| NeuroD is a family of bHLH transcription factors, which play a pivotal role in neuronal differentiation and neuronal development. There are seven members in the family, named NeuroD1～6, and PJA2. As a neuron differentiation factor, NeuroD1 is expressed predominantly in peripheral nerve and genesis of central nervous system. In spite of NeuroD1 is transiently expressed in differentiating neurons in a subset of neural tissues, it also maintains in the cells of the adult mouse cerebellum and hippocampus. However, the expression level gets lower and lower with the mature process of neuron cells. Xenopus ectoderm can be conversed into neurons by NeuroD1. NeuroD1 is also expressed in medulloblastoma, neuroblastoma, and retinoblastoma cells, and once activated and induced to neuron the expression level of NeuroD1 significantly increased. neuroD1 -null mice die shortly after birth. If rescued them by introducing expression plasmid encoding mouse neuroD1 gene, these mice survive to adulthood but appear neuronal deficit in the granule layers of the cerebellum and hippocampus.In eukaryote, histone is an important part of nucleosome. Covalent modifications, such as acetylation occur in the N-terminal amino acids of histones. Histone acetylation is enriched in chromatin structure with where gene transcription is functionally active. Acetylation modification of histone is a reversible dynamic process determined by the accurate balance between the histone acetyltransferases (HAT) and the histone deacetyltransferase (HDAC).In our study, all-trans retinoic acid (RA) treated neuroblastoma SH-SY5Y cells are taken as a model to study mechanisms of neuronal differentiation. The ChIP on Chip assay used here is the association of chromatin immunoprecipitation and promoter DNA array to detect the genome wide distribution of the acetylated residues of both H3k9 and H3K14 with over 20,000 gene promoters covered in one array. We chose to study the acetylation of these lysine residues because they usually stand for an active chromatin structure where transcription machinery is on. Raw data were analyzed by log2 Ratio≥1 or log2 Ratio≤-1, and 597 genes with elevated level of acetylated histone H3 and 647 genes with lowered acetylated histone in the promoter areas were identified. We have selected some genes with changes in acetylation status to check the reliability of the above data and found a consistency of 70%. The significant ones with consistency in both assays are 7 genes, which include the lowered PRKCA and the elevated CGB2, ELF3, CUL7, RARB, FOXH1, JARID1A genes. In contrast, NRG1, MVP and CSF1R promoters showed no marked changes in histone acetylation and thus were inconsistent with the ChIP on Chip results. The most worth mentioning point is that while acetylation of neuroD1 core promoter decreases, its mRNA level elevated. This prompts us to further investigate the mechanism of neuroD1, the neural differentiation marker gene during RA induced neuronal differentiation.To explore the regulation mechanisms of neuroD1 gene during neuronal differentiation, we take RA treated teratocarcinoma P19 cells as a differentiation model specifically for mechanistic studies on neuroD1 gene. We showed that, as the mRNA of neuroD1 increasing upon RA treatment, the acetylation on H3 sites in the promoter region was getting lower and lower, which was exactly in accordance with the situation of SH-SY5Y cells under RA induced differentiation.It has been known for years that NeuroD1 is capable of trans activating its own promoter, and the activation was E1 box dependent in cis. We showed in P19 cells that the trans auto-regulation of neuroD1 gene can be enhanced by RA. Additionally, histone deacetyltransferase HDAC3 showed synergic effect on the self-activation of NeuroD1. In vitro co-IP showed that NeuroD1 and HDAC3 coexisted in the same protein complex, whereas other HDACs (1,2, 4～6) were non-effective. Unsimilar with the HDAC3 effect shown above, an HDAC inhibitor, tricostatin A (TSA) inhibited the NeuroD1 auto-activity effect. Western blotting and Immunofluorescence analyses showed that NeuroD1 was dominantly located in the nucleus. However, the higher level of NeuroD1 protein induced by TSA was concentrated on the nuclear membrane.In conclusion, in neuronal differentiation process, the expression of the marker gene neuroD1 is regulated in several levels and through several pathways accurately. As an auto-regulated gene, NeuroD1 protein enhances its own gene expression, this effect is induced by RA and HDAC3, but inhibited by TSA, which suggests a critical role of HDAC3 both in synergistically induced NeuroD1 expression and to counteract acetylation on H3K9-H3K14 as shown in ChIP on Chip and in the RA induced neuronal differentiation from P19 and SH-SY5Y cells.
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