In vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation

Recent evidence has determined that the conserved X chromosome mega-structures controlled by the Firre and Dxz4 loci are not required for X chromosome inactivation (XCI) in cell lines. Here, we examined the in vivo contribution of these loci by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus-dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology.


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In addition, for validation of the dysregulated genes observed in the double deletion we use independently generated single KO mouse strains (2 or 3 replicates per group).
eLife Sciences Publications, Ltd is a limited liability non-profit non-stock corporation incorporated in the State of Delaware, USA, with company number 5030732, and is registered in the UK with company number FC030576 and branch number BR015634 at the address 1st Floor, 24 Hills Road, Cambridge CB2 1JP | August 2014 2 Each RNA-seq experiment was performed once. In the manuscript we use biological replicates, defined as tissue derived from different individuals, as detailed in the Figures, Figure legends and Material and Methods. We did not conduct technical replicates in this study.
To determine whether the deletion of Firre, Dxz4 or Firre-Dxz4 impact random or imprinted X chromosome inactivation, we collected embryonic day 12.5 brains, placentas and visceral yolk sac from reciprocal F1 crosses between the deletion strains and CAST/EiJ. For the brain and the placenta, we collected samples for all three strains from the forward cross (3 males and 3 females for WT and maternal deletion) and reverse cross (3 females for WT and paternal deletion). For the placenta DKO we added an additional replicate of the maternal and paternal deletion. In addition, we collected visceral yolk sac samples from the DKO forward cross (3 females WT and maternal deletion) and reverse cross (3 females WT and paternal deletion).
Moreover, to test whether DNA methylation levels are altered in the absence of Firre and Dxz4 on either Xa or Xi, we collected placentas from the DKO forward cross (1 female WT and 2 maternal deletion) and reverse cross (2 females WT and 2 paternal deletion) to perform reduced representation bisulfite sequencing (RRBS) sequencing.
For the DKO adult bodymap, we collected the spleen, brain, kidney, heart, lung and liver from 6 weeks old female mice carrying a homozygous double deletion (4 WT and 4 DKO replicates). To validate and classify the dysregulated genes form the DKO, we collected liver and spleen from independent generated female SKO strains (Firre 3 SKO and Dxz4 2 SKO replicates). To test whether the molecular phenotype observed in the female spleen can be uncoupled form X inactivation, we collected 6 weeks old spleens from DKO males (2 WT and 2 DKO replicates).
We displayed outliers in the boxplot figures.
Sequence data and alignments have been submitted to the Gene Expression Omnibus (GEO) database under accession code GSE127554.

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Sequence data and alignments have been submitted to the Gene Expression Omnibus (GEO) database under accession code GSE127554. To review GEO accession GSE127554: Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127554 Enter token mfcvkggovjujfkt into the box