Transcriptional Repression by DrosophilaMethyl-CpG-Binding Proteins

ABSTRACT C methylation at genomic CpG dinucleotides has been implicated in the regulation of a number of genetic activities during vertebrate cell differentiation and embryo development. The methylated CpG could induce chromatin condensation through the recruitment of histone deacetylase (HDAC)-containing complexes by methyl-CpG-binding proteins. These proteins consist of the methylated-DNA binding domain (MBD). Unexpectedly, however, several studies have identified MBD-containing proteins encoded by genes of Drosophila melanogaster, an invertebrate species supposed to be void of detectable m5CpG. We now report the genomic structure of aDrosophila gene, dMBD2/3, that codes for two MBD-containing, alternatively spliced, and developmentally regulated isoforms of proteins, dMBD2/3 and dMBD2/3Δ. Interestingly, in vitro binding experiments showed that as was the case for vertebrate MBD proteins, dMBD2/3Δ could preferentially recognize m5CpG-containing DNA through its MBD. Furthermore, dMBD2/3Δ as well as one of its orthologs in mouse, MBD2b, could function in human cells as a transcriptional corepressor or repressor. The activities of HDACs appeared to be dispensable for transcriptional repression by dMBD2/3Δ. Finally, dMBD2/3Δ also could repress transcription effectively in transfectedDrosophila cells. The surprisingly similar structures and characteristics of the MBD proteins as well as DNA cytosine (C-5) methyltransferase-related proteins in Drosophila and vertebrates suggest interesting scenarios for their roles in eukaryotic cellular functions.

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Identification of a CpG Island in the Human LRP-2 Gene and Analysis of Its Methylation Status in Parathyroid Adenomas

Biological Chemistry ◽

10.1515/bc.2000.056 ◽

2000 ◽

Vol 381 (5-6) ◽

pp. 433-438 ◽

Cited By ~ 4

Author(s):

A. Knutson ◽

P. Lillhager ◽

G. Westin

Keyword(s):

Cell Line ◽

Transcriptional Repression ◽

Cpg Island ◽

Methylation Status ◽

Parathyroid Tissue ◽

Cellular Functions ◽

Gene Encoding ◽

Parathyroid Adenomas ◽

Multiple Ligands

Abstract Methylation of CpG residues in mammalian genomes is a mechanism of vital importance for many cellular functions, which all relate to gene expression. In this study we describe the identification of a CpG island in the 5′-region of the gene encoding human megalin/LRP-2, a receptor capable of binding multiple ligands, which is involved in the regulation of calcium metabolism. Southern blot analysis and genomic bisulfite sequencing revealed that the CpG island is methylated in a non-expressing cell line, largely unmethylated in an expressing cell line and unmethylated in human parathyroid tissue. In addition, we show that artificial methylation of LRP-2 promoter reporter plasmids leads to strong transcriptional repression, in vitro as well as in transfected cells. No evidence for aberrant LRP-2 gene methylation in parathyroid adenomas, in which the LRP-2 protein is generally down-regulated, was found.

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Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1805-1814.1993 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1805-1814

Author(s):

H Wang ◽

D J Stillman

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Fusion Protein ◽

Protein Interactions ◽

Transcriptional Repression ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Predicted Amino Acid Sequence

The yeast SIN3 gene (also known as SDI1, UME4, RPD1, and GAM2) has been identified as a transcriptional regulator. Previous work has led to the suggestion that SIN3 regulates transcription via interactions with DNA-binding proteins. Although the SIN3 protein is located in the nucleus, it does not bind directly to DNA in vitro. We have expressed a LexA-SIN3 fusion protein in Saccharomyces cerevisiae and show that this fusion protein represses transcription from heterologous promoters that contain lexA operators. The predicted amino acid sequence of the SIN3 protein contains four copies of a paired amphipathic helix (PAH) motif, similar to motifs found in HLH (helix-loop-helix) and TPR (tetratricopeptide repeat) proteins, and these motifs are proposed to be involved in protein-protein interactions. We have conducted a deletion analysis of the SIN3 gene and show that the PAH motifs are required for SIN3 activity. Additionally, the C-terminal region of the SIN3 protein is sufficient for repression activity in a LexA-SIN3 fusion, and deletion of a PAH motif in this region inactivates this repression activity. A model is presented in which SIN3 recognizes specific DNA-binding proteins in vivo in order to repress transcription.

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Differential Contributions of DNA-Binding Proteins to Polycomb Response Element Activity at the Drosophila giant Gene

Genetics ◽

10.1534/genetics.119.302981 ◽

2020 ◽

Vol 214 (3) ◽

pp. 623-634

Author(s):

Elnaz Ghotbi ◽

Kristina Lackey ◽

Vicki Wong ◽

Katie T. Thompson ◽

Evan G. Caston ◽

...

