The NeST Long ncRNA Controls
Microbial Susceptibility and Epigenetic
Activation of the Interferon-g Locus
J. Antonio Gomez,1Orly L. Wapinski,2Yul W. Yang,2Jean-Franc ¸ois Bureau,3Smita Gopinath,1Denise M. Monack,1
Howard Y. Chang,2Michel Brahic,1and Karla Kirkegaard1,*
1Department of Microbiology and Immunology
2Program in Epithelial Biology, Howard Hughes Medical Institute
Stanford University School of Medicine, Stanford, CA 94305, USA
3De ´partement de Virologie, Institut Pasteur, 75724 Paris Cedex 15, France
Long noncoding RNAs (lncRNAs) are increasingly
appreciated as regulators of cell-specific gene
expression. Here, an enhancer-like lncRNA termed
NeST (nettoie Salmonella pas Theiler’s [cleanup
Salmonella not Theiler’s]) is shown to be causal
for all phenotypes conferred by murine viral sus-
ceptibility locus Tmevp3. This locus was defined
by crosses between SJL/J and B10.S mice and
contains several candidate genes, including NeST.
The SJL/J-derived locus confers higher lncRNA
expression, increased interferon-g (IFN-g) abun-
dance in activated CD8+T cells, increased Theiler’s
virus persistence, and decreased Salmonella enter-
ica pathogenesis. Transgenic expression of NeST
lncRNA alone was sufficient to confer all phenotypes
of the SJL/J locus. NeST RNA was found to bind
WDR5, a component of the histone H3 lysine 4 meth-
yltransferase complex, and to alter histone 3 methyl-
ation at the IFN-g locus. Thus, this lncRNA regulates
epigenetic marking of IFN-g-encoding chromatin,
expression of IFN-g, and susceptibility to a viral
and a bacterial pathogen.
Bioinformatic analysis of the chromatin marks in intergenic DNA
regions and of expressed sequence tags (ESTs) predicts the
existence of more than 5,000 long noncoding RNA (lncRNA)
genes in the human genome (Guttman et al., 2009; Khalil et al.,
2009; Qureshi et al., 2010). However, it is currently unknown
how many of these RNAs are functional. In a few well-studied
cases, such as AIR, XIST, and HOTAIR, lncRNAs have been
shown to operate at the transcriptional level by binding to
proteins in histone-modifying complexes and targeting them to
particular genes (Chu et al., 2011; Jeon and Lee, 2011; Nagano
et al., 2008; Wang and Chang, 2011). A role for lncRNAs in
mammalian susceptibility to infection or the immune response
to pathogens has not been previously described.
NeST,formally knownasTmevpg1,isanlncRNA genelocated
adjacent to the interferon (IFN)-g-encoding gene in both mice
(Ifng) and humans (IFNG). NeST was originally identified as a
candidate gene in a susceptibility locus for Theiler’s virus
(NeST stands for nettoie Salmonella pas Theiler’s [cleanup
Salmonella not Theiler’s]). In both mouse and human genomes,
for IFN-g, and the two genes are transcribed convergently (Fig-
ure 1A). In the mouse, NeST RNA contains six exons spread
over a 45 kb region (Vigneau et al., 2001, 2003). The most
abundant splice variant is 914 nt long, is expressed in CD4+
T cells, CD8+T cells, and natural killer cells, and contains no
AUG codons in translational contexts that appear functional.
The orientation and location of human NEST are conserved,
but the primary transcript encompasses the opposite strand of
the entire IFNG gene (Figure 1A).
Theiler’s virus, a picornavirus, is a natural pathogen of mice.
from strain to strain, and, because the phenotype can be
conferred by bone marrow transfer (Aubagnac et al., 2002;
Brahic et al., 2005; Vigneau et al., 2003), is likely to result from
different immune responses to the pathogen. A major effect is
conferred by the H2 locus. Two additional loci that affect
Theiler’s virus clearance were mapped by crosses between
H2s-bearing SJL/J and B10.S mice. Whereas B10.S mice
can clear the virus, SJL/J mice become persistently infected
and develop demyelinating lesions similar to those observed
in human multiple sclerosis (Aubagnac et al., 2002; Bureau
et al., 1993).
One of these loci, Tmevp3 (Theiler’s murine encephalitis virus
persistence 3; Figure 1B), was mapped to a 550 kb interval on
lines were developed by crossing SJL/J to B10.S and back-
crossing to each parental line for 10 to 12 generations (Bihl
et al., 1999; Bureau et al., 1993; Levillayer et al., 2007). The
B10.S.Tmevp3SJL/Jline is congenic with B10.S but contains
the Tmevp3 locus from SJL/J and is unable to clear persistent
infections. Conversely, the SJL/J.Tmevp3B10.Sline is congenic
Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc. 743
with SJL/J but contains the Tmevp3 locus from B10.S and
successfully clears infections. Analysis of SNPs in the smallest
introgressed B10.S-derived region revealed a small number of
polymorphic genes, including those that encode Mdm1 (Chang
et al., 2008), the potent immune cytokines IL-22 and IFN-g,
and the lncRNA (Figure 1C).
