Gillian M. Schiralli Lester , Katarzyna Kaczmarek , Andrew J

Mechanisms by which Negative Elongation Factor (NELF)
Establishes
and
Maintains
HIV
Latency.
1
1
1
2
1
Henderson
Gillian M. Schiralli Lester , Katarzyna Kaczmarek , Andrew J. Woerner , Malini Natarajan and Andrew J.
of Medicine, Section of Infectious Disease, Boston University School of Medicine. 2Stowers Institute for Medical Research, Kansas City, MO 64110..
Nelf Limits HIV Transcription and Replication in Primary CD4+ T Cells
F.)
** P < 0.01
800
700
600
500
400
300
200
100
0
NF-κB
SP-1
Sp1
Sp1
Sp1
HELZ – RNA helicase, promotes cell proliferation and translation
initiation.
40
PMA+PHA
30
20
10
HIV Elongated Transcripts
(normalized to β-actin)
RBM12 Transcripts
(normalized to β-actin)
DMSO
0
30
DMSO
25
PMA+PHA
15
10
5
0
PDZD8
1.4
RNAP II
1.2
PMA+PHA
1
0.8
0.6
0.4
0.2
35
DMSO
30
PMA+PHA
25
20
15
5
0
11B10
HELZ
NELF
Ac
RNAP II
TAR
nuc 0
HAT
Tat
P-TEFb
*
*
*
1.2
PMA+PHA
1
0.8
0.6
0.4
0.2
0
DSIF
E.)
60
HIV Elongated Transcripts
(relative to β-actin)
nuc 1
DMSO
50
DMSO
PMA+PHA
4000
50000
0
Side-scatter
shNelfB
8
7
6
5
4
3
2
1
0
D.)
11B10
CA5
Figure 3: Cell lines were transfected with siControl or siNelf-B siRNA or treated with TSA for 72 hours. A.) GFP
expression was measured by flow cytometry. B.) The fold induction of GFP expression over the siControl sample is
graphed for multiple treatments of each cell line. Cell lines were transduced with shPRS Vector or shNelf-B specific
Lentivirus, 72 hours post transduction C.) GFP expression was measured by flow cytometry. D.) The fold induction of
GFP expression over the shPRS Vector sample is graphed for the representative experiment.
Figure 4
Overexpression of Nelf Represses HIV Transcription and Recruits Pcf11 to the HIV LTR.
A.)
B.)
40
35
30
25
20
15
10
** P< 0.01
5
E.)
3.5
3
Bcl11b
2.5
β-Actin
2
1.5
1
0.5
Figure 6: HEK293T cells were transfected with 2ug of pNL43-Luc DNA along with 2ug of pcDNA3Vector or Bcl11b-HA or Flag-Nelf-B or both Bcl11b-HA + Flag-Nelf-B. At 48 hours post transfection A.)
cells were lysed to measure HIV transcription via Luciferase expression. B.) Whole cell lysates were
used to measure protein expression. C.) Viral supernatants were used to measure cell free p24
protein via ELISA. The latent cell line model Ach2 was transfected with siControl or siBcl11b specific
siRNA, at 24 hours post transfection D.) mRNA was isolated and used to measure expression of HIV
elongated transcripts. E.) Whole cell lysates were used to verify protein knockdown of Bcl11b.
Figure 7
Bcl11b Represses HIV Transcription in the CA5 Cell Line Model of
Transcriptional Interference
C.)
FLAG-NELF-B
Vector
**
20
A.)
IB: Pcf11
IB: NELF-D
*
15
10
5
Untreated
siControl
B.)
siBcl11b
CA5
5.9%
6.07%
9.3%
**
GFP Expression
3.5
3
2.5
2
1.5
1
0.5
0
This data has been published in J. Biol. Chem. 2013 Sep 6;288(36):25995-6003. doi: 10.1074/jbc.M113.496489
Figure 4: HEK293T cells were transfected with 5ug HIV-Luc and pcDNA3 vector control or pcDNA3-FLAG-NELF-B. A)
Luciferase assays were performed 48 h post-transfection to measure HIV transcription. B) 48 h post transfection,
ChIPs were performed using FLAG, NELF-D, RNAP II and Pcf11 antibodies as indicated and primers that spanned -45+72 of the HIV LTR. C) Jurkat T cells were lysed and precleared lysates were used for immunoprecipitation using a nonspecific antibody (Control Ig), anti-Pcf11 or anti-NELF-D antibodies. Immunoprecipitated extracts and 10% input
controls were immunoblotted (IB) with Pcf11 and NELF D antibodies. Each IB was run on a single gel and processed as
a single image. Lanes were rearranged for presentation purposes but were not individually modified.
