Table Of ContentExtra ViEw
Nucleus 3:6, 478–486; November/December 2012; © 2012 Landes Bioscience
Telomeres, tethers and trypanosomes
Mark C. Field,1,* David Horn,2 Sam Alsford,2 Ludek Koreny1 and Michael P. Rout3
1Department of Pathology; University of Cambridge; Cambridge, UK; 2London School of Hygiene & Tropical Medicine; London, UK; 3The Rockefeller
University; New York, NY USA
Temporal and spatial organization of expression mechanisms. We discuss these
the nucleus is critical for the con- and other recent findings on the organi-
trol of transcription, mRNA processing zation of nuclei in diverse organisms, and
and the assembly of ribosomes. This the implications of these findings for the
includes the occupancy of specific terri- evolutionary origin of eukaryotes.
tories by mammalian chromosomes, the
presence of subnuclear compartments Overview
such as the nucleolus and Cajal bodies
and the division of chromatin between Even in the earliest explorations of eukary-
active and inactive states. These latter otic ultrastructure it was clear that the
are commonly associated with the loca- nucleus, the defining organelle of eukary-
tion of DNA within euchromatin and otes, can and does differ widely in size and
heterochromatin respectively; critically morphology between different species.
these distinctions arise through modifi- These nuclei do, however, seem to share a
cations to chromatin-associated proteins, number of certain features in the electron
including histones, as well as the pref- microscope: every nucleus is bounded by
erential localization of heterochromatin a double-membraned nuclear envelope
at the nuclear periphery. Most research (NE), perforated by nuclear pore com-
on nuclear organization has focused on plexes (NPCs) and often underlayered on
metazoa and fungi; however, recent tech- the nuclear periphery by a dense lamina.1-4
nical advances have made more diver- Electron dense bodies such as the nucleolus
gent eukaryotes accessible to study, with are commonly found in the nuclear inte-
some surprising results. For example, the rior; and even the earliest light microscopic
organization of heterochromatin is medi- studies recognized that chromatin, the
ated in metazoan nuclei in large part by nucleus’ packaged DNA, seemed to come
lamins, the prototypical intermediate in less and more densely packaged forms,
filament proteins. Despite the presence termed euchromatin and heterochroma-
Keywords: evolution, trypanosoma, anti-
of heterochromatin, detected both bio- tin respectively.5 Though all these features
genic variation, lamina, nuclear envelope
chemically and by EM in most eukary- are clearly present in the model metazoan
Submitted: 08/10/12 otic organisms, until this year lamins organisms, such as vertebrate, fly, and
Revised: 09/03/12 were thought to be restricted to metazoan nematode cells, stark differences emerge
taxa, and the proteins comprising the when we look at another favorite group
Accepted: 09/11/12
lamina in other lineages were unknown. of organisms, the fungi. The model yeast
http://dx.doi.org/10.4161/nucl.22167 Recent work indicates the presence of Saccharomyces lacks both a lamina and
*Correspondence to: Mark C. Field; lamin orthologs in amoeba, while try- obvious heterochromatin, and the NPCs
Email: [email protected] panosomatids possess a large coiled-coil seem far smaller than those in metazoa.6,7
protein, NUP-1, that performs functions Other major differences appear during
Extra View to: DuBois KN, Alsford S, Holden JM, analogous to lamins. These data indicate cell division. Thus, metazoan organisms
Buisson J, Swiderski M, Bart JM, et al. NUP-1 Is a
that the presence of a nuclear lamina generally disassemble their NE and NPCs
large coiled-coil nucleoskeletal protein in try-
is substantially more widespread than completely at the onset of mitosis to allow
panosomes with lamin-like functions. PLoS Biol
2012; 10:e1001287; PMID:22479148; http://dx.doi. previously thought, with major implica- the spindle to assemble and segregate the
org/10.1371/journal.pbio.1001287. tions for the evolution of eukaryotic gene chromosomes. A new NE and the NPCs
478 Nucleus Volume 3 issue 6
Extra ViEw Extra ViEw
Figure 1. Evolutionary histories of known nuclear envelope components. (A) Cartoon of structures at, and associated with, the nuclear envelope.