Here, we show additional phenotypes associated with the
Tmevp3 locus. In addition to the failure to clear Theiler’s virus,
the SJL/J-derived alleles also confer both resistance to lethal
infection with Salmonella enterica Typhimurium and inducible
synthesis of IFN-g in CD8+T cells. We show that NeST lncRNA
is sufficient to confer these disparate phenotypes, demon-
strating its crucial role in the host response to pathogens and
illustrating an integral function for lncRNAs in immune regulation
and susceptibility to infectious disease.
Mapping the Tmevp3 Locus of Mouse Chromosome 10
To refine the borders of the Tmevp3 locus, we utilized the JAX
mouse diversity genotyping array, which employs 623,124
SNPs and 916,269 invariant genomic probes. We also
sequenced complementary DNAs (cDNAs) encoding inter-
leukin-22 (IL-22), IFN-g, and NeST RNA from SJL/J and B10.S
mice, and added these findings to microarray results from the
Figure 1. Genotypes of the Parental and Congenic Strains Used to
Investigate NeST RNA and the Tmevp3 Locus on Murine Chromo-
(A) Schematic of the NeST-encoding genes in mouse chromosome 10 and
human chromosome 12. The bars represent exons, and arrows indicate the
direction of transcription. NeST (previously termed Tmevpg1) is adjacent to
both murine Ifng and human IFNG (Vigneau et al., 2003). The major transcript,
showninred,encodessixexons.Inbothmiceand humans,theNeSTand IFN-
g-encoding transcripts are convergently synthesized; in humans, the tran-
scribed regions overlap.
(B) Diagram of the Tmevp3 locus on murine chromosome 10, as defined by the
differential ability to clear persistent infection by Theiler’s virus observed
between SJL/J and B10.S mice. The SJL/J.Tmevp3B10.Sline (previously
termed C2; Vigneau et al., 2003) is congenic with SJL/J except for the region
shown, from microsatellite marker D10Mit74 to the interval between
D10Mit180 and D10Mit233. The B10.S.Tmevp3SJL/Jstrain is congenic with
B10.S except for the region shown, between the D10Mit151/D10Mit271
interval and the D10Mit233/D10Mit73 interval. The Theiler’s virus (TMEV)
persistence and clearance phenotypes and Nramp1 alleles for all four strains
(C) Finer mapping of the polymorphic regions of the congenic lines. The x axis
indicates the nucleotide number on mouse chromosome 10 (NCBI37/mm9).
The introgressed region of SJL/J in B10.S.Tmevp3SJL/Jis up to 1.6 3 107bp
(top), whereas the introgression in SJL/J.Tmevp3B10.Sis approximately 5.5 3
105bp (middle and bottom). Each bar displays the number of SNPs in the
window size indicated. The most polymorphic region maps to the Tmevp3
locus and coincides with the region of introgression in SJL/J.Tmevp3B10.S; see
Table S1 for lists of all genetic differences between the two Tmepv3 alleles.
The physical locations and direction of transcription of the murine NeST, Ifng,
Il22, and Mdm1 genes are indicated by arrows.
(D) NeST RNA expression in CD3+T cells. The abundance of NeST RNA in
CD3+splenocytes from B10.S mice and B10.S.Tmevp3SJL/Jwas determined
by preparing total cellular RNA and determining the amount of RNA per cell
using qRT-PCR and standard curves of transcribed RNAs. The threshold of
detection was 0.005 molecules of NeST RNA per cell. Mean values are shown
744 Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc.
Jackson Laboratory (Bar Harbor, ME; Figure 1C) and the list of
known polymorphisms in the locus (Table S1 available online).
Our results corroborated the presence of a unique introgressed
region that contained the previously mapped Tmevp3 locus,
and allowed us to refine its boundaries. The maximum sizes of
the introgressed regions were 16 3 106bp and 550 3 103bp,
respectively, for the B10.S.Tmevp3SJL/Jand SJL/J.Tmevp3B10.S
congenic lines (Figure 1C).
These analyses identified Il22, Ifng, and NeST as the most
likely candidates for the gene or genes responsible for the
Tmevp3 locus phenotypes by virtue of their polymorphic
character and their known expression patterns. In Figure 1C,
the top and middle bar graphs represent the number of
SNPs in a series of nonoverlapping 50 kb window regions.
The regions of densest polymorphism between the congenic
and parental lines can be seen in more detail in the bottom
part of Figure 1C. The product of Mdm1 is expressed predom-
inantly in the retina (Chang et al., 2008), making it an unlikely
candidate. The three most polymorphic genes are Ifng,
NeST, and Il22. The polymorphisms corresponding to NeST
are shown in red and all polymorphisms in the locus are listed
in Table S1. We were especially interested in the lncRNA
because of its potential novelty. As shown in Figure 1D,
CD3+T cells from B10.S.Tmevp3SJL/Jmice displayed signifi-
cantly higher amounts of NeST RNA than did those from
B10.S mice. This result differs from that reported by Vigneau
et al. (2003), possibly due to differences in the T cell prepara-
tions used or the use of saturating RT-PCR methods in the
previous study. Here, quantitative RT-PCR (qRT-PCR), the
use of standard curves, and comparisons of RNA abundances
from identical numbers of cells showed repeatedly that spleno-
cytes from mice with an SJL/J-derived Tmevp3 allele accumu-
lated substantially more NeST RNA than those from mice with
a B10.S-derived Tmevp3 allele. Even so, the amount of NeST
RNA that accumulated in total CD3+T cells was, on average,
only 0.15 molecules per cell (Figure 1D). It is known that
many lncRNAs are present at similarly low amounts but still
are sufficient to cause epigenetic changes that are then self-
propagating (reviewed in Guttman and Rinn, 2012). It is also
possible that NeST RNA is more abundant in a subset of the
CD3+T cells. Indeed, a higher abundance of NeST RNA was
observed in CD8+T cells (Figure 3B) than in total CD3+
T cells (Figure 1D).
