Roles Of Histone Acetyltransferase P300 In ALAS2 Gene Transcription Regulation And The Mechanism By Which Deacetylase Inhibitor Butyrate Activates ALAS2 Gene Transcription

Erythroid-specific 5-aminolevulinate synthase (ALAS2) catalyzes the rate-limiting step in heme biosynthesis of erythroid cells. The absence of ALAS2 leads to maturation arrest of primitive erythroid cells. Heritable mutations of ALAS2 gene are responsible for the human X-linked sideroblastic anemia (XLSA). ALAS2 expression is markedly increased during erythropoiesis to meet the demand for heme in hemoglobin production. The expression of ALAS2 gene is controlled at both the transcriptional and translational levels.Experimental data presented in this thesis show that treatments of erythroid K562 cells with HDAC inhibitors NaBu or TSA gave rise to a significant increase in ALAS2 gene transcripts, with a concurrent increase in acetylation level of histone H4 at ALAS2 gene promoter, as revealed by quantitative PCR and chromatin immunoprecipitation (ChIP) analyses. ChIP assays indicated that the histone acetyltransferase p300 bound with ALAS2 gene promoter. Overexpression of p300 increased both the promoter reporter expression and endogenous mRNA level of ALAS2. The histone acetyltransferase (HAT) activity of p300 was necessary for it to activate the ALAS2 gene promoter. Additionally, two functional Sp1 sites located in ALAS2 gene promoter were identified by using EMSA tests. The site mutation analyses revealed that both of the GATA-1 sites at ALAS2 gene promoter contributed to p300 recruitment. The reporter ChIP experiments showed that p300 and GATA-1 worked cooperatively to increase the acetylation level of histone H4 at ALAS2 gene promoter. All the Sp1 sites within the ALAS2 gene promoter also contributed to the transcription synergistic action with p300. This work identified that ALAS2 gene is a novel target gene for p300/CBP action as histone acetyltransferases.Meanwhile, we investigated the mechanisms by which HDAC inhibitor NaBu stimulates ALAS2 gene expression. We found that NaBu stimulated ALAS2 gene expression not only in erythroid K562 cells, but also in non-erythroid 293T cells with a concurrent increase in acetylation level of histone H3 and dimethylation level of K4 at the same histone at ALAS2 gene promoter. HDAC inhibitor NaBu also promotes the expression of ALAS2 gene promoter reporter gene in a dose-dependent manner. Following that 293T cells were transfected with ALAS2 gene promoter site-mutation reporter plasmids and treated with NaBu, and the luciferase reporter activity assays revealed that the GATA-1 binding sites were not responsible for NaBu stimulation, but all of the Sp1 sites located in ALAS2 gene promoter were the responsive elements for NaBu. We believe that, at least in part, NaBu activates ALAS2 gene expression through Sp1 sites. In addition, ChIP assays showed that NaBu treatment increased the binding of Sp1 to ALAS2 gene promoter. Furthermore, the transcription co-repressor and deacetylase HDAC1 down-regulated the transcription activity of ALAS2 gene promoter. It also reversed the activation of ALAS2 gene promoter by Sp1 and p300. ChIP assays indicated that HDAC1 bound to ALAS2 gene promoter and CoIP experiments showed that Sp1 and HDAC1 co-existed in the same protein complex. Therefore, we propose that the transcription factor Sp1 recruits deacetylase HDAC1 to repress ALAS2 gene transcription, and deacetylase...