zooming in on nuclear organization and gene expression control in

Parasitology

cambridge.org/par

Review

Cite this article: Faria JRC (2021). A nuclear

enterprise: zooming in on nuclear organization

and gene expression control in the African

trypanosome. Parasitology 148,1237–1253.

https://doi.org/10.1017/S0031182020002437

Received: 25 October 2020

Revised: 22 December 2020

Accepted: 24 December 2020

First published online: 7 January 2021

Key words:

Antigenic variation; genome architecture;

nuclear bodies; RNA processing; gene

expression; transcription factories;

Trypanosoma brucei; Trypanosomatids; VSG

Author for correspondence:

Joana R. C. Faria,

E-mail: jrcorreia[email protected]

Cambridge University Press. This is an Open

Access article, distributed under the terms of

the Creative Commons Attribution licence

(http://creativecommons.org/licenses/by/4.0/),

which permits unrestricted re-use,

distribution, and reproduction in any medium,

provided the origina l work is properly cited.

A nuclear enterprise: zooming in on nuclear

organizatio n and gene expression control in the

African trypanosome

Joana R. C. Faria

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street,

Dundee DD1 5EH, UK

Abstract

African trypanosomes are early divergent protozoan parasites responsible for high mortality

and morbidity as well as a great economic burden among the world’s poorest populations.

Trypanosomes undergo antigenic variation in their mammalian hosts, a highly sophisticated

immune evasion mechanism. Their nuclear organization and mechanisms for gene expression

control present several conventional features but also a number of striking differences to the

mammalian counterparts. Some of these unorthodox characteristics, such as lack of controlled

transcription initiation or enhancer sequences, render their monogenic antigen transcription,

which is critical for successful antigenic variation, even more enigmatic. Recent technological

developments have advanced our understanding of nuclear organization and gene expression

control in trypanosomes, opening novel research avenues. This review is focused on

Trypanosoma brucei nuclear organization and how it impacts gene expression, with an

emphasis on antigen expression. It highlights several dedicated sub-nuclear bodies that com-

partmentalize specific functions, whilst outlining similarities and differences to more complex

eukaryotes. Notably, understanding the mechanisms underpinning antigen as well as general

gene expression control is of great importance, as it might help designing effective control

strategies against these organisms.

Introduction

Trypanosomes are members of the Euglenozoa, a group of organisms within the Incertae sedis

Eukarya (ex-Excavata) supergroup (Adl et al., 2019). These organisms are likely to have

branched very early during evolution, which may explain the vast number of unorthodox fea-

tures that define their biology (Navarro et al., 2007; Adl et al., 2019). Euglenozoa include

Euglenids, Phytomonads and Trypanosomatids, free-living phagotrophs, plant and animal

parasites, respectively (Adl et al., 2019). Trypanosomatids include several parasitic protozoa

that cause a huge health and economic burden amongst the world’s poorest populations;

these include Leishmania sp, Trypanosoma cruzi, Trypanosoma brucei, Trypanosoma congo-

lense and Trypanosoma vivax. Notably, climate change, increased mobility and mass migration

pose great challenges to our ability to control diseases caused by these organisms, rendering

the need for new drugs to fight new parasite strains and resistance emergence imperative.

Therefore, a detailed molecular understanding of fundamental aspects of their cell biology,

gene expression, metabolism and interaction with the hosts is critical to design effective con-

trol strategies.

Trypanosoma brucei is the causative agent of sleeping sickness and nagana in humans and

cattle, respectively, and has been used for decades as a model organism for this group mostly

given its genetic tractability and available tools for reverse and forward genetics (Djikeng et al.,

2001; Alsford et al., 2011; Dean et al., 2017; Rico et al., 2018).

Trypanosoma brucei is transmitted through the bite of a tsetse fly and rapidly differentiates

into ‘slender’ bloodstream forms (BSFs) in the mammalian host. The slender forms are capable

of sensing the population density, which triggers differentiation into stumpy form s. The latter

are pre-adapted to life in the tsetse, where they will eventually differentiate into the procyclic

forms. In the mammalian host, besides the BSFs, these parasites can occupy multiple tissues

(brain, adipose tissue, skin, etc.), some recently identified as important reservoirs (Capewell

et al., 2016; Trindade et al., 2016).

Mammalian-infective T. brucei undergoes antigenic variation to successfully evade the host

adaptive immune responses (Fig. 1A), similarly to other pathogens such as malaria and giar-

diasis causing parasites (Duraisingh and Horn, 2016). For that purpose, it relies on a vast gen-

etic repertoire of genes that encode for their variant surface glycoprotein (>2500 VSG genes

and pseudogenes), approximately one-third of its genome (Berriman et al., 2005; Muller

et al., 2018). There are two key features for successful antigenic variation: (1) the ability to

express a single antigen from myriad possibilities (monogenic expression); (2) the ability to

switch from one antigen isoform to another (Duraisingh and Horn, 2016). However, despite

the vast genetic repertoire, a VSG gene can only be expressed from a limited subset of sub-

telomeric transcription units known as expression-sites (ESs) (Navarro and Cross,

1996;

https://doi.org/10.1017/S0031182020002437 Published online by Cambridge University Press

Hertz-Fowler et al., 2008 ; Fig. 1B). VSG-ESs are polycistronic

transcription units (PTUs) that share the same DNA elements,

and yet, one is active whereas the remaining are silent – a classic

epigenetic paradigm (Duraisingh and Horn, 2016).

The molecular understanding of the mechanisms underpin-

ning antigenic variation is critical as it sustains persistent infec-

tions and has greatly challenged vaccine development against

these organisms. This review will be focused on nuclear compart-

mentalization and how it affects or might affect both antigen and

global gene expression in the African trypanosome. Overall,

T. brucei nuclear architecture and mechanisms for gene expres-

sion control follow some of the classic conventions but also pre-

sent phenomenal dissimilarities when compared to so-called

model eukaryotes.

Genome organization

Eukaryotic genomes are condensed by several orders of magni-

tude; such compaction is critical to fit into the nucleus of a cell.

This is achieved by coiling the DNA around histones forming

chromatin fibres, which are subsequently arranged into more

complex high-order structures such as loops, domains and com-

partments (Gibcus and Dekker, 2013; Finn and Misteli, 2019).

Several of these architectural features are conserved across the

evolutionary tree, suggesting an elementary role of spatial organ-

ization in genome function and gene expression control (Foster

and Bridger, 2005). Indeed, DNA spatial organization and compart-

mentalization has been found to play a key role in the regulation of

gene expression and recombination in multiple organisms. In mam-

mals on a larger scale, two major sub-nuclear compartments can be

defined, one is transcription-permissive (compartment A) and the

other transcription-repressive (compartment B), roughly corre-

sponding to euchromatin and heterochromatin, respectively

(Gibcus and Dekker, 2013; Finn and Misteli, 2019). Further, within

chromatin domains known as topologically associating domains

(TADs), chromatin loops modulate interactions between promoters

and distal regulatory elements, ultimately impacting gene expression

(Rao et al., 2014; Schoenfelder and Fraser, 2019). TADs are usually

defined by boundary elements containing architectural chromatin

proteins; these include cohesin, CCCTC-binding factor (CTCF)

and histone variants (Millau and Gaudreau, 2011; Merkenschlager

and Odom, 2013).

The core genome of the African trypanosome

Trypanosoma brucei has a diploid genome, the haploid nuclear

genome (32 Mbp) is divided into three classes of linear chromo-

somes: 11 pairs of megabase chromosomes (at least 1 Mbp), one

to five intermediate-sized chromosomes and more than 100 mini-

chromosomes (50–150 kbp) (Wickstead et al., 2004; Berriman

et al., 2005). The megabase chromosomes contain all

RNA-Polymerase-II (Pol-II) transcribed genes and VSG-ESs

(Berriman et al., 2005; Muller et al., 2018). The minichromo-

somes are highly repetitive and many also contain VSG genes

(Wickstead et al., 2004; Fig. 1B). Additionally, T. brucei has an

unknown number of highly repetitive circular extra-chromosomal

DNAs of unknown function (Alsford et al., 2003).

In trypanosomes, electron-dense chromatin regions can be

found close to the nuclear periphery and their arrangement is devel-

opmentally regulated (Belli, 2000;Eliaset al.,

2001;Navarroet al.,

2007). Indeed, in T. brucei, chromosome conformational capture

(Hi-C) revealed that the transcribed chromosome core regions

and the sub-telomeric regions coding for the large reservoir of silent

VSG genes appear to fold into structurally distinct compartments

(Muller et al., 2018), similar to active A and silent B compartments

described in mammalian cells (Schoenfelder and Fraser, 2019).

Further, the relative interaction frequency was substantially higher

across sub-telomeric regions compared to core regions, indicating

that sub-telomeres are more compact than the core region.

Additionally, centromeres and junctions between the core and sub-

telomeres were found to be the most prominent boundaries of DNA

compartments (Muller et al., 2018).

Regarding architectural chromatin proteins, while CTCF

appears to be absent in non-metazoans (Heger et al., 2012), the

major subunit of cohesin is present in T. brucei and its depletion

is lethal (Landeira et al., 2009). Moreover, histone variants (H3V

and H4V) also function as architectural proteins in this organism

(Muller et al., 2018). Indeed, studies in T. brucei H3V and H4V

knockout cell lines revealed changes in global genome architec-

ture and local chromatin configuration, which triggered switches

in VSG expression (Muller et al., 2018).

