Restoration of KMT2C/MLL3 in human colorectal cancer cells reinforces genome-wide H3K4me1 profiles and influences cell growth and gene expression.

BACKGROUND: The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant. RESULTS: Gene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation. CONCLUSIONS: Here, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.

AML displays increased CTCF occupancy associated with aberrant gene expression and transcription factor binding.

CCTC-binding factor (CTCF) is a key regulator of gene expression through organization of the chromatin structure. Still, it is unclear how CTCF binding is perturbed in leukemia or in cancer in general. We studied CTCF binding by chromatin immunoprecipitation sequencing in cells from patients with acute myeloid leukemia (AML) and in normal bone marrow (NBM) in the context of gene expression, DNA methylation, and azacitidine exposure. CTCF binding was increased in AML compared with NBM. Aberrant CTCF binding was enriched for motifs for key myeloid transcription factors such as CEBPA, PU.1, and RUNX1. AML with TET2 mutations was characterized by a particularly strong gain of CTCF binding, highly enriched for gain in promoter regions, while AML in general was enriched for changes at enhancers. There was a strong anticorrelation between CTCF binding and DNA methylation. Gain of CTCF occupancy was associated with increased gene expression; however, the genomic location (promoter vs distal regions) and enrichment of motifs (for repressing vs activating cofactors) were decisive for the gene expression pattern. Knockdown of CTCF in K562 cells caused loss of CTCF binding and transcriptional repression of genes with changed CTCF binding in AML, as well as loss of RUNX1 binding at RUNX1/CTCF-binding sites. In addition, CTCF knockdown caused increased differentiation. Azacitidine exposure caused major changes in CTCF occupancy in AML patient cells, partly by restoring a CTCF-binding pattern similar to NBM. We conclude that AML displays an aberrant increase in CTCF occupancy that targets key genes for AML development and impacts gene expression.

Downregulation of inflammatory markers by conjugated linoleic acid isomers in human cultured astrocytes.

OBJECTIVES: Conjugated linoleic acid (CLA) isomers have been shown to possess anti-inflammatory activity in the central nervous system. In this study, we aimed to evaluate whether modulation of the fatty acid profile by the CLA isomers c9,t11 or t10,c12CLA was associated with changes in the expression of pro-inflammatory molecules in human astrocytes. METHODS: Cultured astrocytes were treated for 6 days with 100 µM fatty acids (c9,t11CLA or t10,c12CLA or oleic acid). Following the treatment, the fatty acid profile of the cell and pro-inflammatory molecule expression were assessed. RESULTS: Only the t10,c12CLA isomer induced a significant decrease in arachidonic acid and increased the ratio of docosahexaenoic acid/eicosapentaenoic acid, which constitutes indirect evidence of peroxisome proliferator-activated receptor alpha activation. Inhibition of tumour necrosis factor-α, interleukin-1ß, and RANTES expression was observed in astrocytes treated with c9,t11CLA and t10,c12CLA. DISCUSSION: Current data demonstrate that CLA isomers, particularly t10,c12, may affect neuroinflammation by reducing the pro-inflammatory molecules in cultured astrocytes, suggesting a potential nutritional role of CLA isomers in modulating the astrocyte inflammatory response.

Cancer-specific changes in DNA methylation reveal aberrant silencing and activation of enhancers in leukemia.

