scholarly journals Methylated cis-regulatory elements mediate KLF4-dependent gene transactivation and cell migration

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jun Wan ◽  
Yijing Su ◽  
Qifeng Song ◽  
Brian Tung ◽  
Olutobi Oyinlade ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromatin Remodeling ◽  
Molecular Mechanisms ◽  
Biological Function ◽  
Morphological Changes ◽  
Methylation Status ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Binding Activity ◽  
New Paradigm

Altered DNA methylation status is associated with human diseases and cancer; however, the underlying molecular mechanisms remain elusive. We previously identified many human transcription factors, including Krüppel-like factor 4 (KLF4), as sequence-specific DNA methylation readers that preferentially recognize methylated CpG (mCpG), here we report the biological function of mCpG-dependent gene regulation by KLF4 in glioblastoma cells. We show that KLF4 promotes cell adhesion, migration, and morphological changes, all of which are abolished by R458A mutation. Surprisingly, 116 genes are directly activated via mCpG-dependent KLF4 binding activity. In-depth mechanistic studies reveal that recruitment of KLF4 to the methylated cis-regulatory elements of these genes result in chromatin remodeling and transcription activation. Our study demonstrates a new paradigm of DNA methylation-mediated gene activation and chromatin remodeling, and provides a general framework to dissect the biological functions of DNA methylation readers and effectors.

scholarly journals Plenary Lecture 2 Transcription factors, regulatory elements and nutrient–gene communication

2009 ◽  
Vol 69 (1) ◽  
pp. 91-94 ◽  
Author(s):  
Robert J. Cousins ◽  
Tolunay B. Aydemir ◽  
Louis A. Lichten
Keyword(s):  
Transcription Factors ◽  
Histone Acetylation ◽  
Intracellular Transport ◽  
Molecular Mechanisms ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Atp Citrate Lyase ◽  
Signalling Molecules ◽  
Citrate Lyase ◽  
Normal Metabolism

Dramatic advances have been made in the understanding of the differing molecular mechanisms used by nutrients to regulate genes that are essential for their biological roles to carry out normal metabolism. Classical studies have focused on nutrients as ligands to activate specific transcription factors. New interest has focused on histone acetylation as a process for either global or limited gene activation and is the first mechanism to be discussed. Nuclear ATP-citrate lyase generates acetyl-CoA, which has been shown to have a role in the activation of specific genes via selective histone acetylation. Transcription factor acetylation may provide a second mode of control of nutrient-responsive gene transcription. The third mechanism relates to the availability of response elements within chromatin, which as well as the location of the elements in the gene may allow or prevent transcription. A fourth mechanism involves intracellular transport of Zn ions, which can orchestrate localized enzyme inhibition–activation. This process in turn influences signalling molecules that regulate gene expression. The examples provided in the present review point to a new level of complexity in understanding nutrient–gene communication.

scholarly journals Transcriptome and DNA Methylation Analyses of the Molecular Mechanisms Underlying with Longissimus dorsi Muscles at Different Stages of Development in the Polled Yak

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 970 ◽  
Author(s):  
Ma ◽  
Jia ◽  
Chu ◽  
Fu ◽  
Lei ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
Expression Patterns ◽  
Methylation Status ◽  
Muscle Growth ◽  
Class Ii ◽  
Epigenetic Mechanism ◽  
Class I ◽  
Longissimus Dorsi ◽  
Growth Stages

DNA methylation modifications are implicated in many biological processes. As the most common epigenetic mechanism DNA methylation also affects muscle growth and development. The majority of previous studies have focused on different varieties of yak, but little is known about the epigenetic regulation mechanisms in different age groups of animals. The development of muscles in the different stages of yak growth remains unclear. In this study, we selected the longissimus dorsi muscle tissue at three different growth stages of the yak, namely, 90-day-old fetuses (group E), six months old (group M), and three years old (group A). Using RNA-Seq transcriptome sequencing and methyl-RAD whole-genome methylation sequencing technology, changes in gene expression levels and DNA methylation status throughout the genome were investigated during the stages of yak development. Each group was represented by three biological replicates. The intersections of expression patterns of 7694 differentially expressed genes (DEGs) were identified (padj < 0.01, |log2FC| > 1.2) at each of the three developmental periods. Time-series expression profile clustering analysis indicated that the DEGs were significantly arranged into eight clusters which could be divided into two classes (padj < 0.05), class I profiles that were downregulated and class II profiles that were upregulated. Based on this cluster analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEGs from class I profiles were significantly (padj < 0.05) enriched in 21 pathways, the most enriched pathway being the Axon guidance signaling pathway. DEGs from the class II profile were significantly enriched in 58 pathways, the pathway most strongly enriched being Metabolic pathway. After establishing the methylation profiles of the whole genomes, and using two groups of comparisons, the three combinations of groups (M-vs.-E, M-vs.-A, A-vs.-E) were found to have 1344, 822, and 420 genes, respectively, that were differentially methylated at CCGG sites and 2282, 3056, and 537 genes, respectively, at CCWGG sites. The two sets of data were integrated and the negative correlations between DEGs and differentially methylated promoters (DMPs) analyzed, which confirmed that TMEM8C, IGF2, CACNA1S and MUSTN1 were methylated in the promoter region and that expression of the modified genes was negatively correlated. Interestingly, these four genes, from what was mentioned above, perform vital roles in yak muscle growth and represent a reference for future genomic and epigenomic studies in muscle development, in addition to enabling marker-assisted selection of growth traits.

Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites

Blood ◽  
2003 ◽  
Vol 101 (12) ◽  
pp. 4894-4902 ◽  
Author(s):  
Stanislas Goriely ◽  
Dominique Demonté ◽  
Séverine Nizet ◽  
Dominique De Wit ◽  
Fabienne Willems ◽  
...  
Keyword(s):  
Cell Line ◽  
Chromatin Remodeling ◽  
Binding Sites ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Micrococcal Nuclease ◽  
Interleukin 12 ◽  
P35 Gene ◽  
Free Region ◽  
Ifn Γ

AbstractTo get insight into the regulation of human interleukin-12 (IL-12) synthesis, we determined the chromatin organization of the IL-12(p35) promoter region. First, we determined positioning of nucleosomes within the IL-12(p35) promoter using the indirect end-labeling technique in the THP-1 monocytic cell line. On stimulation with bacterial lipopolysaccharide (LPS) and interferon-γ (IFN-γ), hypersensitivity to digestion with DNase I, micrococcal nuclease, and specific restriction enzymes was detected in the region encompassing nucleotide (nt) –310 to –160, indicating selective inducible chromatin remodeling involving disruption of a single nucleosome (named nuc-2). Using p35 promoter deletion mutants and reporter gene assays, we demonstrated that the –396/–241 region contained critical cis-acting elements. Within this latter region, we characterized physically and functionally 2 Sp1-binding sites, which were acting as key regulatory elements for both basal and LPS/IFN-γ–inducible p35 gene expression: Sp1#1 lies within the remodeled nuc-2 region and Sp1#2 is located in the nucleosome-free region immediately upstream of nuc-2. Finally, we extended the chromatin structure analysis to dendritic cells (DCs) derived from human monocytes and observed the same nucleosomal organization and remodeling as in the THP-1 cell line. Moreover, we found that in DCs, LPS and IFN-γ synergized in the induction of nucleosomal remodeling and that chromatin remodeling at the p35 locus immediately preceded IL-12(p35) mRNA synthesis. Taken together, our results demonstrate that IL-12(p35) gene activation in the course of DC maturation involves selective and rapid remodeling of a single positioned nucleosome within a region of the promoter containing critical Sp1-binding sites.

scholarly journals Genome wide efficiency profiling reveals modulation of maintenance and de novo methylation by Tets

2020 ◽  
Author(s):  
Pascal Giehr ◽  
Charalampos Kyriakopoulos ◽  
Karl Nordström ◽  
Abduhlrahman Salhab ◽  
Fabian Müller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Epigenetic Modification ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Sequencing Data ◽  
Reduced Representation ◽  
Genome Wide ◽  
Global And Local

