Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site

Jost, Jean-Pierre; Saluz, Hans‐Peter; Pawlak, André

doi:10.1093/nar/19.20.5771

Cited by 52 publications

(30 citation statements)

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“…The binding activity of NHP1 increases more than 2-fold in the liver of estradiol treated immature roosters where it coincides with the active demethylation of CpGs situated within the promoter region of the vitellogenin gene [4]. Similarly, we show here that there is an increase in the NHP1 p75 and p85 subunits during the differentiation of mouse G8 myoblasts.…”

Section: Discussionsupporting

confidence: 76%

“…Since it has been reported that NHP1 DNA binding activity was upregulated by estradiol during the active demethylation of the avian vitellogenin gene [4] we decided to follow the amount of NHP1 during the genome-wide demethylation of mouse myoblasts. As shown in the left-hand panel of Fig.…”

Section: 1 Nhp1 Is" a Member Of The Ku Protein Familymentioning

confidence: 99%

“…Furthermore, at the onset of active demethylation *Corresponding author. Fax: (41) of the avian vitellogenin gene in the liver there was a transient increase in NHP1 binding activity [4]. It has also been suggested that NHP1 could tag the DNA for the subsequent binding of the estradiol receptor [1][2][3].…”

Section: Introductionmentioning

confidence: 99%

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Non‐histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes

1996

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promyelocytes there is an increase in the amount of p85 subunit protein whereas the level of the p75 subunit is unchanged. In differentiating mouse G8 myoblasts there is, however, an upregulation of both the p75 and p85 subunits and of the p85 mRNA. An inhibition of mouse myoblast differentiation by either cAMP, 3-aminobenzamide or sodium butyrate abolishes the upregulation of the p85 subunit. In G8 myoblasts chemical, or physical stress by UV fight or X-rays does not upregulate the level of the p85 subunit. The possible involvement of NHP1 in the active demethylation of bifilarly methylated DNA will be discussed.

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Section: Discussionsupporting

confidence: 76%

Section: 1 Nhp1 Is" a Member Of The Ku Protein Familymentioning

confidence: 99%

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Non‐histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes

1996

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“…Several proteins that bind preferentially to methylated DNA (MeCPs [methylated cytosine-binding proteins] [27,30] and MDBPs [methylated DNA-binding proteins] [20,32,42]) have been discovered. These proteins are thought to be involved in the repression of methylated genes (5,6,21). Furthermore, it has been shown that MeCP-2 is associated with heterochromatin (27), whereas MDBP-2 shares sequence homologies with histone H1 (20), implying a function for both proteins in chromatin formation.…”

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confidence: 99%

Inactive chromatin spreads from a focus of methylation.

Kass¹,

Goddard²,

Adams³

1993

Mol. Cell. Biol.

122

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The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown, but it has become obvious that the formation of chromatin plays an important role in this process. Using an approach enabling us to methylate, in vitro, chosen regions in a plasmid, we now show that specific methylation of nonpromoter sequences results in transcriptional inhibition of a reporter gene construct and that this inhibition is independent of the position of the methylated region within the plasmid. In plasmid minichromosomes containing a short region of methylated DNA, both methylated and unmethylated sequences are protected from MATERIALS AND METHODS Plasmids. Plasmid pVHCk was constructed by cloning a 2-kb KpnI-EcoRI fragment containing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of the SV40 promoter/enhancer and the SV40 terminator region from pVHC1 (7) into pBluescript KSII-. A map of the plasmid is shown in Fig. 1. The promoterless plasmid pPLk was generated by deleting the SV40 promoter/enhancer from * Corresponding author.pVHCk by digestion with K1nI-HindIII, generation of blunt ends with S1 nuclease, and religation. Two constructs containing the SV40 promoter/enhancer at both sides of the CAT gene/terminator region were generated by cloning the BamHI-linkered promoter fragment in both orientations into the BamHI site of pVHCk immediately 3' of the terminator region. They were named pPPk (promoters in same orientation) and pPRk (second promoter in reverse orientation), respectively.Generation of patch-methylated constructs. Phagemid single-stranded DNA (ssDNA) was isolated by the method of Blondel and Thillet (4). Restriction fragments were gel purified and annealed to ssDNA at a molar ratio of 3:1 (fragment DNA:ssDNA) in 20 mM Tris-HCl (pH 7.5)-10 mM MgCl2-50 mM NaCl-1 mM dithiothreitol by heating for 2 min at 95°C and allowing to cool down from 70 to 30°C in 1 h. To prevent binding to and methylation of the singlestranded region by methylase SssI (M.SssI), T4 gene 32 protein (Boehringer Mannheim) was added (10 ,ug/,g of ssDNA), and the reaction mixture was incubated at 37°C for 15 min. The double-stranded DNA patch was methylated for 16 h, using M.SssI (New England Biolabs) under conditions recommended by the manufacturer. After proteinase K treatment and phenol extraction, the unmethylated gap was filled in and ligated by standard procedures (35). For every patch-methylated construct, a mock-methylated control was generated in the same way but omitting M.SssI. To confirm the location of the methylated region, constructs were isolated immediately or 2 days after transfection, and the methylated region was released with the appropriate restriction enzymes and subjected to HpaII digestion. The DNA was then separated on a 1.5% agarose gel, transferred to a nylon membrane (Hybond N+; Amersham), and hybridized to the appropriate labeled fragment. Membranes were reprobed after stripping with boiling sodium dodecyl sulfate (SDS) solution (0.5%).Transfections and analysis of CA...

