Alsford, S; Horn, D (2011) Elongator Protein 3b Negatively

Alsford, S; Horn, D (2011) Elongator Protein 3b Negatively Regulates

Ribosomal DNA Transcription in African Trypanosomes. Molecular

and cellular biology, 31 (9). pp. 1822-32. ISSN 0270-7306 DOI:

10.1128/MCB.01026-10

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Elongator Protein 3b Negatively Regulates Ribosomal DNA

Transcription in African Trypanosomes

䌤

†

Sam Alsford and David Horn*

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

Received 1 September 2010/Returned for modiﬁcation 28 September 2010/Accepted 16 February 2011

Eukaryotic cells limit ribosomal DNA (rDNA) transcription by RNA polymerase I (RNAP-I) to maintain

genome integrity. African trypanosomes present an excellent model for studies on RNAP-I regulation because

they possess a bifunctional RNAP-I and because RNAP-II transcription appears unregulated. Since Elp3, the

catalytic component of Elongator, controls RNAP-II transcription in yeast and human cells, we predicted a role

for a trypanosome Elp3-related protein, ELP3a or ELP3b, in RNAP-I regulation. elp3b null and conditional

strains speciﬁcally exhibited resistance to a transcription elongation inhibitor, suggesting that ELP3b nega-

tively impacts elongation. Nascent RNA analysis and expression of integrated reporter cassettes supported this

interpretation and revealed negative control of rDNA transcription. ELP3b speciﬁcally localized to the nucle-

olus, and ELP3b loss rendered cells hypersensitive to DNA damage and to translation inhibition, suggesting

that anti-Elongator function was important to maintain genome integrity rather than to modulate ribosome

production. Finally, ELP3b displayed discrimination between RNAP-I compartments in the same cell. Our

results establish ELP3b as a major negative regulator of rDNA transcription and extend the roles of the

Elp3-related proteins to RNAP-I transcription units. ELP3b is also the ﬁrst trypanosome protein shown

to distinguish between rDNA and variant surface glycoprotein transcription within different RNAP-I

compartments.

In eukaryotic cells, RNA polymerase II (RNAP-II) tran-

scription regulation appears to operate predominantly at the

level of elongation (18), and several factors that inﬂuence this

process have been described in human cells and in model

organisms (43). Much less is known about RNAP-I control, but

cells do limit ribosomal DNA (rDNA) transcription, and this is

important for the maintenance of genome integrity (22); extra

copies of rDNA allow for reduced transcription, which facili-

tates repair.

Trypanosomatids are protozoa that branched early from the

eukaryotic lineage and are important human and animal patho-

gens which are emerging as model organisms for the study of

epigenetic regulation (13). In trypanosomatids, RNAP-II

transcription of protein-coding genes is polycistronic and

apparently constitutive (10). In the bloodstream-form Afri-

can trypanosome Trypanosoma brucei, RNAP-I is bifunc-

tional, directing transcription of rDNA in the nucleolus and

monotelomeric transcription of a variant surface glycoprotein

(VSG) gene in an expression site body (ESB) that is distinct

from the nucleolus (32). RNAP-I is readily able to contribute

to mRNA production in trypanosomatids (45) because all ma-

ture mRNAs are fused to an RNAP-II-transcribed, trans-

spliced leader sequence (19). The high rate of transcription

achieved by RNAP-I may be important for VSG expression

since T. brucei bloodstream-form cells derive ⬎10% of total

cellular protein from a single VSG gene. These features lead us

to predict that conserved factors involved in RNAP-II tran-

scription elongation control in other eukaryotes, such as Elon-

gator, might function in RNAP-I control in trypanosomes.

Saccharomyces cerevisiae Elongator (49) associates with

elongating RNA polymerase II (RNAP-II) with a hyperphos-

phorylated carboxyl-terminal domain (37). The catalytic sub-

unit of the six-subunit Elongator complex (26), Elp3 (Elonga-

tor protein 3, also called KAT9), appears to provide a direct

link between histone acetylation and transcription by facilitat-

ing RNAP-II elongation in a chromatin- and acetyl coenzyme

A (acetyl-CoA)-dependent manner (56, 57). Human Elongator

also facilitates RNAP-II elongation through chromatin and

displays histone acetyltransferase activity with speciﬁcity for

lysine residues in the N-terminal tail of histone H3 (25). More

recently, S. cerevisiae Elp3 was shown to modulate transcrip-

tional silencing at telomeres and to modulate DNA repair

through an interaction with proliferating cell nuclear antigen

(28). As well as a GNAT-type acetyltransferase domain, Elp3

contains a radical S-adenosylmethionine (SAM) domain with

an iron-sulfur (FeS) cluster. Recent evidence points to a role

for the mouse Elp3 radical SAM domain in DNA demethyla-

tion (36). Surprisingly, S. cerevisiae Elongator (40, 41) and

human Elongator (25) localize predominantly to the cyto-

plasm, and roles have also been reported in exocytosis (41) and

tRNA metabolism in S. cerevisiae (21) and in tubulin acetyla-

tion in mouse neurons (11).

There are two Elp3-related proteins in trypanosomatids,

and we predicted a role for one or both of these proteins in

RNAP-I regulation. Here, we demonstrate that T. brucei

ELP3b negatively regulates rDNA transcription. This conclu-

sion is supported by elongation inhibitor resistance, increased

nascent rDNA transcription, and increased rDNA-integrated

* Corresponding author. Mailing address: London School of Hy-

giene and Tropical Medicine, Keppel Street, London WC1E 7HT,

United Kingdom. Phone: (44) 20 7927 2352. Fax: (44) 20 7636 8739.

