The role of the nucleoporin Nup93 sub-complex in regulating HOXA gene expression

Abstract

The nuclear pore complex (NPCs) is required for the regulated import and export of RNA and proteins. NPC is a megadalton multiprotein channel composed of ~ 30 nucleoporins (Nups). In addition to transport, nucleoporins are involved in the regulation of chromatin organization and gene expression. Here we investigated the role of nucleoporin Nup93 in the regulation of HOXA gene expression. HOXA expression is restricted to early stages of development and differentiation, while its aberrant expression in differentiated cells is associated with cancers. We showed that Nup93 is associated with HOXA1, HOXA3, and HOXA5 promoters and represses HOXA expression, in a manner assisted by its interacting partners Nup188 and Nup205. Single cell imaging by 3D-Fluorescence in situ hybridization (3D-FISH) analyses, revealed that the depletion of Nup93 untethers the HOXA gene locus from the nuclear periphery, which we also ascertained by RNA-FISH. We propose a novel role for the Nup93 sub-complex in repressing HOXA gene locus thereby preventing its untimely expression in differentiated cells. To address the genome-wide role of Nup93 in gene regulation, we performed Chromatin Immunoprecipitation (ChIP) of Nup93 followed by whole-genome sequencing. Analyses of Nup93 ChIP-seq data shows that Nup93 specifically associates with genes involved in development and differentiation. Furthermore, Nup93 associates with genomic regions similar to the repressive histone mark (H3K27me3), suggesting a repressive role of Nup93 in gene regulation. In addition, Nup93 enriched regions also overlap with CTCF - a major genome organizer, suggesting a crosstalk between Nup93 and CTCF in gene regulation. To further understand the role of Nup93 and CTCF during differentiation, we performed Retinoic acid-mediated differentiation of NT2/D1 cells in the background of Nup93 and CTCF depletion. We found that Nup93 depletion in NT2/D1 cells upregulates HOXA gene cluster. In contrast, CTCF depletion downregulates HOXA gene cluster. Surprisingly, we found that retinoic acid treatment in Nup93 depleted cells significantly enhances HOXA gene expression. In contrast, RA treatment in CTCF depleted cells reduces HOXA gene expression. 3D-FISH analyses revealed that HOXA gene locus shows a dynamic repositioning from the nuclear periphery during differentiation to the nuclear interior upon activation and relocates to the nuclear periphery that correlates with repression. We found that the untethering of HOXA locus from the nuclear periphery correlates with the reduced occupancy of Nup93 and increased occupancy of CTCF on the HOXA1 promoter during differentiation. In summary, our results suggest that Nup93 and CTCF have an antagonistic role in the regulation of HOXA gene expression during differentiation.