Arthur D. Riggs

X inactivation, differentiation, and DNA methylation

A model based on DNA methylation is proposed to explain the initiation and maintenance of mammalian X inactivation and certain aspects of other permanent events in eukaryotic cell differentiation. A key feature of the model is the proposal of sequence-specific DNA methylases that methylate unmethylated sites with great difficulty but easily methylate half-methylated sites. Although such enzymes have not yet been detected in eukaryotes, they are known in bacteria. An argument is presented, based on recent data on DNA-binding proteins, that DNA methylation should affect the binding of regulatory proteins. In support of the model, short reviews are included covering both mammalian X inactivation and bacterial restriction and modification enzymes.

5-Methylcytosine, Gene Regulation, and Cancer

Publisher Summary This chapter focuses on 5-methylcytosine, gene regulation, and cancer. The regulation of mammalian gene expression clearly is accomplished by multiple control systems operating at several levels. Some obvious levels of control include chromosome condensation, chromatin structure transcriptional control by repressors and activators, RNA processing, and translational control. The chapter presents the argument for a newly recognized additional component of mammalian gene control and enzymatic DNA methylation and points out the relevance of this gene control system to cancer. Mammalian DNA is modified shortly after replication by the enzymatic conversion of about 3% of cytosines to 5-methylcytosine. 5-Methylcytosine is the only naturally occurring modified base yet found in mammalian DNA. The chapter focuses primarily on the work of the past 2 years, considering first the general aspects of methylation and gene control and then the cancer-related aspects.

X-chromosome inactivation and cell memory

Mammalian X-chromosome inactivation is an excellent example of the faithful maintenance of a determined chromosomal state. As such, it may provide insight into the mechanisms for cell memory, defined as the faithful maintenance of a determined state in clonally derived progeny cells. We review here the aspects of X-chromosome inactivation that are relevant to cell memory and discuss the various molecular mechanisms that have been proposed to explain its occurrence, with emphasis on DNA methylation and a recently proposed mechanism that depends on the timing of replication.

A human B cell methylome at 100−base pair resolution

Using a methylated-DNA enrichment technique (methylated CpG island recovery assay, MIRA) in combination with whole-genome tiling arrays, we have characterized by MIRA-chip the entire B cell “methylome” of an individual human at 100-bp resolution. We find that at the chromosome level high CpG methylation density is correlated with subtelomeric regions and Giemsa-light bands (R bands). The majority of the most highly methylated regions that could be identified on the tiling arrays were associated with genes. Approximately 10% of all promoters in B cells were found to be methylated, and this methylation correlates with low gene expression. Notably, apparent exceptions to this correlation were the result of transcription from previously unidentified, unmethylated transcription start sites, suggesting that methylation may control alternate promoter usage. Methylation of intragenic (gene body) sequences was found to correlate with increased, not decreased, transcription, and a methylated region near the 3′ end was found in approximately 12% of all genes. The majority of broad regions (10–44 kb) of high methylation were at segmental duplications. Our data provide a valuable resource for the analysis of CpG methylation patterns in a differentiated human cell type and provide new clues regarding the function of mammalian DNA methylation.

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.

DNA Methylation and Demethylation in Mammals

Cell type-specific DNA methylation patterns are established during mammalian development and maintained in adult somatic cells. Understanding how these patterns of 5-methylcytosine are established and maintained requires the elucidation of mechanisms for both DNA methylation and demethylation. The enzymes involved in the de novo methylation of DNA and the maintenance of the resulting methylation patterns have been fairly well characterized. However, important remaining challenges are to understand how DNA methylation systems function in vivo and in the context of chromatin. In addition, the enzymes and mechanisms for demethylation remain to be elucidated. There is still no consensus as to how active enzymatic demethylation is achieved in mammalian cells, but recent studies implicate base excision repair for genome-wide DNA demethylation in germ cells and early embryos.

High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer

Changes in DNA methylation patterns are an important characteristic of human cancer. Tumors have reduced levels of genomic DNA methylation and contain hypermethylated CpG islands, but the full extent and sequence context of DNA hypomethylation and hypermethylation is unknown. Here, we used methylated CpG island recovery assay-assisted high-resolution genomic tiling and CpG island arrays to analyze methylation patterns in lung squamous cell carcinomas and matched normal lung tissue. Normal tissues from different individuals showed overall very similar DNA methylation patterns. Each tumor contained several hundred hypermethylated CpG islands. We identified and confirmed 11 CpG islands that were methylated in 80–100% of the SCC tumors, and many hold promise as effective biomarkers for early detection of lung cancer. In addition, we find that extensive DNA hypomethylation in tumors occurs specifically at repetitive sequences, including short and long interspersed nuclear elements and LTR elements, segmental duplications, and subtelomeric regions, but single-copy sequences rarely become demethylated. The results are consistent with a specific defect in methylation of repetitive DNA sequences in human cancer.

Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay.

De novo methylation of CpG islands is a common phenomenon in human cancer, but the mechanisms of cancer-associated DNA methylation are not known. We have used tiling arrays in combination with the methylated CpG island recovery assay to investigate methylation of CpG islands genome-wide and at high resolution. We find that all four HOX gene clusters on chromosomes 2, 7, 12, and 17 are preferential targets for DNA methylation in cancer cell lines and in early-stage lung cancer. CpG islands associated with many other homeobox genes, such as SIX, LHX, PAX, DLX, and Engrailed, were highly methylated as well. Altogether, more than half (104 of 192) of all CpG island-associated homeobox genes in the lung cancer cell line A549 were methylated. Analysis of paralogous HOX genes showed that not all paralogues undergo cancer-associated methylation simultaneously. The HOXA cluster was analyzed in greater detail. Comparison with ENCODE-derived data shows that lack of methylation at CpG-rich sequences correlates with presence of the active chromatin mark, histone H3 lysine-4 methylation in the HOXA region. Methylation analysis of HOXA genes in primary squamous cell carcinomas of the lung led to the identification of the HOXA7- and HOXA9-associated CpG islands as frequent methylation targets in stage 1 tumors. Homeobox genes are potentially useful as DNA methylation markers for early diagnosis of the disease. The finding of widespread methylation of homeobox genes lends support to the hypothesis that a substantial fraction of genes methylated in human cancer are targets of the Polycomb complex.

lac represser binding to non-operator DNA: Detailed studies and a comparison of equilibrium and rate competition methods☆

Competition experiments have been used to measure the interaction of Escherichia coli lac represser with many natural and synthetic DNAs that do not contain the lac operator (non-operator DNA). Two types of experiments were done: (1) equilibrium competition experiments, where the effect of competing DNA on the equilibrium concentration of represser-operator complex was measured, and (2) rate competition experiments, where the effect of competing DNA on the rate of represser-operator complex formation was measured. The second method is an order of magnitude more sensitive than the first. For both types of experiments, we present methods for the estimation of Krd, the apparent equilibrium dissociation constant for represser binding to non-operator DNA. The methods are compared and we find that both yield similar Krd values. Earlier work is confirmed that represser binds preferentially to natural DNAs of high A + T content and that poly[d(A–T)]is a very good competitor. Poly[r(A,U)], tRNA, and rRNA are very poor competitors. The glucosylated DNAs, T2 and T4, compete well, about as expected from their A + T content. Denatured non-operator DNA competes approximately the same as native DNA. Effector ligands could not be shown to affect non-operator binding. For most DNAs, Krd can be calculated only in weight units, but for poly[d(A-T)]we find that Krd = 1–3 × 10−8 m. The effect of reaction conditions (ionic strength, pH, temperature) on Krd was investigated. Contrary to earlier conclusions, non-operator DNA binding is not preferentially reduced at high ionic strength. These results are significant for several areas of current interest: (a) the fractionation and purification of other DNA-binding proteins, (b) the general problem of demonstrating specific DNA binding, and (c) the deatiled mechanism of regulatory protein-DNA interaction.

The histone methyltransferase SETDB1 and the DNA methyltransferase DNMT3A interact directly and localize to promoters silenced in cancer cells

DNA CpG methylation can cooperate with histone H3 lysine 9 (H3-K9) methylation in heterochromatin formation and gene silencing. Trimethylation of H3-K9 by the recently identified euchromatic histone methyltransferase SETDB1/ESET may be responsible for transcriptional repression of certain promoters. Here, we show that SETDB1 associates with endogenous DNA methyltransferase activity. SETDB1 interacts with the de novo DNA methyltransferases DNMT3A and DNMT3B but not with the maintenance methyltransferase DNMT1. The interaction of SETDB1 with DNMT3A was further characterized and confirmed by in vivo and in vitro interaction studies. A direct interaction of the two proteins occurs through the N terminus of SETDB1 and the plant homeodomain of DNMT3A. Co-expression of SETDB1 and DNMT3A was essential for repression of reporter gene expression in a Gal4-based tethering assay and resulted in their recruitment to the artificial promoter. We further demonstrate that the CpG-methylated promoters of the endogenous p53BP2 gene in HeLa cells and the RASSF1A gene in MDA-MB-231 cells are simultaneously occupied by both SETDB1 and DNMT3A proteins, which provides evidence for SETDB1 being at least partly responsible for H3-K9 trimethylation at the promoter of RASSF1A, a gene frequently silenced in human cancers. In summary, our data demonstrate the direct physical interaction and functional connection between the H3-K9 trimethylase SETDB1 and the DNA methyltransferase DNMT3A and thus contribute to a better understanding of the complexity of the self-reinforcing heterochromatin machinery operating at silenced promoters.