The telomeres and sub-telomeres

Genome sequences of T. brucei and Plasmodium revealed that

Pol-II transcribed genes are located in the central core and

Fig. 1. Antigenic variation in T. brucei bloodstream forms. (A) Antigenic variation. Waves of parasitaemia are a hallmark of infections by African trypanosomes in

mammals. This is due to waves of parasites expressing different VSG coats (different colours). VSGs are highly immunogenic, typically triggering an effective and

lasting immune response (immunosuppression can occur later during infection). This illustration is a simplified depiction of the in vivo dynamics, indeed, at any

time point the populations can be much more complex than represented: these may include large numbers of different clonal VSG variants. (B) Genomic organ-

ization of VSG genes. Bloodstream VSG expression-sites (BESs) contain expression-site-associated genes (ESAGs), which are located between the promoter and the

70 bp repeats. The VSG genes are near telomeric repeats. Large extensions of 50 bp repeats are located upstream of all BESs. Metacyclic VSG expression-sites (MESs)

lack ESAGs and are expressed in metacyclic trypomastigotes in the salivary glands of the tsetse fly. Pol-II transcribed genes are organized in long polycistronic

transcription units in the 11 megabase (Mb) size chromosomes. The arrows indicate the direction of Pol-II transcription. VSG genes or pseudogenes are organized

in sub-telomeric regions of megabase chromosomes or at the telomeres of minichromosomes.

1238 Joana R. C. Faria

https://doi.org/10.1017/S0031182020002437 Published online by Cambridge University Press

antigen genes are located in sub-telomeric regions (Berriman

et al., 2005; Otto et al., 2018; Fig. 1B).

The telomere is a special functional complex at the end of lin-

ear chromosomes, consisting of tandem repeat DNA sequences

and associated proteins, which can form a specialized heterochro-

matic structure that suppresses the expression of genes located at

the sub-telomere, known as telomere position effect or telomeric

silencing (Ottaviani et al., 2008). Telomeres are essential for gen-

ome integrity and chromosome stability in eukaryotes and their

synthesis is mainly achieved by the cellular reverse transcriptase

telomerase, an RNA-dependent DNA polymerase that adds telo-

meric DNA to telomeres (Cong et al., 2002). Telomerase activity

was found to be absent in most normal human somatic cells, which

is intimately related with the ageing process, but present in over 90%

of cancerous cells (Cong et al., 2002). Notably, telomere-binding

proteins play critical r oles on the maintenance of telomer e length,

telomere heterochro ma tin formation, regulation of the telomeric

transcript levels, among others (Ottaviani et al., 2008). The mam-

malian telomere comple x has been well chara cterized and contains

six core proteins that include TRF1, TRF2, TIN2, RAP1, TPP1 and

POT1 (de Lange, 2005). Additionally, an integral component of

telomeric heterochromatin is the telomeric repeat-containing RNA

(TERRA), a large non-coding RNA whose transcription occurs at

most or all chromosome ends. Further, R-Loops have been identi-

fied at the telomeres, these are three-str anded nucleic acid structures

that contain a DNA:RNA hybrid. R-Loops can play an important

role in a number of cellular functions but they can also be an

instability factor (T an and Lan, 2020).

In the insect-stage, trypanosome telomer es tend to be close to the

nuclear periphery, but this is much less pronounced in the

mammalian-s t age (DuBois et al., 2012). In T. brucei, besides the tel-

omerase components (Dr eesen et al., 2005;Sandhuet al., 2013),

which are critical for telomere maintenance, sev er al other telomere

pr oteins have been identified. Among these, TbTRF, a functional

homologue of mammalian TRF2, a TbTRF-intera cting factor,

TIF2, RAP1 and TelAP1 (Yang et al., 2009;Jehiet al., 2014a,

2014b;Reiset al., 2018). Except for TelAP1, all the other factors

are essential for cell viability; TbTRF and TbTIF2 are critical for telo-

mere integrity and their depletion leads to an increase in double-

strand breaks and increased VSG switching (Jehi et al., 2014a,

2014b). TbRAP1 intera cts with TbTRF and its depletion leads to

derepression of silent VSG

-ESs in the mammalian- infectiv e stage,

but also in insect-stage cells, where VSG expr ession is developmen-

tally shut down (Yang et al., 2009). Further, TbRAP1-mediated

silencing has a str onger impact on telomere proximal genes (Yang

et al., 2009). Moreover, by associating with telomere chromatin,

TbRAP1 also suppresses the expr ession of the TERRA transcripts

and telomeric R-Loops, consistent with a role on telomere integrity

(Nanavaty et al., 2017). Recent studies on T. brucei ribonuclease H

enzymes, endonuclease enzymes that ca taly se the cleavage of RNA

in an RNA/DNA substrate, also showed that R-loops at the telomere

and the sub-telomer e affect VSG switching frequencies (Briggs et al.,

2018, 2019).

Interestingly, the nuclear phosphatidylinositol 5-phosphatase

(PIP5Pase), part of the inositol phosphate pathway, has been

recently shown to interact with TbRAP1 in a ∼0.9-MDa complex

(Cestari et al., 2019). The inositol phosphate pathway regulates

several cellular processes in eukaryotes including chromatin

remodelling and gene expression, and had been shown to have

a role on telomere silencing and VSG monogenic expression in

T. brucei (Cestari and Stuart, 2015).

In summary, in T. brucei (similarly to Plasmodium), Pol-II

transcribed genes are located in the central core whereas the anti-

gen genes are located in sub-telomeric regions (Berriman et al.,

2005; Otto et al., 2018). This chromos ome partitioning may be

important to fine-tune recombination in regions that encode for

antigens and to ensure that all but one antigen is repressed.

Similarly to Plasmodium, there is a large amount of evidence

that supports a role for telomeric chromatin in VSG gene silen-

cing (Duraisingh and Horn, 2016). Moreover, the sub-telomeric

location of VSG-ESs is thought to favour recombination, since

these sites are rather unstable (Glover et al., 2013).

Recombination-based and transcriptional mechanisms can lead

to VSG switching, but undoubtedly recombination makes the lar-

gest contribution in T. brucei compared to Plasmodium. Indeed,

telomere integrity and stability impacts VSG swit ching frequen-

cies and has been also proven critical to maintain VSG monogenic

expression (reviewed by Saha et al.,

2020). Notably, one of the

many remaining outstanding questions is how the active

VSG-ES escapes telomeric silencing.

Remarkably, the active VSG-ES and the silent VSG-ESs reside

within distinct nuclear compartments; the importance of nuclear

compartmentalization on global gene expression control and VSG

expression, in particular, will be addressed in the next chapter.

Nuclear compartmentalization

The nucleus is a double lipid bilayer enclosed organelle, which

separates genomic DNA from the rest of the cell. Its architecture

shields the genome from the sources of damage whilst providing

opportunities for gene expression regulation (reviewed by Lin and

Hoelz, 2019). There is ample evidence in multiple eukaryotes that

the transcriptional activity of genes is influenced by nuclear

organization, which changes during differentiation and develop-

ment. Indeed, the regulated expression of genes during develop-

ment is influenced by the availability of regulatory proteins and

the accessibility of the DNA to the transcriptional machinery

(Finn and Misteli, 2019). In eukaryotes, heterochromatin, which

is highly compact, is mainly located at the nuclear periphery,

whereas the less compact euchromatin occupies a more interior

nuclear position.

Additionally, key nuclear functions such as transcription, rep-

lication or RNA processing are not homogeneously distributed

throughout the nuc leus and can be compartmentalized. Such

compartmentalization within the nucleopla sm enables functional

specialization, separation of conflicting processes as well as

increasing the concentration of specific factors at their target

point of action (Finn and Misteli, 2019).

Two main models of nuclear organization emerged in the past. A

deterministic model proposed that specific structural elements in

the nucleus assembled into a scaffold that was then used by tran-

scriptional processes, resulting in transcriptional compartmentaliza-

tion, which was independent of active processes. Chromosome

position would therefor e be maintained by intera ctions with the

scaffold (Misteli, 2007). In striking contrast, in a self-organization

model, functional sites were formed depending on the gene activa-

tion status and without the need for predefined structures; chromo-

some position would therefore be established by chromatin itself

and interactions with functional sites. Arguably, experimental data

from many model systems strongly favour self-organization models

over deterministic models. For instance, perturbing nuclear lamins,

one of the prime structural components of the nucleus, has a

modest impact on the spatial organization of transcription and

pre-mRNA splicing sites, arguing against deterministic models

(Spann et al., 1997). Conversely, perturbation of most active nuclear

processes results in rapid chromatin architectural changes, consist-

ent with self-organization models (Misteli, 2007).

The nuclear periphery

At the nuclear periphery, there is a meshwork, designated nuclear

lamina (NL), which in mamm als is composed mainly by nuclear

Parasitology 1239

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lamins. A growing number of nuclear proteins are known to bind

lamins and are implicated in nuclear and chromatin organization,

mechanical and genome stability, cell signalling, gene regulation,

among others (Dechat et al., 2008). Notably, many molecules

must be able to traffic between the nucleus and the cytoplasm,

rendering nucleo-cytoplasmic transport absolutely critical for

cell survival. The trafficking of macromolecules in and out of

the nucleus occurs through nuclear pore complexes (NPCs)

(reviewed by Lin and Hoelz, 2019).

Nuclear pore

NPCs are massive macromolecular assemblies! In humans, each

NPC consists of ∼1000 protein subunits, designated nucleoporins,

rendering it one of the largest protein complexes in nature

(∼110 MDa). Each NPC is located in and stabilizes an ∼800

Å-wide nuclear pore, which is generated by the fusion between

the inner and outer nuclear membranes (reviewed by Lin and

Hoelz, 2019).

NPCs are critical to maintain the nuclear integrity by prevent-

ing macromolecules from freely diffu sing in or out of the nucleus.

Macromolecules smaller than ∼40 kDa can passively diffuse

through the diffusion barrier, whereas larger macromolecules

generally do not. Facilitated transport through NPCs is rapid,

adding up to hundreds to thousands of macromolecules per

second. Notably, NPCs condu ct their cargos in their native

state, allowing macromolecules to act immediately after transport,

for instance during signal transduction (reviewed by Lin and

Hoelz, 2019).

Most NPC proteins typically form a symmetric core that pos-

sesses an 8-fold rotational symmetry (nucleoporins are incorpo-

rated in multiples of eight). This symmetric core surrounds the

central transport channel and functions as the scaffold onto

which asymmetric nucleoporins attach on the cytoplasmic and

nuclear compartments to form structures known as the cytoplas-

mic filaments and nuclear basket, respectively (reviewed by Lin

and Hoelz, 2019). One inner ring that is embedded within the

nuclear envelope, and two outer rings that reside on the inner

or outer nuclear membrane generate the symmetric core itself.

