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Identification of the regulatory elements controlling
the transmission stage-specific gene expression
of PAD1 in Trypanosoma brucei
Paula MacGregor and Keith R. Matthews*
Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research,
School of Biological Sciences, University of Edinburgh, King’s buildings, West Mains Road, Edinburgh,
EH9 3JTU, UK
Received March 23, 2012; Revised May 10, 2012; Accepted May 11, 2012
ABSTRACT
Trypanosomatid parasites provide an extreme
model for the posttranscriptional control of eukary-
otic gene expression. However, most analysis of
their differential gene regulation has focussed on
comparisons between life-cycle stages that exist
in the blood of mammalian hosts and tsetse flies,
the parasite’s vector. These environments differ
acutely in their temperature, and nutritional,
metabolic and molecular composition. In the blood-
stream, however, a more exquisitely regulated
developmental step occurs: the production of trans-
missible stumpy forms from proliferative slender
forms. This transition occurs in the relatively
homogenous bloodstream environment, with
stumpy-specific gene expression being repressed
until accumulation of a proposed parasite-derived
signal, stumpy induction factor. Here, we have
dissected the regulatory signals that repress the
expression of the stumpy-specific surface trans-
porter PAD1 in slender forms. Using transgenic
parasites capable of stumpy formation we show
that PAD1-repression is mediated by its
30-untranslated region. Dissection of this region in
monomorphic slender forms and pleomorphic
slender and stumpy forms has revealed that two
regulatory regions co-operate to repress PAD1
expression, this being alleviated on exposure to
SIF in pleomorphs or cAMP analogues that act as
stumpy induction factor mimics in monomorphs.
These studies identify elements that regulate tryp-
anosome gene expression during development in
their mammalian host.
INTRODUCTION
Cell-type differentiation is usually driven by an external
cue. In the developmental events within multicellular
organisms, soluble signals such as growth factors, cyto-
kines and hormones can generate paracrine and autocrine
signalling systems that trigger cell-type specialisation (1).
Developmental events can similarly be stimulated in uni-
cellular organisms by cell-derived signals, such as the yeast
mating pheromone (2) and the DIF1 stalk cell differenti-
ation signal in Dictyostelium discoideum (3). However,
unicellular organisms also respond to environmental
cues such as pH, temperature and osmolarity. This is
particularly the case for those organisms that encounter
extreme environmental instability such as Chlamydomonas
spp. that undergo sexual development in response to
nitrogen starvation (4), and dimorphic fungi that alternate
between mould and yeast forms dependent on temperature
(5). Transduction of the resulting signals generates specific
changes in gene expression that elicit the cellular events
associated with developmental adaptation.
Kinetoplastid parasites, infectious agents responsible
for a variety of important tropical and subtropical
diseases, provide important models for development and
developmental gene expression for three reasons. First,
these parasites were among the earliest branching eukary-
otic organisms (6), such that their developmental events
can provide insight into the processes underlying the
differentiation of all eukaryotic organisms. Second, the
genome organisation of these parasites is highly unusual
(7). Specifically, genes are arranged in large groups of
co-transcribed cistrons (polycistronic arrays) whereby
pre-mRNAs are transcribed from often-distant upstream
promoters, individual mRNAs being generated after a con-
certed trans splicing and polyadenylation reaction (8,9).
This organisation dictates that differential gene expression
is controlled predominantly at the posttranscriptional
*To whom correspondence should be addressed. Tel: +44 131 651 3639; Fax: +44 131 651 3670; Email: keith.matthews@ed.ac.uk
Published online 7 June 2012 Nucleic Acids Research, 2012, Vol. 40, No. 16 7705–7717
doi:10.1093/nar/gks533
ßThe Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
level, through regulated mRNA stability and translational
mechanisms (10). Third, kinetoplastid parasites undergo
complex developmental pathways, being transmitted
between mammalian hosts by blood feeding arthropods
(11). These developmental events require elaborate
changes in the parasite’s morphology, metabolism, and
surface protein expression, each being governed by
differential gene expression (12). The important cue for
these changes in different kinetoplastid parasites is the
change in temperature associated with passage from
a homoeothermic to poikilothermic carrier (13,14).
Progression from the bloodstream of mammalian hosts
to the alimentary canal of arthropod vectors is also
associated with major changes in available glucose, pH
and osmolarity, as well as exposure to the proteolytic
and immunological environment of the insect gut
(15,16). These environmental changes stimulate altered
gene expression, the best characterized being the regula-
tion of procyclin surface antigens on African trypano-
somes as they establish in their tsetse fly vector (17).
Here, surface protein expression is controlled by
exposure to glycerol or low oxygen content (GPEET
procyclin) (18) or a temperature reduction of 15C, or
more (EP procyclin) (14).
Most studies of developmental gene expression in
trypanosomes have focused on the differentiation from
bloodstream to procyclic forms in culture, using
‘monomorphic’ bloodstream parasite lines selected for
their uncontrolled growth in vitro and in vivo (19).
However, in natural infections, the transition to procyclic
forms from bloodstream forms requires the production of
specialized transmission stages, called stumpy forms,
which arise in the bloodstream from proliferative slender
forms. Slender forms cannot differentiate in the tsetse
midgut because they are rapidly killed by its digestive
environment (20) and because they cannot detect the
differentiation signal, which comprises citrate/cis
aconitate (14,21). This signal is detected in stumpy forms
because they express a carboxylate surface transporter
family, called PAD proteins, of which PAD1 is only
expressed at significant levels in the transmission stage
(22). The transition from slender forms to stumpy forms
is believed to be triggered by a parasite-derived factor,
stumpy induction factor (SIF) (23,24), which has thus
far eluded identification. Nonetheless, in response to
accumulating SIF, slender cells stop proliferating and
differentiate to stumpy forms, which are characterized
by their morphology, limited mitochondrial elaboration,
resistance to proteases and pH and their expression of
PAD proteins (22,25–28). Since this transformation
occurs in the mammalian blood and is triggered by a
parasite-derived factor, it must be stringently regulated
without the extreme environmental cues that characterize
the transition to procyclic forms. As such, it represents an
exquisitely specific form of developmental regulation,
whereby stumpy expressed genes must be held silent
until repression is released by the accumulation, and
detection, of SIF.
