Distinct contributions of DNA methylation and histone acetylation to the genomic occupancy of transcription factors.

Epigenetic modifications on chromatin play important roles in regulating gene expression. Although chromatin states are often governed by multilayered structure, how individual pathways contribute to gene expression remains poorly understood. For example, DNA methylation is known to regulate transcription factor binding but also to recruit methyl-CpG binding proteins that affect chromatin structure through the activity of histone deacetylase complexes (HDACs). Both of these mechanisms can potentially affect gene expression, but the importance of each, and whether these activities are integrated to achieve appropriate gene regulation, remains largely unknown. To address this important question, we measured gene expression, chromatin accessibility, and transcription factor occupancy in wild-type or DNA methylation-deficient mouse embryonic stem cells following HDAC inhibition. We observe widespread increases in chromatin accessibility at retrotransposons when HDACs are inhibited, and this is magnified when cells also lack DNA methylation. A subset of these elements has elevated binding of the YY1 and GABPA transcription factors and increased expression. The pronounced additive effect of HDAC inhibition in DNA methylation-deficient cells demonstrates that DNA methylation and histone deacetylation act largely independently to suppress transcription factor binding and gene expression.

DNA methylation in germ cell tumour aetiology: current understanding and outstanding questions.

Germ cell tumours (GCTs) are a diverse group of neoplasms that can be histologically subclassified as either seminomatous or non-seminomatous. These two subtypes have distinct levels of differentiation and clinical characteristics, the non-seminomatous tumours being associated with poorer prognosis. In this article, we review how different patterns of aberrant DNA methylation relate to these subtypes. Aberrant DNA methylation is a hallmark of all human cancers, but particular subsets of cancers show unusually high frequencies of promoter region hypermethylation. Such a 'methylator phenotype' has been described in non-seminomatous tumours. We discuss the possible cause of distinct methylation profiles in GCTs and the potential of DNA methylation to provide new targets for therapy. We also consider how recent developments in our understanding of this epigenetic modification and the development of genome-wide technologies are shedding new light on the role of DNA methylation in cancer aetiology.

Illuminating the Dark: Highly Selective Inhibition of Serine/Threonine Kinase 17A with Pyrazolo[1,5-a]pyrimidine-Based Macrocycles.

Serine/threonine kinase 17A (death-associated protein kinase-related apoptosis-inducing protein kinase 1─DRAK1) is a part of the death-associated protein kinase (DAPK) family and belongs to the so-called dark kinome. Thus, the current state of knowledge of the cellular function of DRAK1 and its involvement in pathophysiological processes is very limited. Recently, DRAK1 has been implicated in tumorigenesis of glioblastoma multiforme (GBM) and other cancers, but no selective inhibitors of DRAK1 are available yet. To this end, we optimized a pyrazolo[1,5-a]pyrimidine-based macrocyclic scaffold. Structure-guided optimization of this macrocyclic scaffold led to the development of CK156 (34), which displayed high in vitro potency (KD = 21 nM) and selectivity in kinomewide screens. Crystal structures demonstrated that CK156 (34) acts as a type I inhibitor. However, contrary to studies using genetic knockdown of DRAK1, we have seen the inhibition of cell growth of glioma cells in 2D and 3D culture only at low micromolar concentrations.

Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours' biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription-quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.