Acute myeloid leukemia (AML) is characterized by an impaired differentiation process leading to an accumulation of immature blasts in the blood. One feature of cytogenetically normal AML is alterations to the DNA methylome. We analyzed 57 AML patients with normal karyotype by using Illumina's 450k array and showed that aberrant DNA methylation is significantly altered at enhancer regions and that the methylation levels at specific enhancers predict overall survival of AML patients. The majority of sites that become differentially methylated in AML occur in regulatory elements of the human genome. Hypermethylation associates with enhancer silencing. In addition, chromatin immunoprecipitation sequencing analyses showed that a subset of hypomethylated sites correlate with enhancer activation, indicated by increased H3K27 acetylation. DNA hypomethylation is therefore not sufficient for enhancer activation. Some sites of hypomethylation occur at weak/poised enhancers marked with H3K4 monomethylation in hematopoietic progenitor cells. Other hypomethylated regions occur at sites inactive in progenitors and reflect the de novo acquisition of AML-specific enhancers. Altered enhancer dynamics are reflected in the gene expression of enhancer target genes, including genes involved in oncogenesis and blood cell development. This study demonstrates that histone variants and different histone modifications interact with aberrant DNA methylation and cause perturbed enhancer activity in cytogenetically normal AML that contributes to a leukemic transcriptome.

Functional Analyses of the Crohn's Disease Risk Gene LACC1.

BACKGROUND: Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression. METHODS: We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function. RESULTS: FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems. CONCLUSION: FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

Severe gastrointestinal dysmotility developed after treatment with gonadotropin-releasing hormone analogs.

BACKGROUND: Sporadic cases of abdominal pain and dysmotility has been described after treatment with gonadotropin-releasing hormone (GnRH) analogs. The aim of the present study was to scrutinize for patients with severe gastrointestinal complaints after treatment with GnRH analogs, to describe the expression of antibodies against progonadoliberin-2, GnRH1, GnRH receptor (GnRHR), luteinizing hormone (LH), and LH receptor in serum in these patients, and to search for possible triggers and genetic factors behind the development of this dysmotility. METHODS: Patients suffering from prolonged gastrointestinal complaints after treatment with GnRH analogs at the Department of Gastroenterology, Skåne University Hospital, were included. GnRHR and LH receptor (LHCGR) genes were exome-sequenced. Serum was analyzed by enzyme-linked immune sorbent assays for the presence of antibodies. Healthy blood donors and women treated with GnRH analogs because of in vitro fertilization (IVF) were used as controls. RESULTS: Seven patients with severe gastrointestinal complaints after GnRH treatment were identified, of whom six suffered from endometriosis. Several variants were found within the 11 exons of LHCGR. The minor allele G, at the single nucleotide polymorphism rs6755901, was detected in homozygosity in two patients (28.5%) who had developed chronic intestinal pseudo-obstruction and in 5.5% of the IVF controls. Three patients expressed IgM antibodies against progonadoliberin-2 and three against GnRH1 (42.9%) when cut off was set to a titer >97.5th percentile in blood donors. CONCLUSION: A high prevalence of endometriosis, polymorphism in the LHCGR and GnRH1 and progonadoliberin-2 antibodies in serum was found among the patients with severe dysmotility after treatment with GnRH analogs.

Mutations in RAD21 disrupt regulation of APOB in patients with chronic intestinal pseudo-obstruction.

BACKGROUND & AIMS: Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimics a mechanical subocclusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and to identify potential biomarkers. METHODS: We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of messenger RNA (mRNA) and proteins were analyzed by quantitative reverse-transcription polymerase chain reaction, immunoblot, and mobility shift assays. Complementary DNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a). Gut tissues were collected and analyzed. RESULTS: We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a Morpholino zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in the regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 also is overexpressed in sporadic CIPO in sera and gut biopsy specimens. CONCLUSIONS: Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and the development of enteric neurons.

Transcription-coupled recruitment of human CHD1 and CHD2 influences chromatin accessibility and histone H3 and H3.3 occupancy at active chromatin regions.