Additional Pathogen Phenotypes for the Tmevp3 Locus
To determine whether Tmevp3 polymorphisms affected the
outcome of another infection, we monitored their effects on the
pathogenesis of Salmonella enterica Typhimurium, a pathogen
that, like Theiler’s virus, grows in macrophages (Monack et al.,
2004; Rossi et al., 1997) and is extremely sensitive to IFN-g
and CD8+T cell control (Foster et al., 2005; Rodriguez et al.,
2003). We began by comparing SJL/J and SJL/J.Tmevp3B10.S
mice because the size of the introgressed region was smaller
in this pair than in the B10.S and B10.S.Tmevp3SJL/Jpair (Fig-
ure 1B). Both SJL/J mice and SJL/J.Tmevp3B10.Smice are
homozygous for the functional allele of Nramp1, which encodes
an ion channel that facilitates clearance of Salmonella (Frehel
et al., 2002). As expected, both strains were resistant to oral
inoculation (Figure 2A). However, when subjected to the more-
potent intraperitoneal inoculation, both groups were susceptible
but the SJL/J.Tmevp3B10.Smice showed significantly more
mortality (Figure 2B).
Nramp1169Asp/169Asploss-of-function allele, which increases
their susceptibility to Salmonella infection. When inoculated
orally, B10.S mice displayed significantly more mortality than
B10.S.Tmevp3SJL/Jmice at several infectious dosages (Fig-
ure 2C). Intraperitoneal inoculation was rapidly lethal for both
mouse strains (Figure 2D). The differences in phenotypes
between SJL/J and SJL/J.Tmevp3B10.Sand also between
B10.S and B10.S.Tmevp3SJL/Jmice strengthen the hypothesis
that the Tmevp3 polymorphisms initially discovered by analysis
of Theiler’s virus persistence have implications for general
immune function. In subsequent experiments, we focused on
B10.S and B10.S.Tmevp3SJL/Jmice and Salmonella pathogen-
esis, given that oral infection is the natural route.
To determine whether the differences in phenotype resulted
from different bacterial loads, we infected B10.S and B10.S.
Tmevp3SJL/Jmice and monitored the abundance of S. Typhimu-
rium in spleen and feces. B10.S and B10.S.Tmevp3SJL/J
mice were orally inoculated with 106CFU and spleens were
dissected 4, 9, and 14 days after inoculation. No significant
differences in bacterial loads were observed in either spleen or
feces at any time point (Figure 2E). Interestingly, by day 14,
their infections even though mice from both groups continued
to die. To test for differences in Salmonella growth in cultured
macrophages, we infected bone-marrow-derived primary mac-
rophages from B10.S and B10.S.Tmevp3SJL/Jand measured
the amounts of intracellular Salmonella at various times after
infection. No significant differences in bacterial growth within
cells were observed (Figure 2F). All of these data are consistent
with the hypothesis that lethality is not due to the bacterial
load per se, but rather to the inflammatory response to bacterial
infection (Miao and Rajan, 2011; Pereira et al., 2011; Strowig
et al., 2012). In fact, the SJL/J-derived Tmevp3 locus also
conferred increased resistance to the lethal inflammatory
Transgenic Expression of NeST RNA Reproduces the
Phenotype Associated with the SJL/J Tmevp3 Allele
We hypothesized that NeST RNA could play a causal role in the
phenotypes conferred by the Tmevp3 locus. To address this
issue, we developed B10.S transgenic mice that express either
SJL/J- or B10.S-derived NeST RNA under the control of
a promoter that directs constitutive expression in both CD4+
and CD8+T cells (Figure 3A; Sawada et al., 1994). We obtained
two transgenic mouse lines: B10.S.NeSTB10.Sand B10.S.
NeSTSJL/J. To test whether the transgenes had inserted near
the endogenous Tmevp3 locus, we performed genetic crosses
between the B10.S.NeSTB10.Sand the B10.S.NeSTSJL/Jtrans-
genic mice and mice that bore neither marker. For both trans-
genic lines, the NeST transgenes and the endogenous locus
showed no evidence of linkage (data not shown). Both trans-
genic NeST RNAs were expressed in CD8+T cells (Figure 3B),
Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc. 745
although at different amounts. The B10.S NeST transgene was
expressed to an abundance similar to that of the endogenous
NeST gene in the B10S.Tmevp3SJL/Jline, whereas the SJL/
J-derived transgene accumulated to much greater abundance
To test whether the transgenic RNAs conferred protection
against Salmonella pathogenesis, we inoculated B10.S mice,
B10.S mice congenic at the Tmevp3 locus, and B10.S mice
transgenic for each NeST allele orally with Salmonella. Mice
that expressed the NeST B10.S transgene completely recapitu-
lated the Tmevp3SJl/Jsurvival phenotype (Figure 3C). Mice that
expressed the SJL/J NeST transgene also showed protection.
These findings demonstrate that NeST RNA can function in trans
to reduce Salmonella pathogenesis.
