ATRX is a chaperone that loads histone variant H3.3 to direct heterochromatin formation at repeat DNA sequences including telomeres. One striking finding in the area of cancer epigenetics has been the identification of mutated histone genes (oncohistones) in paediatric glioblastomas (pGBMs). Two H3.3 mutations are found. The first mutation replaces lysine 27 with a methionine. The second one replaces glycine 34 with an arginine (G34R). H3.3G34R mutations always overlap with ATRX and Tp53 mutations, and these pGBMs are activated in the Alternative Lengthening of Telomeres (ALT) telomere maintenance pathway.
The ALT pathway is utilised in 15% of cancers for telomere maintenance. The common overlaps of H3.3G34R, ATRX and TP53 mutations in ALT-positive pGBMs suggests that these mutations cooperate to drive ALT. To better understand ALT, we have created ATRX gene knockout embryonic stem (ES) cell ALT models to recapitulate the initial molecular events linked to ALT activation. In these ATRX mutant ALT cell models, we have introduced mutations of H3.3 (a histone gene), TP53 (DNA checkpoint factor) and TERT (a telomerase gene).
Due to the loss of telomerase, the H3.3G34R/ATRX/TP53/TERT ALT mutant shows a gradual loss of telomere and hits a crisis after 12-14 months, but it recovers by activating ALT. We find that H3.3G34R inhibits the function of KDM4 histone demethylase - this acts with ATRX loss to promote aberrant chromatin and transcriptional states at the telomere to promote ALT.
Our success in inducing ALT in H3.3G34R/ATRX/TP53/TERT cell model verifies the roles of H3.3 and ATRX in ALT activation and our hypothesis that ALT activation is a multi-factorial process. We propose KDM4 network as a key driver that promotes ALT process in cancers. Our study provides insights into epigenetic defects linked to ATRX/H3.3 mutations and early molecular events associated with ALT.