PMID- 34694864 OWN - NLM STAT- MEDLINE DCOM- 20220107 LR - 20220107 IS - 1524-4628 (Electronic) IS - 0039-2499 (Linking) VI - 52 IP - 11 DP - 2021 Nov TI - GATA3 (GATA-Binding Protein 3)/KMT2A (Lysine-Methyltransferase-2A) Complex by Increasing H3K4-3me (Trimethylated Lysine-4 of Histone-3) Upregulates NCX3 (Na(+)-Ca(2+) Exchanger 3) Transcription and Contributes to Ischemic Preconditioning Neuroprotection. PG - 3680-3691 LID - 10.1161/STROKEAHA.121.034637 [doi] AB - BACKGROUND AND PURPOSE: NCX3 (Na+-Ca2+ exchanger 3) plays a relevant role in stroke; indeed its pharmacological blockade or its genetic ablation exacerbates brain ischemic damage, whereas its upregulation takes part in the neuroprotection elicited by ischemic preconditioning. To identify an effective strategy to induce an overexpression of NCX3, we examined transcription factors and epigenetic mechanisms potentially involved in NCX3 gene regulation. METHODS: Brain ischemia and ischemic preconditioning were induced in vitro by exposure of cortical neurons to oxygen and glucose deprivation plus reoxygenation (OGD/Reoxy) and in vivo by transient middle cerebral artery occlusion. Western blot and quantitative real-time polymerase chain reaction were used to evaluate transcripts and proteins of GATA3 (GATA-binding protein 3), KMT2A (lysine-methyltransferase-2A), and NCX3. GATA3 and KMT2A binding on NCX3 gene was evaluated by chromatin immunoprecipitation and Rechromatin immunoprecipitation experiments. RESULTS: Among the putative transcription factors sharing a consensus sequence on the ncx3 brain promoter region, GATA3 was the only able to up-regulate ncx3. Interestingly, GATA3 physically interacted with KMT2A, and their overexpression or knocking-down increased or downregulated NCX3 mRNA and protein, respectively. Notably, site-direct mutagenesis of GATA site on ncx3 brain promoter region counteracted GATA3 and KMT2A binding on NCX3 gene. More importantly, we found that in the perischemic cortical regions of preconditioned rats GATA3 recruited KMT2A and the complex H3K4-3me (trimethylated lysine-4 of histone-3) on ncx3 brain promoter region, thus reducing transient middle cerebral artery occlusion-induced damage. Consistently, in vivo silencing of either GATA3 or KMT2A prevented NCX3 upregulation and consequently the neuroprotective effect of preconditioning stimulus. The involvement of GATA3/KMT2A complex in neuroprotection elicited by ischemic preconditioning was further confirmed by in vitro experiments in which the knocking-down of GATA3 and KMT2A reverted the neuroprotection induced by NCX3 overexpression in cortical neurons exposed to anoxic preconditioning followed by oxygen and glucose deprivation plus reoxygenation. CONCLUSIONS: Collectively, our results revealed that GATA3/KMT2A complex epigenetically activates NCX3 gene transcription during ischemic preconditioning. FAU - Guida, Natascia AU - Guida N AUID- ORCID: 0000-0003-0887-3239 AD - IRCCS SDN, Naples, Italy (N.G., S.A., L.A.). FAU - Mascolo, Luigi AU - Mascolo L AD - Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy (L.M., O.C., P.B., G.P., P.M., L.F.). FAU - Serani, Angelo AU - Serani A AUID- ORCID: 0000-0002-8734-6543 AD - Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Genoa, Italy (A.S.). FAU - Cuomo, Ornella AU - Cuomo O AD - Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy (L.M., O.C., P.B., G.P., P.M., L.F.). FAU - Anzilotti, Serenella AU - Anzilotti S AUID- ORCID: 0000-0002-8729-0009 AD - IRCCS SDN, Naples, Italy (N.G., S.A., L.A.). FAU - Brancaccio, Paola AU - Brancaccio P AD - Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy (L.M., O.C., P.B., G.P., P.M., L.F.). FAU - Pignataro, Giuseppe AU - Pignataro G AUID- ORCID: 0000-0002-7290-4397 AD - Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy (L.M., O.C., P.B., G.P., P.M., L.F.). FAU - Molinaro, Pasquale AU - Molinaro P AUID- ORCID: 0000-0003-2770-2267 AD - Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy (L.M., O.C., P.B., G.P., P.M., L.F.). FAU - Annunziato, Lucio AU - Annunziato L AUID- ORCID: 0000-0002-8534-8270 AD - IRCCS SDN, Naples, Italy (N.G., S.A., L.A.). FAU - Formisano, Luigi AU - Formisano L AUID- ORCID: 0000-0001-7294-8595 AD - Division of Pharmacology, Department of Neuroscience, Reproductive and Dentistry Sciences, School of Medicine, "Federico II" University of Naples, Naples, Italy (L.M., O.C., P.B., G.P., P.M., L.F.). LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20211025 PL - United States TA - Stroke JT - Stroke JID - 0235266 RN - 0 (GATA3 Transcription Factor) RN - 0 (Histones) RN - 0 (Sodium-Calcium Exchanger) RN - 0 (histone H3 trimethyl Lys4) RN - EC 2.1.1.43 (Histone-Lysine N-Methyltransferase) SB - IM MH - Animals MH - Brain/blood supply MH - Brain Ischemia/metabolism MH - GATA3 Transcription Factor/*metabolism MH - Gene Expression Regulation/*physiology MH - Histone-Lysine N-Methyltransferase/*metabolism MH - Histones/metabolism MH - *Ischemic Preconditioning MH - Male MH - Mice MH - Neuroprotection/*physiology MH - Rats MH - Rats, Sprague-Dawley MH - Sodium-Calcium Exchanger/*biosynthesis MH - Up-Regulation OTO - NOTNLM OT - histone OT - ischemic preconditioning OT - methyltransferase OT - neuroprotection oxygen EDAT- 2021/10/26 06:00 MHDA- 2022/01/08 06:00 CRDT- 2021/10/25 17:13 PHST- 2021/10/25 17:13 [entrez] PHST- 2021/10/26 06:00 [pubmed] PHST- 2022/01/08 06:00 [medline] AID - 10.1161/STROKEAHA.121.034637 [doi] PST - ppublish SO - Stroke. 2021 Nov;52(11):3680-3691. doi: 10.1161/STROKEAHA.121.034637. Epub 2021 Oct 25.