Transgenic NeST RNA Expression Prevents Clearance
of Theiler’s Virus
To test the role of NeST RNA in Theiler’s virus infection, the
microbial susceptibility phenotype that led to its discovery, we
inoculated B10.S, B10.S.Tmevp3SJL/J, and B10.S.NeSTB10.S
transgenic mice by intracranial injection. Viral loads in the spinal
cord were determined seven and 67 days after inoculation. At
seven days, all strains displayed comparable viral titers (Fig-
ure 4A), suggesting that NeST RNA plays little role during the
acute phase of infection. However, 67 days after inoculation,
infectious virus could only be recovered from mice that carried
the NeST transgene or the B10.S.Tmevp3SJL/Jlocus (Figure 4B).
The amounts of viral RNA in the spinal cords of the transgenic
mice and the B10.S.Tmevp3SJL/Jmice were orders of magnitude
Figure 2. Effect of the Tmevp3 Locus on Salmonella Pathogenesis
(A and B) Strains SJL/J and SJL/J.Tmevp3B10.Swere inoculated via oral (A) and intraperitoneal (B) routes with S. enterica Typhimurium. The Nramp1+/+alleles
expressed by SJL/J and SJL/J.Tmevp3B10.Smice render them relatively resistant to Salmonella infection.
(C and D) Strains B10.S and B10.S.Tmevp3SJL/Jwere also inoculated via oral (C) and intraperitoneal (D) routes with S. enterica Typhimurium at the dosages
indicated and mortality was monitored. The Nramp1169Asp/169Aspalleles render these mice highly sensitive to Salmonella pathogenesis. In both backgrounds, the
SJL/J allele of the Tmevp3 locus reduced mortality after oral inoculation. Statistical significance was determined by the log rank test.
(E) B10.S and B10.S.Tmevp3SJL/Jwere orally inoculated with S. Typhimurium at 106CFU/mouse. Bacteria were monitored in spleen and feces at the
(F) Intracellular bacterial growth was monitored ex vivo in bone-marrow-derived macrophages from B10.S and B10.S.Tmevp3SJL/Jmice. Lines represent the
mean of triplicate experiments, and statistical significance was determined using Student’s t test.
See also Figure S1.
746 Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc.
higher than those found in the spinal cords of the nontransgenic
B10.S parent (Figure 4C). Thus, the susceptibility to Theiler’s
virus persistence in spinal cord associated with the Tmevp3SJL/J
allele was recapitulated by the expression of the NeST RNA
Effect of the Tmevp3 Locus on the Expression of IFN-g
by CD8+T Cells
Several enhancer-like lncRNAs are known to activate neigh-
boring genes, as exemplified by HOTTIP and Jpx (Ørom et al.,
2010; Tianet al., 2010; Wang et al.,2011). The physical proximity
of Il22 and Ifng to NeST inspired us to test for differences in
expression of these two genes in CD4+and CD8+T cells from
B10.S and B10.S.Tmevp3SJL/Jmice. Isolated CD4+and CD8+
T cells were cultured for 1 day, stimulated with phorbol 12-myr-
istate 13-acetate (PMA) and ionomycin, and monitored for both
cytokine secretion (Figures 5A and 5B) and intracellular RNA
abundance (Figure S2). In CD4+T cells, ex vivo stimulation
caused large but similar increases in the secretion of both cyto-
kines in both B10.S and B10.S.Tmevp3SJL/Jmice (Figure 5A).
Similarly, the Tmevp3 allele did not significantly affect the
amounts of IL-22 secreted from CD8+T cells. However, whereas
the amount of IFN-g secreted from CD8+T cells derived from
B10.S mice was barely detectible, IFN-g secretion from
The difference in IFN-g production by CD8+T cells coincided
with the amounts of IFN-g RNA and NeST RNA (Figure S2B).
These results show a strong correlation between the abundance
of NeST RNA and IFN-g RNA and the amount of IFN-g protein in
activated CD8+T cells.
Transgenic Expression of NeST Induces IFN-g Synthesis
in Activated CD8+T Cells
To determine whether the expression of NeST RNA alone could
elicit the observed changes in IFN-g expression in CD8+T cells,
B10.S, B10.S.NeSTSJL/J, and B10.S.NeSTB10.Smice. As before,
CD8+T cells from the parental B10.S mice accumulated very
little cytokine after ex vivo stimulation (Figure 5C). However,
the transgenic expression of either the B10.S or the SJL/J allele
of NeST conferred the ability to induce IFN-g secretion. Interest-
ingly, the SJL/J-derived NeST RNA was less effective than the
B10.S-derived RNA in mediating IFN-g production, but both
alleles caused statistically significant increases in IFN-g expres-
sion upon CD8+T cell activation. Subsequent experiments were
designed to investigate the mechanism of these effects.
Figure 3. Effect of Transgenically Expressed NeST RNA on Salmonella Pathogenesis
(A) Schematic of transgenes introduced into B10.S mice. (S)JL/J NeST cDNA ([S]ea green) and (B)10.S NeST cDNA ([B]lue) were cloned downstream of a CD4+
and CD8+T cell-specific promoter. The promoter-NeST transgene fragments were used to construct transgenic mouse lines in the B10.S background.