Figure 5
Nelf Functionally Interacts with the NCoR1-GPS2-HDAC3 complex which
Binds the Proviral LTR and Limits HIV Transcription
B)
IP-FLAG
MW (kDa) NELF-D
Smrter (NCoR)
250
NELF-A
150
Bcl11b
100
50
C)
NELF-B
FLAG-NELF-D
HDAC3
+
+
+
+
+
+
FLAG-NELF HA-HDAC3 IP
FLAG-NELF +
HA-GPS2 +
IP
IB: α-HA
D)
-
+
+
-
IB:α- HA
** P < 0.01
siControl siBcl11b
Figure 7: The CA5 cell line was transfected with siControl or siBcl11b specific siRNA. At 24
hours post transfection A.) GFP expression of HIV was measured by flow cytometry. B.) mRNA
was isolated to measure elongated HIV transcripts.
SUMMARY
• Proximal pausing of RNAP II on the HIV-LTR is a key checkpoint
controlling HIV transcription within multiple cell models of HIV latency
which include CD4+ primary T cells, cell line models of transcriptional
interference, and T cell latent HIV line Ach2.
• Releasing the paused polymerase by limiting Nelf results in the
rebound of HIV transcription in cell line models of transcriptional
interference irrespective of the control and activity level of the
neighboring gene promoter.
• We have identified a functional interaction with Nelf and NCoR1GPS2-HDAC3 corepressor which binds the transcriptionally silent HIVLTR and are released upon reactivation of transcription.
0.7
37
25
20
20
10
15
0
10
Nelf Binds Latent HIV LTR and is Disassociated Upon Stimulation
2000
GFP Expression
40
30
6000
0
75
HIV
TAR
1.4
B-Actin
8000
shPRS Vec
CA5
A)
10
HIV Induction Correlates with Decrease of Host Gene Expression in 11B10 Cell Line
D.)
RNAP II
5’ LTR
10
9
HIV
DMSO
100000
CA5
20
T Cell Activation Induces HIV but does not Influence Host Gene Expression in BA1 Cell Line
C.)
HELZ Transcripts
(relative to β-actin)
Transcriptional
elongation
11B10
BAI
HIV
50
Nelf-B
Side-scatter
HELZ
CA5
3Me
Ac
11B10
Relative Binding to Background
PDZD8 - moesin-interacting cytoskeletal regulatory protein. Interacts
with Gag and promotes HIV infection. Stabilizes HIV capsid and
supports HIV infection.
HIV Induction Correlates with Induction of Host Gene Expression in CA5 Cell Line
B.)
DSIF
D.)
BAI
Luciferase Units x104
11B10
LBP
3Me
SWI/SNF
D.)
500
RBM12 – contains five RNA binding motifs, two proline-rich regions
and several putative transmembrane domains.
PDZD8
BA1
HDAC
*
shNelfB
1000
RBM12
CA5
nuc 1
nuc 0
shPRS Vec
C.)
PLAP expression
HIV LTR Orientation
Bcl11b
150000
10000
0
0
Cell Line Host Gene
3Me
5’ LTR
GFP Expression
1500
A.)
TAR
nuc 0
C.)
Luciferase units
16%
0
Polymerase
Proximal
Pause
β-actin
BA1
α- FLAG
Promoter
TBP
TATA
NELF-B
14000
12000
*P=0.02
α- FLAG
p65
CD3+ CD28
C.)
200000
CD3 + CD28
10% Input
NF-κB
p50
H.)
1
10% Input
5’ LTR
p65
siNELF
2
BA1
TAFs
p50
G.)
CA5
+TSA
α-FLAG
B.)
0
siNELF
3
HIV Integration into Host Genes
Transcription
Initiation
3Me
0.5
B.)
250000
siCtrl
Ctrl IgG
Enhancer
1
A.)
