nuclear gene transcription and chromatin in trypanosoma brucei

Invited review

Nuclear gene transcription and chromatin in Trypanosoma brucei

David Horn*

London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK

Received 2 February 2001; received in revised form 13 February 2001; accepted 25 April 2001

Abstract

As in other eucaryotes, the nuclear genome in Trypanosoma brucei is organised into silent domains and active domains transcribed by

distinct RNA polymerases. The basic mechanisms underlying eucaryotic gene transcription are conserved between humans and yeast, and

understood in some detail in these cells. Meanwhile, relatively little is known about the transcription machinery, the chromatin templates or

their interactions in trypanosomatids. Here, I discuss and compare nuclear gene transcription in T. brucei with transcription in other

eucaryotes focusing in particular on mono-allelic transcription of genes that encode the variant surface glycoproteins. q 2001 Australian

Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Transcription; Trypanosoma brucei; VSG

1. Introduction

Trypanosoma brucei are mono-¯agellated parasitic proto-

zoa. These African trypanosomes cause diseases known as

sleeping sickness in humans and Nagana in domestic live-

stock. They are spread among mammalian hosts by tsetse

¯ies of the genus Glossina, the limited distribution of which

actually prevents T. brucei from spreading beyond Africa. T.

brucei passes through several dividing and non-dividing

stages during its life cycle.

The molecules involved in eucaryotic transcription

include the DNA templates, the RNA polymerases, general

transcription factors, the histones and additional regulatory

molecules (Kornberg, 1999). These molecules and their

interactions are conserved between humans and yeast.

Trypanosoma brucei and several other trypanosomatids

are parasites of signi®cant medical, veterinary and scienti®c

importance but relatively little is known about the transcrip-

tion machinery or the chromatin templates in these diver-

gent protozoa. See Ersfeld et al. (1999) and Belli (2000) for

recent reviews dealing with nuclear organisation in T.

brucei and chromatin remodelling in trypanosomatids,

respectively.

Genome sequencing is underway for T. brucei (El-Sayed

et al., 2000) as well as for the other pathogenic trypanoso-

matids, Trypanosoma cruzi and Leishmania major. The T.

brucei genome size is ,3.5 £ 107 bp with 11 homologous

chromosome pairs and additional intermediate and minichro-

mosomes of uncertain ploidy. Sequence comparisons

between known rRNAs (Sogin et al., 1989), histones

(Thatcher and Gorovsky, 1994) and RNA polymerases

(Jess et al., 1990) suggest that trypanosomes diverged early

from the eucaryotic lineage and several novel molecular and

biochemical features of trypanosomatids also appear to

re¯ect divergence (Donelson et al., 1999). In trypanosoma-

tids almost all protein-coding genes appear to be transcribed

as polycistrons and trans-splicing leads to the addition of a

common sequence to the 5 0 end of every mRNA. Conse-

quently most genes appear to be transcribed constitutively

suggesting the need for signi®cant regulation of mRNA and/

or protein abundance at the post-transcriptional level.

Transcription of genes encoding abundant surface

proteins in T. brucei, the variant surface glycoproteins

(VSGs) and the repetitive EP/GPEET procyclins, differs

from that of most other protein-coding genes in at least

two important ways. Firstly, they are transcribed in a

stage-speci®c manner. The VSG is expressed during the

bloodstream stage of the life cycle and the EP/GPEET

procyclins in the insect midgut stage. Second, VSG and

EP/GPEET genes are almost certainly transcribed by

RNA polymerase I (Lee and Van der Ploeg, 1997), a poly-

merase con®ned to rRNA transcription in other eucaryotes.

In each cell, of the 1000 or so VSG genes, about 20 are

localised in polycistronic, telomeric transcription units

that contain several other `expression site associated

genes' encoding various cell-surface proteins (Pays and

Nolan, 1998). However, gene organisation does not appear

International Journal for Parasitology 31 (2001) 1157±1165

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(01)00264-8

www.parasitology-online.com

* Tel.: 144-20-7927-2352; fax: 144-20-7636-8739.