AbstractBackgroundDNA methylation is an essential epigenetic modification which is set and maintained by DNA methyl transferases (Dnmts) and removed via active and passive mechanisms involving Tet mediated oxidation. While the molecular mechanisms of these enzymes are well studied, their interplay on shaping cell specific methylomes remains less well understood. In our work we model the activities of Tets and Dnmts at single CpGs across the genome using a novel type of high resolution sequencing data.ResultsTo accurately measure 5mC and 5hmC levels at single CpGs we developed RRHPoxBS, a reduced representation hairpin oxidative bisulfite sequencing approach. Using this method we mapped the methylomes and hydroxymethylomes of wild type and Tet triple knockout mouse embryonic stem cells. These comprehensive datasets were then used to develop an extended Hidden Markov model allowing us i) to determine the symmetrical methylation and hydroxymethylation state at millions of individual CpGs, ii) infer the maintenance and de novo methylation efficiencies of Dnmts and the hydroxylation efficiencies of Tets at individual CpG positions. We find that Tets exhibit their highest activity around unmethylated regulatory elements, i.e. active promoters and enhancers. Furthermore, we find that Tets’ presence has a profound effect on the global and local maintenance and de novo methylation activities by the Dnmts, not only substantially contributing to a universal demethylation of the genome but also shaping the overall methylation landscape.ConclusionsOur analysis demonstrates that a fine tuned and locally controlled interplay between Tets and Dnmts is important to modulate de novo and maintenance activities of Dnmts across the genome. Tet activities contribute to DNA methylation patterning in the following ways: They oxidize 5mC, they locally shield DNA from accidental de novo methylation and at the same time modulate maintenance and de novo methylation efficiencies of Dnmts across the genome.

scholarly journals Chromosomal Elements Regulate Gene Activity and Chromatin Structure of the Human Serpin Gene Cluster at 14q32.1

2003 ◽  
Vol 23 (10) ◽  
pp. 3516-3526 ◽  
Author(s):  
Mark D. Marsden ◽  
R. E. K. Fournier
Keyword(s):  
Gene Cluster ◽  
Chromatin Remodeling ◽  
Gene Activation ◽  
Serine Protease Inhibitor ◽  
Regulatory Elements ◽  
Human Chromosomes ◽  
Functional Tests ◽  
Serpin Gene ◽  
Hepatic Cells ◽  
Number Of Genes

ABSTRACT The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human α1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb −8.1 and −5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.

Contributions of 5’HoxA/D regulation to actinodin evolution and the fin-to-limb transition

2018 ◽  
Vol 62 (11-12) ◽  
pp. 705-716 ◽  
Author(s):  
Robert L. Lalonde ◽  
Marie-Andrée Akimenko
Keyword(s):  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Regulatory Elements ◽  
Endochondral Bone ◽  
Cis Acting ◽  
Advantages And Disadvantages ◽  
Regulatory Strategies ◽  
Deep Homology ◽  
Fish Fins

The evolution of tetrapod limbs from paired fish fins comprised major changes to the appendicular dermal and endochondral skeleton. Fish fin rays were lost, and the endochondral bone was modified and elaborated to form three distinct segments common to all tetrapod limbs: the stylopod, the zeugopod and the autopod. Identifying the molecular mechanisms that contributed to these morphological changes presents a unique insight into our own evolutionary history. This review first summarizes previously identified cis-acting regulatory elements for the 5’HoxA/D genes and actinodin1 that were tested using transgenic swap experiments between fish and tetrapods. Conserved regulatory networks provide evidence for a deep homology between distal fin structures and the autopod, while diverging regulatory strategies highlight potential molecular mechanisms that contributed to the fin-to-limb transition. Next, we summarize studies that performed functional analysis to recapitulate fish-tetrapod diverging regulatory strategies and then discuss their potential morphological consequences during limb evolution. Finally, we also discuss here some of the advantages and disadvantages of using zebrafish to study molecular and morphological changes during the fin-to-limb transition.

scholarly journals SUN-090 Investigation of Imprinting Defects in MKRN3 and DLK1 in Children with Idiopathic Central Precocious Puberty Through Specific DNA Methylation Analysis

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Ana Pinheiro-Machado Canton ◽  
Virginie Steunou ◽  
Marie-Laure Sobrier ◽  
Luciana Ribeiro Montenegro ◽  
Danielle de Souza Bessa ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Precocious Puberty ◽  
Molecular Mechanisms ◽  
Methylation Status ◽  
Uniparental Disomy ◽  
Central Precocious Puberty ◽  
Sequencing Analysis ◽  
Puberty Onset ◽  
Temple Syndrome ◽  
Age At Puberty

