published_or_final_version / Pathology / Master / Master of Medical Sciences

Η παρούσα εργασία στοχεύει στην «χαρτογράφηση» της περιοχής 3’UTR του ανθρώπινου γονιδίου της β-σφαιρίνης, ως προς την παρουσία σ’ αυτήν δομών που έχουν σημασία στο μεταβολισμό του mRNA – την ωρίμανση του 3’ άκρου του μηνύματος, ή / και την σταθεροποίησή του. Η μελέτη έγινε με τον σχεδιασμό μεταλλάξεων στην 3’UTR οι οποίες επιφέρουν μεταβολές στην πρωτοταγή και τη δευτεροταγή αλληλουχία της περιοχής, και την δημιουργία τριών μεταλλαγμένων γονιδίων β-σφαιρίνης στην 3’ UTR τους. Στη συνέχεια μελετήθηκε η έκφραση των μεταλλαγμένων γονιδίων σε διαγονιδιακές κυτταροκαλλιέργειες ερυθρολευχαιμικών κυττάρων ποντικού (mouse erythroleukaemia cells, MEL), με λειτουργικά πειράματα ανάλυσης του RNA με τη μέθοδο της προστασίας από τη νουκλεάση S1. Η έκφραση των μεταλλαγμένων διαγονιδίων προσδιορίστηκε σε σχέση με την έκφραση του φυσιολογικού γονιδίου της β-σφαιρίνης, λαμβάνοντας υπόψη τον αριθμό των αντιγράφων των διαγονιδίων στα κύτταρα, και την έκφραση του ενδογενούς σφαιρινικού γονιδίου βmaj του ποντικού. Τα αποτελέσματα έδειξαν ότι και τα τρία μεταλλαγμένα γονίδια παρουσιάζουν μειωμένα ποσοστά έκφρασης και μειωμένη επάρκεια ωρίμανσης του 3’ άκρου του mRNA, ενώ ο ρυθμός αποδόμησης του –μεταλλαγμένου– mRNA δεν διαφέρει από το φυσιολογικό για κανένα από τα τρία γονίδια. Συμπερασματικά, με την μελέτη αυτή επιβεβαιώνεται ο ρόλος της περιοχής 3’UTR του γονιδίου της β-σφαιρίνης στην διαδικασία ωρίμανσης του 3’ άκρου του mRNA, και η απουσία ειδικού σταθεροποιητικού μηχανισμού για το mRNA στην περιοχή του γονιδίου που μελετήθηκε με τις παρούσες μεταλλάξεις. / The present work aimed at mapping of the 3’UTR of the human β-globin gene as to the possible presence of structures that are involved in mRNA metabolism; in particular effective 3’ end formation or / and stability of the message. The study involved the design of 3’ UTR mutations that alter the primary and secondary structure of the sequence, and the subsequent formation of three mutant β-globin genes. The expression of the mutated genes was studied in transgenic mouse erythroleukaemia cell (MEL) lines, with functional analysis of mRNA expression using S1 nuclease protection assays. The expression of the mutated transgenes was compared to the relevant expression of the normal β-globin transgene, after correcting for transgene copy number and expression of the endogenous murine globin gene βmaj. Our results show that all three studied mutant transgenes have decreased expression levels, and decreased efficiency in 3’ end formation of the mRNA, whereas the stability of the –mutant– mRNA is not affected for any of the three transgenes. We conclude that the β-globin 3’ UTR plays an important role in the efficiency of message 3’ end formation, and that the mutations studied do not disrupt any stabilising mechanism.

Hemogen (Hemgn) is reported as a tissue specific transcriptional regulator in testis as well as hematopoietic tissues. It is known that Hemgn positively regulates erythroid differentiation; however,the underlying molecular mechanism is not well understood. In the current study, using proteomic approach in combination with other molecular biology tools,we have attempted to decipher the role of Hemgn in differentiating Murine erythroblast leukemia (MEL) cells as a model system. Our study reveals that Hemgn predominantly interacts with transcriptional regulators, chromatin modifiers and histones. Furthermore, using Chromatin Immunoprecipitation and knockdown approach, we have demonstrated that Hemgn is recruited to the b-globin locus, which is known to be activated during erythroid differentiation. Based on the results,we speculate that Hemgn acts as a tissue specific histone chaperone that regulates transcription during erythroid differentiation.