E-mail address: [email protected] (D. Horn).

to be optimal as the telomere-proximal VSG, which gener-

ates the cell's most abundant protein, is furthest from the

promoter. The telomeric VSGs are expressed in a mutually

exclusive manner. Low frequency switches in VSG expres-

sion via VSG gene rearrangements or via switching tran-

scription between telomeres allow this extracellular

parasite to undergo antigenic variation such that some T.

brucei cells continually escape host immune responses

(Rudenko et al., 1998). Thus, determination of the molecu-

lar mechanisms and machinery regulating VSG gene expres-

sion remain major goals in the ®eld of the molecular biology

of T. brucei. In addition to the bloodstream VSGs, ,20

telomeric VSGs can be expressed in the insect vector sali-

vary gland and for a few days following mammalian infec-

tion but these `metacyclic' VSGs are in monocistronic

transcription units. The minichromosomes serve as reposi-

tories of additional telomeric VSG genes.

2. RNA polymerases, promoters and terminators

In most eucaryotes, RNA polymerase I is responsible for

the generation of rRNA, RNA polymerase II for mRNA and

small nuclear RNAs and RNA polymerase III for transfer

RNA and other small RNAs. A similar situation appears to

operate in trypanosomes with the major exception that RNA

polymerase I generates some mRNAs (see above). The

genes for the largest sub-units of all three T. brucei RNA

polymerases (Jess et al., 1990) and promoters that recruit

each of them have now been identi®ed (see below). In other

eucaryotes, phosphorylation of the RNA polymerase II C-

terminal domain occurs prior to transcription elongation and

the C-terminal domain couples transcription to mRNA

capping, splicing and polyadenylation (Proudfoot, 2000).

Despite the absence of a typical C-terminal domain on T.

brucei RNA polymerase II (Evers et al., 1989) this protein is

found to be phosphorylated when isolated from elongating

transcription complexes (Chapman and Agabian, 1994) but

a link to mRNA processing has yet to be established.

Unusually, T. brucei has two loci encoding similar RNA

polymerase II genes but the signi®cance is unclear since

disruption of one of these genes has no detectable effect

(Chung et al., 1993).

In other organisms, promoters are composed of core and

regulatory regions. The core includes a sequence ,25 bp

upstream from the start site that is recognised by TATA-

box-binding protein while positive or negative regulatory

sequences can be located nearby or at great distances.

Elements important for the function of the various T. brucei

promoters are illustrated in Fig. 1.

In addition to rRNA promoters, there are three additional

promoters that recruit RNA polymerase I in T. brucei

(Vanhamme and Pays, 1995), the VSG expression site,

metacyclic VSG and EP/GPEET promoters. These RNA

polymerase I promoters have no obvious sequence similar-

ity but there are some common structural features (Fig. 1)

and they all display a similar level of activity in transient

transfection assays (Kim and Donelson, 1997; Vanhamme

and Pays, 1995). In these assays, a region of ,70 bp is

suf®cient for fully active VSG expression site and metacyc-

lic VSG promoters, whereas an equivalent activity from the

EP/GPEET and rRNA promoters requires an additional

upstream control element(s). However, a hybrid VSG

expression site/rRNA promoter does remain functional

(Vanhamme et al., 1995). Thus, the combined `core'

elements from VSG promoters and from these other promo-

ters are not functionally equivalent but the elements prox-

imal to the initiation site may recruit the same factors.

Assays with hybrid promoters con®rm the activity of the

upstream control elements and indicate that EP/GPEET

and rRNA promoter upstream control elements are inter-

changeable (Janz and Clayton, 1994). Previously, it was

unclear whether the putative metacyclic VSG promoter

identi®ed in bloodstream trypanosomes (Kim and Donelson,

1997) could function in metacyclic trypanosomes (Graham

et al., 1999). This now seems likely since 5 0 RACE analysis

indicates that transcription initiation sites for metacyclic

VSGs in tsetse salivary gland-derived cells coincide with

those seen in bloodstream-derived cells (J.D. Barry et al.,

personal communication).