4
α-FLAG
10% Input
Modulatory Element
1.5
Figure 2
NFAT
LSF
NF-κB
11B10
2
Events
11%
TSA
CG17002 (GPS2)
NELF-E
Percent precipitated
LEF1
USF
TATA
SiNELF
2000
HIV Elongated Transcripts
(normalized to β-actin)
COUP
Sp1
2.5
elongated
initiated
Figure 1: Human primary CD4+T cells infected with HIV-LUC were transfected with siControl (siCtrl) or siNELF-B. A.) NELF
depletion was verified at 48 hpk by immunoblot. B.) q-PCR for NELF-B mRNA transcripts. C.) 48hpk luciferase activity was
measured to monitor HIV transcription. D.) mRNA was isolated from HIV-Luc infected cells and used to measure initiated
(+1 to +40) and elongated transcripts (+5396 to +5555), initiated and elongated data were normalized and set equal to 1
in siCtrl treatment, siNELF data reflects increased transcription over normalized siCtrl levels. E.)Cell culture supernatants
from CD4+ T cells were used to measure virus particle release via ELISA 48 hpk. F.) CD4+ T cells were infected with HIVPLAP with VSV-G, 48 h post-infection the cells were transfected with siControl or siNELF-B. 48 h post transfection cells
were stained with anti-PLAP and FACS was used to assess the HIV infected cell population. The MFI value for siControl
treatment is 6624, for siNelf treatment it is 7174. G.) 48hpk HIV-LUC infected CD4+ T cells were activated with anti-CD3
and anti-CD28 antibodies for 4 h, 12-16 hrs post stimulation whole cell lysates were immunobloted to detect NELF-B
protein levels. H.) Luciferase activity was measured to monitor HIV transcription in siCtrl or siNELF treated cells following
CD3 + CD28 activation.
PDZD8 Transcripts
(normalized to β-actin)
5’ LTR
C/EBP
initiated
elongated
3
siNELF
siControl
SiCtrl
B.)
3.5
This data has been published in J. Biol. Chem. 2013 Sep 6;288(36):25995-6003. doi: 10.1074/jbc.M113.496489
NFAT
NFAT C/EBP
0
PLAP expression
Regulation of HIV Transcription Initiation, Polymerase Proximal Pausing,
Chromatin Regulation and Elongation.
A.)
20
Events
P24 (pg/ml)
E.)
BACKGROUND
YY1
40
* P < 0.05
siCtrl
RBM12
ETS
60
A.)
HIV Elongated Transcripts
β-actin
* P< 0.05
80
4
Bcl11b Represses HIV Transcription
Nelf Limits HIV Transcription in Cells that Exhibit Transcriptional Interference
HIV Elongated Transcripts
NELF-B
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
D.)
** P < 0.01
Figure 6
Side-scatter
B.)
Basal Transcription
C.)100
Luciferase units
A.)
Relative NELF-B expression
Background: Understanding events which contribute to HIV latency are critical
for improving current treatments and potentially eradicating HIV infection.
Events that contribute to HIV transcriptional latency include repressive
chromatin structure, transcriptional interference, the inability of Tat to recruit
P-TEFb and poor processivity of RNA polymerase II (RNAP II). Previously, we
have established an important role for promoter proximal pausing in regulating
HIV transcription. In this study we investigate whether RNAP II pausing is
operative in the context of different cell models of latency, including
transcriptional interference. Our data support that RNAP II pausing via NELF
recruits co-repressor complexes and transcription factors which influence T cell
commitment to repress HIV transcription.
Methodology: We have identified novel NELF interacting factors via Mass Spec
analysis and have used immunoprecipitation and ChIP analysis to verify physical
and functional interactions. We utilized multiple infected cell models including
HIV infected Jurkat T cells, the latently infected ACH2 cell line, latently infected
transcriptional interference cell lines and primary CD4+ T cells to further
investigate NELF’s involvement in HIV transcriptional latency.
Results: We have identified novel NELF interacting partners NcoR1-GPS2HDAC3, a known co-repressor complex, and Bcl11b, a T cell commitment factor.
HDAC3, GPS2 and Bcl11b bind latent HIV LTR. When the latent HIV LTRs are
induced with PMA binding of these factors to the LTR is diminished. In addition,
we have investigated NELF’s involvement in regulating HIV transcription in cell
lines where the latent HIV LTR is integrated into a transcriptionally active gene,
resulting in transcriptional interference of the provirus. Treatment of these cell
lines with a HDAC inhibitor such as TSA did not induce HIV transcription,
however, when we knock down NELF in this model HIV transcription is induced.
Furthermore, overcoming HIV proviral transcriptional interference correlates
with diminished NELF binding to the HIV LTR.
Conclusions: Our data indicate that NELF is coordinating multiple mechanisms
responsible for transcriptional silencing of the HIV LTR. Key factors involved in
NELF induced repression of the provirus are NcoR1-GPS2-HDAC3 and Bcl11b.
NELF coordinates chromatin organization through the NcoR1-GPS2-HDAC3
complex and the recruitment of Bcl11b. These data support that RNAP II
pausing is a major checkpoint in the establishment of HIV latency and a
potential target by which to purge latent HIV reservoirs.