In this article, we have exploited our identification of
PAD1 as the first molecular marker for the parasite trans-
mission stage to examine the gene regulatory signals
that are responsible for stumpy-specific gene expression.
We find that PAD1 is regulated through elements in its
30-untranslated region, which repress its expression in
slender forms, this being alleviated in stumpy forms and
when monomorphic slender forms are exposed to cell per-
meable cAMP analogues believed to mimic activation
of the SIF pathway. This provides a first dissection of
gene expression in the parasite’s transmission stage,
and its regulation in response to highly specific, and
parasite-derived, developmental cues.
MATERIALS AND METHODS
Trypanosome culturing and chemicals
Monomorphic bloodstream form trypanosomes were
Trypanosoma brucei Lister 427. Pleomorphic bloodstream
form trypanosomes used to harvest stumpy RNA were
AnTat1.1, whereas those used in transfections were
T. brucei AnTat1.1 90:13 (14). Parasites were grown rou-
tinely in HMI-9 at 37Cin5%CO
2
(29). Slender forms
were harvested from MF1 mice at 3 days post infection
and stumpy forms were harvested from cyclophospha-
mide-treated MF1 mice at 5 and 6 days post infection.
Trypanosomes were purified from blood by DEAE
cellulose purification (30). For parasite transfection,
10–15 mg of linearized DNA was electroporated using
an Amaxa Nucleofector II. Monomorphic parasites were
selected using 0.5 mg/ml puromycin (pHD617 CAT-PAD1
30-UTR), 3 mg/ml hygromycin (pHD617 GUS Actin
30-UTR) or 0.5 mg/ml phelomycin (all CAT449 reporter
constructs). Pleomorphic parasites were selected with
1.5 mg/ml phleomycin.
Differentiation of bloodstream forms to procyclic forms
was induced by addition of 6 mM cis-acconitate and a
temperature change from 37Cto27
C. Differentiation
capacity was assessed by EP procyclin expression,
measured by antibody staining as described in the study
by Dean et al. (22).
8pCPT-cAMP was purchased from Sigma Aldrich
(UK), 8pCPT-20-O-Me-cAMP was purchased from
BioLog Life Science Institute (Germany). Troglitazone
was purchased from Biomol (Germany).
Analysis of mRNA polyadenylation site
To map the site of PAD1 mRNA polyadenylation, reverse
transcriptase–polymerase chain reaction on stumpy-form
RNA was carried out. The 30-end of the PAD1 transcript
was amplified using a gene-specific oligonucleotide
hybridizing close to the 30-end of the coding region
(50-GAC CAA AGG AAC CTT CTT CCT-30) and a
30oligo-dT ADAPT oligonucleotide hybridizing to the
poly (A) tail (50-GGC CAC GCG TCG ACT AGT
ACT TTT TTT TTT TTT TT-30) (31). Amplified
products were then subjected to a second round of
amplification using an oligonucleotide hybridizing to the
50-end of the PAD1 30-UTR (50-TTA GGA TCC GCT
TAG GGG AGC CAG TGG AGG GC-30) and the
primer AUAP (50-GGC CAC GCG TCG ACT AGT
AC-30), which binds to the specific oligonucleotide
sequence incorporated into the 50-end of the ADAPT
7706 Nucleic Acids Research, 2012, Vol. 40, No. 16
primer (31). The resulting products were gel purified and
sequenced to determine the site of polyadenylation.
DNA cloning and trypanosome transfection
Plasmid constructs in this study were based on the tryp-
anosome expression construct pHD617 (32) and a CAT
449 reporter construct previously described (31). Precise
details of each construct and the associated primers used
for their construction and analysis are provided in
the Supplementary Materials.
CAT and GUS reporter assays
CAT protein levels were determined by CAT ELISA assay
(Roche) according to manufacturer’s instructions, with
absorbance being measured using a BioTek ELx808
Absorbance Microplate Reader. Duplicate wells for each
assay were included to ensure consistency. In all cases, a
CAT standard curve was constructed to ensure all sample
readings were within the linear range of the assay. CAT
standard curves had a linear regression value of 0.99 or
greater.
GUS enzyme activity was measured using
4-methylumbelliferyl-b-D-glucopyranosiduronic acid (MUG)
as a fluorescent substrate. Trypanosome cultures were
incubated 1:1 with the MUG substrate (1 mM MUG,
0.82M Tris–HCl pH8, 0.6% SDS, 0.3 mg/ml BSA) at
37C for 2 hours. Fluorescence was measured with an ex-
citation of 355 nm and emission of 460 nm on a BioTek
Flx800 Fluorescent Microplate Reader.
Northern blotting and quantification
RNA was extracted from trypanosomes using an RNeasy
Mini Kit (Quiagen). Between 0.15 and 1.5 mg purified
RNA was resolved on formaldehyde agarose gels in
MOPS buffer and transferred to nylon membranes by
capillary blotting. A digoxigenin (DIG)-labelled riboprobe
was hybridized to the membrane and the transcript was
detected using an anti-DIG antibody (Roche) with a
chemiluminescent CDP-star substrate (Roche). The tran-
script levels were quantified using a Syngene Gbox with
GeneTools software, using rRNA levels to normalize for
loading.
Data analysis
For all analyses, error bars represent the SEM calculated
using two biological replicates (n= 2), whereas the solid
bars represent the mean of those biological replicates, this
providing an intuitive visual representation of the data.
These biological replicates represent two independently
isolated transfectant cell lines derived from transfection
with the same construct. For both CAT and GUS assay,
values were normalized to cell number in all cases.
RESULTS
Identification of the endogenous polyadenylation site
for PAD1
To identify any regulatory regions in the PAD1 30-UTR, it
was first necessary to identify its polyadenylation site and
thereby define the boundaries of regulatory sequences
within the PAD1 mRNA. Initially, we analysed existing
data describing the polyadenylation sites of almost 6000
genes in slender and procyclic form trypanosomes (33).