The major constituent of the outer rings in the NPC is the coat

nucleoporin complex, which serves as a structural scaffold and

docking site for other nucleoporins. The nuclear basket, com-

posed of Nup153, Nup50 and Tpr, also serves as a hub for orga-

nising nuclear architecture and modulating gene transcription,

mRNA processing and export (reviewed by Lin and Hoelz, 2019).

The majority of N PC architecture appears to be conserved

throughout the Eukaryota and was already established in the

last common eukaryotic ancestor (DeGrasse et al., 2009).

However, although the proteins and complexes are rather con-

served, their arrangements can differ substantially between cells

in the same organism or even within the same cell type at the sin-

gle cell level (Ori et al., 2013). Specifically, how the NPC connects

with the lamina and mRNA transport is likely to be highly diver -

gent between different lineages (Rout et al., 2017).

Proteomics analyses of NPC-containing fractions from T. bru-

cei provided a comprehensive inventory of its nucleoporins, which

clearly share a similar fold type, domain orga nization, compos-

ition and modularity in comparison with metazoan and yeast

(DeGrasse et al., 2009). Further, an exhaustive interactome

assigned T. brucei nucleoporins to discrete NPC substructures,

which despite retaining similar protein composition also pre-

sented remarkable architectural differences (Obado et al., 2016;

illustrated in Fig. 2). Briefly, while most elements of the inner

core are conserved, multiple peripheral structures are highly dis-

similar, possibly to accommodate divergent nuclear and cytoplas-

mic functions (Obado et al., 2016). TbNPC is highly symmetric,

with asymmetry only provided by its two nuclear basket Nups

(Obado et al., 2016). Further, orthologues of cytoplasmic Nups

or mRNA remodelling factors are absent in trypanosomes.

Notably, TbNup76, likely the cytoplasm-specific Nup82/88 ortho-

logue, localizes to both faces of the NPC (Obado et al., 2016).

Overall, trypanosomes present substantial variation in the pore

membrane proteins and the absence of critical components

involved in mRNA export in fungi and animals. Additionally,

there is evidenc e supporting a Ran-dependent system for

mRNA export in trypanosomes, which suggests distinct mechan-

isms of protein and mRNA transport (Obado et al., 2016).

TbNup110 and

TbNup92, the two components of the nuclear

basket, are predicted to have predominantly coiled-coil structure

and are likely to represent the Mlp/Tpr proteins of trypanosomes

(Holden et al., 2014). Despite performing similar roles in chromo-

some segregation, TbNup92 has a restricted taxonomic distribu-

tion and appears to have a distinct evolutionary origin than

Mlp. Further, unlike Mlp, there was no evidence for a role on

the creation of transcriptional boundaries, consistent with tryp-

anosome genome organization and gene expression control

(Holden et al., 2014). However, TbNup92-knockout cells differen-

tially expressed genes associated with RNA turnover, raising the

interesting possibility that TbNup92 might associate with a par-

ticular subset of RNA-binding proteins (Holden et al., 2014).

Notably, in T. brucei as well as related organisms, a compre-

hensive analysis on whether there are changes in the NPC com-

position or structure following differentiation into different

developmental stages is yet to be performed (Rout et al., 2017);

and if such changes occur, whether those play a role in gene

expression modulation is yet to be investigated.

Nuclear lamina

In mammals, NL is a meshwork consisting of A- and B-type

lamins and lamin-associated proteins, which lines the inner

nuclear membrane. In differentiated cells, lamin expression is crit-

ical to sustain nuclear architecture, prevent abnormal blebbing of

the nuclear envelope, and position the NPCs (Dechat et al., 2008).

NL can influence transcriptional activity and interact with a wide

range of transcription factors; it is also involved in the compaction

of peripheral chromatin (Shevelyov and Ulianov, 2019).

Eukaryotic heterochromatin, which is mainly located at the

nuclear periphery, is subdivided into densely packed constitutive

heterochromatin, including pericentromeric and telomeric

chromosomal regions, and the less condensed or so-called facul-

tative heterochromatin located in chromosomal arms (Finn and

Misteli, 2019). Chromosomal regions interacting with the NL

are designated lamina-associated domains (LADs) have been

identified in a wide-range of eukaryotes, from nematodes to

humans, and contain mostly silent or weakly expressed genes

(Shevelyov and Ulianov, 2019). This supports the idea that NL

is a repressive nuclear compartment.

Lamin genes were found in metazoa but appeared to be absent

in plants and unicellular organisms. In mammals, two major

A-type lamins (lamin A and C) and two major B-type lamins

(lamin B1 and B2) have been identified and characterized

(Dechat et al., 2008). They are composed of a long central

α-helical rod domain, flanked by globular N-terminal (head)

and C-terminal (tail) domains, which self-assemble into higher-

order structures whose basic subunit is a coiled-coil dimer

(Dechat et al., 2008). Notably, aberrant lamin protein structure

or expression can lead to irregular nuclei and abnormal gene

expression. Indeed, hundreds of mutations have been identified

in human lamins and linked to diseases, collectively known as

laminopathies that include progeria and muscular dystrophies

(Dechat et al., 2008). Interestingly, examples from yeast and

plants suggest that alternative, non-lamin, molecula r systems

can construct an NL (Dechat et al., 2008).

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https://doi.org/10.1017/S0031182020002437 Published online by Cambridge University Press

In T. brucei, an analog ous to vertebrate lamins, NUP-1 is a

major compo nent of the nucleoskeleton and plays a key role on

heterochromatin organization at the nuclear periphery (DuBois

et al., 2012; illustrated in Fig. 2). NUP-1 is a critical component

of a stable network at the inner face of the trypanosome nuclear

envelope, its depletion leads to abnormally shaped nuclei and dis-

rupts NPCs and chromosomes organization (DuBois et al., 2012).

NUP-1 affinity purification led to the identification of a second

coiled-coil protein, designated NUP-2. Following NUP-2 deple-

tion, NUP-1 is mislocalized and vice versa, strongly suggesting

that NUP-1 and NUP-2 form a co-depen dent network

(Maishman et al., 2016). NUP-2 knockdown leads to severe fit-

ness cost and a dramatic impact on nuclear architecture including

severe changes to the nuclear envelope and chromosomal organ-

ization. Moreover, NUP-1 and NUP-2 are conserved across trypa-

nosomes; from a structural and functional perspective, they

behave similarly to lamins (Maishman et al ., 2016).

Notably, while the active VSG-ES resides within a transcription

factory adjacent to the nucleolus (Navarro and Gull, 2001) the

silent VSG-ESs are located at the extra-nucleolar nucleoplasm

but at more peripheral locations in BSFs (Chavez et al., 1998;

Landeira and Navarro, 2007; Fig. 2B and Fig. 3). Further, all

VSG-ESs localize to the nuclear envelope and appear to form con-

stitutive heterochromatin in insect-stage cells (Landeira and

Navarro, 2007). This is consistent with the idea that the NL is a

repressive compartment. Curiously, in Plasmodium, all silenced

var genes localize in a series of clusters at the nuclear periphery,

however, the transcription of the active var gene also occurs at a

specific site at the nuclear periphery, where the activated gene

moves away from the silenced clusters (Duraisingh et al., 2005;

Freitas-Junior et al., 2005; Lemieux et al., 2013).

In T. brucei, NUP-1 plays a role on epigenetic control of devel-

opmentally regulated loci. Indeed, following NUP-1 knockdown,

megabase chromosome telomeres reposition, multiple VSG-ESs

become active, and the frequency of VSG switching increases

(DuBois et al., 2012; Rout et al., 2017). Additionally, the active

VSG-ES promoter fails to migrate to the nuclear periphery

upon differentiation, and metacyclic VSGs are derepressed in

insect-stage cells, both likely associated with the defective forma-

tion and/or maintenance of a repressive heterochromatin com-

partment (DuBois et al., 2012). Heterochromatin-based

silencing in trypanosomes involves several proteins, such as

ISWI, RAP1 and histone deacetylase (DAC) 3 (Hughes et al.,

2007; Yang et al., 2009; Wang et al., 2010), whilst histone H1

participates in maintaining condensed chromatin in silenced

regions (Povelones et al., 2012). Strikingly, T. brucei lacks

H3K9me3, a well-characterized marker for heterochromatin,

and heterochromatin-protein 1 (HP1) (Berriman et al., 2005). It

is noteworthy that the misregulation of VSG and procyclin

genes is quite modest following NUP-1 depletion (up to

10-fold) (DuBois et al., 2012); however, it demonstrates that

NUP-1 and the trypanosome NL integrate a series of possibly

multiple mechanisms that constrain the inactive VSG-ESs and

reinforce their silent state.

Membraneless nuclear bodies

Eukaryotic cells contain membraneless organelles, designated cel-

lular bodies, which compartmentalize essential biochemical reac-

tions and cellular functions. These bodies are generated by phase

separation mediated by cooperative interactions between multiva-

lent molecules (Strom and Brangwynne, 2019; Razin and

Gavrilov, 2020). Well-characterized examples of such organelles

in the nucleus are nucleoli, which are sites of rRNA biogenesis;

Cajal bodies (CB), which are assembly sites for small nuclear ribo-

nucleoproteins (RNPs); and nuclear speckles (NSs), which are

storage compartments for RNA processing factors (Strom and

Brangwynne, 2019; Razin and Gavrilov, 2020). Besides their abil-

ity to move throughout the nucleus, another fascinating feature of

several nuclear bodies is their ability to form within the nuclear

milieu without apparent support structures, again consistently

with a self-organization model. Moreover, these organelles exhibit

properties similar to liquid droplets, being able to undergo fiss ion

and fusion. In fact, mixtures of specific RNA and certain

Fig. 2. Nuclear organization and nuclear bodies. (A) The schematics represents the mammalian nucleus, the lateral boxes highlight differences in T. brucei. (B) The

schematics represents the nuclear organization and nuclear bodies in T. brucei. Trypanosome-specific compartments are highlighted, such as the bloodstream form

(BSF)-specific expression-site body (active-VSG-Expression-Site transcription; extra-nucleolar Pol-I transcription) and the Spliced Leader (SL)-array transcription

compartments.

