The transcriptional co-activator LEDGF/p75
displays a dynamic scan-and-lock mechanism
for chromatin tethering
Jelle Hendrix1,2, Rik Gijsbers3, Jan De Rijck3, Arnout Voet4, Jun-ichi Hotta2,
Melissa McNeely3, Johan Hofkens2, Zeger Debyser3,* and Yves Engelborghs1,*
1Laboratory for Biomolecular Dynamics,2Laboratory for Photochemistry and Spectroscopy,3Laboratory
for Molecular Virology and Gene Therapy and4Laboratory for Biomolecular Modelling, University of Leuven,
Leuven, Flanders, B-3000, Belgium
Received May 2, 2010; Revised September 27, 2010; Accepted September 28, 2010
Nearly all cellular and disease related functions of
the transcriptional co-activator lens epithelium-
derived growth factor (LEDGF/p75) involve tethering
conserved integrase binding domain (IBD), but
little is known about the mechanism of in vivo chro-
matin binding and tethering. In this work we studied
combining different quantitative fluorescence tech-
niques: spot fluorescence recovery after photo-
bleaching (sFRAP) and half-nucleus fluorescence
recovery after photobleaching (hnFRAP), continuous
photobleaching, fluorescence correlation spectros-
copy (FCS) and an improved FCS method to study
diffusion dependence of chromatin binding, tunable
focus FCS. LEDGF/p75 moves about in nuclei of
living cells in a chromatin hopping/scanning mode
typical for transcription factors. The PWWP domain
of LEDGF/p75 is necessary, but not sufficient for
in vivo chromatin binding. After interaction with
HIV-1 integrase via its IBD, a general protein–
protein interaction motif, kinetics of LEDGF/p75
shift to 75-fold larger affinity for chromatin. The
PWWP is crucial for locking the complex on chro-
matin. We propose a scan-and-lock model for
LEDGF/p75, unifying paradoxical notions of tran-
scriptional co-activation and lentiviral integration
to chromatinvia its
inliving HeLa cells
Gene expression is regulated by transcriptional cofactors
that fine-tune the interaction of the general transcription
machinery with gene-specific transcription factors. Lens
epithelium-derived growth factor (LEDGF/p75) was ori-
ginally identified as a 75-kDa transcriptional co-activator
interacting with the VP16 activation domain and with
components of the general transcription machinery (1).
LEDGF/p75 (530 amino acids) shares the first 325
amino acids with p52, an alternative splice variant from
the same PSIP1 gene (1,2) (Figure 1A) and plays an im-
portant role in cell survival (3), oncogenesis (4–7), auto-
immunity (8,9) and integration and replication of the
human immunodeficiency virus type 1 (HIV-1) (10,11).
LEDGF/p75 has an extensive interactome (Figure 1B).
The protein contains multiple DNA/chromatin binding
domains and a conserved protein–protein interaction
domain. The N-terminal PWWP domain of LEDGF/p75
(amino acids 1–93) holds a conserved (though not invari-
ant) Pro-Trp-Trp-Pro motif and belongs to the Tudor
domain ‘Royal Family’ of protein domains regulating
the chromatin function (12,13). This domain is generally
involved in chromatin structure regulation through
protein–protein interactions (14). A tripartite element in
LEDGF/p75 consisting of the two AT-hooks (amino acids
191–197 and amino acids 178–183) and the nuclear local-
ization signal (NLS) (amino acids 148–156) cooperates
with the PWWP domain for interaction with DNA/chro-
matin, as has been shown in vitro (15) and in vivo (16).
LEDGF/p75 allegedly interacts with heat shock elements
(HSE) through its helix-turn-helix (HTH) motifs (amino
acids 421–442 and amino acids 471–492) and with
*To whom correspondence should be addressed. Tel:+32 16 33 21 83; Fax:+32 16 33 63 36; Email: email@example.com and Tel:+32 16
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Nucleic Acids Research, 2011, Vol. 39, No. 4Published online 25 October 2010
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
stress-related regulatory elements (STRE) through the
PWWP domain to specifically promote expression of
stress-related genes (17–21). LEDGF/p75 contains a
high percentageof charged
contains two defined regions with positively charged
residues (amino acids 94–142 and amino acids 208–325),
that are involved in electrostatic interactions with DNA/
chromatin (15). A conserved superhelical domain in
LEDGF/p75 was originally identified as the domain for
binding to HIV-1 integrase (IN), the enzyme that catalyzes
the integration of HIV in the genome of an infected cell
(22,23). By virtue of this interaction LEDGF/p75 biases
lentiviral integration towards actively transcribed regions
in the genome (24–26). The ‘integrase binding domain’
(IBD) of LEDGF/p75 (amino acids 347–429) shows struc-
tural homology to known protein–protein interaction
motifs (27) and has been shown to interact with multiple
other proteins in cells: (i) JPO2, a Myc transcription factor
interacting protein (28), (ii) the menin tumor suppressor,
implicated in cancer and transcriptional regulation as a
component of the MLL-HMT (mixed lineage leukemia
histone methyltransferase) complex (7), (iii) PogZ, pogo
transposable element derived protein with a Zinc finger, a
domesticated transposase (29) and (iv) heterodimeric
Cdc7/ASK (30). Cdc7 is a Ser/Thr kinase essential for
the initiationof DNA replication
S-phase and its activity is controlled via interaction with
a regulatory subunit, activator of S-phase kinase (ASK).
In this work we study the intracellular mobility
of LEDGF/p75, in particular chromatin interactions,
quantitatively and in vivo with a combination of fluores-
cence techniques. We label LEDGF/p75 with eGFP and
perform a detailed characterization of the hybrid protein.
We provide new insight in the transcription factor chro-
matin binding kinetics in vivo. We investigate interaction
partner mediated chromatin tethering of LEDGF/p75 and
demonstrate the role of the PWWP domain for this.
MATERIALS AND METHODS
HeLa cells were obtained from the NIH Reagent program
and were grown in ‘Complete Medium’, high-glucose
Figure 1. The primary structure and interactome of LEDGF/p75. (A) Schematic representation of LEDGF/p75, its alternative splice variant
LEDGF/p52 (13) and a deletion mutant, LEDGF/p75326–530(61). (B) Primary structure and interactome of LEDGF/p75. Important domains of
LEDGF/p75, interacting proteins or DNA-sequences are indicated. NLS, nuclear localization signal; AT, AT-hook domains; HTH, predicted
Helix-Turn-Helix motifs; GSRs, gene specific regulators; GTM, general transcription machinery; STRE, stress-related regulatory element; HIV-1
PIC, pre-integration complex; IN, HIV-1 integrase; Plusses, positively charged regions in the p52 part of LEDGF/p75. (C) Confocal fluorescence
image of HeLa cells expressing eGFP-LEDGF/p75. Scale bar=5mm. (D) Western blot with an anti-LEDGF/p75 antibody of HeLa cells transiently
expressing eGFP-fusions. eL, eGFP-LEDGF/p75; e326-530, eGFP-LEDGF/p75326-530; eLKR, K56D-R74D; L, endogenous LEDGF/p75. (E) Cellular
fractionation assay of HeLa cells transiently expressing eGFP-fusions. Western blot of different fractions is shown using antibodies to indicated
proteins. T, total cell lysate; S1, Triton-soluble cellular fraction; P1, Triton-insoluble cellular fraction; S2, DNase/(NH4)2SO4-soluble cellular fraction;
P2, DNase/(NH4)2SO4-insoluble cellular fraction.