Shown are the nuclear pore complex/karyopherins (red), Sun/Kash (LiNC complex) proteins (blue) and known components of a nuclear lamina (lamins,
green; NUP-1, yellow). (B) the evolutionry distributions of the nuclear pore complex/karyopherins, Sun/Kash proteins and known nuclear lamina pro-
teins are shown colorized and overlaid upon a schematic phylogeny of the eukaryotes, emphasizing the five contemporary-recognized supergroups.
FECa, First eukaryotic common ancestor; LECa, Last eukaryotic common ancestor; KaP, karyopherin; Sar + CCtH, stramenopiles alveolates, and
rhizaria + cryptomonads, centrohelids, telonemids and haptophytes.
reassemble around the daughter chromo- the way of a molecular characterization.11 seem far more conserved than appearances
somes at the end of mitosis. By contrast, Thus, our normal opisthokont-centric suggested from the rather divergent tran-
both the NE and NPCs remain intact view, focusing on those cells and organ- scriptional mechanisms in trypanosomes,
throughout the entire cell cycle of yeast, isms most closely related to our selves, i.e., but that there are crucial differences. These
with the spindle forming inside the unbro- animals and fungi, may be inadequate in advances allow us not only to view the
ken nucleus (see refs. 8 and 9 for reviews). attempting to understand these economi- whole tree of eukaryotic evolution anew,
Yeast and metazoa are actually rather cally important and human-impacting dis- but shed new light on how our own cells
close cousins from the perspective of the ease organisms. Further, as many protists are organized and function, with therapeu-
entire eukaryotic evolutionary tree, both are major components of the biosphere, tic implications not only for these diver-
sharing the opisthokont branch or super- their overall impact on the environment is gent parasites but also for ourselves.
group10 (Fig. 1). However, many unicellu- immense. Here we use one such divergent
lar parasitic protozoa are highly divergent organism, Trypanosoma brucei, as a per- Transcription: Not All the Same
eukaryotes, and many sources have sug- spective on this issue. Remarkably, recent
gested that their nuclear organization is advances have revealed that many of the Trypanosomes are members of the
markedly different, albeit lacking much in underlying structures and systems in nuclei Euglenozoa, a group of organisms that
www.landesbioscience.com Nucleus 479
belongs to the Excavata supergroup than the more usual RNA pol II which is manner and the divergent evolutionary
(Fig. 1). They most likely branched very responsible for transcribing the majority position of trypanosomes suggests the
early during eukaryotic evolution, which of PTUs. Both VSG and procyclin are presence of unusual mechanisms at the
in part may explain some of their more developmentally regulated, with the for- nuclear periphery.
unusual biology.10,12 Euglenozoa have a mer expressed exclusively in mammalian
truly global impact on the environment, infective forms and the latter in insect Conservation and Divergence
economy and public health. Species include vector stages. While procyclin ORFs are at the Nuclear Periphery
the photosynthetic Euglenids, which embedded within the body of the chro-
populate most fresh water environments, mosomes and consist of small loci of a Although it had been suggested from in
free living phagotrophs, such as Bodo handful of ORFs, VSG transcription is silico analysis that the nuclear transport
saltans, and plant and animal parasites, substantially more complex.15,25 system of trypanosomes had little in com-
the Phytomonads and Trypanosomatids, VSG is the molecular basis for anti- mon with that of opisthokonts,29 a com-
respectively.13 Trypanosoma brucei has genic variation, the primary immune eva- bination of proteomics, structural studies,
emerged as the model organism for this sion mechanism in these organisms. The and comparative genomics has revealed
group, on account of both its impor- sequential expression of distinct VSGs, in that the NPC and associated karyopherin
tance as the causative agent of human a semistochastic manner, serves to allow transport factors are an ancient feature,
African trypanosomiasis, and from tech- escape from immune destruction at the and that the overall configuration of this
nical advances that include robust RNAi population level, and which appears to organelle and the transport mechanism
systems, excellent cytology and, most rely on monoallelic expression, i.e., a sin- and diversity of pathways were established
recently, forward genetics based on whole gle VSG expressed in any one cell at any in the last eukaryotic common ancestor
genome RNAi screening.14 Accordingly, one time. Key to this is the restriction of (termed LECA). By contrast the lamina
our understanding of transcription and transcriptionally active VSG ORFs to sub- and associated macromolecules appear to
chromatin organization is comparatively telomeric expression sites (ES), where the be more divergent.
