scholarly journals Epigenetic Reprogramming of OCT4 and NANOG Regulatory Regions by Embryonal Carcinoma Cell Extract

2007 ◽  
Vol 18 (5) ◽  
pp. 1543-1553 ◽  
Author(s):  
Christel T. Freberg ◽  
John Arne Dahl ◽  
Sanna Timoskainen ◽  
Philippe Collas
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
Cell Extract ◽  
Epigenetic Reprogramming ◽  
Proximal Promoter ◽  
Embryonal Carcinoma Cell ◽  
Distal Enhancer ◽  
Regulatory Regions ◽  
Molecular Events

Analyses of molecular events associated with reprogramming somatic nuclei to pluripotency are scarce. We previously reported the reprogramming of epithelial cells by extract of undifferentiated embryonal carcinoma (EC) cells. We now demonstrate reprogramming of DNA methylation and histone modifications on regulatory regions of the developmentally regulated OCT4 and NANOG genes by exposure of 293T cells to EC cell extract. OCT4 and NANOG are transcriptionally up-regulated and undergo mosaic cytosine-phosphate-guanosine demethylation. OCT4 demethylation occurs as early as week 1, is enhanced by week 2, and is most prominent in the proximal promoter and distal enhancer. Targeted OCT4 and NANOG demethylation does not occur in 293T extract-treated cells. Retinoic acid-mediated differentiation of reprogrammed cells elicits OCT4 promoter remethylation and transcriptional repression. Chromatin immunoprecipitation analyses of lysines K4, K9, and K27 of histone H3 on OCT4 and NANOG indicate that primary chromatin remodeling determinants are acetylation of H3K9 and demethylation of dimethylated H3K9. H3K4 remains di- and trimethylated. Demethylation of trimethylated H3K9 and H3K27 also occurs; however, trimethylation seems more stable than dimethylation. We conclude that a central epigenetic reprogramming event is relaxation of chromatin at loci associated with pluripotency to create a conformation compatible with transcriptional activation.

221 EPIGENETIC DYNAMICS OF PLURIPOTENCY GENES IN THE CONTEXT OF DIFFERENTIATION AND NUCLEAR REPROGRAMMING DETERMINED BY Q2ChIP, A QUICK AND QUANTITATIVE CHROMATIN IMMUNOPRECIPITATION TECHNIQUE APPLICABLE TO SMALL CELL SAMPLES

2007 ◽  
Vol 19 (1) ◽  
pp. 227 ◽  
Author(s):  
J. A. Dahl ◽  
C. K. Taranger ◽  
P. Collas
Keyword(s):  
Gene Expression ◽  
Chromatin Immunoprecipitation ◽  
Regulation Of Gene Expression ◽  
Cell Extract ◽  
Proximal Promoter ◽  
Pcr Analysis ◽  
Cross Linking ◽  
Nuclear Reprogramming ◽  
Distal Enhancer ◽  
Developmentally Regulated

