Identification of Genomic Sites That Bind the Drosophila Suppressor of Hairy-wing Insulator Protein

ABSTRACT Eukaryotic genomes are divided into independent transcriptional domains by DNA elements known as insulators. The gypsy insulator, a 350-bp element isolated from the Drosophila gypsy retrovirus, contains twelve degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein. Su(Hw) associates with over 500 non-gypsy genomic sites, the functions of which are largely unknown. Using a bioinformatics approach, we identified 37 putative Su(Hw) insulators (pSIs) that represent regions containing clustered matches to the gypsy insulator Su(Hw) consensus binding sequence. The majority of these pSIs contain fewer than four Su(Hw) binding sites, with only seven showing in vivo Su(Hw) association, as demonstrated by chromatin immunoprecipitation. To understand the properties of the pSIs, these elements were tested for enhancer-blocking capabilities using a transgene assay system. In a complementary set of experiments, effects of the pSIs on transcriptional regulation of genes at the natural genomic location were determined. Our data suggest that pSIs have complex genomic functions and, in some cases, establish insulators. These studies provide the first direct evidence that the Su(Hw) protein contributes to the regulation of gene expression in the Drosophila genome through the establishment of endogenous insulators.

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Definition of the transcription factor TdIF1 consensus-binding sequence through genomewide mapping of its binding sites

Genes to Cells ◽

10.1111/gtc.12216 ◽

2015 ◽

Vol 20 (3) ◽

pp. 242-254 ◽

Cited By ~ 3

Author(s):

Kotaro Koiwai ◽

Takashi Kubota ◽

Nobuhisa Watanabe ◽

Katsutoshi Hori ◽

Osamu Koiwai ◽

...

Keyword(s):

Transcription Factor ◽

Binding Sites ◽

Consensus Binding Sequence ◽

Definition Of ◽

Binding Sequence

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DEPPDB — DNA ELECTROSTATIC POTENTIAL PROPERTIES DATABASE: ELECTROSTATIC PROPERTIES OF GENOME DNA ELEMENTS

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720012410041 ◽

2012 ◽

Vol 10 (02) ◽

pp. 1241004 ◽

Cited By ~ 7

Author(s):

ALEXANDER A. OSYPOV ◽

GLEB G. KRUTININ ◽

EUGENIA A. KRUTININA ◽

SVETLANA G. KAMZOLOVA

Keyword(s):

Physical Properties ◽

Electrostatic Potential ◽

Binding Sites ◽

Metabolic Switch ◽

Electrostatic Properties ◽

Protein Dna Interactions ◽

Dna Elements ◽

Ncbi Refseq ◽

Analytical Tools ◽

Eukaryotic Genomes

Electrostatic properties of genome DNA are important to its interactions with different proteins, in particular, related to transcription. DEPPDB — DNA Electrostatic Potential (and other Physical) Properties Database — provides information on the electrostatic and other physical properties of genome DNA combined with its sequence and annotation of biological and structural properties of genomes and their elements. Genomes are organized on taxonomical basis, supporting comparative and evolutionary studies. Currently, DEPPDB contains all completely sequenced bacterial, viral, mitochondrial, and plastids genomes according to the NCBI RefSeq, and some model eukaryotic genomes. Data for promoters, regulation sites, binding proteins, etc., are incorporated from established DBs and literature. The database is complemented by analytical tools. User sequences calculations are available. Case studies discovered electrostatics complementing DNA bending in E.coli plasmid BNT2 promoter functioning, possibly affecting host-environment metabolic switch. Transcription factors binding sites gravitate to high potential regions, confirming the electrostatics universal importance in protein–DNA interactions beyond the classical promoter–RNA polymerase recognition and regulation. Other genome elements, such as terminators, also show electrostatic peculiarities. Most intriguing are gene starts, exhibiting taxonomic correlations. The necessity of the genome electrostatic properties studies is discussed.

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Allele-Specific Binding of CTCF to the Multipartite Imprinting Control Region KvDMR1

Molecular and Cellular Biology ◽

10.1128/mcb.02036-06 ◽

2007 ◽

Vol 27 (7) ◽

pp. 2636-2647 ◽

Cited By ~ 59

Author(s):

Galina V. Fitzpatrick ◽

Elena M. Pugacheva ◽

Jong-Yeon Shin ◽

Ziedulla Abdullaev ◽

Youwen Yang ◽

...