Abstract Background: Loss of imprinting has been implicated in the pathogenesis of several human diseases. Monogenic causes of central precocious puberty (CPP) were identified in families with loss-of-function mutations affecting mainly the coding region of two paternally expressed imprinted genes: Makorin ring finger 3 (MKRN3) and Delta-like 1 homolog (DLK1). The role of imprinting defects involving these two genes in CPP has not been described so far. Objective: To investigate the methylation status at primary differentially methylated regions (DMR) of MKRN3 and DLK1 in a cohort of children with idiopathic CPP. Patients and methods: One-hundred and twenty CPP patients (112 sporadic, 8 familial; 115 females, 5 males) were selected for analysis. Leukocyte DNA was obtained from all patients. MKRN3 and DLK1 pathogenic allelic variants were first excluded by DNA sequencing analysis. Bisulfite treatment followed by Allele-Specific Methylated Multiplex Real-Time Quantitative PCR was performed with leukocyte DNA, analyzing separately the methylation index (MI) of MKRN3:TSS-DMR and DLK1/MEG3:IG-DMR for each patient. The MI results were compared with controls with normal pubertal development. Results: Mean age at puberty onset was 5.8 ±1.9yr for girls and 7.2 ±2.6yr for boys. Hypomethylation at DLK1/MEG3:IG-DMR was identified in 3 patients (I, II and III) with sporadic CPP: MI 10%, 16% and 11%, respectively. Interestingly, cases II and III were both girls who had been firstly referred to pediatric endocrinology for presenting precocious menarche; while case I was a boy who had been referred for presenting mild growth retardation, and developed CPP during monitoring. In addition, during follow-up, other clinical findings were noticed: being born small for gestational age, prominent forehead, small hands/feet, overweight/obesity and early onset type 2 diabetes in case III. Additional genetic investigation included SNP array in cases I and II, identifying a maternal uniparental disomy at chromosome 14 (upd(14)mat). Meanwhile, case III had normal genomic microarray and microsatellites analysis, excluding copy number variants and upd(14)mat, and indicating a mechanism of epimutation at DLK1/MEG3:IG-DMR. Uniparental disomy and epimutation are molecular mechanisms associated with the imprinting disorder known as Temple syndrome. In the remaining cases, mean MI for DLK1/MEG3:IG-DMR was 49±2%. In all cases, mean MI for MKRN3:TSS-DMR was 49±6%. There were no significant correlations between age at puberty onset and MI for MKRN3 (p=0.69) and DLK1(p=0.45). Conclusion: There was no leukocyte DNA methylation defect at MKRN3 imprinting control region in the idiopathic CPP cohort. DLK1/MEG3:IG-DMR hypomethylation was identified in 3 patients with CPP and additional findings of Temple syndrome, indicating that loss of effective imprinting of DLK1 locus is a mechanism leading to CPP.

Prorenin and gene activation

1991 ◽  
Vol 69 (9) ◽  
pp. 1367-1374 ◽  
Author(s):  
Brian J. Morris ◽  
D. Lynne Smith
Keyword(s):  
Transient Expression ◽  
Hela Cells ◽  
Regulatory Element ◽  
Gene Activation ◽  
Regulatory Elements ◽  
Positive Control ◽  
Binding Activity ◽  
Transient Expression Assay ◽  
Nuclear Extracts

After the discovery of an inactive, putative renin precursor that could be proteolytically activated, and the proteases involved in vivo, Morris and co-workers directly demonstrated that renin is indeed synthesized as a "pro" form, and from genetic coding sequences they provided the structure of human prorenin. The gene is inactive and must be activated in prorenin-synthesizing tissues. To study the mechanism involved, we have performed transient expression analyses of putative regulatory DNA of the human gene (REN). 5′-Flanking DNA, extending from residue −144 to −2400, was linked to a reporter gene, viz. that for chloramphenicol acetyl transferase (CAT), and its ability to drive a heterologous (thymidine kinase, tk) promoter was examined by transfecting plasmid constructs into ceils in culture and measuring CAT activity 48 h later. Because suitable renin-synthesizing cells were not available, choriocarcinoma (JEG-3) and cervical carcinoma (HeLa) cells were used. Although this DNA caused a reduction in CAT activity relative to the positive control, examination of a range of subfragments suggested that the −2400 to −144 region did not contain negative regulatory elements. In contrast, all fragments containing the −149 to +13 DNA segment gave CAT activities that were lower than the promoterless control. Together, the data were consistent with the presence of negative regulatory element(s) in that fragment of DNA that contained the REN promoter. On the basis of mouse gene studies, it has been suggested that the inactivity of the renin gene in tissues that do not synthesize prorenin is not due to repression, but rather, cells that do express the gene may possess unique trans-acting factor(s) that stimulate enhancer(s) in the renin DNA, therefore activating the renin promoter. Nuclear extracts of JEG-3, but not HeLa, cells contained a binding activity for the −340 to −192 segment, but the relationship, if any, of this to the model proposed is unclear. Thus, having demonstrated the synthesis, structure, and activation of prorenin, the mechanism of activation of the gene represents the next challenge.Key words: gene regulation, transient expression assay, JEG-3 ceils, HeLa cells, prorenin.