Interactions between proteins and DNA are essential for cellular functions such as genomic stability, DNA replication and repair, chromosome segregation, transcription, and epigenetic silencing of gene expression. Chromatin immunoprecipitation (ChIP) is a key technique for mapping histone modifications and transcription factor binding on DNA and thereby unraveling the role of epigenetics in the regulation of gene expression. Current ChIP protocols require extensive sample handling and large numbers of cells (5-10 million). primarily owing to ample loss of material during the procedure. We altered critical steps of conventional ChIP to develop a quick and quantitative (Q2) ChIP assay suitable for cell numbers 100- to 1000-fold lower than those required for conventional ChIP. Key modifications of the ChIP procedure include (i) formaldehyde DNA–protein cross-linking in suspended cells, (ii) cross-linking in the presence of 20 mM sodium butyrate to enhance specificity of precipitation of acetylated histones, (iii) transfer of washed precipitated immune complexes to a clean tube ('tube shift') to increase ChIP specificity by virtually eliminating nonspecifically bound chromatin, and (iv) combination of cross-link reversal, protein digestion, and DNA elution into a single 2-h step. We used Q2ChIP to monitor changes in 6 histone H3 modifications on the human developmentally regulated genes OCT4 (POU5F1), NANOG, and LMNA (lamin A) in the context of retinoic acid (RA)-mediated differentiation of embryonal carcinoma cells and upon reprogramming of kidney epithelial 293T cells to pluripotency in carcinoma cell extract (Taranger et al. 2005 Mol. Biol. Cell 16, 5719–5735). Real-time PCR analysis of precipitated DNA unravels an unexpected two-step heterochromatin assembly elicited by RA on the OCT4 proximal promoter, proximal enhancer, and distal enhancer, and on the NANOG promoter, whereby methylation of H3K9 and H3K27 is followed by H3K9 deacetylation. H3K4 di- and trimethylation remain relatively unaffected by RA treatment. In contrast, reprogramming of 293T cells in carcinoma extract promotes assembly of histone marks characteristic of transcriptional induction of OCT4 and NANOG, such as acetylation and demethylation of H3K9. The results argue toward ordered chromatin repackaging at developmentally regulated promoters upon differentiation or, conversely, nuclear reprogramming to pluripotency.

scholarly journals Translation of Maternal TATA-Binding Protein mRNA Potentiates Basal but Not Activated Transcription in Xenopus Embryos at the Midblastula Transition

1999 ◽  
Vol 19 (12) ◽  
pp. 7972-7982 ◽  
Author(s):  
Gert Jan C. Veenstra ◽  
Olivier H. J. Destrée ◽  
Alan P. Wolffe
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Translational Regulation ◽  
Binding Protein ◽  
Cell Extract ◽  
Tata Binding Protein ◽  
Nucleosome Assembly ◽  
Midblastula Transition ◽  
Egg Extract

ABSTRACT Early embryonic development in Xenopus laevis is characterized by transcriptional repression which is relieved at the midblastula stage (MBT). Here we show that the relative abundance of TATA-binding protein (TBP) increases robustly at the MBT and that the mechanism underlying this increase is translation of maternally stored TBP RNA. We show that TBP is rate-limiting in egg extract under conditions that titrate nucleosome assembly. Precocious translation of TBP mRNA in Xenopus embryos facilitates transcription before the MBT, without requiring TBP to be prebound to the promoter before injection. This effect is transient in the absence of chromatin titration and is sustained when chromatin is titrated. These data show that translational regulation of TBP RNA contributes to limitations on the transcriptional capacity before the MBT. Second, we examined the ability of trans-acting factors to contribute to promoter activity before the MBT. Deletion of cis-acting elements does not affect histone H2B transcription in egg extract, a finding indicative of limited trans-activation. Moreover, in the context of the intact promoter, neither the transcriptional activator Oct-1, nor TBP, nor TFIID enable transcriptional activation in vitro. HeLa cell extract, however, reconstitutes activated transcription in mixed extracts. These data suggest a deficiency in egg extract cofactors required for activated transcription. We show that the capacity for activated H2B transcription is gradually acquired at the early gastrula transition. This transition occurs well after the blastula stage when the basal transcription machinery can first be complemented with TBP.

scholarly journals Identification of a novel distal enhancer in human adiponectin gene

2008 ◽  
Vol 200 (1) ◽  
pp. 107-116 ◽  
Author(s):  
Katsumori Segawa ◽  
Morihiro Matsuda ◽  
Atsunori Fukuhara ◽  
Kentaro Morita ◽  
Yosuke Okuno ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Promoter Region ◽  
Luciferase Activity ◽  
Proximal Region ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Adiponectin Gene ◽  
Distal Enhancer

