Exploring Molecular Mechanisms Of γ-globin Expression Induction By The HDACi Apicidin In Human Chronic Myeloid Leukemia Cells

Eukaryotic gene expression regulation is a complicated process. Within the context, cell receives input environmental signals and initiates corresponding signaling pathway to the target gene, which will be activated or repressed spatio-temporally.Additionally, differentially gene expression is essential to organismal activities.Mammalian globin gene clusters are always the typical models for genetic and epigenetic regulation research. Studies of the regulation of the human globin loci have provided powerful insights into human gene expression in general at the molecular level. The human globin gene clusters are among the best characterized in the human genome at the gene and protein levels. The synthesis of hemoglobin is also controlled by the two developmentally regulated multigene clusters: theα-like globin gene cluster on chromosome 16 (5'-ζ-α2-α1-3'),and theβ-like globin locus on chromosome 11 (5'-ε-^Gγ-^Aγ-δ-β-3') producing embryonic (ζ₂ε₂),fetal (α₂γ₂) and adult (α₂β2) hemoglobin. Coordinated expression of the genes in each clusters at all stages of development is dependent on critical regulatory elements, locus control region (LCR) and major regulatory element (MRE), located far upstream of the two gene clusters, respectively.So far, there are more than 1000 naturally occurring inherited mutations affecting either the structure or synthesis of theα- andβ-like globin chains. An imbalance in the synthesis of each cluster will result in thalassemia.α-thalassemia occurs when there is a deficiency inαglobin expression andβ- thalassemia occurs whenβglobin synthesis is downregulated.Thalassemia is the world's most common genetic disorders. The recent report showed that 4.83 percent of the world's population carries globin variants, including 1.67 percent of the population who are heterozygous for a-thalassemia andβ-thalassemia. In addition, 1.92 percent carries sickle hemoglobin, 0.95 percent carry hemoglobin E, and 0.29 percent carry hemoglobin C. Thus, the worldwide birth rate of people who are homozygous or compound heterozygous for symptomatic globin disorders, includingα-thalassemia andβ-thalassemia, is no less than 2.4 per 1000 births, of which 1.96 have sickle cell disease and 0.44 have thalassemias.Based on an understanding of the pathophysiology ofβ-thalassemia and sickle cell disease (SCD), pharmacological induction of fetal hemoglobin (Hb F) will be an effective strategy for the treatment of the diseases. The imbalance in the production ofα- andβ-globin chains was identified as the main factor responsible for the pathophysiology ofβ-thalassemia. Additionally,the polymerization of deoxygenated sickle hemoglobin and the resulting vaso-occlusion were recognized as the most important factors in the pathophysiology of SCD. Induction ofγ-globin gene expression was proposed as a potential therapeutic approach in both disorders. Because inβ-thalassemia the increase inγ-globin chains would be expected to decrease globin chain imbalance, in SCD, the increase in Hb F would be expected to interfere with the polymerization of sickle hemoglobin. Therefore, the understanding of the pathophysiology of both disorders and the induction of Hb F as the therapeutic approach provided the impetus for the drug discoveries for the treatment ofβ-thalassemia and SCD.Additionally, based on the discoveries in globin gene regulation studies, the transcriptionally activeβ-globin genes are hypomethylated and hyperacetylated at the promoter regions, and the transcriptionally inactiveγ-globin genes are hypermethylated at the promoter region with low-level histone modifications in adult life. In contrast, theβ-globin genes have hypermethylated promoters with histone hyperacetylation and theγ-globin genes have hypomethylated regulatory regions with hyperacetylation in fetal life. The results suggested that reactivation and increasing ofγ-globin gene expression should a feasible strategy based on such epigenetic rationale.To date,the DNA methyltransferase inhibitor 5-Azacytidine and the histone deacetylase inhibitors (HDACi), sodium butyrate (SB) and Trichostatin (TSA) have been applied for the pharmacological induction of Hb F. However, concerning about the potential for serious side effects from the long-term