Nucleic Acids Research,2011, Vol.39, No. 41311
Belgium), supplemented with 10% heat-inactivated fetal
Belgium), at 5% CO2and 37?C in a humidified atmos-
phere. The generation of monoclonal HeLa cells stably
suppressing endogenous LEDGF/p75 (HeLa-p75KD)
has been described earlier (31). An average HeLa cell
nuclei volume V ¼4?
measuring the length (L) and width (W) of seven nuclei by
ellipsoidal nuclear shape. Assuming 10–100000 genes per
cell, this roughly corresponds to a gene concentration of
Western blotting, cellular fractionation assays
Modified EagleMedium (GibcoBRL,
? ?2¼ 2165mm3was calculated by
Western blotting was performed on whole cell lysates as
described before (32) with a specific anti-LEDGF/p75
antibody (A300-847A, Bethyl Laboratories, Montgomery,
TX, USA). Cellular fractionation assays were performed
as described earlier (15), with the specific LEDGF/p75
antibody, an anti-RFP antibody for mRFP-IN (AB3216,
Millipore N.V., Brussels, Belgium), an in-house polyclonal
anti-GFP antibody and anti-a-tubulin (T5168, Sigma-
Aldrich, Bornem, Belgium). Briefly, 24h post-transfection
cells were harvested and either lysed for checking overall
expression (T) or extracted with Triton X-100 to separate
the soluble (S1) and insoluble (P1) cellular proteins.
DNaseI-treatment allowed subsequent separation into a
soluble, chromatin binding fraction (S2) and an insoluble,
non-chromatin binding fraction (P2).
For the transient transfections 0.5–1?105cells were
seeded per well in a Lab-TekTMChambered Coverglass
50–70% cell confluency after overnight incubation.
Transfections were performed with a Mirus TransIT?-
HeLaMONSTER?transfection kit (VWR International,
Leuven, Belgium), with 1ml TransIT reagent, a maximum
of 0.5mg plasmid DNA per well and 1ml MONSTER
reagent permg of DNA. After the transfection mixture
was prepared in 50ml fresh OptiMEM per well (Gibco
BRL, Belgium), 450ml prewarmed Complete Medium
was added and this mixture was slowly added to the
cells and the cells were incubated for at least 12h.
Theory: quantifying interactions with immobile structures
Consider a binding equilibrium of a protein L that can
interact with an immobile binding site S on the chromatin:
L+S ? ? ? ? !
? ? ? ?
LS with Kass¼½LS?
Association rate ¼ kon½L?½S? ? ? ? ? ? !ðkonS0Þ½L? ¼ k?
Dissociation rate ¼ koff½LS?
onand tbound¼ 1=koff
with [L] the concentration of free protein L, in this case,
LEDGF/p75, [S] the concentration of free immobile
binding sites, [LS] the concentration of protein L bound
to a binding site, Kassthe association binding constant, kon
the rate constant for association and koffthe rate constant
for dissociation. When the concentration of binding sites
is high, k*on=konS0is the pseudo first order rate constant
for association. The tfree is the average time a protein
spends between two successive binding events. The tbound
is the residence time on the chromatin binding site.
Intracellular binding reactions with immobile structures
(nuclear envelope, chromatin, membrane) can be studied
with fluorescence recovery after photobleaching (FRAP)
(33), continuous photobleaching (CP) (34,35) and fluores-
cence correlation spectroscopy (FCS) (36). Generally,
four dynamic regimes can be thought of:
Fast association (tfree??diff,free) and slow dissociation
(tbound>?diff,free). The protein binds with high affinity.
It will be photobleached in the laser focus when FCS is
performed. In this case, FRAP is a good method to
analyse the binding kinetics: the dynamics will be slow
and a model for reaction and diffusion is necessary
because of the high probability of association (33).
Slow association (tfree>?diff,free) and very slow dissociation
(tbound>100ms). The chance of binding is low but once
bound the fluorochrome will be photobleached when
measured with FCS. The fraction of the signal (from CP
or FCS) that photobleaches is a measure for the
When Equation 1 is solved to [LS] in function of total
concentrations L0and S0, K*ass(Kdiss) and the concentra-
tion of binding sites can be determined by non-linear
least-squares fitting of the percentage photobleaching
versus the total concentration of protein L0:
The total concentration L0can be measured with FCS.
The parameter y0 is a constant accounting for the
constant fraction of photobleaching at high concentra-
tions. Alternatively, diffusion independent FRAP can be
used to study the binding reaction (33).
Fast association (tfree<?diff,free) and fast dissociation
(tbound<?diff,free). The binding reaction is observed as a
diffusion component in FRAP and FCS. The ‘effective’
slow diffusion coefficient defines the affinity (33,36,37):
In this regime, only the ratio of rate constants can be
determined from the ACF. The diffusion coefficient of
1312Nucleic Acids Research, 2011,Vol.39, No. 4
free cellular LEDGF/p75 was calculated with (assuming
Mr being the relative molecular mass and DeGFP=
33.0mm2/s (as measured with FCS).
Slow association (tfree>>?diff), moderate dissociation
observed as an exponential component in FRAP and
FCS (33,36). The fraction of free and ‘apparent slow’ dif-
fusion components in FCS is a measure for the affinity:
In conclusion, in the first two regimes the binding reaction
is not and in the last two regimes the binding reaction is
observed during an FCS experiment.
Spot fluorescence recovery after photobleaching
and half-nuclear fluorescence recovery after
Diffusion rates and binding reactions of cellular proteins
with slow/immobile cellular structures can be quantified
with spot fluorescence recovery after photobleaching
(sFRAP) (33,38). By illuminating a defined region-of-
interest in the sample with a brief high-intensity laser
pulse, the fluorescence is rapidly photobleached. It will
recover due to the exchange of bleached with unbleached
molecules. FRAP measurements were performed on a
LSM510/ConfoCor2 (Carl Zeiss, Jena, Germany) at
488-nm excitation. Since bleaching-while-acquisitioning
(39) and recovery-while-bleaching (40) influence the
shape of the FRAP curve, careful optimization of laser
power and imaging parameters was necessary.
sFRAP measurements were performed in a 1-mm radius
(o) circular spot. For eGFP and eGFP-LEDGF/p75, ac-
quisitioning wasevery 65ms
power (front objective aperture) and bleaching was per-
formed with a single 65-ms scan at 1.6mW. For
eGFP-LEDGF/p75 in the presence of mRFP-IN, acquisi-
tioning was every 5s and bleaching was performed with a
single 170-ms scan. Spot FRAP data represent averages of
at least 10 cells. Analysis was performed with a standard
diffusion model (38), a reaction-dominant model (33) or a
full diffusion-reaction model (33), as described in detail in
the Supplementary Information.
