SAGA/ATAC complexes sustain aberrant chromatin regulation and promote tumorigenesis in diffuse midline glioma [CUT&Run]

Diffuse midline gliomas (DMG) are aggressive pediatric brain tumors characterized by chromatin and transcriptional dysregulation induced by H3K27M mutations. Strategies for overcoming epigenetic dysfunction to reduce DMG tumorigenesis remain limited. We identified multiple components of the SAGA and ATAC chromatin regulatory complexes as DMG genetic dependencies and found that genetic or pharmacological inhibition of the SAGA/ATAC-associated chromatin reader, SGF29, reduces DMG proliferation. Small molecules targeting SAGA/ATAC-associated histone acetylation, ubiquitination, and methylation similarly suppressed DMG growth. Further chromatin profiling and RNAseq analyses reveal that SGF29 controls H3K9ac and H3K4me3 dynamics at both H3K27M-bound and H3K27M-independent target genes linked to proliferation, differentiation, and metabolism. Finally, we find that SAGA/ATAC inhibition may reduce DMG viability by repressing cholesterol metabolism gene expression and show that combinations of cholesterol- and SAGA/ATAC-targeting drugs synergistically reduce DMG growth. These findings reveal a functional link between SAGA/ATAC-dependent chromatin regulation and both transcriptional and metabolic dysregulation underlying DMG malignancy. Overall design: For CUT&RUN, a single cell suspension of 5x105 of control of SGF29KO SU-DIPGXIII+Cas9 cells were immobilized on ConA beads, permeabilized with 0.01% Digitonin, incubated with primary antibodies, and then cut with pAG-MNase to release DNA fragments as previously described. The following antibodies were used in CUT&RUN analyses: rabbit IgG (cat# C15410206, Diagenode), H3K4me3 (cat# C15410003, Diagenode), H3K9ac (cat# 9649, Cell Signaling Technology), and H3K27M (cat# ABE419, Millipore Sigma). Yeast spike-in DNA (S. cerevisiae; Cell Signaling Technology) was added at 0.5% to each reaction following chromatin digestion.