advanced for T. brucei, although major VSG is the most telomere-proximal ORF The basic NPC architecture, in terms
gaps remain in our knowledge.15-17 in a pol I-driven PTU. Selection of a sin- of division into a structural scaffold, con-
As far as we know, all trypanosoma- gle VSG is achieved by repression of most sisting predominantly of protocoatomer-
tids exploit primarily polycistronic tran- VSG ESs within heterochromatin, and related proteins, together with a gating
scription, where large (tens) numbers of the translocation of the active expression system comprising about ten disordered
open reading frames are transcribed in site into a specialized region of the nucleus FG repeat-containing nucleoporins,
tandem. There is little evidence for func- called the expression site body (ESB).15,26,27 is conserved across the eukaryotes.30,31
tional association between ORFs in indi- It is likely that the positioning of VSG ESs Further, the karyopherin family is like-
vidual polycistronic transcription units at the nuclear periphery, by virtue of their wise ancient and, perhaps more remark-
(PTUs), although evidence suggests that subtelomeric location, places the inactive ably, based on an ability to reconstruct the
the position within the PTUs is associ- ESs within the heterochromatin. Both majority of karyopherin clades as being
ated with mRNA copy number.18 The telomere suppression and VSG-specific widely represented across the eukaryotes,
nascent RNAs that are produced from repression mechanisms appear to operate the level of diversity and specificity in
the PTUs are processed by a combination (see below). Further, the procyclin locus, nucleocytoplasmic transport is both well
of trans-splicing and polyadenylation to which is also transcribed by Pol I, is also conserved across the eukaryotic lineage
yield mature mRNAs that, bar an unusual subjected to control via positioning within and a feature of the LECA.32
5' cap structure,19,20 are organizationally the nucleus; when active, i.e., in the insect, What these studies imply is that the
indistinguishable from higher eukaryotes. the locus resides peripherally to the nucle- plural mechanisms of nucleocytoplasmic
However, this mode of transcription does olus.28 While definitive data are lacking, transport, which are intimately wrapped
mean that much of the genome is constitu- all evidence points to a peripheral nuclear up with mRNA processing and translo-
tively transcribed, and mRNA abundance position for the locus in the mammalian cation into the cytoplasm and hence gene
is primarily controlled at the post-tran- infective stages, where it is (relatively) expression, are extremely ancient. Minor
scriptional level,21-24 in sharp contrast to inactive. differences in the compositions between
metazoan and yeast model systems, where While polycistronic transcription is mammalian, yeast, plant and protozoan
the transcription of each gene is usually certainly not unique to trypanosomes, NPCs may signal some diversification/
highly regulated by a dedicated promoter. the high proportion of the genome tran- adaptation of mechanism that reflects the
Notable exceptions to this mechanism scribed by this mechanism is highly differing transcriptional systems in place
are the loci encoding the superabundant unusual. However, taken with the pres- in these various lineages, but at a level
surface antigens, the variant surface gly- ence of pol I-mediated transcription of that is quite detailed and one that is not
coprotein (VSG) and procyclin; these protein-coding genes, exploitation of 4D immediately obvious or accessible from a
molecules are expressed at very high lev- positioning of loci encoding super-abun- study of the proteins comprising the NPC
els, and rather remarkably, their transcrip- dant surface antigens whose expression and in silico approaches alone. In part,
tion is driven by RNA polymerase I, rather is modulated in a life cycle dependent this is due to our rather limited grasp of
480 Nucleus Volume 3 issue 6
This study also provides a salient lesson as
well—identification of divergent proteins
and detecting an ortholog is often chal-
lenging; the tools available have especial
difficulty with coiled coil proteins. This
topic is discussed further in a recent Extra
View article in these pages to which the
interested reader is referred.37
In the remaining supergroups, the
bikonts, there is so far only one example
of a lamina-associated protein for which
convincing functional evidence is avail-
able. This is the trypanosome NUP-1
protein, which actually has structural
similarities with and fulfills many of the
