Independent cell type-specific expression and distal regulation of the 9p21 locus cell cycle regulators: p14ARF, p16INK4A, p15INK4A, and ANRIL [CRISPR and scRNA-seq]

The 9p21 locus encodes tumor suppressors p16INK4A and p14ARF (both encoded by CDKN2A), p15INK4B (CDKN2B), and a long non-coding RNA, ANRIL (CDKN2B-AS1). The ~1 megabase locus is notable for a high density of single nucleotide polymorphisms (SNPs) associated with aging-related diseases and traits. Despite clear importance, our understanding of the the cell-type specific expression dynamics and cis-regulatory mechanisms of the 9p21 transcripts has been constrained by the complexity of the transcript structures, low expression, and severe alterations to the locus in cancer cell lines. Here, we innovated custom computational and molecular tools to overcome these technical challenges and fill critical knowledge gaps in our understanding of 9p21 locus architecture and regulation of the 9p21 transcripts. Across tissues, we found that p15INK4b is the predominant transcript. We deconvolved the expression of the CDKN2A transcripts, p16INK4A and p14ARF, revealing stark differences in the expression of these overlapping but functionally distinct transcripts across tissues and cell types. Comparative analysis in a model of fibroblast senescence revealed a striking switch from preferential p14ARF expression in cycling cells, to p16INK4A and p15INK4b as cells age and senescence. Perturbation of putative cis regulatory elements nominated a network of promoters and enhancers that regulate p16INK4A, p14ARF, and p15INK4b and ANRIL. The identified elements are accessible in vivo, and can be used to guide studies into variant interpretation. Our systematic characterization of 9p21 transcript isoforms, promoters, and distal elements across cell states, in vivo tissues and senescence offers new mechanistic insights and a framework for future studies of this vital but poorly understood locus.