Keyword(s):

Control Region ◽

Binding Sites ◽

Noncoding Rna ◽

Specific Binding ◽

Ctcf Binding ◽

Luciferase Reporter ◽

Imprinting Control Region ◽

Enhancer Blocking ◽

Functional Components

ABSTRACT Paternal deletion of the imprinting control region (ICR) KvDMR1 results in loss of expression of the Kcnq1ot1 noncoding RNA and derepression of flanking paternally silenced genes. Truncation of Kcnq1ot1 also results in the loss of imprinted expression of these genes in most cases, demonstrating a role for the RNA or its transcription in gene silencing. However, enhancer-blocking studies indicate that KvDMR1 also contains chromatin insulator or silencer activity. In this report we demonstrate by electrophoretic mobility shift assays and chromatin immunoprecipitation the existence of two CTCF binding sites within KvDMR1 that are occupied in vivo only on the unmethylated paternally derived allele. Methylation interference and mutagenesis allowed the precise mapping of protein-DNA contact sites for CTCF within KvDMR1. Using a luciferase reporter assay, we mapped the putative transcriptional promoter for Kcnq1ot1 upstream and to a site functionally separable from enhancer-blocking activity and CTCF binding sites. Luciferase reporter assays also suggest the presence of an additional cis-acting element in KvDMR1 upstream of the putative promoter that can function as an enhancer. These results suggest that the KvDMR1 ICR consists of multiple, independent cis-acting modules. Dissection of KvDMR1 into its functional components should help elucidate the mechanism of its function in vivo.

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Factor cooperation for chromosome discrimination in Drosophila

10.1101/414193 ◽

2018 ◽

Author(s):

Christian Albig ◽

Evgeniya Tikhonova ◽

Silke Krause ◽

Oksana Maksimenko ◽

Catherine Regnard ◽

...

Keyword(s):

Dosage Compensation ◽

Binding Sites ◽

Physical Interaction ◽

Functional Sites ◽

Genome Wide ◽

Nucleosome Formation ◽

Dna Elements ◽

Transcription Regulators ◽

Male Specific

AbstractTranscription regulators select their genomic binding sites from a large pool of similar, non-functional sequences. Although general principles that allow such discrimination are known, the complexity of DNA elements often precludes a prediction of functional sites.The process of dosage compensation in Drosophila allows exploring the rules underlying binding site selectivity. The male-specific-lethal (MSL) Dosage Compensation Complex selectively binds to some 300 X-chromosomal ‘High Affinity Sites’ (HAS) containing GA-rich ‘MSL recognition elements’ (MREs), but disregards thousands of other MRE sequences in the genome. The DNA-binding subunit MSL2 alone identifies a subset of MREs, but fails to recognize most MREs within HAS. The ‘Chromatin-linked adaptor for MSL proteins’ (CLAMP) also interacts with many MREs genome-wide and promotes DCC binding to HAS. Using genome-wide DNA-immunoprecipitation we describe extensive cooperativity between both factors, depending on the nature of the binding sites. These are explained by physical interaction between MSL2 and CLAMP. In vivo, both factors cooperate to compete with nucleosome formation at HAS. The male-specific MSL2 thus synergises with a ubiquitous GA-repeat binding protein for refined X/autosome discrimination.

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Nucleosomes and regulation of gene expression. Structure of the HIV-1 5'LTR.

Acta Biochimica Polonica ◽

10.18388/abp.1998_4303 ◽

1998 ◽

Vol 45 (1) ◽

pp. 209-219 ◽

Cited By ~ 1

Author(s):

P Widłak ◽

W T Garrard

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Chromatin Remodeling ◽

Binding Sites ◽

Regulation Of Gene Expression ◽

Dnase I ◽

Chromatin Assembly ◽

Hypersensitive Sites ◽

Hiv 1

Packaging of DNA into chromatin adds complexity to the problem of regulation of gene expression. Nucleosomes affect the accessibility of transcription factors to occupy their binding sites in chromatin of eukaryotic cells. The disruption of nucleosome structure within the enhancer/promoter region of the integrated HIV-1 proviral genome is an instructive example of a chromatin remodeling process during transcriptional activation. To investigate the mechanism responsible for generating nuclease hypersensitive sites that exist in vivo in the promoter/enhancer region of the 5'LTR (long terminal repeat) of integrated HIV-1 we have utilized an in vitro chromatin assembly system with Xenopus oocyte extracts. Chromatin assembly in the presence of Sp1 and NFkappaB transcription factors induces DNase I hypersensitive sites on either side of their binding sites and positions the adjacent nucleosomes. This structure can also be formed in a factor-induced, ATP-dependent chromatin remodeling process and closely resembles the in vivo chromatin structure. The DNase I hypersensitive sites that form within the HIV LTR are probably histone-free and remain after removal of transcription factors.