E-mail: [email protected].

䌤

Published ahead of print on 28 February 2011.

† The authors have paid a fee to allow immediate free access to

this article.

1822

reporter expression in elp3b-depleted strains and ELP3b nu-

cleolar localization. Remarkably, ELP3b selectively controls

rDNA transcription, indicating an ability to distinguish be-

tween different RNAP-I transcription units.

MATERIALS AND METHODS

Trypanosomes. Bloodstream forms of T. brucei, MiTat 1.2 clone 221a, were

maintained, transfected, and differentiated as previously described (3). At least

5 h after transfection, transformants were exposed to puromycin (2 ␮gml

⫺1

;

phosphonoacetic acid [PAA]), blasticidin (10 ␮gml

⫺1

), hygromycin (2.5 ␮g

⫺1

), or phleomycin (2 ␮gml

⫺1

) selection, as appropriate. ELP3b disruption

was conﬁrmed by PCR and Southern blotting, using standard protocols (5). For

conditional strains, pHD1313 (Tet-R) (1), was integrated at the ␤-tubulin locus

and pRP

iGFP

ELP3b was integrated at an rDNA locus in an ELP3b heterozygous

strain prior to disruption of the second native allele of ELP3b. Transformants

were screened by immunoblotting and immunoﬂuorescence to conﬁrm robust

regulated expression, and two strains were transformed with the ﬁnal disruption

construct in the presence of tetracycline (Tet; 1 ␮gml

⫺1

) to generate four

conditional strains. For growth assays, cultures were seeded at ⬃10

⫺1

and

diluted back every 24 h. Cell counts were made using a hemocytometer and

carried out over at least 72 h. Cumulative counts were used to calculate popu-

lation doubling times, assuming exponential growth. For half-maximal effective

concentration (EC

) determinations, cells were seeded at 2 ⫻ 10

⫺1

96-well plates in a 2-fold dilution series of drug. After ⬃3 days of growth, 20 ␮l

of Alamar blue (AbD Serotec) was added to each well, and the plates were

incubated for a further 7 h. Fluorescence was determined using a ﬂuorescence

plate reader (Molecular Devices) at an excitation wavelength of 530 nm, an

emission wavelength of 585 nm, and a ﬁlter cutoff of 570 nm (42). All assays were

carried out in the absence of any additional antibiotics.

Plasmid construction. Enhanced green ﬂuorescent protein (eGFP) and cMYC

fusions were generated using the pRP (ELP3a, ELP3b, RPB6z, and RPB6) and

pNAT (SNAP42 and RPC160) vectors (2). Protein-coding sequences were am-

pliﬁed from T. brucei genomic DNA using Phusion polymerase (New England

BioLabs). Primers were designed using the publicly available T. brucei genome

sequence (www.genedb.org/genedb/tryp/). For the generation of ELP3b disrup-

tion constructs, targeting fragments were ampliﬁed from genomic DNA and

cloned into pBLA (blasticidin S deaminase) and pPAC (puromycin N-acetyl-

transferase). ELP3a targeting fragments were cloned into pPAC; the PAC gene

was subsequently replaced with BLE and HYG to generate additional disruption

constructs. All transcription run-on probes were cloned in pBluescript (Strata-

gene) or pGEM-T Easy (Promega) with the exception of R3, which proved

refractory to cloning. All primer sequences are available upon request.

Protein analysis. Immunoblotting was carried out following SDS-PAGE of

whole-cell lysates and electroblotting using standard protocols (5) and an en-

hanced chemiluminescence kit (Amersham), according to the manufacturer’s

instructions. Immunoﬂuorescence analysis was carried out on ﬁxed cells settled

onto slides pretreated with 3-aminopropyl triethoxysilane (Sigma) and pro-

cessed as previously described (4). eGFP and cMYC fusions were detected

with rabbit polyclonal anti-GFP (Molecular Probes) or mouse monoclonal

anti-GFP (AbCam) and mouse anti-cMYC (9E10; Santa Cruz Biotechnology),

respectively. NOG1 and NUP1 were detected with rabbit anti-NOG1 (38) and

mouse anti-NUP1 (35), respectively. Images were captured using a Nikon Eclipse

E600 epiﬂuorescence microscope in conjunction with a Coolsnap FX (Photo-

metrics) charge-coupled device (CCD) camera and processed in Metamorph 5.0

(Photometrics) and Adobe Photoshop elements 2.0 (Adobe).

Transcript analysis. Total RNA was isolated using an RNeasy kit (Qiagen),

fractionated on 5% polyacrylamide-7 M urea or 1.5% agarose-formaldehyde

gels, and analyzed by Northern blotting according to standard protocols (5).

