Necl1 Protein Expression In Glioma Missing Epigenetic Mechanism

NECL1 (nectin-like molecule 1), also named as CADM3/IGSF4B/TSLL1/ SynCAM3, was first cloned and reported by our lab in 1998, it is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule. NECL1 is a membrane protein containing 398 amino acids, and it includes a extracellular region composed by three Ig-like domains, a single transmembrane region and a short cytoplasmic region. In the previous studies of our lab, we found the cytoplasmic region of NECL1 could recruit 4.1N to the membrane, and resolved the crystal structure of the V domain of NECL1 and found that Phe82 was a key residue for the dimerization of NECL1 V domain. Up to now, studies have reproted that NECL1 plays an important role in homo- or heterophilic interaction, the formation of synapses, axon bundles and myelinated axons, and the morphogenesis of cerebellum. The other lab has reported that NECL1 is abrogated or markedly reduced in glioma cell lines at RNA level, it maybe a potential tumor suppressor in gliomas in 2001. This PhD dissertation is mainly focused on the studies of epigenetic mechanisms of the silence of NECL1 in gliomas.Real-time PCR and immunohistochemistry methods were used to find that compared with the normal brain tissue, NECL1 is abrogated or markedly reduced in 6 human glioma cell lines and 18 human glioma tissues(gradeâ…¡: 8, gradeâ…¢: 5, gradeâ…£: 5), this result suggests that NECL1 is probably a tumor suppressor in glioma at mRNA level and protein level. Generally, DNA methylation of the promoter region and histone deacetylation are often associated with the silence of genes, and DNA demethylation and histone acetylation are often associated with the expression of genes. Based on the above cues, this PhD dissertation is mainly focused on the epigenetic regulation of NECL1 abrogation in gliomas.After the transcription start site (named as +1) of NECL1 is determined by 5'RACE, we ligated a series of deletion fragments of NECL1 promoter region into reporter gene vector and carried out dual-luciferase reporter gene experiment in 293ET, T98G and U251 cell lines. This experiment identified the basic promoter region of NECL1.Bioinformatics analysis indicated that there is a CpG island in the promoter region of NECL1. Bisulfite sequencing was used to find that there are no hypermethylation of NECL1 promoter region in glioma cell lines and glioma tissues. The above results indicated that the abrogation of NECL1 in glioma is not due to the direct hypermethylation of NECL1 promoter region.Based on the above result, we detected the activity of histone deacetylase(HDAC) in glioma tissues and found the activity of HDAC was higher than that in normal tissues, and the expression of NECL1 was restored in glioma cell lines after treated with the inhibitor of histone deacetylase TSA, these results indicated that the abrogation of NECL1 was associated with histone deacetylation. Chromatin immunoprecipitation assay showed a significant increase of acetyl histone H3 binding to the promoter of NECL1 in T98G and U251 cell lines treated with TSA. Paper has reported TSA could activate the expression of some genes through Sp1 binding sites. Bioinformatics analysis indicated there are two potential Sp1 binding sites in the promoter region of NECL1. Luciferase reporter gene assay indicated that the transcription activity of NECL1 is markedly increased after the treatment of TSA, and it is only the Sp1 binding site located upstream of transcription start site that could response to the stimulation of TSA. DNA pulldown assay clarifies the exact binding of Sp1 on NECL1 basic promoter region. Dual-luciferase reporter gene assay was used to find that the transcription activity of NECL1 was not increased after this Sp1 binding site mutated. Taken all these results together, TSA can activate the expression of NECL1 through Sp1 binding site.Based on clues of pertinent literatures, Sp1 may recruit HDAC corepressor complex to repress the expression of genes before the treatment of TSA, but after the treatment of TSA, Sp1 recruits p300/CBP coactivator complex to activate the expression of genes. So, ChIP assay and Co-IP assay were used to detect the interactions between Sp1 and HDAC1 or p300/CBP before and after the treatment of TSA. The results indicated that Sp1 can interact with HDAC1 but not with p300/CBP before the treatment of TSA; but after the treatment of TSA, Sp1 can interact with p300/CBP, the interaction between Sp1 and HDAC1 is markedly decreased. Based on the results of this part, we constructed the epigenetic regulation model of how TSA activated the expression of NECL1.In addition, the bioinformatics analysis, N-Glycosidase F treatment experiment, site-specific mutagenesis and cell aggregation assay were used to firstly demostrate that NECL1 is a glycoprotein with a single N-glycosylation site which can influence the cell-cell adhesion activity.