(B)TheabundanceofNeST RNA wasmeasured inCD8+splenocytesfrom B10.Smicecongenicforthe SJL/J-derivedTmevp3 locus (B10.S.Tmevp3SJL/J),B10.S
of RNA per cell was determined using qRT-PCR; in vitro transcribed NeST RNA was used to construct standard curves. Mean values are shown with SE.
(C) B10.S, B10.S.Tmevp3SJL/J, B10.S.NeSTSJL/J, and B10.S.NeSTB10.Smice were orally inoculated with S. Typhimurium at the dosages indicated and mortality
was monitored. All experiments with the 107CFU/mouse were performed at the same time; the B10.S control is shown in these panels for clarity. Statistical
significance was determined by the log rank test.
See also Figure S1.
Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc. 747
NeST Is a Nuclear lncRNA that Can Function in trans to
Affect its Neighboring Locus
We hypothesized that, like several lncRNAs, NeST RNA affects
IFN-g accumulation at the transcriptional level by interacting
with chromatin modification complexes. Consistent with this
idea, most of the NeST RNA in either congenic or transgenic
mice was found in the nuclear fraction of CD8+T cells, cofractio-
nating with unspliced (but not with spliced) actin mRNA
The finding that two different transgenic NeST RNAs that were
notgenetically linkedtotheIfnglocusconferred thepropertiesof
the Tmevp3SJL/Jlocus to B10.S mice (Figures 3, 4, and 5) argues
that this lncRNA can function in trans. To determine whether
NeST RNA can indeed function in trans from its normal position
of synthesis, we took advantage of the fact that NeST RNA is
expressed in stimulated CD8+T cells of B10.S.Tmevp3SJL/J
mice but not in CD8+T cells of B10.S mice (Figure 3B and
S2B). We developed a PCR assay to distinguish between the
SJL/J- and B10.S-derived IFN-g alleles (Figure 6B). CD8+
T cells from two heterozygous B10.S/B10.S.Tmevp3SJL/Jmice
were stimulated, RNA was extracted, and the allele from which
the RNA was transcribed was determined from the RT-PCR
shown in Figure 6B. Approximately equal amounts of IFN-g
mRNA from the B10.S and SJL/J alleles accumulated following
stimulation (Figure 6C), arguing that the single functional NeST
gene in the heterozygous mice could stimulate transcription
from the Ifng genes on both chromosomes.
NeST RNA Binds to a Subunit of the MLL/SET1 H3K4
Methylase Complex and Increases Chromatin
Modification at the Ifng Locus
If NeST RNA were to have a direct effect on the expression of
IFN-g, via chromatin modification, it should be an activating
effect. Recently, a new class of enhancer-like lncRNAs was
discovered (Ørom et al., 2010; Wang et al., 2011). Among these,
HOTTIP lncRNA was found to bind WDR5 protein to recruit
complexes that facilitate transcription (Wang et al., 2011).
WDR5 is a core subunit of MLL1-4 and SET1A/1B complexes,
which catalyze the methylation of histone H3 at lysine 4,
a mark of active gene expression. To test whether NeST RNA
physically interacts with WDR5, the epitope-tagged protein
was coexpressed in combination with a variety of RNAs via tran-
sient transfection of 293T cells (Figure 7A). Extracts were then
prepared and WDR5 protein was immunoprecipitated and
tested for associated RNAs by qRT-PCR. HOTTIP served as
a positive control, and both HOTAIR lncRNA and U1 nuclear
RNA served as negative controls. Immunoprecipitation (IP) of
WDR5 specifically retrieved both NeST RNAs and HOTTIP, but
not U1 or HOTAIR RNAs (Figures 7A and 7B). The physical inter-
action between NeST and WDR5 raises the intriguing possibility
that NeST may control H3K4 methylation at the Ifng locus.
To examine the contribution of NeST RNA to IFN-g production
during immune challenge, we used a well-characterized mouse
model of sepsis: intraperitoneal injection of LPS. B10.S mice
as well as B10.S.NeSTB10.Sand B10.S.NeSTSJL/Jtransgenic
mice were injected with LPS. By 6 hr postinjection, the presence
of either transgenic NeST allele increased the amount of IFN-g in
splenic tissue compared with the B10.S control (Figure 7B). An
increase in H3K4me3 occupancy at the Ifng locus preceded
this increased IFN-g synthesis by 2 hr (Figure 7B). Transgenic
mice with the SJL/J-derived allele, which accumulate much
more NeST RNA than those that express the B10.S allele (Fig-
ure 3B), showed a larger amount of IFN-g-encoding DNA with
H3K4me3 modifications (Figure 7B). Thus, increased NeST
RNA abundance can result in more extensive H3K4me3 modifi-
cation. Still, NeST RNA is extremely potent even at low abun-
dance, either because the epigenetic effects persist in its
absence or because activation of only a subset of cells is neces-
sary for the observed phenotypes.
