Site Of Expression Research Articles

DNA double-strand breaks and telomeres play important roles in trypanosoma brucei antigenic variation.

Human-infecting microbial pathogens all face a serious problem of elimination by the host immune response. Antigenic variation is an effective immune evasion mechanism where the pathogen regularly switches its major surface antigen. In many cases, the major surface antigen is encoded by genes from the same gene family, and its expression is strictly monoallelic. Among pathogens that undergo antigenic variation, Trypanosoma brucei (a kinetoplastid), which causes human African trypanosomiasis, Plasmodium falciparum (an apicomplexan), which causes malaria, Pneumocystis jirovecii (a fungus), which causes pneumonia, and Borrelia burgdorferi (a bacterium), which causes Lyme disease, also express their major surface antigens from loci next to the telomere. Except for Plasmodium, DNA recombination-mediated gene conversion is a major pathway for surface antigen switching in these pathogens. In the last decade, more sophisticated molecular and genetic tools have been developed in T. brucei, and our knowledge of functions of DNA recombination in antigenic variation has been greatly advanced. VSG is the major surface antigen in T. brucei. In subtelomeric VSG expression sites (ESs), VSG genes invariably are flanked by a long stretch of upstream 70-bp repeats. Recent studies have shown that DNA double-strand breaks (DSBs), particularly those in 70-bp repeats in the active ES, are a natural potent trigger for antigenic variation in T. brucei. In addition, telomere proteins can influence VSG switching by reducing the DSB amount at subtelomeric regions. These findings will be summarized and their implications will be discussed in this review.

SUMOylation by the E3 ligase TbSIZ1/PIAS1 positively regulates VSG expression in Trypanosoma brucei.

Bloodstream form trypanosomes avoid the host immune response by switching the expression of their surface proteins between Variant Surface Glycoproteins (VSG), only one of which is expressed at any given time. Monoallelic transcription of the telomeric VSG Expression Site (ES) by RNA polymerase I (RNA pol I) localizes to a unique nuclear body named the ESB. Most work has focused on silencing mechanisms of inactive VSG-ESs, but the mechanisms involved in transcriptional activation of a single VSG-ES remain largely unknown. Here, we identify a highly SUMOylated focus (HSF) in the nucleus of the bloodstream form that partially colocalizes with the ESB and the active VSG-ES locus. SUMOylation of chromatin-associated proteins was enriched along the active VSG-ES transcriptional unit, in contrast to silent VSG-ES or rDNA, suggesting that it is a distinct feature of VSG-ES monoallelic expression. In addition, sequences upstream of the active VSG-ES promoter were highly enriched in SUMOylated proteins. We identified TbSIZ1/PIAS1 as the SUMO E3 ligase responsible for SUMOylation in the active VSG-ES chromatin. Reduction of SUMO-conjugated proteins by TbSIZ1 knockdown decreased the recruitment of RNA pol I to the VSG-ES and the VSG-ES-derived transcripts. Furthermore, cells depleted of SUMO conjugated proteins by TbUBC9 and TbSUMO knockdown confirmed the positive function of SUMO for VSG-ES expression. In addition, the largest subunit of RNA pol I TbRPA1 was SUMOylated in a TbSIZ-dependent manner. Our results show a positive mechanism associated with active VSG-ES expression via post-translational modification, and indicate that chromatin SUMOylation plays an important role in the regulation of VSG-ES. Thus, protein SUMOylation is linked to active gene expression in this protozoan parasite that diverged early in evolution.

FACT plays a major role in histone dynamics affecting VSG expression site control in Trypanosoma brucei

Chromatin remodelling is involved in the transcriptional regulation of the RNA polymerase I transcribed variant surface glycoprotein (VSG) expression sites (ESs) of Trypanosoma brucei. We show that the T. brucei FACT complex contains the Pob3 and Spt16 subunits, and plays a key role in ES silencing. We see an inverse correlation between transcription and condensed chromatin, whereby FACT knockdown results in ES derepression and more open chromatin around silent ES promoters. Derepressed ESs show increased sensitivity to micrococcal nuclease (MNase) digestion, and a decrease in histones at silent ES promoters but not telomeres. In contrast, FACT knockdown results in more histones at the active ES, correlated with transcription shut-down. ES promoters are derepressed in cells stalled at the G2/M cell cycle stage after knockdown of FACT, but not in G2/M cells stalled after knockdown of cyclin 6. This argues that the observed ES derepression is a direct consequence of histone chaperone activity by FACT at the G2/M cell cycle stage which could affect transcription elongation, rather than an indirect consequence of a cell cycle checkpoint. These experiments highlight the role of the FACT complex in cell cycle-specific chromatin remodelling within VSG ESs.