Three potential polyadenylation sites were predicted
downstream of the PAD1 gene in procyclic forms, these
being positioned at 428, 572 and 835 nt after the PAD1
termination codon (33). Contrasting with this data,
a 273 nt EST sequence was available (accession number
W00184) from a T. brucei rhodesiense cDNA library
which aligned from positions 437 to 709 in the intergenic
region downstream of PAD1, suggesting that the PAD1
30-UTR polyadenylation site extended at least 709 nt after
the termination codon. In order to resolve this apparent
discrepancy, and to identify the polyadenylation site used
in the relevant life-cycle stage, the PAD1 polyadenylation
site was mapped experimentally using bloodstream
stumpy form cDNA as a template, as previously described
(31). This analysis confirmed positions 707–710 as a PAD1
polyadenylation site, with a further site at positions
722–723 being identified (Figure 1A). This sequence is
contained within a region also found downstream of
PAD5 and PAD7. These intergenic regions contain
several potential alternative processing sites that may
generate non-coding intergenic region-derived RNAs
responsible for the sites mapped in whole transcriptome
studies. The entire region downstream of the PAD1 coding
region to its polyadenylation site did not contain any
motifs previously observed as being present in some
stumpy-enriched transcripts identified by microarray
analysis (34).
The PAD1 30-UTR controls stumpy-specific gene
expression
To identify whether the PAD1 30-UTR was responsible
for stage-specific expression in stumpy forms, it ability
to appropriately regulate a heterologous marker gene
encoding chloramphenicol acetyl transferase was
investigated. To achieve this, a variant of the expression
vector, pHD617 (32), lacking the tetracycline operator
sequence and conferring resistance to puromycin, was
created, to allow transgene expression independently
of the presence of tetracycline induction. Thereafter, the
CAT coding region linked to the PAD1 30-UTR construct
was integrated, and the resulting construct, CAT-PAD1
30-UTR (Figure 1B), transfected into the T. brucei
AnTat1.1 90:13 cell line (a kind gift of Michael Boshart
and Markus Engstler) (14). This is a pleomorphic cell line,
capable of generating uniform populations of stumpy
forms when grown in rodents.
To determine whether the AnTat1.1 90:13 CAT-PAD1
30-UTR reporter cell line exhibited developmentally
regulated CAT expression, two mice were infected with
the cell line. Trypanosomes were harvested from one
mouse on Day 3 post-infection when the population was
predominantly slender, whereas trypanosomes were har-
vested from the second mouse on Day 6 post-infection
when the population was predominantly stumpy in
morphology. Figure 1C demonstrates that slender form
AnTat1.1 90:13 CAT-PAD1 30-UTR cells generated very
Nucleic Acids Research, 2012, Vol. 40, No. 16 7707
little CAT mRNA, consistent with PAD1 30-UTR-
mediated repression of gene expression at this life-cycle
stage. In contrast, the transgenic stumpy forms expressed
a large amount of CAT mRNA at equivalent cell
numbers. This demonstrated that in pleomorphic trypano-
somes, the PAD1 30-UTR is capable of directing the ap-
propriate life-cycle stage regulation of a heterologous
reporter gene.
Mapping of the PAD1 30-UTR silencing elements in
monomorphic slender forms
Given the difficulty in generating transgenic cell lines from
trypanosomes capable of developing to stumpy forms, we
initially investigated the regulatory elements in the PAD1
30-UTR in more detail using transgenic monomorphic
slender lines. These cell lines are readily grown in culture
and transfected and were expected to repress gene expres-
sion of the CAT reporter when linked to the PAD1
30-UTR, matching the scenario in pleomorphic slender
cells. Thus, the full-length PAD1 50-UTR, PAD1
30-UTR, as well as nine sequential deletions of the
PAD1 30-UTR were coupled to the chloramphenicol
acetyl transferase (CAT) reporter gene and incorporated
into an expression vector [CAT449 (31), derived from
pHD449 (32)] designed to integrate into the trypanosome
tubulin locus, ensuring constitutive read-through tran-
scription within a polycistronic polII transcription unit
(Figure 2A). The sequence of the 30-UTR is shown in
Supplementary Figure 1, whereas the predicted Sfold
structures of the intact and deletions of the PAD1
30-UTR are shown in Supplementary Figures S2 and S3.
These constructs were then transfected into monomorphic
trypanosomes, and two stable cell lines derived for each.
Our expectation was that as sequences were deleted in the
PAD1 30-UTR, an alleviation of reporter gene repression
would be observed that would identify elements that
repress gene expression in slender forms.
Figure 2B shows the CAT mRNA levels derived from
each construct, values representing the mean and S.E.M.
of two independent cells lines for each of 11 distinct PAD1
UTR-linked constructs and normalized to the CAT449
control, in which the CAT gene is linked to a truncated
Aldolase 30-UTR. First, matching the earlier observations
using a pHD617-based construct in pleomorphic cells,
inclusion of the full length PAD1 30-UTR caused an
approximately 3.7-fold decrease in CAT mRNA abun-
dance compared with the control construct (i.e. 27.2%
CAT with respect to CAT449, normalized to 100%). In
contrast, replacement of the Aldolase 50-UTR in the
CAT449 control construct with the PAD1 50-UTR did
not repress mRNA expression, with mRNA levels being
164% of control levels. These results confirmed that
repression was mediated in the 30-UTR of the PAD1
transcript.
Progressive deletions into the PAD1 30-UTR from the
50-end initially caused very little effect on CAT mRNA
abundance, such that deletion of the first 207 nt of the
PAD1 30-UTR generated CAT mRNA levels equivalent
to the intact PAD1 30-UTR. Further deletions beyond
207 nt, however, resulted in a progressive increase in
CAT mRNA levels with deletion of 1-502 nt of the
PAD1 30-UTR causing an approximately 8.1-fold
Figure 1. Polyadenylation and devlopmental regulation of PAD1. (A) Identification of the PAD1 polyadenylation sites in stumpy form T. brucei.
Sites mapped experimentally by reverse transcriptase–polymerase chain reaction are shown by black dots, whereas the site indicated by EST analysis
of T. brucei rhodesiense is shown by a white dot. The diagram is not drawn to scale. (B) Developmental regulation of gene expression by the PAD1
30-UTR. Representation of the expression construct used to analyse PAD1 30-UTR effects on gene expression in pleomorphic cells (Figure 2B and C)
or during evaluation of reported chemical inducers of stumpy formation (Figure 6). (C) Slender and stumpy populations of the AnTat1.1 90:13
CAT-PAD1 30-UTR cell line were isolated from mouse infections either on Day 3 (‘Slender’) or Day 6 (‘Stumpy’) post infection. CAT mRNA levels
were elevated in the stumpy population. rRNA labelled with ethidium bromide is shown in the bottom panel to demonstrate relative loading.
‘P’ untransfected trypanosomes, ‘SL’ slender forms, ‘ST’ stumpy forms.
7708 Nucleic Acids Research, 2012, Vol. 40, No. 16
increase in reporter mRNA abundance compared to the
full-length PAD1 30-UTR, consistent with sequences
between 207 and 502nt containing a slender-form repres-
sion element. The final PAD1 30-UTR deletion construct
(1-776 nt) removed the sequences containing the experi-
mentally mapped polyadenylation site for the PAD1 gene
in stumpy forms. Consistent with this, by northern blot
analysis (Supplementary Figure S4), it was observed that
the progressive deletions of the full length PAD1 30-UTR
up to 609 nt caused equivalent decreases of the CAT tran-
script size, indicating use of the same polyadenylation
sites. In contrast, transcripts from the PAD1 30-UTR
1–776 construct were longer than those from the
previous deletion. This likely represents the use of an
alternative polyadenylation site and indicates that the
dominant polyadenylation sites used in monomorphic
slender forms coincides with those mapped in stumpy
forms, rather than those reported in earlier RNAseq
analyses (33).
To assess regulation at the protein level, at least two
CAT ELISA assays were carried out for each of the two
replicate cell lines described earlier, and these normalized
to the CAT449 control (Figure 2C). Matching the analysis
at the mRNA level, replacement of the Aldolase 50-UTR
with the PAD1 50-UTR caused an approximately 1.6-fold
increase in CAT protein expression, eliminating a function
for this element in silencing gene expression in slender
forms. In contrast, CAT protein was almost undetectable
when the aldolase 30-UTR of the original CAT449
construct was replaced by the full-length PAD1 30-UTR.
Figure 2. Deletion analysis of regulatory elements in the PAD1 30-UTR. (A) Schematic diagram of the CAT449 PAD1 30-UTR deletion constructs
used to map control elements in the PAD1 30-UTR. The ‘A’ in the PAD1 30-UTR depicts the 707-710 nt and 722-723 nt polyadenylation sites. This
diagram is not to scale. (B) Quantification of the mRNA abundance in two independent clones for each cell line represented in panel A. Values are
normalized to the CAT mRNA abundance derived from the CAT449 control construct. Deletion 1–776 nt deletes beyond the mapped
polyadenylation sites. (C) Quantification of the CAT protein in two independent clones for each cell line represented in panel A. Values are
normalized to the CAT protein abundance derived from the CAT449 control construct. Deletion 1–776 nt deletes beyond the mapped
polyadenylation sites.
Nucleic Acids Research, 2012, Vol. 40, No. 16 7709
Indeed, while there was a 3.7-fold decrease in mRNA
expression, there was an almost 180-fold decrease in
protein expression using the PAD1 30-UTR, indicating
that repression operates at both the level of transcript
abundance and protein expression in slender forms.
Protein level control was also more stringent as deletion
constructs of the PAD1 30-UTR were analysed. As with
the mRNA analysis, deletion of the first 207 nt of the
PAD1 30-UTR caused no discernable effect on reporter
protein expression compared with the full-length PAD1
30-UTR. Hence, it appears that the first 207 nt is not
necessary for repression of mRNA abundance or protein
expression in slender forms at least in the presence of
distal regions of the 30-UTR. Discrepancy between
mRNA and protein levels was observed for deletions
between 207 and 354 nt, however, whereby mRNA levels
reached 59% (1–265) and 80% (1–354) of CAT449
levels while CAT protein levels were 1.4% and 7.1% of
CAT449, respectively (Figure 2C). This indicates the
presence of a dominant repressive element operating at
the protein level between 207 and 354 nt in the PAD1
30-UTR. Deletion from 354 to 502 nt generated further
increases in CAT protein levels, similar in overall trend
to the mRNA profile. This indicated the presence of a
further repressive element at the distal end of the 30-UTR.
Operation of identified regulatory regions in isolation
To further investigate the contributions of subregions
within the PAD1 30-UTR, two constructs containing
internal deletions were created to specifically remove
387–564 nt and 354–624 nt from the intact PAD1
30-UTR (Figure 3A). Both deletions were also designed
Figure 3. Analysis of specific deletions in the PAD 30-UTR for their effects on gene silencing in slender forms. (A) Representation of the expression
construct used to analyse specific PAD1 30-UTR elements for their contribution to gene silencing in slender forms. (B) A northern blot of the cell
lines used to analyse the contribution of specific PAD1 30-UTR elements to reporter gene expression. rRNA stained with ethidium bromide is shown
in the bottom panel to demonstrate relative loading. Lane 1: CAT449 Original; Lane 2: CAT449 PAD1 30-UTR; Lane 3: CAT449 387–564 PAD1
30-UTR; Lane 4: CAT449 354–624 PAD1 30-UTR. The size of the relevant bands (marked with an asterisk) are Lane 1, 862 nt; Lane 2, 1449 nt;
Lane 3, 1278 nt and Lane 4, 1185 nt. (C) Quantitation of the CAT mRNA generated from the PAD1 30-UTR deletion constructs detailed in Panels A
and B. The data represent a quantitation based on independent northern blots of each of two cell lines derived for each expression construct. (D)
Quantitation of the CAT protein generated from the PAD1 30-UTR deletion constructs detailed in Panels A and B. The data represents a
quantitation based on independent assays of each of two cell lines derived for each expression construct.
7710 Nucleic Acids Research, 2012, Vol. 40, No. 16
to preserve the overall secondary structure of the entire
PAD1 30-UTR predicted using S-fold (Supplementary
Figure S5). From the analysis of two independent cell
lines, the PAD1 30-UTR 387–564 deletion generated no
detectable loss of repression of the mRNA with respect
to the full length PAD1 30-UTR (Figure 3B and C).
In contrast, the PAD1 30-UTR 354–624 construct
generated a 2-fold increase (56% of CAT499) in CAT
mRNA abundance over the full-length PAD1 30-UTR
(26% of CAT 449) (Figure 3B and C), although this was
considerably less than observed when individual sequen-
tial deletions were analysed in this region (e.g. PAD1
30-UTR 1–502 was 219.73% of CAT449, Figure 2B).
However, when the CAT protein levels were assayed,
negligible expression was detected with either the PAD1
30-UTR 386–564 or PAD1 30-UTR 354–624 deletion
constructs, demonstrating that in isolation neither
could relieve the PAD1 30-UTR-mediated repression
(Figure 3D). This indicated that a proximal element
must also operate in the PAD1 30-UTR to silence expres-
sion even in the absence of the distal silencing element
positioned beyond nt 354.
Having generated evidence for proximal and distal
silencing elements in the PAD1 30-UTR, we investigated
whether either region could function when inserted inde-
pendently into the 30-UTR of a constitutively expressed
gene. Therefore, the proximal region (1–354 nt) and
distal region (354–624 nt) of the PAD1 30-UTR were
integrated into the truncated aldolase 30-UTR of the
CAT449 control construct, directly after the CAT gene
termination codon (Figure 4A). Two transgenic cell lines
were then isolated for each construct and these cell lines
analysed for their CAT mRNA and protein expression.
Figure 4B shows that the integration of each element did
not alter the preferred polyadenylation site in the aldolase
30-UTR because the size increase of the generated CAT
mRNA was consistent with the size of each integrated
element (Figure 4B, compare lanes 1, 3 and 4).
Nonetheless, whilst neither the proximal or distal insertion
elements caused repression of CAT mRNA abundance
from the reporter construct (Figure 4B and C), the expres-
sion of CAT protein was reduced to 21% and 29% of
the CAT449 using the proximal and distal element,
respectively (Figure 4D). Apparently in the context of
the aldolase 30-UTR both proximal and distal elements
can act to repress upstream gene expression at the
protein level, but neither can act independently to reduce
mRNA abundance.
Mapping elements that respond to inducers of stumpy
formation
Upon differentiation to stumpy forms, the silencing
of stumpy-specific proteins is alleviated in response to
the parasite-derived signal, stumpy induction factor.
However, this factor is unidentified and monomorphic
cell lines are not responsive to it. Therefore, to investigate
the gene regulation on activation of the stumpy induction
pathway we examined the response of the PAD1 reporter
lines to treatment with compounds proposed to mimic the
SIF response.
Initially, we assayed the ability of the cell permeable
cAMP analogues, pCPTcAMP (24), 8pCPT-20-O-
Me-cAMP (35) or troglitazone (36), to induce hallmarks
of stumpy formation in monomorphic lines, namely the
inhibition of cell growth and improved efficiency of
differentiation to procyclic forms. These validation experi-
ments were carried out using a monomorphic reporter
line transfected with both the pHD617-CAT-PAD1
30-UTR expression vector and pHD617-GUS-actin
30-UTR (Figure 1A), enabling gene repression and allevi-
ation via the PAD1 30-UTR to be monitored in the context
of a constitutively expressed Actin 30-UTR control.
Matching the observation in pleomorphic slender cells
(Figure 1C) and the monomorphic lines harbouring the
CAT PAD1 30-UTR deletion series (Figure 2), the levels
of CAT protein expression were very low in the resulting
transgenic monomorphic line (Figure 5D–F, 0 h time
point), confirming repression by the PAD1 30-UTR.
The reporter line was then exposed to 100 mM
pCPTcAMP, 10 mM 8pCPT-20-O-Me-cAMP or 5 mM
troglitazone (36) doses being determined via titration
experiments or literature analysis (data not shown).
After treatment with each compound, the cells were
investigated for different parameters of stumpy formation,
including cell growth arrest (Figure 5A–C), their capacity
for differentiation to procyclic forms (Figure 5J–L;
Supplementary Figure S6) and their expression of the
CAT (Figure 5D–F) and GUS (Figure 5G–I) reporter
proteins. Figure 5 shows that both cAMP analogues
induced a potent growth arrest within 24 h (Figure 5A
and B) and more efficient differentiation to procyclic
forms after treatment with cis-aconitate, such that 75%
(pCPTcAMP) and 83% (8pCPT-20-O-Me-cAMP) of cells
expressed procyclin within 6 h compared with <2% in the
untreated populations (Figure 5J and K; Supplementary
Figure S6). 8pCPT-20-O-Me-cAMP induced some mor-
phological transformation in the treated cells, although
there also appeared to be ‘unhealthy’ cells in the popula-
tion. Nonetheless, when the response of the reporter genes
was analysed after treatment with either cAMP analogue,
the level of the constitutive GUS reporter was essentially
unchanged or somewhat decreased (Figure 5G and H),
whereas the expression of the PAD1 30-UTR linked
CAT gene was strongly elevated (pCPTcAMP, 24-fold,
Figure 5D; 8pCPT-20-O-Me-cAMP, 18-fold, Figure 5E)
after 24h. This indicated that both compounds promoted
some stumpy form characteristics in the treated
monomorphic cells, supportive of an (incomplete) devel-
opmental response that also relieves repression of the
CAT-PAD1 30-UTR reporter. In contrast, troglitazone
did not activate CAT (Figure 5F) or induce growth
arrest (Figure 5C) under the assay conditions used. This
indicates that this compound may not act on the stumpy
formation pathway, despite its reported anti-proliferative
and morphological effects (36).
Having validated the ability of cAMP analogues to
derepress CAT PAD1 30-UTR in monomorphic forms,
we used the cell lines transfected with the panel of
PAD1 30-UTR deletion constructs (Figure 2) to investigate
whether the regions responsible for repression in slender
forms coincided with those responsible for the alleviation
Nucleic Acids Research, 2012, Vol. 40, No. 16 7711
of repression in response to these compounds. Initially,
treatment of cells containing the control CAT449
construct with 10 mM 8pCPT-20-O-Me-cAMP was found
to cause a small decrease in CAT protein expression (as
was also seen with this compound in Figure 5H), perhaps
caused by the cells arresting growth after treatment,
or exhibiting some translational repression (37)
(Figure 6A). Interestingly, however, replacement of the
aldolase 50-UTR with the PAD1 50-UTR eliminated this
effect, perhaps because the PAD1 50-UTR contributes to
stabilizing the transcript, or assisting translation, as the
cells progress to ‘stumpy-like’ forms. As expected, the
PAD1 30-UTR responded effectively to 8pCPT-20-O-
Me-cAMP treatment such that CAT protein became
elevated at least 40-fold over untreated cells. Hence both
repression and the alleviation of that repression are
each dependent on the 30-UTR of PAD1.
When cells containing constructs with deletions of the
PAD1 30-UTR were analysed, the overall CAT expression
caused by 8pCPT-20-O-Me-cAMP treatment diminished
progressively with removal of the first 207 nt of the
PAD1 30-UTR, with even a deletion of the first 21 nt
generating a reproducible reduction (Figure 6A).
Nonetheless, the CAT expression was in all cases clearly
inducible in response to the treatment. Deletions beyond
position 265 nt, however, reduced the effect of 8pCPT-20-
O-Me-cAMP, with an 11-fold increase in CAT protein
expression in the 1-265 cell lines and a 3- and 2-fold
increase in the 1-354 and 1-391 cell lines, respectively.
Deletion beyond position 502 nt actually caused decreased
Figure 4. Out of context regulation of gene silencing by PAD1 30-UTR control elements. (A) Representation of the expression construct used to
analyse specific PAD1 30-UTR elements inserted into the aldolase 30-UTR for their contribution to gene silencing in slender forms. (B) A northern
blot of the cell lines used to analyse the contribution of specific PAD1 30-UTR elements to reporter gene expression. The sizes of the CAT transcripts
demonstrate that the endogenous aldolase polyadenylation site is preserved after insertion of the PAD1 30-UTR elements. rRNA stained with
ethidium bromide is shown in the bottom panel to demonstrate relative loading. Lane 1: CAT449 Original; Lane 2: CAT449 PAD1 30-UTR;
Lane 3: CAT449 1-354 PAD1 30-UTR; Lane 4: CAT449 354-624 PAD1 30-UTR. The relevant band in each lane is marked with an asterisk. The
predicted size of each band is Lane 1, 862 nt; Lane 2, 1449 nt; Lane 3, 1216 nt and Lane 4, 1132 nt. (C) Quantitation of the CAT mRNA generated
from the PAD1 30-UTR deletion constructs details in Panels A and B. The data represent a quantitation based on independent northern blots of each
of two cell lines derived for each expression construct. (D) Quantitation of the CAT protein generated from the PAD1 30-UTR deletion constructs
detailed in Panels A and B. The data represent a quantitation based on independent assays of each of two cell lines derived for each expression
construct. In some samples, the presence of DMSO apparently altered the basal level of CAT protein expression, although the effect did not alter the
overall interpretation of the analysis.
7712 Nucleic Acids Research, 2012, Vol. 40, No. 16
protein expression on 8pCPT-20-O-Me-cAMP treatment,
indicating that at this point, responsiveness had been
completely lost. Combined, these data indicate that the
element (s) responsible for up-regulation of protein
expression on stumpy induction is located 50to position
391 nt, essentially coincident with the proximal region
responsible for repression in slender forms. Supporting
this, constructs where there was specific deletion of
nucleotides 387–564 and 354–624 from the PAD1
30-UTR remained responsive to 8pCPT-20-O-Me-cAMP
(Figure 6B, PAD1 30-UTR 387–564 and PAD1 30-UTR
354–624), although the overall CAT protein expressed
was less than with the intact PAD1 30-UTR.
To dissect in more detail the regions responsible for
the responsiveness to 8pCPT-20-O-Me-cAMP within the
PAD1 30-UTR and their ability to operate out of
context, cell lines in which the proximal (nucleotides
1–354) and distal silencing elements (nucleotides
354–564) were inserted individually into the aldolase
30-UTR (described in Figure 4) were exposed to this
compound. Figure 6B (PAD1 30-UTR 1–354, PAD1
30-UTR 354–624) demonstrates that the distal element
alone was not responsive to 8pCPT-20-O-Me-cAMP,
whereas the proximal element was strongly inducible.
We conclude that in monomorphic forms the proximal
region of the PAD1 30-UTR contributes to mRNA and
Figure 5. Effect of chemical inducers of stumpy formation on monomorphic PAD1 reporter lines. Analysis of the response of the CAT-PAD1
30-UTR, GUS-actin 30-UTR reporter line to 100 mM pCPTcAMP (panels A, D, G and J), 10 mM 8pCPT-20-O-Me-cAMP (panels B, E, H and K) and
5mM troglitazole (panels C, F, I and L). Panels A–C show the growth of cells under each drug regimen, Panels D–F represent the CAT protein
expression over 48 h of drug treatment relative to zero hours, Panels G–I show the GUS protein expression after 48 h of treatment whereas Panels J
and Krepresent the percentage of cells expressing EP procyclin protein determined by flow cytometry after exposure to each drug for 48 h followed
by incubation with 6 mM cis-aconitate for 6 or 24 h.
Nucleic Acids Research, 2012, Vol. 40, No. 16 7713
protein repression in slender forms and the activation of
PAD1 expression on treatment with a cAMP analogue,
whereas the distal region contributes only to gene
repression.