use of thses drugs stimulated the continuing search for potentially safer inducers of Hb F.Recently, another kind of HDACi apicidin was identified as the potential induction of Hb F 10-fold at nanomolar concentration in embryonic/fetal cell line, K562. In our studies, we found that apicidin has mild effect on cellular proliferation. Additionally, similar to previously identified SB, TSA and HU, apicidin can activate signaling through p38 MAP kinase pathway. After blockade of p38 pathway, the effect of apicidin in stimulating Hb F was largely diminished. However, it is not clear how these signaling effects lead to the transcriptional activation of theγ-globin genes. Further understanding the molecular mechanisms of these drug actions are central to their therapeutic application to the patient populationIn our current studies, we firstly detected the effects of apicidin on cellular proliferation, differentiation, cell cycle retardation and apoptosis with MTT and FACS assays. Benzidine staining and RT-PCR were applied for detect the induction of Hb F by apicidin. At the same time, we detect the roles of apicidin in expression of erythroid transcription factors, GATA-1,GATA-2, NF-E2 P45 and P18, as well as histone 3 (H3) and H4 covalent modification states. The further Chromatin immunoprecipitation (ChIP) assays analyzed the active histone modification states along the humanβ-globin gene locus by apicidin treatment before and after the blockade of p38 signaling pathway. Finally, the DNA immuno-fluorescence in situ hybridization (FISH) was employed to detect apicidin's role in nuclear localization ofβ-globin locus.Experimental results are mainly included: (i) Compared with SB, TSA and 5-Azacytidine, apicidin has mild effect on cellular proliferation.(ii) Apicidin can induce the erythroid differentiation of K562 cell line with the elevation of 7-10 folds ofγ-globin mRNA expression level. Activated p38 plays indispensable role in the induction ofγ-globin expression and K562 cell differentiation.(iii) The levels of both H3 and H4 hyperacetylation and, unexpectedly, specific hypermethylation of H3 lysine 4 induced by the apicidin were higher than ones in control when exposed to apicidin in culture. In general, the active histone modification state of K562 cells induced by apicidin is independent on p38 activity. ChIP assays showed that acetylation state of H3 on the intergenic regions and LCR were strongly increased by apicidin treatment when compared with promoter region ofγ-globin gene, which the active state is selectively dependent on p38 activity.(iv) P38 signaling pathway cooperates with apicidin to regulate GATA-1, GATA-2 expression at mRNA and protein level, but these genes are partially dependent on p38 activity for full expression. ChIP analysis of GATA-1 occupancies showed that GATA-1 was recruited to theγ-globin promoter dramatically with 12-hour apicidin treatment. After blockade of p38 pathway, the GATA-1 occupancies are largely diminished.(v) ChIP analysis of polâ…¡and ubiquitous transcription factor sp1 occupancies showed that polâ…¡and sp1 dramatically bound to LCR,ε- andγ-promoter regions by apicidin treatment, which are p38 pathway independent.(vi) DNA FISH results showed that theβ-globin locus tends to loop out of the chromosome territories under the induction of apicidin.Based on the results described above and our further analysis, several conclusions should be drawn.(i) Considering the regulatory complexity of clustered genes such as the humanβ-globin locus, the traditional drug-screening method only withγ-globin gene promoter might not be satisfied the needs for Hb F inducer finding.(ii) In combination with the recent progress in the long-range regulatory mechanism at theβ-globin locus, we proposed that the hyperacetylated intergenic regions provide a preferential looping site and subsequently activatedγ-globin gene expression augmentation. Additionally, p38 pathway regulates the active histone modification states, which induced by apicidin treatment. The enhanced chromatin looping functions are p38 pathway dependent.(iii) GATA-1, sp1 and polâ…¡coordinately contributed to the elevation of Hb F expression.(iv) Apicidin is a kind of effective Hb F inducer with several advantages as we discussed above. Therefore, apicidin shoul be a potential drug in clinics for the treatment ofβ-thalassemia and SCD.