O'Carroll, Donal

Cook, Atlanta

Dias Mirandela, Madeleine

2024-03-11T12:15:09Z

2024-03-11T12:15:09Z

2024-03-11

The survival of the germline is essential for the survival of a species. Yet, the integrity of those cells is threatened by transposons during foetal development. In fact, failure to silence transposons results in male infertility in mammals. The PIWI-interacting RNA (piRNA) pathway is the primary germline defence system which places CpG DNA methylation at transposon loci. piRNAs recruit the PIWI protein MIWI2 to nascent transposon transcripts via complementary base pairing and instruct DNA methylation through SPOCD1. In this work, I defined the interaction of SPOCD1 with three downstream factors, SPIN1, C19ORF84 and TPR. C19ORF84 interacts with a c-terminal α-helix of SPOCD1 and TPR interacts with the SPOCD1 TFIIS-M domain. In addition, I solved the structure of the SPOCD1 SPOC domain, whose function remains to be determined, at 1.7 Å. I then focused on the SPOCD1-SPIN1 interaction. I showed that SPOCD1 directly interacts with SPIN1, a chromatin reader that recognises H3K4me3 and H3K9me3. I found that SPIN1 and SPOCD1 are interacting before MIWI2 is expressed which challenges the current view of all molecular events required for piRNA-directed DNA methylation occurring after the engagement of MIWI2. In addition, I saw that young LINE1 transposon copies are marked by the SPIN1-associated chromatin marks before the initiation of piRNA-directed DNA methylation. I then generated a Spocd1 separation-of-function allele in the mouse encoding a SPOCD1 variant that can no longer interact with SPIN1. I showed that the SPOCD1-SPIN1 interaction is in fact essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 transposons. In summary, this work defines the interaction of SPOCD1 with SPIN1, C19ORF84 and TPR, gives an insight into the SPOCD1 SPOC domain structure and defines the importance of the SPOCD1-SPIN1 interaction which challenges the current model and proposes a novel two-factor authentication system to form the basis of precision.

https://hdl.handle.net/1842/41609

http://dx.doi.org/10.7488/era/4341

en

The University of Edinburgh

SPOCD1

de novo DNA methylation

piRNA-directed de novo DNA methylation

PIWI-interacting RNA (piRNA) pathway

CpG DNA methylation

Understanding the role and function of SPOCD1 in piRNA-directed de novo DNA methylation

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy