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In eukaryotes, the process of transcriptional regulation, DNA recombination, condensation and replication relies on changes in chromatin structure that do not involve alterations in the DNA sequence but are nevertheless inherited1. Such regulation is said to be beyond genetics (epigenetics) and has been extensively studied in recent years, starting a hunt for factors that act as modulators of epigenetic information. In the nucleus of every eukaryotic cell, a module of approximately 147 bp of DNA is wrapped around an octamer of core histone proteins, forming a nucleosome, which is the basic repetitive unit of chromatin (FIG. 1). Histone 1 (H1) binds to the DNA loop between two neighbouring nucleosomes. Histones and DNA can undergo several types of modification, which can be faithfully transmitted to cell progeny. The Allis labora tory proposed a hypothesis known as the histone code, in which post-translational modifications (PTMs) of histones act in a combinatorial or sequential manner to specify unique downstream activities that affect transcription activation, chromatin condensation and signalling for DNA repair2. The code is ‘written’ by histonemodifying enzymes and ‘read’ by histonebinding proteins (FIG. 2).
Trypanosoma brucei is a unicellular eukaryotic parasite that causes sleeping sickness in humans and nagana in cattle in subSaharan Africa. Sleeping sickness is a vectorborne disease that affects 70 million people and is responsible for approximately 50,000 deaths annually3. The study of this substantial economical and medical threat has been facilitated by the development of genetic tools that allow functional studies and genetic screens (BOX 1). Therefore, T. brucei is a useful model
organism to study basic biological processes of other Kinetoplastida and of higher eukaryotes.
T. brucei inhabits the bloodstream and interstitial spaces of the mammalian host and is covered by a dense coat of a glycosylphosphatidylinositol (GPI)anchored variant surface glycoprotein (VSG) (TABLE 1). Throughout its life cycle, T. brucei changes forms and must adapt to different host environments. By unknown means, a fraction of the proliferating slender bloodstream form commits to a differentiation process that generates a quiescent, cell cyclearrested population (stumpy form) that is reactivated after ingestion by their insect vector, the tsetse. In the midgut of the fly, the parasite seems to sense both the drop in temperature and a chemical cue that trigger its differentiation into a proliferative, pro cyclic form4,107. The procyclic form does not express VSGs, but its surface is covered by GPIanchored proteins called EP (rich in GluPro repeats) or GPEET (rich in GlyProGluGluThr repeats) procyclins. After migrating to the salivary glands, the parasites further differentiate into and proliferate as epimastigotes, the surface of which seems to consist of alaninerich protein (BARP)5. When epimastigotes differentiate into the nonreplicative metacyclic form, BARP is replaced by VSG. When the tsetse takes another meal, the metacyclics are injected into the mammal, where they differentiate into the bloodstream form. Throughout its life cycle, T. brucei alternates in a tightly regulated manner between cell proliferation and cell differentiation. Differentiation in T. brucei is probably a result of responses to different environments and involves a coordinated cascade of signalling events and transcriptional and posttranscriptional machineries. These features probably exist in all biological systems.
*Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, New York 10065, USA. ‡University of Munich, Biocenter, Department of Genetics, Groβhardener Straβe 2‑4, 82152, Martinsried, Germany. Correspondence to L.M.F. e‑mail: [email protected]:10.1038/nrmicro2149
Post-translational modificationAn enzymatic modification of an amino acid residue that occurs after a protein is synthesized, and often modifies the function or lifespan of a protein.
Variant surface glycoprotein(VSG). The most abundant protein (approximately 10 million identical copies on individual cells) at the surface of the bloodstream slender, stumpy and metacyclic life cycle stages of Trypanosoma brucei. Periodic change of the VSG surface coat is a crucial part of the evasion mechanism known as antigenic variation.
Epigenetic regulation in African trypanosomes: a new kid on the blockLuisa M. Figueiredo*, George A. M. Cross* and Christian J. Janzen‡
Abstract | Epigenetic regulation is important in many facets of eukaryotic biology. Recent work has suggested that the basic mechanisms underlying epigenetic regulation extend to eukaryotic parasites. The identification of post-translational histone modifications and chromatin-modifying enzymes is beginning to reveal both common and novel functions for chromatin in these parasites. In this Review, we compare the role of epigenetics in African trypanosomes and humans in several biological processes. We discuss how the study of trypanosome chromatin might help us to better understand the evolution of epigenetic processes.
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Nature Reviews | Microbiology
Nucleosome
Histones
DNA
Spacer DNA
Histoneoctamerplus 147 bp of DNA
H1
TsetseThe insect vector (Glossina spp.) that ensures the transmission of Trypanosoma brucei between two mammalian hosts. The word tsetse means fly and it originates from the Tswana language.
ProcyclinThe most abundant protein at the surface of the procyclic life cycle form. There are two types of procyclin: EP and GPEET. These glycosylated proteins are not subject to allelic exclusion.
EpimastigoteThe Trypanosoma brucei life cycle stage that colonizes the tsetse salivary glands.
MetacyclicThe Trypanosoma brucei life cycle stage in the tsetse salivary glands that reinfects the mammalian host.
Determination of stem cell fate6 and T cell development7 are two wellknown examples.
The genome of T. brucei contains approximately 8,000 genes that are mostly transcribed polycistronically by RNA polymerase II (Pol II)8. unlike bacterial operons, most genes in a polycistronic unit are not functionally related, and gene regulation occurs mainly at the posttranscriptional level9. The only known exceptions are procyclin and VSG genes, which are regulated at the transcriptional level. These genes are transcribed by Pol I, which is used exclusively for the transcription of ribosomal DNA (rDNA) in other organisms.
