The transcriptional activator SNF2PH regulates VSG and promotes infective form surface protein expression in trypanosoma brucei

Resumen

Trypanosoma brucei is an extracellular parasite belonging to Trypanosomatidae family (Protist kingdom). In the mammalian host, the bloodstream form of T. brucei causes sleeping sickness or Human African Trypanosomiasis (HAT) and nagana in cattle. The parasite undergoes morphological changes between the insect and the mammalian host over the course of its life cycle. The mammalian bloodstream form expresses the Variant Surface Glycoproteins (VSG) required for antigenic variation. Changes in the expression of different types of VSGs allow the parasite to elude the mammalian host adaptive immune response, leading to a persistent infection. In the insect host, the VSG is replaced by a different glycoprotein family named procyclins when the parasite differentiates to procyclic form in the tsetse fly. During the bloodstream form of the parasite, a single VSG is expressed from a telomeric locus known as the VSG-Expression Site, (VSG-ES). The transcription of this locus is mediated by RNA Polymerase I (RNA Pol I) in a nuclear body called the Expression Site Body (ESB), which is located outside the nucleolus where this polymerase normally resides. In the genome of T. brucei there are about 1000 genes for different types of VSG genes although they can only be expressed from a VSG-ES of approximately 15 different telomeric loci, of which only one is transcribed at any given time. The expression of a VSG on the surface of the parasite can be changed to another either by recombination of another VSG gene in the active VSG-ES, or by a in situ transcriptional switch to another of the 15 VSG-ESs by epigenetic mechanisms not very known. During differentiation to the procyclic form, no VSG is expressed and the RNA pol I in the nucleolus transcribes the genes of the procyclins, while the telomeric VSG-ES is repressed. Previous published work from our group described SUMOylation (Small Ubiquitin-like MOdifier, SUMO) as a Post Translational Modification (PTM) that regulates VSG expression (Lopez-Farfan et al., 2014). SUMOylation is a large and reversible post-translational modification (PTM) that regulates many critical processes in eukaryotes, including transcription, DNA replication, repair and protein signaling. Transcription factors are well known SUMO targets, whose activity can be modulated in both gene silencing and activation. In bloodstream forms trypanosomes, SUMO-conjugated proteins are enriched at the highly-SUMOylated focus (HSF), which co-localized to the nuclear body ESB and are also abundant in the active VSG-ES chromatin (Lopez-Farfan et al., 2014). Thus, it is possible that SUMO acts as an epigenetic chromatin mark to positive regulate VSG expression. In this PhD thesis, we first identify major SUMO-conjugated proteins in the mammalian infective form by proteomic analyses as a starting point to uncover new factors related to VSG expression. This study yielded to the identification of several SUMO targets, some related to VSG expression and probably linked to developmental regulation, in addition to proteins involved in well-known process regulated by SUMO as DNA repair, replication, etc. Out of the SUMO-modified proteins, we identified a transcriptional activator (Tb927.3.2140) which contains a Sucrose Non Fermentative-2 (Snf2) chromatin remodeler domain and a Plant Homeodomain (PH); we thus named SNF2PH. The SNF2 domain functions by modulating chromatin accessibility, while the PH domain is involved in recognizing epigenetic chromatin marks involved in gene expression regulation of development. Our data suggested that the PH domain of SUMOylated SNF2PH changes the protein interaction interface that reads histone tail modifications and thus regulates VSG ES transcription by increasing chromatin accessibility. Importantly, ectopic expression of SNF2PH in the insect form, where it is normally downregulated, triggers the transcription of bloodstream stage-specific genes of surface proteins, while the expression of a truncated version of the SNF2PH (SNF2∆PH) that lacked the PH domain did not induce the transcription of these genes. Altogether, these data suggest that SNF2PH SUMOylation positively regulates VSG monoallelic transcription while the PH domain is required for the expression of bloodstream-specific surface protein genes. Thus, SNF2PH functions as a positive transcriptional activator, and is required to ensure coordinated expression of surface stage-specific proteins. Altogether our results suggest that SNF2PH is a central regulator of VSG monoallelic expression and maintenance infective stage-specific surface protein integrity, acting as a major regulator of pathogenicity. Analysis of gene expression in bloodstream form cell lines where SNF2PH was depleted using RNA interference detect silencing of the active VSG-ES and the activation of the expression of the developmentally regulated genes markers, such as PADs (Protein associated with the differentiation) and procyclins, suggesting a role in development. In addition, of SNF2PH protein levels in stumpy-like cells obtained after AMPKα1 activation by AMP together with detection of SNF2PH downregulation in stumpy form where AMPKα1 activation occurs, suggests AMPK pathway negatively regulates SNF2PH expression by an undefined mechanism. We propose a model whereby SNF2PH has distinct activities in a PTM-dependent manner, since SNF2PH chromatin remodeling protein requires SUMO modification to maintain active monoallelic VSG expression while the PH domain functions to ensure coordinated transcription of surface stage-specific protein genes and thus adaptation to the mammalian host. This finding shows a novel link between the epigenetic inheritance of a single active allele, among a multiallelic VSG family by the PTM of SNF2PH, and subnuclear body dynamics by SUMOylation. All of which provide a new framework to unravel the molecular mechanisms underlying inheritance of monoallelic expression of the VSG.