The Histone Modification Characterization Of Lineage-affliated Genes During Hematopoietic Stem Cells Differentiating To Myeloid Cells

BackgroudHematopoietic stem cells(HSCs) are multipotent stem cells capable of self-renewal and multi-lineage differentiation.HSCs can differentiate to all types of hemapoietic cell lineages including erthryocytes, granulocytes,megakarocytes and T,B lymphocytes.When HSCs give rise to all the blood cell types,there was activation of lineage-specific genes and non-lineage specific genes accompanied by loss of multipotentiation.However,detailed mechanisms behind it are still unclear.Epigenetics research studies regulation of gene expression due to changes of genetic modifications which including DNA methylation, histone modification,genomic imprinting and RNA interference,while no DNA sequence changing.Modifications including DNA methylation, histone acetylation and histone methylation,which are closely related to euchromatin and heterochromatin formation,were studied most recently. Currently,it was shown that epigenetics are of great important in maintaining pluripotency of embryonic stem(ES) cells and can regulate lineage differentiation of ES cells by forming "bivalent" domains.But how it works in HSCs is still unclear.In this study,through investigating histone modifications,which are closely relevant to the chromatin formation,in different cell subgroups,like CD34~+CD38~- cells and differentiatied cells,we try to reveal possible mechanisms of how chromatin structures would maintain HSCs' multipotent characteristics and regulate relevant genes expression during differentiating,thus providing new perspectives in hemapoiesis,expansion in vitro and transplantation of HSCs,also mechanisms of leukemia generation.This study includes following three parts:Part 1 Sorting of CD34~+CD38~- cells from umbilical cord blood and inducing differentiation in vitroObjective:To establish a feasible method to sort CD34~+CD38~- cells from umbilical cord blood,and stabilize induction system for granulocytes,erythrocytes and megakaryocytes differentiation in vitro, thus providing cell samples for next step experiments.Methods:①Using MACS to positively sort CD34~+ cells,followed by negatively sorting of CD34~+CD38~- cells.FACS and trypan blue methods were used to determine the purity and survive rate of cells.②Using SCF+IL-3+G-CSF or EPO or TPO cytokines to induce CD34~+CD38~- cells differentiate into granulocytes,erythrocytes and megakaryocytes.Cell growth curve was drawn and FACS was adopted to determine the differentiation efficiency.Results:By using CD34 magnetic beads to sort CD34~+ cells,the purity reached up to 95.24±1.03%;After second round sorting,ratio of CD34~+/CD38~- was 90.23±2.52%.The cell survival rate reached up to 99%either before or after sorting.②After induced-differentiation in vitro for 14 days,the number of granulocytes increased up to 1186.67±106.01 folds,erythrocytes increased up to 894.67±48.22 folds and megakaryocytes increased up to 627±49.65 folds.③FACS data show that after induction for 14 days,the ratio of CD15~+ positive cells was 91.49%,CD235a~+ cells was 95.55%and CD41a~+ cell was 86.52%.Conclusion:Efficient MACS sorting and induced-system were established,which provide a reliable source of cells for further experiments.Part 2 Establishment of mini-Chromatin Immunoprecipitation (miniChIP) MethodObjective:ChIP is the most powerful tool currently used in protein-DNA interaction research.However,the drawback of traditional ChIP method is that it needs tremendous number of cells.To collect huge number of primary cells from umbilical cord blood is obviously not easy. Thus,our aim is to establish a ChIP method which requires less cells.Method:Based on the literatures,we established miniChIP method by improving conventional ChIP method.Traditional ChIP method and miniChIP method were both used to compare their efficiency in detecting histone H4 acetylation level inβglobin genes in MEL cells.The feasibility and reliability of miniChIP method was confirmed.Differentβglobin gene loci include HS2,promoter region ofβmaj and Ey.Results:By using miniChIP method,in untreated MEL cells a certain level of