FACT-seq: profiling histone modifications in formalin-fixed paraffin-embedded samples with low cell numbers.

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing ∼4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.

Glioblastoma stem cells express non-canonical proteins and exclusive mesenchymal-like or non-mesenchymal-like protein signatures.

Glioblastoma (GBM) cancer stem cells (GSCs) contribute to GBM's origin, recurrence, and resistance to treatment. However, the understanding of how mRNA expression patterns of GBM subtypes are reflected at global proteome level in GSCs is limited. To characterize protein expression in GSCs, we performed in-depth proteogenomic analysis of patient-derived GSCs by RNA-sequencing and mass-spectrometry. We quantified > 10 000 proteins in two independent GSC panels and propose a GSC-associated proteomic signature characterizing two distinct phenotypic conditions; one defined by proteins upregulated in proneural and classical GSCs (GPC-like), and another by proteins upregulated in mesenchymal GSCs (GM-like). The GM-like protein set in GBM tissue was associated with necrosis, recurrence, and worse overall survival. Through proteogenomics, we discovered 252 non-canonical peptides in the GSCs, i.e., protein sequences that are variant or derive from genome regions previously considered non-protein-coding, including variants of the heterogeneous ribonucleoproteins implicated in RNA splicing. In summary, GSCs express two protein sets that have an inverse association with clinical outcomes in GBM. The discovery of non-canonical protein sequences questions existing gene models and pinpoints new protein targets for research in GBM.