The spliced-leader RNA is encoded by ,100 genes

arranged in tandem arrays. These genes are transcribed by

RNA polymerase II (Gilinger and Bellofatto, 2001) and they

provide the 39-nt fragment that is trans-spliced to the 5 0 end

of every mRNA. The spliced-leader RNA gene promoter

(GuÈnzl et al., 1997) is illustrated in Fig. 1. Although no

other con®rmed RNA polymerase II promoter has been

identi®ed in trypanosomatids, RNA polymerase II transcrip-

tion as measured by UV treatment followed by nuclear run-

on does re¯ect the pattern of gene organisation on L. major

chromosome 1 (P.J. Myler et al., personal communication).

This chromosome appears to contain two large divergent

polycistronic transcription units with 29 genes encoded on

one DNA strand and the remaining 50 genes encoded on the

opposite strand (Myler et al., 1999). Thus, these experi-

ments provide tentative evidence that transcription initiates

at the single `strand in¯ection point' on this chromosome

and proceeds bi-directionally towards each telomere. It will

certainly be interesting to see how these results relate to the

situation in T. brucei and T. cruzi and to see whether the

putative promoter fragments can function on plasmids or at

other chromosomal loci.

A number of genes transcribed by RNA polymerase III

have been characterised in trypanosomatids including those

encoding the U2, U3 and U6 snRNA and the 7SL RNA. The

promoters for these genes require extragenic A and B blocks

that are the intragenic control regions for a divergently

oriented tRNA gene. The tRNAThr/U6 snRNA transcription

unit (Nakaar et al., 1997) is illustrated in Fig. 1.

Some T. brucei promoter elements, such as the upstream

control element of the rRNA promoter (Laufer et al., 1999),

domain IV of the EP/GPEET promoter (Laufer and GuÈnzl,

D. Horn / International Journal for Parasitology 31 (2001) 1157±11651158

2001), some portions of the spliced-leader RNA promoter

(GuÈnzl et al., 1997) and the B block of the U6 snRNA gene

(Nakaar et al., 1997) have been shown to be more important

for transcription activity in vivo than in vitro. This suggests

that these elements (Fig. 1) are speci®cally involved in

chromatin organisation or the recruitment of factors in a

chromosomal context.

Due to the relative lack of clearly de®ned transcription

units in trypanosomatids transcription termination has not

been investigated at many loci. Transcription termination

speci®c for RNA polymerase I occurs within the terminal

2 kbp of a ,10-kbp EP/GPEET transcription unit (Berberof

et al., 1996). DNA fragments from this region also terminate

transcription from VSG expression site and rRNA promoters

in an orientation dependent manner and the sequence

involved appears to consist of several attenuator elements.

A 9-bp motif within these elements may function in attenua-

tion by recruiting a protein that also binds promoters and

telomeres (Berberof et al., 2000). RNA polymerase II tran-

scription is terminated at spliced-leader RNA loci by a string

of .six T bases at the 3 0 end of the gene (Sturm et al., 1999).

3. General transcription factors

Synthesis of mRNA requires the combined activities of a

large number of polypeptides. In most eucaryotes, TATA-

box-binding protein is central to complex assembly at core

promoter elements prior to transcription initiation by all three

eucaryotic RNA polymerases (Green, 2000). Although tran-

D. Horn / International Journal for Parasitology 31 (2001) 1157±1165 1159

Fig. 1. T. brucei promoters. Schematic diagram of the RNA polymerase I promoters; VSG expression site (ES), metacyclic (M) VSG, EP/GPEET and rRNA, an