The high occupancy of H3K4me3 at the Ifng locus in the
B10.S.NeSTSJL/Jtransgenic mice allowed us to measure chro-
matin modification in isolated primary CD8+cells in the presence
and absence of NeST RNA. Following activation of B10.S- and
B10.S.NeSTSJL/J-derived CD8+T cells, we found that the pres-
ence of NeSTSJL/JRNA caused an increase in H3K4me3 at the
Ifng locus (Figure 7C). The NeST RNA-dependent increase in
H3K4me3 activation in both total splenic cells and CD8+T cells
strongly suggests that, via binding to WDR5, NeST RNA is
Figure 4. Effect of NeST RNA on Theiler’s
B10.S mice, B10.S mice congenic for the Tmevp3
locus from SJL/J mice (B10.S.Tmevp3SJL/J), and
B10.S mice containing the B10.S NeST transgene
(B10.S.NeSTB10.S) were inoculated by intracranial
injections of 107plaque-forming units (pfu) of
(A and B) Spinal cords wereharvested at 7days (A)
and 57 days (B) postinoculation and viral load was
measured by plaque assay on BHK-21 cell
(C) The abundance of viral RNA in spinal cord from
B10.S, B10.S.Tmevp3SJL/Jand B10.S.NeSTB10.S
mice was determined by preparing total cellular
RNA from homogenized tissue and determining
the amount per gram of tissue using qRT-PCR.
TMEV RNA was transcribed from cDNA-contain-
ing plasmid to construct standard curves. Means
and SE are shown.
748 Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc.
required to program an active chromatin state that confers
inducibility to the Ifng gene.
In this work, we performed a genetic analysis of an lncRNA
expressed in T cells. Mice that express NeST RNA, either in its
natural chromosomal environment or by transgenic delivery,
displayed increased resistance to Salmonella-induced patho-
genesis but increased susceptibility to Theiler’s virus persis-
tence. These disparate effects illustrate the role of balanced
polymorphisms in susceptibility to infectious disease (Dean
et al., 2002; Liu et al., 1996; Williams et al., 2005; Wang et al.,
2010; Cagliani et al., 2011). Genes of the immune system are
under purifying selection by challenges from a plethora of path-
ogens, and mutations that protect against one microbe may
increase susceptibility to another. In the case of autoimmunity,
the rs2076530-G allele of BTNL2, a major histocompatibility
complex (MHC) II-linked gene, confers increased susceptibility
to rheumatoid arthritis and type 1 diabetes but decreased sus-
ceptibility to multiple sclerosis and autoimmune thyroiditis
(Orozco et al., 2005; Sirota et al., 2009; Valentonyte et al., 2005).
A potential explanation for the disparate effects of NeST RNA
on Theiler’s virus persistence and Salmonella pathogenesis is
that it alters the magnitude or timing of inflammatory responses.
CD8+T cell populations are extremely heterogeneous (Davila
et al., 2005; Joosten et al., 2007; Xystrakis et al., 2004); for ex-
ample, the CD8+Tregpopulation is important in resolving inflam-
mation and preventing autoimmunity (Frisullo et al., 2010; Sun
et al., 2009; Trandem et al., 2011). Alternatively, NeST-depen-
dent activation of basal inflammation could serve to attenuate
subsequent inflammatory events. Finally, NeST RNA may have
targets in addition to the Ifng gene that contribute to its appar-
ently anti-inflammatory effect (Figure S2).
The fact that the effects of NeST can be conferred by trans-
genic expression from ectopic loci, and to Ifng alleles on both
chromosomes when NeST is expressed heterozygously, argues
that NeST function, even on the adjacent IFN-g-encoding locus,
can be provided in trans. Although many lncRNAs, such as Xist
and HOTTIP, exert their function on neighboring genes exclu-
sively in cis, trans-acting lncRNA function has precedent in
HOTAIR, linc-p21, and Jpx lncRNAs (reviewed in Guttman and
Rinn, 2012). Notably, Jpx is required to activate the expression
of the adjacent Xist gene on the presumptive inactive X chromo-
in cis or trans (Tian et al., 2010). Thus, there is increasing recog-
nition in the field that lncRNA regulation of nearby genes can
occur by trans-acting mechanisms. The increased demands
made on these lncRNAs for target specificity are currently under
In the vicinity of the Ifng locus, many of the distal regulatory
elements map to regions now known to encode NeST (Sekimata
etal.,2009).Forexample, acetylation ofhistone 4(H4Ac),amark
of active transcription, has been observed in discrete regions
surrounding Ifng in activated CD4+and CD8+T cells. One peak
in particular, which correlates well with the differentiation of
both CD8+and CD4+T cells (Chang and Aune, 2005; Zhou
et al., 2004), is located 59 kb downstream of Ifng and coincides
Figure 5. Effect of Tmevp3 Locus and Transgenically Expressed
NeST RNA on Cytokine Expression by T Cell Subsets
(A and B) Splenic (A) CD4+and (B) CD8+T cells were isolated from three B10.S
(black circles) and three B10.S.Tmevp3SJL/J(white circles) mice and stimu-
lated ex vivo with PMA and ionomycin. The abundance of IFN-g and IL-22
protein secreted was determined by ELISA from supernatants collected at the
indicated times. Means and SE are indicated for each time point. Statistical
significance was determined using a two-way ANOVA test; asterisks denote
values that differ significantly between T cells derived from B10.S and T cells
derived from B10.S.Tmevp3SJL/Jmice.
(C) Splenic CD8+T cells were isolated from B10.S (black), B10.S.NeSTSJL/J
(sea green), and B10.S.NeSTB10.S(blue) mice and stimulated ex vivo with PMA
and ionomycin. The abundance of secreted IFN-g was determined by ELISA.
Asterisks and p values refer to the comparisons between T cells derived from
B10.S and T cells derived from each transgenic line.