To validate these observation in the physiologically
relevant pleomorphic cells, the proximal and distal
regions were analysed after treatment with 8pCPT-20-O-
Me-cAMP and in true slender and stumpy forms. Thus,
transgenic pleomorphic trypanosome cell lines were
generated transfected with the CAT-449 construct,
CAT-PAD1 30-UTR, CAT-PAD1 30-UTR387–564,
CAT-PAD1 30-UTR 354–624 and the CAT-PAD1
30-UTR 354–624 element inserted into the truncated
aldolase 30-UTR. Each construct was inserted into the
pleomorphic AnTat1.1 90:13 line and two independent
cell lines were isolated for each construct. Once these
transfectants were derived, each was inoculated into
mice and infections allowed to progress for either 3 or
4 days, to derive predominantly slender cell populations.
For 8pCPT-20-O-Me-cAMP treatment, the slender cells
were then maintained in culture by daily passage to
110
5
cells/ml and then exposed to drug (or DMSO)
for 48 h. Alternatively, infections in mice were maintained
for 6 and 7 days, to isolate true stumpy-enriched cell
populations. Thereafter, protein samples were derived
and assayed for their CAT protein expression in either
slender forms, 8pCPT-20-O-Me-cAMP treated slender
forms (‘stumpy-like’ forms) or in vivo generated stumpy
forms (‘true stumpy forms’).
Figure 7 shows CAT protein levels from 8pCPT-20-O-
Me-cAMP treated slender forms (Figure 7A) or in vivo
generated slender and stumpy populations (Figure 7B)
of pleomorphic cells transfected with each construct. In
each case, CAT-PAD1 30-UTR demonstrated a strongly
elevated CAT expression in 8pCPT-20-O-Me-cAMP
treated slender forms or stumpy forms. The response of
true stumpy forms was considerably greater than the
response of either pleomorphic slender or monomorphs
to 8pCPT-20-O-Me-cAMP, indicating a more robust
response in the pleomorphic lines generated in vivo than
in response to drug. When the distal deletion constructs
were analysed (PAD1 30-UTR387–564; 354–624),
the repression of CAT expression was preserved in true
slender forms and the induction of expression in true
stumpy forms was also retained (most strongly in the
354–624 construct), confirming that the proximal
element alone can provide stumpy-specific expression.
Insertion of the 354–624 element of the PAD1 30-UTR
into the aldolase 30-UTR also revealed elevated expres-
sion in true stumpy forms compared to slender forms,
contrasting with the weak inducibility of the same
construct in pleomorphic or monomorphic cells
exposed to 8pCPT-20-O-Me-cAMP (Figures 6B and
7A). This, however was less than the change seen with
the intact 30-UTR or deletions removing the distal
element.
Combined, this indicated that the response of
monomorphic and pleomorphic slender cells to cAMP
analogues broadly mimicked that during the in vivo tran-
sition from pleomorphic slender to stumpy cells, confirm-
ing the physiological relevance of assays using this
compound. However, the overall amplitude of differential
response was greatly enhanced in true stumpy forms and
the distal element remained able to confer stage-specific
expression, supporting the concept that the response
to cAMP analogues is incomplete with respect to in vivo
developmental responses that generate true stumpy forms.
Overall, the proximal element provided the most stringent
regulation, but both proximal and distal elements
cooperate to achieve developmental gene expression.
Figure 6. Effect of PAD1 30-UTR control elements on inducibility in response to 8pCPT-20-O-Me-cAMP. Response of different deletion constructs
of the PAD1 30-UTR (A), or specific 30-UTR deletions or 30-UTR element inserts into the aldolase 30-UTR (B) to incubation with 8pCPT-20-O-
Me-cAMP or with an equivalent amount of DMSO as control. Individual graphs show the CAT protein expression normalized to the control
CAT449 construct, each bar representing the mean and standard error of the mean of the analysis of two independent cell lines for each construct.
7714 Nucleic Acids Research, 2012, Vol. 40, No. 16
DISCUSSION
When trypanosomes prepare for transmission they
undergo a precise developmental programme involving
cell cycle arrest, morphological transformation and the
expression of proteins specific to the stumpy form. Of
these, PAD1 is linked to transmission competence, being
the transporter through which the citrate/cis-aconitate
signal is transduced (22), with this stimulating the trans-
formation to procyclic forms. Unlike differentiation in the
tsetse midgut, however, the generation of stumpy forms
occurs in the mammalian bloodstream, a relatively stable
environment in which a parasite-derived signal provides
the essential cue for the development of transmission
stages. Hence, the gene expression changes that accom-
pany stumpy formation are driven by a soluble signal in
many ways analogous to the precisely controlled and
programmed differentiation responses characteristic of
metazoan cell specialisation. This contrasts with the
differentiation of parasites when they enter an arthropod
vector, where extreme changes in the external environ-
ment, such as in temperature, might be predicted to
have wide-ranging consequences for gene expression at
the post transcriptional level, for example by altering the
respective conformation of different RNA secondary
structures (14).
PAD1 expression represents the first molecular
hallmark of stumpy cells, tightly regulated at both the
mRNA and protein level (22,26). It therefore provided
an excellent tool to dissect, for the first time, gene expres-
sion control signals regulating transmission competence,
the gene being tightly repressed in slender forms but highly
expressed in stumpy forms. Our analysis in both pleo-
morphic and monomorphic parasite lines established
that PAD1 expression is controlled through sequences in
its 30-untranslated region, matching the paradigm for
most regulated gene expression identified thus far in
these parasites (38). Moreover, dissection of the sequences
within the 30-UTR has identified a distal repression
element, which operates in concert with a proximal
element to repress PAD1 expression in slender forms.
In addition to identifying repression elements that
prevent PAD1 expression in slender forms, our analyses
also exploited chemical inducers to drive the production of
‘stumpy-like’ forms in monomorphic parasites. Several
studies in the literature have used a number of chemical
agents to provoke the generation of ‘stumpy-like’ forms.
