Aims Our previous study demonstrated that Ca2+ influx through the Orai1 store-operated Ca2+ channel in macrophages contributes to foam cell formation and atherosclerosis via the calcineurin–ASK1 pathway, not the classical calcineurin–nuclear factor of activated T-cell (NFAT) pathway. Moreover, up-regulation of NFATc3 in macrophages inhibits foam cell formation, suggesting that macrophage NFATc3 is a negative regulator of atherogenesis. Hence, this study investigated the precise role of macrophage NFATc3 in atherogenesis. Methods and results Macrophage-specific NFATc3 knockout mice were generated to determine the effect of NFATc3 on atherosclerosis in a mouse model of adeno-associated virus-mutant PCSK9-induced atherosclerosis. NFATc3 expression was decreased in macrophages within human and mouse atherosclerotic lesions. Moreover, NFATc3 levels in peripheral blood mononuclear cells from atherosclerotic patients were negatively associated with plaque instability. Furthermore, macrophage-specific ablation of NFATc3 in mice led to the atherosclerotic plaque formation, whereas macrophage-specific NFATc3 transgenic mice exhibited the opposite phenotype. NFATc3 deficiency in macrophages promoted foam cell formation by potentiating SR-A- and CD36-meditated lipid uptake. NFATc3 directly targeted and transcriptionally up-regulated miR-204 levels. Mature miR-204-5p suppressed SR-A expression via canonical regulation. Unexpectedly, miR-204-3p localized in the nucleus and inhibited CD36 transcription. Restoration of miR-204 abolished the proatherogenic phenotype observed in the macrophage-specific NFATc3 knockout mice, and blockade of miR-204 function reversed the beneficial effects of NFATc3 in macrophages. Conclusion Macrophage NFATc3 up-regulates miR-204 to reduce SR-A and CD36 levels, thereby preventing foam cell formation and atherosclerosis, indicating that the NFATc3/miR-204 axis may be a potential therapeutic target against atherosclerosis.

Epstein-Barr virus nuclear antigen 2 (EBNA2) is a transactivator of viral and cellular gene expression, which plays a critical role in the Epstein-Barr virus-associated diseases. It was reported that EBNA2 regulates gene expression by reorganizing chromatin and manipulating epigenetics. Recent studies showed that liquid-liquid phase separation plays an essential role in epigenetic and transcriptional regulation. Here we show that EBNA2 reorganized chromatin topology to form accessible chromatin domains (ACDs) of the host genome by phase separation. The N-terminal region of EBNA2, which is necessary for phase separation, is sufficient to induce ACDs. The C-terminal domain of EBNA2 promotes the acetylation of accessible chromatin regions by recruiting histone acetylase p300 to ACDs. According to these observations, we proposed a model of EBNA2 reorganizing chromatin topology for its acetylation through phase separation to explain the mechanism of EBNA2 hijacking the host genome by controlling its epigenetics.

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra–Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.

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Background: Chromatin physical interactions provide essential information for understanding the regulation of ciselements like enhancers, promoters, and insulators in cell development and differentiation. The Hi-C assay is a technique detecting chromatin structures of the whole genome, but not sensitive to interactions of regulatory elements. Several methods, like HiChIP, DNase-C, and OCEAN-C, have been developed for enriching interactions of regulatory regions, but all of them have some shortcomings. New simple, efficient, and robust methods are still in need for detecting interactions of regulatory regions. Results: We developed a new, simple, and robust assay called CoP (Column Purified chromatin) for profiling of open chromatin regions by directly purifying fragmentized crosslinked chromatin with a DNA purification column. The accessible chromatin regions, including active enhancers, promoters, and insulators, were significantly enriched in CoP chromatin. The CoP-seq assay can efficiently detect open chromatin regions, especially active promoters, with a high signal-to-noise ratio. We integrated the CoP-seq and Hi-C technique (HiCoP) to detect interactions of accessible chromatin regions, which represent active cis-regulatory elements in cells. We observed that the HiCoP captured the peaks in the promoters-associated enhancer regions. HiCoP detected more promoter-enhancer (P-E), promoter-promoter (P-P), and enhancer-enhancer (E-E) interactions within 20 kb-5 Mb than Hi-C. Most of the loops identified by HiCoP are associated with the expressed genes. Conclusion: CoP assay can efficiently enrich open chromatin regions. When CoP assay was integrated with Hi-C assay, it provides a simple, robust, alternative technique for profiling accessible chromatin regions and chromatin conformation simultaneously.