Transcription run-on analysis was carried out using an adapted lysolecithin

permeabilization protocol (51). Brieﬂy, ⬃2 ⫻ 10

cells were washed in transcrip

tion buffer (TB; 20 mM

L-glutamic acid monopotassium salt, 3 mM MgCl

, 150

mM sucrose, 1 mM dithiothreitol [DTT], 10 ␮g ml 1-leupeptin [Sigma]) and

incubated for1min0.4mlTBcontaining 0.2 mg lysolecithin (

L-␣-lysophos-

phatidylcholine palmitoyl [Sigma]) on ice. Permeabilized cells were washed in TB

and then placed in labeling mix (20 mM

L-glutamic acid [monopotassium salt], 3

mM MgCl

, 1 mM DTT, 10 ␮gml

⫺1

leupeptin, 25 mM creatine phosphate, 0.6

␮gml

⫺1

creatine phosphokinase [type I, rabbit muscle; Sigma], 2 mM ATP, 1

mM CTP, 1 mM GTP [Fermentas], 100 ␮Ci [␣-

P]UTP [Perkin-Elmer]) in a

ﬁnal volume of 200 ␮l for 15 m at 37°C. Total RNA was isolated using an RNeasy

kit (Qiagen). Slot blots were generated in sets of three and included seven rDNA

probes: R1 (rDNA promoter; ⫺244 to ⫹255), R2 (including the rDNA promoter

and the ﬁrst 400 bp of small-subunit [SSU] rDNA; RH6), R3 (⫹1 to 3310,

including SSU rDNA), R4 (⫹3339 to 6389, including 5.8S and large-subunit ␣

[LSU␣] rDNA), R5 (⫹6444 to 9455, including LSU␤ rDNA), R6 (nontran-

scribed rDNA spacer), and 5S rDNA; four mRNA-associated probes, VSG2,

␤-tubulin, Procyclin, and spliced-leader RNA (SL-RNA); and a pBluescript neg-

ative control. Three micrograms of each plasmid (or 1.5 ␮g of puriﬁed R3 PCR

product) was denatured in 0.4 M NaOH, transferred to nylon membrane under

vacuum, and UV cross-linked. Total labeled RNA was hybridized to the slot blots

overnight at 65°C and subsequently washed and processed according to standard

protocols (5). Hybridization signals were detected using a Typhoon phosphor-

imager (Amersham), quantiﬁed using ImageQuant (Amersham), and analyzed in

MS Excel.

RESULTS

Trypanosomatid genomes encode two Elp3-related proteins.

Elp3 orthologues are conserved from archaea to humans. Pairs

of Elp3 orthologues were identiﬁed in T. brucei, Trypano-

soma cruzi, and Leishmania major (23), but we found no

organism beyond the trypanosomatids with more than one

Elp3 orthologue. As expected from the position in the Ex-

cavata, the trypanosomatid Elp3 orthologues, designated

ELP3a and ELP3b, are diverged relative to Elp3 orthologues

from the Opisthokonta, i.e., metazoa, plants, fungi, and alveo-

lates (Fig. 1A). In addition, the ELP3a and ELP3b paralogue

groups are monophyletic, suggesting a single gene duplication

that predates divergence of the trypanosomatids. Despite the

divergence, the residues required for substrate binding within

both the radical SAM and acetyltransferase domains are con-

served in both T. brucei ELP3a and ELP3b (Fig. 1B). Our

phylogenetic and sequence analysis indicated that ELP3a and

ELP3b have similar features regardless of the trypanosomatid

under consideration; we have focused on the T. brucei proteins.

elp3b mutants are resistant to transcription elongation in-

hibition and hypersensitive to DNA-damaging agents. To ex-

plore the function of the trypanosomatid Elp3-related pro-

teins, we generated elp3a (Fig. 2A) and elp3b (Fig. 2B) null

strains. Double-null elp3a/elp3b strains were also generated

(Fig. 2A and B). These strains were indistinguishable from wild

type in relation to cell cycle phase distribution and differenti-

ation to the insect stage (data not shown), but the elp3b strains

displayed a growth defect relative to elp3a and wild-type try-

panosomes (Fig. 2C). Interestingly, repeating the analysis after

further growth suggested that the cells were adapting to the

defect, with population doubling time approaching that of

wild-type cells (Fig. 2C). Double-null elp3a/elp3b strains also

displayed a growth defect and partial reversal of this pheno-

type after prolonged growth. Transcription elongation defects

increase sensitivity to depleted nucleoside triphosphate (NTP)

substrate pools. Indeed, S. cerevisiae elp3 null strains were

hypersensitive to 6-azauracil (6AU) (57), which inhibits IMP

dehydrogenase (IMPDH) and depletes pools of GTP and UTP

(46). Surprisingly, elp3b cells displayed speciﬁc and signiﬁcant

resistance to 6AU relative to that of elp3a and wild-type try-

panosomes (Fig. 2D), suggesting that ELP3b negatively con-

trols transcription elongation. This phenotype, like the growth

phenotype, was diminished after further growth (Fig. 2E).

Double-null elp3a/elp3b strains also displayed 6AU resistance

and partial reversal of this phenotype after prolonged growth.

Thus, elp3b null cells display an adaptation phenomenon char-

acterized by partial reversal of the growth and 6AU resistance

phenotypes, and ELP3a is not required for this adaptation.

VOL. 31, 2011 ELONGATOR REGULATES rDNA IN T. BRUCEI 1823

The data above are consistent with the idea that ELP3b

negatively controls transcription, and preliminary analysis of

nascent RNA did indeed reveal increased rDNA transcription

in elp3b cells (data not shown). Consistent with a link between

the growth and 6AU resistance phenotypes described above,

the rDNA transcription derepression phenotype was also un-

stable (data not shown but see below). Saccharomyces cerevi-

siae strains lacking Elp3 (28) or with increased rDNA tran-

scription (22) are hypersensitive to DNA-damaging agents,

and notably, elp3b null cells were also hypersensitive to phleo-

mycin (Fig. 2F), an agent that damages DNA via a mechanism

involving chelation of metal ions and the generation of free

radicals.