Half-nuclear FRAP (hnFRAP) measurements (41,42)
were performed by photobleaching half of the nucleus
with a single scan (1.4s, 1.6mW) and monitoring the sub-
sequent fluorescence redistribution (every 1.4s, 5mW).
Average nuclear pixel intensities per horizontal image
line were calculated for each image with an ImageJ
plug-in. Next, each post-bleach data point was normalized
by the total average cell intensity, to account for
whole-cell photobleaching. Next, each normalized data
point was normalized by the corresponding pre-bleach
inhomogeneities. Finally, vertical pixel distance was con-
verted to an absolute distance scale (1 pixel=90nm) and
data were plotted in a graph and fitted with (43):
with Fluonorm(x) the normalized fluorescence intensity as a
function of the position x (mm), Fbleachthe bleach depth,
erf an error function describing the shape of the bleach
profile, with b (mm) the position of the bleach border and
s(t) the displacement of the proteins as a function of time
t (s). HnFRAP data for a single cell were fitted globally
with Fbleachas a global fit parameter. From the slope of
the mean squared displacement s2(t) the diffusion coeffi-
cient D (mm2/s) was calculated (44):
FluonormðxÞ ¼ 1 ?Fbleach
b ? x
?2ðtÞ ¼ 4Dt
Time zero was approximated with the moment during ac-
quisition where half a bleaching iteration had been
performed. HnFRAP data represent averages of at least
FCS and fluorescence cross-correlation spectroscopy
With FCS, diffusion and binding reactions of fluorescently
labeled molecules can be studied in solution and in cells
(45,46). On a confocal microscope the fluorescence in
the sub-micrometer (<10?15l) sized confocal volume is
measured and then autocorrelated. The low time reso-
lution decaying fluorescence trace contains information
about diffusion and binding reactions, the high time reso-
provides the absolute protein concentration. For normal
FCS/[fluorescence cross-correlation spectroscopy (FCCS)]
measurements a commercial FCS/FCCS microscope
(LSM510/ConfoCor2, Carl Zeiss, Jena, Germany) was
used. Enhanced GFP (47) was excited at 488nm (3mW)
and mRFP1 (48) was excited at 543nm (9mW). The exci-
tation light was reflected by a dichroic mirror (HFT 488/
543) and focused through a C-Apochromat 40?/1.2 W
Korr/0.13–0.17 objective. The fluorescence emission light
was split by a second dichroic mirror (NFT 570) into two
separate beam paths and passed through a 505–530nm
bandpass filter and 70-mm pinhole for eGFP fluorescence
and a 600–650nm bandpass filter and 78-mm pinhole for
mRFP1 fluorescence. The radial radius of the confocal
volume at 1/e2times its maximal intensity, o1, was
determined by measuring the ?diffof rhodamine 6G in
water (D=426mm2/s)(49). In each cell cytoplasm or
nucleus, 10 consecutive fluorescence intensity (detected
photons per second) and correlation measurements of
20s were performed. Correlation functions were fitted
with a one- or two-component model, a normal or anom-
alous diffusion model (50), a reaction-dominant model
(36) or a confined diffusion model (43), as described in
detail in the Supplementary Information.
CP traces were also recorded on the FCS set-up.
Individual CP measurements were normalized to the first
Nucleic Acids Research,2011, Vol.39, No. 4 1313
datapoint, averaged and fitted either to a biexponential
decay (51) or to a model for a binding reaction with
medium dissociation rate (35), as described in detail in
the Supplementary Information.
Tunable focus FCS
Tunable focus FCS (TFFCS) was performed on a
homebuilt set-up (52). The 30-mW 488-nm line of a Ar–
Kr laser(Newport Spectra-Physics,
Netherlands) was split 50/50. One beam was maximally
focused at the back-aperture of the objective (Olympus
UPLFLN 100?/NA1.3/Oil, Olympus Belgium N.V.,
Aartselaar, Belgium) to create wide-field excitation for
observing the fluorescence of the cells through the
eyepiece of the microscope (Olympus IX). The other
beam was carefully expanded, collimated and directed
centrally through an adjustable diaphragm, to allow for
TFFCS measurements. A fixed 50-mm pinhole was used
for confocal detection on an avalanche photodiode
(SPCM-AQR-15, Perkin-Elmer, Wiesbaden, Germany).
The excitation power at each diaphragm setting was
attenuated to give a constant power/area in the focal
spot suitablefor cellular
was achieved by measuring the residual power behind
the diaphragm. TFFCS measurements were analysed
with a 2-component model for normal diffusion.
Structure based selection of PWWP residues important
for DNA binding
To select residues important for the interaction of the
PWWP domain of LEDGF/p75 with DNA we used
biomolecular modeling. First, we modeled the structure
of the PWWP domain of LEDGF/p75 using the latest
available version of Modeller (53), with the NMR struc-
ture (PDB 2B8A) of the HDGF-PWWP as a template
(54). Second, we predicted putative DNA binding
residuesusing the HotPatch
analysis indicated residues K56 and R74 to be solvent
exposed and not required for the stabilization of the
tertiary structure of the protein, minimizing the chance
of perturbing the PWWP folding. Of concern, these
residues in the LEDGF/p75-PWWP are conserved in the
HDGF-PWWP sequence and were already shown to be
important for DNA binding of the HDGF-PWWP (54).
Fluorescent protein labeling of LEDGF/p75 preserves
To study the dynamics of LEDGF/p75 in living HeLa
cells with fluorescence techniques, we first expressed
and characterized the fusion of LEDGF/p75 and the
enhanced green fluorescent protein (eGFP). This protein,
eGFP-LEDGF/p75, displayed a heterogeneous nuclear
distribution characteristic of endogenous LEDGF/p75
(Figure 1C) (22). Western blotting revealed no protein deg-
radation of eGFP-LEDGF/p75 (Figure 1D). Chromatin
binding properties of the fusion were similar to that of
endogenous LEDGF/p75 as demonstrated by in vitro
cell fractionation (Figure 1E): following extraction of the
cells with Triton X-100, eGFP-LEDGF/p75 was present
in the insoluble pellet (P1), whereas a DNaseI treatment of
the P1 pellet released the protein into the supernatant (S2),
as demonstrated earlier for endogenous LEDGF/p75 (15).
In summary, an N-terminal fluorescent tag does not affect
the functional properties of LEDGF/p75 analyzed in this
study. In order to minimize the effects of endogenously
expressed LEDGF/p75, we made use of HeLa cells stably
depleted of endogenous LEDGF/p75 (HeLa-p75KD) (31).