Given that Pol II transcription in T. brucei seems to be essentially constitutive and regulation is mainly posttranscriptional, it might be expected that, in contrast to the situation in higher eukaryotes, there will be a paucity of histone PTMs and chromatinmodifying and chromatinremodelling enzymes dedicated to repressing Pol II transcription. Consistent with this prediction, T. brucei has a smaller set of histone PTMs10,11 and fewer proteins that write and read the histone code. Therefore, T. brucei is a useful model system in which to study epigenetic phenomena. In this Review, we discuss the current knowledge of the role of epigenetic mechanisms in T. brucei, specifically in the regulation of Pol I transcription, cell differentiation and the cell cycle.
The repertoire of epigenetic regulatorsThe physiological role of chromatin at a particular locus depends on the activity of a set of epigenetic regulators that act at different levels, from nucleosomal positioning and remodelling to writing and reading histone modifications of single amino acids. DNA nucleotide modifications also carry epigenetic information.
Chromatin-remodelling enzymes. Chromatin remodellers are specialized complexes that alter the structure, composition and positioning of nucleosomes and thereby facilitate access of the transcription machinery to nucleosomal DNA12. Remodellers are multiprotein complexes containing a core ATPase that functions as a motor to alter histone–DNA contacts. Most eukaryotes have several classes of remodellers, including the SWI/SNF (switch/sucrose nonfermentable) complex, and the ISWI (imitation SWI), NuRD/MI2/CHD, INo80, SWR1 and RAD54related proteins. These complexes are involved in a wide range of biological functions, including gene expression, splicing, DNA repair or signalling12. SWI/SNF and ISWI are the beststudied remodellers and, in general, SWI/SNF remodellers alter nucleosome positioning to promote transcription factor binding and transcription13, whereas members of the ISWI family primarily mediate transcriptional repression14.
In each class of remodeller, the catalytic subunit has a SNF2like domain and a unique domain that interacts with specific chromatin substrates. SWI/SNF has a bromo domain, CHD has a chromodomain and ISWI has a SANT and/or SlIDE domain15. T. brucei has 13 genes with a putative SNF2 domain (TABLE 2), of which two have been characterized: ISWI16 and jBP2 (base j binding protein 2)17. ISWI has a SlIDE motif,
previously labelled as a MyBlike domain by earlier versions of the 3DjIGSAW database (G. Rudenko, personal communication). jBP2 lacks any recognizable chromatinbinding domain, but sequence homology of the SNF2 domain suggests that jBP2 belongs to the RAD54 class (R. Sabatini, personal communication). As discussed below, both proteins affect the chromatin structure of specialized subtelomeric loci. A second RAD54related enzyme has been recently identified on the basis of sequence homology.
Histone-modifying enzymes. Every eukaryote has a conserved set of enzymes that methylate, acetylate, phosphorylate and ubiquitylate histones. Genome searches suggest that African trypanosomes have a smaller set of histonemodifying enzymes than mammalian cells (TABLE 2). All putative genes were named according to new nomenclature18; previously characterized genes will be referred to using both new and old nomenclature.
Five lysine acetyltransferases (KATs) and seven histone deacetylases (HDACs) have been found in T. brucei19–21. The KATs belong to two classes: three are members of the MyST family (HAT1–3; also known as KAT1–3), which localize in the nucleus20,21, and two are uncharacterized homologues of the human histone acetyltransferase ElP3 (ElP3a (also known as KAT9a) and ElP3b (also known as KAT9b)). T. brucei HDACs share homology with class I (HDAC1–2) or class II (HDAC3–4) histone deactetylases found in yeast and mammals19. SIR2related histone deacetylases are also present in T. brucei. SIR2rp1 possesses histone deacetylase activity and localizes in the nucleus, whereas SIR2rp2 and SIR2rp3 are mitochondrial proteins22,23.
Figure 1 | Chromatin structure. The nucleosomes are the basic units of chromatin. Each nucleosome is composed of approximately 147 bp of DNA wrapped around an octamer of histones (two copies of H2A, H2B, H3 and H4). Histone H1 binds to the DNA that links the two nucleosomes.
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Nature Reviews | Microbiology
NH
O
O
NH3+
Non-modifiedlysine (H3K76me0)
NH
O
O
NH2+H3C
Monomethyl lysine(H3K76me1)
NH
O
O
NH+
CH3
H3C
Dimethyl lysine(H3K76me2)
NH
O
O
N+
CH3
CH3H3C
Trimethyl lysine(H3K76me3)
a
b
KMT KMT KMT
Me
Me
Edman degradationA method developed by P. Edman to sequence peptides by sequential labelling and cleavage of single residues from the amino-terminal end of polypeptides.
In humans and trypanosomes, there are two classes of lysine methyltransferase (KMT) that can add one, two or three methyl groups onto lysine residues: those that contain the SET (su(var)3–9, enhancer of zeste, trithorax) domain and the DoT1 (disruptor of telomeric silencing) homologues. Twenty to twentyseven genes encoding a SET domain can be found in the T. brucei genome, but none has been characterized. Two DoT1 (also known as KMT4) homologues have been described in T. brucei, DoT1A (also known as KMT4A) and DoT1B (also known as KMT4B), the substrate specificity and function of which will be addressed below. Mammals have two types of lysine demethylase: the jumonji family and the lSD1 homologues24. Although lSD1 homologues have not been found in T. brucei, four genes have been identified that encode putative jmjC domaincontaining proteins (jumonji family).
Arginine methylation occurs in various proteins and is controlled by protein arginine methyltransferases (PRMTs)25. In histones, arginine methylation has been associated with both transcriptional activation and repression26. Five candidate PRMTs are present in the T. brucei genome, three of which are homologues of mammalian PRMT1, PRMT5 and PRMT7 (REFS 27–29). Although recombinant forms of the three PRMTs from T. brucei methylate arginines of bovine histones in vitro,
arginine methylation of trypanosome histones has not been detected. Therefore, it remains unclear whether arginine methylation has a role in epigenetic regulation in T. brucei.
Histone modifications. The histone repertoire of T. brucei consists of the four canonical histones (H2A, H2B, H3 and H4) and four histone variants (H2Az, H2Bv, H3v and H4v)30–32. As in other eukaryotes, T. brucei also has divergent H1 linker histones (FIG. 1). Phylogenetic analyses and the structure and function of trypanosome core histones and their variants have been described in detail elsewhere32,33. Although core histones are among the most evolutionarily conserved proteins, there are substantial differences between trypanosome histones and those of higher eukaryotes, especially in the highly modified aminoterminal tails11,34. A survey of T. brucei PTMs using Edman degradation and mass spectrometry revealed some unexpected results: a striking absence of many wellconserved modifications and some unusual and apparently trypanosomespecific PTMs10,11 (FIG. 3). Similar observations were made for histone H4 in a closely related species, Trypanosoma cruzi 35. However, some PTMs are conserved between T. brucei and humans and might represent an ancient basal repertoire of histone modifications. As T. brucei mainly regulates gene expression posttranscriptionally, the functions of the few PTMs that have been described and whether they can be predicted by analogy to higher eukaryotes are important areas of research.
In T. brucei, the first amino acid (alanine) of histone H2A can be either methylated or acetylated, whereas the serine at position 1 in human H2A can be phosphorylated, although the function of these modifications is unknown. Perhaps the most surprising differences between human and T. brucei H2A are the modifications found at the carboxyl terminus, where T. brucei H2A is heavily acetylated in a complex pattern. The function (or functions) of these acetylation marks remains a mystery, but they might contribute to various levels of accessibility of the modified chromatin for protein complexes to promote processes such as transcription, replication or recombination. Homologous modifications to the other human H2A marks have not been identified to date.
of the trypanosome histones, H2B is one of the least conserved, and only four PTMs have been detected: A1 is methylated and K4, K12 and K16 are acetylated. No clear homologues of these PTMs can be found in human H2B, suggesting that these marks have T. bruceispecific functions. Surprisingly, T. brucei H2B lacks one of the most conserved lysine residues, K120, which is ubiquitylated in various organisms. Saccharomyces cer-evisiae H2BK123ub (monoubiquitylated histone H2B on lysine 123) is a wellinvestigated example of PTMs that influence the level of other modifications, socalled PTM crosstalk36, because it is required for the efficient methylation of H3K4 and H3K79 (REFS 37,38). Although there seems to be a homologous lysine in the trypanosome H2B variant (H2BvK129), it was not observed to be ubiquitylated, and mutation of K129 caused no change in phenotype. H2Bv, however, specifically associates
Figure 2 | The histone code hypothesis. a | The amino-terminal histone tails protrude from the core of nucleosomes and undergo multiple post-translational modifications (PTMs), which define the role of chromatin for that genomic locus. The enzymes that write this code are called histone-modifying enzymes. For example, a lysine methyltransferase (KMT; light blue) transfers a methyl group (yellow) from a donor molecule (green) to a lysine in the histone tail. Some PTMs are read by a histone-binding protein (purple), which will subsequently interact with other factors to produce the appropriate biological response. b | Lysines can be mono-, di- or trimethylated by the same or different KMTs. Part b adapted, with permission, from Nature Reviews Molecular Cell Biology REF. 105 (2007) Macmillan Publishers Ltd. All rights reserved.
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EuchromatinA form of chromatin that was first defined as lightly stained nuclear regions by light microscopy and is usually associated with transcriptionally competent chromosome loci.
HeterochromatinA more compact form of chromatin that, when using light or electron microscopy, appears as darker regions of the nucleus. Heterochromatin is usually associated with transcriptionally silent chromosome loci.
with H3 that was enriched in H3K4 and H3K76 trimethylation39, suggesting that trypanosomes regulate H3K4 and H3K76 methylation by incorporating H2Bv into nucleosomes.
