Defining the essential function of FBP/KSRP proteins: Drosophila Psi interacts with the mediator complex to modulate MYC transcription and tissue growth
Nucleic Acids Research
Defining the essential function of FBP/KSRP proteins: Drosophila Psi interacts with the mediator complex to modulate MYC transcription and tissue growth
Linna Guo 2
Olga Zaysteva 2
Zuqin Nie 1
Naomi C. Mitchell 2
Jue Er Amanda Lee 2
Thomas Ware 0
Linda Parsons 2
Rodney Luwor 0
Gretchen Poortinga 5
Ross D. Hannan 3 4
David L. Levens 1
Leonie M. Quinn 2
0 Department of Surgery, University of Melbourne, Royal Melbourne Hospital , Parkville, VIC 3010 , Australia
1 Center for Cancer Research, National Cancer Institute, NIH , Bethesda, MD 20892 , USA
2 School of Biomedical Sciences, University of Melbourne , Parkville, VIC 3010 , Australia
3 Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University , Canberra City, ACT 2600 , Australia
4 Department of Medicine, St. Vincent's Hospital, University of Melbourne , Parkville, VIC 3010 , Australia
5 Peter MacCallum Cancer Centre , St. Andrews Place, East Melbourne, VIC 3002 , Australia
Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.
The FBP-family of KH domain proteins were first described
20 years ago, however, the core function of these
proteins in vivo remains unclear and controversial. KH motifs
have been attributed RNA-binding functions (
), but can
also engage single stranded DNA (ssDNA) with an
affinity equal to RNA (at least in vitro) (3). Thus, there is a lack
of consensus in the literature regarding the key
physiological role of these single stranded nucleic acid binding factors.
The weight of reports favour roles in RNA metabolism, with
FBP proteins being ascribed multiple roles in RNA
processing: both as negative and positive regulators of mRNA
splicing, mRNA stability, mRNA export and mRNA
). On the other hand the FBP1 protein has been
implicated in modulating activated transcription via ssDNA
binding and interaction with the general transcription
factor complexes, particularly upstream of the promoter for
the MYC oncogene (
Added to this confusion, mammalian genetics has not
resolved the major molecular function of the FBP-family
proteins most critical to development. In Drosophila the
three FBP proteins (FBP1-3) are represented by one
ortholog, P-element somatic inhibitor (Psi). Psi was originally
ascribed the function of modulating splicing of
transposable P-elements in Drosophila (
). However, given that
Pelements are a recent addition to Drosophila melanogaster,
only entering the genome within the past 50 years (
function cannot reflect evolutionary pressures. To clarify
the primary role of the FBP family in development we took
advantage of Drosophila genetics, and the lack of
redundancy in these KH domain proteins, and here demonstrate
that Psi is essential for cell and tissue growth.
Co-immunoprecipitation (Co-IP) mass spectrometry
data (DPiM (
)) provided clues into possible mechanism(s)
for growth control. Psi was predominantly detected in
association with both core and gene-specific RNA Pol II
transcriptional machinery; 63% of the 65 strongest
Psiinteractors have been directly implicated in RNA Pol II
activity. Strikingly, Psi was found in complex with most
subunits of the transcriptional Mediator (MED) complex,
which interacts with the RNA Pol II machinery to
modulate transcription in all eukaryotes (
). Although the
MED complex is required for most (if not all) RNA Pol II
dependent transcription, the MED/CDK8 module can act
as a sensor of developmental and environmental cues to
activate specific transcriptional programs (
studies in flies suggested that kohtalo and skuld, which encode
Drosophila homologs of the MED12 and MED13 subunits
of the kinase module, are essential for the transcription of
Wg/Wnt and Notch pathway targets and, thus, required
to establish compartment boundaries of the wing imaginal
). More recently, a specific reduction in the
expression of genes involved in wing margin formation was
observed for MED26 null mutant wing disc clones (23). At
the level of promoters, direct interplay between
gene/tissuespecific Hox transcription factors and MED19 is
essential for regulating expression of both embryonic and larval
imaginal disc patterning genes (
). Here, we demonstrate
that the interaction between Psi and the MED complex
underlies Psi’s essential function in cell and tissue growth.