Figure 2. relative sizes of metazoan and trypanosomatid nuclei and lamina monomers. Blue ovals reported functions of lamins. NUP-1
indicate the relative sizes of a fibroblast and trypanosome nucleus, at 10 μm and 1.5 μm in diam- forms a fenestrated, apparently fibrillar,
eter. the molecular weight of the unikont lamins is approximately 60 kDa while NUP-1 is 450 kDa;
network on the inner face of the nucleus,
these are indicated as white bars close to the indicator lines for each nucleus. the inference is that
and is implicated in functions includ-
the structural arrangement of the lamina in these systems is potentially very different.
ing maintaining the structural integrity
of the nucleus, positioning of NPCs and
the precise functions of many individual requirement for structural support of the telomeres, and mediating telomeric silenc-
nucleoporins themselves, and it may well nucleus (though yeast does manage with- ing. Interactome analysis also indicates a
be that some functionality is redundant, out apparent support), a knowledge of the physical association between the NPC and
such that the presence of a specific subunit molecular basis was restricted to meta- NUP-1 (Obado S, MCF, MPR and Chait
from one organism has little or no clear zoa, where the lamin proteins have been BT, unpublished). In trypanosomes, the
impact on functionality; this is certainly well characterized.35,36 Despite extensive role in telomere positioning is particularly
an area where more investigation is war- searches of the genomes of yeasts and critical as monoallelic VSG expression is
ranted. Remarkably, the conservation many other organisms, no evidence for dependent on heterochomatinization of
within NPC functionality even extends lamin orthologs had been forthcoming, subtelomeric VSG expression sites, and
to moonlighting nucleoporins in trypano- leading to the suggestion that lamins rep- indeed there is good evidence that this
somes. We have evidence for a nucleopo- resent a metazoan innovation. process is disrupted by knockdown of
rin, TbNUP92, that appears functionally Two studies published in 2012 have, NUP-1.39
similar to the mitotic spindle-associated however, overturned this paradigm. While the parallels between NUP-1
TPR nuclear basket proteins, despite an First, through a serendipitous route, a and unikont lamins are striking, there
absence of obvious sequence-based homol- clear lamin ortholog was identified for are some critical differences. First, there is
ogy and also an FG-repeat nucleoporin, Dictyostelium discoideum, which even no obvious sequence relatedness and the
TbNUP53b, potentially involved in tran- includes a C-terminal prenylation signal, amino acid repeats in T. brucei are 144
scriptional control and hence analogous heptad repeat signatures and a poten- residues, significantly larger than the hep-
to the NUP98 proto-oncogene from tial CDK-1 phosphorylation site con- tad repeats of the lamins. Second, NUP-1
Drosophila, albeit again with no obvious sensus;37,38 orthologs are also present in is a huge protein of 407 kDa, compared
primary structural similarity (Holden J, additional Amoebozoa genomes. The with ~60 kDa for lamins, which means
MPR and MCF, unpublished data, and significance of this discovery is far reach- that NUP-1 can reach across a substantial
refs. 9, 33 and 34). It is unclear if these ing. With lamins restricted to Metazoa, proportion of the trypanosome nuclear
functional similarities in the absence of lineage-specific innovation is the obvious volume, and hence may indicate a rather
evidence for sequence relatedness are a explanation, but the new data suggest that distinct structural organization to the
result of convergent evolution or a fail- in fact lamins arose several hundred mil- lamins (Fig. 2). However, this may in
ure in search algorithms to detect such lion years earlier, at least as early as the ori- part be specific to trypanosomes as the
similarity. gin of the unikont lineage, which includes orthologs in Leishmania are considerably
In sharp contrast to the NPC/karyo- the Opisthokonta and Amoebozoa. smaller, albeit still predicted to contain
pherin system, there is good evidence Clearly then, the absence from fungi can over 2,000 residues. Hence, while there is
for significant divergence in the nuclear now be interpreted as a secondary loss, no evidence for any relationship or com-
lamina. While a nuclear lamina has and has profound implications for our mon ancestry between NUP-1 and the
been clearly detectable by ultrastruc- views on gene expression mechanisms and lamins, we remain agnostic on this issue.
