Siegel

 

Research

Fig 1: Scanning electron micrograph of a long slender bloodstream form T. brucei parasite. (Courtesy of Thierry Blisnick, Philippe Bastin laboratory, Institut Pasteur, Paris.)
Fig 2: During a blood meal the tsetse injects the infective metacyclic form into the bloodstream of the mammalian host. Inside the bloodstream, the parasite differentiates into the prolific long slender form, a process that is accompanied by significant morphological changes and the expression of a characteristic surface coat. Over time a pleomorphic population develops containing some short stumpy bloodstream forms that are pre-adapted for uptake by the tsetse. Inside the vector, the parasite again undergoes significant morphological changes and differentiates into the parasite-specific procyclic form. After migrating to the salivary glands, the parasite differentiates into the dividing epimastigote form and further into the non-dividing, infective metacyclic form.
Fig 3: Divergent strand switch regions contain RNA pol II transcription start sites. Convergent-SSRs contain RNA pol II transcription termination sites. Blue boxes represent open reading frames and green arrows indicate the direction of transcription.

What are trypanosomes?

Trypanosomes are small unicellular eukaryotic parasites of insects, birds, fish, and mammals that have been around for more than 300 million years. Most species of trypanosomes are non-pathogenic but infamous exceptions exist: Trypanosoma brucei (Fig.1) causes sleeping sickness in Sub-Saharan Africa, Trypanosoma cruzi causes Chagas in Central and South America and related Leishmania parasites are responsible for leishmaniasis, a disease considered endemic in 88 countries.

T. brucei branched early in evolution from the eukaryotic lineage and causes African trypanosomiasis, commonly referred to as sleeping sickness in humans and nagana in livestock. Transmission of sleeping sickness is restricted to regions where its vector, the tsetse (Glossina sp.), is endemic and occurs almost exclusively in Sub-Saharan Africa (for life cycle, see Fig. 2). If left untreated, sleeping sickness is generally lethal. Soon after the parasite was discovered more than hundred years ago by Surgeon-Captain David Bruce, its great evolutionary distance from other organisms and its many unique solutions to ‘biological problems’ started to fascinate researchers. Unfortunately, despite decades of research, no vaccine has been developed, and effective drugs only exist against the early stages of the disease before the parasite has crossed the blood-brain barrier of its host. However, the intense research has made T. brucei an important model organism that has led to discoveries like GPI-mediated anchoring of proteins, RNA-editing, and trans splicing of RNA. Yet, many aspects of trypanosome biology are still not well understood, including the regulation of gene expression – the research interest of our group.

Epigenetic regulation of gene expression

The basis of transcription initiation in eukaryotes is the recruitment of transcription factors (TFs) and other components of the transcription complex to a specific DNA sequence element, yet most eukaryotic DNA cannot be readily accessed by TFs as it is packaged in a nucleoprotein complex called chromatin. The fundamental unit of chromatin is the nucleosome: 147 bp of DNA wrapped around an octamer of core histones H2A, H2B, H3 and H4. The packaging of DNA into chromatin is highly regulated, leaving some regions of the genome more accessible to TFs than others.

To facilitate access to DNA, the structure of nucleosomes can be altered by posttranslational modification of histones or replacement of canonical histones with histone variants. For example, genome-wide analyses of H2AZ distribution, a variant of the canonical histone H2A, revealed an enrichment of H2AZ at promoters or close to transcription start sites (TSSs) in several organisms. At TSSs, H2AZ appears to contribute, in combination with other factors, to nucleosome destabilization, which may lead to increased nucleosome eviction and thereby facilitate access of the transcription complex to specific DNA sequence elements.

Histone modifications and histone variants are often referred to as ‘epigenetic factors’ because both can influence gene expression in a heritable fashion without altering the underlying DNA sequence. Intense research during the last years has demonstrated the importance of epigenetics in regulation of gene expression for biological mechanisms ranging from the differentiation of embryonic stem cells to antigenic variation of human infective parasites. Today there is extensive knowledge about enzymes adding or removing specific histone modifications or replacing canonical histones with histone variants, but it is less well understood why some genomic loci contain certain histone modifications or specific histone variants and others do not. Interestingly, in some instances non-coding RNA (ncRNA) transcripts have been shown to target histone-modifying enzymes to specific genomic loci.

Atypical for a eukaryote, genes transcribed by RNA polymerase II (RNA pol II) in T. brucei are arranged in polycistronic transcription units (PTUs), sometimes spanning more than 100 genes. Within a PTU, genes are transcribed from the same strand, but transcription of two neighboring PTUs located on opposite strands can either be convergent or divergent. The regions between PTUs located on opposite strands are referred to as strand-switch regions (SSRs) (Fig. 3). Several pieces of evidence suggest that transcription initiates at divergent SSRs but to date, no RNA pol II promoter for protein-encoding genes has been identified. In fact, it had been hypothesized that no RNA pol II promoter motifs exist and that an ‘open’ chromatin structure may be sufficient for RNA pol II transcription initiation.

Our Research

While transcription factors have remained elusive in T. brucei, much evidence suggests that chromatin structure plays an important role in gene regulation. Although the primary structure of trypanosome core histones diverges significantly relative to other eukaryotes, trypanosomes do contain several histone modifications, including an extensively acetylated H4 tail. T. brucei also has one variant of each of the four core histones: H2AZ, H2BV, H3V and H4V. Using ChIP-seq, we were able to show that RNA pol II TSSs in T. brucei are strongly enriched in H4K10ac, H2AZ and H2BV, while transcription termination sites are marked by variants of histone H3 and H4.

Our group is interested in how epigenetic factors like posttranslational histone modifications, histone variants and ncRNA interact to form chromatin structures that modulate transcription.

More specifically, we plan to investigate why histone variants and modified histones are enriched at some genomic loci and not at others and what role ncRNA plays in targeting these different epigenetic factors