Calcium-responsive Transactivator CREST Promotes Neuronal Differentiation Through P300 To Regulate Acetylation Of P53

The calcium-responsive transactivator(CREST)is a nuclear protein highly expressed in the brain.It promotes gene transcription through calcium-responsive transactivation.CREST plays an important role in promoting dendritic growth and development,as well as in maintaining the morphology and function of mature neurons.Expression of CREST emerges at the late stage of the embryonic period,peaks early after birth and remains at a relatively low level throughout adulthood.CREST was only expressed in the postmitotic cells of the cortical plate,not in the proliferating neuroblasts in the ventricular zone.The neurons of CREST-knockdown mice displayed abnormal dendritic growth and development,with significantly lowered average length and number of branches.Our previous work has showed an increased level of CREST expression during the induction of mice neuroblastoma cell(N2a)differentiation.Overexpression of CREST stabilised p53 and enhanced its activity through the promotion of p53 acetylation in N2 a cells,thus upregulating the expression of p21,inhibiting the phosphorylation of retinoblastoma protein(Rb)and further restraining the expression of cyclins.The series of events resulted in cell cycle arrest,promoted the outgrowth of neurites in N2 a cells,and upregulated several neuronal differentiation biomarkers.In the induction of the differentiation of mouse embryonic neural stem cells(NSCs)cultured with low serum level,CREST immunoreactivity increased gradually in the cells whose expression of neuronal specific nuclear protein(NeuN)also increased gradually.The levels of CREST mRNA and protein in NSCs rose with increasing induction time.When inducing the differentiation of CREST-silenced NSCs with low-level fetal bovine serum(FBS),the number of cells expressing NeuN decreased,while the number of cells expressing glial fibrillary acidic protein(GFAP)increased.The mRNA levels of dendritic markers,axonal markers as well as neuron maturity markers were remarkably less than the control cells.These results indicate that CREST is related to the regulation of neuronal differentiation,but its regulatory mechanism in neuronal differentiation remains poorly understood.The activation of downstream gene expression by CREST has been related closely to its recruitment of CREB-binding protein CBP and histone acetyltransferase p300,both of which enhance the acetylation of p53,activate the expression of neuronal differentiation markers and promote neurite outgrowth.This study aims to determine whether CREST activates its downstream genes and promotes neuronal differentiation by promoting the acetylation of p53 via CBP and/or p300.We first confirmed the relationship between CREST and neuronal differentiation.Based on this foundation,we analysed the relationship between p53 and CBP/p300,leading to a deeper understanding of how CREST promotes neuronal differentiation.CREST regulates neuronal differentiation To further verify the relationship between CREST and neuronal differentiation,we first investigated the influence of serum withdrawal culture on CREST expression in N2 a and NSC cells.In N2 a cells,we discovered that both the CREST mRNA and protein levels were increased in a time-dependent manner after serum withdrawal.In differentiated NSCs induced with low(5%)FBS,Real-Time PCR(qPCR)showed that CREST mRNA level increased with time.Immunofluorescent microscopy demonstrated that the immunoreactivity and the ratio of nestin-positive cells reduced significantly after induction.On the contrary,the immunoreactivity and the ratio of CREST-positive cells increased with prolonged serum withdrawal.This implies that the induction of neuronal differentiation by serum withdrawal could upregulate CREST expression.To gain further insight into the influence that CREST has on N2 a cells,we used immunofluorescence and Western blotting in N2 a cells overexpressing CREST to detect the expression and distribution of neurite markers(?-Tub3,the marker of specific mature neuronal skeleton;Map2,the marker of dendrites and NF-H,the marker of axons).Results showed that the protein levels of these three neurite markers increased significantly,concentrating in the cell processes.We further detected the mRNA levels of specific neuronal markers with qPCR,including neuron maturity markers(?-Tub3,NeuN,NeuroD),dendritic markers(Map2,Dbn1)and axonal markers(NF-H,GAP43,Syn1).The results showed that the overexpression of CREST promoted the transcription of the above biomarker genes.In the differentiated N2 a cells induced by serum withdrawal,the silencing of CREST resulted in a complete reversal of the above results,indicating that in N2 a cells,CREST could in general upregulate the genes related to neuronal differentiation.CREST interacts with p53 middle domain through its N-terminus Our previous work indicated that CREST could increase p53 acetylation.To clarify the mechanism of CREST-regulated p53 acetylation,we investigated the interaction characteristics between CREST and p53 as well as their regions of interaction.Double-staining immunofluorescence showed that there was little amount of CREST protein colocalized with p53 in undifferentiated N2 a cells.After their differentiation was induced by serum withdrawal,the immunoreactivities of both CREST and p53 enhanced and the colocalisation ratio increased significantly.Co-immunoprecipitation(Co-IP)was used to detect any in vitro interaction in differentiated N2 a cells induced by serum withdrawal,while in vivo interaction was measured from the brain cortex