Analysis of Chromatin-Immunopurified MeCP2-Associated Fragments

doi:10.1006/bbrc.2001.6023

Biochemical and Biophysical Research Communications

Volume 289, Issue 3, 7 December 2001, Pages 733-737

https://doi.org/10.1006/bbrc.2001.6023 Get rights and content

Abstract

As molecular biologists, we are continuing to unravel the interactions by which DNA binding proteins mediate the expression of genes. The chromatin immunoprecipitation (ChIP) technique provides us with an exquisite tool to investigate the interplay between chromatin structure and its role in regulating transcription, replication, and recombination in vivo. We describe a robust assay used to identify the molecular determinants associated with chromatin. In this article we illustrate the ChIP technique and use the transcriptionally silent–hypermethylated multidrug resistance (MDR1) gene as the platform for methyl-CpG binding protein 2 (MeCP2) localization on chromatin. Driven by the hypothesis that repression is strongly dependent on the methylation profile of the endogenous promoter, we demonstrate that MDR1 is targeted by MeCP2. Methylated MDR1 chromatin is highly enriched with MeCP2 and is in striking contrast to localization observed in cells in which MDR1 is transcriptionally active. In a distinct model system we discuss experimental methods used to immunopurify the MeCP2 repressor complex on chromatin and quantify protein–DNA association by competitive PCR approach.

References (12)

X. Nan et al.
Cell
(1997)
F. Magdinier et al.
Proc. Natl. Acad. Sci. USA
(2001)
P.L. Jones et al.
Nat. Genet.
(1998)
X. Nan et al.
Nature
(1998)
B. Hendrich et al.
Mol. Cell. Biol.
(1998)

There are more references available in the full text version of this article.

Cited by (22)

High-Content Fluorescence-Based Screening for Epigenetic Modulators
2006, Methods in Enzymology
Citation Excerpt :
ChIP to measure histone acetylation at the integrated silenced promoter. As has been described by others (El Osta and Wolffe, 2001; Lambert and Nordeen, 2001), chromatin immunoprecipitation assays can be useful in identifying the modification status of particular histones at a given genomic loci. Cells can be treated with classical epigenetic modulators as described earlier in this chapter and with candidate compounds in parallel for various lengths of time, as determined by GFP induction time courses, taking into consideration that histone modifications presumably are a relatively early event in the induction process.
Epigenetic processes have gained a great amount of attention in recent years, particularly due to the influence they exert on gene transcription. Several human diseases, including cancer, have been linked to aberrant epigenetic pathways. Consequently, the cellular enzymes that mediate epigenetic events, including histone deacetylases and DNA methyltransferases, have become prime molecular targets for therapeutic intervention. The effective and specific chemical inhibition of these activities is a top priority in cancer research and appears to have therapeutic potential. This chapter describes the development of mammalian cell‐based fluorescent assays to screen for epigenetic modulators using an innovative combination of approaches. Detailed protocols for the use of the assays in drug screens, as well as for the initial characterization of hits, are provided. Furthermore, options for evaluating the mechanism of action of these compounds are presented and principles to govern the choice of hit compounds for the development of leads are discussed.
Identification of the molecular requirements for an RARα-mediated cell cycle arrest during granulocytic differentiation
2004, Blood
Citation Excerpt :
C/EBPϵ-ER MPROs were treated with 4-OH-tamoxifen for 0, 2, and 4 hours. ChIP was carried out essentially as described with some modifications29 (Upstate Biotechnology, Lake Placid, NY). All assays were performed in triplicate using 3 × 106 cells per time point.
Retinoids are potent inducers of cell cycle arrest and differentiation of numerous cell types, notably granulocytes. However the mechanisms by which retinoids mediate cell cycle arrest during differentiation remain unclear. We have used myeloid differentiation to characterize the molecular pathways that couple cell cycle withdrawal to terminal differentiation. Using primary cells from mice deficient for either the cyclin-dependent kinase inhibitor (CDKi) p27^Kip1, the Myc antagonist Mad1, or both Mad1 and p27^Kip1, we observed that signals mediated through retinoic acid receptor α (RARα), but not RARβ or γ, required both Mad1 and p27^Kip1 to induce cell cycle arrest and to accelerate terminal differentiation of granulocytes. Although RARα did not directly regulate Mad1 or p27^Kip1, the RARα target gene C/EBPϵ directly regulated transcription of Mad1. Induction of C/EBPϵ activity in granulocytic cells led to rapid induction of Mad1 protein and transcript, with direct binding of C/EBPϵ to the Mad1 promoter demonstrated through chromatin immunoprecipitation assay. These data demonstrate that cell cycle arrest in response to RARα specifically requires Mad1 and p27^Kip1 and that Mad1 is transcriptionally activated by CCAAT/enhancer-binding protein ϵ (C/EBPϵ). Moreover, these data demonstrate selectivity among the RARs for cell cycle arrest pathways and provide a direct mechanism to link differentiation induction and regulation of the Myc antagonist Mad1.
Rett syndrome and MeCP2: Linking epigenetics and neuronal function
2002, American Journal of Human Genetics
Citation Excerpt :
For example, the leukosialin gene, which is differentially methylated depending on the tissue, is repressed by MeCP2 in a transfection assay when the promoter is methylated, although whether MeCP2 binds to this promoter in vivo remains to be determined (Kudo 1998). In addition, the multidrug resistance gene promoter in drug-sensitive cells is hypermethylated and bound by MeCP2, whereas in drug-resistant cells, the promoter is hypomethylated, transcriptionally active, and relatively free of MeCP2 (El-Osta and Wolffe 2001; El-Osta et al. 2002). MeCP2 has also been implicated in the regulation of imprinted genes.
FMR1 silencing and the signals to chromatin: A unified model of transcriptional regulation
2002, Biochemical and Biophysical Research Communications
Citation Excerpt :
This concept is currently being explored using chromatin immunoprecipitaton (ChIP) and DNA microarray technologies with exquisite resolution [49,50]. The ChIP technique offers a powerful platform to directly identify molecular determinants associated with chromatin [51]. Capturing the molecular events involved in FMR1 silencing will identify regions on the allele that are resistant to 5aC treatment, this could conceivably lead to landmark genetic sequences that are susceptible to methyl-CpG protein release.
Understanding the effect of DNA methylation requires an appreciation of the determinants responsible for gene expression. To this end, we and others have demonstrated the exquisite silencing properties of methylation-dependent repressors that belong to the methyl-CpG binding domain (MBD) protein family. It is crucial, to establish a direct connection between the remodelling events observed with the fragile X mental retardation gene 1 (FMR1) promoter if we are to understand the mechanism of control. In this paper contrasting but not mutually exclusive models are discussed, which conceptually and experimentally reflects an appreciation of the surrounding chromatin environment. Examining general transcription systems controlled by DNA methylation, proteins that recognize the methyl-CpG moiety and the enzyme that maintains methylation, a unified model of FMR1 gene silencing are discussed.
Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells
2016, Oncotarget
Chimeric microRNA-1291 biosynthesized efficiently in Escherichia coli is effective to reduce target gene expression in human carcinoma cells and improve chemosensitivity
2015, Drug Metabolism and Disposition

View all citing articles on Scopus

¹: To whom correspondence and reprint requests should be addressed. E-mail: [email protected].

²: Deceased.

View full text

Regular ArticleAnalysis of Chromatin-Immunopurified MeCP2-Associated Fragments

Abstract

Cell

Proc. Natl. Acad. Sci. USA

Nat. Genet.

Nature

Mol. Cell. Biol.

Regular Article
Analysis of Chromatin-Immunopurified MeCP2-Associated Fragments