br SUV H low cell
2.4. SUV39H1low cell populations within cervical tumours show features of enhanced migration and a bulk depletion of H3K9me3
We observed gene expression signatures of migration in SUV39H1-low tumours. The SUV39H1-low state of these tumours may be due to a global depletion of SUV39H1 in Dynasore within tumours, or due to varying proportions of cells with low and high SUV39H1 expression. To ex-amine whether SUV39H1low cell populations are observed within tu-mours, and whether these putative SUV39H1low populations show features of migration, we examined individual tumour biopsies using Immunohistochemistry (IHC). We examined SUV39H1 staining in-
tensity within sections, and broadly characterised cell populations as SUV39H1high and SUV39H1low. As a loss of cell-cell adhesion and proliferation programs and a gain of an elongated, “sarcomatoid-like” morphology are key facets of migration, we next examined for regions of serial sections showing depletion of CDH1 (ECAD) and P63. ECAD and P63 mark cell-cell adhesion and keratinocyte proliferation, re-spectively, and a depletion of these markers indicates a migratory state [16–18]. 5 out of 12 SCC biopsies showed the presence of SUV39H1-veECAD-veP63-ve cell populations (Fig. 3, Fig. S4, Fig. S5), and cells in these populations also showed a sarcomatoid-like morphology. Other SCC sections showed less drastic depletion of SUV39H1, nonetheless, populations of cells showing lower SUV39H1 also showed lower ECAD and P63.
Since SUV39H1 is known to mediate transcriptional regulation through H3K9me3, we also assessed bulk H3K9me3 distribution in se-rial sections. We observed that SUV39H1low populations within tu-mours were also H3K9me3low, suggesting an altered chromatin state in these populations (Fig. 4a, Fig. S5). Interestingly, in normal cervix sections, in sections showing the entire spectrum of differentiation, SUV39H1-ve and H3K9me3-ve cells were located in the basal stem layer,
whereas the more differentiated, proliferative parabasal layer was SUV39H1high and H3K9me3high (Fig. 4b), suggesting a possible link
between a SUV39H1low chromatin state and stemness. Thus, SUV39H1low cell populations are observed within tumours, and these populations show features of cell migration and a bulk loss of H3K9me3.
2.5. Migrated populations of cervical cancer cells show SUV39H1-linked migratory gene expression signatures
chromatin state may drive migratory populations. We sought to de-termine whether migrated populations showed 1. SUV39H1-linked transcriptional changes and 2. linked changes in H3K9me3 distribution, using migrated populations of SiHa (sorted using a transwell setup, Fig. 1a). First, we sought to determine whether migrated populations showed reflections of migration and EMT (Epithelial-Mesenchymal Transition) at a transcriptional level, and whether these transcriptional changes showed parallels with transcription profiles of SUV39H1-low tumours. The transcriptome of in vitro migrated cell populations was examined by RNA-Seq (Fig. S6a, c). Migrated populations showed an upregulation of 944 genes and a downregulation of 531 genes, relative to non migrated populations. RNA-Seq analysis revealed the following findings: 1. Migrated populations showed enrichment of cell motion and cell migration Gene Ontology (GO) terms, along with DNA re-plication signatures (at p < 10-4) (Fig. 5a, b). Genes downregulated in migrated populations, on the other hand, showed enrichment for apoptosis and cell death GO processes (at p < 10-4). 2. An upregulation of known apex EMT transcription factors TWIST1, SNAIL2, and ZEB1 was observed in the migrated population (Fig. 5c). Differential ex-pression of downstream effectors/ regulators of migration was also noted, including a downregulation of cell-cell adhesion marker CDH1/ ECAD and an upregulation of FN1 (Fibronectin), CD44, MMP14 and MMP28 (Fig. 5d). 3. Transcription factors such as AP1, TGIF, STAT3, and SOX9 showed overrepresented target genes in migrated popula-tions (Fig. 5e), indicating that these regulators may effect the tran-scriptional changes observed in migrated populations. 4. A significant overlap was noted between genes upregulated in migrated populations and genes upregulated in TCGA SUV39H1-low tumours (p = 2.5 × 10-4) (Fig. 5f), highlighting a link with SUV39H1. Common genes upre-gulated in both these sets included migration-associated genes such as TWIST1, ZEB1, MMP2, and PKCA. Lastly, differential expression of SUV39H1 itself was not observed at the transcription level in migrated populations, indicating that altered levels of SUV39H1 protein observed in SiHa migrated populations are regulated post-transcriptionally. Thus, migrated populations of cervical cancer cells show reflections of mi-gratory processes at a transcriptional level, and these signatures show a link with a SUV39H1 transcriptional signature.