Complementation of these cells with wild-type LEDGF/
p75 rescues HIV replication and lentiviral vector transduc-
tion (31). Likewise, we complemented LEDGF-depleted
cells with eGFP-LEDGF/p75 and rescued viral replication
and vector transduction to wild-type levels (data not
shown), demonstrating that the eGFP label does not
affect LEDGF/p75 functionality.
In vivo eGFP-LEDGF/p75 exists in a chromatin bound
state, a slow state and a fast state
We performed sFRAP experiments in HeLa cells. The
fluorescence of the eGFP control exhibited fast and
complete recovery, as expected for a free diffusing, inert
protein (Figure 2A black). The fluorescence recovery of
eGFP-LEDGF/p75 was also complete but much slower
(Figure 2A, magenta). A pure diffusion model provided
a good fit of the experimental data and a diffusion coeffi-
cient D=0.41±0.06mm2/s was calculated (Table 1),
which is 53-fold lower than expected for free diffusion
(Figure 2A, green, with D from Equation 8). Of
concern, while diffusion time of eGFP (tdiff=97ms)
could not be determined correctly due to recovery-
LEDGF/p75 (tdiff=2.13s) were slow enough to be
measured accurately. A reaction-dominant model did
not fit the data well and a full diffusion-reaction model
did not provide a better fit than a pure diffusion model
(Supplementary Figure S1A–C).
Next, we applied CP (39). The CP signal from eGFP in
HeLa cells decayed slowly and biexponentially (Figure 2B,
black), as has been described before for eGFP (35). For
eGFP-LEDGF/p75 (Figure 2B, magenta) a biphasic
decay was also observed (Figure 2B, black). Resuming
the CP measurement after a pause resulted in a similar
decay (data not shown), arguing for the absence of an
immobile fraction. It has been shown that slow diffusion
in a finite compartment can indeed lead to a biphasic
eGFP-LEDGF/p75 (D=0.41mm2/s) in a HeLa nucleus
Information), which agrees quite well with the completion
of the fast exponential decay we observed (Figure 2B,
dashed line). Of concern, fitting the same data with
(Figure 2B, green) did not result in a better description
of the experimental data than a simple biexponential
decay (35), proving that diffusion and binding are neces-
sary to describe the observed photobleaching.
the dynamicsof eGFP-
Settlingof diffusion of
1314 Nucleic Acids Research, 2011,Vol.39, No. 4
Interestingly, in the <500nM concentration range the
fraction of the fast exponential was concentration depend-
ent (Figure 2C), which suggests the presence of a low con-
centration of higher affinity binding sites. In line with this,
a full reaction-diffusion model for analyzing the sFRAP
data also suggested a low fraction of higher affinity
binding sites (Supplementary Table S1). With Equation
6 the data from Figure 2C could be fitted to determine
an average concentration of binding sites S0=61±
The fluorescence signal after the initial photobleaching
was divided in 20-s intervals which were autocorrelated
individually (Figure 2B-top) and an average ACF was
calculated with FCS (56). The eGFP exhibited free diffu-
sion in the nucleus (Figure 2D, black and Table 1) with
D=33.0±3.5mm2/s (Table 1), which is 2.9 times slower
than measured in solution (49) and in good agreement
with previous findings in cells if the correct D of the cali-
bration probe (426mm2/s versus 280mm2/s) is taken into
account (57). The eGFP-LEDGF/p75 ACF was again
strongly shifted into a slower time scale (Figure 2D,
magenta) with respect to the calculated ACF for free
diffusion (Figure 2D, green). A one-component (normal
or anomalous) diffusion model did not describe the
ACF well (Supplementary Figure S2A and B). A
two-component normal diffusion model described the
ACF very well (Supplementary Figure S2C): a fast com-
ponent (35±3%) with apparent D=22.3±4.1mm2/s
and a slow component (65±3%) with D=0.5±
0.1mm2/s was determined (Table 1). The slow D is in
good accordance with the D obtained from FRAP. A
Figure 2. Dynamic subpopulations of LEDGF/p75 revealed by sFRAP, CP and FCS. (A) sFRAP experiment of eGFP and eGFP-LEDGF/p75 in
living HeLa cells. The diffusion coefficient we calculated for eGFP after fitting with a pure diffusion model was only 10.28mm2/s (Table 1). For eGFP
the observed recovery was lower than expected (solid orange curve) because of unavoidable recovery-while-photobleaching, which occurred because
the diffusion time of eGFP (tdiff=97ms) was too small with respect to the acquisitioning time of the microscope (65ms per iteration on our setup)
(40). For eGFP-LEDGF/p75, the arrow indicates the decrease in mobility of eGFP-LEDGF/p75 due to chromatin interactions. DeGFP=33mm2/s is
the D we measured with FCS, DeGFP-LEDGF/p75=22mm2/s is calculated from DeGFPwith Equation 8. See also Supplementary Figure S1. (B) CP
experiment of eGFP and eGFP-LEDGF/p75. Upper panel is a 80–100-s zoom. (C) Concentration dependence of the amount of photobleaching of
eGFP-LEDGF/p75 during the first 20s. Solid line=fit with Equation 6. (D) FCS experiment of eGFP and eGFP-LEDGF/p75. Solid
lines=two-component normal diffusion model. See also Supplementary Figure S2. Error bars=SD.
Nucleic Acids Research,2011, Vol.39, No. 41315
reaction-dominant fit model did not describe the FCS data
well, implying that the slow component in the ACF cannot
be described solelywith
component diffusion models that described confined
(Supplementary Figure S2E and F) than a normal
2-component diffusion model, suggesting the latter is ne-
cessary and sufficient to describe the observed dynamics.
Finally, we performed control measurements to verify
that the dynamics we observed for LEDGF/p75 can
indeed be related to chromatin interactions. First, we per-
formed similar experiments on known chromatin binding
proteins to verify that chromatin interaction can indeed be
observed in the experimental setup (Supplementary Figure
S3A). Next, we deleted the N-terminal p52 region of
LEDGF/p75 and performed similar experiments on this
protein, eGFP-LEDGF/p75326–530. The CP curve of this
protein resembled that of free eGFP and the ACF shifted
almost completely back to free diffusion (Supplementary
Figure S3B). This proves that chromatin interactions of
LEDGF/p75 via its N-terminal p52 domain contribute
significantly and protein–protein interactions of the IBD
do not contribute significantly to the observed overall
dynamics of LEDGF/p75.
In summary, we combined sFRAP, CP and FCS to
study the mobility of eGFP-LEDGF/p75 in the nucleus
of living HeLa cells. Overall, LEDGF/p75 does not
interact strongly with chromatin, as implied from the
absence of a permanent immobile fraction in sFRAP.
Rather, we observed different dynamic states: (i) a slow
state with D=0.4–0.5mm2/s, observed with sFRAP and
FCS, (ii) a presumed bound state with ?60nM binding
sites and a dissociation constant Kdiss?100nM, suggested
from sFRAP and CP measurements and (iii) a fast state,
observed only with FCS.