To facilitate studies of ELP3b function in a controlled en-

vironment, we generated strains with a conditional (Tet-on)

copy of

GFP

ELP3b in an elp3b null background. Cells in which

GFP

ELP3b expression was inactivated (Fig. 3

A) displayed de-

creased growth rate (Fig. 3B), reduced 6AU sensitivity (Fig.

3C), and increased sensitivity to methyl methanesulfonate

(MMS) (Fig. 3D), an alkylating agent that damages DNA and

is thought to stall replication forks. These results recapitulate

the elp3b null phenotypes and demonstrate that

GFP

ELP3b

functionally complements the elp3b defect.

ELP3b negatively controls ribosomal DNA transcription

elongation. We next measured nascent transcripts emanating

from different loci in cells expressing

GFP

ELP3b or in cells

FIG. 1. Phylogenetic and sequence analysis of Elp3 orthologues. (A) ELP3a and ELP3b were identiﬁed in T. brucei (Tb), T. cruzi (Tc), and

Leishmania major (Lm). The unrooted neighbor-joining tree was generated using CLUSTAL 1.8X and TreeView. Where excellent (ⱖ99.9%, open

circles) or very good (ⱖ90%, closed circles), branching conﬁdence is indicated. Hs, Homo sapiens; Xl, Xenopus laevis; Dm, Drosophila melanogaster;

Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Tt, Tetrahymena thermophila; At, Arabidopsis thaliana.

All accession numbers are indicated. The GeneIDs for the T. brucei proteins are as follows: TbELP3a, Tb927.8.5770; TcELP3a,

Tc00.1047053503851.10; LmELP3a, LmjF16.0240; TbELP3b, Tb927.8.3310; TcELP3b, Tc00.1047053509769.110; LmELP3b, LmjF23.1350.

(B) Schematic representation of the predicted structures of T. brucei ELP3a and ELP3b compared with human Elp3. The radical SAM domains

(black boxes) and GNAT-type acetyltransferase domains (gray boxes) are indicated with the FeS cluster and motif A, respectively. The sequences

were aligned using ClustalW, and the conserved domains (boxed) are indicated. Residues that are shared between all three proteins are white on

a black background, and residues shared among any pair of proteins are on a gray background. Arrowheads indicate the Cys residues that form

part of the FeS cluster. Asterisks indicate the conserved residues of motif A, QHXGXG, in all Elp3 orthologues.

1824 ALSFORD AND HORN M

OL.CELL.BIOL.

where

GFP

ELP3b expression had been inactivated for 3 or 7

days (Fig. 4). In this case, we used a series of ﬁve probes (R1

to R5) covering the length of the rDNA transcription unit (Fig.

4A). Slot blots were also loaded with probes for VSG2, the

active VSG in all clones analyzed (RNAP-I), ␤-tubulin genes

(RNAP-II), spliced-leader (SL-RNA) genes (RNAP-II), 5S

rDNA genes (RNAP-III), and several negative controls:

nontranscribed rDNA intergenic spacer, an insect-stage-

FIG. 2. ELP3b loss is associated with unstable resistance to transcription inhibition and hypersensitivity to a DNA-damaging agent. (A) South-

ern blot indicating ELP3a disruption. Genomic DNA was digested with HindIII. The map details the HindIII sites (h), deleted regions, and probes

used (horizontal bars). The deleted region was replaced with the selectable marker BLE, HYG,orPAC; the HYG and PAC cassettes each contain

a HindIII site. The elp3aPAC/HYG strain was used for the assay shown in panel F. (B) Southern blot indicating ELP3b disruption. The deleted

region was replaced with the selectable marker PAC or BLA. Other details are as in panel A. (C) Population doubling time in wild-type strain and

elp3a, elp3b, and elp3a/elp3b strains. elp3b* and elp3a/elp3b* strains were maintained in culture for more than 8 weeks. Standard deviations are

indicated, and P values were derived using an unpaired Student t test.

***

, P ⬍ 0.001;

, P ⬍ 0.01;

, P ⬍ 0.05; ns, not signiﬁcant.

(D) Half-maximal effective concentrations (EC

) for 6AU. All data were derived from two wild-type samples and two independent null strains.

Other details are as in panel C above. (E) 6AU sensitivity (50 ␮gml

⫺1

). Other details are as in panel C above. (F) EC

for phleomycin. Other

details are as in panel C above.

OL. 31, 2011 ELONGATOR REGULATES rDNA IN T. BRUCEI 1825

speciﬁc transcript that is not expressed in the bloodstream-

form cells used for this analysis (procyclin), and plasmid

vector DNA. The transcription run-on analysis indicated

signiﬁcantly increased transcription through the rDNA unit

following

GFP

ELP3b inactivation (Fig. 4B). In the 7-day

samples, transcription was signiﬁcantly increased (by ⬎60%)

across a region encompassed by probes in the middle and at

the distal end of the rDNA unit (Fig. 4B). The equivalent 3-day

samples also displayed increased transcription across this re-

gion but without achieving statistical signiﬁcance. Probes en-

compassing the proximal end of the rDNA unit failed to show

an increase in transcription, suggesting little attenuation in this

region in wild-type cells. Thus, consistent with increased 6AU

resistance, cells depleted for ELP3b displayed a relative in-

crease in nascent rDNA promoter-distal transcripts; the large

increase is remarkable given the major contribution that rDNA

genes are thought to make to total transcription in unper-

turbed cells. Interestingly, VSG2 transcription was signiﬁcantly

reduced (by ⬃20%) in these cells (Fig. 4B), which could reﬂect

depletion of the extranucleolar pool of RNAP-I. No negative-

control transcript, including nontranscribed rDNA intergenic

spacer, was signiﬁcantly above background (data not shown),

and neither the RNAP-II (SL-RNA) or RNAP-III (5S rRNA)

transcripts displayed signiﬁcant change (Fig. 4B).

