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The Firre Locus Produces a Trans-Acting RNA Molecule That Functions in Hematopoiesis

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The Firre Locus Produces a Trans-Acting RNA Molecule That Functions in Hematopoiesis ARTICLE https://doi.org/10.1038/s41467-019-12970-4 OPEN The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis Jordan P. Lewandowski1, James C. Lee 1,2, Taeyoung Hwang1,3, Hongjae Sunwoo 4, Jill M. Goldstein1,5, Abigail F. Groff1,6, Nydia P. Chang1, William Mallard 1, Adam Williams7, Jorge Henao-Meija8, Richard A. Flavell 9, Jeannie T. Lee 4,10,11, Chiara Gerhardinger1, Amy J. Wagers1,5,12 & John L. Rinn 1,3* RNA has been classically known to play central roles in biology, including maintaining telo- 1234567890():,; meres, protein synthesis, and in sex chromosome compensation. While thousands of long noncoding RNAs (lncRNAs) have been identified, attributing RNA-based roles to lncRNA loci requires assessing whether phenotype(s) could be due to DNA regulatory elements, tran- scription, or the lncRNA. Here, we use the conserved X chromosome lncRNA locus Firre,asa model to discriminate between DNA- and RNA-mediated effects in vivo. We demonstrate that (i) Firre mutant mice have cell-specific hematopoietic phenotypes, and (ii) upon expo- sure to lipopolysaccharide, mice overexpressing Firre exhibit increased levels of pro- inflammatory cytokines and impaired survival. (iii) Deletion of Firre does not result in changes in local gene expression, but rather in changes on autosomes that can be rescued by expression of transgenic Firre RNA. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis. 1 Department of Stem Cell and Regenerative Biology and Harvard Stem Cell Institute, Harvard University, Cambridge, MA, USA. 2 Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, UK. 3 BioFrontiers Institute, University of Colorado Boulder, Boulder, CO, USA. 4 Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA. 5 Paul F. Glenn Center for the Biology of Aging, Harvard Medical School, 77 Louis Pasteur Avenue, Boston, MA, USA. 6 Department of Systems Biology, Harvard Medical School, Boston, MA, USA. 7 The Jackson Laboratory, JAX Genomic Medicine, Farmington, CT, USA. 8 Department of Pathology and Laboratory Medicine, Perelman School of Medicine University of Pennsylvania, Philadelphia, PA, USA. 9 Department of Immunobiology and Howard Hughes Medical Institute, Yale University, School of Medicine, New Haven, CT, USA. 10 Department of Genetics, Harvard Medical School, Boston, MA, USA. 11 Howard Hughes Medical Institute, Boston, MA, USA. 12 Joslin Diabetes Center, Boston, MA, USA. *email: [email protected] NATURE COMMUNICATIONS | (2019) 10:5137 | https://doi.org/10.1038/s41467-019-12970-4 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12970-4 ranscription occurs at thousands of sites throughout the RNA-seq on eight different WT embryonic tissues and plotted the mammalian genome. Many of these sites are devoid of expression of noncoding and coding transcripts. Consistent with T 32–35 protein-coding genes and instead contain long noncoding previous reports , we observed that noncoding transcripts RNAs (lncRNAs). While lncRNA loci have been implicated in a were generally less abundant than protein-coding transcripts variety of biological functions, comparatively few lncRNA loci (Fig. 1b). Despite most lncRNAs being expressed at low levels, we have been genetically defined to have RNA-based roles. Indeed, found that Firre, like Malat1 (refs36–38), is an abundant transcript deletions of entire lncRNA loci have uncovered a number of (Fig. 1b). Next, since Firre is located on the X chromosome and in vivo phenotypes1–5; however, this approach alone is con- escapes X-inactivation15,22–24, we investigated whether Firre has founded because in addition to the lncRNA transcript, lncRNA different expression levels in male and female WT tissues. While loci can also exert function through DNA regulatory elements6–8, levels of Firre RNA varied across embryonic tissue types, within the promoter region9, as well as by the act of transcription10,11. individual tissues, male and female samples exhibited similar Thus, attributing RNA-based role(s) to lncRNA loci requires expression levels of Firre, despite escaping X-inactivation testing whether other regulatory modes potentially present at the (Fig. 1c). locus have molecular activity that could contribute to phenotypic effects3,12,13. In this study, we use the functional intergenic repeating RNA Firre knockout and overexpression mice are viable and fertile. element, (Firre) locus as a model to discriminate between DNA- To investigate the in vivo role of Firre and assess DNA- and and RNA-mediated effects in vivo. We selected this locus for our RNA-mediated