Slow eGFP-LEDGF/p75 corresponds to hopping
The slow moving fraction of LEDGF/p75 (D=0.5mm2/s,
?diff=26ms) is too fast to represent chromatin controlled
movement of associated LEDGF/p75, which is reported
to be 2–3 orders-of-magnitude slower (58). We performed
TFFCS, where the excitation volume can be tuned in size,
to verify the scaling of the observed dynamics with the
radius of the measurement spot (33,59,60): if the
observed diffusion time (?obs) does not scale with the
size of the excitation volume, then ?obsis solely determined
by the residence time tbound(=1/koff) on the immobile
structure (Figure 3A). If however ?obs does scale with
the size of the excitation volume, then the binding
process is much faster than the diffusion time scale
(Figure 3B). Put differently, the relaxation time of the
binding process [=1/(k*on+koff)] will be smaller than
?obsif ?obsvaries with the focal spot size. We first per-
formed detailed in vitro control experiments to character-
ize this set-up (Figure 3C). In aqueous solution, both the
apparent particle number (Supplementary Figure S4A)
and the average diffusion time (Supplementary Figure
S4B) of the standard probe rhodamine 6G increased
when tuning the laser beam to a smaller diameter (and
hence the focal spot to a larger diameter) (Figure 3D
black). In a next step, we used eGFP as a probe. Both
in phosphate buffered saline (PBS) (Figure 3D, magenta)
and in a buffer with an index of refraction matching the
intracellular environment (PBS, 23.5% w/w sucrose,
n=1.37) (Figure 3D, green) increasing the laser focus
again shifted the experimental ACF to a slower time
scale, as expected. The TFFCS set-up thus performs well
for determining the dependence of the ACF on the size of
the focus. We next performed similar measurements in
living HeLa cells (Figure 3E). The diffusion time of
eGFP was clearly dependent on the size of the focal spot
Table 1. Dynamics of eGFP and eGFP-LEDGF/p75 measured with sFRAP, FCS, CP, TFFCS and hnFRAP
sFRAP FCS/CPTFFCS sFRAP FCS/CPhnFRAP TFFCS
Values represent averages of at least 10 (sFRAP), 20 (FCS/CP), 10 (TFFCS) or 5 (hnFRAP) measurements and are ±SD.
aThis value is an underestimation, because the shortest bleach iteration (65ms) was still too long to avoid recovery-while-photobleaching.
bThis parameter likely does not represent a diffusion process, see Figure 3F and ‘Discussion’ section.
cThe contribution of each component did not depend significantly on the concentration.
dThe fractional contribution of each component did not depend on the focus spot size.
eThis parameter was obtained by fitting with a one-component anomalous diffusion fit.
fFit not satisfying, see Supplementary Figure S2B.
gFfreeis calculated from the apparent diffusion law, see ‘Materials and Methods’ section, Equation 7, with DeGFP-LEDGF/p75=22.3mm2/s.
Fimm, immobile fraction; Dfastand Dslow, diffusion coefficient from fitting the FCS curves; DFRAP, diffusion coefficient obtained from Supplementary
Equations S1 and S2; Ffast, fractional contribution of the fast component observed with FCS; a, anomaly parameter; Ffree, fraction of the protein
population observed with FCS that is freely diffusing.
1316Nucleic Acids Research, 2011,Vol.39, No. 4
Figure 3. TFFCS shows fast chromatin binding of LEDGF/p75. (A) The observed slow diffusion time (?obs=25.9ms, D=0.5mm2/s) is the binding
time of LEDGF/p75 with chromatin, tbound=1/koff. In this case ?obsdoes not scale with the laser focus diameter, indicated in blue. (B) LEDGF/p75
continuously associates with and dissociates from the chromatin while diffusing through the measurement spot. The ?obsscales with the laser focus
diameter. (C) Schematic representation of the home built TFFCS set-up. The beam expander and diaphragm are used to vary the diameter of the
collimated laser beam. ND, neutral density attenuation filter; APD, avalanche photodiode. The diameter of the diaphragm is controlled by measuring
the power of the excitation light passing through it. Higher power through the diaphragm means a broader beam will enter the objective back
aperture. The objective focuses a broader beam in a smaller focal point, since the beam diameter determines the effective numerical aperture of the
objective (NA=n?sina with n the refractive index and a the angular aperture). For further details, see the ‘Materials and Methods’ section. (D)
In vitro TFFCS measurements of rhodamine 6G in water (black), eGFP in PBS (magenta) and PBS/sucrose with a viscosity and refractive index
similar to that of the intracellular environment (n=1.37) (green). o1: radial radius of the excitation spot. (E) Intracellular TFFCS measurements of
eGFP (black), eGFP-LEDGF/p75 (magenta) and eGFP-LEDGF/p75 K56D-R74D (green). Error bars=SD. (F–H) Plot of the diffusion time from
the experimental ACF versus the squared radial radius of the confocal excitation spot for (F) eGFP, (G) the fast and (H) the slow component of
Nucleic Acids Research,2011, Vol.39, No. 41317
(Figure 3E, black and Figure 3F), consistent with the free
intracellular movement of the protein. Overall, the ACF
of eGFP-LEDGF/p75 also shifted in a larger focal spot
(Figure 3E, magenta). More specifically, while the
apparent diffusion time (?diff) of the fast component did
not significantly shift to a slower time scale if the focal
spot was increased, the ?diffof the slow component scaled
perfectly with the focal spot size. In other words, the fast
component likely does not represent a diffusion process,
while the slow component clearly does. Moreover, the
D we calculated from the slope of the linear fit was
D=36.4±3.1mm2/s for eGFP and 0.95±0.01mm2/s
for eGFP-LEDGF/p75, in good accordance with the cor-
responding values obtained with sFRAP and normal FCS.
To further corroborate that the dynamics of the slow
FCS component are indeed diffusion controlled, we per-
formed hnFRAP. After photobleaching half a nucleus, we
monitored the nuclear fluorescence equilibration over
time, perpendicular to the bleach border, as illustrated in
Figure 4A. The bleach profile changed slowly, but grad-
ually over time, typical for a diffusion controlled binding
reaction (Figure 4B), as has been described before for
other known chromatin binding proteins. When we
plotted the mean squared displacement, obtained by
fitting the hnFRAP curves, versus time, we again
obtained a nearly perfect linear relation and the D that
was calculated from the slope was D=1.02±0.01mm2/s,
in perfect agreement with our TFFCS data. Furthermore,
the linear MSD-timerelationship
Brownian-like diffusion FCS fit model. In conclusion,
LEDGF/p75 likely exhibits a hopping mechanism in the
nucleus, characteristic of non-specific, frequent chromatin
interactions (Figure 3B).
LEDGF/p75-PWWP contributes to chromatin binding
We set out to verify the previously documented contribu-
tion of the PWWP domain to the in vivo chromatin
binding properties of LEDGF/p75 (15,16). To affect the
overall protein structure as little as possible, we sought to
specifically alter the affinity of this domain for chromatin.