ELP3b suppresses transcription of a reporter integrated

within ribosomal DNA. We next sought an independent ap-

proach to conﬁrm negative control of transcription by ELP3b.

Trypanosomes are unusual in that all mature mRNAs are fused

to an RNAP-II-transcribed, trans-spliced leader sequence (19).

This allows RNAP-I to transcribe the protein-coding segment

of mRNA (45), as is the case for VSG genes. We took advan-

FIG. 3. ELP3 downregulation phenocopies ELP3b knockout.

(A) Western blotting with anti-GFP conﬁrmed Tet-on (1 ␮gml

⫺1

)

regulation of

GFP

ELP3b in an elp3b background. The lower panel

shows an equivalent Coomassie blue-stained gel as a loading con-

trol. Data from one representative cell line are shown. (B and C)

Population doubling times (B) and 6AU (50 ␮gml

⫺1

) sensitivities

GFP

ELP3b strains. (D) Methyl methane

sulfonate (MMS; 0.0004%) sensitivity in

GFP

ELP3b strains (EC

⫹Tet, 0.00036% ⫾ 0.000015%; ⫺Tet, 0.00024% ⫾ 0.000016%).

The ⫺Tet,

GFP

ELP3b-depleted population was assessed 4 to 7 days

after Tet removal. Data in panels B to D were derived from four

independent clones. Standard deviations are indicated, and P values

were derived using a paired Student t test.

***

, P ⬍ 0.001;

, P ⬍

0.01.

FIG. 4. ELP3b negatively regulates transcription elongation at

rDNA loci. (A) Schematic of a T. brucei rDNA transcription unit and

the location of the R1 to R5 probes (horizontal bars) for nascent

transcript analysis. Each rDNA unit is approximately 10 kbp in length

(55). The promoter (ﬂag) and the rDNA subunit coding regions are in-

dicated. (B) Transcription run-on analysis during depletion of

GFP

ELP3b. Phosphorimager signals were corrected against ␤-tubulin

transcript abundance and expressed relative to the ⫹Tet value (set to

1). The inset shows a sample slot blot. V2, VSG2; ␤T, ␤-tubulin. VSG2

is a single-copy gene ⬃60 kbp from its promoter that is transcribed by

RNAP-I. The spliced-leader RNA (SL-RNA) is derived from a tan-

dem gene array and contributes a fragment that is trans-spliced to the

5⬘ end of every mRNA in trypanosomes. This RNA and ␤-tubulin, used

as a loading control and also derived from a tandem gene array, are

transcribed by RNAP-II. 5S rRNA is also derived from a tandem gene

array and is transcribed by RNAP-III. Data were derived from four

independent

GFP

ELP3b strains. Error bars represent one standard

deviation, and P values were derived using a paired Student t test.

P ⬍ 0.05.

1826 ALSFORD AND HORN M

OL.CELL.BIOL.

tage of this feature and used a selectable marker as a reporter

of transcription through rDNA (Fig. 5A). A neomycin phos-

photransferase (NPT) gene was inserted between the 5.8S and

LSU␣ genes in cells engineered for conditional expression of

GFP

ELP3b in an elp3b null background, and three independent

clones were analyzed (Fig. 5B); correct integration was con-

ﬁrmed using PCR assays (data not shown). NPT expression

was increased 3 and 7 days after inactivation of

GFP

ELP3b

expression, achieving statistical signiﬁcance in the 3-day sam-

ples (Fig. 5B). The results conﬁrm negative control of tran-

scription through rDNA by ELP3b and suggest suppression of

transcription through individual rDNA units rather than com-

plete transcription blockade in a subset.

Having established that ELP3b negatively controls transcrip-

tion at rDNA loci, we now revisited the adaptation phenome-

non in the elp3b strains described above. We predicted that

adaptation would involve reduced rDNA transcription, either

through change in rDNA gene copy number or through the

action of a second negative regulator. There are nine complete

rDNA units annotated in the haploid T. brucei genome se-

quence, with one unit each on chromosomes 1 and 7, three on

chromosome 2, and four on chromosome 3 (6). Using elp3b

null cells grown in culture for several weeks (elp3b*), we saw

no evidence for altered rDNA gene dosage (data not shown),

but as predicted, transcription run-on analysis revealed re-

duced rDNA transcription (Fig. 6B). In elp3b* cells, transcrip-

tion was reduced by ⬎50% compared to the wild type in a

region encompassed by three probes at the proximal end of the

rDNA unit. This does not reﬂect more transcription in the

distal region but is readily explained by a two-stage process

involving increased elongation after ELP3b loss which is com-

pensated for by less initiation (compare Fig. 6B and Fig. 4B).

Consistent with adaptation in the absence of ELP3a (in elp3a/

elp3b double-null strains [Fig. 2C and E]), nascent transcript

analysis in elp3a strains did not reveal changes in rDNA tran-

scription that were statistically signiﬁcant (Fig. 6C), implicating

another, unknown factor in reduced initiation and adaptation

in elp3b null cells.