Expression site attenuation mechanistically links antigenic variation and development in Trypanosoma brucei.

We have discovered a new mechanism of monoallelic gene expression that links antigenic variation, cell cycle, and development in the model parasite Trypanosoma brucei. African trypanosomes possess hundreds of variant surface glycoprotein (VSG) genes, but only one is expressed from a telomeric expression site (ES) at any given time. We found that the expression of a second VSG alone is sufficient to silence the active VSG gene and directionally attenuate the ES by disruptor of telomeric silencing-1B (DOT1B)-mediated histone methylation. Three conserved expression-site-associated genes (ESAGs) appear to serve as signal for ES attenuation. Their depletion causes G1-phase dormancy and reversible initiation of the slender-to-stumpy differentiation pathway. ES-attenuated slender bloodstream trypanosomes gain full developmental competence for transformation to the tsetse fly stage. This surprising connection between antigenic variation and developmental progression provides an unexpected point of attack against the deadly sleeping sickness.DOI: http://dx.doi.org/10.7554/eLife.02324.001.

Inositol phosphate regulation of variant surface glycoprotein expression in Trypanosoma brucei (232.3)

Trypanosoma brucei evades the host immune system by antigenic variation, expressing only one of >1,000 variant surface glycoprotein (VSG) genes through transcription at one of ~20 subtelomeric expression sites (ESs), thereby producing a homogenous surface coat. These coats vary periodically via VSG gene recombination or transcriptional ES switching, and the mechanisms regulating these processes are unknown. T. brucei has an inositol phosphate pathway encompassing 26 genes for inositol phosphate (IP) and phosphatidylinositol (PI) kinases, phosphatases, and metabolic enzymes. We generated T. brucei bloodstream form (BF) conditional null lines for genes in this pathway and found that PI(4) 5‐kinase and IP3 5‐phosphatase are essential for growth and infection of mice. Conditional repression of PI(4) 5‐kinase or IP3 5‐phosphatase, or overexpression of phospholipase C, resulted in expression of VSGs from multiple ESs, including BF and metacyclic ESs. Expression of procyclins or other genes encoded by RNA polymerase I, II or III were not affected, and repression of other IP pathway genes did not induce VSG expression. Repression of PI(4)5‐kinase or IP3(5)‐phosphatase also caused telomere repositioning in the nucleus; re‐expression of these enzymes generated cells that switched VSGs. Thus, T. brucei possesses an IP pathway which appears to be involved in regulation of VSG expression and antigenic switching.

TDP1 is an HMG chromatin protein facilitating RNA polymerase I transcription in African trypanosomes

Unusually for a eukaryote, Trypanosoma brucei transcribes its variant surface glycoprotein (VSG) gene expression sites (ESs) in a monoallelic fashion using RNA polymerase I (Pol I). It is still unclear how ES transcription is controlled in T. brucei. Here, we show that the TDP1 architectural chromatin protein is an essential high mobility group box (HMGB) protein facilitating Pol I transcription in T. brucei. TDP1 is specifically enriched at the active compared with silent VSG ES and immediately downstream of ribosomal DNA promoters and is abundant in the nucleolus and the expression site body subnuclear compartments. Distribution of TDP1 at Pol I-transcribed loci is inversely correlated with histones. Depletion of TDP1 results in up to 40–90% reduction in VSG and rRNA transcripts and a concomitant increase in histones H3, H2A and H1 at these Pol I transcription units. TDP1 shares features with the Saccharomyces cerevisiae HMGB protein Hmo1, but it is the first architectural chromatin protein facilitating Pol I-mediated transcription of both protein coding genes as well as rRNA. These results show that TDP1 has a mutually exclusive relationship with histones on actively transcribed Pol I transcription units, providing insight into how Pol I transcription is controlled.

