scholarly journals A Luciferase-EGFP Reporter System for the Evaluation of DNA Methylation in Mammalian Cells

2021 ◽  
Author(s):  
X. X. Wang ◽  
H. J. Jia ◽  
Y. R. Lv ◽  
H. H. Sun ◽  
X. L. Wei ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mammalian Cells ◽  
Reporter System ◽  
Egfp Reporter

scholarly journals Control of the DNA Methylation System Component MBD2 by Protein Arginine Methylation

2006 ◽  
Vol 26 (19) ◽  
pp. 7224-7235 ◽  
Author(s):  
Choon Ping Tan ◽  
Sara Nakielny
Keyword(s):  
Dna Methylation ◽  
Complex Formation ◽  
Histone Deacetylases ◽  
Mammalian Cells ◽  
Genome Stability ◽  
Epigenetic Modification ◽  
Arginine Methylation ◽  
System Component ◽  
Transcription Repression ◽  
Mbd Proteins

ABSTRACT DNA methylation is vital for proper chromatin structure and function in mammalian cells. Genetic removal of the enzymes that catalyze DNA methylation results in defective imprinting, transposon silencing, X chromosome dosage compensation, and genome stability. This epigenetic modification is interpreted by methyl-DNA binding domain (MBD) proteins. MBD proteins respond to methylated DNA by recruiting histone deacetylases (HDAC) and other transcription repression factors to the chromatin. The MBD2 protein is dispensable for animal viability, but it is implicated in the genesis of colon tumors. Here we report that the MBD2 protein is controlled by arginine methylation. We identify the protein arginine methyltransferase enzymes that catalyze this modification and show that arginine methylation inhibits the function of MBD2. Arginine methylation of MBD2 reduces MBD2-methyl-DNA complex formation, reduces MBD2-HDAC repression complex formation, and impairs the transcription repression function of MBD2 in cells. Our report provides a molecular description of a potential regulatory mechanism for an MBD protein family member. It is the first to demonstrate that protein arginine methyltransferases participate in the DNA methylation system of chromatin control.

A novel reporter system for bacterial and mammalian cells based on the non-ribosomal peptide indigoidine

2012 ◽  
Vol 14 (4) ◽  
pp. 325-335 ◽  
Author(s):  
Marius Müller ◽  
Simon Ausländer ◽  
David Ausländer ◽  
Christian Kemmer ◽  
Martin Fussenegger
Keyword(s):  
Mammalian Cells ◽  
Reporter System

scholarly journals DNA methylation directs microRNA biogenesis in mammalian cells

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Ohad Glaich ◽  
Shivang Parikh ◽  
Rachel E. Bell ◽  
Keren Mekahel ◽  
Maya Donyo ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Sequence Conservation ◽  
Mirna Biogenesis ◽  
Coding Sequence ◽  
Microrna Biogenesis ◽  
Flanking Regions

AbstractMicroRNA (miRNA) biogenesis initiates co-transcriptionally, but how the Microprocessor machinery pinpoints the locations of short precursor miRNA sequences within long flanking regions of the transcript is not known. Here we show that miRNA biogenesis depends on DNA methylation. When the regions flanking the miRNA coding sequence are highly methylated, the miRNAs are more highly expressed, have greater sequence conservation, and are more likely to drive cancer-related phenotypes than miRNAs encoded by unmethylated loci. We show that the removal of DNA methylation from miRNA loci leads to their downregulation. Further, we found that MeCP2 binding to methylated miRNA loci halts RNA polymerase II elongation, leading to enhanced processing of the primary miRNA by Drosha. Taken together, our data reveal that DNA methylation directly affects miRNA biogenesis.

scholarly journals Establishment of a Rabbit Oct4 Promoter-Based EGFP Reporter System

PLoS ONE ◽  
2014 ◽  
Vol 9 (10) ◽  
pp. e109728 ◽  
Author(s):  
Longquan Quan ◽  
Yongqiang Chen ◽  
Jun Song ◽  
Quanmei Yan ◽  
Quanjun Zhang ◽  
...  
Keyword(s):  
Reporter System ◽  
Egfp Reporter

scholarly journals Stress-Inducible Alternative Translation Initiation of Human Cytomegalovirus Latency Protein pUL138