Based on the molecular model of the PWWP domain of
HDGF in complex with DNA (54), we predicted that posi-
tively charged residues K56 and R74 in LEDGF/p75 are
most likely interacting with the phosphates of the host
DNA. Next, we constructed and purified two single
mutant proteins of LEDGF/p75, K56D and R74D and
the double mutant K56D-R74D. Correct overall folding
was corroborated with circular dichroism spectroscopy
(Supplementary Table S2). Next, eGFP-fusions of the
same proteins were transiently expressed in HeLa cells.
The proteins were characterized by a more diffuse
nuclear localization (Figure 5A–C), compared with the
wild-type protein (22) (Figure 1C), suggesting that chro-
matin binding properties were affected. Next, we used
sFRAP, CP and FCS to study the dynamics of the
mutants. All mutants were considerably faster (4- to
8-fold) than wild-type LEDGF/p75 (Figure 5D and F
and Table 2). TheR74D
mutationhad a more
pronounced effect than the K56D mutation (Figure 5D
and F green and orange, respectively) while the double
mutant did not show an additive effect compared with
the R74D single mutation (Figure 5D and F, gray). All
mutants were still considerably slower than expected for
free diffusion, indicating they still have some residual
interactions with chromatin. Importantly, the difference
in cellular dynamics between the K56D and R74D
mutants could not be inferred from a differential distribu-
tion of the proteins, but was only evidenced by our
sFRAP and FCS measurements, demonstrating the
sensitivity of these techniques. Finally, the ACF of
Figure 4. LEDGF/p75 shows normal diffusion dependent dynamics.
(A) Illustration of half-nucleus FRAP. Half of the nucleus is
photobleached and recovery perpendicular to the bleach border is
monitored versus time. (B) Nuclear intensity profile versus time along
the arrow designated ‘position’ in (A) Solid lines=Equation 10. (C)
Mean squared displacement – time plot. Error bar=SD. Solid
1318Nucleic Acids Research, 2011,Vol.39, No. 4
eGFP-LEDGF/p75 K56D-R74D still varied with the
focus size, implying that this protein also moves by diffu-
sion (Figure 3E). In summary, by mutating a single amino
acid residue in LEDGF/p75-PWWP, R74D, chromatin
binding of the protein was severely impaired, as evidenced
by an up to 8-fold increase in dynamics with respect
to wild-type LEDGF/p75. The PWWP domain of
LEDGF/p75 is thus important but not sufficient for
in vivo chromatin binding.
LEDGF/p75 locks the IN-LEDGF/p75 complex on
chromatin through its PWWP domain
LEDGF/p75 has been shown to tether IN to chromatin by
virtue of a direct protein–protein interaction (15,22,61–64).
We performed CP measurements of eGFP-tagged IN in
HeLa cells to show tethering in vivo: we observed
variable photobleaching of eGFP-IN from cell to cell
in wild-type HeLa cells (Figure 6A), but no signifi-
cant photobleaching of eGFP-IN was observed in
HeLa-p75KD cells (Figure 6B). When HeLa-p75KD cells
were back-complemented with 500nM of RNAi-resistant
mRFP-LEDGF/p75, a large fraction of eGFP-IN was
again photobleached (Figure 6C). The dynamics of IN
are thus strongly influenced by LEDGF/p75 mediated
chromatin tethering. We next quantified the effect of IN
on in vivo chromatin binding kinetics of LEDGF/p75.
Upon co-expression of mRFP-labeled IN the overall
dynamics of LEDGF/p75 shifted to much slower time
scale (Figure 6D). When fitting the sFRAP curve with a
normal diffusion model (Supplementary Equation S1), a
good fit was obtained (Figure 6D and Supplementary
FigureS5A) and D=0.0055±0.001mm2/s
calculated, about 75 times lower than in cells without
co-expression of mRFP-IN
reaction-dominant model (Supplementary Equation S3)
did not give a good fit (Supplementary Figure S5B) and
a full diffusion-reaction model (Supplementary Equation
S4) did not result in a better fit than a normal diffusion
model (Supplementary Figure S5C). With hnFRAP a
similar decrease in dynamics was observed (D=0.021±
0.004mm2/s) (Figure 6E and F).
Next, we verified with sFRAP whether the mutations in
the PWWP domain of LEDGF/p75 affected chromatin
tethering of IN. Co-expression of mRFP-IN decreased
the dynamics of the K56D-R74D mutant about six
times, as calculated from the difference in D (Tables 2
and 3 and Figure 6G), which is much less than wild-type
eGFP-LEDGF/p75 (note the difference in time scale in
Figure 6D and G). To verify that the absence of chromatin
tethering was not due to a loss of the IN-LEDGF/p75
interaction, we used cellular FCCS. A high relative
we published before for IN-LEDGF/p75326–530 and
IN-LEDGF/p75-K150A (61,62), was observed (Figure
6H), indicating that the interaction is not affected by the
mutations. In conclusion, chromatin tethering of IN by
LEDGF/p75can be observed
showed for the first time in vivo that IN-LEDGF/p75
complexes have a 75-fold lower mobility as compared
with LEDGF/p75. Since IN alone did not appear to
(Tables1 and 3).A
comparable with what
Figure 5. The PWWP domain of LEDGF/p75 contributes to high
affinity chromatin binding and is crucial for chromatin tethering of
HIV-1 integrase. (A–C) Confocal fluorescence images of HeLa cells ex-
pressing (A) eGFP-LEDGF/p75 K56D, (B) R74D and (C) K56D-R74D.
(D) sFRAP experiment. (E) CP experiment of eGFP-LEDGF/p75
K56D-R74D. The CP curves of eGFP and eGFP-LEDGF/p75 are
shown as a reference. (F) FCS experiment of eGFP-LEDGF/p75
mutants. The ACFs of eGFP and eGFP-LEDGF/p75 are shown as a
reference. Error bars=SD.
Nucleic Acids Research,2011, Vol.39, No. 4 1319
interact strongly with chromatin, LEDGF/p75 thus
tethers IN strongly to chromatin. Rational mutation of
the PWWP domain resulted in a strongly decreased
ability to tether IN, indicating that the PWWP domain
in LEDGF/p75 constitutes a ‘protein lock’ that strongly
tethers IN to chromatin.
Fluorescent LEDGF/p75 displays wild-type properties
Our experimental approach combines complementary
fluorescence techniques to study fluorescently labeled
LEDGF/p75 in living cells. It had been shown that the
interactions with cellular partners through the IBD are
not hampered by the presence of the eGFP label (28–
30,63). Unlike a C-terminal eGFP (data not shown),
an N-terminal fusion displayed a proper intracellular
distribution. In addition, the overall chromatin binding
characteristics of eGFP-LEDGF/p75 are not altered
(Figure 1E); since LEDGF/p75 lost chromatin binding
by mutating the PWWP domain, we are confident that
chromatin binding of eGFP-LEDGF/p75 is conserved.
