Transcriptional and post-transcriptional gene regulatory mechanisms in the malaria parasite, Plasmodium falciparum

Malaria is a devastating disease which affects almost half of the world’s population. Since the description of the malaria genome sequence, various aspects of the parasite have been studied, including drug resistance mechanisms, epidemiology and surveillance systems. Alarmingly, very little is known about the basic biological processes such as the regulation of the expression of parasite genes. The parasite, Plasmodium falciparum, has developed highly specialized methods of regulating the transcription of genes, starting at the regulation of genes controlling basic cellular processes such as protein synthesis and erythrocyte invasion, followed by the transcriptional regulation of more specialized genes, such as those aiding in immune evasion and pathogenesis. The description of the P. falciparum transcriptome by Bozdech et al. in 2003 revealed a complex, just-in-time and tightly regulated transcription profile of P. falciparum genes. This suggests that the most probable Achilles heel for Plasmodium may be its unique mechanisms of regulating gene expression. Various cis- and trans-regulatory sequences have been identified in P. falciparum, along with possible DNA (and RNA) binding proteins. The first part of this research focussed on transcriptional regulatory mechanisms in which an in silico search identified cis-regulatory sequences in the 5’ untranslated region of the antigenically variant var gene family. Electrophoretic mobility shift assays (EMSA) were used to identify protein binding partners of these sequences, which could ultimately act as transcription factors in regulating the expression of this essential gene family. The second part of the research investigated the involvement of post-transcriptional regulatory mechanisms in the polyamine biosynthetic pathway of P. falciparum. Polyamines have been proven to be crucial for the parasite’s development and therefore, an RNA interference knock-down strategy was used to verify the importance of the polyamine biosynthetic enzymes S-Adenosylmethionine decarboxylase (AdoMetDC), Ornithine decarboxylase (ODC) and Spermidine synthase. It is clear that various mechanisms for gene regulation are used by the parasite and that this is critical for the survival of this organism. The results of this study suggest the potential presence of both double-stranded and single-stranded DNA regulatory proteins within P. falciparum nuclear extract. As controversial as RNA interference remains in P. falciparum, this technique was used as a plausible knock-down strategy of parasite specific genes and certain trends, regarding the visible decreases in gene transcript level after double-stranded RNA treatment, were observed. However, final conclusions as to the feasibility of using RNA interference in P. falciparum remain to be elucidated. This study therefore ultimately lends insight into the transcriptional and post-transcriptional levels of P. falciparum gene regulation.Dissertation (MSc)--University of Pretoria, 2010.Biochemistryunrestricte

A large-scale investigation on the Enhancer-Promoter interaction mediated by transcription factors

Contesto Il differenziamento dei tessuti durante lo sviluppo embrionale dei mammiferi richiede una complessa regolazione dell'espressione genica che può essere modulata da enhancer distali. Geni coinvolti nello sviluppo sono fiancheggiati da sequenze altamente conservate che potrebbero essere enhancer distali di tali geni. Uno dei possibili meccanismi per la regolazione della trascrizione è la formazione di un'ansa tra enhancer e promotore mediata da fattori di trascrizione specifici. Metodologie/Risultati Per studiare l'interazione enhancer-promotore abbiamo generato un set di 1011 Coppie Enhancer-Promotore (CEP) accoppiando 702 enhancer, coinvolti nello sviluppo e validati sperimentalmente, ai 621 promotori più prossimi. Su enhancer e promotori accoppiati sono stati predetti Siti di Legame dei Fattori di Trascrizione (SLFT) specifici per l'organismo umano. Utilizzando metodi statistici, partendo da un set di 2837 Coppie di Fattori di Trascrizione (CFT), sono state filtrate 364 CFT significative, composte da 196 Fattori di Trascrizione (FT). Queste sono state successivamente validate consultando diverse fonti di informazione disponibili e abbiamo osservato che il 98% dei FT significativi sono presenti nell'interattoma umano. L'analisi delle loro interazioni indica che il 96% delle CFT sono separate da due o meno proteine intermedie. Il 99% delle CFT sono composte da FT espressi in tessuti comuni. Dallo stesso set di CEP e SLFT sono stati identificati 48 bicluster significativi composti da 22 FT e 222 CEP e osservato che il 92% di questi FT sono condivisi con le CFT. I bicluster contenevano da 2 a 6 FT e dopo una comparazione con le CFT ipotizziamo che i bicluster potrebbero evidenziare meccanismi di regolazione più fini. Conclusioni L'analisi computazionale integra informazioni provenienti da diverse risorse sperimentali, supporta la nozione che l'accoppiamento/raggruppamento di FT è alla base di interazioni a lungo raggio biologicamente rilevanti di enhancer dello sviluppo e contribuisce alla caratterizzazione dei meccanismi di regolazione genica dello sviluppo dei mammiferi.Background Development of mammalian tissues rely on complex regulations of tissue specific gene expression, possibly due to distant-acting transcriptional enhancers. Flanking regions of important developmental genes contain highly conserved sequences, supporting the notion that these regions are distal enhancers of their flanking genes. One possible mechanism of gene transcription regulation is the “enhancer-promoter” looping, mediated by sequence-specific transcription factors. Methodology/Principal Findings In order to investigate mechanisms of enhancer-promoter interaction, we generated 1011 Enhancer-Promoter Pairs (EPP) by coupling 702 in vivo validated developmental enhancers and their nearest 621 promoters. We predicted human-specific Transcription Factors Binding Sites (TFBSs) to the paired enhancers and promoters. From an initial set of 2837 Transcription Factor Pairs (TFPs) and by adopting a statistical approach, we filtered 364 significant TFPs, comprising 196 transcription factors (TFs). We then validated our finding by extracting information from presently available databases and found that 98% of significant TFs are part of the human interactome. The analysis of their interaction network indicates that 96% of the interacting pairs are from zero to two proteins apart. Moreover, 99% of TFPs are composed by TFs expressed in the same tissues. From the same sets of EPPs and TFBSs we also identified 48 significant biclusters composed by 22 TFs and 222 EPPs and found that 92% of these TFs are shared with the TFPs. Biclusters contained from 2 to 6 TFs and after a comparison with TFPs we speculate that biclusters could point out mechanisms of a finer regulation. Conclusions Our computational analysis, integrating information from different experimental resources, supports the notion that transcription factor pairing/grouping is at the basis of biologically relevant long-distance interactions of developmental enhancers and add to the characterization of complex gene regulation mechanisms in mammal development