RNA polymerase II promoter for the spliced-leader (SL) RNA and an RNA polymerase III promoter for the tRNAThr/U6 snRNA. Transcription start-sites are

indicated as arrows while boxes indicate elements shown to be important for promoter activity in vitro and/or in vivo. Grey boxes represent regions that appear

to be speci®cally important in vivo (see text). A longer box does not necessarily indicate a larger element, but rather that ®ne mapping within that region has not

been done. The structure of the M VSG promoter was determined by mutagenesis (Kim and Donelson, 1997) but this has not been veri®ed by linker-scanning

analysis. Some promoter elements are labelled and the positions of the VSG promoters relative to telomeres (end) are indicated (see text).

scription can occur in the absence of TATA-box-binding

protein, this protein has an ancient evolutionary origin even

appearing in the archea. Neither TATA-box-binding protein

nor general transcription factors that are well conserved

between human and yeast appear to have orthologues in

trypanosomatids suggesting that these factors are either

poorly conserved or have been replaced by a distinct

complex.

Using a combination of in vitro activity assays, gel mobi-

lity shift assays and footprinting assays with extracts from

trypanosomatids including Leptomonas spp., speci®c bind-

ing activities have been characterised for the RNA polymer-

ase I promoters (see Laufer and GuÈnzl, 2001), the spliced-

leader RNA RNA polymerase II promoter (see Gilinger and

Bellofatto, 2001) and RNA polymerase III (Bell and Barry,

1995) promoters. Results regarding the functional signi®-

cance of factors that bind to T. brucei RNA polymerase I

promoters appear to be rather controversial. For example,

factors bind double-stranded DNA from the VSG expression

site promoter (Pham et al., 1997) and a common factor binds

single-stranded DNA from the VSG expression site promoter,

telomeric repeats and the EP/GPEET terminator (Berberof et

al., 2000). However, transcription from the VSG expression

site promoter appears to require only factors that bind

double-stranded DNA (Laufer and GuÈnzl, 2001). The VSG

expression site, EP/GPEET and rRNA promoters recruit at

least one common factor and the rRNA and spliced-leader

RNA promoters, which recruit different RNA polymerases,

also appear to recruit a common factor (Laufer and GuÈnzl,

2001). This second factor does not bind the VSG expression

site or EP/GPEET promoters. In some cases it appears that

promoter elements co-operate in factor recruitment since

altered spacing of the elements disrupts factor binding. In

addition, a trans-activating transcription factor speci®c for

the EP/GPEET promoter appears to be developmentally

regulated, expressed only at the insect midgut stage (Laufer

et al., 1999).

4. The histones

Chromosomal DNA is folded with nucleosomes that

contain two molecules of each core histone, H2A, H2B,

H3 and H4. In other eucaryotes, nucleosomes repress tran-

scription in vitro and in vivo whereas transcription factors

counteract nucleosome-mediated repression. Chromatin

structure is regulated by covalent modi®cation of the

DNA or the histones or by a variety of other factors. For

example, histone N-terminal tails that extend beyond the

nucleosome core can be acetylated, phosphorylated, ubiqui-

tinated, methylated or ADP ribosylated. TATA-box-binding

protein associated factor (TAF) II250 alone can catalyse the

®rst three of these modi®cations (Mizzen and Allis, 2000)

resulting in altered chromatin structure and transcription.

T. brucei core histone sequences (Fig. 2) were identi®ed

by combining partial peptide sequences (Bender et al.,

1992) with expressed sequence tags (El-Sayed et al.,

1995). Although trypanosomatid histones (Galanti et al.,

1998) are among the most divergent known (Thatcher and

Gorovsky, 1994), the level of sequence conservation

suggests that the overall structure of the T. brucei nucleo-

some is probably similar to that in other eucaryotes. Thus,

lysine residues, substrates for acetylation or methylation

(Strahl and Allis, 2000), appear to be conserved within the

N-terminal tails of the core histones (Fig. 2). Phosphoryla-

tion of serine 10 in histone H3 is involved in chromosome

condensation (Wei et al., 1999) in other eucaryotes. The

absence of this residue in the T. brucei histone may be

functionally signi®cant since chromosomes do not condense

during mitosis in trypanosomatids. Genes encoding histone

`variants' that may in¯uence chromatin structure, organisa-

tion or metabolism are also present in the T. brucei genome

but the function of these proteins is currently unknown.

