Paul A. Wade

Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Methylation of DNA at the dinucleotide CpG is essential for mammalian development and is correlated with stable transcriptional silencing. This transcriptional silencing has recently been linked at a molecular level to histone deacetylation through the demonstration of a physical association between histone deacetylases and the methyl CpG-binding protein MeCP2 (refs 4,5). We previously purified a histone deacetylase complex from Xenopus laevis egg extracts that consists of six subunits, including an Rpd3-like deacetylase, the RbA p48/p46 histone-binding protein and the nucleosome-stimulated ATPase Mi-2 (ref. 6). Similar species were subsequently isolated from human cell lines, implying functional conservation across evolution. This complex represents the most abundant form of deacetylase in amphibian eggs and cultured mammalian cells. Here we identify the remaining three subunits of this enzyme complex. One of them binds specifically to methylated DNA in vitro and molecular cloning reveals a similarity to a known methyl CpG-binding protein. Our data substantiate the mechanistic link between DNA methylation, histone deacetylation and transcriptional silencing.

Rhythmic histone acetylation underlies transcription in the mammalian circadian clock

In the mouse circadian clock, a transcriptional feedback loop is at the centre of the clockwork mechanism. Clock and Bmal1 are essential transcription factors that drive the expression of three period genes (Per1–3) and two cryptochrome genes (Cry1 and Cry2). The Cry proteins feedback to inhibit Clock/Bmal1-mediated transcription by a mechanism that does not alter Clock/Bmal1 binding to DNA. Here we show that transcriptional regulation of the core clock mechanism in mouse liver is accompanied by rhythms in H3 histone acetylation, and that H3 acetylation is a potential target of the inhibitory action of Cry. The promoter regions of the Per1, Per2 and Cry1 genes exhibit circadian rhythms in H3 acetylation and RNA polymerase II binding that are synchronous with the corresponding steady-state messenger RNA rhythms. The histone acetyltransferase p300 precipitates together with Clock in vivo in a time-dependent manner. Moreover, the Cry proteins inhibit a p300-induced increase in Clock/Bmal1-mediated transcription. The delayed timing of the Cry1 mRNA rhythm, relative to the Per rhythms, is due to the coordinated activities of Rev-Erbα and Clock/Bmal1, and defines a new mechanism for circadian phase control.

Histone acetylation: chromatin in action

Histone acetylation acts as a landmark and determinant for chromatin function. Active roles in the transcription and assembly of chromatin have been discovered for histone acetyltransferases and deacetylases. This review highlights these roles and discusses their significance for the maintenance of cell differentiation.

MTA3, a Mi-2/NuRD Complex Subunit, Regulates an Invasive Growth Pathway in Breast Cancer

Estrogen receptor is a key regulator of proliferation and differentiation in mammary epithelia and represents a crucial prognostic indicator and therapeutic target in breast cancer. Mechanistically, estrogen receptor induces changes in gene expression through direct gene activation and also through the biological functions of target loci. Here, we identify the product of human MTA3 as an estrogen-dependent component of the Mi-2/NuRD transcriptional corepressor in breast epithelial cells and demonstrate that MTA3 constitutes a key component of an estrogen-dependent pathway regulating growth and differentiation. The absence of estrogen receptor or of MTA3 leads to aberrant expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transitions. Aberrant Snail expression results in loss of expression of the cell adhesion molecule E-cadherin, an event associated with changes in epithelial architecture and invasive growth. These results establish a mechanistic link between estrogen receptor status and invasive growth of breast cancers.

