Histone variant H3.3 is incorporated into DNA by ATRX to form heterochromatin structure at DNA repeats (i.e., telomere). One striking finding in cancer epigenetics has been the identification of mutated histone genes in paediatric glioblastomas (pGBMs). A glycine 34 to arginine substitution (H3.3G34R) is commonly observed and found to consistently overlap with ATRX mutations. These pGBMs are found to be activated in the Alternative Lengthening of Telomeres (ALT) telomere maintenance pathway. We recently discovered that H3.3G34R inhibits the histone demethylase: KDM4 (removes methyl marks from H3K9me3/H3K36me3) and KDM4B inactivation is the missing factor that cooperates with ATRX mutations to activate the ALT pathway (Udugama et al., 2021).
In eukaryotic cells, there are ~200 copies of ribosomal DNA repeats and only a small subset of them is actively transcribed at any given time. RNA polymerase I (Pol I)-driven transcription of rDNA produces rRNA, which is a crucial component of the ribosome. We previously showed that the inactivation of ATRX led to rDNA repeat loss and instability, leading to reduced rRNA transcription and sensitivity to the RNA Pol I inhibitor: CX-5461. Here, we investigate if H3.3G34R also affects rDNA integrity and rRNA transcription. To achieve this, we assess rDNA copy number, rRNA transcription, chromatin status, and sensitivity to RNA Polymerase I inhibitors in both mouse and human cell models carrying H3.3G34R and ATRX mutations. We find that:
1) H3.3G34R inhibits the function of KDM4B at rDNA, leading to reduced rRNA transcription
2) rRNA transcription is further reduced in double H3.3G34R and ATRX mutant cells.
3) Human pGBM cells show reduced rRNA transcription.
3) Mouse and human H3.3 mutant cells show increased sensitivity to CX-5461
Together, our studies suggest that the RNA Pol I inhibitor: CX-5461 could be used as a potential therapeutic strategy for treatment of ATRX and H3.3 mutated cancers.