A Novel Regulatory Region of the p27 Locus Is Required for Normal Erythroid p27 Expression, and Produces a Long Noncoding RNA with No Detectable Function

Mammalian cells express thousands of lineage-restricted long noncoding RNAs (lncRNAs), although the functions of most are unknown. "Knockout" studies to investigate lncRNA function by gene deletion in cells or whole animals have been informative. However, since most lncRNAs show open chromatin and transcription factor (TF) binding at their promoters as well as elsewhere in their genomic loci, it is essential to distinguish the roles of the RNA transcript from those of potential cis-regulatory elements, both of which are eliminated in deletion studies. We identified a novel mouse lncRNA named Drop27 (Downstream of p27) that is transcribed 4 kb downstream of the Cdkn1b gene encoding the cell-cycle inhibitor p27, which is strongly upregulated during erythroid maturation. Drop27 is conserved between mice and humans, has two exons separated by a 21 kb intron, and produces a 400 bp spliced, polyadenylated RNA that is abundantly transcribed and highly enriched in mouse erythroblasts compared to other tissues. We used the CRISPR/Cas system to delete the entire Drop27 locus in the G1E erythroid cell line. Microarray analysis showed reduced Cdkn1b mRNA in Drop27-deleted cells with very few other changes in the transcriptome. Quantitative PCR verified this finding by demonstrating that heterozygous deletion of Drop27 caused reduced Cdkn1b mRNA by 35%, while homozygous deletion led to a 70% reduction. This effect also occurred at the primary transcript level, demonstrating that deletion of the Drop27 reduced Cdkn1b transcription. To distinguish the effects of loss of the lncRNA transcribed from Drop27 from the potential loss of a cis-acting regulatory module in the Drop27 genomic locus, we used homologous recombination to insert a bovine growth hormone (BGH) polyadenylation (polyA) cassette into the first exon of Drop27, 80 bp downstream of the transcription start site. This caused premature termination of the lncRNA transcript while maintaining all genomic sequences, including potential cis-regulatory elements. Analysis of multiple clones showed that homozygous polyA cassette integration reduced full-length Drop27 transcript levels by more than 99%, although Cdkn1b mRNA levels were unaffected. These findings demonstrate that Drop27 lncRNA is dispensable for Cdkn1b transcription, while its genomic sequences are required, indicating that the Drop27 gene locus contains an erythroid cis-regulatory element (enhancer) for the Cdkn1b gene. Several strong candidates for the proposed enhancer are found in the Drop27 gene. Multiple epigenetic features strongly associated with enhancers map in several distinct locations in the Drop27 locus in erythroid and other hematopoietic cells, including DNase hypersensitivity, p300 binding, and multiple transcription factor sites. A functional role for the Drop27 lncRNA is not identified by this experiment, and it is possible that it arose as a byproduct of enhancer activity. Our findings provide new insight into mechanisms of hematopoietic gene expression and are of more broad relevance to the lncRNA field in general. In particular, we demonstrate that the genomic loci of some lncRNA genes may function as cis elements, irrespective of the transcripts arising from them.