Identification and characterization of ADNP as a novel heterochromatin component

Abstract

In eukaryotic cells genetic information in the form of long linear DNA fibers is stored in the cell nucleus. Time and tissue specific gene expression patterns are manifested by the combination of DNA with proteins resulting in a structure called chromatin. The basic repeating element of chromatin is the nucleosome, where DNA is wrapped around an octamer of histone proteins, interconnected by sections of linker DNA. Both DNA and histones are subject to chemical modifications, which are associated with certain chromatin states. Several factors that set or recognize such modifications have been described in the recent years. Heterochromatin is a chromatin form that is characterized by compaction, transcriptional inactivity and late replication in S-phase. It is associated with methylation of DNA and histones. Trimethylation of histone H3 lysine 9 (H3K9me3) is one hallmark of heterochromatin, and is mediated by the methyltransferase Suv39 and recognized by heterochromatin protein 1 (HP1), which has three isoforms in mammalian cells. How these and other enzymes and factors establish and maintain distinct chromatin states is not yet completely understood. For a better understanding of factors involved with heterochromatin I used stable isotope labeling by aminoacids in cell culture (SILAC) in a H3K9me3 pull-down experiment to identify new interaction partners. Activity dependent neuroprotector (ADNP) was one such factor that had not been described in a heterochromatin context before. Association of ADNP with H3K9me3 was verified with independent experiments and the factor was further characterized. Cell based and in vitro assays suggested that ADNP does not bind to H3K9me3 directly but is targeted to this modification by HP1. This recruitment of ADNP to H3K9me3 could be mediated by all three isoforms of HP1. Mapping of the interaction interface revealed a major contribution of the HP1 chromoshadow domain binding to a PxVxL motif within the ADNP homeodomain. Mutation of the PxVxL motif caused partial delocalization from heterochromatin. An additional mutation of an ARKS motif, which is also present in the ADNP homeodomain, enhanced this effect. However, involvment of a possible lysine methylation in that motif in HP1 binding was not detected. To determine the function of ADNP I used knock-down in cells by siRNA. I analyzed whether ADNP influences typical heterochromatin features such as distribution of histone modifications as well as HP1 localization as well as DNA methylation state. However, no effect on these properties could be detected. In luciferase reporter assays ADNP displayed transcriptional silencing potential. Knock-down and overexpression experiments suggested, that ADNP is specifically involved in silencing of major satellite repeats in pericentromeric heterochromatin. Further studies are needed to address by which mechanism ADNP exerts this silencing function. Altogether, in this work I identified ADNP as a novel component of pericentromeric heterochromatin, which is recruited by HP1 and acts in silencing of major satellite repeats. This study deepens the understanding of how the Suv39/H3K9me3/HP1 pathway impacts chromatin function.