Abstract
Thousands of sense-antisense mRNA-lncRNA gene pairs occur in the mammalian genome. While there is usually little doubt about the function of the coding transcript, the function of the lncRNA partner is mostly untested. Here we examine the function of the homeotic Evx1-Evx1as gene locus. Expression is tightly co-regulated in posterior mesoderm of mouse embryos and in embryoid bodies. Expression of both genes is enhanced by BMP4 and WNT3A, and reduced by Activin. We generated a suite of deletions in the locus by CRISPR-Cas9 editing. We show EVX1 is a critical downstream effector of BMP4 and WNT3A with respect to patterning of posterior mesoderm. The lncRNA, Evx1as arises from alternative promoters and is difficult to fully abrogate by gene editing or siRNA approaches. Nevertheless, we were able to generate a large 2.6 kb deletion encompassing the shared promoter with Evx1 and multiple additional exons of Evx1as. This led to an identical dorsal-ventral patterning defect to that generated by micro-deletion in the DNA-binding domain of EVX1. Thus, Evx1as has no function independent of EVX1, and is therefore unlikely to act in trans. We predict many antisense lncRNAs have no specific trans function, possibly only regulating the linked coding genes in cis.
Highlights
To be co-regulated with the HoxA cluster during gastrulation
We found that disruption of EVX1 results in upregulation of anterior visceral endoderm (AVE) and definitive endoderm genes including Cer[1] and Sox[17], at the expense of posterior genes, such as Mixl[1] and Kdr (Flk-1)
Whole mount in situ hybridization (WISH) of E7.5 and E9.5 embryos demonstrates the Evx[1] and Evx1as are co-expressed in the primitive streak during gastrulation (Fig. 1c)
Summary
To be co-regulated with the HoxA cluster during gastrulation. In Xenopus and zebrafish, the homologs of EVX1 (xhox[3], eve1) play roles in ‘posteriorization’ of nascent mesoderm[8,9]. There are other well studied lncRNAs associated with the HoxA cluster, including Haunt/Halr[1] which is located ~40 kb upstream of Hoxa[124], and HOTTIP which is immediately downstream of Hoxa[13], the most 3′member of the cluster[25] (Fig. 1) Both have complex functions in global regulation or fine tuning of expression of the HoxA cluster. To test whether Evx1as has a function independent of EVX1, we generated a suite of CRISPR-Cas[9] mediated deletions using a similar approach to that recently reported for Haunt[24]. With all of these DNA manipulations, we were not able to disrupt expression of Evx1as without disrupting expression of Evx[1]. We cannot rule out a function for Evx1as in the regulation of Evx[1] in cis
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