The observation that Psi interacts with core RNA
Polymerase II transcriptional machinery to maintain cell and
tissue growth is of great interest given (i) the human
Psirelated KH protein FBP1 has been implicated in
modulating activated transcription from the MYC promoter
), and (ii) the proficiency of the MYC
transcription factor in driving cellular growth programs (
These data do not exclude roles in RNA processing, but
suggest dysregulation of dMYC transcription is key to
phenotypic outcome, potentially as a consequence of the capacity
of MYC to act as a context dependent global amplifier of
elaborate developmental transcription programs (
the context of rapidly proliferating wing disc cells the
major program of MYC-modulated transcription will include
genes required for cell and tissue growth.
MATERIALS AND METHODS
Unless otherwise stated, the Drosophila strains were
obtained from the Bloomington Stock Centre. We used two
non-overlapping PsiRNAi lines: PsiRNAi 1 (chromosome
2) PsiRNAi 2 (chromosome 3). The UAS-Psi RNAi 1 line
(V105135), UAS-Psi RNAi 2 line (V28990), UAS-CDK8
RNAi (V31264), UAS-CycC RNAi (V27937) and
UASdMYC RNAi (V2948) lines were obtained from the
Vienna Drosophila RNAi Center (
). UAS-MED18 RNAi
from the Bloomington TRIP collection (BL42634).
UASMED4 (767), UAS-MED7 (2735), UAS-MED17 (302),
UAS-MED18 (2188), UAS-MED19 (1396), UAS-MED20
(2076), UAS-MED30 (1060), UAS-MED31 (988) were
obtained from the DPiM transgenic resource.
Adult wing analysis
Adult wing size was determined for male wings imaged with
Olympus SZ51 binocular at 4.5x magnification using
Olympus DP20 camera. Wing size was measured in pixels for the
area posterior to wing vein L5 using Photoshop software
CS5. For wing hair counts adult male wings were imaged
with Olympus BX 61 microscope at 20x magnification
using the Olympus DP70 camera. Wing cell size was assessed
using wing hair counts in a defined area (200 × 100 pixels)
at the central region posterior of wing vein L5.
Co-IP and Western analysis
Co-IP for Drosophila was performed using 25 wild type 3rd
instar larval heads dissociated in cold lysis buffer (50 mM
Tris pH 7.5, 1.5 mM MgCl2, 125 mM NaCl, 0.2% NP40,
5% glycerol, 1x Protease inhibitor cocktail, Roche).
Following homogenization, protein was collected by
centrifugation at 12 000 rpm for 10 min at 4◦C. The extract was
pre-cleared by incubation with nProtein A Sepharose TM
beads (GE Healthcare Life Science) for 1 h at 4◦C with
rotation and the supernatant collected by centrifugation at
12 000 rpm. Equal amounts of pre-cleared protein lysate
were incubated with either guinea pig anti-MED17 (Gift
from Michael Marr), mouse anti-Cdk8 (Abcam, ab52779)
or anti-Psi (custom rabbit polyclonal antibody, Biomatik)
antibodies overnight at 4◦C. Beads were washed with
lysis buffer 5 times and the eluent resolved using 10%
SDSPAGE/Western with anti-Psi antibody (1/1000) prior to
detection with Li-Cor Odyssey IR detection.
Larval imaginal tissues were fixed for 20 min in 4%
paraformaldehyde (PFA) and blocked in 5% goat serum in
Phosphate buffered saline PBS with 0.1% Tween (PBST) at
room temperature for 1 h, followed by incubation overnight
at 4◦C with anti-Psi antibody (1/1000, rabbit polyclonal,
Biomatik) and detection with fluorescently tagged
secondary antibody. After placing in 80% glycerol, wing
imaginal discs were dissected and imaged with the Zeiss Imager Z
confocal microscope using Zen Meta software. Z-series with
1 m sections were performed at 40x magnification.
