Highly Conserved And Cis-Acting Lncrnas Produced From Paralogous Regions In The Center Of Hoxa And Hoxb Clusters In The Endoderm Lineage

Author summary Each of the four Hox clusters in vertebrate genomes encodes up to 11 transcription factors whose activity is extensively regulated spatially and temporally, and which help determine the developmental and adult transcriptome in space and time. These Hox transcription factors belong to 13 homology groups, and Hox clusters also encode various noncoding transcripts, including microRNAs and long noncoding RNAs (lncRNAs). We characterize in detail two lncRNAs, HOXA-AS3 and HOXB-AS3, which are transcribed from matching regions in the HOXA and HOXB clusters, respectively. These lncRNAs are highly conserved in vertebrate evolution and transcribed antisense to Hox protein-coding genes from groups 5-7. Beyond the matching positions, the promoters of HOXA-AS3 and HOXB-AS3 share sequence similarity, their expression patterns are correlated with each other, mostly in the endoderm lineage, and they positively regulate the expression of the Hox protein-coding genes that they overlap. Regulation by lncRNAs thus appears to be an ancestral feature of HOX clusters, likely pre-dating the duplication of the Hox clusters at the root of the vertebrate lineage.Long noncoding RNAs (lncRNAs) have been shown to play important roles in gene regulatory networks acting in early development. There has been rapid turnover of lncRNA loci during vertebrate evolution, with few human lncRNAs conserved beyond mammals. The sequences of these rare deeply conserved lncRNAs are typically not similar to each other. Here, we characterize HOXA-AS3 and HOXB-AS3, lncRNAs produced from the central regions of the HOXA and HOXB clusters. Sequence-similar orthologs of both lncRNAs are found in multiple vertebrate species and there is evident sequence similarity between their promoters, suggesting that the production of these lncRNAs predates the duplication of the HOX clusters at the root of the vertebrate lineage. This conservation extends to similar expression patterns of the two lncRNAs, in particular in cells transiently arising during early development or in the adult colon. Functionally, the RNA products of HOXA-AS3 and HOXB-AS3 regulate the expression of their overlapping HOX5-7 genes both in HT-29 cells and during differentiation of human embryonic stem cells. Beyond production of paralogous protein-coding and microRNA genes, the regulatory program in the HOX clusters therefore also relies on paralogous lncRNAs acting in restricted spatial and temporal windows of embryonic development and cell differentiation.