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Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states.

ABSTRACT: Metabolism negotiates cell-endogenous requirements of energy, nutrients and building blocks with the immediate environment to enable various processes, including growth and differentiation. While there is an increasing number of examples of crosstalk between metabolism and chromatin, few involve uptake of exogenous metabolites. Solute carriers (SLCs) represent the largest group of transporters in the human genome and are responsible for the transport of a wide variety of substrates, including nutrients and metabolites. We aimed to investigate the possible involvement of SLC-mediated solutes uptake and cellular metabolism in regulating cellular epigenetic states. Here, we perform a CRISPR-Cas9 transporter-focused genetic screen and a metabolic compound library screen for the regulation of BRD4-dependent chromatin states in human myeloid leukaemia cells. Intersection of the two orthogonal approaches reveal that loss of transporters involved with purine transport or inhibition of de novo purine synthesis lead to dysfunction of BRD4-dependent transcriptional regulation. Through mechanistic characterization of the metabolic circuitry, we elucidate the convergence of SLC-mediated purine uptake and de novo purine synthesis on BRD4-chromatin occupancy. Moreover, adenine-related metabolite supplementation effectively restores BRD4 functionality on purine impairment. Our study highlights the specific role of purine/adenine metabolism in modulating BRD4-dependent epigenetic states.

SUBMITTER: Li KC

PROVIDER: S-EPMC7612075 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe complexity of the neurovascular unit (NVU) poses a challenge in the investigations of drug transport across the blood-brain barrier (BBB) and the function of the brain capillary endothelium. Several in vitro models of the brain capillary endothelium have been developed. In vitro culture of primary endothelial cells has, however, been reported to alter the expression levels of various brain endothelial proteins. Only a limited number of studies have addressed this in detail. The aim of the present study was to investigate mRNA levels of selected BBB transporters and markers in in vitro models of the BBB based on bovine primary endothelial cells and compare these to the levels estimated in freshly isolated bovine brain capillaries.MethodsBrain capillaries were isolated from bovine cerebral cortex grey matter. Capillaries were seeded in culture flasks and endothelial cells were obtained using a brief trypsinization. They were seeded onto permeable supports and cultured in mono-, non-contact- or contact co-culture with/without primary rat astrocytes. mRNA-expression levels of the selected BBB markers and transporters were evaluated using qPCR and monolayer integrity of resulting monolayers was evaluated by measuring the transendothelial electrical resistance (TEER).ResultsThe capillary mRNA transcript profile indicated low expression of ABCC1 and CLDN1. The mRNA expression levels of TPA, OCLN, ABCB1, SLC2A1, SLC16A1 and SLC7A5 were significantly decreased in all culture configurations compared to freshly isolated bovine brain capillaries. ALP, VWF, ABCC1 and ABCC4 were upregulated during culture, while the mRNA expression levels of F11R, TJP1, CLDN5, CLDN1 and ABCG2 were found to be unaltered. The mRNA expression levels of VWF, ALP, ABCB1 and ABCC1 were affected by the presence of rat astrocytes.ConclusionThe endothelial mRNA transcript profile in bovine capillaries obtained in this study correlated nicely with profiles reported in mice and humans. Cultured endothelial cells drastically downregulated the mRNA expression of the investigated SLC transporters but maintained expression of efflux transporter and junctional protein mRNA, implying that the bovine in vitro BBB models may serve well to investigate basic barrier biology and in vivo permeation of passively permeating compounds and efflux transporter substrates but may be less well suited for investigations of SLC-mediated transport.

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Cell-surface SLC nucleoside transporters and purine levels modulate BRD4-dependent chromatin states.

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