Fig. 6: XBP1s condensates enhance the interaction between the MGRN1 promoter and distal enhancer especially under copper stress.

A Schematic view across the MGRN1 gene locus (chr16:4, 250,000–5,000,000) with genomic and epigenetic information. Graphic active regulatory regions were generated from CUT&Tag data of XBP1s, P300, MED1, BRD4 and H3K27ac, respectively treatment with ES at different concentrations (Vector, 40 nM and 100 nM). Delineate potential super-enhancer sites through the identification of shared enrichment domains across overlapping image regions. The BgIII digestion sites and corresponding fragments are numbered below the graph. B CUT&Tag sequencing results depicting the genomic elements enriched with XBP1s under ES 100 nM treatment. The pie chart illustrates the distribution of XBP1s-binding regions across various genomic features, including promoters, enhancers, 5’ U TRs, 3’ UTRs, exons, and intergenic regions. C Workflow diagram of Chromatin conformation capture (3 C), illustrating DNA crosslinking, digestion, ligation, and DNA purification steps. P1 and P2 represent the two ends of the MGRN1 promoter, while E1 and E2 represent the two ends of the potential enhancer regions. Four possible primer combinations (P1E1, P1E2, P2E1, P2E2) were designed to assess physical interactions between the promoter and enhancer regions. If a physical interaction exists, PCR amplification of the 3 C products will yield DNA fragments of the expected size (about 250–350 bp), which can be visualized as distinct bands using agarose gel electrophoresis. D Agarose gel electrophoresis of 3 C products from A549 cells, targeting 8 potential regions identified based on (A) and BglII digestion sites. Each region was tested for interaction with the MGRN1 promoter using primer combinations P1E1, P1E2, P2E1, and P2E2. The results indicate that region 2 produces bands of the expected size for all primer combinations (P1E1, P1E2, P2E1, and P2E2), while region 3 only produces a band for P2E1. Other regions were excluded due to the absence of bands of the correct size. E 3 C experiments repeated in PC9 cells for regions 2 and 3. Results reveal that only region 2 exhibits correctly sized positive bands for P1E2 and P2E2, while no such bands are observed for region 3. F Knockdown of XBP1 significantly reduced the interaction between region 2 and the MGRN1 promoter, as demonstrated by diminished PCR band intensity in both A549 and PC9 cells. G, H qPCR analysis of the 3 C products under various treatment conditions (Vector, 40 nM and 100 nM) revealed that copper stress significantly enhanced the interaction intensity between the MGRN1 promoter and region 2 in both A549 and PC9 cells. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns not significant. Error bars represent mean ± SD from three independent experiments. I A refined depiction of an enhancer cluster architecture, facilitated by XBP1s-mediated phase separation, encompasses the integration of coactivators and is reinforced by distal enhancers, collectively orchestrating the potentiation of MGRN1 gene transcription.