A 100kD Fragment Of P300 Is Truncated By ?-calpain And Could Promote Differentiation Of Neuronal Cells

Proper neural development requires very precise temporal and spatial expression of a large number of genes and perturbations in these processes lead to disorders. One key mechanism regulating precise gene expression is chromatin structure, which can integrate both cell intrinsic and environmental information to induce specific phenotypic outcomes. Histone acetyltransferases (HATs) are one family of proteins that modulate expression levels of genes in a space- and timedependent manner. p300 was identified as a HAT that regulates transcription of various gene, particularly during cell cycle progression and cellular differentiation. Except HAT domain, p300 also contains several conserved functional domains through which it interacts with other cellular proteins. the basic functional mode of p300 is to serve as a HAT or a FAT, and to act as a scaffold or bridge for transcription factors and other components of the basal transcription machinery to facilitate chromatin remodeling and to activate gene transcription, such as acetylates p53. Neuronal acetylation of p53 at specific lysines in its C-terminus promotes neurite and axonal outgrowth in permissive conditions and is required for axonal regeneration in vivo. While the similar transcriptional coactivator CBP, they have been implicated in processes as diverse as cell cycle regulation, apoptosis, embryonic development, cellular differentiation and cancer, but the role of p300 in neural differentiation could not be replaced by CBP.Though p300 is essential for embryonic stem cell and F9 cell in differentiation, the mRNA level increase while protein level decrease when embryonic stem cell is induced by embryoid body formation. Moreover, the acetyltransferase p300 is strongly and selectively degraded during RA-induced F9 differentiation. Concomitantly, the HAT-specific enzymatic activity of p300 dramatically increases during differentiation. p300 can be degraded by proteasome pathway. But it is unconfirmed that the degradation of p300 and the increase of HAT activaty during differentiation wheter has relationship with proteasome pathway. A specific inhibitor of calpains can block destruction of CBP/p300 in acute myeloid leukemia. It illustrates that p300 can be cleavage by calpain.In this study prove that during differentiation development process of fetal mouse tissues and mouse neuroblastoma cells (N2a cell). Based on the increase of p300 mRNA with the reduce of full length p300 protein, p300 can be cleavaged by mu-calpain and produce a large number of N terminal fragment sized 100kD. it is first detected in the process of neuronal differentiation, and preliminary evidenced that the fragment has stronger effect on promoting neural cell differentiation than full length p300Decrease in p300 protein level of embryionic mouse brain and N2a cell during differentiation In order to prove the phenomenon that p300 protein decrease with p300 mRNA increase during differentiation. We extract the protein of mouse embryos brain from E9.5 to E16.5, and test the trends of p300 protein level during mice embryonic development. We found that p300 protein levels significantly decreased during mouse embryos E9.5 to E15.5. Then, we induced N2a cells differentiation by retinoic acid RA from 0 to 2 days, using RT-PCR and western blot to test the trend of p300 mRNA level and protein level. We found that p300 protein decrease with p300 mRNA increase during differentiation.Those results illustrate the phenomenon that p300 protein decrease with p300 mRNA increase during differentiation also present in neuron systerm differentiation.Decrease in full length p300 while increase in 100kD fragment of p300 during differentiation of embryonic mouse brain tissues and N2a cell The phenomenon that p300 protein decrease with p300 mRNA in embryionic mouse brain and N2a cell increase during differentiation tell us that p300 is degraded during differentiation. So we using the p300 N-terminal antibody tested full length p300 and its degradation products protein level in embryonic mice brain and RA-induced N2a cells by western blot. We found a new segment named lOOkD fragment of p300 (100kD p300). During embryonic brain development (E9.5 to E 12.5) and RA differentiation (from 0 to 2 days), 100kD p300 protein level increases along with the full length p300 (FL-p300) protein expression level decreases,while lOOkD p300 and FL-p300 degraded together after E13.5.p300 is cleavaged to generate 100kD p300 between amino acids 726 to 1020 lOOkD p300 can be tested by p300 N-terminal