See also Figure S2.
Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc. 749
that is critical for IFN-g expression in CD4+T cells maps 66 kb
downstream of murine Ifng and, in humans, 166 kb downstream
of IFNG. This regulatory element is also located in the NeST
gene. It contains a lineage-specific DNase I hypersensitive site
found in Th1 but not Th2 CD4+T cells (Balasubramani et al.,
2010). During lineage-specific induction of IFN-g, the proteins
CTCF, T-bet, and cohesin all localize to these sequences.
Indeed, arecentstudy (Collier etal.,2012)related theexpression
of NeST RNA (Tmevpg1) to the expression of IFN-g in CD4+
T cells by a mechanism that depends on the simultaneous
expression of T-bet. Simultaneous binding of cohesin, T-bet,
and CTCF results in a complex three-dimensional conformation
that is predicted to bring the NeST and IFN-g coding regions
into direct proximity (Hadjur et al., 2009; Ong and Corces,
2011; Sekimata et al., 2009).
Humans express an RNA species homologous to NeST that
also appears to be noncoding and is transcribed adjacent to
the IFNG locus from the opposite DNA strand. Interestingly,
polymorphisms that correlate with autoimmune and inflamma-
tory diseases such as rheumatoid arthritis, Crohn’s disease,
and multiple sclerosis have been found in the DNA sequence
that encodes the first intron of the IFN-g-encoding gene (Goris
et al., 2002; Latiano et al., 2011; Silverberg et al., 2009); this
DNA region also encodes the fifth intron of the overlapping
NeST RNA-encoding gene. As they do in mice, variations in
NeST RNA expression in humans could contribute to differences
in T cell response and disease susceptibility. Whether and how
disease-associated SNPs alter human NeST expression or func-
tion will be addressed in future studies.
Naturalpolymorphisms, both inhumansandin animalmodels,
can yield subtle quantitative allelic effects that are more difficult
to study but are more relevant to human medicine than the
effects of gene knockout or other loss-of-function genetic tech-
niques. The discovery of NeST RNA was the result of classical
forward genetics. Our analysis of NeST RNA was based on the
conceptual framework that regions that are thought to be ‘‘inter-
genic’’ encode functional RNA elements. Many genome-wide
association studies have also pointed to intergenic regions as
heritable causes of human disease (Libioulle et al., 2007; Sotelo
et al., 2010). This study establishes that some of these intergenic
regions may encode functional lncRNAs that are critical for
proper gene regulation. The promise of individualized medicine
relies on our understanding of as many genetic polymorphisms
Figure 6. NeST RNA Localization and IFN-g trans Activation
(A) Nuclear and cytoplasmic RNA from CD8+T cells from B10.S.Tmevp3SJL/J,
B10.S.NeSTB10.S, and B10.S.NeSTSJL/Jmice were fractionated by differential
centrifugation (Huarte et al., 2010). NeST RNA, unspliced actin RNA (nuclear),
and spliced actin RNA (cytoplasmic) from the nuclear and cytoplasmic frac-
tions were assessed by RT-PCR and gel electrophoresis.
(B) Quantitation of expression ratios of IFN-g mRNA from the B10.S and the
B10.S.Tmevp3SJL/Jalleles. A natural SNP in the IFN-g mRNA (coordinate
117882772; see Table S1) was amplified by RT-PCR (top and left panel).
cDNAs from B10.S and B10.S.Tmevp3SJL/Jwere subjected to a B10.S allele-
specific TaqI restriction digest (bottom, left) and fragments were analyzed by
(C) Splenic CD8+T cells were isolated from two B10.S 3 B10.S.Tmevp3SJL/J
heterozygous mice and stimulated withPMA and ionomycin.The proportionof
B10.S and B10.S.Tmevp3SJL/J-derived IFN-g mRNA was determined by
densitometry of the allele-specific restriction fragments. Mixtures of in vitro
transcribed RNAs at 1:10 and 1:1 ratios were used as controls.
750 Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc.
as possible in the context of individual immunological and other
Viral and Bacterial Strains and Culture
The DA strain of Theiler’s virus was produced by transfecting the PTM762
plasmid into BHK-21 cells as previously described (McAllister et al., 1989).
Salmonella enterica serovar Typhimurium SL1344 (Subbaiah and Stocker,
1964) strain was used. See Extended Experimental Procedures for inoculation
Mouse Strains and Transgenic Line Design
Congenic B10.S.Tmevp3SJL/Jand SJL/J.Tmevp3B10.Smice were imported
from the Pasteur Institute. B10.S and SJL/J mice were obtained from the
Jackson Laboratory (Bar Harbor, ME). (Balb/c x 129) F1 pseudopregnant
female mice were provided by Drs. Hugh McDevitt and Grete Sonderstrup
(Stanford, CA). All mice were bred at the Stanford Research Animal Facility
(RAF) except for the SJLJ/J mice, which were purchased at 4 weeks of
age and housed in the Stanford RAF for at least 6 weeks prior to all
B10.S mice that express NeST RNA transgenically were developed by
pronuclear microinjections. NeST cDNA was cloned downstream of a CD4+
and CD8+T cell-specific promoter and upstream of an SV40 polyadenylation
cell embryos, microinjection procedures, and transfer to pseudopregnant
females were performed as previously described (Singer et al., 1998).
