE) Rb-interaction motif. This binding is strongly reduced by mutation of the conserved Cys-470 of ZmRb-1. ZmRb-1 binds human and Drosophila E2F, and inhibits transcriptional activation of human E2F. We also show that ZmRb-1 is a good in vitro substrate for all human G1/S protein kinases. The functional conservation of proteins that control the G1/S transition in mammals and plants points to the existence of plant E2F homologues. We conclude that evolution of Rb and cyclin D proteins occurred after separation of the fungi from the higher eukaryotic lineage, but preceded the divergence of plant and animal kingdoms.

The HMG-box transcription factor HBP1 is targeted by the pocket proteins and E1A.

A yeast two-hybrid screen has identified HBP1 as a transcription factor capable of interacting with the pocket protein family. We show that HBP1 can interact with one of these, RB, both in vitro and in mammalian cells. Two distinct RB binding sites are present within HBP1--a high affinity binding site, mediated by an LXCXE motif and a separate low affinity binding site present within an activation domain. GAL4-fusion experiments indicate that HBP1 contains a masked activation domain. Deletion of two independent N- and C-terminal inhibitor domains unmasks an activation domain which is 100-fold more active than the full length protein. The released activation capacity is repressed by RB, p130 and p107. In addition, E1A can repress the activity of HBP1 via conserved region 1 sequences in a manner independent of the CBP co-activator. We show by stable expression in NIH3T3 cells that HBP1 has the capacity to induce morphological transformation of cells in culture.

The atrial natriuretic peptide gene and essential hypertension in African-Caribbeans from St Vincent and the Grenadines.

Atrial natriuretic peptide (ANP) which alters sodium balance, blood volume and vascular tone represents an important candidate for investigating the genetic basis of essential hypertension (EH). Accordingly, we have studied Bgl1 and Xho1 restriction fragment length polymorphisms (RFLPs) of the ANP gene in 147 hypertensive, 141 normotensive and 67 population-based control subjects from a homogenous population of West African origin from St Vincent and the Grenadines. We found no association of either Bgl1 and Xho1 RFLPs with EH. This study suggests that the ANP locus may not exert a major gene effect on EH amongst the black people of St Vincent and the Grenadines.

Angiotensinogen in human essential hypertension.

The candidacy of angiotensinogen for a role in the genetic basis of hypertension is supported by the observation that plasma angiotensinogen levels track with raised blood pressure through families. In addition, transgenic mice with overexpression of a rat angiotensinogen gene develop hypertension, and knockout mice with a disrupted gene and absent angiotensinogen production develop low blood pressure. There are now two studies in populations of white European origin and one in African Caribbeans providing support for a role of the angiotensinogen gene locus in human essential hypertension.

Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans.

The renin-angiotensin system regulates blood pressure and sodium balance. The angiotensinogen gene which encodes the key substrate within this system has been linked to essential hypertension in White Europeans. It has been suggested that people of West African ancestry may have a different genetic basis for hypertension. In this study we have tested whether there is linkage of the angiotensinogen gene to essential hypertension in African Caribbeans from St. Vincent and the Grenadines. DNA from 63 affected sibling pairs with hypertension was tested for linkage by analyzing whether there was excess allele sharing among siblings genotyped using an angiotensinogen dinucleotide repeat sequence. There was significant support for linkage (T = 3.07, P = 0.001) and association of this locus to hypertension (chi 2 = 50.2, 12 degrees of freedom, P << 0.001). A DNA polymorphism which alters methionine to threonine at position 235 (M235T) within the angiotensinogen peptide has been associated previously with hypertension. However, we found no association of this variant with hypertension in this study. These findings provide support for linkage and association of the angiotensinogen locus to hypertension in African Caribbeans and suggest some similarities in the genetic basis of essential hypertension in populations of different ethnicity.

An alternatively spliced human insulin-like growth factor-I transcript with hepatic tissue expression that diverts away from the mitogenic IBE1 peptide.

An alternatively spliced transcript of the human insulin-like growth factor-I (IGF-I) gene is described. The transcript was identified in human liver RNA by reverse transcriptase-polymerase chain reaction, cloning, and sequencing. It contained IGF-I exons 3 and 4, 49 basepairs of exon 5, then exon 6 (exon 4-5-6). The 5'-donor site at the exon 5-6 junction was a cryptic 5'-donor splice site (IGF633). The 3'-acceptor site of the splice was the usual intron-exon 6 junction. A second pair of primers across the exon 5-exon 6 junction was used to confirm the presence of the transcript by reverse transcriptase-polymerase chain reaction. Cloning and sequencing this second fragment confirmed the presence of this splice in human liver. The exon 4-5-6 transcript was quantified at about 10% relative to the exon 4-6 transcript in human livers (n = 7 subjects), but was not detected in other tissues. The exon 4-5-6 transcript was found in cultured human hepatoma HepG2 cells and increased, relative to exon 4-6 transcripts, in response to GH, but not in cultured human lymphoblast IM-9 cells. The exon 4-5-6 splice predicts a prepro-IGF-I of 158 amino acid residues, with an E-peptide sequence of 24 residues (Ec). The deduced Ec peptide sequence is 73% homologous to the rat Eb-peptide sequence. The predicted final residues of the Ec peptide are frameshifted exon 6 codons ending in an in-frame stop codon. The predicted peptide sequences of Ec and Eb differ at the cleavage site of the Eb-peptide fragment (IBE1), which has been shown to have mitogenic activity. These data suggest that 1) the exon 4-5-6 splice has hepatic tissue expression and occurs by the use of a cryptic 5'-donor consensus splice site (IGF633) in exon 5; 2) exon 4-5-6 can be hormonally regulated in cultured human HepG2 cells; 3) exon 4-5-6 is the human counterpart of the rat IGF-IEb, because the complementary DNA and predicted sequences are homologous; and 4) the production of IBE1 is potentially regulated by alternative splicing.

