HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205

Abstract

BackgroundThe nuclear pore complex (NPC) mediates nuclear transport of RNA and proteins into and out of the nucleus. Certain nucleoporins have additional functions in chromatin organization and transcription regulation. Nup93 is a scaffold nucleoporin at the nuclear pore complex which is associated with human chromosomes 5, 7 and 16 and with the promoters of the HOXA gene as revealed by ChIP-on-chip studies using tiling microarrays for these chromosomes. However, the functional consequences of the association of Nup93 with HOXA is unknown.ResultsHere, we examined the association of Nup93 with the HOXA gene cluster and its consequences on HOXA gene expression in diploid colorectal cancer cells (DLD1). Nup93 showed a specific enrichment ~1 Kb upstream of the transcription start site of each of the HOXA1, HOXA3 and HOXA5 promoters, respectively. Furthermore, the association of Nup93 with HOXA was assisted by its interacting partners Nup188 and Nup205. The depletion of the Nup93 sub-complex significantly upregulated HOXA gene expression levels. However, expression levels of a control gene locus (GLCCI1) on human chromosome 7 were unaffected. Three-dimensional fluorescence in situ hybridization (3D-FISH) analyses revealed that the depletion of the Nup93 sub-complex (but not Nup98) disengages the HOXA gene locus from the nuclear periphery, suggesting a potential role for Nup93 in tethering and repressing the HOXA gene cluster. Consistently, Nup93 knockdown increased active histone marks (H3K9ac), decreased repressive histone marks (H3K27me3) on the HOXA1 promoter and increased transcription elongation marks (H3K36me3) within the HOXA1 gene. Moreover, the combined depletion of Nup93 and CTCF (a known organizer of HOXA gene cluster) but not Nup93 alone, significantly increased GLCCI1 gene expression levels. Taken together, this suggests a novel role for Nup93 and its interactors in repressing the HOXA gene cluster.ConclusionsThis study reveals that the nucleoporin Nup93 assisted by its interactors Nup188 and Nup205 mediates the repression of HOXA gene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-016-0106-0) contains supplementary material, which is available to authorized users.

Nup93 and CTCF co-modulate spatiotemporal dynamics and function of the HOXA gene cluster during differentiation

“…We determined levels of HOXA genes upon siRNA mediated knockdown of Nup93, CTCF and their combined knockdowns in NT2/D1 cells ( Figure 4A; Supplementary Figure S5A). While the entire HOXA gene cluster is upregulated in differentiated cells, upon Nup93 Kd (29), HOXA expression was differentially regulated in undifferentiated cells. In undifferentiated NT2/D1 cells, 3' HOXA genes (HOXA1 and HOXA5) were significantly upregulated, while the 5' HOXA genes (HOXA9 and HOXA13), were downregulated ( Figure 4A).…”

“…ChIP was performed as described previously (29). In brief, cells (~1.0 X10 7 ) were cross-linked using 1% formaldehyde for 10 min at RT.…”

“…The Nup93 sub-complex associates with and represses HOXA gene cluster in differentiated cells (29). We examined the role of Nup93 in gene regulation by performing ChIP-Sequencing (ChIP-Seq) analyses of Nup93 in diploid colorectal cancer cells -DLD-1 (Supplementary Figure S1A and B).…”

“…In addition, Nup93 represses HOXA genes in differentiated cells (29), whereas CTCF regulates HOXA gene expression as it functions as an insulator demarcating the HOXA gene cluster. We determined the role of Nup93 and CTCF in HOXA gene regulation during differentiation.…”

A Lucifer‐Based Environment‐Sensitive Fluorescent PNA Probe for Imaging Poly(A) RNAs

“…As little as 0.25 μ m of the PNA probe produced visible nuclear and cytoplasmic staining (green), which was found to increase with increasing probe concentration (Figures and S6). A characteristic punctate nuclear and uniform cytoplasmic staining, resembling the poly(A) RNA staining pattern reported in the literature, was observed (Figure S7) –. As a positive control, cells were incubated with 5′‐Cy5‐(dT) 30 (0.50 μ m ), a commercially available fluorescent DNA ON for detection of poly(A) RNAs .…”