SIRT6 Regulates The Function Of NeuroD6 Through Deacetylation And Affects The Growth Of Neuronal Neurites

Background:Sirtuins are a family of NAD+(nicotinamide adenine dinucleotide)-dependent deacetylases and have also been identified as class III histone deacetylases(HDAC).Sirtuins are also known as mammalian silencing regulators.This family is composed of seven members(SIRT1-SIRT7)and they are involved in many cell functions,including regulating cell proliferation and metabolism,maintaining genome stability,and regulating the stress response of cells.At the same time,these family members of Sirtuins differ in their subcellular localizations.SIRT6(silent mating type information regulation 2 homolog 6)that has single ADP-ribosyltransferase activity and deacetylase activityplays an important role in regulating genome stability,cell metabolism,stress response and aging processWhen SIRT6 is used as a deacetylase,it deacetylates a variety of protein substrates including histones and non-histones.For example,SIRT6 can deacetylate H3K9ac.At the same time,SIRT6 also inhibits the transcription of NF-?B and hypoxia-inducible factor la(HIF-1?)target genes.Other studies have shown that SIRT6 determines the diferentiation of stem cells and neural differentiation.SIRT6 is also closely related to the generation of nerve cells in the brain.Studies have shown that mice in which that SIRT6 is over-expressed in adult hippocampus show an increase in the number of immature neurons and a decrease in the number of mature neurons,but do not affect the differentiation and production of glial cells,suggesting that SIRT6 may be involved in neuronal differentiation and maturation in the hippocampus.In addition,SIRT6 may function as a regulator of cell fate during hippocampal neurogenesis.Based on the above studies,we found that SIRT6 is closely related to various brain functions,especially it can affect the differentiation and proliferation of neural cells.However,how SIRT6 expression is regulated remains still unknown.NeuroD6(Neurogenic Differentiation 6),also known as Nexl,contains bHLH(basic helix-loop-helix)DNA binding domain,so it binds to the upstream promoter E-box(CANNTG)sequence of the gene as a nerve-specific transcription factor to promote expression of related downstream genes.It was found that the expression of NeuroD6 gene is parallel to the process of neural differentiation and synapse formation.At the same time,BDNF(brain-derived neurotrophic factor)is the most known neurotrophic factor.It binds with its TrkB(tyrosine kinase B)receptor to activate a series of downstream pathways to increase synaptic plasticity and promote neurogenesis.According to the results of previous research in the laboratory,there is a certain interaction between SIRT6 and NeuroD6.Based on this,we continued to explore the relationship between the two,and provided new insights for regulation of neurite outgrowth by SIRT6.Purpose:Therefore,the purpose of this experiment was to further verify and explore the relationship between SIRT6 and NeuroD6.First,we tested whether SIRT6 binds to NeuroD6 and regulates its acetylation level.Next,we identified whether NeuroD6 regulates the expression of brain-derived neurotrophic factor BDNF.Finally,a series of experiments were performed to test whether SIRT6 regulates the expression level of BDNF and whether SIRT6 has a certain effect on the neurite outgrowth.Methods:(1)We first verified the localization of SIIRT6 and NeuroD6 in cells using immunofluorescence staining,and then used Western Blot to determine their distributions in the nuclear and cytoplasm.Co-IP experiments were performed to test whether SIRT6 and NeuroD6 were combined.At the same time,a Flag tag vector was constructed,and the IP experiment was performed to test the interaction between SIRT6 and NeuroD6 again.Finally,in vitro expression vectors were constructed and expressed..Pulldown experiment was used to verify whether the two proteins have an interaction in vitro.(2)The Ch-IP experiment was used to verify whether NeuroD6,as a transcription factor,binds to the E-box region of BDNF to promote its expression.When the content of NeuroD6 decreased,the binding ability of NeuroD6 with the BDNF E-box region also decreased.At the same time,a biotin probe containing the BDNF promoter region was constructed,and the DNA-pulldown experiment was used to pull NeuroD6 in the opposite direction to detect the binding ability of the two.(3)After verifying that NeuroD6 has a transcription-promoting effect on BDNF,I studied the effect of SIRT6 on BDNF.A mutant plasmid of SIRT6 and a lentivirus encoding SIRT6 shRNA were constructed,and the luciferase reporter assay was used to verify whether SIRT6 affects the activity of the E-box region of BDNF.(4)Finally,verified whether SIRT6 can regulate neurite outgrowth based on the deacetylation of NeuroD6.Primary mouse cortical neurons were cultured,transfected with empty vector,SIRT6 overexpression vector,and SIRT6 mutants,and Western Blot was used to detect the level of acetylated NeuroD6.Simultaneous immunofluorescence staining was performed to study the effect of SIRT6 on the neurite outgrowth.Results:(1)We verified the localization of SIRT6 and NeuroD6 in the nucleus by Western blot and immunofluorescent staining.Co-IP experiments confimed that SIRT6 was indeed combined and interacted with NeuroD6 in vitro.(2)Ch-IP experiments verified that NeuroD6 as a transcription factor combined with both the E-box region and enhancer region of BDNF to promote the expression of BDNF.When the content of NeuroD6 was reduced,its binding ability with the promoter region and enhancer region of the bound BDNF also decreased.At the same time,DNA-pulldown experiments showed that there was a large amount of bindings between the two in the promoter region of BDNF.(3)The luciferase reporter assay showed thatreduced enzyme activity of SIRT6 caused areduction in the activity of the downstream BDNF promoter region indicating that the ability of NeuroD6 to bind BDNF promoter region also decreased.(4)Finally,after transfection of cultured primary cortical neuron with vectors encoding flag,SIRT6-flag or SIRT6 mutant-flag.Western blot showed that SIRT6 overexpressioncaused an increase in the level of the acetylated NeuroD6 and an increase in complexity of neurite tree in the primary cortical neuron compared with control neurons expressing flag only.There was not difference in neurite outgrowth between control and mutant groups.Conclusion:Based on the above results,we found that SIRT6 and NeuroD6 were combined and interacted in vitro.The ability of NeuroD6 to bind the downstream BDNF promoter was depending on the content and the transcriptional activity of NeuroD6.SIRT6 regulated the neurite outhrowth through deacetylating NeuroD6.