Linker histones contribute to higher order chromatin

structure by binding the linker DNA ¯anking the nucleo-

some core. Although the linker histone H1 is non-essential


Fig. 2. T. brucei core histones. Predicted protein sequences of T. brucei (Tb) and S. cerevisiae (Sc) core histones were aligned using ClustalW followed by

manual adjustment. Identical residues are white on a black background and dashes indicate gaps introduced to optimise the alignment. Since lysine (K) residues

within the N-terminal tails of histones H3 and H4 are well-characterised substrates for covalent modi®cation (see text), these tails have been speci®cally

adjusted to optimise lysine alignments. Possible substrates for acetylation are indicated (*) on T. brucei histones H3 and H4.

in Tetrahymena, Xenopus or yeast (Patterton et al., 1998) it

does appear to regulate transcription in these organisms

(Wolffe, 1997). For example, histone H1 regulates core

histone acetylation (Gunjan et al., 2001) and in Tetrahy-

mena, phosphorylation of histone H1 affects transcription

by mimicking H1 removal (Dou et al., 1999).

T. brucei histone H1 sequence was identi®ed by compar-

ing peptide sequences (Burri et al., 1995) with expressed

sequence tags (El-Sayed et al., 1995). Like other protozoa,

T. brucei has atypical linker histones lacking a globular

domain. Histone H1 from T. brucei appears to have a rather

simple, repetitive sequence composed almost entirely of ®ve

amino acid repeats, (X3K2)11±14 where X is a neutral amino

acid (Ala, Val, Pro or occasionally Leu or Gly) and K is

lysine. Similar to the situation in other eucaryotes, H1 phos-

phorylation in T. brucei may be inversely related to chromo-

some compaction (Burri et al., 1995).

5. Histone acetylation and chromatin structure

The link between transcription and acetylation of the 1-

amino group of speci®c lysine residues in the N-terminal tails

of the core histones is well-documented (Strahl and Allis,

2000) and conserved from protozoa to mammals. In general,

histone acetylation increases the rate of transcription so

histone acetyltransferases (Brown et al., 2000) and histone

deacetylases (Ng and Bird, 2000) activate and repress tran-

scription, respectively. These enzymes have no observable

preference for a speci®c DNA sequence environment so

targeting is via association with DNA-binding transcription

factors within large multi-component complexes. A protein

module known as a bromodomain binds to acetylated lysines

on histones H3 and H4 and appears to provide a direct link to

transcription activation (Jacobson et al., 2000) as illustrated

by TAFII250 which has two bromodomains. In yeast, acety-

lation of lysine residues 9, 14, 18 and 23 in histone H3 and 5,

8, 12 and 16 in histone H4, can in¯uence transcription

(Waterborg, 2000). The corresponding lysines in T. brucei

may be residues 10, 16, 19 and 23 in histone H3 and 2, 5, 10

and 14 in histone H4 (Fig. 2).

There are several deacetylases, acetyltransferases and

bromodomain proteins that clearly target histones in

human cells and in the yeast Saccharomyces cerevisiae.

Searches of the T. brucei genome database reveals genes

encoding proteins related to all three of these classes

(Table 1). All three classes of protein also have orthologues

in T. cruzi and L. major. There is ample evidence that such

proteins act in transcription regulation in humans and in

yeast so their function in trypanosomatids merits investiga-

tion. It should also be noted that histone acetylation has

consequences beyond transcription such as in chromatin

assembly and DNA recombination and some of these

enzymes can also modify substrates other than histones.

Therefore, these proteins may function beyond the context

discussed here.