Histone H1 Plays a Role in Heterochromatin Formation and VSG Expression Site Silencing in Trypanosoma brucei

The African sleeping sickness parasite Trypanosoma brucei evades the host immune system through antigenic variation of its variant surface glycoprotein (VSG) coat. Although the T. brucei genome contains ∼1500 VSGs, only one VSG is expressed at a time from one of about 15 subtelomeric VSG expression sites (ESs). For antigenic variation to work, not only must the vast VSG repertoire be kept silent in a genome that is mainly constitutively transcribed, but the frequency of VSG switching must be strictly controlled. Recently it has become clear that chromatin plays a key role in silencing inactive ESs, thereby ensuring monoallelic expression of VSG. We investigated the role of the linker histone H1 in chromatin organization and ES regulation in T. brucei. T. brucei histone H1 proteins have a different domain structure to H1 proteins in higher eukaryotes. However, we show that they play a key role in the maintenance of higher order chromatin structure in bloodstream form T. brucei as visualised by electron microscopy. In addition, depletion of histone H1 results in chromatin becoming generally more accessible to endonucleases in bloodstream but not in insect form T. brucei. The effect on chromatin following H1 knock-down in bloodstream form T. brucei is particularly evident at transcriptionally silent ES promoters, leading to 6–8 fold derepression of these promoters. T. brucei histone H1 therefore appears to be important for the maintenance of repressed chromatin in bloodstream form T. brucei. In particular H1 plays a role in downregulating silent ESs, arguing that H1-mediated chromatin functions in antigenic variation in T. brucei.

Cell-cycle-regulated control of VSG expression site silencing by histones and histone chaperones ASF1A and CAF-1b in Trypanosoma brucei

Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference viability screen. Using this approach, we demonstrate roles in VSG ES silencing for two histone chaperones. Defects in S-phase progression in cells depleted for histone H3, or either chaperone, highlight in particular the link between chromatin assembly and DNA replication control. S-phase checkpoint arrest was incomplete, however, allowing G2/M-specific VSG ES derepression following knockdown of histone H3. In striking contrast, knockdown of anti-silencing factor 1A (ASF1A) allowed for derepression at all cell cycle stages, whereas knockdown of chromatin assembly factor 1b (CAF-1b) revealed derepression predominantly in S-phase and G2/M. Our results support a central role for chromatin in maintaining VSG ES silencing. ASF1A and CAF-1b appear to play constitutive and DNA replication-dependent roles, respectively, in the recycling and assembly of chromatin. Defects in these functions typically lead to arrest in S-phase but defective cells can also progress through the cell cycle leading to nucleosome depletion and derepression of telomeric VSG ESs.

Cytokinesis of Trypanosoma brucei bloodstream forms depends on expression of adenylyl cyclases of the ESAG4 or ESAG4‐like subfamily

Antigenic variation of the parasite Trypanosoma brucei operates by monoallelic expression of a variant surface glycoprotein (VSG) from a collection of multiple telomeric expression sites (ESs). Each of these ESs harbours a long polycistronic transcription unit containing several expression site-associated genes (ESAGs). ESAG4 copies encode bloodstream stage-specific adenylyl cyclases (AC) and belong to a larger gene family of around 80 members, the majority of which, termed genes related to ESAG4 (GRESAG4s), are not encoded in ESs and are expressed constitutively in the life cycle. Here we report that ablation of ESAG4 from the active ES did not affect parasite growth, neither in culture nor upon rodent infection, and did not significantly change total AC activity. In contrast, inducible RNAi-mediated knock-down of an AC subfamily that includes ESAG4 and two ESAG4-like GRESAG4 (ESAG4L) genes, decreased total AC activity and induced a lethal phenotype linked to impaired cytokinesis. In the Δesag4 line compensatory upregulation of apparently functionally redundant ESAG4L genes was observed, suggesting that the ESAG4/ESAG4L-subfamily ACs are involved in the control of cell division. How deregulated adenylyl cyclases or cAMP might impair cytokinesis is discussed.