tural analysis in various eukaryotes, and lifestyles of rather early eukaryotes close to Also, as mitosis is closed in trypano-
might be expected, in the sense of a basic the radiation of the modern supergroups. somes, the NUP-1 network does not break
www.landesbioscience.com Nucleus 481
down as does the mammalian lamin net- in trypanosomatids as well as elsewhere. VSGs are thought to be associated with
work, but rather it is maintained through- This is perhaps consistent with the restric- the nuclear periphery while the active
out mitosis, where it may participate in tion of the NUP-1/NUP-2 lamina to VSG is found in the ESB.26 However, no
chromosomal segregation (Holden J and Trypanosoma and perhaps argues against ESB-specific factor has been described,
MCF, unpublished data). The absence convergent evolution from a common and cells depleted for the telomeric-repeat
of a massive re-assortment of the nuclear lamin/SUN/KASH system. If this is the DNA binding protein, RAP1, that is
envelope during mitosis is quite signifi- result of loss from a LECA possessing a required for full telomeric VSG silenc-
cant in terms of maintaining epigenetic lamin-based system, or reflects differential ing, form multiple ESBs.46 This then
marks and the mechanisms by which evolution of the lamina in the trypano- provides support for the idea that RNA
specific DNA sequences are incorporated somatid and unikont lineages remains to Pol I recruitment and ESB formation are
into heterochromatin, as in metazoa sev- be determined. It is also possible that, as consequences of activation. Thus, current
eral lamin-dependent mechanisms that trypanosomes likely branched early from observations are consistent with a tether-
deliver loci to heterochromatin occur as the eukaryotic lineage,11 that evolution of ing-untethering model as outlined above,
a component of mitosis and reassembly the lamin/SUN/KASH system postdates but it remains to be seen whether activa-
of the nuclear envelope at late anaphase, their speciation event. However, the dis- tion and untethering, if it happens, occur
which clearly cannot be the case for try- covery of the D. discoideum lamin is a fur- in a specific order or if they occur con-
panosomes.40 Hence it is unclear just how ther salient lesson in relying too heavily on comitantly. Significantly here, no increase
many of the proteins that control entry, in silico data alone, if any more are really in the number of ESBs was observed fol-
exit and maintenance of heterochromatin needed at this juncture, and resolution of lowing NUP-1 knockdown, suggesting a
are shared across the eukaryotes. However, this issue will require deeper experimental distinct mechanism to RAP1.
it seems to us a key point that peripheral probing. In summary, the nuclear periph- There is increasing evidence that basal
organization of developmentally-regu- ery of the trypanosomes appears to be a telomeric silencing in T. brucei and VSG
lated chromatin (kept inactivated as het- mixture of highly conserved elements, gene silencing are mechanistically distinct.