Parasitology 1241

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RNA-binding proteins are able to form phase-separated bodies in

vitro (Guo et al., 2019; Hondele et al., 2019).

Nucleolus

The nucleolus is likely to be the most distinctive nuclear compart-

ment, certainly the largest and the site of ribosome biogenesis

where the 45S ribosomal repeats are clustered. Indeed,

pre-rRNA transcription and processing as well as the assembly

of the 40S and 60S complexes take place in this nuclear body

(Hernandez-Verdun et al., 2010). In animals and plants, the

nucleolus presents a tripartite substructure, which can be

observed by electron microscopy. This tripartite substructure

includes fibrillar centres (FC) surrounded by a dense fibrillar

component (DFC); both embedded in the granular component,

the biggest nucleolar subdomain composed of RNP granules.

FC stores inactive rRNA genes, whereas DFC is electron dense

given the high concentration of RNPs and is involved in early

rRNA processing (Hernandez-Verdun et al., 2010).

Eukaryotic ribosomes are composed of 18S, 5.8S, 28S and 5S

rRNA subunits and approximately 80 associated proteins. The

four rRNA molecules are the main structural and catalytic com-

ponents of the ribosome. In most eukaryotes, genes encoding

for 18S, 5.8S, 28S are organized in tandem repeats, which are tran-

scribed by RNA Polymerase I (Pol-I) into a primary transcript

further processed into the mature 18S, 5.8S, 28S rRNAs

(Hernandez-Verdun et al., 2010). Transcription occurs in the

boundary between FC and DFC. 5S rRNA genes, on the other

hand, are transcribed in the nucleoplasm by Pol-III

(Hernandez-Verdun et al., 2010).

Similarly to other eukaryotes, the nucleolus is the most dis-

tinctive membraneless sub-nuclear body in trypanosomes and

Leishmania parasites that can be easily observed by light and elec-

tron microscopy (Ogbadoyi et al., 2000; Nepomuceno-Mej ía

et al., 2010). Presently, FCs have not been identified in the nucle-

olus of these organisms, which presents a bipartite structure, simi-

larly to other protozoa, yeast, invertebrates, fish and amphibians

(Ogbadoyi et al., 2000; Nepomuceno-Mejía et al., 2010; illustrated

Fig. 3. Nuclear organization and VSG expression in T. brucei bloodstream forms. The single active-VSG establishes a stable inter-chromosomal interaction with one

of the SL-arrays. VEX2 orchestrates this spatial integration, which is critical to (1) sustain monog enic expression, (2) enhance RNA processing (Faria et al., 2020). The

active-VSG gene is transcribed at very high levels by Pol-I generating the most abundant protein in the cell. Proximity to the SL-array likely leads to a high local

concentration of SL-RNA therefore facilitating trans-splicing. It is possible that several factors associated with RNA processing (splicing, polyadenylation, etc .) are

concentrated in this sub-nuclear compartment as well. The SL-array appears to function as a post-transcriptional enhancer and such control might extend beyond

VSG genes (Faria et al., 2020). The active VSG-ES lies within a highly SUMOylated focus (López-Farfán et al., 2014); TDP1 is a high mobility group box protein that

facilitates Pol-I transcription and is enriched at the active-ES (Narayanan and Rudenko, 2013 ). VEX2 and VEX1 form discrete protein condensates that associate with

the active-VSG and the SL-array, respectively. The VEX complex, especially VEX2, sustains the exclusive interaction between a single VSG-ES and the SL-array; fol-

lowing its depletion, all VSG-ESs can access the SL-arrays and are derepressed (Faria et al., 2019, 2020). The silent VSG-ESs have more peripheral locations; tran-

scription by Pol-I is initiated at the same rate as at the active-locus but transcription elongation is unsuccessful; these sites also have restricted access to RNA

processing factors and substrates (Vanhamme et al., 2000; Kassem et al., 2014); several repressing factors associated with heterochromatin formation (red circles)

sustain their inactive state. For instance, ISWI (Hughes et al., 2007), FACT (Denninger and Rudenko, 2014), CAF-1 (Alsford and Horn, 2012) or DAC3 (Wang et al., 2010)

repress transcription near the promoter in silent ESs. Telomeric ES proteins, such as RAP1 (Yang et al., 2009) or PIP5Pase (Cestari et al., 2019), repress transcription

of the whole ES, the repressive gradient is stronger near the telomeres (indicated by the darker line). Other repressive proteins include DOT1B (Figueiredo et al

2008), bromodomain proteins (BDFs) (Schulz et al., 2015) and PIP5K and PLC, marked with an asterisk because they are the only ones that do not localize to the

nucleus (Cestari and Stuart, 2015). Moreover, the integrity of the nuclear lamina is critical to maintain this repressive state (DuBois et al., 2012).

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in Fig. 2). Unlike more complex eukaryotes, during cell division in

T. brucei, the nuclear envelope is preserved, chromatin does not

condense and the nucleolus does not disassemble. As mitosis pro-

gresses, the nucleolus stretches, is pulled via the spindle fibres to

opposite poles of the nucleus and ultimately divided into two

independent structures (Ogbadoyi et al., 2000). This process

occurs in the absence of intermediate structures such as prenu-

cleolar bodies, found in other organisms (Hernandez-Verdun

et al., 2010).

In T. brucei and similarly to other organisms, the biogenesis of

ribosome subunits starts in the nucleolus and ends in the cyto-

plasm. The 5S rRNA is imported to the nucleolus very early in

the biogenesis process and incorporated into the 90S pre-

ribosome as an RNP complex; it later undergoes spatial rearrange-

ment to facilitate subsequent maturation steps of the 60S subunit

(Prohaska and Williams, 2009; Liu et al., 2016). The pre-60S par-

ticle is translocated from the nucleus to the cytoplasm through

interactions between P34 and P37and exportin 1 and Nmd3, as

well as r-proteins uL3 and uL11 (Prohaska and Williams, 2009).

The biogenesis of the 40S subunit in T. brucei occurs very similar

to what has been described in yeast (Ferreira-Cerca et al., 2007).

Interestingly, this subunit contains a trypanosomatid-specific hel-

ical structure that has been proposed to participate in translation

initiation by interacting with the SL-sequence and its unusually

modified cap (Hashem et al., 2013).

In humans, the nucleolus has been associated with multiple

functions that extend beyond ribosome biogenesis, one being a

cellular stress sensor (Rubbi and Milner, 2003). Studies in trypa-

nosomes suggest this may be the case in trypanosomatid parasites

as well (Elias et al., 2001; Barquilla et al., 2008). Moreover, the

nucleolus appears as a largely self-organized structure. Indeed,

its integrity relies on both active Pol-I transcription and high

interactivity between ribosomal components (Raska et al.,

2006). Interestingly, ectopic expression of rRNA leads to the

formation of micronucleoli in Drosophila (Karpen et al., 1988),

again consistently with a model of self-organized nuclear com-

partmentalization. In trypanosomes, specifically, depletion of

Pol-I-specific subunits leads to abnormal nucleoli (Devaux

et al., 2007) and depletion of TOR1 kinase leads to Pol-I and

nucleolar dispersion, most likely as a consequence of Pol-I tran-

scription inhibition (Barquilla et al., 2008 ). In T. cruzi, develop-

ment from a proliferative to non-proliferative stage, which is

associated with a pronounced drop in transcriptional activity, is

also accompanied by nucleolar dispersion (Elias et al., 2001).

Further details on nucleolar structure and function in trypano-

somatid parasites have been recently reviewed by (Martínez-

Calvillo et al., 2019).

Nuclear speckles and Cajal bodies

In complex eukaryotes such as animals and plants, CBs are

involved in the post-transcriptional maturation of small nuclear

(snRNAs) and small nucleolar RNAs (snoRNAs) and the biogen-

esis of nuclear RNPs, including some nucleolar proteins,

snoRNPs and snRNPs (Sawyer et al., 2016). The number of

CBs varies across cell types and at a single-cell level within the

same cell type (in mammalian cells typically 0–10 CBs per

nucleus, ranging 0.1–2 μm in diameter). CBs are more abundant

in cells with high transcriptional activity and are highly dynamic

but structurally stable structures. They continuously exchange

components into and out of the domain in response to changes

in the cellular environmental (Sawyer et al., 2016). Interesting ly,

components of the SNAPc complex were reported to be enriched

within the CB, suggesting a strong link between snRNA gene tran-

scription and CBs. Several studies also indicate that CBs influence

the levels and processivity of factors crucial for efficient RNA

splicing; indeed CBs may influence splicing kinetics through dif-

ferent pathways (Sawyer et al., 2016).

Coilin and the nucleolar protein Nopp140 are the two key

markers of CBs. Coilin has been implicated in the link between

the nucleolus and CBs; indeed CBs are frequently detected at

the nucleolar periphery and even within nucleoli. Coilin is a key

structural component of CBs, is involved in RNP metabolism

within these nuclear bodies and it also appears to have a role

on general chromatin organization (Machyna et al., 2015). Its

N-terminal domain is responsible for the self-oligomerization

activity, truncation or mutation of phosphorylation sites in the

conserved C-terminal region leads to a dramatic alteration in

the number of CBs (Shpargel et al., 2003). On the other hand,

Nopp140 does not localize strictly to CBs and it appears to

serve generally as a chaperone for RNPs; it moves between the

nucleolus and the CBs, but also between the nucleolus and the

cytoplasm (Isaac et al., 1998). Indeed, it not only inte racts with

coilin, but also associates with several nucleolar proteins (Isaac

et al., 1998).