In humans, the Nterminal tail of histone H3 has almost two dozen PTMs, many of which are involved in transcriptional regulation. In T. brucei, only a few amino acids of the Nterminal tail of human H3 are conserved. H3K4 is trimethylated in T. brucei and, although H3K4 methylation is a hallmark of transcriptional activation in many organisms, the function of this PTM in T. brucei is unknown. Homologues of wellconserved amino acids, such as K9 and S10, seem to be absent from T. brucei H3, and the only other PTMs detected in the Nterminal tail are acetylation of K23 and trimethylation of K32. on the basis of sequence alignment, the T. brucei homologues of human H3K23, H3K27 and H3K36 are H3K19, H3K23 and H3K32, respectively. Acetylation of H3K27 is associated with transcription in several organisms and is catalysed by GCN5 (also known as KAT2A) in humans40. Human H3K36 can be methylated by SET2 (also known as KMT3) and might be involved in transcriptional elongation of Pol II41. Despite the presence of acetylated H3K23, homologues of GCN5 or SET2 have not been described in trypanosomes, and the function of the corresponding PTMs is unknown. one of the bestcharacterized PTMs in T. brucei is methylation of H3K76, the homologue of H3K79, which is mainly monomethylated or dimethylated in humans. Methylation of H3K79 is mediated by DoT1 methyltransferases and is associated with transcribed chromatin42. Furthermore, DoT1 is involved in meiotic checkpoint control and the DNA damage response43,44. In T. brucei, methylation of H3K76 is mediated by two enzymes: DoT1A and DoT1B, which are exclusively responsible for dimethylation or trimethylation, respectively45. Although the two enzymes and their target seem to be conserved, trypanosomes have developed additional functions for this PTM. one of the main functions of DoT1A and DoT1B may be to influence the trypanosome cell cycle by regulating the level of H3K76 methylation (see below). Additionally, cells in which DoT1B has been depleted have defective VSG switching and differentiation (see below). In addition to these PTMs, mass spectrometry has revealed the presence of other modification states on
the lysinerich Nterminal tail of H3, but pinpointing their localization to individual lysine residues was not possible11.
H4 is the most conserved trypanosome histone and, as for H2A and H2B, H4 is also highly methylated at A1 (REFS 10,11). H4K2 can be either methylated or acetylated, which could possibly have antagonistic functions, as is the case for H3K9me (methylated H3K9) and H3K9ac (acetylated H3K9) in humans46,47. H4K4 of T. brucei is acetylated by HAT3 (also known as KAT3), and might be the homologue of H4K5 in other organisms (the acetylation of H4K5 promotes the incorporation of newly synthesized histones into chromatin48). Although H4K4 is highly acetylated (80–100%), the deletion of HAT3 and the subsequent loss of K4 acetylation, with no compensatory change in the modification status of K5, has no impact on viability21. Another welldescribed and conserved PTM is the acetylation of H4K12, for which the T. brucei homologue may be H4K10. Acetylation of K12 is mediated by different KATs in humans (HAT1 (also known as KAT1), TIP60 (also known as PlIP or KAT5) and HBo1 (also known as MyST2 or KAT7) and is involved in histone deposition, DNA repair and transcriptional activation (reviewed in REF. 49). In T. brucei, H4K10 is acetylated exclusively by HAT2 (also known as KAT2), which is essential for growth. RNA interference (RNAi)mediated depletion of HAT2 results in an accumulation of cells before cytokinesis20. Genomewide studies revealed that this PTM is enriched at probable Pol II transcription start sites, defining an open chromatin structure that may be necessary for initiating transcription50. The fact that trypanosomes use distinct acetyltransferases for H4K4 and H4K10 suggests little redundancy in acetyltransferase activity and supports the idea of a simplified nonredundant histone code. There seems to be a T. brucei homologue (H4K14ac) of human H4K16ac, another euchromatin marker in humans, but neither the corresponding acetyltransferase nor the function of this PTM are known in trypanosomes. There is also a clear T. brucei homologue (H4K18me3) of human H4K20me3. In mammals, H4K20 is dimethy lated or trimethylated by SuV420H1 (also known as KMT5B) and SuV420H2 (also known as KMT5C) and is involved in heterochromatin formation, DNA repair, gene regulation and development51–53. However, a T. brucei KMT that is responsible for H4K18 methylation has not been found. Identification of this enzyme should be straightforward, given the few potential SET domaincontaining proteins in the T. brucei genome (TABLE 2).
Readers of the histone code. The histone code suggests that at least some of the histone PTMs are read by effector molecules and their associated complexes, which define a functional state of the chromatin and eventually trigger a physiological response. Effector proteins can alter the properties of chromatin by interconnecting two or more nucleosomes, by promoting the recruitment of RNA polymerase and transcription factors, by recruiting chromatin remodellers or by promoting downstream chemical modfications54. Readers of the
Box 1 | Genetic tools to study Trypanosoma brucei
T. brucei has several properties that make it amenable to genetic manipulation.• It is easy to culture and clone in liquid medium93 or on agar plates94.
• Homologous recombination is efficient and specific, which allows deletion or epitope tagging of endogenous genes after transfection by electroporation.
• T. brucei has an intrinsic RNAi system that can be exploited for mRNA knockdown95,96.
• Several markers for drug resistance are available for positive and negative selection.
• The ‘cre-lox’ system works efficiently and can be used for various purposes, principally to allow a marker for drug resistance to be removed and reused, or to tag genes without materially affecting upstream or downstream regulatory sequences97.
• Inducible expression can be used to obtain conditional gene knockouts.
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histone code use different domains to recognize and bind to specific PTMs55. Bromodomains bind to acetyl groups and are usually associated with active chromatin. Chromodomains, MBT (malignant brain tumour) fingers and PHD (plant homeo domain) bind to methyl lysine, and Tudor domains recognize methylated arginine or lysine. T. brucei has three classes of putative histone reader: bromodomain, PHD finger domain and Tudor domaincontaining proteins (TABLE 2), none of which has been characterized. unlike many proteins in higher eukaryotes54, all putative T. brucei readers seem to have a single PTMbinding domain. The absence of complex binding properties further supports the idea of a simplified histone code.
