X-linked deletion of Crossfirre, Firre, and Dxz4 in vivo uncovers diverse phenotypes and combinatorial effects on autosomes

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. Here, we characterized the epigenetic profile of these loci, revealing them as the most female-specific accessible regions genome-wide. To address their in vivo role, we performed one of the largest X-linked knockout studies by deleting Crossfirre, Firre, and Dxz4 individually and in combination (TKO). Despite their distinct epigenetic features observed on the X chromosome, our allele-specific analysis uncovers these loci as dispensable for imprinted and random X chromosome inactivation. However, we provide evidence that Crossfirre affects autosomal gene regulation but only in combination with Firre. To shed light on the functional role of these sex-specific loci, we performed an extensive standardized phenotyping pipeline and uncovered diverse knockout and sex-specific phenotypes. Collectively, our study provides the foundation for exploring the intricate interplay of conserved X-linked loci in vivo. RNA-seq from Crossfirre, Crossfirre-Firre and Crossfirre-Firre-Dxz4 deletion mouse strains (extraembryonic and adult organs). scRNA-seq from heterozygous Crossfirre-Firre-Dxz4 deletion mouse strains (adult spleen). RNA-seq from heterozygous Crossfirre-Firre-Dxz4 deletion mouse strains (adult spleen).长链非编码RNA（lncRNA）Crossfirre被鉴定为一种印记X连锁基因，其反义转录自反式作用长链非编码RNA Firre。Firre基因座与Dxz4形成失活X染色体特异性互作，二者共同构成最大的保守染色质结构的平台。本研究对这些基因座的表观遗传谱进行了表征，发现它们是全基因组范围内雌性特异性最高的开放染色质区域。为探究这些基因座的体内功能，我们开展了目前规模最大的X连锁基因敲除研究之一，分别及联合敲除Crossfirre、Firre与Dxz4（三重敲除，TKO）。尽管在X染色体上观察到它们具有独特的表观遗传特征，但等位基因特异性分析显示，这些基因座对于印记X染色体失活和随机X染色体失活均非必需。不过，本研究证实Crossfirre可影响常染色体基因调控，但该作用仅在与Firre联合存在时才会显现。为阐明这些性别特异性基因座的功能，我们采用了大规模标准化表型分析流程，发现了多种敲除相关及性别特异性表型。综上，本研究为探索保守X连锁基因座在体内的复杂相互作用奠定了基础。来源于Crossfirre、Crossfirre-Firre及Crossfirre-Firre-Dxz4敲除小鼠品系的RNA测序（RNA-seq）数据（涵盖胚外组织与成年器官）。来源于杂合子Crossfirre-Firre-Dxz4敲除小鼠品系的单细胞RNA测序（scRNA-seq）数据（成年脾脏样本）。来源于杂合子Crossfirre-Firre-Dxz4敲除小鼠品系的RNA测序（RNA-seq）数据（成年脾脏样本）。