Trypanosoma brucei appears to lack a coilin homologue and

TbNopp140 is strictly nucleolar, strongly suggesting that CBs, in

the strict sense of the definition, are absent in these parasites

(Berriman et al., 2005; Kelly et al., 2006)(Fig. 2). Additionally,

T. brucei possesses two homologues of Nopp140, a canonical

Nopp140 and a Nopp140-like protein, both are phosphorylated

and co-immunoprecipitate with Pol-I and might play a role on

nucleoplasmic snoRNPs shuttling (Kelly et al., 2006). Given the

absence of CBs, it has been proposed that in T. brucei RNPs are

probably assembled in analogous bodies: a possible candidate

was a compartment identified as Spliced-leader-associated RNA

(SLA1)-containing subnuclear site that did not colocalize with

SL-RNA (Hury et al., 2009). SLA1 guides the pseudouridylation

at position −12 (relative to the 5

′

splice site) of the SL-RNA in

all trypanosomatid species.

NSs or splicing speckles were originally discovered as sites for

splicing factor storage and modification and were later revealed to

play a general role in RNA metabolism. Subsequently, numerous

proteins involved in epigenetic regulation, chromatin organiza-

tion, DNA repair and RNA modifications were found in NSs

(Galganski et al., 2017). Similar to other membraneless bodies

with liquid-like properties, NSs are characterized by the dynamic

exchange of components within the nucleoplasm, sharing some

proteins with other nuclear bodies (Galganski et al., 2017).

In trypanosomes, trans-splicing occurs for every single mRNA,

there are only two known cis-spliced introns in T. brucei; both

mechanisms seem to require the spliceosome. Notably, trypano-

somes encode for all snRNA and many spliceosomal proteins

described in other eukaryotes but also encode for a few specific

factors (Palfi et al., 2000; Ambrósio et al., 2009; Preusser et al.,

2009; also reviewed by Günzl, 2010; Michaeli, 2011 and

Clayton, 2019). Interestingly, splicing factors such as Prp31,

SmE, SSm2-1, PRP19 and SPF27 have a speckl e-like organization

and appear to be compartmentalized in specific nuclear areas

(Liang et al., 2006; Tkacz et al., 2007; 2010; Ambrósio et al.,

2015; illustrated in Fig. 2B).

Recent advances in more complex eukaryotes suggested that

NSs facilitate integrated regulation of gene expression

(Galganski et al., 2017). A substantial fraction of the mammalian

genome is preferentially organized around nuclear bodies such as

the nucleolus and NSs; these bodies have been proposed to act as

inter-chromosomal hubs that shape the overall packaging of DNA

in the nucleus (Quinodoz et al., 2018). Additionally, many active

genes reproducibly position near NSs, but the nature of such asso-

ciations had remained unclear until recently, when a study linked

them to stochastic gene expression amplification (Kim et al.,

2020). Whether similar associations are present and play a role

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in genome organization and gene expression in trypanosomes and

related organisms remains to be explored.

In summary, compartmentalization within the nucleoplasm

enables functional specialization; in fact, key nuclear functions

such as transcription or RNA processing are not homogeneously

distributed throughout the nucleus. In the next chapter, I will spe-

cifically cover the current knowledge on transcription regulation

and compartmentalization in trypanosomes.

Transcription regulation

To our knowledge, all trypanosomatids employ primarily polycis-

tronic transcription, where multiple open reading frames with no

functional association are transcribed in tandem. Evidence sug-

gests that the posi tion within the PTU is associated with messen-

ger RNA (mRNA) copy number (Kelly et al., 2012). The nascent

RNAs are processed into mature mRNAs, through a combination

of trans-splicing and polyadenylation (reviewed by Günzl, 2010;

Michaeli, 2011; Clayton, 2019). Notably, mature mRNAs bear

an unusual hypermethylated 5

′

cap structure (Bangs et al.,

1992). The genome is therefore constitutively transcribed and

mRNA abundance is primarily controlled at the post-

transcriptional level in striking contrast with more complex

eukaryotes, where a specific promoter usually regulates the tran-

scription of each gene (Koumandou et al., 2008).

Exceptions to this mechanism are the genomic loci encoding

for highly abundant surface-exposed antigens, VSGs and procy-

clins: these loci are transcribed at very high levels by Pol-I and

not Pol-II, which transcribes the majority of PTUs. Both VSGs

and procyclins expression is developmentally regulated, the for-

mer expressed in the mammalian-stage and the latter in the

insect-stage (Navarro et al., 2007; Daniels et al., 2010).

RNA polymerase I (Pol-I)

Trypanosoma brucei is the only organism known to have evolved

a multifunctional Pol-I system that is used for rRNA synthesis

and for the expression of highly abundant antigens (Günzl

et al., 2003). As previously mentioned, VSGs and procyclins are

strongly developmentally regulated and therefore Pol-I transcrip-

tion in T. brucei is intimately linked to differentiation between dif-

ferent life cycle stages as well as antigenic variation in the

mammalian host, which is critical to sustain persistent infections.

In the vast majority of eukaryotes, Pol-I is recruited to simple

promoters, which contain an upstream element located 100 bp

from the transcription start site. Such promoters are exclusively

used for rRNA gene expression, specifically the 45S rRNA precur-

sor, further processed into 18S, 5.8S and 28S rRNA. Two protein

complexes, the selectivity factor 1 (SL1) and the upstream binding

factor (UBF), are essential for Pol-I recruitment to the rRNA pro-

moter (Russell and Zomerdijk, ). The interaction between Pol-I

and SL1 is mediated by a single polypeptide named RRN3 in

humans; a UBF dimer is further required to activate rRNA tran-

scription. In yeast, RRN3 is conserved, whereas the three subunits

of the core factor (the functional equivalent of SL1) and the six

UBF subunits share no sequence similarity with the mammalian

counterparts (Russell and Zomerdijk, ). Recent CryoEM studies

suggest that, unlike the Pol-II system, promoter specificity relies

on a distinct ‘bendability’ and ‘meltability’ of the promoter

sequence that enables contacts between initiation factors, DNA

and polymerase (Engel et al., 2017). In eukaryotic cells, although

the number of rRNA genes is much lower than the number of

protein-coding genes, Pol-I transcription usually accounts for

more than 50% of the total transcriptional activity, which results

from impressively high transcription initiation rates. Notably,

mammalian Pol-I is unable to synthesize functional mRNA

(Russell and Zomerdijk, ).

Similarly to all eukaryotes, T. brucei Pol-I transcribes the 45S

rRNA precursor in the nucleolus; however, it also transcribes pro-

cyclins and VSGs mRNAs from perinucleolar and extra-nucleolar

locations, respectively (Navarro and Gull, 2001)(Figs 2B and 3).

The rRNA, VSG and procyclin gene promoters are structurally

different, suggesting that they recruit different transcription fac-

tors. Since the last two promoters are absent in related organisms

T. cruzi and Leishmania spp., one would expect to find T.

brucei-specific proteins for VSG and procyclin gene transcription.

In T. brucei, both bioinformatics and biochemical analyses

have unravelled 10 out of 12 Pol-I subunits: RPA1, RPA2,

RPC40, RPB5z, RPB6z, RPB8, RPC19, RPB10z and RPA12

(Walgraffe et al., 2005; Nguyen et al., 2006). RPB5z, RPB6z and

RPB10z are RPB5, RPB6 and RPB10 paralogs, respectively.

Further, T. brucei presents functional diversification of isoforms

that are conventionally shared RNA polymerase subunits

(Devaux et al.,

2007). Trypanosoma brucei also has a specific

component (or a divergent orthologue of yeast RPA43), RPA31,

which is critical for Pol-I transcription and cell viability

(Walgraffe et al., 2005; Nguyen et al., 2007). Although conceiv-

able, there is no evidence that RPA31, RPB5z, RPB6z and

RPB10z play a specific role on mRNA transcription by Pol-I in

T. brucei.

The class I transcription factor A (CITFA) has been identified

in T. brucei; its purification led to the identification of seven novel

subunits, termed CITFA-1 to -7, plus the dynein light chain

DYNLL1 (also known as LC8) (Brandenburg et al., 2007;

Nguyen et al., 2012). CITFA binds rRNA, VSG and procyclin pro-

moters and therefore is a general Pol-I transcription factor in T.

brucei; its depletion is unsurprising ly lethal (Brandenburg et al.,

2007). Further, TDP1, a high motility group box containing pro-

tein, which facilitates Pol-I transcription, is highly enriched at the

active VSG -ES (compared to silent) and in the nucleolus; a block-

ade in TDP1 synthesis results in a pronounced reduction of

Pol-I-derived transcripts (Narayanan and Rudenko, 2013).

TDP1 overexpression was sufficient to open the chromatin of

silent VSG-ESs and disrupt VSG monogenic expression

(Aresta-Branco et al., 2019). Moreover, ELP3B was identified as

a specific negative regulator of rRNA transcription (no impact

on VSG transcription); these observations extend the roles of

the Elp3-related proteins to Pol-I transcription units, as they are

usually associated with Pol-II transcription in humans and yeast

(Alsford and Horn, 2011).

Notably, all proteins involved in T. brucei Pol-I transcription

identified so far are conserved among all trypanosomatids, sug-

gesting that they fulfil general Pol-I functions (Walgraffe et al.,

2005; Nguyen et al., 2006; Devaux et al., 2007). However, it is

entirely possible that these common factors evolved specific func-

tions for protein-coding gene transcription in T. brucei.

Nevertheless, how T. brucei Pol-I acquired the ability to transcribe

mRNA remains mysterious.

RNA polymerase II (Pol-II)

Pol-II synthesizes pre-mRNAs and U-rich short nuclear RNAs

(snRNAs). The latter form the core of the spliceosome, involved

in processing pre-mRNAs into mature mRNAs. In both cases,

the 5

′

ends are capped, which requires adding m

G to the 5

′

tri-

phosphate end of the primary transcript and takes place

co-transcriptionally (Proudfoot et al., 2002). There are several

other co-transcriptional activities, which are assigned to specific

subunits or domains within these subunits (Proudfoot et al.,

2002). Trypanosoma brucei Pol-II produces both pre-mRNAs

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and the spliced leader (SL)-RNA, the latter is detrimental for

trans-splicing.

Eukaryotic Pol-II enzymes usually contain 12 subunits, desig-

nated RPB1 to RPB12. Specifically, RPB1, RPB2, RPB3 and

RPB11 are considered the functional and structural core subunits.