ELP3b localizes to the nucleolus but not to the VSG expres-

sion site body. We proceeded to explore the subcellular

location of

GFP

ELP3a and

GFP

ELP3b in bloodstream-form

trypanosomes engineered for tetracycline (Tet)-inducible

expression (3). Microscopic analysis of these strains did not

reveal any substantial GFP signal in uninduced cultures,

while ﬂuorescence or immunoﬂuorescence analysis of induced

cells indicated speciﬁc accumulation in distinct subnuclear

compartments (data not shown). To directly compare these

compartments, we established trypanosomes constitutively

expressing both

GFP

ELP3a and

MYC

ELP3b (Fig. 7

A). Im-

munoﬂuorescence analysis revealed little overlap in the lo-

cation of the two proteins; the subnuclear compartment

occupied by

GFP

ELP3a is punctate and typically at the nu

clear periphery, while

MYC

ELP3b occupies a more central

compartment (Fig. 7B). We obtained similar results using

insect-stage, procyclic trypanosomes (data not shown). Inter-

estingly, the accumulation of

MYC

ELP3b was speciﬁcally dis

rupted after transcription inhibition (Fig. 7B), which could

indicate engagement with active transcription factors.

Diploid T. brucei nuclei contain a single nucleolus, the site of

rDNA transcription driven by RNAP-I, and apparent ELP3b

accumulation at this site is consistent with the phenotypes

described above. However, bloodstream-form T. brucei try-

panosomes are unusual in that they also use RNAP-I to tran-

scribe VSG mRNA at an extranucleolar site known as the

expression site body (ESB) (32). To examine ELP3b localiza-

tion with respect to both RNAP-I compartments, we estab-

FIG. 5. ELP3b suppresses transcription of an mRNA reporter integrated at an rDNA locus. (A) Schematic of a T. brucei rDNA transcription

unit showing the location of the neomycin phosphotransferase (NPT) reporter. The ﬂanking 5⬘-procyclin and 3⬘-aldolase untranscribed regions are

represented by gray boxes. (B) Northern analysis of NPT mRNA expression following

GFP

ELP3b depletion. One representative Northern blot is

shown. An ethidium bromide-stained gel is included to show loading. Phosphorimager signals were processed as described for Fig. 4B. Data were

derived from three independent

GFP

ELP3b (rDNA::NPT) strains. Error bars represent 1 standard deviation, and P values were derived using a

paired Student t test.

, P ⬍ 0.05.

OL. 31, 2011 ELONGATOR REGULATES rDNA IN T. BRUCEI 1827

lished trypanosomes expressing

MYC

ELP3b and

GFP

RPB6z

(Fig. 7A), the latter being a well-characterized RNAP-I sub-

unit found in both RNAP-I compartments (12, 33); N-termi-

nally tagged RPB6z was previously shown to be fully functional

and does not interfere with RNAP-I activity in vitro (33). Im-

munoﬂuorescence staining of

GFP

RPB6z revealed the nucleo

lus and the ESB as expected (Fig. 7C). Dual detection of

GFP

RPB6z and

MYC

ELP3b revealed a strong

MYC

ELP3b signal

at the nucleolus, but no detectable

MYC

ELP3b signal that co

incided with the smaller ESB, the compartment involved in

VSG transcription (Fig. 7C).

The data above indicated nucleolar sequestration of ELP3b

and little or no association with the ESB. However, we de-

tected some nuclei with a second compartment of

MYC

ELP3b

staining, and we speculated that these represented nascent

nucleoli. This was conﬁrmed using cells expressing

GFP

ELP3b

(Fig. 3A). Dual immunoﬂuorescence detection of

GFP

ELP3b

and NOG1, a nucleolar G protein (38) that is not detected in

the ESB, indicated that the second focus of

GFP

ELP3b staining

always colocalized with NOG1 (Fig. 7D). In addition, nuclear

and mitochondrial (kinetoplast) DNAs, stained with DAPI

(4⬘,6-diamidino-2-phenylindole), provide excellent cytological

markers that deﬁne the position in the cell cycle (58), and as

expected, two nucleoli and two

GFP

ELP3b foci in a single nucleus

were observed in trypanosomes only in the G

/M phases. Thus,

ELP3b was sequestered in the nucleolus and showed little or no

association with the ESB regardless of whether the protein was

fused to GFP or a MYC epitope. The results show that nucle-

olus-enriched

GFP

ELP3b complemented the growth, 6AU re

sistance, and transcription phenotypes seen in elp3b cells (see

above). We also demonstrated that elp3b cells were indistin-

guishable from the wild type in relation to ELP3a, NOG1, and

RPB6z localization (data not shown).

We used a similar approach to further examine the compart-

ment(s) occupied by the other Elp3-related protein, ELP3a.

GFP

ELP3a was coexpressed with other tagged transcription

factors, and expression of proteins of the predicted size was

conﬁrmed by Western blotting (Fig. 8A). ELP3a appeared to

occupy a compartment that was distinct from all three major

RNA polymerases (Fig. 8B). A peripheral nuclear localization

appeared more pronounced during mitosis (Fig. 8C), and

partial colocalization with NUP-1, a putative nuclear lamina

component (44), supported an association with the nuclear

envelope (Fig. 8D). We also examined spindle microtubule

acetylation, telomere position-effect repression (16), and VSG

expression site silencing (54) in elp3a cells, as well as cytosine

methylation (31) in elp3a and elp3b cells (data not shown), but

detected no signiﬁcant differences from the wild type.