Adiponectin is exclusively expressed in adipose tissue and secreted from adipocytes, and shows anti-diabetic and anti-atherogenic properties. However, the precise transcriptional mechanism of adiponectin remains elusive. In this study, the 5′ flanking promoter region of human adiponectin gene was analyzed using UCSC genome browser, and a 10 390-bp fragment, containing an evolutionally conserved region among species, was investigated. The luciferase reporter assay using this fragment identified a novel distal enhancer of human adiponectin gene. Promoter constructs with the distal enhancer exhibited high promoter activities in 3T3-L1 mature adipocytes. However, no such activity was observed in other types of cell lines. The distal enhancer is highly conserved, and contains two completely conserved CCAAT boxes. In 3T3-L1 mature adipocytes, deletion or each point mutation of these CCAAT boxes markedly reduced luciferase activity driven by adiponectin promoter. Knockdown of CCAAT/enhancer-binding protein α (CEBPA; also known as C/EBPα) using small interfering RNA diminished adiponectin mRNA expression and luciferase activity driven by adiponectin promoter with the distal enhancer. However, adiponectin promoter with each mutation of two CCAAT boxes in the distal enhancer did not respond to knockdown of CEBPA expression. Furthermore, CEBPA bound to the distal enhancer both in vitro and in vivo. We also identified a proximal promoter region responsible for transcriptional activation by the distal enhancer in human adiponectin gene. Our results indicate that CEBPA plays a pivotal role in the transcription of human adiponectin gene via the distal enhancer and proximal region in its promoter.

scholarly journals MYC-driven epigenetic reprogramming favors the onset of tumorigensis by inducing a stem cell-like state

2017 ◽  
Author(s):  
Vittoria Poli ◽  
Luca Fagnocchi ◽  
Alessandra Fasciani ◽  
Alessandro Cherubini ◽  
Stefania Mazzoleni ◽  
...  
Keyword(s):  
Stem Cell ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Epigenetic Reprogramming ◽  
Breast Cancers ◽  
Cell Of Origin ◽  
Tumor Initiating Cells ◽  
Oncogenic Pathways

AbstractBreast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes resulted difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Over-expression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.

X chromosome reactivation in mouse embryonal carcinoma cells

1985 ◽  
Vol 5 (10) ◽  
pp. 2705-2712
Author(s):  
G D Paterno ◽  
C N Adra ◽  
M W McBurney
Keyword(s):  
X Chromosome ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
S Phase ◽  
Dna Demethylation ◽  
Embryonal Carcinoma Cell ◽  
X Chromosomes ◽  
Embryonal Carcinoma Cell Line ◽  
Inactive X Chromosome

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

scholarly journals X chromosome reactivation in mouse embryonal carcinoma cells.

1985 ◽  
Vol 5 (10) ◽  
pp. 2705-2712 ◽  
Author(s):  
G D Paterno ◽  
C N Adra ◽  
M W McBurney
Keyword(s):  
X Chromosome ◽  
Dna Sequences ◽  
Transcriptional Repression ◽  
Embryonal Carcinoma ◽  
S Phase ◽  
Dna Demethylation ◽  
Embryonal Carcinoma Cell ◽  
X Chromosomes ◽  
Embryonal Carcinoma Cell Line ◽  
Inactive X Chromosome

The embryonal carcinoma cell line, C86S1, carries two X chromosomes, one of which replicates late during S phase of the cell cycle and appears to be genetically inactive. C86S1A1 is a mutant which lacks activity of the X-encoded enzyme, hypoxanthine phosphoribosyltransferase (HPRT). Treatment of C86S1A1 cells with DNA-demethylating agents, such as 5-azacytidine (5AC), resulted in (i) the transient expression in almost all cells of elevated levels of HPRT and three other enzymes encoded by X-linked genes and (ii) the stable expression of HPRT in up to 5 to 20% of surviving cells. Most cells which stably expressed HPRT had two X chromosomes which replicated in early S phase. C86S1A1 cells which had lost the inactive X chromosome did not respond to 5AC. These results suggest that DNA demethylation results in the reactivation of genes on the inactive X chromosome and perhaps in the reactivation of the entire X chromosome. No such reactivation occurred in C86S1A1 cells when the cells were differentiated before exposure to 5AC. Thus, the process of X chromosome inactivation may be a sequential one involving, as a first step, methylation of certain DNA sequences and, as a second step, some other mechanism(s) of transcriptional repression.