Additionally, RPB4 to RPB10 and RPB12 usually contribute to

Pol-II ability to respond to activators and tightly bind promoter

regions (Proudfoot et al., 2002). The 12 Pol-II subunits could

be identified in T. brucei; RPB1, RPB2, RPB3 and RPB11 were

also considered the functional and structural core (Das et al.,

2006; Devaux et al., 2006). Interestingly, trypanosomes have two

isoforms of R PB5 and RPB6 (Das et al., 2006; Devaux et al.,

2006). RPB1 is the large st subunit in the T. brucei enzyme and

also the most fascinating (Evers et al., 1989). One of the most

remarkable characteristics is the non-structured carboxyl end of

the polypeptide, which deviates from the heptapeptide repeat of

YSPTSPS of varying length that is characteristic of yeast and

mammalian proteins. This repeat is generally involved in the

modulation of multiple co-transcriptional processes that include

capping, splicing, elongation, polyadenylation and nuclear export,

through coordinated kinetic alterations in the phosphorylation of

its serines and threonines (Proudfoot et al., 2002). Despite being

non-repetitive, the trypanosome carboxyterminal is phosphory-

lated and essential for transcription (Evers et al., 1989).

Trypanosoma brucei cdc2-related kinase 9 (CRK9) was found to

be responsible for RPB1 phosphorylation, however, surprisingly,

when silencing CRK9, there was no impact on Pol-II transcription

or co-transcriptional m

G capping. Instead it led to a block of

trans-splicing caused by hypomethylation of the SL-RNA unique

cap4 (Badjatia et al., 2013).

In many organisms, a crucial regulatory point of gene expres-

sion is transcription initiation, which requires the formation of a

pre-initiation complex that includes multiple proteins that inter-

act with Pol-II. Such transcription factors include TFIIA, TFIIB,

TFIID, TFIIE, TFIIF and TFIIH, which recruit and position

Pol-II at promoter sequences (Hahn, 2004). The only canonical

Pol-II promoter in T. brucei is the SL-RNA promoter. In this organ-

ism, the identification of general transcription factors was chal-

lenged by their extremely divergent amino acid sequences from

those of their eukaryotic counterparts. The first transcription factor

purified and characterized was a trimeric SNAPc that formed a lar-

ger complex with TATA-binding protein, the small subunit of

TFIIA (TFIIA2), and a sixth protein (TFIIA1) (Das et al., 2005;

Schimanski et al., 2005). This was followed by the identification

of TFIIB, TFIIH, TFIIE; later, a TFIIH-associated complex of

nine subunits was discovered, and despite exhibiting no motif or

sequence conservation that could reveal its identity, it structurally

resembled the head module of the much larger mediator complex

of other eukaryotes (Schimanski et al., 2006;Leeet al., 2007,

2010). More recently, a TFIIF-like or TFL complex has been iden-

tified, strongly indicating that trypanosomatids possess a full set of

RNA Pol-II general transcription factors, only very divergent from

their mammalian and yeast counterparts (Srivastava et al., 2018).

All these factors are required for SL-RNA transcription and tryp-

anosome viability, but their role, if any, on the transcription of

protein-coding genes remains unknown.

In T. brucei, ubiquitously expressed genes lack well-defined

Pol-II promoter motifs, with the exception of the spliced-leader

RNA promoter. Indeed, the so-called Pol-II disperse promoters

lack conserved sequence motifs and tight regulation; however,

they are defined by specific chromatin structures. In T. brucei

for instance, GT-rich promoters were recently proposed to drive

transcription and promote the targeted deposition of the histone

variant H2A.Z, showing that even highly dispersed, unregulated

promoters might contain specific DNA elements that are able

to induce transcription (Wedel et al., 2017).

Additionally, Pol-II transcription termination is a tightly

regulated process and critical to prevent the elongating Pol-II

complex from interfering with the transcription of downstream

genes. In kinetoplastid flagellates, the modified base

β-D-glycosyl-hydroxymethyluracil (J) replaces a small percentage

of thymine residues, mostly in telomeric regions and is synthe-

sized at the DNA level via the precursor 5-hydroxymethyluracil.

In T. brucei for instance, base J is exclusively present in the

BSF. Notably, in T. brucei and Leishmania major, base J and

H3.V are enriched at sites involved in Pol-II termination. Loss

of base J and H3.V led to transcription read-through (Reynolds

et al., 2016; Schulz et al., 2016). Recently, a novel base

J-binding protein complex involved in Pol-II transcription ter-

mination has been identified (Kieft et al., 2020).

Overall, trypanosomes appear to have limited control over

Pol-II transcription initiation, and therefore most of the gene

expression control is thought to be post-transcriptional.

RNA polymerase III (Pol-III)

Pol-III is responsible for the transcription of a number of small

non-coding RNAs that play a role in translation (tRNA and 5S

rRNAs) and other cellular processes (7SL RNA). In T. brucei,

tRNA genes can be found widely spread throughout large direc-

tional gene clusters on megabase chromosomes, 5S rRNA genes

are clustered in chromosome 8 (Berriman et al.,

2005).

Expression factories

The SL-RNA expression factory

Given that SL-RNA must be added to the 5

′

end of every single

mRNA in T. brucei, trans-splicing relies on large quantities of

SL transcripts generated by Pol-II transcription from a diploid

tandem-repeat locus. Indeed, Pol-II largest subunit is highly con-

centrated at the SL-RNA genomic loci (illustrated in Fig. 2B). In

T. cruzi and Leishmania tarentolae, a single focus is observed pos-

sibly due to pairing of both alleles (Dossin and Schenkman,

2005). In contrast, in T. brucei, two distinct foci could be detected

in G1 cells indicating that the two SL-arrays occupy distinct

chromosome territories (Uzureau et al., 2008). In T. cruzi, the

Pol-II focus disperses following treatment with transcription inhi-

bitors (Dossin and Schenkman, 2005), suggesting that the high

concentration and organization of Pol-II around the SL-arrays

depends on active-transcription and therefore is not a predefined

nuclear structure.

Moreover, SL-RNA transcripts concentrate in a nuclear area

that colocalizes with the snRNP protein SmE and SLA1 RNA,

an RNA involved in the SL-RNA modification. This strongly sug-

gests that there is a spatially defined SL-RNP factory in the

nucleoplasm (Tkacz et al., 2007). When labelling active transcrip-

tion through BrdU incorporation, a broader distribution of extra-

nucleolar transcriptional activity can be observed apart from the

SL-RNA arrays (although that accounts for Pol-III as well)

(Daniels et al., 2010; illustrated in Fig. 2B). One would expect

that capping enzymes and cap methyltransferases would concen-

trate at SL-RNP factories, which is difficult to extrapolate from the

localization data currently available and will therefore require a

more detailed analysis, possibly with higher resolution

microscopy.

The VSG expression factory

African trypanosomes and their VSGs are a fine example of

extreme biology and have led to several groundbreaking discover -

ies, such as trans-splicing, mRNA transcription by Pol-I or GPI

anchors (Navarro et al., 2007; Duraisingh and Horn, 2016).

Notably, recent studies on VSG expression in T. brucei have

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revealed interesting features regarding genome architecture and

nuclear compartmentalization that hint to unknown layers of

gene expression control in these organisms.

The single active VSG gene generates the most abundant pro-

tein in the cell (approximately 10% of the total proteome), which

results from a combination of high levels of transcription by Pol-I

and multiple mechanisms of post-transcriptional control

(Navarro and Gull, 2001; Günzl et al., 2003; do Nascimento

et al., 2020; Viegas et al., 2020). This renders trypanosomes and

their VSGs an amenable model system to study mechanisms

underpinning single gene choice, which are not fully understood

in any eukaryote. Indeed, monogenic expression is one of the

greatest outstanding mysterie s of eukaryotic gene expression.

For instance, it also underpins singular expression of antigen

and olfactory receptors, responsible for the specificity of the

immune response and the sense of smell in mammals, respect-

ively (Monahan and Lomvardas, 2015; Outters et al., 2015).

Interestingly, it was unclear whether genome architecture and

specifically genome position played a role in gene expression con-

trol in trypanosomes and related organisms. However, T. brucei

somehow employs a mechanism of monogenic antigen transcrip-

tion in the absence of controlled transcription initiation and

canonical enhancer sequences. Indeed, Pol-I transcription is

initiated at the same rate at all VSG-ESs, however transcription

elongation is restricted to the active-VSG-ES (Vanhamme et al.,

2000; Kassem et al., 2014). Additionally, RNA maturation

seems to be somehow restricted to the active VSG-ES suggesting

that access to RNA processing factors or substrates might be limit-

ing (Vanhamme et al., 2000; Kassem et al., 2014).

Notably, while the silent VSG-ESs were located at more per-

ipheral locations (Chaves et al., 1998; Landeira and Navarro,

2007), the active VSG-ES was included within an extra-nucleolar

structure (although in close proximity to the nucleolus), desig-

nated the expression-sit e body (ESB), a transcription factory

that contains a local reservoir of Pol-I (Navarro and Gull, 2001)

(Fig. 3). This exclusion from the nucleolus is independent of

the promoter, as swapping the VSG promoter by an rRNA pro-

moter did not lead to nucleolar incorporation (Chaves et al.,

1998), suggesting that other DNA elements/factors are required

for targeting.

The ESB emerged as the def ining structure that sustained VSG

monogenic expression, accommodating a single VSG-ES at a time.

In fact, if two VSGs were simultaneously active, a dynamic colo-

calization with the ESB was observed (Chaves et al., 1999; Budzak

et al

., 2019). However, the mechanisms for targeting the

active-VSG ES to the ESB as well as the protein composition

and the exact DNA sequences incorporated within this structure

have rem ained elusive. Although the complete molecular under-

standing is yet to be achieved, several major advances have

taken place in the recent years.