erochromatin until needed) at a nuclear together with other elements that may be The first evidence in support of this dis-
lamina composed of coiled-coil proteins is lineage specific, or are at the very least, tinction came from studies on telomeres
a shared feature of representative unikont extremely divergent (Fig. 1). themselves, which could be removed,
and bikont organisms. disrupting basal telomeric silencing, but
A final aspect here are the LINC Telomeres and Gene Silencing without disrupting VSG silencing.47 A
complex proteins.41 LINC complexes second piece of evidence derives from
are comprised of trimers of SUN and Given the evidence that NUP-1 is involved studies on the histone deacetylase, Silent
KASH domain proteins; these trimers in the positioning of telomeres, NPCs and Information Regulator 2 related protein 1
then interact within the lumenal space also the repression of both the VSG and (SIR2rp1), which is required to maintain
of the nuclear envelope.42,43 These trans- procyclin loci, it is important to consider basal telomeric silencing in many organ-
membrane domain proteins are respon- how the T. brucei lamina impacts singu- isms but had no major impact on VSG
sible for providing a physical tether for the lar, mono-telomeric VSG expression and silencing.48 Subsequent reports, including
lamina to the nuclear envelope, as well as organizes chromatin in a broader con- the NUP-1 study, have reinforced this dis-
a connection between the nuclear lamina text. It seems unlikely that a lamina itself tinction.45 Interestingly, studies on NUP-1
and the cytoskeleton. Significantly, SUN would directly control allelic exclusion, suggest that it is only VSG silencing that is
and/or KASH domain proteins with the but tethering could certainly be important dependent on the intact nuclear lamina.39
same architecture as those present at the for maintaining the silencing of all but one It remains possible that basal telomeric
nuclear envelope of metazoa are found of the telomeric VSG genes. For example, silencing is dependent upon another com-
in all eukaryotic lineages (including a single telomeric VSG may “untether” as ponent of the nuclear lamina but charac-
Excavata lineages such as the diplomo- part of the process that allows transcrip- terization of these other components will
nads and heterolobosids), with the excep- tional activation. A swap in tethering be required to determine whether this is
tion of the trypanosomatids (Fig. 3). By could then allow transcription to switch indeed the case.
contrast, SUN-like proteins, have dif- from one telomeric VSG to another, a It is clear that the identification of
ferent architecture to classical SUN pro- process known as in situ switching.45 It is regulatory factors provides a “handle” to
teins (Fig. 3) and their function is likely worth considering what is known about start to dissect the process in question.
distinct from the classical SUN proteins the sub-nuclear context of the active and The lamina component, NUP-1, is now
of the LINC complex,44 and are found silent telomeric VSGs in this regard. Silent known to play a role in VSG silencing, and
Figure 3 (See opposite page). Maximum likelihood phylogenetic tree of the SUN domains. the values supporting the separation into two main
subfamilies are: approximate likelihood ratio test calculated with PhyML 3.0/non-parametric bootstrap calculated in raxML 7.2.6/posterior probabili-
ties from PhyloBayes 3.3b. the right panel depicts the domain structures of SUN-domain containing proteins of the representative species used in the
phylogenetic analyses. Position of the SUN domain is symbolized by the blue box. red rectangles represent hydrophobic patches predicted as trans-
membrane domains by tMHMM Server v. 2.0. Dashed red rectangles represent hydrophobic patches, which were not predicted as transmembrane
domains. red taxa are species where considerable characterization of the SUN proteins has been achieved.
482 Nucleus Volume 3 issue 6
Figure 3. For figure legend, see page 482.
www.landesbioscience.com Nucleus 483
other observations are consistent with the DNA content of the nucleus,57 but have of the differential positioning and kinet-
idea that the T. brucei nuclear lamina is no known function. In common with the ics of replication of the ESB. In contrast
important for organizing and reducing the MCs, they are highly repetitive, being to the MBCs and MCs, there is no evi-
mobility of many subtelomeric VSG genes made up of short repeats of variable size. dence that the NR-elements interact with
within the confines of the nuclear space. These different classes of nuclear DNA the nuclear lamina. Indeed, their circular
It will be important to identify lamina- exhibit significant variation in segrega- structure and consequent lack of telomeres
associating sequences and lamina-associ- tion during mitosis and their subsequent may preclude any such interaction, result-
ated domains49 in T. brucei and structural organization in the interphase nucleus. ing in their dispersal during interphase.56
or regulatory RNAs should probably also The MBCs are segregated by a spindle-
be considered in possible models of VSG kinetochore interaction via microtubules Perspectives
expression control.50 Ultimately, any peripheral to the main central spindle,58
model that seeks to explain mono-telo- so enabling the telomeric regions of the Our understanding of the evolution of
meric and switchable VSG transcription, MBCs to maintain their association with the NE has advanced considerably in the
a system that requires communication the nuclear lamina during mitosis and past five years, with 2012 being a particu-
among telomeric genes, will likely require remain within the established heterochro- larly good year for moving forward in our
the identification of new key regulators matin throughout the cell cycle, although understanding of the evolutionary biol-
and will also need to deal with issues relat- to date components of the trypanosome ogy of the lamina. The old paradigm of
ing to cause and effect. Whatever comes kinetochore remain uncharacterized. This lamins as metazoa specific is now clearly
next, we suspect that further characteriza- arrangement may serve to prevent inter- obsolete, and there is robust evidence for
tion of the T. brucei lamina will make an action with the active VSG ES, which a lamina in at least one branch of the
important contribution to improving our localizes to the extranucleolar expression Excavata. Many gaps remain, and we
understanding of this complex process. site body, the site of RNA polymerase I currently lack definitive information on
driven expression of the active VSG,36,59 the compositions of the lamina of plants,
Chromosome Positioning and which replicates later during the cell Chromalveolates, Stramenopiles and sev-
and Genome Segregation cycle.27 eral Excavata lineages, despite the clear
By contrast the MCs and NR-elements evidence for heterochromatin in these
The structure and segregation of the segregate along the central spindle and taxa. In the case of Plasmodium, a mem-
T. brucei nuclear genome, suggests the rapidly migrate to the poles of the daugh- ber of the chromalveolates, a role for telo-
possibility of differential interactions with ter nuclei, after which the MCs maintain meric positioning in antigenic variation
the nuclear lamina, with implications an asymmetric distribution during inter- and with gross similarities to African try-
for recombination and gene expression. phase, and the NR-elements disperse panosomes is well known, but the absence
The 32 Mbp haploid nuclear genome of throughout the interphase nucleus.54,58 of a molecular definition of a lamina is a
T. brucei is distributed across more than Compared with the MBCs, the interac- significant gap in comprehending the
120 linear chromosomes,51 divided into tion of the MCs with the nuclear lamina virulence mechanisms of a major patho-
three classes: 11 pairs of megabase chro- therefore appears episodic, with the asso- gen.60-62 Much novel biology and many
mosomes (MBC) of at least a megabase in ciation breaking down during mitosis as surprises likely await the investigations
size, one to five intermediate sized chro- they migrate along the central spindle, of the lamina in these organisms. The
mosomes (IC), and more than 100 mini- and then becoming re-established upon rather unusual minichromosomes, which
chromosomes (MC) of 50 to 150 kbp.52,53 completion of mitosis, as indicated by a contribute to antigenic variation, are an
The MBCs contain all of the housekeep- persistent asymmetric distribution during important aspect of the biology of try-
ing genes, as well as about 15 telomere- interphase. The VSG genes on the MCs panosomes, and may have had a strong
proximal VSG ESs.54 The MCs are highly are not in ESs meaning that they can- influence of the evolution of the nuclear
repetitive in nature and constructed not be expressed from these loci, so any lamina in these taxa, potentially to avoid
around a palindromic arrangement of potential loss of their heterochromatin promiscuous recombination, reactiva-
a characteristic 177 bp direct repeat;53 state during mitosis will presumably not tion and other defects that could disrupt
many also contain VSG genes, providing lead to expression, negating a need for antigenic variation. However, genome
a library of distinct variants. The inter- this level of repression. Instead, periodic structure alone cannot explain the high
mediate chromosomes also carry VSG dissociation from the nuclear lamina may divergence between lamin-based and
genes, though in contrast to the MCs, allow for recombination between MCs, NUP-1-based lamina as, for example,
these are present in ESs.55 In addition to potentially serving to generate further minichromosomes are absent from T.
