3 resultados para Rubisco small subunit gene ( rbcS) Promoter
em RUN (Repositório da Universidade Nova de Lisboa) - FCT (Faculdade de Cienecias e Technologia), Universidade Nova de Lisboa (UNL), Portugal
The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium
Resumo:
Dissertation presented to obtain the Ph.D degree in Biology
Resumo:
Dissertation presented to obtain the Ph.D degree in Biology
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RESUMO:O glicosilfosfatidilinositol (GPI) um complexo glicolipdico utlizado por dezenas de protenas, o qual medeia a sua ancoragem superfcie da clula. Protenas de superfcie celular ancoradas a GPI apresentam vrias funes essenciais para a manuteno celular. A deficincia na sntese de GPI o que caracteriza principalmente a deficincia hereditria em GPI, um grupo de doenas autossmicas raras que resultam de mutaes nos genes PIGA, PIGL, PIGM, PIGV, PIGN, PIGO e PIGT, os quais sao indispensveis para a biossntese do GPI. Uma mutao pontual no motivo rico em GC -270 no promotor de PIGM impede a ligao do factor de transcrio (FT) Sp1 sua sequncia de reconhecimento, impondo a compactao da cromatina, associada hipoacetilao de histonas, e consequentemente, impedindo a transcrio de PIGM. Desta forma, a adio da primeira manose ao GPI comprometida, a sntese de GPI diminui assim como as protenas ligadas a GPI superficie das clulas. Pacientes com Deficincia Hereditria em GPI-associada a PIGM apresentam trombose e epilesia, e ausncia de hemlise intravascular e anemia, sendo que estas duas ltimas caractersticas definem a Hemoglobinria Paroxstica Nocturna (HPN), uma doena rara causada por mutaes no gene PIGA. Embora a mutao que causa IGD seja constitutiva e esteja presente em todos os tecidos, o grau de deficincia em GPI varia entre clulas do mesmo tecido e entre clulas de tecidos diferentes. Por exemplo nos granulcitos e linfcitos B a deficincia em GPI muito acentuada mas nos linfcitos T, fibroblastos, plaquetas e eritrcitos aproximadamente normal, da a ausncia de hemlise intravascular. Os eventos transcricionais que esto na base da expresso diferencial da ncora GPI nas clulas hematopoiticas so desconhecidos e constituem o objectivo geral desta tese. Em primeiro lugar, os resultados demonstraram que os nveis de PIGM mRNA variam entre clulas primrias hematopoiticas normais. Adicionalmente, a configurao dos nucleossomas no promotor de PIGM mais compacta em clulas B do que em clulas eritrides e tal est correlacionado com os nveis de expresso de PIGM, isto , inferior nas clulas B. A presena de vrios motivos de ligao para o FT especfico da linhagem megacarioctica-eritride GATA-1 no promotor de PIGM sugeriu que GATA-1 desempenha um papel regulador na sua transcrio. Os resultados mostraram que muito possivelmente GATA-1 desempenha um papel repressor em vez de activador da expresso de PIGM. Resultados preliminares sugerem que KLF1, um factor de transcrio restritamente eritride, regula a transcrio de PIGM independentemente do motivo -270GC. Em segundo lugar, a investigao do papel dos FTs Sp demonstrou que Sp1 medeia directamente a transcrio de PIGM em ambas as clulas B e eritride. Curiosamente, ao contrrio do que acontece nas clulas B, em que a transcrio de PIGM requer a ligao do FT geral Sp1 ao motivo -270GC, nas clulas eritrides Sp1 regula a transcrio de PIGM ao ligar-se a montante e no ao motivo -270GC. Para alm disso, demonstrou-se que Sp2 no um regulador directo da transcrio de PIGM quer nas clulas B quer nas clulas eritrides. Estes resultados explicam a ausncia de hemlise intravascular nos doentes com IGD associada a PIGM, uma das principais caractersticas que define a HPN. Por ltimo, resultados preliminares mostraram que a represso da transcrio de PIGM devida mutao patognica -270C>G est associada com a diminuio da frequncia de interaces genmicas em cis entre PIGM e os seus genes vizinhos, sugerindo adicionalmente que a regulao de PIGM e desses genes partilhada. No seu conjunto, os resultados apresentados nesta tese contribuem para o conhecimento do controlo transcricional de um gene housekeeping, especfico-detecido, por meio de FTs genricos e especficos de linhagem.-------------ABSTRACTC: Glycosylphosphatidylinositol (GPI) is a complex glycolipid used by dozens of proteins for cell surface anchoring. GPI-anchored proteins have various functions that are essential for the cellular maintenance. Defective GPI biosynthesis is the hallmark of inherited GPI deficiency (IGD), a group of rare autosomal diseases caused by mutations in PIGA, PIGL, PIGM, PIGV, PIGN, PIGO and PIGT, all genes indispensable for GPI biosynthesis. A point mutation in the -270GC-rich box in the core promoter of PIGM disrupts binding of the transcription factor (TF) Sp1 to it, imposing nucleosome compaction associated with histone hypoacetylation, thus abrogating transcription of PIGM. As a consequence of PIGM transcriptional repression, addition of the first mannose residue onto the GPI core and thus GPI production are impaired; and expression of GPI-anchored proteins on the surface of cells is severely impaired. Patients with PIGM-associated IGD suffer from life-threatening thrombosis and epilepsy but not intravascular haemolysis and anaemia, two defining features of paroxysmal nocturnal haemoglobinuria (PNH), a rare disease caused by somatic mutations in PIGA. Although the disease-causing mutation in IGD is constitutional and present in all tissues, the degree of GPI deficiency is variable and differs between cells of the same and of different tissues. Accordingly, GPI deficiency is severe in granulocytes and B cells but mild in T cells, fibroblasts, platelets and erythrocytes, hence the lack of intravascular haemolysis.The transcriptional events underlying differential expression of GPI in the haematopoietic cells of PIG-M-associated IGD are not known and constitute the general aim of this thesis. Firstly, I found that PIGM mRNA levels are variable amongst normal primary haematopoietic cells. In addition, the nucleosome configuration in the promoter of PIGM is more compacted in B cells than in erythroid cells and this correlated with the levels of PIGM mRNA expression, i.e., lower in B cells. The presence of several binding sites for GATA-1, a mega-erythroid lineage-specific transcription factor (TF), at the PIGM promoter suggested that GATA-1 has a role on PIGM transcription. My results showed that GATA-1 in erythroid cells is most likely a repressor rather than an activator of PIGM expression. Preliminary data suggested that KLF1, an erythroid-specific TF, regulates PIGM transcription but independently of the -270GC motif. Secondly, investigation of the role of the Sp TFs showed that Sp1 directly mediates PIGM transcriptional regulation in both B and erythroid cells. However, unlike in B cells in which active PIGM transcription requires binding of the generic TF Sp1 to the -270GC-rich box, in erythroid cells, Sp1 regulates PIGM transcription by binding upstream of but not to the -270GC-rich motif. Additionally, I showed that Sp2 is not a direct regulator of PIGM transcription in B and erythroid cells. These findings explain lack of intravascular haemolysis in PIGM-associated IGD, a defining feature of PNH. Lastly, preliminary work shows that transcriptional repression of PIG-M by the pathogenic -270C>G mutation is associated with reduced frequency of in cis genomic interactions between PIGM and its neighbouring genes, suggesting a shared regulatory link between these genes and PIGM. Altogether, the results presented in this thesis provide novel insights into tissuespecific transcriptional control of a housekeeping gene by lineage-specific and generic TFs.