DNA modifications. DNA methylation is one of the bestcharacterized epigenetic markers. It occurs in higher eukaryotes, bacteria and archaea but is absent from some organisms, including S. cerevisiae and Caenorhabditis elegans56. DNA methylation plays an important part in the control of gene expression, but it is also involved in the regulation of DNA replication, repair and transposition, and in chromatin packaging57. Although DNA methylation has been observed in T. cruzi 58, for many years it was thought that DNA methylation was absent from T. brucei. Prompted by the discovery of a putative cytosine5 DNA methyltransferase gene, 5methylcytosine was recently identified in nuclear DNA of T. brucei in both the bloodstream and procyclic form59.
The bloodstream form of T. brucei and other related species have a unique DNA modification: βdglucopyranosyloxymethyluracil or base j60,61. This is a bulkier DNA modification than 5methylcytosine (FIG. 4), and it is found predominantly at telomeric repeats and, to a lesser extent, in other repetitive sequences. It also occurs in silent telomeric VSG genes, but the importance of this observation remains unclear61. Two base jbinding proteins (jBP1 and jBP2) were recently shown to be necessary for the synthesis of base j62,63. Because jBP2 requires the SNF2 ATPase domain for its activity, it has been proposed that this potential remodelling domain might recognize distinct chromatin elements and thereby determine the site specificity of base j deposition17. Base j is absent from jBP1 and jBP2 null cells64, but the functional consequences of a lack of base j remain to be determined. By contrast, base j is essential in Leishmania tarentolae65, another member of the Kinetoplastida.
Monoallelic expression of surface proteinsIn most eukaryotes, Pol I exclusively transcribes ribosomal RNA (rRNA) genes. In T. brucei, Pol I also transcribes the genes encoding two major life cyclespecific surface proteins: VSGs and procyclins.
The rRNA gene family in humans contains hundreds to thousands of copies, and their transcription is under epigenetic control; individual genes exist in either an active or silent transcriptional state, and this transcriptional state is inheritable and reversible66. Recent studies have revealed that the transcriptional state of rDNA depends on a complex interplay of DNA and histonemodifying enzymes that act together with chromatinremodelling complexes (reviewed in REF. 66).
T. brucei has eight rRNA genes67 dispersed among four chromosomes, and indirect evidence suggests that they are not all equivalent. When ectopic inducible genes are introduced into the nontranscribed region of T. brucei rDNA, the expression of the inducible gene is tightly regulated in some but not all cell lines68. This variability may reflect different epigenetic states of adjacent rRNA genes. Species of Plasmodium, the eukaryotic parasite that causes malaria, also have a small rRNA gene family (four genes). Remarkably, these genes belong to two distinct classes, the expression of which is regulated in a stagespecific manner69,70. It remains to be seen whether T. brucei rRNA genes also fall into differently regulated categories.
Trypanosomes process RNA transcripts in a fundamentally different way to most other eukaryotes. The mRNA molecule has a 39nucleotide splicedleader capped RNA at the 5′ end, which is transferred from a longer precursor by transsplicing (reviewed in REF. 9). Therefore, capping and mRNA synthesis are uncoupled in trypanosomes, in contrast to most eukaryotes, in which the mRNA capping machinery associates with Pol II71. This feature enables T. brucei to efficiently process Pol I transcripts that encode proteins such as VSGs and procyclins72,73 (TABLE 1).
VSGs are key molecules for antigenic variation, which allows the parasite to escape the mammalian immune
Table 1 | Developmental stages of the Trypanosoma brucei life cycle*
life cycle and parasitic form Surface glycoprotein
gene rNA polymerase
Allelic exclusion?
Bloodstream of mammalian host
Slender (proliferative)
VSG Pol I Yes
Stumpy (non-dividing)
VSG Not applicable
Not applicable
Midgut and salivary gland of insect vector (tsetse)
Procyclic (proliferative)
GPEET, EP procyclins
Pol I No
Epimastigote (proliferative)
BARP Unknown Unknown
Metacyclic (non-dividing)
VSG Pol I Yes
* T. brucei is extracellular in both the bloodstream of its mammalian host and in its transmission vector, the tsetse (Glossina species). Its life cycle alternates between proliferating stages and non-proliferating stages that precede the switch between host and vector. The parasite surface in each stage consists of characteristic glycosylphosphatidylinositol-anchored proteins, the genes of which are mainly transcribed by RNA polymerase I (Pol I). Although transcription is strongly reduced in stumpy forms106, it is unclear whether the variant surface glycoprotein (VSG) detected at the surface originates from weak de novo transcription or if it remains from the previous life cycle stage. For simplicity, the developmental stages in the proventriculus of the insect vector are not included. BARP, alanine-rich protein.
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Antigenic variationThe process by which an infectious organism alters its surface to evade the host immune response; common in several pathogens, such as Trypanosoma brucei, the malaria parasite Plasmodium falciparum and Giardia lamblia.
Monoallelic expressionThe expression of a single allele from a gene family.
system by periodically switching the makeup of its surface coat proteins. Although there are hundreds of VSG genes in the genome, only one is transcribed at any time from a specialized subtelomeric locus called a bloodstream expression site (BES)74. This monoallelic expression occurs in a specialized nonnucleolar structure called the expression site body75,76 (BOX 2). The other BESs are excluded from this compartment, which may be necessary to keep them silent. Allelic exclusion is also used for regulating the expression of VAR genes in the malaria parasite and for regulating the odorant receptor (OR) gene family in olfactory sensory neurons77,78. Therefore, understanding the mechanism of monoallelic expression would be a fundamental achievement not only in the trypanosome field, but also for research into other infectious diseases and neurobiology.