BACKGROUND: CHD1 and CHD2 chromatin remodeling enzymes play important roles in development, cancer and differentiation. At a molecular level, the mechanisms are not fully understood but include transcriptional regulation, nucleosome organization and turnover. RESULTS: Here we show human CHD1 and CHD2 enzymes co-occupy active chromatin regions associated with transcription start sites (TSS), enhancer like regions and active tRNA genes. We demonstrate that their recruitment is transcription-coupled. CHD1 and CHD2 show distinct binding profiles across active TSS regions. Depletion of CHD1 influences chromatin accessibility at TSS and enhancer-like chromatin regions. CHD2 depletion causes increased histone H3 and reduced histone variant H3.3 occupancy. CONCLUSIONS: We conclude that transcription-coupled recruitment of CHD1 and CHD2 occurs at transcribed gene TSSs and at intragenic and intergenic enhancer-like sites. The recruitment of CHD1 and CHD2 regulates the architecture of active chromatin regions through chromatin accessibility and nucleosome disassembly.

The landscape of DNA repeat elements in human heart failure.

BACKGROUND: The epigenomes of healthy and diseased human hearts were recently examined by genome-wide DNA methylation profiling. Repetitive elements, heavily methylated in post-natal tissue, have variable methylation profiles in cancer but methylation of repetitive elements in the heart has never been examined. RESULTS: We analyzed repetitive elements from all repeat families in human myocardial samples, and found that satellite repeat elements were significantly hypomethylated in end-stage cardiomyopathic hearts relative to healthy normal controls. Satellite repeat elements are almost always centromeric or juxtacentromeric, and their overexpression correlates with disease aggressiveness in cancer. Similarly, we found that hypomethylation of satellite repeat elements correlated with up to 27-fold upregulation of the corresponding transcripts in end-stage cardiomyopathic hearts. No other repeat family exhibited differential methylation between healthy and cardiomyopathic hearts, with the exception of the Alu element SINE1/7SL, for which a modestly consistent trend of increased methylation was observed. CONCLUSIONS: Satellite repeat element transcripts, a form of non-coding RNA, have putative functions in maintaining genomic stability and chromosomal integrity. Further studies will be needed to establish the functional significance of these non-coding RNAs in the context of heart failure.

Effect of acute administration of Pistacia lentiscus L. essential oil on rat cerebral cortex following transient bilateral common carotid artery occlusion.

BACKGROUND: Ischemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the administration of Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma. METHODS: Adult Wistar rats underwent BCCAO for 20 min followed by 30 min reperfusion (BCCAO/R). 6 hours before surgery, rats, randomly assigned to four groups, were gavaged either with E.O. (200 mg/0.45 ml of sunflower oil as vehicle) or with the vehicle alone. RESULTS: BCCAO/R triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly polyunsaturated fatty acid most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led further to decreased levels of the enzyme cyclooxygenase-2 (COX-2), as assessed by Western Blot. In plasma, only after BCCAO/R, E.O. administration increased both the ratio of DHA-to-its precursor, eicosapentaenoic acid (EPA), and levels of palmytoylethanolamide (PEA) and oleoylethanolamide (OEA). CONCLUSIONS: Acute treatment with E.O. before BCCAO/R elicits changes both in the frontal cortex, where the BCCAO/R-induced decrease of DHA is apparently prevented and COX-2 expression decreases, and in plasma, where PEA and OEA levels and DHA biosynthesis increase. It is suggested that the increase of PEA and OEA plasma levels may induce DHA biosynthesis via peroxisome proliferator-activated receptor (PPAR) alpha activation, protecting brain tissue from ischemia/reperfusion injury.

A mixture of oleic, erucic and conjugated linoleic acids modulates cerebrospinal fluid inflammatory markers and improve somatosensorial evoked potential in X-linked adrenoleukodystrophy female carriers.