African trypanosomes: the genome and adaptations for immune evasion

The African trypanosome Trypanosoma brucei is a flagellated unicellular parasite transmitted by tsetse flies that causes African sleeping sickness in sub-Saharan Africa. Trypanosomes are highly adapted for life in the hostile environment of the mammalian bloodstream, and have various adaptations to their cell biology that facilitate immune evasion. These include a specialized morphology, with most nutrient uptake occurring in the privileged location of the flagellar pocket. In addition, trypanosomes show extremely high rates of recycling of a protective VSG (variant surface glycoprotein) coat, whereby host antibodies are stripped off of the VSG before it is re-used. VSG recycling therefore functions as a mechanism for cleaning the VSG coat, allowing trypanosomes to survive in low titres of anti-VSG antibodies. Lastly, T. brucei has developed an extremely sophisticated strategy of antigenic variation of its VSG coat allowing it to evade host antibodies. A single trypanosome has more than 1500 VSG genes, most of which are located in extensive silent arrays. Strikingly, most of these silent VSGs are pseudogenes, and we are still in the process of trying to understand how non-intact VSGs are recombined to produce genes encoding functional coats. Only one VSG is expressed at a time from one of approximately 15 telomeric VSG ES (expression site) transcription units. It is becoming increasingly clear that chromatin remodelling must play a critical role in ES control. Hopefully, a better understanding of these unique trypanosome adaptations will eventually allow us to disrupt their ability to multiply in the mammalian bloodstream.

Identification of Trypanosoma brucei RMI1/BLAP75 Homologue and Its Roles in Antigenic Variation

At any time, each cell of the protozoan parasite Trypanosoma brucei expresses a single species of its major antigenic protein, the variant surface glycoprotein (VSG), from a repertoire of >2,000 VSG genes and pseudogenes. The potential to express different VSGs by transcription and recombination allows the parasite to escape the antibody-mediated host immune response, a mechanism known as antigenic variation. The active VSG is transcribed from a sub-telomeric polycistronic unit called the expression site (ES), whose promoter is 40–60 kb upstream of the VSG. While the mechanisms that initiate recombination remain unclear, the resolution phase of these reactions results in the recombinational replacement of the expressed VSG with a donor from one of three distinct chromosomal locations; sub-telomeric loci on the 11 essential chromosomes, on minichromosomes, or at telomere-distal loci. Depending on the type of recombinational replacement (single or double crossover, duplicative gene conversion, etc), several DNA-repair pathways have been thought to play a role. Here we show that VSG recombination relies on at least two distinct DNA-repair pathways, one of which requires RMI1-TOPO3α to suppress recombination and one that is dependent on RAD51 and RMI1. These genetic interactions suggest that both RAD51-dependent and RAD51-independent recombination pathways operate in antigenic switching and that trypanosomes differentially utilize recombination factors for VSG switching, depending on currently unknown parameters within the ES.

An expanded family of proteins with BPI/LBP/PLUNC-like domains in trypanosome parasites: an association with pathogenicity?

Trypanosomatids are protozoan parasites that cause human and animal disease. Trypanosoma brucei telomeric ESs (expression sites) contain genes that are critical for parasite survival in the bloodstream, including the VSG (variant surface glycoprotein) genes, used for antigenic variation, and the SRA (serum-resistance-associated) gene, which confers resistance to lysis by human serum. In addition, ESs contain ESAGs (expression-site-associated genes), whose functions, with few exceptions, have remained elusive. A bioinformatic analysis of the ESAG5 gene of T. brucei showed that it encodes a protein with two BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelium clone)-like domains and that it belongs to a multigene family termed (GR)ESAG5 (gene related to ESAG5). Members of this family are found with various copy number in different members of the Trypanosomatidae family. T. brucei has an expanded repertoire, with multiple ESAG5 copies and at least five GRESAG5 genes. In contrast, the parasites of the genus Leishmania, which are intracellular parasites, have only a single GRESAG5 gene. Although the amino acid sequence identity between the (GR)ESAG5 gene products between species is as low as 15-25%, the BPI/LBP/PLUNC-like domain organization and the length of the proteins are highly conserved, and the proteins are predicted to be membrane-anchored or secreted. Current work focuses on the elucidation of possible roles for this gene family in infection. This is likely to provide novel insights into the evolution of the BPI/LBP/PLUNC-like domains.

NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei

Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

Epigenetics and transcriptional control in African trypanosomes

The African trypanosome Trypanosoma brucei is a unicellular parasite which causes African sleeping sickness. Transcription in African trypanosomes displays some unusual features, as most of the trypanosome genome is transcribed as extensive polycistronic RNA Pol II (polymerase II) transcription units that are not transcriptionally regulated. In addition, RNA Pol I is used for transcription of a small subset of protein coding genes in addition to the rDNA (ribosomal DNA). These Pol I-transcribed protein coding genes include the VSG (variant surface glycoprotein) genes. Although a single trypanosome has many hundreds of VSG genes, the active VSG is transcribed in a strictly monoalleleic fashion from one of approx. 15 telomeric VSG ESs (expression sites). Originally, it was thought that chromatin was not involved in the transcriptional control of ESs; however, this view is now being re-evaluated. It has since been shown that the active ES is depleted of nucleosomes compared with silent ESs. In addition, a number of proteins involved in chromatin remodelling or histone modification and which play a role in ES silencing {including TbISWI [T. brucei ISWI (imitation-switch protein)] and DOT1B} have recently been identified. Lastly, the telomere-binding protein TbRAP1 (T. brucei RAP1) has been shown to establish a repressive gradient extending from the ES telomere end up to the ES promoter. We still need to determine which epigenetic factors are involved in 'marking' the active ES as part of the counting mechanism of monoallelic exclusion. The challenge will come in determining how these multiple regulatory layers contribute to ES control.

The FACT subunit TbSpt16 is involved in cell cycle specific control of VSG expression sites in Trypanosoma brucei.

The African trypanosome Trypanosoma brucei monoallelically expresses one of more than 1000 Variant Surface Glycoprotein (VSG) genes. The active VSG is transcribed from one of about 15 telomeric VSG expression sites (ESs). It is unclear how monoallelic expression of VSG is controlled, and how inactive VSG ESs are silenced. Here, we show that blocking synthesis of the T. brucei FACT subunit TbSpt16 triggers a G2/early M phase cell cycle arrest in both bloodstream and insect form T. brucei. Segregation of T. brucei minichromosomes in these stalled cells is impaired, implicating FACT in maintenance of centromeres. Strikingly, knock-down of TbSpt16 results in 20- to 23-fold derepression of silent VSG ES promoters in bloodstream form T. brucei, with derepression specific to the G2/M cell cycle stage. In insect form T. brucei TbSpt16 knock-down results in 16- to 25-fold VSG ES derepression. Using chromatin immunoprecipitation (ChIP), TbSpt16 was found to be particularly enriched at the promoter region of silent but not active VSG ESs in bloodstream form T. brucei. The chromatin remodeler FACT is therefore implicated in maintenance of repressed chromatin present at silent VSG ES promoters, but is also essential for chromosome segregation presumably through maintenance of functional centromeres.

Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes.

African trypanosomes evade the host immune response through antigenic variation, which is achieved by periodically expressing different variant surface glycoproteins (VSGs). VSG expression is monoallelic such that only one of approximately 15 telomeric VSG expression sites (ESs) is transcribed at a time. Epigenetic regulation is involved in VSG control but our understanding of the mechanisms involved remains incomplete. Histone deacetylases are potential drug targets for diseases caused by protozoan parasites. Here, using recombinant expression we show that the essential Trypanosoma brucei deacetylases, DAC1 (class I) and DAC3 (class II) display histone deacetylase activity. Both DAC1 and DAC3 are nuclear proteins in the bloodstream stage parasite, while only DAC3 remains concentrated in the nucleus in insect-stage cells. Consistent with developmentally regulated localization, DAC1 antagonizes SIR2rp1-dependent telomeric silencing only in the bloodstream form, indicating a conserved role in the control of silent chromatin domains. In contrast, DAC3 is specifically required for silencing at VSG ES promoters in both bloodstream and insect-stage cells. We conclude that DAC1 and DAC3 play distinct roles in subtelomeric gene silencing and that DAC3 represents the first readily druggable target linked to VSG ES control in the African trypanosome.