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Functional divergence between the half-sites of the DNA-binding sequence for the yeast transcriptional regulator Rap1p

Biochemical Journal ◽

10.1042/bj3410477 ◽

1999 ◽

Vol 341 (3) ◽

pp. 477-482 ◽

Cited By ~ 10

Author(s):

Fátima-Zahra IDRISSI ◽

Benjamin PIÑA

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Functional Divergence ◽

Consensus Sequence ◽

Transcriptional Regulator ◽

Dna Complexes ◽

Dna Binding Sites ◽

Binding Sequence ◽

Half Site

The yeast transcriptional regulator Rap1p binds to the DNA consensus sequence ACACCCAYACAYYY. We have previously shown that DNA-binding sites in which all four Y (Y = T or C) positions were Ts (UASrpg sequences) synergized more efficiently to activate transcription than sequences in which all Ys were Cs (telomere sequences) [F.-Z. Idrissi, J. Fernández-Larrea and B. Piña (1998) J. Mol. Biol. 284, 925-935]. Here we provide evidence that the DNA consensus sequence for Rap1p behaves as a combination of two ACAYYY half-sites with different functionality, the presence of Ts in the second half-site being the determinant for the transcriptional behaviour of the UASrpg sequences. DNA structure in the different complexes with Rap1p varied from being relatively uniform to appear rather distorted, this also being dependent on the presence of Ts in the second half-site. These distortions did not cause sharp bends or kinks in the DNA molecule. Computer analysis suggests that high-affinity binding of Rap1p to UASrpg sequences requires a rearrangement of the C-terminal Myb domain of the protein. We propose that the structural alterations in Rap1p-DNA complexes, both in the DNA and in the protein, affect the transcription potential of the complex in an allosteric manner. We also propose that the dimeric nature of the Rap1 DNA-binding domain is a key structural feature that explains the disparate functions of its DNA-binding sites in vivo.

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Rasgrf1 Imprinting Is Regulated by a CTCF-Dependent Methylation-Sensitive Enhancer Blocker

Molecular and Cellular Biology ◽

10.1128/mcb.25.24.11184-11190.2005 ◽

2005 ◽

Vol 25 (24) ◽

pp. 11184-11190 ◽

Cited By ~ 64

Author(s):

Bongjune Yoon ◽

Herry Herman ◽

Benjamin Hu ◽

Yoon Jung Park ◽

Anders Lindroth ◽

...

Keyword(s):

Binding Sites ◽

Repeated Sequence ◽

Ctcf Binding ◽

Paternal Allele ◽

Maternal Allele ◽

Binary Switch ◽

Blocking Activity ◽

Enhancer Blocking

ABSTRACT Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5′ of the promoter. A repeated sequence 3′ of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.

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Connections between transcriptional activators, silencers, and telomeres as revealed by functional analysis of a yeast DNA-binding protein.

Molecular and Cellular Biology ◽

10.1128/mcb.8.12.5086 ◽

1988 ◽

Vol 8 (12) ◽

pp. 5086-5099 ◽

Cited By ~ 132

Author(s):

A R Buchman ◽

N F Lue ◽

R D Kornberg

Keyword(s):

Dna Sequences ◽

Binding Sites ◽

Consensus Sequence ◽

Regulatory Sequences ◽

Regulatory Factor ◽

Yeast Protein ◽

In Vitro System ◽

Dna Elements

General regulatory factor I (GRFI) is a yeast protein that binds in vitro to specific DNA sequences at diverse genetic elements. A strategy was pursued to test whether GRFI functions in vivo at the sequences bound by the factor in vitro. Matches to a consensus sequence for GRFI binding were found in a variety of locations: upstream activating sequences (UASs), silencers, telomeres, and transcribed regions. All occurrences of the consensus sequence bound both crude and purified GRFI in vitro. All binding sites for GRFI, regardless of origin, provided UAS function in test plasmids. Also, GRFI binding sites specifically stimulated transcription in a yeast in vitro system, indicating that GRFI can function as a positive transcription factor. The stimulatory effect of GRFI binding sites at UASs for the PYK1 and ENO1 genes is significantly enhanced by flanking DNA elements. By contrast, regulatory sequences that flank the GRFI binding site at HMR E convert this region to a transcriptional silencer.

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