In this report, we provided ample evidence demonstrating that ADDATRX is a novel H3K9me3 recognition module, which senses the methylation states at both H3K9 and H3K4. Co-crystal structures at atomic resolution revealed a fascinating composite and atypical H3K9me3 binding pocket formed between the GATA- and the PHD- fingers of ADDATRX. Point mutations in this binding pocket substantially compromised AD-DATRX/H3K9me3 binding in vitro and the PCH localization of ATRX in vivo. Complementing the structural analysis, genetic studies provided further support that H3K9me3 is critical for the PCH localization of Atrx. Importantly, ATRX patient mutations in ADDATRX impaired the PCH localization, further indicating the importance of the ADD domain as a critical determinant of proper ATRX localization in vivo.
PCH is fundamental for sister chromatid cohesion, and proper chromatid cohesion indeed requires the H3K9me3 HMTases37,38
. Loss of Atrx has been shown to result in a chromosome segregation defect due to the attenuated cohesion and congression of sister chromatids39
, which may lead to increased apoptosis and a reduced number of neurons in the brain39,40
. Thus, our findings suggest that Atrx is an important component in the Suv39h/H3K9me3 pathway that is required for faithful chromosome segregation ().
Model of the mechanism underlying ATR-X syndrome caused by the mutations in ADDATRX
While chromosome missegregation would suggest a role for Atrx during M-phase, compromised heterochromatin replication in S-phase could also cause mitotic catastrophe as observed for the loss of other chromatin modifying proteins41,42
. Atrx is recruited to the telomere during S-phase and loss of Atrx results in a DNA damage response and de-repression of telomere-associated RNA18,43
. Notably, Atrx coordinates deposition of a variant histone with a histone H3.3-specific chaperone Daxx18,44,45
. H3.3 deposition at PCH and telomeres44,45
may contribute to heterochromatin formation. Taken together, Atrx may be required for faithful replication of both telomeric and pericentromeric heterochromatin.
Importantly, our structural research has identified an unanticipated mode of histone mark recognition, namely the utilization of integrated modules to generate a composite and atypical binding pocket at the interface between the GATA and PHD fingers of ATRX for Kme3 recognition. The concept of integrated modules acting as a single functional unit was recently highlighted in the structures of DPF3b double PHD finger bound to the H3K14ac mark46
and the PHD-Bromo cassette of TRIM24 bound to the H3K23ac mark47
, though in each case the Kac mark was recognized solely by a single module. Taken together, these findings call attention to the role of integrated modules and our newly proposed concept of composite reader pockets, which we anticipate will be increasingly identified amongst various epigenetic regulators associated with histone mark recognition and readout.
In summary, we have uncovered the function of ADDATRX as a recognition motif for the distinctive histone methylation pattern at PCH, representing the first report linking a novel histone methylation sensing mechanism to human brain function. Our study may also open a new avenue for therapeutic intervention of ATR-X syndrome.