TOPO3α Influences Antigenic Variation by Monitoring Expression-Site-Associated VSG Switching in Trypanosoma brucei

Homologous recombination (HR) mediates one of the major mechanisms of trypanosome antigenic variation by placing a different variant surface glycoprotein (VSG) gene under the control of the active expression site (ES). It is believed that the majority of VSG switching events occur by duplicative gene conversion, but only a few DNA repair genes that are central to HR have been assigned a role in this process. Gene conversion events that are associated with crossover are rarely seen in VSG switching, similar to mitotic HR. In other organisms, TOPO3α (Top3 in yeasts), a type IA topoisomerase, is part of a complex that is involved in the suppression of crossovers. We therefore asked whether a related mechanism might suppress VSG recombination. Using a set of reliable recombination and switching assays that could score individual switching mechanisms, we discovered that TOPO3α function is conserved in Trypanosoma brucei and that TOPO3α plays a critical role in antigenic switching. Switching frequency increased 10–40-fold in the absence of TOPO3α and this hyper-switching phenotype required RAD51. Moreover, the preference of 70-bp repeats for VSG recombination was mitigated, while homology regions elsewhere in ES were highly favored, in the absence of TOPO3α. Our data suggest that TOPO3α may remove undesirable recombination intermediates constantly arising between active and silent ESs, thereby balancing ES integrity against VSG recombination.

Biological Variation Among African Trypanosomes: I. Clonal Expression of Virulence Is Not Linked to the Variant Surface Glycoprotein or the Variant Surface Glycoprotein Gene Telomeric Expression Site

The potential association of variant surface glycoprotein (VSG) gene expression with clonal expression of virulence in African trypanosomes was addressed. Two populations of clonally related trypanosomes, which differ dramatically in virulence for the infected host, but display the same apparent VSG surface coat phenotype, were characterized with respect to the VSG genes expressed as well as the chromosome telomeric expression sites (ES) utilized for VSG gene transcription. The VSG gene sequences expressed by clones LouTat 1 and LouTat 1A of Trypanosoma brucei rhodesiense were identical, and gene expression in both clones occurred precisely by the same gene conversion events (duplication and transposition), which generated an expression-linked copy (ELC) of the VSG gene. The ELC was present on the same genomic restriction fragments in both populations and resided in the telomere of a 330-kb chromosome; a single basic copy of the LouTat 1/1A VSG gene, present in all variants of the LouTat 1 serodeme, was located at an internal site of a 1.5-Mb chromosome. Restriction endonuclease mapping of the ES telomere revealed that the VSG ELC of clones LouTat 1 and 1A resides in the same site. Therefore, these findings provide evidence that the VSG gene ES and, potentially, any cotranscribed ES-associated genes do not play a role in the clonal regulation of virulence because trypanosome clones LouTat 1 and 1A, which differ markedly in their virulence properties, both express identical VSG genes from the same chromosome telomeric ES.

A newly discovered role of telomeres in an ancient organism

Trypanosoma brucei expresses Variant Surface Glycoprotein (VSG) genes in a strictly monoallelic fashion in its mammalian hosts, and the regulation of this important virulence mechanism has been the research focus for decades. Telomere position effect (TPE), an epigenetic phenomenon, has been proposed to play a critical role in VSG regulation, yet no telomeric protein was identified whose disruption led to VSG derepression. We recently identified tbRAP1 as an intrinsic component of the T. brucei telomere complex and a major regulator for silencing VSG expression sites (ESs). Knockdown of tbRAP1 led to derepression of all ES-linked VSGs but not VSGs located elsewhere, and resulted in stronger derepression of telomere-proximal genes than telomere-distal genes. This tapered silencing pattern further argues that telomere integrity plays a key role in tbRAP1-dependent silencing and for the first time provides direct evidence indicating that telomeres are important for VSG expression regulation. Whether chromatin remodeling is important for tbRAP1-mediated silencing as in classical TPE will also be discussed.

Cohesin regulatesVSGmonoallelic expression in trypanosomes

Antigenic variation allows Trypanosoma brucei to evade the host immune response by switching the expression of 1 out of ∼15 telomeric variant surface glycoprotein (VSG) expression sites (ESs). VSG ES transcription is mediated by RNA polymerase I in a discrete nuclear site named the ES body (ESB). However, nothing is known about how the monoallelic VSG ES transcriptional state is maintained over generations. In this study, we show that during S and G2 phases and early mitosis, the active VSG ES locus remains associated with the single ESB and exhibits a delay in the separation of sister chromatids relative to control loci. This delay is dependent on the cohesin complex, as partial knockdown of cohesin subunits resulted in premature separation of sister chromatids of the active VSG ES. Cohesin depletion also prompted transcriptional switching from the active to previously inactive VSG ESs. Thus, in addition to maintaining sister chromatid cohesion during mitosis, the cohesin complex plays an essential role in the correct epigenetic inheritance of the active transcriptional VSG ES state.