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Transcriptional Repression ◽

Binding Proteins ◽

Target Genes ◽

Dna Binding Proteins ◽

Polycomb Group ◽

Link Type ◽

Element Activity

Polycomb-group (PcG) proteins are evolutionarily conserved epigenetic regulators whose primary function is to maintain the transcriptional repression of target genes. Recruitment of Drosophila melanogaster PcG proteins to target genes requires the presence of one or more Polycomb Response Elements (PREs). The functions or necessity for more than one PRE at a gene are not clear and individual PREs at some loci may have distinct regulatory roles. Various combinations of sequence-specific DNA-binding proteins are present at a given PRE, but only Pleiohomeotic (Pho) is present at all strong PREs. The giant (gt) locus has two PREs, a proximal PRE1 and a distal PRE2. During early embryonic development, Pho binds to PRE1 ∼30-min prior to stable binding to PRE2. This observation indicated a possible dependence of PRE2 on PRE1 for PcG recruitment; however, we find here that PRE2 recruits PcG proteins and maintains transcriptional repression independently of Pho binding to PRE1. Pho-like (Phol) is partially redundant with Pho during larval development and binds to the same DNA sequences in vitro. Although binding of Pho to PRE1 is dependent on the presence of consensus Pho-Phol-binding sites, Phol binding is less so and appears to play a minimal role in recruiting other PcG proteins to gt. Another PRE-binding protein, Sp1/Kruppel-like factor, is dependent on the presence of Pho for PRE1 binding. Further, we show that, in addition to silencing gene expression, PcG proteins dampen transcription of an active gene.

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1,6-hexanediol rapidly immobilizes and condenses chromatin in living human cells

Life Science Alliance ◽

10.26508/lsa.202001005 ◽

2021 ◽

Vol 4 (4) ◽

pp. e202001005 ◽

Cited By ~ 2

Author(s):

Yuji Itoh ◽

Shiori Iida ◽

Sachiko Tamura ◽

Ryosuke Nagashima ◽

Kentaro Shiraki ◽

...

Keyword(s):

Liquid Droplet ◽

Chromatin Condensation ◽

Human Cells ◽

Dependent Manner ◽

Liquid Droplets ◽

Cellular Functions ◽

Spatiotemporal Regulation ◽

Dose Dependent ◽

Liquid Liquid Phase Separation

Liquid droplets formed inside the cell by liquid–liquid phase separation maintain membrane-less condensates/bodies (or compartments). These droplets are important for concentrating certain molecules and facilitating spatiotemporal regulation of cellular functions. 1,6-hexanediol (1,6-HD), an aliphatic alcohol, inhibits weak hydrophobic protein–protein/protein-RNA interactions required for the droplet formation (droplet melting activity) and is used here to elucidate the formation process of cytoplasmic/nuclear condensates/bodies. However, the effect of 1,6-HD on chromatin in living cells remains unclear. We found that 1,6-HD drastically suppresses chromatin motion and hyper-condenses chromatin in human cells by using live-cell single-nucleosome imaging, which detects changes in the state of chromatin. These effects were enhanced in a dose-dependent manner. Chromatin was “frozen” by 5%, or higher, concentrations of 1,6-HD. 1,6-HD greatly facilitated cation-dependent chromatin condensation in vitro. This 1,6-HD action is distinct from its melting activity of liquid droplets. Alcohols, such as 1,6-HD, appear to remove water molecules around chromatin and locally condense chromatin. Therefore, liquid droplet results obtained using 1,6-HD should be carefully interpreted or reconsidered when these droplets are associated with chromatin.

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Transcriptional repression in Saccharomyces cerevisiae by a SIN3-LexA fusion protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.3.1805 ◽

1993 ◽

Vol 13 (3) ◽

pp. 1805-1814 ◽

Cited By ~ 68

Author(s):

H Wang ◽

D J Stillman

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Fusion Protein ◽

Protein Interactions ◽

Transcriptional Repression ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Predicted Amino Acid Sequence

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RNA Binding Proteins Shape Latent KSHV Genomic Structure: Identification of KSHV Episome Tethering Sites on Host Chromosomes

10.21203/rs.3.rs-136834/v1 ◽

2021 ◽

Author(s):

Ashish Kumar ◽

Yuanzhi Lyu ◽

Yuichi Yanagihashi ◽

Chanikarn Chantarasrivong ◽

Vladimir Majerciak ◽

...