Notably, the single active-VSG displays a specific inter-

chromosomal interaction with a major mRNA splicing locus,

one of the SL-RNA arrays, and this specific nuclear arrangement

is critica l to sustain VSG monogenic expression (Faria et al.,

2020). Specifically, the single active-VSG is expressed within a

dedicated sub-nuclear compartment harbouring the Pol-I tran-

scribed antigen-coding gene and the Pol-II transcribed SL-array

and their respective associated factors to ensure (1) monogenic

antigen transcription and (2) efficient mRNA splicing (Faria

et al., 2020)(Fig. 3). The VSG exclusion proteins 1 and 2

(VEX1 and VEX2), which form discrete protein condensates in

the nucleus in BSFs specifically, associate with the SL-RNA

array and the active-VSG ES, respectively (Faria et al., 2020)

(Fig. 3). VEX1 was identified through a genetic screening

(Glover et al., 2016) and VEX2 through VEX1 affinity purification

(Faria et al., 2019). From the two proteins, VEX2, an

RNA-helicase, has the most critical role on VSG monogenic

expression: following its depletion, the ESB collapses and trypano-

somes simultaneously transcribe all VSG-ESs, subsequently

exposing multiple VSGs on their surface (Faria et al., 2019).

Further, following VEX2 knockdown, all VSG-ESs can access

the SL-RNA arrays, showing that VEX2 somehow sustains this

dedicated sub-nuclear compartment and an exclusive association

between the single active-VSG and the SL-array (Faria et al.,

2020). Additionally, besides maintaining an exclusive interaction

between the active-VSG and the SL-array, VEX2 appears to fine-

tune gene expression at the active-VSG locus (Faria et al., 2019,

2020). It is tempting to speculate that it orchestrates a specific

chromatin configuration that maximizes the interaction between

the VSG gene itself (not the promoter or the ES-associated

genes) and the

SL-array.

Phase separation and transcriptional control

More recently, liquid–liquid phase separation (LLPS) has been

proposed (opinion piece by Hnisz et al., 2017) and later demon-

strated (Guo et al., 2019) to be a major regulatory mechanism for

enhancer-mediated transcriptional control in mammalian cells.

Enhancers are short (50–1500 bp) DNA regulatory elements

that activate the transcription of specific genes to a much higher

level than would be the case in their absence; they function as a

platform for the recruitment of activators, transcription factors

and the RNA polymerase components. These DNA elements

have a distal location and are brought in proximity to the target

gene through chromatin loops. Notably, nucleation of phase-

separated multi-molecular assemblies at enhancer sequences can

explain the formation of super-enhancers (clusters of enhancers;

sometimes hundreds), their high sensitivity to transcription

inhibition, enhancer-mediated patterns of transcriptional bursts

and simultaneous activation of multiple genes by the same enhan-

cer (Hnisz et al., 2017). Notably, computational simulations have

shown that LLPS can explain experimental observations that trad-

itional models for transcriptional control cannot (Hnisz et al.,

2017).

Enhancer sequences have never been found in trypanosomes

and related parasites, and given their polycistronic transcription

and overall lack of controlled transcription initiation, such

mechanisms were thought to be unlikely to operate. But is this

really the case? Indeed, it was unclear whether and how genome

architecture and genome position played a role in gene expression

in these parasites. Could it be that trypanosomes evolved uncon-

ventional enhancers? This will be addressed in the ‘Discussion’

section.

Discussion

Despite the many open questions, previous studies following the

depletion of several chromatin-associated factors (reviewed by

Cestari and Stuart, 2018) and the recently unveiled association

between the active-VSG and the SL-array unequivocally demon-

strate that genome architecture does play a role in VSG mono-

genic transcription in T. brucei. Further, spatial proximity to

RNA-processing centres might be a conserved mechanism for

post-transcriptional enhancement of gene expression but this

had not previously been linked to inter-chromosomal

interactions.

It is possible that all VSG-ESs are able to stochastically interact

with the SL-arrays and compete for a limited pool of VEX2, which

will then stabilize an exclusive interaction between a single VSG

locus and the SL-array. This would render VEX2 a limiting factor,

which is supported by its low abundance and tight regulation

(Faria et al., 2019). Interestingly, this could be explained by an

1246 Joana R. C. Faria

https://doi.org/10.1017/S0031182020002437 Published online by Cambridge University Press

LLPS model (Hnisz et al., 2017;Guoet al., 2019); indeed, phas e-

separating proteins were shown to be capable of generating stable

sub-nuclear structures from dynamic interactions in mammals

(Shin et al., 2018). Multiple studies have shown that high local

concentrations of specific proteins and nucleic acids (where

RNAs appear to be major players) and cooperative interactions

among these molecules are implicated in the formation of phase-

separated bodies (Shin et al., 2018; Guo et al., 2019). Recently, a

family of RNA helicases has been identified as major regulators

of the assembly of sub-nuclear compartments through LLPS

(Hondele et al., 2019); therefore, it is tempting to speculate this

might be the case of VEX2. In fact, specific post-translational

modifications can trigger nucleation of phase-separated bodies;

curiously, the active-ES resides within a hot spot of highly

SUMOylated proteins (López-Farfán et al., 2014). Notably, the

global role, if any, of LLPS and phase-separating proteins on gen-

ome organization in Trypanosomatids is yet to be investigated.

Inter-chromosomal interactions were thought to have a sto-

chastic nature, indeed the existence of stable inter-chromosomal

interactions has been a subject of debate as they were thought

to be difficult to re-establish following cell division, possibly rely-

ing on error-prone mechanisms (Finn and Misteli, 2019).

Consequently, their role on gene expression was rather dubious.

The only other known stable interaction occurs in a terminally

differentiated cell, and very interestingly, in another system sub-

ject to allelic exclusion. Indeed, olfactory neurons possess a multi-

chromosomal super-enhancer that associates with the single

active olfactory receptor gene (Monahan et al., 2019). In trypano-

somes, the association of the active-VSG with the SL-array

appears reminiscent, but classic transcriptional enhancement

was replaced by what appears to be post-transcriptional enhance-

ment instead. Despite the attractive theoretical reasons for the

presence of such an enhancer in malaria-causing parasites,

Hi-C analysis was unable to identify such an element in the P. fal-

ciparum genome (Lemieux et al., 2013).

In trypanosomes, proximity to the SL-array is likely to provide

post-transcriptional enhancement due to a high local concentra-

tion of SL-RNA. A substantial amount of SL-RNA is therefore

hijacked, so that RNA processing can keep pace with the high

rate of transcription provided by Pol-I (Fig. 3). Notably, it will

be interesting to identify other active VSG-ES-associated factors

that take part in this antigen expression factory: it is entirely con-

ceivable that a number of splicing factors and enzymes involved

in polyadenylation might be concentrated in this compartment.

This certainly adds a layer of post-transcriptional control that

had not been previously characterized. Moreover, this association

is also reminiscent of those between highly transcribed chromo-

some regions and NSs in mammals (Quinodoz et al., 2018; Kim

et al., 2020). Whether the high transcription rate is the cause or

a consequence of such association remains debatable. Similarly

in T. brucei, whether the association with the SL-RNA array pre-

cedes the activation of the VSG locus, or whether it occu rs after-

wards merely providing post-transcriptional enhancement,

remains unclear. Notably, in other organisms, co-transcriptional

RNA processing can affect transcription elongation rates

(Kornblihtt et al., 2004). How a specific VSG gene is activated

over the other possible alleles remains a mystery, and those

early events underpinning the establishment of an active tran-

scriptional state are incredibly difficult to capture. In

Plasmodium, for instance, antisense long-non-coding-RNAs

play a key role in regulating

var gene activation and mutually

exclusive expression (Amit-Avraham et al., 2015).

Certainly several mechanisms simultaneously operate to con-

strain the inactive VSG-ESs and prevent their derepression. For

instance, heterochromatin-based silencing in trypanosomes

involves, among others, ISWI, RAP1 and histone deacetylase

(DAC) 3 (Hughes et al., 2007; Yang et al., 2009; Wang et al.,

2010; reviewed by Duraisingh and Horn, 2016; reviewed by

Cestari and Stuart, 2018)(Fig. 3). The histone tri-

methyltransferase DOT1B that targets H3K76, for instance, is

required for rapid VSG-ES silencing and for an efficient transition

from an active to a silent state (Figueiredo et al., 2008). Also,

both the integrity of the NL and histone H1 are critical to

maintain condensed chromatin in silenced regions (DuBois

et al., 2012; Povelones et al., 2012). Strikingly, T. brucei lacks

H3K9me3, a well-characterized marker for heterochromatin,

and HP1 (Berriman et al., 2005), which plays a key role in var

gene silencing in Plasmodium (Brancucci et al., 2014).

Further, in Plasmodium, the histone methyltransferase SET10

colocalizes with the active var gene (Volz et al., 2012) and

NAD(+)-dependent histone deacetylases, Sir2A and Sir2B, are

required for silenci ng of different var gene subset s (Tonkin

et al., 2009), but these histone modifiers do not appear to affect

VSG silencing (Alsford et al., 2007). Indeed, in both trypano-

somes and malaria-causin g parasites, repressive heterochromatin

plays a critical role in silencing all but one antigen-coding gene

for successful antigenic variation. However, different chromatin

remodellers, histone readers/erasers and histone chaperones

appear to be involved in this process in trypanosomes and

Plasmodium (reviewed by Duraisingh and Horn, 2016).

In a broader perspective, post-transcriptional enhancement of

gene expression through spatial proximity to RNA-processing

centres might be particularly relevant in less complex eukaryotes,

where canonical transcriptional enhancers have not been identi-

fied, and particularly in Trypanosomatids, where transcriptional

regulation is limited. Nonetheless, it remains to be investigated

whether this type of regulation extends beyond VSGsinT. brucei,

and whether it plays a broader role in gene expression control in

kinetoplastids.

