Molecular Characterisation of the Regulatory HARE-HTH and PHD domains of Additional Sex Combs-like 1-3
Post-translational modification of Histone proteins plays a major role in Histone-DNA packaging. Attachment of Ubiquitin to the C-terminal tail of Histone H2A (H2AK119Ub) represses gene expression and is removed by the Polycomb Repressive Deubiquitinase (PR-DUB) complex. The core PR-DUB is comprised of a catalytic deubiquitinase, BRCA1-associated protein 1 (BAP1), and the regulatory proteins Additional Sex Combs-like 1-3 (ASXL1-3).
While the basis for ASXL1-3 in activating BAP1 is well-established, the roles of the HARE-HTH and Plant Homeodomain (PHD) of ASXL proteins are poorly understood. Truncating mutations in ASXL1-3 cause loss of the PHD domain and often occur in leukemia, being mutated in ~11% of all hematopoietic cancers.
Typically, one would expect a PHD domain to bind histone modifications for nucleosomal localisation. Instead, we found that the ASXL PHD is atypical and was previously mischaracterized. As such, the ASXL PHD finger was not able to bind any of the Histone marks that we tested. Later, an AlphaFold model of the ASXL2 PHD domain was derived.
This model revealed that the ASXL PHD has diverged significantly from domains of a similar fold, to attain a unique structure and an undescribed functional role. An interactome for the ASXL PHD was generated by fusion to the TurboID promiscuous biotin ligase, expression in HEK293 cells, comparative Mass Spectrometry, and direct interaction studies, thereafter. Finally, chromatin localization-sequencing was used to study the DNA-binding capabilities of the N-terminal ASXL HARE-HTH.
From this data, we were able to identify a consensus binding-motif and target gene- binding. Taken together, these studies identify several unanticipated mechanisms by which the PHD and HARE-HTH domains of ASXL1-3 direct PR-DUB activity with both protein- and DNA-interactions.
Meeting ID: 977 5670 4741
|Date||Tuesday, 13 September 2022|
|Time||12:00pm - 1:00pm|
|Audience||Undergraduate students,Postgraduate students,Staff|
Online and in-person
|Location||Biochemistry Seminar Room G.13 (BIG13) and via Zoom, Dunedin|
|Contact Name||Department of Biochemistry|