Cell Culture, Infection, and Stimulation
Macrophage culture and infection were performed as previously described
(Arpaia etal.,2011;Martinat etal.,2002).ForTcellcultureexvivo,splenocytes
were prepared and CD3+T cell, CD4+T cell, or CD8+T were isolated with the
use of kits from Miltenyi Biotec (Auburn, CA). Nuclei were enriched as previ-
ously described (Huarte et al., 2010). For T cell stimulation assays, cells
were cultured for 10 hr prior to stimulation with 50 ng/ml PMA and 1.5 mM
ionomycin (Sigma-Aldrich, St. Louis, MO).
Figure 7. NeST RNA’s Physical Association with WDR5 Protein and
Effect on Histone 3 Lysine 4 Trimethylation at the Ifng Locus
(A) RNA preparations from 293T cells that were cotransfected with FLAG-
tagged WDR5 cDNA and either B10.S-derived NeST cDNA (blue) or SJL/J-
derived NeST cDNA (sea green) were analyzed after IP with either anti-FLAG
antibodies or anti-immunoglobulin G (anti-igG) control antibodies. NeST RNA
retrieval was determined by measuring RNA input levels normalized to glyc-
eraldehyde 3-phosphate dehydrogenase (GAPDH; bottom left panel). Specific
RNA retrieval was determined by subtracting NeST RNA retrieval with anti-igG
antibodies from the retrieval with anti-FLAG antibodies, followed by normali-
zation to the amount of input RNA. Immunoblot analysis (bottom right panel)
confirmed FLAG-WDR5 expression following transfection and the specificity
of the anti-FLAG and anti-igG antibodies.
(B) IFN-g production and H3K4me3 occupancy in spleen following immune
challenge. B10.S, B10.S.NeSTB10.S, and B10.S.NeSTSJL/Jmice were injected
intraperitoneally with 50 mg of LPS, and spleens were dissected 4 and 6 hr
later. The abundance of IFN-g protein was determined by ELISA in tissue
homogenates (top panel) and the occupancy of histone 3 lysine 4 trimethyla-
tion at the Ifng gene was determined by ChIP-qPCR analysis (bottom panel). A
schematic diagram of the positions of primers used for H3K4me3 is shown.
Specific DNA retrieval was measured by normalization to the amount of input
DNA and ChIP signal at GAPDH loci.
(C) ChIP-qPCR analysis of H3K4me3 at the Ifng locus in CD8+T cells from
B10.S and B10.S.NeSTSJL/Jtransgenic mice. CD8+T cells were isolated from
four B10.S and four B10.S.NeSTSJL/Jmice, and stimulated ex vivo with PMA
and ionomycin. Occupancy of H3K4me3 at the Ifng gene was assayed 24 hr
after stimulation by ChIP-qPCR at four different regions. For all pooled data,
means and SE are shown.
Cell 152, 743–754, February 14, 2013 ª2013 Elsevier Inc. 751
WDR5 and Chromatin IP
NeSTB10.S, NeSTSJL/J, HOTTIP, HOTAIR, and U1-encoding cDNAs were
cloned into a eukaryotic gene expression plasmid and cotransfected with
pcDNA3.1 that did or did not express FlagWDR5. Cells were lysed and immu-
noprecipitated as previously described, with modifications (Wang et al., 2011).
Chromatin IP (ChIP) and qPCR were carried out according to the Farnham
protocol (O’Geen et al., 2011).
RNA and Cytokine Quantitation
Protein quantitation was performed using commercially available ELISA kits
(R&D Systems, Minneapolis, MN) or Luminex (Affymetrix, Santa Clara, CA) ac-
cording to the manufacturer’s instructions. qRT-PCR was performed with total
RNA from cells or tissues of interest and serial dilutions of known quantities of
RNA. The three B10.S and 19 SJL/J polymorphisms (Table S1) were intro-
duced by site-directed mutagenesis.
Mean values and significance were determined using Student’s t test. Survival
curves were analyzed with the log rank test. The null hypothesis (that
the strains compared were not different) was rejected when p values
were %0.05. Instances when the observed differences could be reported
with a confidence of 95% (*), 99% (**), or 99.9% (***) are denoted.
Additional methods are described in Extended Experimental Procedures.
Supplemental Information includes Extended Experimental Procedures, two
figures, and one table and can be found with this article online at http://dx.
We thank Holden Maecker, Roberto Mateo, and Peter Sarnow for comments
on the manuscript. We also thank Laura Attardi for experimental advice, Nigel
Killeen for plasmids, Mary Vadeboncoeur and Grete Sonderstrup for their
transgenics expertise, Shyamalia Roy for Salmonella titering and the Human
Immune Monitoring Core for Luminex assays. Individuals involved in this
work were supported by scholarships from the Gates Foundation and the
Stanford University DARE program (J.A.G.), the National Science Foundation
(O.L.W.), the Stanford Medical Science Training Program (Y.W.Y.), and an
NIH Training Grant (S.G.). H.Y.C. is an Early Career Scientist of the Howard
Hughes Medical Institute. Research funding was provided by the National
Society for Multiple Sclerosis (K.K. and M.B.), the Scleroderma Research
Foundation (H.Y.C.), and an NIH Director’s Pioneer Award (K.K.).
Received: March 1, 2012
Revised: July 28, 2012
Accepted: January 7, 2013
Published: February 14, 2013
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