effects, we generated both Firre loss-of-function study because it is syntenically conserved in a number of mam- and Firre overexpression mice. To delete the Firre locus in vivo, mals, including human14–17, and because studies have reported we generated a mouse line containing a floxed allele (Firrefloxed) diverse biological and molecular roles. Early characterization of from a previously targeted mouse embryonic stem cell line15 and the FIRRE locus in human cell lines identified it as a region that mated to a CMV-Cre deleter mouse39. This produced a genomic interacts with the X-linked macrosatellite region, DXZ4,ina deletion (81.8 kb) that removed the entire Firre gene body and CTCF-dependent manner18–21. Further analyses of the Firre locus promoter (henceforth called ΔFirre) (Fig. 1d). We confirmed the demonstrated that it produces a lncRNA that escapes X- deletion of the Firre locus by genotyping (Supplementary Fig. 1) inactivation15,22–24, although it is predominately expressed from and examined Firre RNA expression. As expected, we did not the active X chromosome21,25. Studies using cell culture models detect Firre RNA in ΔFirre embryos by whole-mount in situ suggest that the Firre locus has biological roles in multiple pro- hybridization or by RNA-seq (Fig. 1a, d). cesses, including adipogenesis26, nuclear architecture15,19,21, and Since Firre is found on the X chromosome, we first sought to in the regulation of gene expression programs15,27. In addition, determine if deletion of the locus had an effect on the expected there is some evidence for roles of the FIRRE locus in human sex ratio of the progeny. Matings between ΔFirre mice produced development and disease28–31. Collectively, these studies viable progeny and had a normal frequency of male and female demonstrate the diverse cellular and biological functions for the pups (Supplementary Table 1) that did not exhibit overt Firre locus. However, the biological roles of Firre as well as dis- morphological, skeletal, or weight defects (Supplementary Fig. 2). entangling DNA- and RNA-mediated function(s) for the Firre Moreover, deletion of Firre did not impact expression levels of locus have not been explored in vivo. Xist RNA in embryonic tissues or perturb Xist RNA localization Using multiple genetic approaches, we describe an in vivo role during random X chromosome inactivation (XCI) in mouse for the Firre locus during hematopoiesis. We report that Firre embryonic fibroblasts (MEFs) (Supplementary Fig. 3A–C). mutant mice have cell-specific defects in hematopoietic popula- Because the ΔFirre allele removes the entire gene body, this tions. Deletion of Firre is accompanied by significant changes in model does not allow us to distinguish between DNA- and RNA- gene expression in a hematopoietic progenitor cell type, which mediated effects. Therefore, in order to be able to investigate the can be rescued by induction of Firre RNA from an autosomal role of Firre RNA, we generated a doxycycline (dox)-inducible transgene within the Firre knockout background. Mice over- Firre overexpression mouse. This mouse model was engineered to expressing Firre have increased levels of pro-inflammatory cyto- contain a Firre cDNA downstream of a tet-responsive element kines and significantly impaired survival upon exposure to (henceforth called tg(Firre)), and was mated to mice that lipopolysaccharide (LPS). Finally, the Firre locus does not contain constitutively express the reverse tetracycline transcriptional cis-acting RNA or DNA elements (including the promoter) that activator (rtTA) gene (combined alleles henceforth called FirreOE) regulate neighboring gene expression on the X chromosome (nine (Fig. 1e). This approach enabled systemic induction of Firre RNA biological contexts examined), suggesting that Firre does not in a temporally controllable manner by the administration of dox. function in cis. Collectively, our study provides evidence for a Moreover, by combining the FirreOE and ΔFirre alleles (hence- trans-acting RNA-based role for the Firre locus that, thus far, has forth called Firrerescue), we could test whether Firre RNA physiological importance for hematopoiesis. expression alone is sufficient to rescue phenotypes arising in the ΔFirre mice, thereby distinguishing DNA- and RNA-based effects. To confirm expression of transgenic Firre RNA, tg(Firre) Results females were mated with rtTA males and placed on a dox diet the The Firre locus produces an abundant lncRNA.Wefirst sought day a copulatory plug was detected, and embryos were collected to investigate the gene expression properties for Firre RNA at E11.5 for analyses. Compared with sibling control embryos, we in vivo. To determine potential spatial and temporal aspects of detected increased Firre RNA in
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