Using disease-causing and population variants to dissect ATRX protein function

Rare diseases are often caused by mutations in a single gene. Using the power of Mendelian genetics, we can analyse how disease-causing mutations might impact protein function. Change of a single amino acid in a functional domain (e.g. at a protein-protein interaction interface or an enzyme active site) may be sufficient to disrupt its function. When disruption of a functional domain causes disease, it highlights the biological importance of this domain. Conversely, neutral variants present in the general population may also be sufficient to impact domain function, revealing instead that a domain may be dispensable for protein function.

Project aim: 

Use disease-causing and population variants to dissect the biological importance of functional domains in ATRX protein

ATRX is a chromatin remodelling ATPase that is involved in several cellular processes including heterochromatin maintenance, transcriptional regulation and DNA damage repair. ATRX protein is 2492 amino acids in length and multiple functional domains have been mapped. It is encoded by the X-linked ATRX gene. To dissect ATRX protein function, the student will analyse four types of variants:

1. Partial loss-of-function (hypomorphic) variants: these cause a rare neurological disorder called ATR-X syndrome in hemizygous males. Patient mutations are usually inherited from heterozygous carrier females who are protected by skewed X chromosome inactivation (XCI). Strikingly, missense and short in-frame insertions/deletions (indels) found in patients cluster in two domains: the N-terminal ADD (ATRX-DNMT3-DNMT3L) domain that recognises H3K9me3 and the C-terminal ATPase domain.
2. Complete loss-of-function variants: these are incompatible with mammalian life (knockout mice die early in embryogenesis). They may be present in heterozygous females in the general population (with skewed XCI). They may also be present in cancer, where mutation of ATRX leads to activation of the ALT (Alternative Lengthening of Telomeres) pathway.
3. Neutral variants: these are found in hemizygous males in the general population.
4. Structure-based variants

The student will use a combination of in vitro and in vivo techniques to investigate variant function. In vitro assays will include the assessment of protein-protein interactions and chromatin remodelling activity. In vivo assessment of variants will be conducted in cultured cells using both transient transfection and CRISPR/Cas9-generated knock-in cell lines. They will determine how variants impact ATRX protein level, subcellular localisation, chromatin binding, transcriptional regulation, DNA methylation and suppression of the ALT pathway. Overall, this project will further our understanding of the biological importance of ATRX protein domains in both development and cancer.