A Comprehensive View of the Epigenetic Landscape Part I: DNA Methylation, Passive and Active DNA Demethylation Pathways and Histone Variants

In multicellular organisms, all the cells are genetically identical but turn genes on or off at the right time to promote differentiation into specific cell types. The regulation of higher-order chromatin structure is essential for genome-wide reprogramming and for tissue-specific patterns of gene expression. The complexity of the genome is regulated by epigenetic mechanisms, which act at the level of DNA, histones, and nucleosomes. Epigenetic machinery is involved in many biological processes, including genomic imprinting, X-chromosome inactivation, heterochromatin formation, and transcriptional regulation, as well as DNA damage repair. In this review, we summarize the recent understanding of DNA methylation, cytosine derivatives, active and passive demethylation pathways as well as histone variants. DNA methylation is one of the well-characterized epigenetic signaling tools. Cytosine methylation of promoter regions usually represses transcription but methylation in the gene body may have a positive correlation with gene expression. The attachment of a methyl group to cytosine residue in the DNA sequence is catalyzed by enzymes of the DNA methyltransferase family. Recent studies have shown that the Ten-Eleven translocation family enzymes are involved in stepwise oxidation of 5-methylcytosine, creating new cytosine derivatives including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Additionally, histone variants into nucleosomes create another strategy to regulate the structure and function of chromatin. The replacement of canonical histones with specialized histone variants regulates accessibility of DNA, and thus may affect multiple biological processes, such as replication, transcription, DNA repair, and play a role in various disorders such as cancer

A comprehensive view of the epigenetic landscape part I : DNA methylation, passive and active DNA demethylation pathways and histone variants

In multicellular organisms, all the cells are genetically identical but turn genes on or off at the right time to promote differentiation into specific cell types. The regulation of higher-order chromatin structure is essential for genome-wide reprogramming and for tissue-specific patterns of gene expression. The complexity of the genome is regulated by epigenetic mechanisms, which act at the level of DNA, histones, and nucleosomes. Epigenetic machinery is involved in many biological processes, including genomic imprinting, X-chromosome inactivation, heterochromatin formation, and transcriptional regulation, as well as DNA damage repair. In this review, we summarize the recent understanding of DNA methylation, cytosine derivatives, active and passive demethylation pathways as well as histone variants. DNA methylation is one of the well-characterized epigenetic signaling tools. Cytosine methylation of promoter regions usually represses transcription but methylation in the gene body may have a positive correlation with gene expression. The attachment of a methyl group to cytosine residue in the DNA sequence is catalyzed by enzymes of the DNA methyltransferase family. Recent studies have shown that the Ten-Eleven translocation family enzymes are involved in stepwise oxidation of 5-methylcytosine, creating new cytosine derivatives including 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Additionally, histone variants into nucleosomes create another strategy to regulate the structure and function of chromatin. The replacement of canonical histones with specialized histone variants regulates accessibility of DNA, and thus may affect multiple biological processes, such as replication, transcription, DNA repair, and play a role in various disorders such as cancer

Analysis of expression of LGALS3BP gene in thyroid tissues and peripheral blood lymphocytes in patients with papillary thyroid cancer

Introduction: Galectin-3 (Gal-3), coding by LGALS3BP gene, is a protein of the lectin family that has been associated with neoplastic processes and seems to play an important role in a variety of cell biological processes. In the thyroid gland, the high expression of this protein has been described in differentiated carcinomas, especially in papillary thyroid cancer (PTC). Aim: Analysis of Gal-3 protein expression in PTC and nodular goiters; investigation of Gal-3 mRNA expression in peripheral blood lymphocytes (PBL) in patients with PTC and nodular goiter; analysis of correlation between Gal-3 protein expression in PTC and Gal-3 mRNA in PBL of the same patient. Material and methods: Gal-3 protein was evaluated by immunohistochemisty in benign (27 multinodular goiters) and malignant (30 papillary carcinomas) thyroid tissues and galectin-3 mRNA expression by real-time PCR in peripheral blood lymphocytes (PBL) from 90 patients with multinodular goiter (n = 27), papillary carcinoma (n = 30) and healthy controls (n = 33). Results: In PTC we observed increased expression of Gal-3 protein (all 30 cases) in cytoplasm, nucleus and cell membranes of cancer cells. 23 of 27 benign thyroid nodular goiters were negative for Gal-3 expression. In all examined blood samples we observed higher LGALS3BP gene expression than GAPDH (house keeping gene) with no difference between both groups, without relation to the Gal-3 expression in PTC. Conclusions: There is no difference in Gal-3 expression in peripheral blood lymphocytes in patients with papillary thyroid cancer in relation to nodular goiter.Wstęp: Choroba nowotworowa wywołuje miejscowe i ogólnoustrojowe zmiany w układzie odpornościowym człowieka. Skomplikowane, trudne do śledzenia mechanizmy mobilizacji komórek układu immunologicznego oraz wytwarzanie swoistych klonów zwalczających komórki z obcym antygenem to jedno z głównych zadań tego układu. Limfocyty T i B odgrywają znaczącą rolę w procesie obrony przeciwnowotworowej. Galektyna-3 (Gal-3), kodowana przez gen LGALS3BP, jest białkiem należącym do grupy lektyn, związanym z procesami nowotworzenia oraz odgrywającym ważną rolę w wielu czynnościach życiowych komórki. Jej zwiększoną ekspresję obserwuje się w wysoko zróżnicowanych rakach tarczycy, a szczególnie w raku brodawkowatym tarczycy (PTC, papillary thyroid cancer). Wykazano, iż Gal-3 występuje w komórkach układu immunologicznego aktywowanych antygenami nowotworu. Jak do tej pory, nie przeprowadzono badań dotyczących analizy ekspresji Gal-3 mRNA w limfocytach krwi obwodowej u pacjentów z PTC. Cel pracy: Analiza ekspresji białka Gal-3 w PTC oraz wolu guzowatym, sprawdzenie, czy w limfocytach krwi obwodowej pobranych od pacjentów z wolem guzowatym i PTC występuje ekspresja mRNA Gal-3, jeżeli tak, czy występuje jakakolwiek zależność pomiędzy ekspresją białka Gal-3 w tarczycy i mRNA Gal-3 w limfocytach krwi obwodowej u tego samego pacjenta. Materiał i metody: Przeprowadzono immunohistochemiczną analizę bloczków parafinowych na obecność białka Gal-3 w 27 zmianach łagodnych o charakterze wola guzkowego obojętnego i 30 zmianach złośliwych (30 PTC). W drugiej części pracy przeprowadzono badanie PCR w czasie rzeczywistym dla mRNA Gal-3 w limfocytach krwi obwodowej u 90 osób: 27 z wolem guzowatym obojętnym, 30 z PTC i 33 bez zmian w zakresie gruczołu tarczowego (grupa kontrolna). Wyniki: We wszystkich 30 próbkach z PTC stwierdzono wysoką lub podwyższoną ekspresję białka Gal-3. W 23 na 27 analizowanych łagodnych zmian tarczycy - wola guzowate - nie wykazano dodatniej reakcji z przeciwciałem anty Gal-3. W drugiej części pracy u 30 pacjentów z PTC i 27 z wolem guzkowym we wszystkich przypadkach obserwowano w limfocytach krwi obwodowej zwiększoną ekspresję genu LGALS3BP w stosunku do genu referencyjnego GAPDH (gen metabolizmu podstawowego). Nie stwierdzono zależności pomiędzy ekspresją genu LGALS3BP na poziomie białka w komórkach PTC a jego ekspresją na poziomie mRNA w limfocytach krwi obwodowej u tych samych pacjentów. Wartości względnej ekspresji genu LGALS3BP w odniesieniu do genu referencyjnego u chorych z rakiem brodawkowatym tarczycy oraz u chorych z wolem guzowatym nie różniły się między sobą w sposób istotny. Wnioski: W limfocytach krwi obwodowej nie ma różnic w ilości mRNA Gal-3 u chorych z PTC i zwykłym wolem

MicroRNAs: Diverse Mechanisms of Action and Their Potential Applications as Cancer Epi-Therapeutics