ELP3b-depleted cells are hypersensitive to translation inhi-

bition. We considered the possible beneﬁts of limiting tran-

scription through rDNA. This could facilitate DNA replication

and DNA damage tolerance (Fig. 2F and 3D) (22) or could

contribute to modulating rRNA synthesis to satisfy cellular

demands for translation capacity. Indeed, rDNA transcription

elongation is the rate-limiting step for rRNA synthesis in hu-

man cells (48). To explore a role for ELP3b in regulating

rRNA synthesis, we examined the downstream consequences

of increased rDNA transcription in ELP3b-deﬁcient cells (Fig.

9). The trypanosomatid rDNA transcription unit is unusual in

that it encodes several small rRNAs (55), but no major change

in the relative steady-state abundance of any of these tran-

scripts was seen in elp3a, elp3b*,orelp3a/elp3b null cells

(Fig. 9A). Furthermore, Northern blot analysis revealed no

major change in relative SSU or LSU transcript abundance

in ELP3b-depleted cells (Fig. 9B). To ask whether increased

rDNA transcription is reﬂected at the level of sensitivity to

translation inhibition, we assessed growth in G418. Surpris-

ingly, ELP3b-depleted cells were hypersensitive to G418 (Fig.

9C). This was also the case in elp3b* cells, while elp3a cells

were indistinguishable from the wild type (data not shown).

This result could reﬂect disruption of the ribosome assembly

process, causing limiting factors to be channeled into a non-

productive pathway, or ELP3b could play an additional role in

tRNA modiﬁcation (21). Taken together, the results are con-

sistent with a role for rDNA transcription control by ELP3b in

maintaining genome stability rather than in modulating trans-

lation capacity.

FIG. 6. Adaptation to the elp3b defect involves downregulation of

rRNA transcription. (A) Schematic of a T. brucei rDNA transcription

unit reproduced from Fig. 4A. (B) Transcription run-on analysis of

adapted elp3b* null strains. Data were derived from two independent

clones. Other details are as in Fig. 4B, except that corrected values are

expressed relative to the wild type (WT). (C) Transcription run-on

analysis of elp3a null strains. Data were derived from four independent

clones. Other details are as in panel B above.

1828 ALSFORD AND HORN M

OL.CELL.BIOL.

DISCUSSION

rDNA genes encode the core components of the ribosomes,

the molecular machines that drive mRNA translation into pro-

tein. Sixty to 80 percent of transcription in rapidly growing

yeast cells is at rDNA loci mediated by RNAP-I. The elonga-

tion rate has been estimated to be ⬃60 nucleotides/s with a

reinitiation rate of ⬍1 s (14). Despite the important link to

genome stability (22), the balance between silencing and acti-

vating complexes and their contributions to the formation of

alternative chromatin states at these loci remain poorly under-

stood (30). A number of factors that typically control multiple

classes of RNA polymerase have been shown to exert positive

(7, 59) and negative (30) control on RNAP-I elongation at

rDNA genes. In addition, structural analysis reveals that S.

cerevisiae RNAP-I contains a built-in elongation factor related

to the RNAP-II-associated factor TFIIF (27), and a mutated

phosphorylation site on S. cerevisiae RNAP-I increases resis-

tance to 6AU, consistent with a role in negative control of

elongation (15). We have demonstrated negative control by

ELP3b that is speciﬁc to nucleolar rDNA genes in trypano-

somes, and our reporter analysis suggests suppression of indi-

vidual rDNA transcription units rather than complete block-

ade of a subset.

Nucleosomes are depleted or disordered at actively tran-

scribed rDNA loci (30). Thus, ELP3b may generate a more

stable or “closed” chromatin state that promotes premature

termination. How might this be achieved? rDNA gene regula-

tion involves histone modiﬁcation and DNA methylation (50),

and ELP3b, like other Elp3 orthologues, has two major do-

mains, an acetyltransferase domain and a radical SAM do-

main. Histone acetylation is important for transcription initi-

ation and elongation, and a speciﬁc enrichment of histone

H4K10 acetylation, H2AZ and H2BV histone variants, and the

BDF3 bromodomain factor is seen at probable RNAP-II tran-

scription start sites in trypanosomes (47). These same variants

are depleted within the nontranscribed rDNA spacer, and

FIG. 7. ELP3b localizes to the nucleolus but is not detected in the ESB in bloodstream-form T. brucei. (A) Western analysis of strains used for

ELP3 localization studies. Blots were incubated with anti-MYC or anti-GFP. A Coomassie blue-stained gel is included to show loading. Predicted

molecular masses of the fusions:

GFP

ELP3a, 104 kDa;

MYC

ELP3b, 77 kDa;

GFP

RPB6z, 42 kDa. The lower anti-GFP panel is a shorter exposure.

(B) Dual localization of

GFP

ELP3a and

MYC

ELP3b. The effect of actinomycin D treatment is also shown. The regions outlined in the phase images

indicate the regions shown in the immunoﬂuorescence panels. The nucleus (n) and kinetoplast (k) were stained with the DNA intercalating dye

4⬘,6-diamidino-2-phenylindole (DAPI), and these images were merged with the immunoﬂuorescence images. Bar, 5 ␮m. (C)

MYC

ELP3b localizes

speciﬁcally to the nucleolus but not to the smaller ESB (arrowhead), as revealed by

GFP

RPB6z. (D) Colocalization of

GFP

ELP3b and the nucleolar

protein, NOG1, through the cell cycle. G

/S cells have a single nucleolus, while a second nucleolus can be seen during G

/M (identiﬁed by two

kinetoplasts). Postmitotic (Post-M) cells have a single nucleolus in each nucleus. Other details are as in panel B above.