However the interpretation of the cellular outcomes has
been restricted by the limitation of morphology as a
marker for stumpy formation, even when coupled with
mitochondrial activation, or cell-cycle perturbation, both
of which are common in stressed cells. In the study used
here we showed that a cell permeable cAMP analogue,
pCPTcAMP, and a hydrolysable cAMP analogue,
8pCPT-20-O-Me-cAMP, were both capable of inducing
cell-cycle arrest and promoting synchronized differenti-
ation to procyclic forms in response to cis aconitate as
measured by EP procyclin expression. More importantly,
however, both compounds were also shown to activate the
expression of the PAD1 reporter gene, revealing that they
are likely operating through, or intersecting with, a
physiologically relevant pathway to generate stumpy-like
responses. Despite this, neither compound generated
populations of cells that had a fully stumpy morphology
and both produced high levels of misshapen and likely
‘unhealthy’ cells. Moreover, the amplitude of PAD1 regu-
lation in response to cAMP regulation was less than
during the transition from slender to stumpy forms
in vivo. Hence, although useful for mapping inducible
control regions in the PAD1 30-UTR, neither compound
could be used to reproducibly generate populations of
stumpy cells representative of those generated in vivo
using pleomorphic populations.
Figure 8 shows a model for the regulation of gene
expression mediated via the PAD1 gene 30-UTR based
on our analysis of its component sequences in mono-
morphic and pleomorphic trypanosomes. At the distal
end of the 30-UTR, within 300 nt of the mapped
polyadenylation site, is an mRNA silencing element that
limits both mRNA and protein expression. However,
Figure 7. Effect of PAD1 30-UTR control elements on reporter expres-
sion in pleomorphic slender and stumpy forms. (A) AnTat1.1 90:13
pleomorphic transfectants were harvested from mice infections when
a slender parasitaemia was apparent (Days 3 or 4 post infection).
Thereafter, cells were maintained in culture and passaged daily to
110
5
/ml. Cells were treated with 10 mM 8pCPT-20-O-Me-cAMP, or
with an equivalent volume of DMSO, for 48 h before the expression of
CAT protein was determined. Error bars represent the SEM calculated
from two biological replicates (n= 2). (B). AnTat1.1 90:13 pleomorphic
lines were transfected with each reporter construct and then inoculated
in to mice. Thereafter, the expression of CAT protein was determined
in slender (Day 3 post-infection) and stumpy (Days 6–7 post-infection)
cells. Values represent the mean and SEM of two cell lines analysed in
independent infections. Values are normalized to the expression of the
CAT-449 construct in slender cells.
Nucleic Acids Research, 2012, Vol. 40, No. 16 7715
specific deletion of this section of the 30-UTR does not
alleviate protein expression when the remainder of the
30-UTR is intact, suggesting a contribution from gene
proximal regions of the 30-UTR. Consistent with this,
when the proximal or distal region of the PAD1 30-UTR
were integrated independently into the 30-UTR of a
constitutively expressed gene, neither reduced mRNA
levels although both reduced the levels of expressed
reporter protein. Therefore, repression is exerted through-
out distinct regions of the PAD1 30-UTR and protein
control is more stringent than mRNA repression. This
matches analysis of PAD1 mRNA and protein in
pleomorphic cells, whereby PAD mRNA is detected
early in intermediate forms (26) whereas protein
expression is restricted to fully differentiated stumpy
forms (22).
In contrast to the repression of the gene expression
mediated by proximal and distal regions within the
PAD1 30-UTR, regulatable expression induced by
8pCPT-20-O-Me-cAMP was only associated with the
proximal 354 nt, although the distal region was
regulatable during development to stumpy forms in vivo.
This highlights the proximal region as being more respon-
sive to cAMP, whereas both proximal and distal regions
can each contribute to the developmental control of
stumpy-specific gene expression in pleomorphs. This
further emphasises that the exposure to cAMP analogues
does not fully reproduce the complete development to
stumpy forms. In terms of the mechanisms of stumpy-
specific gene regulation, in the simplest scenario, the
proximal and distal regions would be the target of a
single negative factor (or mRNP) responsible for repres-
sion through binding to the PAD1 30-UTR, with this
binding being reversed by the response to 8pCPT-20-O-
Me-cAMP or SIF (for example, via phosphorylation
as the product of a signal transduction pathway).
Alternatively, there may be negative and positive factors,
such that an 8pCPT-20-O-Me-cAMP-induced or SIF-
induced activator displaces any silencing factors through
competing at the same binding site (s). Changes in the
affinity of components of an mRNP spanning several
regions of the 30-UTR could also relieve mRNA silencing,
this potentially explaining the different sensitivities of the
proximal and distal regions after 8pCPT-20-O-Me-cAMP.
Although the resolution of the precise mechanisms
awaits the isolation of the responsible protein factors,
the identification of the regulatable defined elements in
the PADI 30-UTR allows the pursuit of experimental
approaches using such ligands to dissect the gene expres-
sion processes necessary for parasite transmission.
Through manipulating these processes by targeted drug
approaches, novel routes to prevent disease spread or to
limit trypanosome virulence through accelerated parasite
development (25) are possible.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Figures 1–6, Supplementary Methods
and Supplementary References [31,39–41].
ACKNOWLEDGEMENTS
We are grateful to Deborah Hall and Julie Wilson for
assistance with mouse infections for the generation of
pleomorphic slender and stumpy forms, Dr Sam Dean
for the modification of the pHD617 vector and Prof
Isabel Roditi for advice on the GUS assay.
FUNDING
Wellcome Trust [080460 to P.M.], [088293 to K.R.M.],
[095831MA to K.R.M.]; Centre for Immunity, Infection
and Evolution, this being supported by a strategic award
Figure 8. A model of the regulation by the PAD1 30-UTR based on the deletion constructs generated in this study. The PAD1 30-UTR contains
proximal and distal silencing elements that repress gene expression at both the RNA and protein level in slender forms. In the absence of the
proximal region, silencing is achieved through mRNA regulation, but more prominently through protein regulation. In the absence of the distal
region, control is predominantly exerted through the proximal region at the protein level. The proximal region is responsible for regulation in
response to 8pCPT-20-O-Me-cAMP, this generating an alleviation of repression that must also operate to alleviate repression mediated through the
distal region. In pleomorphic cells, both proximal and distal elements exhibit an alleviation of repression in stumpy forms.
7716 Nucleic Acids Research, 2012, Vol. 40, No. 16
from the Wellcome Trust [095831MA]. Funding for open
access charge: Wellcome Trust.
Conflict of interest statement. None declared.
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