in vitro characterization of a novel cis-acting element (nce) in the cd4 locus
TRANSCRIPT
In Vitro Characterization of a Novel Cis-acting
Element (NCE) in the Cd4 Locus
Alexis Grant1, Yordan Penev1, Gregory Swan2, Sophia Sarafova3
Biology Department, Davidson College 1: Equal contributors; 2: Currently at NCI, Bethesda, Maryland; 3. Secondary affiliation: Immunology Department, Duke University
Novel Cis-acting Element (NCE)
Figure 2. NCE contains a region of high homology across vertebrate species. The positions of the
known cis-elements in the Cd4 gene (abbreviations listed in Fig. 1) are indicated by colored boxes. Arrows
indicate the orientation of transcriptional units in the locus. UCSC’s genome browswer 30-way Multiz
alignment shows sequence conservation at the silencer and a downstream region that was knocked out along
with the silencer in (6) Zou, et al. and (7) Leung, et al.
0.0 5.0 10.0
Pr + PE
Pr
Empty
GFP rMFI
SS
C
pdsRED
101
102
103
104
105
106
107
0
649017
1298034
1947051
2596068
55%
300000 3650000 700000010
1
102
103
104
105
106
7-A
AD
300K 500K 700K
FSC
30% **
*
*p < 0.05
**p < 0.005
Empty Pr Pr + PE
B
Figure 3. Methods (modified from (8) Wildt, et al. 2007 and Wang, et al. 2010) A) Seven reporter constructs were
created using PCR to amplify the known Cd4 promoter (Pr), the known proximal enhancer (PE), the 1.6 Kb region of
interest (NCE) and the conserved fragments 5 and 8 from the C57BL/6 genomic DNA and ligating them into the multiple
cloning site (MCS) or Afl-II site of the GFP reporter plasmid pd2eGFP-1. B) FACS analysis of transiently transfected
RLM-11 cells. Five million RML-11 murine thymoma cells were transfected with 5-20 μg of one of the above reporters
and 5-20 μg of pRed or CD8 using electroporation (BTX). After 18 hours, the cells were stained forCD8 and 7-AAD. CD8
and eGFP expression were analyzed using an Accurri6 flow cytometer and the FCS Express V3 software. Cells that
were efficiently transfected were identified as 7-AAD-RFPhi. The Mean Fluorescence Intensity (MFI) of eGFP
fluorescence of the 7-AAD-RFPhi was recorded and compared to promoter construct to calculate the relative eGFP MFI
and determine the presence of promoter or enhancer activity.
INTRODUCTION
We thank Dr. Yuan Zhuang for hosting us in his lab during the summer, Dr. Rémy Bosselut for the generous donation of
the RLM11 cell line, Dr. Sue Sharrow of the NCI for FACS analysis advice, and Dr. Alfred Singer for advice and the
generous donation of antibodies. The research was supported by an Educational Enhancement Grant from North
Carolina Biotechnology Center and an NIH AREA grant 1R15AI098001-01A1.
ACKNOWLEDGEMENTS
Figure 4. NCE enhances Cd4 promoter driven eGFP fluorescence in a developmental-stage-specific manner. NCE
enhancer function was tested in 5 mouse cell lines, immortalized at different developmental stages (Fig. 1), and a CD4-SP
human cell line. The proximal enhancer (PE) was used as a positive control. All bars represent at least three trials. Error bars
represent the standard error of the mean. Using a one-tailed T-Test ,* = <0.05, ** = < 0.005. NCE has significant enhancer
function only in the CD4+ intermediate and single positive cell lines. The decrease of proximal enhancer function with
increasing maturity is readily observable, confirming that the transfection conditions used were suitable for each cell line.