Transcription of the VSG gene family needs to be tightly controlled, not only to achieve monoallelic expression, but also to ensure developmental regulation during the life cycle of the parasite (TABLE 1). So far, only one Pol I trans cription complex has been identified, and it is essential for both rRNA and VSG transcription79. VSG transcriptional control is inherited from one generation to the next, and VSG switching
does not necessarily involve DNA rearrangements, suggesting that it is regulated epigenetically. As in higher eukaryotes, histone PTMs are thought to carry the epigenetic information necessary for VSG regulation. Genome database searches in T. brucei have led to the identification of candidate epigenetic factors that might have a role in the regulation of Pol I transcription. Some of them belong to the chromatinremodelling or histonemodifying enzyme families (TABLE 2).
ISWI is an essential gene that encodes the only ISWI family member of chromatin remodellers found in T. brucei 16. The T. brucei ISWI has an Nterminal ATPase domain (although it is not known whether this domain is important for the function of ISWI) and a SlIDE domain at the C terminus, which is thought to be involved in the interaction with DNA and histone tails80. ISWI has been shown to have a repressive role in other systems, and its RNAimediated depletion in T. brucei leads to a derepression of the promoterproximal region of a silent BES. Derepression ranges from 30fold to 60fold in the bloodstream form and from 10fold to 17fold in the procyclic form, suggesting that BES silencing requires ISWI in both stages of the life cycle.
Table 2 | Chromatin-remodelling enzymes, histone-modifying and histone-binding proteins*
Class Name Number of putative homologues in humans‡
Chromatin-remodelling families
ISWI ISWI15 Pfam, 2
RAD54 relative JBP2 (REF. 14) and a RAD54 homologue
Pfam, 0; SMART, 0
Other SNF2 domain-containing homologues
Ten candidate genes Pfam, 91
Histone-modifying enzymes
Lysine acetyltransferase (KAT) HAT1–3 (MYST family)20,21 Pfam, 14 of MYST family; (5 characterized)
Elp3a–b (1 characterized)
Lysine deacetylase (HDAC) HDAC1–4 (REF. 19) Pfam, 47; (11 characterized)
SIR2rp1–3 (REFS 22,23) Pfam, 11; (7 characterized)
Lysine methyltransferase (KMT) DOT1A–B45,83 Pfam, 1
20–27 SET domain-containing proteins (KMTs)
Pfam, 99; SMART, 62
Lysine demethylases (KDM) KDM1–4 (JmjC domain) Pfam, 53; SMART, 38
Arginine methyltransferases PRMT1 (REF.27), PRMT5 (REF.28) and PRMT7 (REF.28) and two other candidate genes
(9 highly related members)
Histone-binding proteins
Bromodomain BDF1–4 Pfam, 132; SMART, 82
PHD finger PHD1–5 Pfam, 214; SMART, 132
Tudor TDR1 Pfam, 35; SMART, 28
*List of candidate chromatin-remodelling enzymes, histone-modifying and histone-binding proteins found in the Trypanosoma brucei and human genomes or predicted by Pfam or SMART databases. ‡The number of characterized human homologues is indicated in brackets. DOT1, disruptor of telomeric silencing protein 1; Elp3, elongator protein 3; HDAC, histone deacetylase; JBP2, J base binding protein 2; JmjC, Jumonji C; ISWI, imitation SWI (switching deficient); MYST, human MOZ (monocytic leukaemia zinc finger protein), yeast Ybf2 (renamed Sas3; something about silencing 3), yeast Sas2 and mammalian TIP60 (HIV Tat-interacting protein 60 kDa); PHD, plant homeo domain; PRMT, protein arginine methyltransferase; RAD54, radiation sensitive 54; SET, su(var)3–9, enhancer of zeste, trithorax; SIR2rp1–3, silencing information regulator 2-related proteins (also known as sirtuins) 1–3; SNF2, sucrose non-fermenting 2.
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Human H2A
T. brucei H2A 1
1
4
4
4 9 23 27 36
3219 23
**
**
*
5
* *
K115
K119
K120
K122
K125
K128
134
129
125
112
136
132
102
99
120
K120
41
***
12 16
Human H2B
T. brucei H2B
Human H3
T. bruceiH3
Human H4
T. bruceiH4
α1 α2 α3
α1 α2 α3
α1 α2
α2
α3
α1
α1
α1
α2
α2
α3
α3
α3
α1 α2 α3
α1 α2 α3
K79
K76
5 12 16 20
181714105421
Acetylation
Methylation
Phosphorylation
Ubiquitylation
Triton acid urea gel electrophoresisA polyacrylamide gel-based electrophoresis technique that allows the efficient separation of core histones as a result of their association with Triton X-100.
Among the ten histonemodifying enzymes that have been characterized so far in T. brucei (HAT1–3, HDAC1–4, SIR2rp1, DoT1A and DoT1B), only a subset has been tested for a potential role in Pol I regulation in the bloodstream form. HAT1, SIR2rp1 and DoT1B are all involved in either telomeric silencing and/or VSG trans cription. The depletion of HAT1 by RNAi results in the derepression of a telomeric reporter promoter, but does not affect transcription from a BES promoter20. SIR2rp1 is a histone deacetylase that is involved in maintaining subtelomeric regions that are silent in S. cerevisiae81 and in the malaria parasite Plasmodium falciparum82. In SIR2rp1 mutants of T. brucei, although a Pol I reporter construct becomes derepressed in both the bloodstream and pro cyclic stages of the life cycle, VSG genes are not derepressed. Moreover, the frequency of transcriptional switching between two BESs is not altered, suggesting that antigenic variation is not dramatically affected by the absence of SIR2rp1 (REF. 22).
