Embryonic stem cell factors DPPA2/4 amplify active H3K4me3-H2AK119ub chromatin domains in non-small cell lung cancer (BS-seq)

Embryonic regulators are often re-expressed in cancers, however the functional and molecular significance of this is not always understood. The epigenetic priming factors Developmental Pluripotency Associated 2 and 4 (DPPA2/4) have crucial roles in early development and are implicated in cancer pathogenesis. We reveal in non-small cell lung cancer (NSCLC), DPPA2/4 co-expression is associated with poorly differentiated tumours and impaired patient outcomes. Biochemically, human DPPA2/4 multimerise for their protein stability and enhanced nucleosome binding activity. In NSCLC cells, DPPA2/4 bind CG-rich sequences including promoters of developmental, Wnt signaling and catabolic genes. Chromatin state modelling revealed DPPA2/4 preferentially bind active H3K4me3 and H3K27ac domains that were intriguingly also enriched for PRC1 and its product H2AK119ub, validated by H3K4me3-H2AK119ub sequential ChIP. Knockdown experiments revealed DPPA2/4 were required to maintain RING1B and H2AK119ub at these domains. Surprisingly, despite the presence of PRC2.1, these regions lacked any detectable H3K27me3, suggesting an uncoupling between the recruitment of PRC2 to chromatin and its catalytic product. When exogenously over-expressed in NSCLC cells where they are not normally present, DPPA2/4 bind to and promote active chromatin states, resulting in an increase in vivo xenograft tumour growth. Our results demonstrate how in NSCLC cells, DPPA2/4 act as molecular amplifiers of active and poised chromatin. Together, this highlights how aberrant re-activation of embryonic factors in cancers may take on new functions, promoting tumourigenesis. Overall design: The NCI-H661 cell line is derived from lung large cell carcinoma (subtype of non-small cell lung cancer) metastases and expresses both DPPA2 and DPPA4 highly at the mRNA and protein level. In order to investigate the functional consequence of peturbing DPPA2/4 we performed siRNA-mediated knockdown of DPPA2 or/and DPPA4 in NCI-H661 cells followed by whole genome bisulfite sequencing (WGBS) analyses. Analyses were conducted for siRNA models following 96h of knockdown with either control siRNA, siDPPA2, siDPPA4 or both siDPPA2+4. All conditions were done in biological triplicates. We additionally assessed DPPA4 knockdown in the COV318 ovarian cancer cell line.