Figure 1. Increasing CD4 levels on developing MHC-II specific thymocytes cannot be explained by
the activity of the known cis-elements. Three developmental stages of MHC-II specific T-cells are
illustrated in increasing order of maturity (blue wedge) with corresponding levels of cell-surface CD4. Above
are the representative cell lines used in the study. The activity of the known cis-elements in the Cd4 gene
(DE: distal enhancer, PE: proximal enhancer, Pr: promoter, and Sil: silencer) is indicated by arrow thickness.
Figure 6. PMA and Ionomycin stimulation and
transfection with NCE. Cells from the RLM11,
2b4, and EL-4 cell lines were transfected with
NCE. Half of each population was stimulated with
13nM PMA and .7uM Io 5 hours after the
transfection. After 18 hours, the cells were stained
for CD69 and analyzed by flow cytometry.
Upregulation of CD69 indicates successful
stimulation. No dramatic effect on NCE expression
was observed, as determined by levels of the
reporter eGFP fluorescence intensity.
Figure 7. Transcription factor homology
and expression. A) NCE sequence
analysis. The minimal functioning conserved
sequence of NCE was analyzed for sequence
identity with transcription factor binding sites
using NCBI’s Blast tool. Sequences with the
greatest homology were ranked & mapped
using color-coding. B) Real-time PCR
confirmation of transcription factor
expression levels. RNA was extracted from
cells of the AKR (DP), RLM11 (Int.), and 2B4
(SP) cell lines using Trisol reagent. CDNA
was synthesized using qScriptTM cDNA
Reaction Mix from Quanta Biosciences.
QPCR primers were designed to span exon
boundaries (with the exception of NFAT5)
and ordered using the PrimeTime qPCR tool
by Integrated DNA Technologies. Results
were quantified via SYBR green real time
eppendorf PCR and compared to previously
reported data on the Gene Skyline database
from the Immunological Genome Project. No
notable differences were observed. The
experiment was repeated 3 times in triplicate.
Transcription factor expression levels were
normalized to the expression levels of a
housekeeping gene, GAPDH.
A
B
Figure 5. Fragment 5 is a functional component of NCE. A. Enzyme digest of plasmids and map of digestion sites.
Plasmids with fragment 5 and two instances of fragment 5 (5x2) were digested using the enzymes KpnI, SpeI, BglII, and
Bam HI because the digestion sites for these enzymes have been mapped in each plasmid (5B). For each digest, the
observed band size correlated to the expected size of single-stranded DNA fragments according to the digestion sites of the
plasmid, confirming the identities of both fragments. B. GFP expression relative to promoter. NCEx2 and fragment 5
showed a two-fold increase in GFP expression compared to promoter. Fragment 5x2 showed a three-fold increase from the
promoter. Fragment 5x2 showed a three-fold increase from the promoter. Results are based on one trial.
• We have isolated and characterized the minimum functional conserved
sequence of a novel positive regulatory element (NCE) in the Cd4 locus.
• NCE exhibits developmental stage specificity in murine cells as it functions in
the intermediate developmental stage cell line RLM11 and the CD4+ single
positive developmental cell lines EL-4 and 2B4, but not in the CD8/CD4 double
positive stage cell lines AKR1G1 and VL3-3M2.
• The assignment of developmental stage to the cell lines matches expected
levels of characteristic transcription factor expression, with the possible
exception of ThPok in the 2B4 cell line.
• Based on a single assay, we conclude no correlation between TCR stimulation
and NCE function, suggesting that the element might operate in a TCR-
independent manner.
CONCLUSIONS
METHODS
RESULTS
1. Ellmeier W, Sawada S, Littman DR. The regulation of CD4 and CD8 coreceptor gene expression during T cell development. Annu Rev Immunol. 1999; 17:
523-554.
2. Singer A, Adoro S, Park JH. Lineage fate and intense debate: Myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat Rev Immunol.
2008 Oct; 8(10): 788-801.
3. Kioussis D, Ellmeier W. Chromatin and CD4, CD8A and CD8B gene expression during thymic differentiation. Nat Rev Immunol. 2002 Dec; 2(12): 909-19.