6. Regulation of VSG gene transcription in T. brucei

Although T. brucei has ,1000 VSGs, it is generally

accepted that only one telomeric VSG is expressed by

each bloodstream-form cell and that essentially no VSG is

expressed in cells in the insect midgut. Some mechanism


Table 1

Possible components of the T. brucei histone acetylation/recognition machinery

T. brucei accession no. Peptide lengtha Orthologous sequencesb

Human Accession no. Expectation score Yeast Accession no. Expectation score

SIR2-like deacetylases

AF102869 351 SIR2-like 3 NP_036371 6250 HST2 P53686 1237

AL486573 273 SIR2-like 4 NP_036372 1234 HST3 P53687 2210

AC079933 244 SIR2-like 5 NP_036373 4236

Class I/II deacetylases

AQ650462 388 HDAC1 Q13547 7274 RPD3 P32561 3271

AQ953308 223c HDAC1 Q13547 6210 HOS2 P53096 428

AQ647789 586 HDAC6 Q9UBN7 5231 HDA1 P53973 3230

AC008031 685 HDAC7 AAF63491 9224 HDA1 P53973 1218

Acetyltransferases

AQ643368 466 TIP60 AAH00166 1250 ESA1 Q08649 4254

AQ649641 604 MOF AAF72665 1222 ESA1 Q08649 2226

Bromodomain factor

AQ640513 188c BRD4d Q15059 426

a A single accession number is used but the length of each polypeptide represents additional sequence from TIGR, The Sanger Center or this laboratory (D.H.

and Alexandra K. Ingram).b Human and S. cerevisiae sequences with the lowest expectation score using the BLAST software.c These ORFs are incomplete.d Homology does not appear to extend beyond the ,50 residue bromodomain motif.

operates to prevent transcription (elongation) at VSG loci

that appear to represent ,20% of the genome. This includes

40 or so telomeric VSG expression sites with associated

promoters, several hundred chromosome-internal VSGs

and the minichromosomes. The chromosome-internal

VSGs appear to lack promoters but these genes are not

expressed even if a promoter is inserted immediately

upstream (Horn and Cross, 1997).

Transcription from VSG expression site, EP/GPEET and

rRNA promoters is repressed when they are inserted at an

`inactive' VSG expression site in bloodstream form cells

(Horn and Cross, 1995; Horn and Cross, 1997; Rudenko

et al., 1995). A rRNA promoter replacing a VSG expression

site promoter can be switched on and off (Rudenko et al.,

1995) and rRNA and EP/GPEET promoters show clear

developmental regulation at these loci (Horn and Cross,

1997). These results indicate that VSG expression site

switching is independent of the VSG expression site promo-

ter but dependent upon some other property of the locus in

bloodstream-form cells. In addition, an EP/GPEET promo-

ter upstream control element can overcome repression of a

VSG expression site promoter in chromatin (Qi et al., 1996)

presumably via transcription factor recruitment. These data

are consistent with a model in which chromatin components

regulate accessibility of the DNA template to transcription

(elongation) factors. By demonstrating that promoters are

repressed when inserted into an `inactive' VSG expression

site, other studies also support a role for chromatin in VSG

regulation at the bloodstream-stage (Graham et al., 1998)

and the insect midgut-stage (Navarro et al., 1999; Qi et al.,

1996; Rudenko et al., 1994).

At yeast telomeres, histone deacetylases (Imai et al.,

2000) and the histones themselves (Wyrick et al., 1999)

in¯uence gene silencing (Grunstein, 1998) extending at

least 20 kbp from the telomere. In T. brucei, VSG silencing

appears to depend upon epigenetic events that are reversible

and locus-dependent (Horn and Cross, 1997). Thus, telo-

meric silencing in yeast may share parallels with silencing

of telomeric VSG genes. Micrococcal nuclease treatment

indicated that T. brucei DNA, including sequences encoding

the expressed and non-expressed VSG genes (Greaves and

Borst, 1987) is organised into nucleosomal arrays (Hecker et

al., 1989). Therefore, chromatin-modifying enzymes in T.

brucei, those that acetylate core histones and those that

phosphorylate histone H1 for example, may be involved

in developmental regulation and VSG transcription regula-

tion. Indeed, isoforms of histone H1 vary, electron dense

chromatin is more prominent and chromatin ®bres are more

condensed in bloodstream-stage cells relative to the insect

midgut-stage (Ersfeld et al., 1999; Hecker et al., 1995).

