The lncRNA Crossfirre was identified as an imprinted X-linked gene and is transcribed antisense to the trans-acting lncRNA Firre. The Firre locus forms an inactive-X-specific interaction with Dxz4, both loci providing the platform for the largest conserved chromatin structures.
Scientists from the Institute of Pharmacology and Toxicology, Technical University Munich, Germany, in collaboration with an international team of researchers, shed light on the in-vivo role of the most female-specific accessible regions genome-wide. They perform one of the largest X-linked knockout studies deleting the IncRNAs Crossfirre, Firre, and Dxz4 individually and in combination. This work was recently published in Nature Communications.
Mice carrying a Crossfirre single deletion or in combination with Firre and Dxz4 are viable and undergo normal development. Despite their distinct epigenetic features observed on the X chromosome, the allele-specific analysis uncovers these loci as dispensable for imprinted and random X chromosome inactivation.
While the Crossfirre, Firre, and Dxz4 KO (TKO) animals showed no obvious impairment in fertility, viability, and sex ratios, the results of molecular analyses revealed gene dysregulation in multiple organs. This provides evidence that Crossfirre affects autosomal gene regulation but only in combination with Firre. Extensive phenotypic investigation of mutants lacking three X-linked lncRNAs, provides comprehensive insights into the putative role of the topmost sex-specific loci. The screening analysis revealed a broad range of phenotypic changes, highlighting the complex influence of these loci on various physiological processes. While 32.14% (n = 9) of the phenotypes were specific to the knockout condition, the majority displayed sex-specific characteristics (male TKO specific: 46.43%, n = 13; female TKO-specific: 21.43%, n = 6). Based on the parameters tested significant knockout- and sex-specific phenotypes affect immunology/allergy, behavior, neurology, cardiovascular, clinical chemistry, dysmorphology, metabolism, and pathology. The nine phenotypes that were specific to male and female TKO included increased locomotor and exploratory activity and decreased acoustic startle reactivity. Moreover, mutant mice exhibited alterations in red blood cell morphology, mild effects on iron metabolism, and increased secondary follicles in the Peyer’s patches of the intestine.
The complex study provides an in-depth characterization of lncRNA loci that serve as a platform for the largest chromatin structures in mammals with prominent sex- and allele specific epigenetic signatures. Interestingly, despite the unique characteristics observed on the X chromosome, the team of scientists uncovered an interplay of Crossfirre and Firre in regulating autosomal genes, rather than affecting X inactivation biology.
Hasenbein TP, Hoelzl S, Smith ZD, Gerhardinger C, Gonner MOC, Aguilar-Pimentel A, Amarie OV, Becker L, Calzada-Wack J, Dragano NRV, da Silva-Buttkus P, Garrett L, Hölter SM, Kraiger M, Östereicher MA, Rathkolb B, Sanz-Moreno A, Spielmann N, Wurst W, Gailus-Durner V, Fuchs H, Hrabě de Angelis M, Meissner A, Engelhardt S, Rinn JL, Andergassen D. X-linked deletion of Crossfirre, Firre, and Dxz4 in vivo uncovers diverse phenotypes and combinatorial effects on autosomes. Nat Commun. 2024 Dec 5;15(1):10631. doi: 10.1038/s41467-024-54673-5.