Usually, miRNAs function post-transcriptionally, by base-pairing with the 3′UTR of target mRNAs, repressing protein synthesis in the cytoplasm. Furthermore, other regions including gene promoters, as well as coding and 5′UTR regions of mRNAs are able to interact with miRNAs. In recent years, miRNAs have emerged as important regulators of both translational and transcriptional programs. The expression of miRNA genes, similar to protein-coding genes, can be epigenetically regulated, in turn miRNA molecules (named epi-miRs) are able to regulate epigenetic enzymatic machinery. The most recent line of evidence indicates that miRNAs can influence physiological processes, such as embryonic development, cell proliferation, differentiation, and apoptosis as well as pathological processes (e.g., tumorigenesis) through epigenetic mechanisms. Some tumor types show repression of tumor-suppressor epi-miRs resulting in cancer progression and metastasis, hence these molecules have become novel therapeutic targets in the last few years. This review provides information about miRNAs involvement in the various levels of transcription and translation regulation, as well as discusses therapeutic potential of tumor-suppressor epi-miRs used in in vitro and in vivo anti-cancer therapy

Genetic polymorphisms and the metabolic effects of olanzapine

Olanzapina to atypowy lek przeciwpsychotyczny (neuroleptyk) stosowany przede wszystkim w leczeniu schizofrenii. Lek metabolizowany jest głównie przez wątrobowe enzymy cytochromu P450, CYP1A2 i CYP2D6. W metabolizm zaangażowane są również enzymy glukuronidowe, UGT1A i UGT2B10. Olanzapina u niektórych pacjentów wywołuje metaboliczne efekty uboczne, takie jak przyrost masy ciała, a także wzrost poziomu: glukozy, lipidów oraz prolaktyny. Badania farmakogenetyczne wskazują, że polimorfizmy w genach kodujących enzymy metabolizujące lek, transportery oraz inne białka zaangażowane w procesy metaboliczne mogą pomóc wyjaśnić zróżnicowaną odpowiedź pacjentów na leczenie olanzapiną. Zatem profilowanie genetyczne mogłoby być przydatne w praktyce klinicznej, głównie w celu wytypowania pacjentów z podwyższonym ryzykiem wystąpienia działań niepożądanych. Możliwość spersonalizowania terapii ułatwiłaby osiągnięcie pożądanego efektu terapeutycznego i zminimalizowała efekty uboczne.Olanzapine is an atypical antipsychotic (neuroleptic) used primarily to treat schizophrenia. The drug is mainly metabolized by the hepatic cytochrome P450 enzymes, CYP1A2 and CYP2D6. The glucuronide enzymes UGT1A and UGT2B10 are also involved in its metabolism. In some patients, olanzapine causes metabolic side effects, such as weight gain, an increase in glucose and lipid levels, and an increase in prolactin levels. Pharmacogenetic studies indicate that polymorphisms in genes encoding drug-metabolizing enzymes, transporters, and other proteins involved in metabolic pathways, may help explain the interindividual differences in response to olanzapine treatment. Thus, genetic profiling could be useful in clinical practice, mainly to identify patients with an increased risk of adverse events. The ability to personalize therapy would facilitate maximizing therapeutic efficacy and minimizing side effects

Identification and In Silico Characterization of a Novel COLGALT2 Gene Variant in a Child with Mucosal Rectal Prolapse

Rectal prolapse is influenced by many factors including connective tissue dysfunction. Currently, there is no data about genetic contribution in the etiology of this disorder. In this study, we performed trio whole-exome sequencing in an 11-year-old girl with mucosal rectal prolapse and her parents and sibling. Genetic testing revealed a novel heterozygous missense variant c.1406G>T; p.G469V in exon 11 of the COLGALT2 gene encoding the GLT25 D2 enzyme. Sanger sequencing confirmed this variant only in the patient while the mother, father and sister showed a wild-type sequence. The pathogenicity of the novel variant was predicted using 10 different in silico tools that classified it as pathogenic. Further, in silico prediction, according to Phyre2, Project HOPE, I-Mutant3.0 and MutPred2 showed that the missense variant can decrease protein stability and cause alterations in the physical properties of amino acids resulting in structural and functional changes of the GLT25D2 protein. In conclusion, the present study identifies a previously unknown missense mutation in the COLGALT2 gene that encodes the enzyme involved in collagen glycosylation. The clinical features observed in the patient and the results of in silico analysis suggest that the new genetic variant can be pathogenic

Identification and In Silico Characterization of a Novel <i>COLGALT2</i> Gene Variant in a Child with Mucosal Rectal Prolapse