Similarly, in T. cruzi a decrease in the rate of transcription

in non-proliferative cells correlates with an increase in the

amount of electron-dense heterochromatin (Elias et al.,

2001). Nucleases and heterologous RNA polymerases are

commonly used to probe the accessibility of DNA within

chromatin. Such studies in bloodstream-stage T. brucei cells

show increased nuclease sensitivity at the telomeric end of

an active VSG expression site (Greaves and Borst, 1987) but

show no differential sensitivity between active and

repressed transcription states in the regions surrounding

VSG expression site promoters (Navarro and Cross, 1998).

In contrast T7 RNA polymerase accessibility is increased at

both ends of the expression site at the insect-stage relative to

the bloodstream-stage (Navarro et al., 1999). These results

certainly suggest a role for developmentally regulated chro-

matin remodelling in VSG regulation but it is not clear if

speci®c chromatin structures are required for VSG gene

regulation at the bloodstream-stage.

Since native VSG promoters appear to lack proximal

upstream control elements it seems likely that mutually

exclusive activation of one of these promoters in vivo is

achieved via a more distal positive 'control element'. The

telomere itself which is composed of TTAGGG repeats is a

candidate for such an element since all VSG expression site

and metacyclic VSG promoters (,40 in total) appear to be

located proximal to telomeres and telomeric loci are the

exclusive expression sites for VSG genes. Thus, T. brucei

promoters may be unable to participate in the generation of

processive transcription in a chromosomal context unless a

control element recruits additional factors. Developmen-

tally regulated expression of factors speci®c for the meta-

cyclic VSG and VSG expression site promoter sequences,

the presence of 50-bp repeats upstream of VSG expression

site promoters and/or differences in distance from the telo-

mere (Fig. 1) could explain the activity of these promoters at

different life cycle stages.

Several other factors, apart from those discussed above,

could be involved in mutually exclusive VSG expression at

the bloodstream-stage and VSG inactivation in the insect-

stage. Although DNA methylation has not been observed in

T. brucei (Gommers-Ampt and Borst, 1995), nucleotide

base glucosylation forms the J-base speci®cally in the

bloodstream-stage. This J base may strengthen silencing

and/or help to stabilise repeats in the genome by acting as

a `¯ag' to target speci®c factors to repetitive regions (van

Leeuwen et al., 2000). Sub-nuclear organisation may be

important although VSG activation does not appear to be

based upon RNA polymerase I recruitment through locali-

sation in the nucleolus. The active VSG actually appears to

be transcribed at a discreet site outside the nucleolus

(Chaves et al., 1998). Productive VSG transcription may

depend upon speci®c recruitment of a transcription elonga-

tion factor or RNA processing machinery (Vanhamme et al.,

2000). This is supported by the observation of truncated

transcription at repressed VSG expression sites. Since the

VSG lies ,50 kbp from its promoter transcription

complexes with low processivity will not produce VSG

mRNA. However, at least two observations appear to favour

a model in which productive transcription depends upon the

speci®c recruitment of an elongation factor rather than the

RNA processing machinery. First, inhibition of trans-spli-

cing does not affect transcription of the a-tubulin genes


(Tschudi and Ullu, 1990) suggesting that the two processes

are not coupled at least in the case of genes transcribed by

RNA polymerase II. Second, repressed rRNA promoters

inserted at VSG loci generate processive transcription

complexes in bloodstream form cells (Horn and Cross,

1997) suggesting that reduced processivity is speci®cally

associated with the VSG expression site promoter in this

life cycle stage.