Thesis (M.Sc. (Biochemistry))--Mahidol University, 1983.

Thesis (Ph. D.)--Duke University, 2007.

The HbS gene had a limited number of origins during history, and these can be defined by the haplotype (a set of DNA polymorphisms inherited together) of the associated β-globin gene. Five major haplotypes have been identified, and associated with different ethnic groups. These are the Arab Indian haplotype, the Benin haplotype, the Cameroonian haplotype, the Central African Republic (CAR) or Bantu haplotype, and the Senegal haplotype. The polymorphisms defining these haplotypes are associated with fetal hemoglobin, the major modifier of sickle cell disease phenotype and severity. The Arab Indian haplotype, in particular, is associated with unusually high HbF levels (20%), and a significantly less severe clinical presentation. We found a novel (C>T) SNP -68 bp 5' to HBD in this region, expressed in patients with the Arab Indian haplotype, but not sickle cell disease patients expressing other β-globin cluster haplotypes. There is evidence that this -68 (C>T) polymorphism may play a functional role in the hemoglobin expression of these patients, and its effect on globin levels and disease severity is the long-term interest of this study. A previous reporter assay in K562 cells determined that the -68 SNP was associated with decreased δ-globin gene expression. This study aims to clone the HBD region of a patient positive for this SNP into a lentiviral firefly luciferase reporter vector, for use in more physiologically accurate CD34+ erythropoietic progenitor cells. If the mutations in these β-globin gene haplotypes, such as the HBD mutation described in this study, are responsible for the protective effects seen in patients, perhaps some of these genetic locations can serve as targets for therapeutics in sickle cell disease or other blood disorders.

In eukaryotes, DNA is wrapped around a group of proteins termed histones that are required to precisely control gene expression during development. The amino acids of both the globular domains and unstructured tails of these histones can be modified by chemical moieties, such as methylation, acetylation and ubiquitination. The ‘histone code’ hypothesis proposes that specific combinations of these and other histone modifications contain transcriptional information, which guides the cell machinery to activate or repress gene expression in individual cell types. Chromatin immunoprecipitation (ChIP) experiments using undifferentiated stem cell populations have identified the genomic co-localisation of histone modifications reported to have opposing effects on transcription, which is known as bivalency. The human α-globin promoter, a well-established model for the study of transcriptional regulation, is bivalent in embryonic stem (ES) cells and this bivalency is resolved once the ES cells terminally differentiate (i.e. only activating or repressing marks remain). In a humanised mouse model, the deletion of a bone fide enhancer within the human α-globin locus results in heterogeneous expression patterns in primary erythroid cells. Notably, this correlates with an unresolved bivalent state at this promoter in terminally differentiated cells. Using this mouse model it is not feasible to ascertain whether the transcriptional heterogeneity observed in the cells lacking an α-globin enhancer is reflective of epigenetic heterogeneity (i.e. a mixed population of cells) rather than co-localisation of bivalent histone modifications within the same cells. Furthermore, the functional contribution of bivalency to development has yet to be described. To address these difficulties, I aimed to generate a fluorescent reporter system for human α-globin to facilitate the separation of transcriptionally heterogeneous erythroid cells. This model will provide material for ChIP studies on transcriptionally active and inactive populations to determine whether the epigenetic bivalency is reflective of a mixed cell population or true bivalency. In addition, I aimed to produce epigenetic editing tools to target bivalent promoters, which in combination with in vitro differentiation assays would provide an interesting framework to test the function of bivalency during development. In this study, I extensively tested gene-editing strategies for generating a fluorescent reporter knock-in in humanised mouse ES cells. I validated the suitability of humanised mouse ES cell lines for gene targeting studies and optimised a robust in vitro differentiation protocol for studying erythropoiesis. I utilised both recombineering and CRISPR/Cas9 gene editing tools in tandem with PiggyBac transposon technology, to knock-in the reporter gene. I made significant steps in gene targeting and successfully inserted the reporter downstream of the α-globin gene. I also generated a cloning system to express site-specific DNA-binding domains (TALEs) fused to epigenetic regulators with the aim to resolve bivalent histone modifications in vitro. From preliminary tests using these fusion proteins targeting Nrp1, a bivalent promoter in mES cells, I observed mild but significant changes in gene expression although histone modifications were unchanged. The various tools generated and tested in this study provide a solid foundation for future development of genetic and epigenetic editing at the human α-globin and other bivalent loci.

