Readers of DNA and Histone modifications in Development

Abstract

The development of an adult organism from a fertilized egg is accompanied by the generation of some 200 different cell types. Gene expression is highly regulated, such that each cell type expresses a specific subset of genes. The genome in all these cells stays the same during cellular differentiation, which leads to the question: How does one genotype produce so many different phenotypes? Epigenetics is at least part of the answer. It is defined as DNA-sequence independent changes in gene expression and phenotype. In higher eukaryotes this is mainly achieved through methylation of DNA on cytosines and by post-translational modifications of histones. The patterns of these modifications are dynamic during development and the modifications thus help to create cell-type-specific gene expression profiles. The modifications can recruit regulatory proteins that can than exert their function at the site of recruitment. The specific binding of these so-called chromatin ‘readers’ therefore significantly contributes to the biological function of each individual epigenetic modification.

In this thesis, we have studied readers for novel DNA modifications: hydroxymethylcytosine, formylcytosine and carboxylcytosine. We also identified many proteins that preferentially bind non-methylated DNA. We therefor propose that DNA methylation is a context-dependent binding signal for many proteins, whereas only a few proteins recognize the mark in a sequence-independent manner. Furthermore we found multiple tissue-specific readers for the histone modifications H3K4me3 and H3K9me3 in kidney, liver and brain. Finally this thesis focuses on one of the protein complexes that specifically recognizes DNA as well as histone modifications. The Nucleosome Remodeling and Deacetylase (NuRD) complex is a multi-subunit complex for which the biology is not fully understood. The complex is thought to repress transcription as it contains deacetylase activity. It plays an important role in development and many mutations of NuRD subunits are found in different types of cancer. We identified CDK2AP1 (also called DOC-1) as a bona fide subunit of the complex, although its role in the complex is still unclear. Since the mechanism of NuRD recruitment to target genes is largely unknown, we also focused on ZMYND8 and its zinc-finger containing interaction partners. These are probably capable of sequence-specific DNA binding. Since ZMYND8 also interacts with the NuRD complex, this could be one of the mechanisms of NuRD recruitment to promoters lacking DNA methylation. The NuRD subunit MBD2 is a reader for DNA methylation that can recruit the complex to methylated promoters.

In conclusion, understanding the function and the mechanisms that underlie epigenetic regulation of gene expression is very important, because epigenetics is at the basis of every process in a cell. Studying this phenomenon will result in insights about possible intervention points in diseases such as cancer. Research into the basic working mechanisms of cells is thus very important for future development of medication.