antibody, so the cleavage site is a third of p300 N terminal calculated by weight. And p300 homologous protein CBP didn't has simple phenomenon during differentiation. Though they have 60% identify in sequence, the similarity in a third of N terminal is little. So we use CBP as a control, constructed plasmids as competitive substrate of FL-p300 cleavaged site. After transfecting plasmids into N2a cell, we used RA inducing N2a cell differentiation 36h. Western blot results show, controled with HA group, CBP Short fragment (aa 777-974,CBP S) and CBP Long fragment (aa726-1020, CBP L) had no effect on the generation of 100kD p300. While p300 Short fragment (aa 777-974,p300 S) and p300 Long fragment (aa726-1020, p300 L) could inhibit the generation of 100kD p300, and the p300 L is better. Controled the role of RA in inducing N2a cells' nutrite growth, which save as a sign of neuron differentiation. It showed that p300 S and p300 L depress the neutrite growth, and the p300 L is better. The results show that the cleavage site that generating lOOkD p300 is between the amino acids 726-1020,and imply that cleavaging p300 in the site could promote neuron differentiation.?-calpain is the cleavage protease that generates100kD p300 In order to find the cleavage enzyme that generating 100kD p300, we do research as follows. First, we observed the effect of proteasome inhibitor MG132 and calpain inhibitor calpeptin on the generation of lOOkD p300 during RA induced N2a cell differentiation. Results show that, MG132 and calpeptin had no effect on degradation of p300 and 100kD p300 in the undifferentiated group. In RA differentiation group, MG132 could block the degradation of p300, but it could not affect the generation of lOOkD p300, while calpeptin could inhibite the degradation of FL-p300 and the generation of 100kD p300. So calpain is the cleavage enzyme that generating 100kD p300. calpain family contains ?-calpain(micromole) and m-calpain(millimole), which need different calcium concentration to active.we used ?-calpain and m-calpain to digeste brain protein of mice embryos E13.5 in vitro,and the calcium concentration is 5?mol/L and 5mmol/L. We found that m-calpain could reduce FL-p300 but had no effect on the generation of 100kD p300. While ?-calpain could promote the reduce FL-p300 and the generation of lOOkD p300, and the best enzyme concerntration is between 0.01U/ul and 0.02 U/?l. Adding 10 ?mol/L calpeptin and ?-calpain (0.003U/?l or 0.01 U/?l) could inhibit the cleavage of ?-calpain,especially in 0.01 U/?l. Therefore, ?-calpain is the cleavage enzyme that generating 100kD p300.We transfected HA-p300 to N2a cells, after 36h extracted protein And using HA tags antibody detection, we found that 100kD p300 had HA tag and the generation could be inhibited by calpeptin. So the cleavage site that generating 100kD p300 is determined at p300 N-terminal 1/3.Our further study in the activity of ?-calpain in mouse embryonic brain during development showed that, the level of ?-calpain activity large subunit is increased during E9.5 to E15.5. It illustrates that the increasing generation of 100kD p300 is correlated with the increasing activity of ?-calpain.100kD N-terminal fragment of cleavaged p300 could promote differentiation of N2a cell In order to understand the role of p300 cleavaged into 100kD p300 in differentiation, and the different effect of p300 fragments on N2a cell differentiation. Results show overexpression p300 N terminal fragment (aa9-1007, p300 N) could promot the growth of neutrite and the expression level of GAP43. Overexpression p300 C terminal fragment (aa1006-2414, p300 C) could promot p53 actylation but had no effect on the growth of neutrite and the expression level of GAP43. And overexpression p300 N and p300 C together, the role in promoting the growth of neutrite and the expression level of GAP43 is weaker tha p300 N, just like p300. and the role in promoting p53 actylation is stronger than p300, but weaker than p300 C. The results show that the function of p300 in promoting neuron differentiation is completed by p300 N terminal 100kD fragment. p300 C has stonger role in promoting actylation.and the role of full length p300 in promoting differentiation and acytilation is weaker.Conclusion:p300 is cleavaged by ?-calpain at the N terminal 1/3 and generate a 100kD fragment, the cleavage site is in the area of amino acids 726 to 1020. The function of p300 in promoting neuron differentiation is completed by p300 N terminal 100kD fragment. p300 C has stonger role in promoting actylation.and the role of full length p300 in promoting differentiation and acytilation is weaker.