upstream deletion leads to dysregulation of and autosomal gene expression.

long noncoding RNA is the master regulator of the X-Chromosome inactivation (XCI) process. is expressed from the inactive X and coats the inactive X to facilitate XCI. -regulation of expression remains poorly understood in the context of maintenance of XCI. Here, we have explored the role of the upstream sequences (∼6 kb) lying between and in the regulation of and XCI in mouse extra-embryonic endoderm stem cells (XEN), which represent the maintenance phase of imprinted XCI. Here, we show that the deletion of this upstream sequence in the inactive X leads to the upregulation of expression accompanied by the dispersal of the cloud. Notably, we find the loss of enrichment of repressive marks such as H3K27me3, H4K20me1, and MacroH2A, except that of H2AK119ub, in dispersed nuclei. However, X-linked genes remain silent despite dispersal and loss of enrichment of repressive marks. Notably, we find that many autosomal genes, including cohesin , are dysregulated in -upstream-deleted cells. Additionally, we demonstrate that -upstream deletion leads to alterations of topological contacts of the locus with its upstream positive regulator and across the inactive X and autosomes. Finally, we show genome-wide alterations of the occupancy of architectural proteins CTCF/RAD21, including at many loci of the inactive X such as the upstream regions and the locus, which is critical for maintaining inactive X conformation. Taken together, we demonstrate that the upstream sequence imparts a multifaceted role in genome regulation beyond the XCI.