Rectal prolapse is influenced by many factors including connective tissue dysfunction. Currently, there is no data about genetic contribution in the etiology of this disorder. In this study, we performed trio whole-exome sequencing in an 11-year-old girl with mucosal rectal prolapse and her parents and sibling. Genetic testing revealed a novel heterozygous missense variant c.1406G>T; p.G469V in exon 11 of the COLGALT2 gene encoding the GLT25 D2 enzyme. Sanger sequencing confirmed this variant only in the patient while the mother, father and sister showed a wild-type sequence. The pathogenicity of the novel variant was predicted using 10 different in silico tools that classified it as pathogenic. Further, in silico prediction, according to Phyre2, Project HOPE, I-Mutant3.0 and MutPred2 showed that the missense variant can decrease protein stability and cause alterations in the physical properties of amino acids resulting in structural and functional changes of the GLT25D2 protein. In conclusion, the present study identifies a previously unknown missense mutation in the COLGALT2 gene that encodes the enzyme involved in collagen glycosylation. The clinical features observed in the patient and the results of in silico analysis suggest that the new genetic variant can be pathogenic

Damage to the Brain Serotonergic System Increases the Expression of Liver Cytochrome P450

Genes coding for cytochrome P450 are regulated by endogenous hormones such as the growth hormone, corticosteroids, thyroid, and sex hormones. Secretion of these hormones is regulated by the respective hypothalamus-pituitary-secretory organ axes. Since the brain sends its serotonergic projections from the raphe nuclei to the hypothalamus, we have assumed that damage to these nuclei may affect the neuroendocrine regulation of cytochrome P450 expression in the liver. Thereby, 5,7-dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, was injected into the dorsal and median raphe nuclei of male Wistar rats. Ten days after the neurotoxin injections, the brain concentrations of neurotransmitters, serum hormone, and cytokine levels, as well as the expression of cytochrome P450 in the liver were measured. Injection of 5,7-DHT decreased serotonin concentration in the brain followed by a significant rise in the levels of the growth hormone, corticosterone, and testosterone, and a drop in triiodothyronine concentration in the serum. No changes in interleukin (IL) levels (IL-2 and IL-6) were observed. Simultaneously, the activity and protein level of liver CYP1A, CYP3A1, and CYP2C11 rose (the activity of CYP2A/2B/2C6/2D was not significantly changed). Similarly, the mRNA levels of CYP1A1, CYP1A2, CYP2C11, and CYP3A1 were elevated. This is the first report demonstrating the effect of intracerebral administration of serotonergic neurotoxin on liver cytochrome P450. The obtained results indicate involvement of the brain serotonergic system in the neuroendocrine regulation of liver cytochrome P450 expression. The physiologic and pharmacological significance of the findings is discussed

The role of brain noradrenergic system in the regulation of liver cytochrome P450 expression

The aim of the present study was to examine the effect of the brain noradrenergic system on the expression of cytochrome P450 in the liver. The experiment was carried out on male Wistar rats. Intracerebroventricular injection of the noradrenergic neurotoxin DSP-4 diminished noradrenaline level in the brain. Simultaneously, significant decreases in the serum concentration of the growth hormone, testosterone and the thyroid hormone thyroxine, as well as an increase in corticosterone level were observed. The concentrations of triiodothyronine and the cytokines interleukine 2 (IL-2) and 6 (IL-6) were not changed by DSP-4. The neurotoxin produced complex changes in the functioning of cytochrome P450. Significant decreases in the activity of liver CYP2C11 (measured as a rate of the 2α- and 16α-hydroxylation of testosterone) and CYP3A (measured as a rate of the 2β- and 6β-hydroxylation of testosterone) were found. In contrast, the activity of CYP1A (measured as a rate of caffeine metabolism) rose, while that of CYP2A (measured as a rate of the 7α-hydroxylation of testosterone), CYP2C6 (measured as a rate of the 7-hydroxylation of warfarin) and CYP2D (the 1'-hydroxylation of bufuralol) remained unchanged. The changes in the activity of CYP1A, CYP2C11 and CYP3A correlated positively with those in CYP protein levels and with the CYP mRNA levels of CYP1A1, CYP2C11 and CYP3A1/2 genes, respectively. The obtained results indicate an important role of the brain noradrenergic system in the neuroendocrine regulation of liver cytochrome P450 expression, which may be of significance in pathological states involving this system, or during pharmacotherapy with drugs affecting noradrenergic transmission