Orientador: Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-06T00:23:27Z (GMT). No. of bitstreams: 1 Andrade_TiagoGomesde_D.pdf: 2136964 bytes, checksum: be76264a603d3b420701714d5a04072b (MD5) Previous issue date: 2006 / Resumo: Persistência Hereditária de Hemoglobina Fetal (PHHF) consiste num grupo heterogêneo de alterações hereditárias, sem manifestações clínicas significativas, onde ocorrem falhas na mudança perinatal normal de hemoglobina fetal para hemoglobina adulta, resultando em altos níveis de Hb F durante a vida adulta. Nos tipos delecionais ocorre normalmente aumento de ambas as cadeias, ? A e ? G, até 30%, estando associadas a deleções de seqüências de DNA dentro do grupo de genes ß. As (dß)º - talassemias também se originam a partir de deleções, em muitos casos bastante similares às que originam as formas delecionais de PHHF, entretanto com níveis de Hb F aumentados em menor proporção (5-20%), sendo acompanhadas nos heterozigotos por hipocromia e microcitose nas hemácias. Três hipóteses principais foram propostas para explicar a relação destas deleções com a ausência de supressão normal dos genes ? na fase adulta: competição entre promotores pelo LCR (Locus Control Region); justaposição de elementos acentuadores, normalmente encontrados a 3' do cluster, nas proximidades dos genes ?; remoção de elementos silenciadores, localizados entre ? A e d. Neste trabalho, realizamos estudos funcionais com uma região intergênica, aqui denominada R/PYR, potencialmente envolvida com o silenciamento de ? e a troca na síntese das cadeias de globinas. Os resultados obtidos com transfecção estável em células MEL apontam uma possível participação deste elemento na regulação dos genes de globina. Também investigamos o possível envolvimento de fatores de transcrição, construindo Bibliotecas de cDNA Subtrativas Supressivas para identificar transcritos diferencialmente expressos em reticulócitos de um sujeito com PHHF-2. A análise dos perfis diferenciais de expressão gênica em reticulócitos de PHHF-2 e normais mostram alterações em larga escala e que podem estar associadas com o fenótipo de PHHF. Estas modificações podem ser decorrentes de perturbações nas interações cromossômicas e/ou nos níveis de RNAs regulatórios não codificantes e ajudarem a melhor compreender a disposição e organização dos genes transcricionalmente ativos no genoma, e de que maneira são regulados. A presença de fatores de transcrição e remodeladores de cromatina, como ARID1B, TSPYL1 e ZHX2, dentre outros, com expressão alterada indicam fortemente que outros mecanismos genéticos estão envolvidos com o aumento da síntese de gama globina no estágio adulto, além dos modelos previamente descritos na literatura. Além disto, são candidatos potenciais a reguladores de globina que ainda não haviam sido descritos, e podem ajudar a compreender as diferenças fenotípicas encontradas entre dß-talassemia e PHHF delecional. Identificamos ainda outros genes com expressão diferencial, além de seqüências sem similaridade a genes categorizados, como ORFs, ESTs e seqüências genômicas, que podem ter papel importante na fisiologia normal das células vermelhas ou nas hemoglobinopatias hereditárias. Além disto, a observação de que os níveis de RNAm de alfa globina estão diminuídos em PHHF-2 e dß-Talassemia Siciliana sugere um mecanismo de regulação pós-transcripcional compensatório para as cadeias de globina ou de regulação gênica por interação física direta entre os cromossomos 11 e 16. Ao nosso conhecimento, esta é a primeira descrição demonstrando as implicações na expressão gênica em larga escala em células contendo alterações no DNA como as deleções apresentada / Abstract: The genetic mechanisms underlying the continued expression of the ?-globin genes during the adult stage in deletional hereditary persistence of fetal hemoglobin (HPFH) and dß-thalassemias are not completely understood. For deletional HPFH, three main hypotheses were proposed to explain the relationship between these deletions and the non-suppression of ? -genes in the adult; 1- the removal of competitive regions that interact with the LCR; 2- the juxtaposition of enhancer elements located downstream from the breakpoint region; and 3- the removal of gene silencer elements. Herein, we studied a region, called R/PYR, that lies 1-3 Kb upstream from the d gene. This region has been implicated in ? globin repression and globin gene switching. Stable transfections in MEL cells suggest a possible involvement of R/PYR in globin regulation. We also investigated the possible involvement of transcription factors, using the SSH method to identify differentially expressed transcripts in reticulocytes from a normal and a HPFH-2 subject. Some of the detectable transcripts may participate in globin gene regulation. Quantitative RT-PCR experiments confirmed the downregulation of ZHX2, a transcriptional repressor, in two HPFH-2 subjects and in a carrier of the Sicilian dß-thalassemia trait. The chromatin remodeling factors ARID1B and TSPYL1 had a very similar pattern of expression with an incremental increase in HPFH and decreased expression in dß-thalassemia. These differences suggest a mechanism to explain the heterocellular and pancellular distribution of F cells in dß-thalassemia and deletional HPFH, respectively. Interestingly, a-globin mRNA levels were decreased, similar to ß-globin in all reticulocyte samples analyzed, which may be the result of a common regulatory process affecting a-like and ß-like globin synthesis. Part of the altered gene expression detected within the subtracted libraries may be an indirect effect due to the increased concentration of HbF in the cells. However, genetic alterations similar to those presented in this work may be implicated in the perturbation of chromosome interactions and/or disruption of the expression of regulatory non-coding transcripts with functional consequences for cellular gene expression. Additionally, other genes may have modifications of their expression, but were not detected in our experiments. To our knowledge, this is the first description of wide-ranging gene expression alterations in cells containing deletional HPFH and thalassemia. Finally, the dissection of the differential expression data presented in this study may help elucidate important pathways of erythroid differentiation and maintenance of red cells in peripheral circulation, as well as aid in the understanding of genetic mechanisms involved in erythrocyte pathologies such as sickle cell disease and thalassemias / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutor em Fisiopatologia Medica