Notably, in T. brucei

, Hi-C and ChIP-Seq analyses revealed

that other highly transcribed loci (e.g. tandem arrays that encode

for histones, tubulin, heat shock proteins, etc.) can interact with

the SL-RNA array in the mammalian-infective stage (Faria

et al., 2020). Moreover, in insect-stage T. brucei, procyclin coding

loci also interact with the SL-RNA array (Faria et al., 2020). Given

the fact that Hi-C is a very sensitive technique, it can capture

strong and stable but also stochastic and transient interactions

(Finn and Misteli, 2019); therefore, it will be interesting to

investigate whether the interactions above are stochastic or

whether they are associated with stable and heritable structures

at the single-cell level. In other words, are there any other tran-

scription/splicing factories in T. brucei and possibly in other

related parasites? Could the SL-array act as an unconventional

and post-transcriptional enhancer? The fact that the tubulin

gene loci in T. cruzi do not colocalize with the SL-RNA arrays

(Dossin and Schenkman, 2005) does not completely rule out

this idea. This is not inconsistent with such interactions being

transient and therefore more difficult to capture my microscopy,

but could also mean that strong and stable interactions might be

restricted to specific gene families and specific developmental

stages, possibly depending on transcriptional activity. Additionally,

it is very inter esting that in T. brucei, the two SL-arra ys occupy dis-

tinct chromosome territories, essentially there are two SL-RNA

expression factories: is it because one is permanently used to sustain

the expression of the active-VSG gene?

In mammals, a high degree of heterogeneity in genome organ-

ization has been observed, suggesting that individual cells in a

population can assume many distinct, albeit related, spatial con-

formations (Finn et al., 2019). Notably, such variability does

not mean that chromatin organization has no functional rele-

vance, but rather suggests that structural heterogeneity may be

another layer impacting gene expression (Finn and Misteli,

Parasitology 1247

https://doi.org/10.1017/S0031182020002437 Published online by Cambridge University Press

2019). In Plasmodium for instance, the 3D genome structure

appears to be strongly connected with the transcriptional activity

of specific gene families throughout the life cycle (Bunnik et al.,

2018). Whether such variability can be observed in

Trypanosomatid parasites and how that might modulate gene

expression at the single-cell level and in different developmental

stages remains to be unravelled.

Future directions

Trypanosoma brucei genome sequencing was a phenomenal turn-

ing point that marked the beginning of a new era of research.

Since then, sequencing technology, gene-editing tools, imaging

and affinity purification techniques have massively evolved, allow-

ing us to experimentally tackle long-standing questions that had

been previously untrackable.

Similarly to many pathogens, in T. brucei, the highly repetitive

nature and heterozygosity of the antigen-gene arrays had pre-

cluded a complete genome assembly. Recently, through a combin-

ation of PacBio single-molecule real-time sequencing technology

and Hi-C, the haplotype-specific assembly and scaffolding of

the long antigen-gene arrays has been successful (Muller et al.,

2018). This refined genome assembly has been proven critical

to perform further analyses on chromatin organization and

gene expression, especially regarding VSG genes. Among several

downstream analyses, which largely benefitt ed from a refined gen-

ome assembly, is Hi-C.

Hi-C and other chromosome conformational capture techni-

ques are a set of powerful molecular biology methods based on

proximity labelling, which enable the analysis of chromatin spatial

organization. These methods quantify the interaction frequency

between genomic loci that are nearby in the 3D nuclear space,

but may be far in the linear genome, allowing the identification

of enhancer–promoter contacts or chromatin loops for instance

(reviewed by Kempfer and Pombo, 2020). Hi-C studies in T. bru-

cei have identified key architectural proteins and that a specific

chromatin configuration is critical to fine-tune recombination

events; indeed, perturbation of that specific architecture triggers

switches in antigen expression (Muller et al., 2018). Further, vir-

tual 4C analyses survey the interaction frequencies between a bait

locus of interest and any other loci in the genome. In T. brucei,

such analyses have demonstrated that the active-VSG ES (but

not the silent) as well as genes encoding for other highly abundant

proteins interact with the SL-array, uncovering a potential

enhancer-like mechanism (Faria et al., 2020).

Next-generation sequencing techniques including RNA-Seq

(transcript abundance), ChIP-Seq (chromatin-association) and

CLIP-Seq (RNA-binding) have now been amply used in trypano-

somes and other Trypanosomatids. More recently, ATAC-Seq

(chromatin accessibility) and single-cell RNA-Seq have been per-

formed in T. brucei (Muller et al., 2018). The latter opens unpre-

cedented opportunities to investigate differential gene expression

during developmental transitions and inherent single-cell vari-

ability within a particular life cycle stage.

Huge improvements have been made to imaging techniques

and the fast pace of development is truly remarkable. In T. brucei,

protein and DNA loci have been recently tracked at high res o-

lution, using confocal-based or structured illumination micros-

copy (XY resolution 100–120 nm), which has been critical to

characterize specific sub-nuclear compartments (Glover et al.,

2016; Budzak et al., 2019; Faria et al., 2019, 2020). But there is

a growing need for methods that can image chromosomes with

greater genomic and optical resolution; super resolution micros-

copy can now allow an XY resolution as low as 20–30 nm. To

understand how the genome functions and regulates several key

biological processes, it is necessary to visualize many genomic

regions simultaneously, not just a few. Recently, there have been

huge breakthroughs in other systems, such as OligoFISSEQ, a

combination of three methods that employ fluorescence in situ

sequencing (FISSEQ) of barcoded Oligopaint probes to enable

the rapid visualization of multiple targeted genomic regions

(Nguyen et al., 2020). Another powerful technique is electron

cryotomography, an imaging technique used to produce high-

resolution 3D views of samples, typically biological macromole-

cules and cells. In trypanosomatids, it has been used to study fla-

gellar and mitochondrial structures but to my knowledge, not to

study supramolecular sub-nuclear complexes. For instance in

humans, it was extensively used to study the human NPC

(reviewed by Lin and Hoelz, 2019

The clustered regularly interspaced short palindromic repeats

(CRISPR)/CRISPR-associated system (Cas) technology has revo-

lutionized molecular biology; indeed, it is a powerful tool that

allows highly efficient and reproducible manipulation of genomic

sequences for both locus-specific and genome-wide approaches.

But its huge potential is not exclusively linked to the site-directed

nuclease activity. A catalytically inactive Cas9 (dCas9) can be used

as a universal recruitment platform in order to control transcrip-

tion, visualize DNA sequences or investigate in situ proteomes

(Anton et al., 2018; Martens et al., 2019). Indeed, for the identi-

fication of locus-associated proteins, dCas9 can be fused to a

FLAG-tag and targeted to a locus of interest; chromatin is then

crosslinked and fragmented; dCas9-bound chromatin fragments

are subsequently isolated by FLAG-specific antibodies and ana-

lysed via mass spectrometry (enChIP) (Anton et al., 2018).

Unlike enChIP, CasID requires the expression of dCas9 fused to

the promiscuous biotin ligase BirA*. After the culture medium

has been supplemented with exogenous biotin, BirA* catalyses

the addition of biotin to lysine residues of proteins that are in

close proximity to the dCas9-BirA* fusion protein. Lysis of the

cells and denaturation of proteins is then followed by affinity puri-

fication of biotinylated peptides, which are identified via tandem

mass spectrometry (Anton et al., 2018; Trinkle-Mulcahy, 2019).

Indeed, this DNA-centric system can be used to pull-down pro-

teins that associate with a specific locus; taking the VSG-ESs as

an example, this system could help identifying factors specifically

associated with the active or silent-ESs and factors involved in

gene activation or gene silencing.

Additionally, several different dCas9-based systems have been

developed to perform programmable control of spatial genome

organization, among those is the CRISPR-genome organization

(CRISPR-GO) system. It delivers a highly efficient and versatile

control over the spatial positioning of genomic loci relative to spe-

cific nuclear compartments, including the nuclear periphery, CBs

and promyelocytic leukaemia bodies to study how nuclear struc-

ture affects gene regulation and cellular function (Wang et al.,

2018). For example, in T. brucei, this could be used to bring gen-

omic loci in proximity to the SL-array or the NL and assess how

that impacts gene expression. Recently, a CasDrop system was

designed to study the formation of phase-separated compart-

ments in the nucleus by enabling liquid condensation of tran-

scriptional regulators at target loci (Shin et al., 2018). For

example, in T. brucei, this could be used to investigate the forma-

tion of VEX2 protein condensates at the active VSG-ES. CRISPR/

Cas9 technology has been successfully adapted to trypanosomes

(reviewed by Bryant et al., 2019) and proven highly versatile; it

will be interesting to see the future developments.

In summary, huge technological advances have been accom-

plished in the recent years and certainly many more will in the

near future. This burst of technological breakthroughs will hope-

fully pave the way for future discoveries on nuclear and genome

organization as well as gene expression control in African trypa-

nosomes and related organisms.

1248 Joana R. C. Faria

https://doi.org/10.1017/S0031182020002437 Published online by Cambridge University Press

Concluding remarks

Trypanosoma brucei nuclear organization and gene expression

present several striking differences when compared to more com -

plex eukaryotes. Multiple lines of evidence strongly support that

its monogenic antigen transcription, which is critical for success-

ful antigenic variation, is enforced and facilitated by a key nuclear

architecture that involves specific inter-chromosomal interactions

and compartmentalization (possibly also modification) of specific

factors.

The molecular understanding of the mechanisms underpin-

ning gene expression control in different developmental stages

of these parasites is of great importance, as it might aid future vac-

cine and drug development efforts. For instance, acoziborole, a

single-dose oral drug to treat trypanosomiasis, was shown to tar-

get cleavage and polyadenylation specificity factor 3 (Wall et al.,

2018). Therefore, RNA process ing is now established as a clinic-

ally validated drug target in the African trypanosome.

Understanding the context within which drugs work can greatly

facilitate the drug discovery process.

Notably, recent technological advances on sequencing,

imaging and affinity purification techniques have led to important

discoveries and paved the way to novel research avenues regarding

nuclear organization and gene expression control in trypano-

somes. Indeed, we live in exciting times where the pace of tech-

nology development is phenomenal and hopefully will allow us

to address long-standing questions in infection biology that

were previously inaccessible.

Acknowledgements. I would like to thank David Horn for the many excit-

ing discussions about this subject and I would like to apologize to those whose

work was not cited because of content and length constraints.

Financial support. J.R.C.F. is a senior research associate funded by a

Wellcome Trust Investigator Award to Professor David Horn (100320/Z/12/Z).

Conflict of interest. None.

Ethical standards. Not applicable.

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