Understanding chromatin dynamics during antigenic variation in trypanosoma brucei

Branco, Francisco Maria dos Santos e Silva Aresta, 1985-

http://hdl.handle.net/10451/27186

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Authors

Branco, Francisco Maria dos Santos e Silva Aresta, 1985-

Advisor(s)

Figueiredo, Luísa Miranda

Abstract(s)

African trypanosomiasis is a disease restricted to sub-Saharian Africa and it assumes two forms: Human African trypanosomiasis (HAT or sleeping sickness) or Animal African trypanosomiasis (AAT or n’gana), both being fatal if untreated. While HAT causes an estimated number of 15000 new cases each year and 70 million people are at risk, AAT causes an enormous economic burden due to livestock elimination [1]. HAT is caused by the unicellular Trypanosoma brucei parasite, which is transmitted by the tsetse transmitting vector. Upon a tsetse blood meal, parasites are delivered into a mammalian host where they proliferate in the bloodstream, lymphatic system and inside preferred tissues, such as the adipose tissue [2]. The parasites can progressively migrate to the central nervous system, leading to neurological disorders, coma and death [3]. Each Trypanosoma brucei parasite expresses a single type of Variant Surface Glycoprotein (VSG) at its cell surface. To avoid being eliminated by the host immune system, parasites use antigenic variation, which consists in periodically changing to a new VSG coat, obligating the immune system to reinitiate a new adaptive response and generate new antibodies. VSG genes are transcribed by RNA polymerase I (Pol I) from specialized subtelomeric loci termed Bloodstream Expression Sites (BESs) [4]. Although there are around 15 BESs in the genome, only one is active at any given time, thus ensuring VSG monoallelic expression. Replacement of the VSG coat starts in the nucleus by changing the actively transcribed VSG. This process takes place through: i) switching by recombination, which involves DNA recombination between the active and a silent BES encompassing the VSG gene or by copying a VSG sequence from archival copies present in the genome into the active BES [5, 6]; ii) in situ switching, where the active BES becomes silenced with the concomitant activation of a silent BES [7, 8]. VSG in situ switching is very poorly understood. We do not know the key players or the sequence of events that regulate it. As the actively transcribed BES possesses a very open chromatin conformation relative to transcriptionally silent BESs [9, 10], we proposed to understand how chromatin and transcription interweave during an in situ switching. Another event in which the active BES becomes silenced is during differentiation from bloodstream to procyclic (present in the tsetse) forms [11]. In this situation, we observed that transcriptional silencing precedes chromatin changes. Thus, we postulated that during an in situ switching event transcription is probably also halted prior to closing chromatin structure. We chose to use a tetracycline-inducible BES transcriptional silencing system from the Horn lab that induces VSG silencing [12], resulting in an increase of the switching frequency to 8%. Blockage of transcription was confirmed by the absence of Pol I in the active BES and led to a rapid decrease in transcript levels of this locus within the first 8 hr after BES silencing. Despite such significant changes at the transcriptional level, chromatin of the previously active BES remained in an open conformation. We hypothesized that the cell-cycle was necessary to induce chromatin remodeling as in Pol I-transcribed ribosomal DNA (rDNA) genes in yeast, but this revealed unfruitful. We observed that TDP1 (Trypanosome DNA-binding protein 1), an essential high mobility group box protein [13, 14], remained in the active BES even after BES silencing had been triggered. Depletion of TDP1 in these conditions led to closure of chromatin indicating that TDP1 is a key factor in stabilizing open chromatin conformation under transcriptional switching. We showed that during in situ switching, cells probe more than one silent BES, inclusively expressing other VSGs. We determined that after 24 hr of BES silencing, roughly half of the cells were committed to switch while the remaining could revert to transcribe the initial BES. Interestingly, cells that at 24 hr of BES silencing were not probing a specific BES (and VSG) did not switch to that BES. Overall, these results led us to propose a model in which under an in situ switching, transcription of the active BES is halted but its chromatin is maintained in an open conformation by TDP1. This allows parasites to probe silent BES to make a decision of switching or returning to the initial BES. The importance of TDP1 in Pol I transcription and in the switching process led us to ask if TDP1 overexpression could interfere with VSG monoallelic expression. Using a TDP1-overexpressing cell-line, we observed that genome-wide chromatin becomes more open (at different extent) except in the active BES and rDNA genes, suggesting that these two loci already have the chromatin fully open. Furthermore, the mRNA transcript levelsonly increased in silent BES, MES (Metacyclic Expression Sites) and procyclin loci confirming the role of TDP1 as a Pol I transcription facilitator [14]. Importantly, we observed the expression of a silent VSG upon TDP1 overexpression confirming the disruption of monoallelic expression. Overall, this dissertation enlightens the relevance of chromatin during an in situ switching and elucidates the roles of TDP1 as key factor for antigenic variation.