4. Siu G. Controlling CD4 gene expression during T cell lineage commitment. Semin Immunol. 2002 Dec; 14(6): 441-51.
5. Sarafova SD, Van Laethem F, Adoro S, Guinter T, Sharrow SO, Feigenbaum L, Singer A. Upregulation of CD4 expression during MHC class II-specific
positive selection is essential for error-free lineage choice. Immunity. 2009 Sep 18; 31(3): 480-90.
6. Zou YR, Sunshine MJ, Taniuchi I, Hatam F, Killeen N, Littman DR. Epigenetic silencing of CD4 in T cells committed to the cytotoxic lineage. Nat Genet.
2001 Nov; 29(3): 332-336.
7. Leung RK, Thomson K, Gallimore A, Jones E, Van den Broek M, Sierro S, Alsheikhly AR, McMichael A, Rahemtulla A. Deletion of the CD4 silencer
element supports a stochastic mechanism of thymocyte lineage commitment. Nat Immunol. 2001 Dec; 2(12): 1167-1173.
8. Wildt KF, Sun G, Grueter B, Fischer M, Zamisch M, Ehlers M, Bosselut R. The transcription factor Zbtb7b promotes CD4 expression by antagonizing
Runx-mediated activation of the CD4 silencer. J Immunol. 2007 Oct 1; 179(7): 4405-14.
REFERENCES
Conditions like HIV infection, chemotherapy, and congenital defects will deplete
patients’ CD4 T-cells. CD4 T-cells are the coordinators of the adaptive immune response,
and the strength of a person’s immune system depends on the amount and timing of
production of functional CD4 T-cells. In the process of developing into CD4 T-cells, the
progenitor cells in the thymus (thymocytes) go from the double positive (DP) stage
(CD4+CD8+) to the intermediate (INT) stage (CD4+CD8lo), to the single positive (SP)
stage (CD4+CD8-), with progressive increase of CD4 expression that is essential for
making the correct lineage choice based on the specificity of their T cell receptor (TCR)
(1,5). The alternative lineage that stems from the INT stage is the CD8 cytotoxic T cell,
which is responsible for killing host cells infected with intracellular pathogens (2). Our
overall goal is to understand how the upregulation of CD4 expression is regulated during
development.
Previous studies have identified a promoter, two enhancers, and a silencer
responsible for Cd4 gene expression (4). However, the activity of the known enhancer
elements decreases with maturation, which is inconsistent with the simultaneous
increase in CD4 levels as the cells develop from DP to SP (3). Thus, current knowledge
of the function of the regulatory elements involved in Cd4 expression is not sufficient to
explain its tight regulation during the intermediate phase between the double positive
and single positive (CD4+) stages of T cell development (Figure 1). To address this
inconsistency, we are investigating the newly identified cis-transcriptional regulatory
element (NCE) for enhancer function dependent on developmental stage. Previous
experiments have shown that NCE, a highly conserved sequence on intron 1 of the Cd4
locus (Figure 2), functions as an enhancer independent of its position and orientation.
We used cells from the AKR, RLM11, EL-4 and 2B4 cell lines to represent the double
positive, intermediate, and single positive stages of thymocyte development. Here we
describe the differences in NCE function between developmental stages based on these
cell line models. Additionally, we show our initial attempts at identifying the minimum
functional unit of NCE and the transcription factors that bind it, as well as the element’s
T-cell receptor responsiveness.