the linear chromosomes, the T. brucei VSG variants. Establishment of putative cruzi, a related trypanosomatid that has a
nucleus contains an unknown number MC-lamina interactions may also allow clear NUP-1 ortholog.
of circular extra-chromosomal DNAs recombination with silent VSG ESs also In terms of defining the functions of
termed NlaIII repeat (NR)-elements of present in the peripheral heterochromatin, the trypanosomatid NUP-1 lamina, a pre-
up to 400 kbp in size.56 The NR-elements but likely also prevents disruption of the requisite for deeper comprehension of how
contribute approximately 6% of the total active VSG during interphase on account related the mechanisms of organization of
484 Nucleus Volume 3 issue 6
chromatin truly are in trypanosomes and expression is not needed, or perhaps more biosphere that contributes to stabilizing
unikonts, it will be essential to probe the critically, would be detrimental if allowed Earth’s modern climates.65 It is also pos-
interactions of these proteins in greater to occur; the dynamic range of hetero- sible that the specific location that LECA
detail. This will necessitate interrogation chromatin suppression is very much found itself in had specific challenges that
of how trypanosome lamina components greater than simple promoter-based inac- necessitated periodic modulations of gene
operate in the context of specific DNA tivation. Hemoglobin genes are thus nor- expression patterns, for example salinity
sequences, determination of the nature mally inactive in the nuclear peripheral or seasonal changes. We speculate that
and distribution of lamina-targeting heterochromatin of all but hemopoetic perhaps LECA evolved to change its form
sequences (if such exist), how the NUP-1 cells while cell adhesion molecules that and function to adapt to these shifting
lamina organizes chromatin-modifying specify cell type and position are similarly environments. An ability to store great
activities, and how this system integrates inactivated during metazoan embryo- amounts of genetic information to be
with the nuclear envelope and spindle. genesis.63,64 But LECA was undoubtedly used upon need could have been the path
Answering these questions is most likely unicellular, and therefore these sophisti- to the eukaryotic colonization of diverse
a long journey, but the identification of cated multicellular organism-related pro- land and sea environments, and the basis
NUP-1 as a lamina component has finally cesses cannot explain the need for such for the multicellularity required for the
provided a doorway through which we tight regulation. Likewise, the parasitic complex lifeforms that enabled such
can begin that journey. lifestyle of trypanosomes necessitates colonization.
Lastly, we can ask: Why do eukary- massive remodeling to accommodate
otes seem to share, as a most fundamental drastically different insect and vertebrate Acknowledgments
mechanism of genetic control, develop- host environments, but again LECA pre- We thank Sam Obado and Jenny Holden
mental regulation by packaging of huge dates multicellularity, and hence such for discussions and access to unpublished
blocks of chromatin at the nuclear periph- parasitic lifestyles. However, the inference data. Work described in this article that
ery, a periphery organized in many cases that LECA possessed such a developmen- has arisen from the authors’ laboratories
by an extensive coiled-coil protein net- tal regulation mechanism indicates that has been funded by the Wellcome Trust
work? Such developmental heterochro- it could undergo significant changes in (082813 to M.C.F., 093010 to D.H.),
matin in “higher” eukaryotes is used to its lifestyle. The primordial environment the National Institutes of Health (R21
store the genetic information needed to LECA faced was potentially much more AI096069, U54 GM103511 to M.P.R.)
build tissues. Cohorts of tissue-specific unstable and chaotic than most organ- and by a Marie Curie fellowship (to LK).
genes are packaged away in lineages where isms face nowadays, as it is our extensive This support is gratefully acknowledged.
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