Structural Insights Into The Recognition Of ?-globin Gene Promoter By BCL11A & The Structural And Functional Investigations Of BAF180 With Nucleosome

The thesis is composed of two chapters.Chapter one:the mechanism by which BCL11A repress y-globin expression;Chapter two:the structural and functional investigations of the first BAH domain of BAF180 and the interaction with nucleosome.Chapter one:hemoglobin can transport oxygen to various tissues and organs of the body to maintain normal life activities of human beings.Hemoglobin consists of two a-like and two P-like globin subunits which are located on human chromosomes 16 and 11,respectively.The ?-like globin genes are encoded from a single locus comprising five globin genes(?-,G?-,A?-,?-,and ?-globin in sequence)and their expression is under critical developmental control.Within the first trimester of pregnancy the dominant ?-like globin chain is ?-globin.During the fetal life,the duplicated fetal y-globin G?-and A?-)genes are upregulated.Shortly after birth,the adult ?-globin becomes the major ?-like globin with less than 1%residual fetal hemoglobin(HbF or?2?2).?-hemoglobin disorders are caused by the mutations in the adult ?-globin gene which led to the abnormal structure or production of ?-globin,such as sickle cell disease and ?-thalassemia.The current treatments for p-hemoglobin disorders include hydroxyurea therapy,chronic blood transfusion,bone marrow transplantation and gene therapy.The individuals with "hereditary persistence of fetal hemoglobin(HPFH)"syndrome do not complete normal hemoglobin switching and have higher residual level of fetal hemoglobin.The individuals with HPFH syndrome have no obvious impact on their health,but for the patients with ?-hemoglobin disorders,it can alleviate the clinical phenotype.Thus,reactivating the expression of developmentally silenced y-globin genes is an attractive treatment strategy for P-hemoglobin disorders.Genome wide association studies(GWAS)of HPFH identified the transcription factor B-cell lymphoma/leukemia 11A(BCL11A)as the major HbF silencer.Down-regulation of BCL11A expression by siRNA in primary adult erythroid cell CD34+led to robust HbF expression.Conditional inactivation of BCL11A in sickle cell disease transgenic mice can correct the hematologic and pathologic defects associated with sickle cell disease through high-level pancellular HbF induction.Although there were many experiments which have demonstrated BCL11 A can regulate the expression of y-globin,whereas the mechanism by which BCL11A represses y-globin expression has not been fully elucidated.Recently,two groups independently reported that BCL11A could bind to the promoter of y-globin located approximately 115bp upstream of the transcription start sites through its C-terminaGilman,J.G.,Mishima,N.,Wen,X.J.,Stoming,T.A.,Lobel,J.,and Huisman,T.H.(1988b).Distal CCAAT box deletion in the A gamma globin gene of two black adolescents with elevated fetal A gamma globin.Nucleic Acids Res 16,10635-10642.Grevet,J.D.,Lan,X.,Hamagami,N.,Edwards,C.R.,Sankaranarayananl three tandem C2H2 zinc fingers(Znf4-6).Coincidentally,the naturally occurring mutations which contributed to high fetal hemoglobin level located in approximately 115bp upstream region of the y-globin including single base substitutions at-117(G-117A)and-114(C-114A/T/G),whereas the mechanisms remain unclear.We cloned and purified the Znf4-6 of BCL11A and designed the DNA with different length to ctystallize.Finally,the BCL11A-DNA complex structure was refined to 2.50 A resolution.Based on the structural analysis and ITC experiments,we found HPFH associated mutations including G-117A and C-114A/T/G substantially weakened the interaction between BCL11A and DNA,which are consistent with our observations of solid contacts of Znf4-5 with G-117 and G-114.Furthermore,we also confirmed that the other C2H2 zinc finger protein ZBTB7A could bind to the promoter of y-globin located approximately 200bp upstream of the transcription start sites.Interestingly,there are some mutations which have been identified associated with HPFH syndrome.Moreover,we performed the ITC assay and illustrated the binding affinity is approximately 120 nM.Chapter two:chromatin remodeling is one of the important components of epigenetic regulation,which relies on the chromatin remodeling complex to function.In human,the SWI/SNF family can be further divided into two protein subfamilies called BAF and PBAF complex,which both consist of several core subunits and accessory subunits.BAF180 is a unique subunit belonged to PBAF subfamily,which plays important roles in maintaining genomic stability,promoting mammalian heart chamber maturation,repairing damage during DNA transcription,and inhibiting breast cancer and renal cell carcinoma.BAF 180 consists of N-terminal six tandem Bromo domains,two tandem BAH domains in the middle and a C-terminal HMG box domain which can bind to DNA unspecifically.However,the structural and functional investigations of the BAH domain of BAF 180 have not been determined.It has been reported that BAH domain was involved in the recognition of post-translational modification of histones to regulate gene expression.In addition,it also could be involved in binding nucleosome directly to regulate gene silencing and protein-protein interaction.It is important to investigate the structure and function of the BAH domain of BAF180 to understand the biological function of BAF180.Firstly,we determined the crystal structure of the first BAH domain of BAF180,and demonstrated that the BAH domain can directly bind to nucleosome assembled in vitro or extracted from 293T cells by GST pull-down,EMSA and ITC experiments.Furthermore,we identified the binding surface on BAH domain which could interact with nucleosome by a number of point mutation assays,further confirmed by the following GST pull-down and NMR titration experiments.We hope that the interaction interface on nucleosome could be obtained through subsequent NMR titration experiments,and then we can solve the complex structure of BAH domain and nucleosome through the model building.