Absence of mutations in the MEN2A region of the ret proto-oncogene in non-MEN 2A phaeochromocytomas.

OBJECTIVE: To determine the presence of abnormalities of the MEN2A region of the ret proto-oncogene in phaeochromocytomas/paragangliomas (PHAEO) of different aetiologies. DESIGN: Total RNA was extracted from tumours and used as templates for reverse transcriptase polymerase chain reactions. A ret primer pair, which encompasses the region which is mutated in the germ-line of patients with MEN 2A, was used. The resulting 262-bp product was sequenced. PATIENTS: Ten PHAEOs were examined. Four tumours were from von Hippel-Lindau disease patients; five were sporadic, isolated tumours; one from a patient with multiple endocrine neoplasia type 2A (MEN 2A). The medullary thyroid cancer from the single MEN 2A patient was also examined. RESULTS: A heterozygous TGC to CGC mutation of codon 634 (cysteine to arginine) was found in the PHAEO and medullary thyroid cancer from the MEN 2A patient. The 262-bp ret fragment was not found in two tumours (one malignant PHAEO and one secretory paraganglioma), although the intra-cellular ret tyrosine kinase domain was detected in these tumours. The cysteine codons were normal in all other non-MEN 2A PHAEOs. CONCLUSION: Mutations of key cysteine codons of the ret proto-oncogene may be specific to MEN 2A.

Linkage of the angiotensinogen gene to essential hypertension.

BACKGROUND: The renin-angiotensin system is a powerful pressor system with a major influence on salt and water homeostasis. Angiotensinogen (also called renin substrate) is a key component of this system; it is cleaved by renin to yield angiotensin I, which is then cleaved by angiotensin-converting enzyme to yield angiotensin II. The observation that plasma angiotensinogen levels correlate with blood pressure and track through families suggests that angiotensinogen may have a role in essential hypertension. We therefore investigated whether there is linkage between the angiotensinogen gene on chromosome 1q42-43 and essential hypertension. METHODS: Samples of DNA from 63 white European families in which two or more members had essential hypertension were tested for linkage of the angiotensinogen gene to this disorder. Affected cousins, nephews, nieces, and half-siblings were included when possible. To test for linkage, we used as a marker a dinucleotide-repeat sequence flanking this gene, and we employed the affected-pedigree-member method of linkage analysis. Two molecular variants of the angiotensinogen gene, one encoding threonine instead of methionine at position 235 (M235T) and the other encoding methionine rather than threonine at position 174 (T174M), were also tested for possible association with essential hypertension. RESULTS: We found significant linkage (t = 5.00, P < 0.001) and association (chi-square = 53.3, P < 0.001) of the angiotensinogen-gene locus to essential hypertension in the 63 multiplex families. This linkage was consistently maintained in the subgroup of subjects with diastolic pressure above 100 mm Hg and in the subgroups classified according to sex. It has been proposed previously that T174M and M235T are associated with essential hypertension. However, we found no association in our population between either polymorphism and this disorder. CONCLUSIONS: This study provides strong and consistent support for the linkage to essential hypertension of regions within or close to the angiotensinogen gene. Precisely how mutations in this region may result in hypertension remains to be determined.

Homodimer formation is rate-limiting for high affinity DNA binding by glucocorticoid receptor.

The glucocorticoid receptor (GR) is a hormone-inducible transcription factor which activates transcription of specific genes by binding to a DNA sequence present in the promoters of inducible genes. These glucocorticoid response elements (GREs) have a conserved palindromic sequence. Each half-GRE palindrome binds one subunit of GR. We have assessed the relative affinity of GR monomers and homodimers for GRE and determined whether homodimer formation is rate-limiting for high affinity GRE binding. The in vitro affinity of GRE binding by GR homodimers was approximately 2 x 10(-10) M, whereas it was approximately 1 nM for GR monomers. While homodimer:GRE complexes were very stable, monomer:GRE complexes appeared less stable in vitro. At low receptor concentration, GR preferentially bound GRE as a homodimer. Prior dilution of GR (equilibrium shifted to monomers) before addition to a GRE binding reaction resulted in slower kinetics of binding by comparison to parallel reactions in which concentrated (largely homodimeric) GR was added first. Taken together, these experiments suggest that homodimer formation is rate-limiting for high affinity GRE binding. A GRE mutant which contained only a half-binding site and which was unable to bind GR homodimers was also unable to confer glucocorticoid-inducible transcription. Taken together with previous work, these experiments support the model that GR homodimers are required for hormone-dependent activation of transcription and that receptor homodimer formation is rate-limiting for GRE binding.

Variable methylation of the 5'-flanking DNA of the human pro-opiomelanocortin gene.

The pro-opiomelanocortin gene is widely expressed in human tissues, although both transcriptional initiation sites and regulation appear to be tissue specific. In order to determine how promoter and enhancer choice is effected, we have studied the methylation pattern of the gene in a number of normal tissues, tumours and cell lines. Variability of this pattern was observed in the 5'-flanking DNA, particularly at the HpaII site located at -304 bp upstream from the pituitary CAP site. This site was generally methylated in tissues likely to express the predominant extrapituitary (800 nucleotide) message, while in tissues known to express the normal pituitary (1150 nucleotide) message and longer species, a tendency towards undermethylation was observed. Although the sites at which variable methylation occurs did not correspond to established binding sites for regulatory proteins, many of these regions remain to be determined and thus it is possible that methylation may be influential in the tissue-specific regulation of this gene.