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“…This could be caused by competition between MVBP and one or the other VBP for DNA binding. Replacement of transcription factors by methyl cytosine-binding proteins has been observed for some methylated animal promoters (36,55). Evidence for competition could be deduced from our experiments with extracts from suspension cells with apparently lower MVBP activity in which formation of the VBP1 complex with methylated oligonucleotides appeared to be efficient, whereas in extracts from rice shoots with higher MVBP activity, it was reduced.…”

Section: Discussionmentioning

confidence: 55%

Sequence-specific and Methylation-dependent and -independent Binding of Rice Nuclear Proteins to a Rice Tungro Bacilliform Virus Vascular Bundle Expression Element

Fütterer

Höhn

2001

Journal of Biological Chemistry

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Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE). One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG potential methylation motifs. Binding by these proteins was determined to be cytosine methylation-independent. However, a novel protein present in all analyzed extracts bound specifically to the methylated VBE. A region of at least 49 nucleotides overlapping the VBE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBEbinding protein (MVBP).Promoters for genes transcribed by RNA polymerase II are composed of different cis elements that provide binding sites for trans-acting factors (1-2). The interactions of these transacting factors with each other, with associated cofactors, and with the basal transcription machinery determine the expression characteristics of a promoter. Promoter activity is also influenced by epigenetic control mechanisms that may modulate the accessibility of promoter sequences for the transcription machinery in various ways. Plant promoter studies have elucidated several molecular components involved in tissuespecific gene expression (1-2). These include cis-acting DNA elements and trans-acting protein factors involved in regulating gene expression in vascular tissue (3-8).The rice tungro bacilliform virus (RTBV) 1 promoter ( Fig. 1) has been analyzed in both transformed rice plants (9 -11) and transfected rice protoplasts (12-15). It was found that sequences downstream of the transcription start site are required for promoter activity in protoplasts (13-15). In transgenic plants, the RTBV promoter containing the first 250 transcribed nucleotides showed activity in the epidermis and in all cells of the vascular bundle, not just the phloem (9). In the latter study, the most important promoter element for this vascular bundle activity was localized to the region between Ϫ100 and Ϫ165 (9), whereas previous analyses indicated sequences termed box II (Ϫ53 to Ϫ39), ASL box (Ϫ98 to Ϫ79), and GATA-like motif (Ϫ143 to Ϫ135) as phloem specificity elements (10,11,16). Proteins binding to all these regions were detected in nuclear extracts from rice. It was also found that the vascular bundle activity was suppressed in some transgenic rice lines with methylated promoter sequences, 2 indicating that methylation directly or indirectly alters the assembly of transcription factors on the promoter.In this report, we have further analyzed DNA-protein interactions of nuclear proteins with the vascular bundle expression element located in the region between Ϫ165 and Ϫ100 with a detailed mutagenesis analysis and have compared factor-binding to methylated and nonmethylated sequences. Our results show that independent of the met...

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Estradiol down regulates the binding activity of an avian vitellogenin gene repressor (MDBP-2) and triggers a gradual demethylation of the mCpG pair of its DNA binding site

Abstract: A negative regulating protein (MDBP-2) from rooster liver nuclear extracts binds preferentially to a methylated promoter region 5'TTCACCTTmCGCTATG-

Cited by 52 publications

References 30 publications

Non‐histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes

Non‐histone protein 1 (NHP1) is a member of the Ku protein family which is upregulated in differentiating mouse myoblasts and human promyelocytes

Inactive chromatin spreads from a focus of methylation.

Sequence-specific and Methylation-dependent and -independent Binding of Rice Nuclear Proteins to a Rice Tungro Bacilliform Virus Vascular Bundle Expression Element

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