acH4 in promoter region of HS2 andβmaj can be detected, but acH4 level in the promoter region of Ey gene was even lower.After MEL cells were induced,the acH4 level in promoter region of HS2 andβmaj gene increased to 2.73 and 2.27 folds,respectively.The acH4 level in Ey gene promoter was almost unchanged.This result was consistent with traditional ChIP method,also as reported before.Conclusion:The traditional ChIP method was improved,which can only use little quantity of cells as sample source.By validating in improved ChIP method,we confirmed its feasibility and reliability.Chapter 3 Characterization of histone modifications during differentiation of HSCs to myeloid cellsObjective:To observe the histone modifications of related transcription factors and lineage specific genes in CD34~+CD38~- cells and commitment cells.Understanding the role of chromatin structure played in maintaining HSCs' multipotent characterization and differentiation preferences.Method:①Quantitive PCR was adopted to detect the mRNA expression levels of relevant transcription factors and lineage specific genes in CD34+CD38~- cells,CD15~+ cells,CD235a~+ cells and CD41a~+ cells.Transcription factors and genes include early hematopoiesis-related transcription factor HOXA9;granulocyte transcription factor PU.1, granulocyte specific gene MPO and CD11b;erythrocyte/megakaryocyte transcription factor GATA-1,erythrocyte specific gene EPOR and megakaryocyte specific gene CD41a;lymphocyte transcription factor GATA-3 and PAX5,lymphocyte specific gene CD3,CD79a.②miniChIP-qPCR was used to detect six types of histone medications in hematopoiesis relevant transcription factors and lineage specific genes promoter regions,including active modifications acH4,H3K4me2, H3K4me3 and repressive modifications H3K9me3 and H3K27me3.Results:①Differentiating-related transcription factors and lineage specific genes had very low level of expression in CD34~+CD38~- cells while HOXA9,which is relevant to early stage of hemapoiesis,had high mRNA expression;After CD34~+CD38~- cells differentiated,the expression of lineage specific genes increased significantly,while non-lineage specific genes expression can't be detected,at the same time,the expression of HOAX9 decreased drastically.②A certain level of H4 acetylation and to a lesser extent H3 acetylation together with high level of H3K4me2 and low level of H3K4me3 were present in lineage specific genes in CD34~+CD38~- cells.③Lineage specific genes have low level of H3K9me3 and H3K27me3 modifications in CD34~+CD38~- cells.④As CD34~+CD38~- differentiated,the modifications of acH3,acH4 and H3K4me2 level of lineage specific genes have a little increased,while H3K4me3 level increased greatly.At the same time,H3K9me3 and H3K27me3 modifications maintained at original low level.In non-lineage specific genes,the acH3 and acH4 levels decreased,and H3K4me3 level maintained at low level,at the same time H3K9me3 and H3K27me3 levels increased.⑤After CD34~+CD38~- cells commited,the promoter region of HOXA9,which is relevant to early stage of hematopoiesis,showed decreased active histone modifications including acH3,acH4,H3K4me2 and H3K4me2,while increased repressive histone modifications including H3K9me3 and H3K27me3.Conclusion:①Certain degree of acH3,acH4 and high level of H3K4me2 together with low level of H3K4me3 and low extent of repressive histone modifications(H3K9me3 and H3K27me3) were observed at the promoter of lineage specific genes in HSC-riched CD34~+CD38~- cell population.The expression of these genes shows low or undetected.②After CD34~+CD38~- cells differentiated,active histone modifications of lineage specific genes increased,especially H3K4me3 increased significantly,at the same time maintain low level of repressive histone modifications,leading to high transcriptional activity.While non-lineage specific genes had active histone modifications decreased, but repressive histione modifications level increased,lead to transcription silence.③After CD34~+CD38~- ceils differentiated,the promoter region of HOXA9 had low level of active histone modifications and high level of repressive histone modifications,leading to the gene transcription silencing.