MafA, NeuroD1, and HNF1β synergistically activate Slc2a2 (Glut2) gene in β-cells

2021 ◽  
Author(s):  
Yuka Ono ◽  
Kohsuke Kataoka
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Glucose Transporter ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Distal Enhancer ◽  
Β Cells ◽  
Β Cell ◽  
Target Sites

Glucose transporter type 2 (GLUT2), encoded by the SLC2A2 gene, is an essential component of glucose-stimulated insulin secretion in pancreatic islet β-cells. Like that of the gene encoding insulin, expression of the SLC2A2 gene expression is closely linked to β-cell functionality in rodents, but the mechanism by which β-cell-specific expression of SLC2A2 is controlled remains unclear. In this report, to identify putative enhancer elements of the mouse Slc2a2 gene, we examined evolutional conservation of the nucleotide sequence of its genomic locus, together with ChIP-seq data of histone modifications and various transcription factors published in previous studies. Using luciferase reporter assays, we found that an evolutionarily conserved region located approximately 40 kbp downstream of the transcription start site of Slc2a2 functions as an active enhancer in the MIN6 β-cell line. We also found that three β-cell-enriched transcription factors, MafA, NeuroD1, and HNF1β, synergistically activate transcription through this 3’ downstream distal enhancer (ECR3’) and the proximal promoter region of the gene. Our data also indicate that the simultaneous binding of HNF1β to its target sites within the promoter and ECR3’ of Slc2a2 is indispensable for transcriptional activation, and that binding of MafA and NeuroD1 to their respective target sites within the ECR3’ enhances transcription. Co-immunoprecipitation experiments suggested that MafA, NeuroD1, and HNF1β interact with each other. Overall, these results suggest that promoter-enhancer communication through MafA, NeuroD1, and HNF1β is critical for Slc2a2 gene expression. These findings provide clues to help elucidate the mechanism of regulation of Slc2a2 gene expression in β-cells.

scholarly journals Dynamic histone acetylation in floral volatile synthesis and emission in petunia flowers

2021 ◽  
Author(s):  
Ryan M Patrick ◽  
Xing-Qi Huang ◽  
Natalia Dudareva ◽  
Ying Li
Keyword(s):  
Transcriptional Activation ◽  
Metabolic Pathways ◽  
Transcriptional Repression ◽  
Metabolic Networks ◽  
Underlying Mechanism ◽  
Expression Of Genes ◽  
Genome Wide ◽  
Chemical Inhibitors ◽  
Volatile Organic ◽  
Floral Volatile

Abstract Biosynthesis of secondary metabolites relies on primary metabolic pathways to provide precursors, energy, and cofactors, thus requiring coordinated regulation of primary and secondary metabolic networks. However, to date, it remains largely unknown how this coordination is achieved. Using Petunia hybrida flowers, which emit high levels of phenylpropanoid/benzenoid volatile organic compounds (VOCs), we uncovered genome-wide dynamic deposition of histone H3 lysine 9 acetylation (H3K9ac) during anthesis as an underlying mechanism to coordinate primary and secondary metabolic networks. The observed epigenome reprogramming is accompanied by transcriptional activation at gene loci involved in primary metabolic pathways that provide precursor phenylalanine, as well as secondary metabolic pathways to produce volatile compounds. We also observed transcriptional repression among genes involved in alternative phenylpropanoid branches that compete for metabolic precursors. We show that GNAT family histone acetyltransferase(s) (HATs) are required for the expression of genes involved in VOC biosynthesis and emission, by using chemical inhibitors of HATs, and by knocking down a specific HAT gene, ELP3, through transient RNAi. Together, our study supports that regulatory mechanisms at chromatin level may play an essential role in activating primary and secondary metabolic pathways to regulate VOC synthesis in petunia flowers.

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