In summary, T. brucei telomeres may recruit relatively

abundant negative regulatory factors that can repress tran-

scription (elongation) over long distances, possibly via

DNA folding or looping while a single telomere may bind

a dominant positive regulatory factor that would counteract

silencing at that locus and allow switching between telo-

meres. The proposed positive factor could be a specialised

nuclear pore. Such an association could facilitate processive

transcription and allow rapid export of VSG mRNA explain-

ing why mature VSG transcripts are not detected in isolated

nuclei (Vanhamme et al., 2000). A precedent for such a

model exists in the yeast S. cerevisiae where telomeres

interact with nuclear pore complexes (Galy et al., 2000)

and telomere folding can facilitate gene silencing (de

Bruin et al., 2000) or gene activation (de Bruin et al., 2001).

7. Concluding remarks

As expected for an early-branching eucaryote, the tran-

scription machinery and certain aspects of gene expression

appear divergent in T. brucei relative to those observed in

later-branching eucaryotes. However, as in other eucaryotes,

T. brucei chromosomes are organised into distinct transcrip-

tion domains, which involves the speci®c targeting of tran-

scription factors. It is interesting that there are no clear

examples of regulated transcription in T. cruzi or Leishma-

nia. Distinct from the situation in T. brucei, transcription

occurs on transiently transfected plasmids containing a

trans-splice acceptor site and an adjacent polypyrimidine

tract but no additional trypanosomatid sequence (Curotto

de Lafaille et al., 1992). These same plasmid DNA sequences

may be unable to recruit RNA polymerase II on chromo-

somes while the `promoters' that can, may fail to act as

promoters when moved from their native context and placed

on plasmids. Thus, it is unclear how or to what extent RNA

polymerase II is targeted to speci®c DNA sequences or chro-

mosomal domains in trypanosomatids. Since RNA polymer-

ase I promoter regulation in T. brucei appears to be dependent

upon chromosomal context it may be necessary to dissect

promoter control elements at their native loci. However,

such experiments with VSG expression site promoters did

not reveal speci®c regulatory sequences proximal to the

core promoter (Blundell and Borst, 1998; Navarro and

Cross, 1998).

In other organisms, transcription regulation is understood

in some detail and histone modi®cation plays a major role.

In T. brucei, histone and DNA modi®cation, nuclear orga-

nisation and interactions between the RNA polymerases and

RNA processing machinery all have the potential to regulate

transcription initiation and/or elongation events. Other

important regulatory sequences are those involved in tran-

scription termination (Berberof et al., 1996; Sturm et al.,

1999) and the `insulators' (Bell et al., 2001) that form

boundaries between transcription domains.

Trypanosomatid genome sequence allows us to clarify

and improve our understanding of the relationship between

a variety of processes in these organisms and other eucar-

yotes. As the T. brucei genome sequencing project

approaches completion it will be interesting to see how

VSGs and other genes, known promoters and repetitive

elements are organised and what additional candidate tran-

scription regulatory molecules will be identi®ed. Since the

transcription machinery in trypanosomatids appears to be

divergent, direct characterisation of promoter-binding

factors and other interacting factors will be necessary but

genome sequence data certainly offers new approaches to

experimental design. Combined with established and emer-

ging tools, such as genetic manipulation (Clayton, 1999),

RNA interference (Shi et al., 2000), analysis of transcription

in vitro (Laufer and GuÈnzl, 2001) and `functional genomic'

analysis, these data will continue to facilitate progress in

characterising transcription regulation in the trypanosoma-

tids.

Acknowledgements

I would like to thank John Kelly and Martin Taylor for

critical reading of the manuscript, Vivian Bellofatto, Dave

Barry, Arthur GuÈnzl and Peter Myler for communicating

unpublished data and Alexandra K. Ingram for providing

sequence data. Preliminary sequence data was obtained

from The Insitute for Genomic Research website (www.ti-

gr.org.) and The Sanger Centre website (www.sanger.a-

c.uk). Research in D.H.'s laboratory is supported by a

Research Career Development Fellowship from The Well-

come Trust (052323).

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