X-linked adrenoleukodystrophy is a rare inherited demyelinating disorder characterized by an abnormal accumulation of very long chain fatty acids, mainly hexacosanoic acid (26:0), due to a mutation of the gene encoding for a peroxisomal membrane protein. The only available, and partially effective, therapeutic treatment consists of dietary intake of a 4:1 mixture of triolein and trierucin, called Lorenzo's oil (LO), targeted to inhibit the elongation of docosanoic acid (22:0) to 26:0. In this study we tested whether, besides inhibiting elongation, an enhancement of peroxisomal beta oxidation induced by conjugated linoleic acid (CLA), will improve somatosensory evoked potentials and modify inflammatory markers in adrenoleukodystrophy females carriers. We enrolled five heterozygous women. They received a mixture of LO (40 g/day) with CLA (5 g/day) for 2 months. The therapeutic efficacy was evaluated by the means of plasma levels of 26:0, 26:0/22:0 ratio, modification of cerebrospinal fluid (CSF) inflammatory markers and somatosensory evoked potentials. Changes of fatty acid profile, and in particular CLA incorporation, were also evaluated in CSF and plasma. The results showed that CLA promptly passes the blood brain barrier and the mixture was able to lower both 26:0 and 26:0/22:0 ratio in plasma. The mixture improved somatosensory evoked potentials, which were previously found unchanged or worsened with dietary LO alone, and reduced IL-6 levels in CSF in three out of five patients. Our data suggest that the synergic activity of CLA and LO, by enhancing peroxisomal beta-oxidation and preventing 26:0 formation, improves the somatosensory evoked potentials and reduces neuroinflammation.

Effect of dietary krill oil supplementation on the endocannabinoidome of metabolically relevant tissues from high-fat-fed mice.

BACKGROUND: Omega-3 polyunsaturated fatty acids (ω-3-PUFA) are known to ameliorate several metabolic risk factors for cardiovascular disease, and an association between elevated peripheral levels of endogenous ligands of cannabinoid receptors (endocannabinoids) and the metabolic syndrome has been reported. We investigated the dose-dependent effects of dietary ω-3-PUFA supplementation, given as krill oil (KO), on metabolic parameters in high fat diet (HFD)-fed mice and, in parallel, on the levels, in inguinal and epididymal adipose tissue (AT), liver, gastrocnemius muscle, kidneys and heart, of: 1) the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), 2) two anandamide congeners which activate PPARα but not cannabinoid receptors, N-oleoylethanolamine and N-palmitoylethanolamine, and 3) the direct biosynthetic precursors of these compounds. METHODS: Lipids were identified and quantified using liquid chromatography coupled to atmospheric pressure chemical ionization single quadrupole mass spectrometry (LC-APCI-MS) or high resolution ion trap-time of flight mass spectrometry (LC-IT-ToF-MS). RESULTS: Eight-week HFD increased endocannabinoid levels in all tissues except the liver and epididymal AT, and KO reduced anandamide and/or 2-AG levels in all tissues but not in the liver, usually in a dose-dependent manner. Levels of endocannabinoid precursors were also generally down-regulated, indicating that KO affects levels of endocannabinoids in part by reducing the availability of their biosynthetic precursors. Usually smaller effects were found of KO on OEA and PEA levels. CONCLUSIONS: Our data suggest that KO may promote therapeutic benefit by reducing endocannabinoid precursor availability and hence endocannabinoid biosynthesis.

Krill oil significantly decreases 2-arachidonoylglycerol plasma levels in obese subjects.

We have previously shown that krill oil (KO), more efficiently than fish oil, was able to downregulate the endocannabinoid system in different tissues of obese zucker rats.We therefore aimed at investigating whether an intake of 2 g/d of either KO or menhaden oil (MO), which provides 309 mg/d of EPA/DHA 2:1 and 390 mg/d of EPA/DHA 1:1 respectively, or olive oil (OO) for four weeks, is able to modify plasma endocannabinoids in overweight and obese subjects.The results confirmed data in the literature describing increased levels of endocannabinoids in overweight and obese with respect to normo-weight subjects. KO, but not MO or OO, was able to significantly decrease 2-arachidonoylglycerol (2-AG), although only in obese subjects. In addition, the decrease of 2-AG was correlated to the plasma n-6/n-3 phospholipid long chain polyunsaturated fatty acid (LCPUFA) ratio. These data show for the first time in humans that relatively low doses of LCPUFA n-3 as KO can significantly decrease plasma 2-AG levels in obese subjects in relation to decrease of plasma phospholipid n-6/n-3 LCPUFA ratio. This effect is not linked to changes of metabolic syndrome parameters but is most likely due to a decrease of 2-AG biosynthesis caused by the replacement of 2-AG ultimate precursor, arachidonic acid, with n-3 PUFAs, as previously described in obese Zucker rats.

