Molecular mechanism of KAP1-dependent transcriptional silencing

Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for at least half of the human genome. Transcription of transposable elements is tightly regulated to restrict their proliferation and prevent toxic gene expression. A major factor contributing to the repression of potentially harmful retroelements is KRAB-associated protein 1 (KAP1, also known as TRIM28 or TIF1β). Following its recruitment to retrotransposons by sequence-specific KRAB domain-containing zinc finger proteins (KRAB-ZFPs), KAP1 induces the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. To understand the molecular basis of KAP1-dependent transcriptional regulation I determined the crystal structure of the RBCC domain of KAP1. My structural and biophysical data demonstrate that KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevented higher-order oligomerization and resulted in significant defects in KAP1-dependent transcriptional repression, suggesting that self-assembly may contribute to KAP1 function. Furthermore, I characterized the interaction of KAP1 with the KRAB domain of KRAB-ZFPs, which is crucial for recruitment of KAP1 to its genomic targets. My data show that each KAP1 dimer can only bind a single KRAB domain, resulting in a 2:1 stoichiometry. Moreover, my crystal structure of the KAP1 RBCC dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 RBCC domain responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Stoll, Guido Alexander

Advisor dc:contributor.advisor

• Modis, Yorgo

• transcriptional repressor
• epigenetic silencing
• endogenous retrovirus
• transposable element
• ubiquitin E3 ligase

Licence

• https://www.repository.cam.ac.uk/bitstreams/1f90dd8e-988d-4327-a70f-ca9e2a494909/download
• https://creativecommons.org/licenses/by/4.0/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.59801

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/312703