Keyword(s):

Dna Binding ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genomic Structure ◽

Gene Activation ◽

Lytic Replication ◽

Structure Identification ◽

Viral Gene

Abstract In previous studies, we have shown that expression of a viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we identify viral lncRNA binding proteins, and show that an underlying molecular mechanism regulating the KSHV latency-lytic replication switch is via a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Proximity biotin labeling, single cell transcriptomics, and siRNA screening along with complementation studies identified that CHD4's enzymatic activity silences viral gene expression by preventing transcription factory formation. Furthermore, Capture Hi-C, Cleavage Under Targets and Release Using Nuclease (CUT&RUN), and proteomics approaches together identify KSHV episome docking sites on host chromosomes and colocalization with a CHD4 protein complex, ChAHP, at epigenetically active genomic regions. PAN RNA binds and competes with CHD4 DNA binding in vitro, and KSHV episomes detached from these host genomic loci sites when reactivation is triggered. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible genomic looping, and is therefore important for the ability of KSHV to establish and maintain latency in a "poised" state at selected host genomic loci.

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NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

Nucleic Acids Research ◽

10.1093/nar/gkt1311 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3177-3193 ◽

Cited By ~ 16

Author(s):

Bhagyashri D. Burgute ◽

Vivek S. Peche ◽

Anna-Lena Steckelberg ◽

Gernot Glöckner ◽

Berthold Gaßen ◽

...

Keyword(s):

Transcriptional Repression ◽

Messenger Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Speckles ◽

Rna Helicases ◽

Hematopoietic Stem ◽

Basic Domain

Abstract NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS–NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.

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Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100116620 ◽

1975 ◽

Vol 33 ◽

pp. 600-601

Author(s):

John C. Garancis ◽

Robert O. Hussa ◽

Michael T. Story ◽

Donald Yorde ◽

Roland A. Pattillo

Keyword(s):

Electron Microscopy ◽

Cellular Proliferation ◽

Morphological Changes ◽

Culture Fluid ◽

Cellular Functions ◽

Human Trophoblast ◽

Transmission Electron ◽

Marked Activity ◽

Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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Identification of Differentially Expressed Genes Using cDNA-AFLP During Six Days In Vitro Tuberization of Potato

Zuriat ◽

10.24198/zuriat.v14i1.6751 ◽

2015 ◽

Vol 14 (1) ◽

Author(s):

Nono Carsono ◽

Christian Bachem

Keyword(s):

Gene Expression ◽

Developmental Process ◽

Expression Patterns ◽

Induction Medium ◽

In Vitro Tuberization ◽

Storage Organ ◽

Developmentally Regulated ◽

Study Gene Expression ◽

Differential Pattern

Tuberization in potato is a complex developmental process resulting in the differentiation of stolon into the storage organ, tuber. During tuberization, change in gene expression has been known to occur. To study gene expression during tuberization over the time, in vitro tuberization system provides a suitable tool, due to its synchronous in tuber formation. An early six days axillary bud growing on tuber induction medium is a crucial development since a large number of genes change in their expression patterns during this period. In order to identify, isolate and sequencing the genes which displaying differential pattern between tuberizing and non-tuberizing potato explants during six days in vitro tuberization, cDNA-AFLP fingerprint, method for the visualization of gene expression using cDNA as template which is amplified to generate an RNA-fingerprinting, was used in this experiment. Seventeen primer combinations were chosen based on their expression profile from cDNA-AFLP fingerprint. Forty five TDFs (transcript derived fragment), which displayed differential expressions, were obtained. Tuberizing explants had much more TDFs, which developmentally regulated, than those from non tuberizing explants. Seven TDFs were isolated, cloned and then sequenced. One TDF did not find similarity in the current databases. The nucleotide sequence of TDF F showed best similarity to invertase ezymes from the databases. The homology of six TDFs with known sequences is discussed in this paper.

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Investigation of the effect of loratadine and calcium ions on oxidoreductase activity of catalase enzyme

Current Enzyme Inhibition ◽

10.2174/1573408016999200817173859 ◽

2020 ◽

Vol 16 ◽

Author(s):

Edhem Hasković ◽

Safija Herenda ◽

Zehra Halilović ◽

Snežana Unčanin ◽

Denis Hasković ◽

...

Keyword(s):

Calcium Ions ◽

Enzyme Reaction ◽

Analytical Techniques ◽

Competitive Inhibitor ◽

Cellular Functions ◽

Oxidoreductase Activity ◽

H1 Antihistamines ◽

Activity Of Enzymes ◽

The Given

Background: The Spectrophotometric method is one of the most suitable analytical techniques for testing the activity of enzymes under the influence of various factors. Methods: The effect of H1-antihistamines of loratadine and calcium ions on enzyme catalase under in vitro conditions was investigated in this paper. Results and Discussion: It has been shown that loratadine isa partial inhibitor of catalase, but this effect is diminished in the presence of calcium ions. Calcium as well as other cations are important for many biological and cellular functions. The kidneys play a central role in the homeostasis of these ions. The activity of the catalase enzyme under the given conditions, the type of inhibition,and the kinetic parameters of the enzyme reaction were determined. Conclusion: We concluded that loratadine is a partially competitive inhibitor.

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