of 1wk-old mice.It was found that there was indeed interaction between endogenous CREST and p53 both in differentiated N2 a cells and in mice cortex tissue.For N2 a cells,the interaction was enhanced with prolonged serum withdrawal.To determine the interaction regions of CREST and p53 respectively,we introduced various recombinant plasmids into N2 a cells,including pEGFP-CREST,mCherry-p53,as well as a number of mutants with different structural domains deleted.Co-IP assays indicated that the interaction regions were located in the N-terminus(aa1-148)of CREST and in the middle domain(aa101-256)of p53.CREST promotes p53 acetylation at K382 which upregulates downstream gene expression and promotes neuronal differentiation p53 can be activated by enhancing the acetylation of its two acetylation sites K373 and K382,which in turn upregulates neuronal differentiation related genes.To better understand the mechanism behind CREST-promoted neuronal differentiation,we further investigated the influence CREST had on p53 acetylation and subsequently its function in neuronal differentiation.qPCR and Western blotting were used in N2 a cells whose results showed that the overexpression of CREST had no detectable effect on the expression of p53 mRNA but did increase the level of p53 protein.We further determined the protein levels of p53 at different time points after cycloheximide(CHX)treatment,and found that the metabolic stability of p53 was significantly increased after the overexpression of CREST.We then measured the acetylation level of the two acetylation sites K373 and K382 of p53,and came to the conclusion that CREST promoted p53 acetylation at K382 significantly,but had no identifiable effect on the acetylation at K373.Meanwhile upregulation was detected for both the mRNA and the protein levels of p21,downstream of p53.Immunofluorescent microscopy further showed that the distribution of p53 in the nuclei increased significantly during CREST-promoted N2 a cell differentiation.Luciferase reporter gene assay indicated that the overexpression of CREST in undifferentiated N2 a cells could also enhance the transcriptional activities downstream of p53.On the other hand,the silencing of CREST could markedly inhibit the p53-activation of downstream transcription in differentiated N2 a cells induced by serum withdrawal.Hence,it was further confirmed that CREST could enhance p53 stability and promote its downstream transcriptional activities by elevating its acetylation at K382.The acetylated p53 can interact with the promoters of neuronal differentiation related genes,which activate their transcription and thus promote neuronal differentiation.To determine whether the promotion of neuronal differentiation by CREST is indeed through the above pathway,Co-IP and qPCR were applied to measure the effect CREST had on the interactions between p53 and the promoters of neural differentiated marker genes,including those of Map2,GAP43,Coronin 1b,Rab 13,TrkA,and Wnt7 b.The results suggested that CREST could strongly enhance those interactions.On the other hand,the subsequent silencing of p53 can completely reverse the above enhancements.Immunofluorescent staining of ?-Tub3,a neurite marker,indicated that both the silencing of p53 and the overexpression of K382-mutant p53(p53 K382 R,using plasmids)could inhibit CREST-promoted neurite outgrowth.Western blotting showed that silencing p53 after CREST overexpression could downregulate the expression of different neuronal differentiation markers like Map2,GAP43,Coronin 1b,Rab 13,?-Tub3,NF-H and NeuroD.CREST promotes p53 acetylation via p300,thus upregulating the transcription of related genes and promoting neuronal differentiation CREST itself does not possess acetyltransferase activity but it can interact with CBP and p300,both of which could catalyse p53 acetylation at K382.To determine whether CREST promotes neuronal differentiation by promoting p53 acetylation through CBP or p300,we analysed,using Co-IP,the interaction amongst CREST,CBP,p300 and p53 in differentiated N2 a cells.We also examined the influence on CREST-promoted neuronal differentiation when silencing CBP,p300 or p53 respectively.Co-IP results showed that CREST promoted the binding of p53 to p300 rather than to CBP.Triple immunofluorescent staining showed that compared to undifferentiated N2 a cells,in differentiated N2 a cells induced by serum withdrawal,more CREST-p300-p53 colocalized bodies could be seen.Therefore,it could be seen from these results that increased CREST in neuronal differentiation could indeed promote the interaction between p300 and p53.Further studies were performed on CBP and p300 under CREST-promoted neuronal differentiation via increased p53 acetylation.It was observed that the silencing of CBP had no influence on the upregulation of p53 acetylation at K382,on the promotion of neurite outgrowth,or on the increase of protein levels of neuronal differentiation related genes,while silencing p300 could weaken those effects.As a result,p300 may play an important role in CREST-promoted neuronal differentiation via increased p53 acetylation.Conclusion: The expression of CREST is increased during neuronal differentiation.A high level of CREST may lead to the recruitment of p300 and p53 proteins,thus forming a new CREST-p300-p53 protein complex that may promote p300-catalysed p53 acetylation at K382.Acetylated p53 could then activate downstream transcriptional activities,leading to an enhanced transcription of various target genes involved in neuronal differentiation.This mechanism might be the molecular underpinning of CREST-promoted neuronal differentiation.