AP-1
Figure 3
Fold Induction of GFP
expression
Figure 1
Relative Light Units
RESULTS
Fold Induction of GFP
expression
ABSTRACT
Relative HIV transcripts
1Department
650 Albany Street
EBRC, room 640
Boston, MA. 02118
[email protected]
[email protected]
Phone: (617) 414-5240
Fax: (617) 414-5280
p24 (pg/ml)
Poster Session Number: P-F1:
Abstract: 1360
0.6
DMSO
PMA
0.5
0.4
** P < 0.01
0.3
• We have identified a novel Bcl11b interaction with the paused NelfRNA polymerase complex and have found Bcl11b to be a repressive
factor in HIV transcription.
** P < 0.01
0.2
0.1
ACKNOWLEDGEMENTS
0
20
15
10
2.5
BA1 stimulated w/ PMA
2
Percent Input
Transcription Schematic: Regulation of HIV transcription initiation and elongation. A.) HIV LTR organization. This only
represents a small subset of cis-elements and transcription factors, which bind these sites. B.) Cellular transcription factors
are recruited to LTR elements and initiation complex forms at the transcriptional start site. Nucleosomes are
posttranslationally modified favoring a condensed chromatin structure that impedes RNAP II transcriptional elongation. C.)
RNAP II processes a short distance downstream from the transcriptional start site when DSIF and NELF induce a pause in
transcription. Pcf11 reinforces this block in elongation by prematurely terminating the transcription of the short nascent RNA
product. HDAC recruitment to the paused complex reinforces a transcriptionally repressed chromatin state. The red asterisk
depicts phosphorylation of RNAP II CTD at serine 5 position. D.) RNAP II elongation complex is released from the
transcriptional pause by the recruitment of P-TEFb, which mediates hyperphosphorylation of the CTD at serine 2 position and
phosphorylation of DSIF, which induces NELF disassociation from the complex (red asterisks indicate key phosphorylation
events). The recruitment of chromatin remodeling machinery such as HATs and PBAF SWI/SNF facilitates acetylation of
nucleosomes, which displaces the blocking nucleosome and supports transcription elongation.
Percent Input
25
BA1 unstimulated
1.5
1
5
0.5
0
0
Figure 2: A.) Schematic of HIV provirus integration sites relative to neighboring cellular promoter for CA5, 11B10
and BA1 latently infected Jurkat cells. B.) – D.) Cell lines were treated with DMSO vehicle control or PMA (10ng/ml)
+ PHA (2ug/ml) for 24 hours, mRNA samples were collected and analyzed for gene expression of specific host gene
and elongated HIV. E.) BA1 cell line was left untreated or treated with PMA (10ng/ml) + PHA (2ug/ml) for 8 hours,
cells were then subject to formaldehyde crosslinking and sonication. Sheered chromatin was immunoprecipitated
with a non-specific antibody (Flag) or specific antibodies for RNAP II and NelfD. ChIP samples were analyzed via
qPCR with HIV specific transcriptional start site primers.
This data has been published in J. Biol. Chem. 2013 Sep 6;288(36):25995-6003. doi: 10.1074/jbc.M113.496489
Figure 5: A.) Nuclear extracts were prepared from FLAG-NELF-D transgenic Drosophila embryos and the
epitope tag was used to immunoprecipitate NELF complexes. Proteins were resolved by SDS-PAGE on 4-20%
gels (Invitrogen), and visualized by Coomassie Blue staining. Bands were excised and digested with trypsin and
proteins were identified by mass spectrometry. Bands identified are indicated by arrowheads with human
orthologs in parenthesis. B.) & C.) HEK293T cells were transfected with indicated vectors or pcDNA3 control
vector. Whole cell extracts were used for immunoprecipitation using a non-specific antibody and anti-FLAG
antibody, or FLAG resin which pulls down NELF. Immunoprecipitates were immunoblotted with anti-HA
antibody, which detects HA-HDAC3 and HA-GPS2. Data represent three or more independent experiments.
D.) ChIP using chromatin prepared from untreated or PMA treated ACH-2 cells. Antibodies are indicated below
the abscissa. Data are from a single experiment performed in triplicate and error bars represent the standard
error between these data points. These data are representative of at least three independent ChIP
experiments. * P < 0.05, ** P < 0.01.
We would like to thank Dr. Olaf Kutsch (University of Alabama at
Birmingham) for generously supplying the CA5, 11B10 and BA1 cell
lines along with his insightful comments and critical data analysis.
We would like to thank our long-term collaborator Dr. David Gilmour
(Penn State) for his expertize and continual support. We would like to
thank Dr. Chanhyo Lee for his experimental and intellectual efforts on
the project. Flow cytometry was performed with assistance of the
Boston University Medical Campus Flow Cytomety Core Facility.
Mass Spec was performed at the Boston University Medical Campus
Proteomics Core under the direction of Dr. Martin Steffen. This work
was supported by NIH grants AI062467 and AI077463