Sickle cell disease and β-thalassemia affect millions of people worldwide. γ-globin is the fetal counterpart to the adult β-globin. Research has shown that affected patients with higher than normal γ-globin show less severe symptoms. Therefore, reversing or preventing the hemoglobin switch from γ- to β- globin is a promising avenue of research for treating these diseases. KLF1 is an erythroid transcription factor involved in hemoglobin switching. Herein, we show that KLF1 directly regulates the γ-globin repressor gene LRF in both the mouse and human systems. KLF1 may also directly activate γ-globin expression by binding the promoter. In human HUDEP-2 cells, an increase in γ-globin expression is seen upon modest knockdown (~50%) of KLF1, whereas normal amounts of KLF1 are observed upon robust knockdown (>75%) of KLF1. The data suggest that KLF1 plays both a positive and negative role in γ-globin expression.

Hemogen (Hemgn) is reported as a tissue specific transcriptional regulator in testis as well as hematopoietic tissues. It is known that Hemgn positively regulates erythroid differentiation; however,the underlying molecular mechanism is not well understood. In the current study, using proteomic approach in combination with other molecular biology tools,we have attempted to decipher the role of Hemgn in differentiating Murine erythroblast leukemia (MEL) cells as a model system. Our study reveals that Hemgn predominantly interacts with transcriptional regulators, chromatin modifiers and histones. Furthermore, using Chromatin Immunoprecipitation and knockdown approach, we have demonstrated that Hemgn is recruited to the b-globin locus, which is known to be activated during erythroid differentiation. Based on the results,we speculate that Hemgn acts as a tissue specific histone chaperone that regulates transcription during erythroid differentiation.