scholarly journals Reciprocal Nuclear Shuttling of Two Antagonizing Zn Finger Proteins Modulates Tup Family Corepressor Function To Repress Chromatin Remodeling

2006 ◽  
Vol 5 (12) ◽  
pp. 1980-1989 ◽  
Author(s):  
Kouji Hirota ◽  
Charles S. Hoffman ◽  
Kunihiro Ohta
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Repression ◽  
Molecular Mechanisms ◽  
Glucose Repression ◽  
Regulatory Elements ◽  
Initial Stage ◽  
Extracellular Glucose Concentration ◽  
Extracellular Glucose ◽  
Nuclear Shuttling

ABSTRACT The Schizosaccharomyces pombe global corepressors Tup11 and Tup12, which are orthologs of Saccharomyces cerevisiae Tup1, are involved in glucose-dependent transcriptional repression and chromatin alteration of the fbp1 + gene. The fbp1 + promoter contains two regulatory elements, UAS1 and UAS2, one of which (UAS2) serves as a binding site for two antagonizing C2H2 Zn finger transcription factors, the Rst2 activator and the Scr1 repressor. In this study, we analyzed the role of Tup proteins and Scr1 in chromatin remodeling at fbp1 + during glucose repression. We found that Scr1, cooperating with Tup11 and Tup12, functions to maintain the chromatin of the fbp1 + promoter in a transcriptionally inactive state under glucose-rich conditions. Consistent with this notion, Scr1 is quickly exported from the nucleus to the cytoplasm at the initial stage of derepression, immediately after glucose starvation, at which time Rst2 is known to be imported into the nucleus. In addition, chromatin immunoprecipitation assays revealed a switching of Scr1 to Rst2 bound at UAS2 during glucose derepression. On the other hand, Tup11 and Tup12 persist in the nucleus and bind to the fbp1 + promoter under both derepressed and repressed conditions. These observations suggest that Tup1-like proteins recruited to the fbp1 + promoter are controlled by either of two antagonizing C2H2 Zn finger proteins. We propose that the actions of Tup11 and Tup12 are regulated by reciprocal nuclear shuttling of the two antagonizing Zn finger proteins in response to the extracellular glucose concentration. This notion provides new insights into the molecular mechanisms of the Tup family corepressors in gene regulation.

scholarly journals Tet-Mediated DNA Demethylation Is Required for SWI/SNF-Dependent Chromatin Remodeling and Histone-Modifying Activities That Trigger Expression of the Sp7 Osteoblast Master Gene during Mesenchymal Lineage Commitment

2017 ◽  
Vol 37 (20) ◽  
Author(s):  
Hugo Sepulveda ◽  
Alejandro Villagra ◽  
Martin Montecino
Keyword(s):  
Dna Methylation ◽  
Histone Modification ◽  
Chromatin Remodeling ◽  
Mammalian Cells ◽  
Gene Activation ◽  
Dna Demethylation ◽  
Histone Demethylase ◽  
Lineage Commitment ◽  
Histone Deacetylase 1 ◽  
Leading Role

ABSTRACT Here we assess histone modification, chromatin remodeling, and DNA methylation processes that coordinately control the expression of the bone master transcription factor Sp7 (osterix) during mesenchymal lineage commitment in mammalian cells. We find that Sp7 gene silencing is mediated by DNA methyltransferase1/3 (DNMT1/3)-, histone deacetylase 1/2/4 (HDAC1/2/4)-, Setdb1/Suv39h1-, and Ezh1/2-containing complexes. In contrast, Sp7 gene activation involves changes in histone modifications, accompanied by decreased nucleosome enrichment and DNA demethylation mediated by SWI/SNF- and Tet1/Tet2-containing complexes, respectively. Inhibition of DNA methylation triggers changes in the histone modification profile and chromatin-remodeling events leading to Sp7 gene expression. Tet1/Tet2 silencing prevents Sp7 expression during osteoblast differentiation as it impairs DNA demethylation and alters the recruitment of histone methylase (COMPASS)-, histone demethylase (Jmjd2a/Jmjd3)-, and SWI/SNF-containing complexes to the Sp7 promoter. The dissection of these interconnected epigenetic mechanisms that govern Sp7 gene activation reveals a hierarchical process where regulatory components mediating DNA demethylation play a leading role.

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