The only histonemodifying enzyme that is clearly involved in VSG transcriptional regulation and BES switching is DoT1B, which is responsible for trimethylation of H3K76 (REF. 83). Deletion of this gene results in a tenfold derepression of various silent VSG genes. DoT1B is also necessary for rapid transcriptional switching between two BESs. In a DoT1B null mutant, switching between two BESs occurred more slowly than in wildtype cells, and stable intermediates expressing two VSGs simultaneously at the surface could be detected. In these intermediates, the two BESs are transcribed from the promoter through the subtelomeric VSG, but only one BES is transcribed at the normal level, suggesting that other epigenetic factors are responsible for ensuring that only one BES is maximally transcribed. Despite the clear reliance on
DoT1B, the precise mechanistic role of H3K76me3 in VSG regulation remains unclear.
The proximity of BES to telomeres suggests that telomeric chromatin is involved in VSG regulation. However, the first two telomerebinding proteins described in T. brucei (H3 variant30 and TRF84) did not reveal a role in VSG transcription. By contrast, depletion of RAP1, a TRFinteracting protein, resulted in an approximately 100fold derepression of silent VSGs85. Moreover, genes located close to the telomeres were found to be more derepressed than genes located further upstream, suggesting that telomere structure is essential for silencing. Even though mechanistic details are lacking, it is clear that telomeric proteins play an important part in the regulation of surface protein expression. Future studies should identify other factors, which will help us to understand how this regulation is achieved.
Cell differentiationDifferentiation of mammalian cells is accompanied by substantial alterations of chromatin structure (reviewed in REF. 86). Is there any evidence that chromatin structure is involved in regulating gene expression during the trypanosome life cycle? Studies during the early 1990s that compared histones from two T. brucei life cycle stages using Triton acid urea gel electrophoresis revealed differences in the number and pattern of bands of the core histones and linker histone H1, suggesting stagespecific PTM differences87,88. Studies of chromatin condensation in vitro showed that the level of condensation was higher in the bloodstream than in the procyclic form87. These observations were confirmed by electron microscopy, which revealed that the electrondense (heterochromatic) regions in the nucleus of the bloodstream form are large and mostly associated with the envelope, whereas nuclei in the procyclic form contain a
Figure 3 | Sequence alignment and histone modifications of Trypanosoma brucei and human core histones. Important regions of homology are shaded by grey boxes. Residues that might be modified are marked with circles, colour-coded as indicated. Modifications that were found only in the bloodstream form are indicated by asterisks. The three helices of the histone core are shown as coloured boxes. The post-translational modification (PTM) inventory of human histones is not complete. Only abundant or well-characterized PTMs are shown.
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N
NH2
ON
O
O
O
NH
O
ON
O
O
O
O
OHO
HO OH
HO
5-methylcytosine Base J
KaryokinesisThe process that partitions of the nucleus into the daughter cells during cell division.
higher number of small heterochromatin areas that are scattered throughout the nucleus89.
Is there any evidence that histone PTMs are responsible for these stagespecific differences in chromatin structure, and do they have a role in differentiation? There are few differences in the inventory of histone PTMs between the bloodstream and procyclic forms of T. brucei 10,11 and, although abundant PTMs are present in similar amounts, there are few interesting differences. For example, H4K18me3 seems to be enriched almost fourfold in the procyclic form. As H4K18me3 may be the homologue of the mammalian heterochromatin marker H4K20me3, it might be interesting to explore a possible role for H4K18 methylation in stagedependent heterochromatin formation in trypanosomes. other PTMs that were found in the bloodstream form but not in the procyclic form were acetylation of H2A (K4 and K122), H2B (K4, K12 and K16), H3 (K23) and H4 (K2, K5 and K14) and methylation of H3K32 and H4K2. However, these variations might be attributable to technical differences between the studies of the bloodstream and procyclic forms. For example, different protocols needed to be used to isolate and purify histones from different life cycle stages and different mass
spectrometry techniques were applied to analyse PTMs.DoT1B is the only histonemodifying enzyme that
has been studied in the context of differentiation from the bloodstream form to the procyclic form in vitro45. When wildtype cells from the bloodstream form are exposed to differentiation signals in vitro, they differentiate to the procyclic form and grow normally. By contrast, DoT1Bdeficient cells died several days after attempting differentiation. Cell cycle profiles of ∆DoT1B cells, determined by flow cytometry, were indistinguishable from those of proliferating wildtype cells, suggesting that growth was not arrested at a specific phase of the cell cycle. A cell cycle phenotype during differentiation is of special interest because only G1arrested trypanosomes can enter the differentiation process90. Therefore, there seems to be an unknown connection between cell cycle control and commitment to differentiation. For reasons of tractability, however, these experiments were performed in a T. brucei strain, for which the differentiation kinetics differ from the synchronous transition that occurs in less laboratoryadapted strains. Experiments to unravel the role of chromatin structure during developmental differentiation of T. brucei are still in the early stages, and it remains to be seen what can be learnt from differentiation processes in other eukaryotic cells.