A multiple subunit Mi-2 histone deacetylase from Xenopus laevis cofractionates with an associated Snf2 superfamily ATPase

Chromatin structure plays a crucial regulatory role in the control of gene expression. In eukaryotic nuclei, enzymatic complexes can alter this structure by both targeted covalent modification and ATP-dependent chromatin remodeling. Modification of histone amino termini by acetyltransferases and deacetylases correlates with transcriptional activation and repression [1-3], cell growth [4], and tumorigenesis [5]. Chromatin-remodeling enzymes of the Snf2 superfamily use ATP hydrolysis to restructure nucleosomes and chromatin, events which correlate with activation of transcription [6,7]. We purified a multi-subunit complex from Xenopus laevis eggs which contains six putative subunits including the known deacetylase subunits Rpd3 and RbAp48/p46 [8] as well as substoichiometric quantities of the deacetylase-associated protein Sin3 [9-13]. In addition, we identified one of the other components of the complex to be Mi-2, a Snf2 superfamily member previously identified as an autoantigen in the human connective tissue disease dermatomyositis [14,15]. We found that nucleosome-stimulated ATPase activity precisely copurified with both histone deacetylase activity and the deacetylase enzyme complex. This association of a histone deacetylase with a Snf2 superfamily ATPase suggests a functional link between these two disparate classes of chromatin regulators.

The human Mi-2/NuRD complex and gene regulation

The Mi-2/nucleosome remodeling and deacetylase (NuRD) complex is an abundant deacetylase complex with a broad cellular and tissue distribution. It is unique in that it couples histone deacetylation and chromatin remodeling ATPase activities in the same complex. A decade of research has uncovered a number of interesting connections between Mi-2/NuRD and gene regulation. The subunit composition of the enzyme appears to vary with cell type and in response to physiologic signals within a tissue. Here, we review the known subunits of the complex, their connections to signaling networks, and their association with cancer. In addition, we propose a working model that integrates the known biochemical properties of the enzyme with emerging models on how chromatin structure and modification relate to gene activity.

MTA3 and the Mi-2/NuRD Complex Regulate Cell Fate during B Lymphocyte Differentiation

The transcriptional repressor BCL-6 regulates B lymphocyte cell fate during the germinal center reaction by preventing terminal differentiation of B lymphocytes into plasma cells until appropriate signals are received. Here, we report a cofactor, MTA3, a cell type-specific subunit of the corepressor complex Mi-2/NuRD, for BCL-6-dependent cell fate determination. MTA3 is expressed in the same pattern in germinal centers as BCL-6. BCL-6 physically interacts with Mi-2/NuRD and this interaction is sensitive to BCL-6 acetylation status. Depletion of MTA3 by RNAi impairs BCL-6-dependent repression and alters the cell-specific transcriptional pattern characteristic of the B lymphocyte. Remarkably, exogenous expression of BCL-6 in a plasma cell line leads, in an MTA3-dependent manner, to repression of plasma cell-specific transcripts, reactivation of the B cell transcriptional program, expression of B lymphocyte cell surface markers, and reprogramming of cell fate.

Aberrant Expression of the Transcription Factors Snail and Slug Alters the Response to Genotoxic Stress

ABSTRACT Snail and Slug are closely related transcriptional repressors involved in embryonic patterning during metazoan development. In human cancer, aberrant expression of Snail and/or Slug has been correlated with invasive growth potential, a property primarily attributed to their ability to directly repress transcription of genes whose products are involved in cell-cell adhesion, such as E-cadherin, occludin, and claudins. To investigate the molecular mechanisms of alterations in epithelial cell fate mediated by aberrant expression of Snail or Slug, we analyzed the consequences of exogenous expression of these factors in human cancer cells. Aberrant expression of either Snail or Slug led to changes in cell morphology, the loss of normal cell-cell contacts, and the acquisition of invasive growth properties. Snail or Slug expression also promoted resistance to programmed cell death elicited by DNA damage. Detailed molecular analysis revealed direct transcriptional repression of multiple factors with well-documented roles in programmed cell death. Depletion of endogenous Snail by RNA interference led to increased sensitivity to DNA damage accompanied by increased expression of the proapoptotic factors identified as targets of Snail. Thus, aberrant expression of Snail or Slug may promote tumorigenesis through increased resistance to programmed cell death.