2010 ◽  
Vol 84 (18) ◽  
pp. 9472-9486 ◽  
Author(s):  
Lora Grainger ◽  
Louis Cicchini ◽  
Michael Rak ◽  
Alex Petrucelli ◽  
Kerry D. Fitzgerald ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Translation Initiation ◽  
Rna Splicing ◽  
Mammalian Cells ◽  
Open Reading Frames ◽  
Reporter System ◽  
Entry Site ◽  
Sequence Element ◽  
Low Passage

ABSTRACT We have previously characterized a 21-kDa protein encoded by UL138 (pUL138) as a viral factor inherent to low-passage strains of human cytomegalovirus (HCMV) that is required for latent infection in vitro. pUL138 is encoded on 3.6-, 2.7-, and 1.4-kb 3′ coterminal transcripts that are produced during productive and latent infections. pUL138 is encoded at the 3′ end of each transcript and is preceded by an extensive 5′ sequence (∼0.5 to 2.5 kb) containing several putative open reading frames (ORFs). We determined that three putative ORFs upstream of UL138 (UL133, UL135, and UL136) encode proteins. The UL138 transcripts are polycistronic, such that each transcript expresses pUL138 in addition to the most-5′ ORF. The upstream coding sequences (CDS) present a significant challenge for the translation of pUL138 in mammalian cells. We hypothesized that sequences 5′ of UL138 mediate translation initiation of pUL138 by alternative strategies. Accordingly, a 663-nucloetide (nt) sequence overlapping the UL136 CDS supported expression of a downstream cistron in a bicistronic reporter system. We did not detect cryptic promoter activity or RNA splicing events that could account for downstream cistron expression. These data are consistent with the sequence element functioning as an internal ribosome entry site (IRES). Interestingly, pUL138 expression from the 3.6- and 2.7-kb transcripts was induced by serum stress, which concomitantly inhibited normal cap-dependent translation. Our work suggests that an alternative and stress-inducible strategy of translation initiation ensures expression of pUL138 under a variety of cellular contexts. The UL138 polycistronic transcripts serve to coordinate the expression of multiple proteins, including a viral determinant of HCMV latency.

MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway

2010 ◽  
Vol 88 (3) ◽  
pp. 445-450 ◽  
Author(s):  
Pei Liang ◽  
Yongqi Wan ◽  
Yan Yan ◽  
Yuequn Wang ◽  
Na Luo ◽  
...  
Keyword(s):  
Metal Binding ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Reporter System ◽  
Biochemical Assays ◽  
Metal Binding Domain ◽  
Erk Signal Pathway ◽  
Gene Maps ◽  
Putative Zinc Finger ◽  
Two Hybrid

Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4’s ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.

Methylation Profile of B-Chronic Lymphocytic Leukemia Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2951-2951
Author(s):  
Jun Fan ◽  
Asou Norio ◽  
Masao Matsuoka
Keyword(s):  
Dna Methylation ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Mononuclear Cells ◽  
Cpg Island ◽  
Methylation Status ◽  
Lymphocytic Leukemia ◽  
Dna Hypomethylation ◽  
Peripheral Blood Mononuclear

Abstract DNA methylation plays an important role in the development and aging of mammalian cells, and its dysregulation has been frequently observed in cancer cells. The purpose of this study is to investigate the involvement of aberrant DNA methylation in B chronic lymphocytic leukemia (B-CLL) cells. We compared methylation status of B-CLL cells isolated from patients with that of normal CD19+ cells isolated from health donors by methylated CpG island amplification/representative difference analysis method. 5 hypermethylated and 27 hypomethylated DNA regions were identified in B-CLL sample. Among the 27 hypomethylated regions, 5 located on chromosome 9q34, 3 on 10q25-26 and 4 on 19q13. Methylation status was confirmed by sequencing using sodium bisulfite-treated DNA samples. By comparing DNA samples from same patients at different clinical stages, we found that lower methylation density in these regions is linked with disease progression. Expression of 15 genes surrounding hypomethylated regions was studied by RT-PCR. Expression of laminin beta3 gene and melanotransferrin gene was found to be upregulated in all B-CLL cell lines as well as lymphoma cell lines comparing with normal CD19+ peripheral blood mononuclear cells. B-cell CLL/lymphoma 11b gene showed increased expression in only 2 B-CLL cell lines. For other genes, no transcriptional change was found regardless of changed DNA methylation. This study showed the predominance of DNA hypomethylation in B-CLL cells compared with hypermethylation. Hypomethylated regions clustered in a limited number of chromosomes and methylation density appeared to be inversely correlated with disease progress. Figure Figure

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