Cell cycle controlCell cycle control in trypanosomes is characterized by several unusual features. Mitosis is closed, meaning that the nuclear envelope persists during all stages of the cell cycle, and the chromosomes do not condense91. Although many orthologues of conserved cell cycle regulators are identifiable in the T. brucei genome, some key enzymes appear to be missing and trypanosomes seem to use different checkpoints from those of other model eukaryotes (reviewed in REF. 92). What is known about the role of PTMs in cell cycle control? Genetic manipulations of several histonemodifying enzymes result in cell cycle defects in trypanosomes. For example, HDAC4 is required for normal cell cycle progression19. In both bloodstream and procyclic forms, HDAC4depleted cells have a mild growth defect that is caused by a delay in the G2 to Mphase transition. By contrast, cell cycle analysis of cells with depleted HAT1 and HAT2 showed an increase in the number of postmitotic cells20. It is not yet known how different acetylation levels influence cell cycle progression. The most striking effects on cell cycle regulation were obtained by depleting DoT1A and DoT1B45. Dimethylated H3K76 can be detected only during mitosis and karyokinesis, whereas trimethylated H3K76 is present throughout the cell cycle. Deletion of DoT1B leads to cell cycleindependent dimethylation of H3K76, suggesting that the mitosisspecific regulation of H3K76me2 is mediated by the presence or absence of DoT1B. Depletion of DoT1A by RNAi generates a population of cells that contain half the normal (diploid) amount of DNA, suggesting that H3K76me2 has an important role in the regulation of cell cycle progression. The exact mechanism by which PTMs regulate cell cycle progression in trypanosomes remains to be determined. Better understanding of the regulation of
Figure 4 | Structure of the DNA modifications in Trypanosoma brucei. The genomes of both bloodstream and procyclic forms of T. brucei contain 5-methylcytosine.At least 1 in every 10,000 cytosines is methylated. Base J, (β-d-glucopyranosyloxymethyluracil) is found only in the bloodstream form, but its absence is not lethal. Two proteins (JBP1 and JBP2) have been found that bound to base J in the context of DNA, and either catalysed or modulated the synthesis of base J.
Box 2 | Monoallelic expression: a gene regulation dilemma
Some multigene families need to be tightly regulated because only one allele must be expressed at a time. The best-studied examples are the VSG gene family in Trypanosoma brucei98, the VAR gene family in the human malaria parasite Plasmodium falciparum99 and the odorant receptor (OR) gene family in the mammalian olfactory sensory neurons100. To achieve monoallelic expression, cells must successfully perform several steps: choose which member is going to be activated, count the number of actively transcribed genes and, perhaps, retain some form of cellular memory to remember which members were recently activated. We still know very little about the molecular machineries that mediate these processes, although recent data raised testable working hypotheses: one model suggests that the active allele locates to a unique subcompartment in the nucleus75,101; a second model proposes that chromatin plays a part in maintaining the epigenetic state of each member of the family16,83,102; a third model proposes that cis-regulatory elements within members of the gene family may determine which allele is activated103; and a fourth model suggests a role for cis-linked promoter activity in maintaining all except one allele silent104.
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histonemodifying enzymes and identification of PTMbinding proteins should help to unravel fundamental biological processes.
ConclusionIn the past few years, efforts to find and characterize histone PTMs and the corresponding chromatin modifying enzymes have been initiated in trypanosomes. It is already apparent that trypanosomes have a smaller enzymatic repertoire than higher eukaryotes and, presumably, a less complicated histone code. Considering that trypanosomes diverged several hundred million years ago from the main eukaryotic lineage, better understanding of T. brucei epigenetic regulation could help to answer three basic questions in the chromatin field: what does a simple histone code look like, what are the minimum essential functions of chromatin and what drives histonemodifying
enzymes to acquire new chromatinrelated functions? Answering these questions will help us to understand the evolution of the histone code and its functions. Because Pol II transcription in trypanosomes seems to be constitutive, there are probably few PTMs that are dedicated to Pol II silencing. T. brucei is therefore a potentially useful model organism in which to study the PTMs that regulate only Pol I transcription and other evolutionarily conserved chromatinmediated processes. Future studies have to focus on the dynamic aspects of PTMs and chromatin structure in trypanosomes. For example, how is the deposition and removal of these marks and the activity of histonemodifying enzymes regulated? Furthermore, PTMbinding proteins must be identified to unravel how different histone modifications mediate important biological processes such as transcriptional regulation and cell cycle control in this fascinating organism.
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AcknowledgementsThe authors thank S. Hake and N. Siegel for their critical read-ing of the manuscript; G. Rudenko and R. Sabatini for helpful comments; and P. Bastin and B. Rotureau for providing micro-graphs of the different stages of the T. brucei life cycle, which were used, in part, to draw the cartoons in Table 1.
DATABASESEntrez Genome Project: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeprjCaenorhabditis elegans | Saccharomyces cerevisiae | Trypanosoma bruceiUniProtKB: http://www.uniprot.orgELP3 | GCN5 | H2BK123ub | HAT1 | HAT2 | HBO1 | JBP1 | PRMT1 | PRMT5 | PRMT7 | RAD54 | SUV420H1 | SUV420H2 | TIP60 | TRF
FURTHER INFORMATIONGeorge A. M. Cross’s homepage: http://tryps.rockefeller.eduPfam: http://pfam.sanger.ac.ukPlasmoDB: http://plasmodb.org/plasmoSmart: http://smart.embl-heidelberg.deTriTrypDB: http://tritrypdb.org/tritrypdb
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