Endocannabinoids may mediate the ability of (n-3) fatty acids to reduce ectopic fat and inflammatory mediators in obese Zucker rats.

Dietary (n-3) long-chain PUFA [(n-3) LCPUFA] ameliorate several metabolic risk factors for cardiovascular diseases, although the mechanisms of these beneficial effects are not fully understood. In this study, we compared the effects of dietary (n-3) LCPUFA, in the form of either fish oil (FO) or krill oil (KO) balanced for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content, with a control (C) diet containing no EPA and DHA and similar contents of oleic, linoleic, and alpha-linolenic acids, on ectopic fat and inflammation in Zucker rats, a model of obesity and related metabolic dysfunction. Diets were fed for 4 wk. Given the emerging evidence for an association between elevated endocannabinoid concentrations and metabolic syndrome, we also measured tissue endocannabinoid concentrations. In (n-3) LCPUFA-supplemented rats, liver triglycerides and the peritoneal macrophage response to an inflammatory stimulus were significantly lower than in rats fed the control diet, and heart triglycerides were lower, but only in KO-fed rats. These effects were associated with a lower concentration of the endocannabinoids, anandamide and 2-arachidonoylglycerol, in the visceral adipose tissue and of anandamide in the liver and heart, which, in turn, was associated with lower levels of arachidonic acid in membrane phospholipids, but not with higher activity of endocannabinoid-degrading enzymes. Our data suggest that the beneficial effects of a diet enriched with (n-3) LCPUFA are the result of changes in membrane fatty acid composition. The reduction of substrates for inflammatory molecules and endocannabinoids may account for the dampened inflammatory response and the physiological reequilibration of body fat deposition in obese rats.

Changes in conjugated linoleic acid and palmitoleic acid are correlated to retinol levels in chronic renal failure in both hemodialysis and conservative treatment patients.

An increase in conjugated linoleic acid (CLA), a natural fatty acid present in our diet, which possesses anticarcinogenic and antiatherogenic activities in experimental models, has been found in both the plasma and adipose tissue of end-stage chronic renal failure (ESCRF) patients. Increased levels of retinol have also been found in those patients, due to a reduced excretion of the retinol-binding protein. Since retinol is known to influence lipid metabolism, we evaluated whether changes in retinol, CLA, and other fatty acids are correlated in the plasma of CRF patients. We measured CLA, retinol, and unsaturated fatty acids in the plasma of the following groups: (A) 35 ESCRF patients; (B) 20 hemodialysis (HD) patients; (C) 20 healthy controls. Subjects with total cholesterol and/or triglycerides higher than 250 mg/dL were excluded. We found a significant increase in CLA, retinol, palmitoleic (16:1), and oleic (18:1) acids in ESCRF patients. In HD patients we found a similar pattern, however, CLA increase was not significant. No changes were observed in the other fatty acids measured. In the groups of ESCRF and HD patients, a positive correlation between the levels of plasma retinol and CLA, and between retinol and 16:1 was found. These correlations were not detected in controls. The abnormal levels of plasma retinol in CRF patients might partly explain the changes in CLA and 16:1. The influence of retinol levels on these fatty acids might be due to an induction of delta 9 desaturase. In fact, 16:1 is known to be produced, partly, by delta 9 desaturation of palmitic acid. Moreover, the formation of CLA from delta 9 desaturation of vaccenic acid-a trans-monounsaturated fatty acid present in our diet-has recently been demonstrated in humans. Nevertheless, our data do not represent direct evidence supporting an increased delta 9 desaturase activity in CRF patients. Another possible explanation might be a variation in the exogenous intake.