Ly49 Pro1 activity is associated with gene activation, not gene expression: Pro1 transcripts are not present in mature Ly49-expressing NK cells.

Abstract

Abstract Recent studies of the Ly49 loci have challenged the traditional view of promoters, since transcriptional start sites for the variegated Ly49 genes were not found to be associated with regions possessing transcriptional activity in in vitro promoter assays. The upstream Pro1 bidirectional promoter previously associated with probabilistic activation of variegated Ly49 genes by immature bone marrow NK cells was shown to be active in mature NK cells and function as an enhancer element, suggesting that it may play a role in Ly49 transcript initiation in mature NK cells. To directly assess if Pro1 transcripts are present in mature Ly49-expressing NK cells, RNA sequencing of the total transcript pool was performed on freshly isolated splenic NK cells sorted for expression of either Ly49G or Ly49I. No Pro1 transcripts were detected from the Ly49a, Ly49c or Ly49i genes in mature Ly49+ve NK cells that contained high levels of Pro2 or Pro3 transcripts. Ly49g Pro1 transcripts were found at low levels in both Ly49G+ve and Ly49G-ve populations, consistent with the presence of a small population of mature NK cells undergoing Ly49g gene activation, as previously demonstrated by culture of splenic NK cells in IL-2. Furthermore, Ly49 gene reporter constructs containing Pro1 failed to show any enhancer activity of Pro1 on the downstream Ly49 promoter active in mature Ly49-expressing NK cells. Taken together, the results are consistent with Pro1 transcription playing a role in gene activation in developing NK, and argue against a role for Pro1 in Ly49 gene transcription by mature NK cells.