Dissecting the gene regulatory function of the long non-coding RNA Maenli

• Applicant: Professor Dr. Stefan Mundlos
• Subject Area: General Genetics and Functional Genome Biology ; Developmental Biology
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 577404682

The non-coding genome of vertebrates is pervasively transcribed. In fact, it has been found that approx. 80% of the genome is transcribed and the majority of these sequences are thought to be involved in gene regulation. A subset of these transcripts are long non-coding RNAs (lncRNAs), originally defined as transcripts longer than 200 bps that do not contain an open reading frame. Like mRNAs, these transcripts are spliced and polyadenylated. In spite of being poorly conserved, lncRNAs often have critical evolutionary conserved functions and are important components of the gene regulatory network. In a previous study we identified the lncRNA Maenli as the major regulatory of En1 expression in the limb. Mutations affecting Maenli results in a complex limb phenotype in humans and mice (Allou et al. Nature 2021). Our studies showed that Maenli functions in cis within the En1 TAD but it remains unclear how it activates En1 and how its function is embedded within the 3D chromatin landscape of the locus. We will study the gene regulatory function of lncRNAs using Maenli as an example in vivo in the mouse. For this purpose, we use cutting edge technologies of genome engineering and synthetic biology. In Aim 1 we will use Transformation-Associated Recombination (TAR) to synthetically assemble the Maenli locus of approx. 25 kb in different variations (constructs 1-7) in yeast. The TAR system uses homologous recombination of 2-3 kb fragments to produce synthetic DNA fragments of up to 50 kb. The synthesized sequence is then transferred from yeast to bacteria in a BAC with which it can be propagated. In Aim 2 we will insert the synthetically produced fragment containing the entire Maenli locus in a Maenli deletion mouse ES cell clone. This will be performed by first inserting a landing pad and then inserting the fragment with Bxb1 integrase. Mice will be produced from these cells. In Aim 3 we will investigate the locus specificity of the Maenli lncRNA. We will insert Maenli outside of the En1 TAD to see if Maenli function depends on the 3D conformation of the locus. In Aim 4 we will test if the human MAENLI locus can rescue the mouse phenotype. For this purpose, we will synthesize the human 27 kb deletion identified in our initial study in patients and insert it into the mouse genome. In Aim 5 we aim at dissecting the individual components of Maenli function, i.e. the promoter, splicing and the presence of poly-A sites. First, we will replace the Maenli promoter with a BMP2 enhancer to test if we can direct En1 expression in a BMP2-like fashion. Second, we will create constructs without introns and without polyadenylation. These constructs are again inserted in the wt locus. Readout is for all constructs En1 expression at E10.5 and phenotype at E17.5. Mice will be produced by our established ES cell aggregation system.

• DFG Programme: Research Grants