OL. 31, 2011 ELONGATOR REGULATES rDNA IN T. BRUCEI 1829

BDF3 is depleted in the transcribed rDNA region, revealing a

distinct chromatin architecture at these loci (see the region

encompassing Tb927.3.3421 to Tb927.3.3455 at http://tritrypdb

.org/). Importantly, histone acetylation has also been associated

with negative control of transcription (9, 24, 29, 53). Thus, acet-

ylation by ELP3b may negatively control rDNA transcription

elongation. The radical SAM domain could equally be responsi-

ble for negative control, possibly via DNA demethylation (36).

The nucleus is highly heterogeneous, containing euchro-

matic and heterochromatic compartments thought to be per-

missive and repressive for transcription, respectively. RNAP-I

transcribes rDNA genes in the nucleolar compartment, and, in

T. brucei, RNAP-I also synthesizes a subset of abundant pre-

mRNAs. In fact, T. brucei is the only known eukaryote with a

multifunctional RNAP-I and presents a unique opportunity to

study RNAP-I regulators. The trypanosomatid RNAP-I com-

plex (34, 52) contains three “specialized” subunits, RPB5z,

RPB6z, and RPB10z (12), as well as RPA31 (33) and the RPB7

subunit, typically associated with RNAP-II (39). Trypanosome

RNAP-I transcription also depends upon a novel complex

known as class I transcription factor A (8). ELP3b has not been

identiﬁed in fractions containing T. brucei RNAP-I (8, 33, 34,

52), possibly due to weak or little direct interaction or because

the majority of RNAP-I is engaged in processive transcrip-

tion. Remarkably though, ELP3b speciﬁcally impacts rela-

tively short RNAP-I transcription units (rDNA, 10 kbp) and

appears to be excluded from a much longer RNAP-I transcrip-

tion unit at the ESB (VSG, 60 kbp). Several additional nucle-

olar factors are undetectable in the ESB, but ELP3b is the ﬁrst

of these factors shown to distinguish between rDNA and VSG

mRNA synthesis. The distinct promoters that operate at these

loci could determine this differential association.

FIG. 8. ELP3a occupies nuclear territories distinct from those occupied by the transcription machinery. (A) Western analysis of strains used

for ELP3 localization studies. Predicted molecular masses of the fusions:

6MYC

RPB6z, 24 kDa;

6MYC

RPB6, 25 kDa;

GFP

RPB6, 43 kDa;

SNAP42

12MYC

, 60 kDa; RPC160

12MYC

, 188 kDa. (B)

GFP

ELP3a and the tagged polymerase subunits occupy distinct nuclear territories.

6MYC

RPB6z, RNAP-I;

6MYC

RPB6 and SNAP42

12MYC

, RNAP-II (colocalization of these factors is associated with sites of SL-RNA transcription

[30, 52]); RPC160

12MYC

, RNAP-III. (C) Localization of

GFP

ELP3a and SNAP42

12MYC

through the cell cycle. (D)

GFP

ELP3a shows partial

colocalization with NUP1. Other details are as in Fig. 7B.

1830 ALSFORD AND HORN M

OL.CELL.BIOL.

Microarray analysis of steady-state transcripts in Elongator-

defective S. cerevisiae revealed 52 genes that were downregu-

lated and 44 genes that were upregulated (26). Under the

conditions examined here, ELP3b negatively controls rDNA

transcription and can be considered an “anti-Elongator.” We

suggest that this may also be the case at the upregulated loci in

S. cerevisiae. Indeed, Elp3 was recently shown to have a role in

maintaining silencing at telomeres and at mating-type loci in S.

cerevisiae (28). Our results indicate a negative role for ELP3b

in processivity, while, at this stage, the role of ELP3a remains

unknown.

The purpose of limited transcription through rDNA appears

to be to improve genome stability (22). Our results are consis-

tent with the idea that unregulated transcription of rDNA

genes is indeed toxic and compromises the capacity for DNA

repair at these loci. Our ﬁndings could reﬂect a conserved role

for Elp3 proteins in this process. Indeed, Elp3 is concentrated

in nucleoli in HeLa cells (20). We favor a model whereby

ELP3b, like Elp3 in S. cerevisiae, associates with elongating

RNAP and modiﬁes chromatin structure, but we cannot rule

out other possible scenarios to explain negative control of

RNAP-I processivity at this stage. Targeted meganuclease

cleavage (17) could now be used to further explore the impact

of ELP3b transcription control on double-strand break repair

within rDNA transcription units. It will also be important to

determine what contributions the ELP3b acetyltransferase and

radical SAM domains make to the novel anti-Elongator func-

tion described here.

ACKNOWLEDGMENTS

This work was supported by The Wellcome Trust (project grants

079457 and 083648).

We also thank John Kelly (LSHTM) for critical reading of the

manuscript, Marilyn Parsons (Seattle Biomedical Research Institute)

for NOG1 antisera, Klaus Ersfeld (University of Hull, United King-

dom) for NUP1 antisera, and Elisabetta Ullu (Yale University) for the

transcription run-on protocol.

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