Understanding the molecular mechanism of KAP1-dependent transcriptional silencing
- Department of Medicine, University of Cambridge, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
- Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Approximately half of the human genome consists of transposable elements (TEs), some of which are still replication-competent. TEs represent major drivers of evolution and there is mounting evidence that they fulfil important functions in early embryonic development. At the same time, they have the potential to severely damage the host genome if allowed to replicate unchecked. The activity of retrotransposons consequently must be tightly controlled. A major factor contributing to the repression of potentially harmful retroelements in mammals is the transcriptional regulator KRAB-associated protein 1 (KAP1). Following its recruitment to retrotransposons by KRAB domain-containing zinc finger proteins (KRAB-ZFPs), KAP1 induces epigenetic silencing of these elements by coordinating the assembly of a large repressor complex comprising the histone methyltransferase SETDB1, heterochromatin-associated protein 1 (HP1) and the Nucleosome Remodelling and Deacetylase (NuRD) complex. Despite the essential role of KAP1 in preserving genome integrity, structural and biophysical data on this protein is scarce and consequently, many aspects of this process remain poorly understood. To elucidate the molecular basis of KAP1-dependent transcriptional regulation, we characterized assembly and stoichiometry of KAP1 complexes. Multi-angle light scattering (MALS) and analytical ultracentrifugation (AUC) revealed that KAP1 is dimeric in solution but assembles into higher-order oligomers with increasing protein concentration. We subsequently determined the affinity of KAP1 self-association and mapped the protein domains mediating oligomerization. Possible functional consequences of this behavior are discussed. Furthermore, we present our progress towards high-resolution structures of KAP1-containing repressor complexes using a combination of X-ray crystallography and electron cryomicroscopy (cryo-EM).