Results are in agreement with Turlure et al. (16), who
showed that eGFP-PWWP still functionally interacts
with chromatin. All experiments in cells were performed
at relevant concentrations of fluorescent LEDGF/p75. On
average, the concentration
eGFP-LEDGF/p75 was similar to that of endogenous
LEDGF/p75 (Figure 1D) and measurements were per-
formed in a 1nM–2.5mM concentration range to study
potential concentration dependent effects (Figure 1D).
Furthermore, the ACF of eGFP-LEDGF/p75 was con-
centration independent (data not shown) and our investi-
gation of chromatin tethering of IN (Figure 6A–C) led
us to assume that the intracellular concentration of
LEDGF/p75 lies in submicromolar range. In line with
this, LEDGF/p75 has been shown to be a much more
abundant cellular protein than its alternative splice
variant p52 (22,65).
Dynamic in vivo chromatin interactions of LEDGF/p75
DNA binding and chromatin fractionation assays have
been used in the past to study interactions of LEDGF/
p75 with chromatin but these methods only provide a
static and average view on interactions with DNA/chro-
matin (15,16,21). Also, irreconcilable notions of in vivo
chromatin binding of LEDGF/p75 exist. On the one
hand, LEDGF/p75 has been shown to be a ‘site-specific’
transcriptional co-activator, interacting with promoter
elements of stress-related genes. On the other hand,
LEDGF/p75 has been shown to be the factor that
targets lentiviral integration
regions of active transcription (21,24). We used sFRAP,
CP, FCS, TFFCS and hnFRAP to monitor in vivo
chromatin interactions of LEDGF/p75 for the first time.
We showed that LEDGF/p75 does not permanently
take part in immobile protein/chromatin complexes
The slow state was observed both with all techniques
and represents low-affinity, likely non-specific chromatin
scanning/hopping, a common phenomenon of transcrip-
tion factors (66). By this mechanism, LEDGF/p75 likely
targets lentiviral integration to random (actively trans-
cribed) regions (24–26). On the other hand, the concentra-
tion dependent CP measurements strongly suggest the
existence of low-concentration higher-affinity chromatin-
bound states (Figure 2A–C and Table 1), which might
responsive genes (21,67) or more generally, as has
recently been shown, from the association downstream
of transcription start sites (TSS) of active transcription
units (68). The concentration of binding sites we
calculated (61nM), indeed
order-of-magnitude as the concentration of TSS in a eu-
karyotic cell (?5–100nM, see ‘Materials and Methods’
To our surprise, we only observed the fast state
(Figure 2D and Table 1) with FCS. This state did not
appear to represent translational motion of the protein
(Figure 3G). The time scale and fraction of the fast
process did not appear to be concentration or cell depend-
ent (data not shown), ruling out free/bound protein frac-
tions or a post-translationally modified fraction. This state
might represent any process that changes the emissive
state of the eGFP fluorophore, such as protonation (69),
quenching or possibly rotational diffusion of chromatin
bound eGFP-LEDGF/p75. In that respect, it has been
shown that mRFPs can reside in dark states even in the
milliseconds time scale under 1P excitation (70).
Table 2. Dynamics of LEDGF/p75 PWWP mutants measured with sFRAP and FCS
eGFP-LEDGF/p75 K56DeGFP-LEDGF/p75 R74D eGFP-LEDGF/p75 K56D R74D
sFRAPFCS sFRAPFCS sFRAPFCS
Values represent averages of at least 10 (sFRAP) or 20 (FCS) measurements and are ±SD. Parameters Dfast, Ffastand Dsloware obtained from a
two-component normal diffusion fit. Parameter a is obtained from a one-component anomalous fit.
aFit not satisfying. For more details on the parameters, see Table 1.
1320 Nucleic Acids Research, 2011,Vol.39, No. 4
Quantitative information on in vivo chromatin
hopping with TFFCS
We showed with hnFRAP that diffusion of eGFP-
LEDGF/p75 in the slow (seconds) time scale seems
normal in absence (Figure 4C) or presence (Figure 6F)
of HIV-1 IN. By adapting a standard FCS microscope,
we could also clearly distinguish the contribution of dif-
fusion to the observed chromatin binding dynamics of
LEDGF/p75 and showed that even in the fast (millisec-
onds) time scale diffusion still seems normal (Figure 3E
and H). This implies that association with and dissociation
from chromatin occurs faster than the transit time of the
protein through the laser focus, as illustrated in Figure 3B.
As such, the strength by which LEDGF/p75 is slowed
down could be used to quantify the low-affinity chromatin
binding of LEDGF/p75. Based on Dobserved=0.5mm2/s
Figure 6. LEDGF/p75-PWWP locks IN-LEDGF/p75 on chromatin. (A–C) CP experiment of eGFP-IN in (A) wild-type HeLa cells, (B)
HeLa-p75KD cells and (C) HeLa-p75KD cells back-complemented with 500nM mRFP-LEDGF/p75. (D) sFRAP experiment of eGFP-LEDGF/
p75 in HeLa cells, without (magenta) and with (green) co-expression of mRFP-IN. Solid lines represent a fit to a normal diffusion model. Results
from the fitting are presented in Table 3. See also Supplementary Figure S5. (E) HnFRAP experiment in nuclei expressing eGFP-LEDGF/p75 and
mRFP-IN. Solid lines=Equation 10. (F) MSD-time plot. Solid line=Equation 11. (G) FRAP measurement of eGFP-LEDGF/p75 K56D-R74D in
HeLa cells, without (magenta) and with (green) co-expression of mRFP-IN. Solid lines=Supplementary Equation S1. Results from the fitting are
presented in Table 1. (H) FCCS measurements in cells expressing eGFP-LEDGF/p75 K56D-R74D and mRFP-IN. The high amplitude of the CCF
means a strong protein–protein interaction. Error bars=SD.
Nucleic Acids Research,2011, Vol.39, No. 41321
and the theoretical Dfree=22mm2/s, we calculated with
Equation 8 that LEDGF/p75 remains 98% of the time
bound to chromatin and moves during the remaining
2% of the time to a new binding site. The average tfree
and tboundshould thus be considerably shorter than the
observed diffusion time (25.9ms) and k*on ?43 times
larger than koff(Equation 7). In other words, koffshould
be bigger than 38.6/s and k*on>1.7?103/s. This kinetic
‘effective diffusion’ regime of non-specific chromatin
binding has been simulated before (36) and implies that
transcription factor chromatin binding might be even
more dynamic than currently appreciated (33,36,42,59).