300bp
Fragment 5 Fragment 5x2
1k
b la
dd
er
Kp
n1
Sp
e1
Bam
HI
Bg
lII
Un
cu
t
1kb
3kb
10kb 4kb 1
kb la
dder
10kb 1k
b la
dd
er
Kp
n1
Sp
e1
Bam
HI
Bg
lII
Un
cu
t
300bp
1kb
3kb 4kb
Key
KpnI (K)
SpeI (S)
BamHI (BH)
BglII (B)
GFP
BH S S B
K 450bp K 540bp
GFP
BH S S S S
K K 540bp
1.6kb
450bp 450bp 0 0.5 1 1.5 2 2.5 3 3.5
Pr
NCEx1
NCEx2
Frag 5
Frag 5x2
GFP expression relative to promoter
Afl-
II
Pr eGFP SV40
Poly A 1
PE Pr eGFP SV40
Poly A
PE
Pr
2
6 Pr eGFP SV40
Poly A
4 Pr eGFP SV40
Poly A
3 Pr eGFP SV40
Poly A NCE
NCE
NCE NCE
7 Pr eGFP SV40
Poly A
eGFP
5 Pr eGFP SV40
Poly A
NCE with conserved
region (red)
Cd4 Promoter (550bp)
Cd4 Proximal
Enhancer (300bp)
Enhanced Green
Fluorescent Protein
Pr
PE + Pr
NCEx1 + Pr
NCEx2 + Pr
Fragment 5 + Pr
Fragment 5x2 + Pr
Fragment 8 + Pr
A
CD69
RLM11
EL4
2B4
Cell line
eGFP
RLM
not stimulated stimulated
CD6910
110
210
310
410
510
610
7
0
388
776
1163
1551
RLM
not stimulated stimulated
GFP-A
Cou
nt
101
102
103
104
105
106
107
0
19
39
58
77
EL-4
not stimulated stimulated
GFP-A
Cou
nt
101
102
103
104
105
106
107
0
3
6
9
12
2B4
not stimulated stimulated
GFP-A
Cou
nt
101
102
103
104
105
106
107
0
57
114
170
227
101
102
103
104
105
106
107
0
1097
2194
3290
4387
101
102
103
104
105
106
107
0
1101
2202
3303
4404
Not stimulated Stimulated Isotype control
TTCCCAGGGGTGACGTACACAGGATGCTGCGCTTACTCTTTATGAGATATAGC
GCCTCTGGGGAGGCTGAGGATGTGGGAATCAAAGGCCAGGTCTCTGGGGT
AGCAGCGGAAACCTGAAGCACCAATGCAGGCTGTGGCTGGAAACCCAC
Binding sites:
1)KLFS: Kruppel like transcription factors
2)IKRS: Ikaros zinc family finger
3)ETSF: ETS1 factors (ELK-1)
4) RBPF: RBPJ – kappa
5) LEFF: LEF1/TCF
6) C2H2 zinc finger transcription factors 2 (ThPOK)
7) CATT: CCAAT binding factors
8) CEBP: Ccaat/Enhancer Binding Protein
9) ETSF: Human and murine ETS1 factors
10) NFAT: Nuclear factor of activated T-cells
60 70 80 90 100
110 120 130 140 150
1 2
10 20 30 40 50
3
4 5
9 13
7 10
6 8
0
100
200
300
400
DP Int. SP
Rel
ativ
e ex
pre
ssio
n
no
rmal
ized
to
GA
PD
H
NFAT5
0
100
200
300
400
DP Int. SP
Rel
ativ
e ex
pre
ssio
n
no
rmal
ized
to
GA
PD
H
RBPJκ
0
200
400
600
800
DP Int. SP
Rel
ativ
e ex
pre
ssio
n
no
rmal
ized
to
GA
PD
H
ThPok
0
100
200
300
400
DP Int. SP
Rel
ativ
e ex
pre
ssio
n
no
rmal
ized
to
GA
PD
H
CEBPζ
Expected Observed
0
1
2
3
4
5
6
7
8
9
AKR1G1 VL3-3M2 RLM11 EL-4 2B4 Jurkat
GFP
rM
FI
Pr
Pr + PE
Pr + NCE
Pr + NCEx2
**
** **
* *
* * *
*
DP (mouse) Int (mouse) SP (mouse) SP (human)
*
*