Importantly, TFFCS as such would not be able to
discern between slow movement of a (big) protein
complex and frequent chromatin interactions of a
(small) protein. Therefore, one has to verify that chroma-
tin interactions are indeed contributing to the observed
dynamics, which we did, both in vitro (Figure 1E) and in
cells (Supplementary Figure S3). Furthermore, by rational
mutation of the PWWP domain, a known chromatin
interaction motif, we did prove that the PWWP contrib-
utes to the observed affinity of LEDGF/p75 for chromatin
in vivo. Additionally, we could show cargo-mediated chro-
matin tethering by LEDGF/p75, which has been shown
many times to be an important function of LEDGF/p75
for HIV replication. Finally, it must be noted that the
‘chromatin hopping’ we describe for LEDGF/p75 could
also represent chromatin hopping of a larger complex con-
taining LEDGF/p75 and other nuclear components and
this complex would experience a larger effective viscosity
in the chromatin matrix. Further research will provide
more insight in this. In line with our kinetic regime of
hopping, fast chromatin binding kinetics have recently
also been experimentally observed for the Drosophila
Hox gene Sex combs reduced transcription factor with
DNA in vivo (60) and DNA-bound and slow apparent
diffusion two-state chromatin binding has recently also
been observed for Heterochromatin protein 1 (43), a
protein we also studied as a control for chromatin
binding (Supplementary Figure S3).
Extremely slow, yet diffusion controlled dynamics of
It has been shown that LEDGF/p75 can act as a molecu-
lar tether coupling IN to the chromatin (15,22,61–64) and
IN has been suggested to be present in a paracrystalline
form in the viral particle (71). In the presence of its viral
cargo, IN, LEDGF/p75 formed a 75-fold slower dynam-
ical complex. Since interaction with IN likely does not
decrease k*on of LEDGF/p75, the observed dynamics
should still be diffusion controlled, which is what we
observed (Supplementary Figure S5 and Figure 6E and
F). The effective diffusion law (Equation 7) allowed us
to calculate that while for LEDGF/p75, k*on/koff?43,
for IN-LEDGF/p75, k*on/koff ?2.7?103. The k*on is
thus at least as large as for LEDGF/p75 alone and the
kofflikely decreases considerably after interaction with IN.
The IN is known to be present at least in a dimeric form
atnM–mM concentrations (72) and already a IN dimer
contains two binding sites for LEDGF/p75 (73). As we
have shown, IN does not bind strongly to chromatin
(Figure 6A–C). Probably thus, IN-LEDGF/p75 com-
plexes contain at least two LEDGF/p75 units that can
both tether IN cooperatively to chromatin, resulting in
the apparent diffusion controlled increase in affinity. An
allosteric effect of IN on the intrinsic affinity of LEDGF/
p75 for chromatin is unlikely, since the IBD and chroma-
tin binding domains of LEDGF/p75 are located in distinct
structural parts of the protein. It might be argued that the
IN-LEDGF/p75 complex senses, due to its oligomeric
size, a much larger viscosity in cells than does LEDGF/
p75 alone and this might explain the observed 75-fold
decrease in dynamics. As we have shown in Figure 6G
and H, PWWP-mutated LEDGF/p75 still has wild-type
affinity for IN, but its dynamics are only slowed down
6-fold by IN co-expression. Therefore, we are confident
that the observed dynamical shift of IN-LEDGF/p75
does represent an increased affinity for chromatin.
The linker histone H1, which we also used as a control
for our measurements (Supplementary Figure S3), has
recently also been studied quantitatively and similar
slow overall dynamics were observed (74). Because
dynamics of H1 were described best with a full diffu-
sion/reaction model while a simple diffusion model
sufficed for IN-LEDGF/p75, this suggests that while for
H1 very slow dissociation from chromatin is the cause
of the slow overall dynamics, very fast association with
chromatin is rather the cause of the slow dynamics for
The PWWP domain is the key to the chromatin lock
In recent years, LEDGF/p75 has become a protein of
interest to understand various diseases such as leukemia,
autoimmunity and AIDS (4,7,8,22). Nearly all normal and
disease related functions of LEDGF/p75 involve tethering
of interaction partners to chromatin through its conserved
IBD. We investigated the role of the PWWP domain in the
chromatin binding of LEDGF/p75 in vivo. The conserved
PWWP domain of LEDGF/p75 contains a recently
described general protein fold, present in proteins that
carry chromatin-binding motifs and has been proposed
to bind DNA in several independent structural biology
studies (54,75). Other studies however suggested, albeit
without experimental evidence so far, that the PWWP
fold is implicated in non-DNA chromatin interactions
Table 3. Effect of HIV-1 integrase co-expression on the dynamics of
LEDGF/p75 and LEDGF/p75 K56D-R74D measured with sFRAP
Values represent averages of at least 10 (sFRAP) or 5 (hnFRAP)
measurements and are ±SD. For more details on the parameters, see
1322 Nucleic Acids Research, 2011,Vol.39, No. 4
(12,14). Although eGFP-PWWP did not show any DNA
binding in vitro, it was shown to be localized on mitotic
chromosomes, much alike full length LEDGF/p75 (16).
Our rational mutagenesis of the PWWP domain of
LEDGF/p75 resulted in a protein with up to 8-fold
increase in overall mobility as compared with wild-type
LEDGF/p75 (Figure 5D and F). The mutant protein
(Figures 3E and 5F and Table 2), which could either
result from residual interactions with the chromatin (76)
or from interactions with other cellular cofactors, leading
to obstructed diffusion (77). Nonetheless, we confirm that
interactions of the PWWP domain with chromatin are
necessary but not sufficient to convey wild-type affinity
of LEDGF/p75 for chromatin.
Importantly, we are the first to show that upon
mutation of the PWWP-domain, tight chromatin tethering
of IN disappears, while the tight interaction of LEDGF/
p75 with IN is conserved. This strongly argues for a sole
role of LEDGF/p75, in particular the PWWP-domain, in
associating an IBD-bound cargo to chromatin. The
HDGF-PWWP has been shown to be able to dimerize
(78) and this might be an important step in chromatin
cargo-induced locking is a general phenomenon for
tethering cargos to chromatin. Being the key for locking
cargos to chromatin, the PWWP-domain might be a
promising novel target for blocking LEDGF/p75 malfunc-
tioning in AIDS but also oncogenesis.
are investigating whether
Supplementary Data are available at NAR Online.
The authors acknowledge Davide Mazza (Laboratory for
Receptor Biology and Gene Expression, National Cancer
Institute, Bethesda, MD) for providing the Matlab code
for analyzing the FCS data with the binding models,
Antoine Delon (Universite ´ Joseph Fourier, France) for
(Laboratory for Biomolecular Dynamics) for writing the
ImageJ script for hnFRAP analysis.
Funding for open access charge: University of Leuven
(GOA/2006/02, IOF/KP/07/008); the Institute for the
Technology in Flanders (CellCoVir SBO grant 60813);
G.0530.08); the European Commission project Targeting
HIV Integration Co-factors (grant HEALTH-F3-2008-
201032); FWO postdoctoral fellowship (to J.Hendrix.);
Mathilde-Krim postdoctoral fellowship (The Foundation
for AIDS Research) (to J.D.R.).
through Science and
Conflict of interest statement. None declared.
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