Fluorophores were imaged using band-pass filters to remove
cross-detection between channels. Images were processed
and prepared using Image J 1.43u, and Adobe Photoshop
CS4 Version 11.0.2.
Luciferase reporter assays
GAL4 fusion protein expression plasmids were generated
for full-length Psi and Psi deletion constructs by cloning
cDNA encoding the fragments (shown in Figure 6A) in
frame with the GAL4 DNA binding domain in pSG424.
The GAL4:VP16 and GAL4:E1a plasmids were used as
positive controls for transactivation. The reporter plasmid
(pLGB2) contains a minimal E1b TATAA (13 bp) and a
single Gal4 site upstream of luciferase. Plasmids were
transfected into 2 × 106 Hela cells (per well in a 6-well dish) using
Lipofectamine, cells were harvested after 24–30 h, extracts
prepared and assayed for luciferase. Assays were conducted
as described previously (
cDNA synthesis and q-RT PCR
RNA was isolated from equivalent numbers of wing
imaginal discs (10 pairs for each genotype) using the Promega
ReliaPrep RNA Cell miniprep system and eluted in 20 l
nuclease-free water. RNA purity and integrity was assessed
using an automated electrophoresis system (2200
TapeStation, Agilent Technologies). A total of 6 l of the eluted
RNA samples were used for cDNA synthesis (Bioline Tetro
cDNA Synthesis kit). qPCR was performed using KAPA
SYBR FAST qPCR Master Mix (Geneworks). Each qPCR
reaction contained 0.1 l of cDNA, 5 l KAPA SYBR
Master Mix and 0.2 M of each gene specific primer in a
final volume of 10 l. qPCR reactions (95◦C for 2 min,
followed by 40 cycles of: 90◦C/5 s and 60◦C/15 s) were run
using the Viia7 Real-Time PCR System and Sequence
Detection Systems in 384-well plates (Applied Biosystems).
Amplicon specificity was verified by melt curve analysis.
Average Ct values for three technical replicates were
calculated for each sample. Multiple internal control genes were
analysed for stability as described (35) and target gene
expression was normalised to the geometric mean of cyp1
and tubulin, selected for having high expression and little
sample-to-sample variability. Fold change was determined
using the 2- CT method.
The primers used were:
MYC - 5 GTGGACGATGGTCCCAATTT 3
5 GGGATTTGTGGGTAGCTTCTT 3 ;
PSI 5 CGATGGCATCCCATTTGTTTGT 3
Tubulin 5 TGGGCCCGTCTGGACCACAA 3
5 TCGCCGTCACCGGAGTCCAT 3
CYP1 – 5 TCGGCAGCGGCATTTCAGAT 3
5 TGCACGCTGACGAAGCTAGG 3
Cdk8 5 GGACATGGACAATCCGGTGC 3
5 GCTTGTCTCCTTCCATTTCGC 3
Chromatin immunoprecipitation (ChIP) assays were
carried out essentially as described previously (
). For each
ChIP sample, 30 larval heads were fixed in 4% PFA for 20
min. Larval heads were then mashed and chromatin sheared
in 0.4% sodium dodecyl sulphate (SDS) using a Covaris S2
(10 min duration, 10% DUTY, 200 cycles per burst,
Intensity 4, achieving average DNA fragment sizes 200–600 bp).
ChIP was performed in IP buffer containing 0.1% SDS and
3 g of antibody was used for each IP (anti-RNA Pol II
phospho S5 antibody (ab5131), or anti-RNA Pol II
phospho S2 (ab5095). Analysis was performed in triplicate using
KAPA SYBR FAST qPCR Master Mix (Geneworks) on a
Viia7 Real-Time PCR System and Sequence Detection
Systems in 384-well plates (Applied Biosystems). To calculate
the percentage of total DNA bound, non-immuno
precipitated input samples from each condition were used as the
qPCR reference for all qPCR reactions.
The primers for qPCR were:
MYC1 - 5 GGCGATCGTTTCTGGCCTACGG 3
5 GCAGGCGCATTTGACTCGGC 3 ;
MYC2 - 5 ACTACTACTAACAACTGTCACAAGCC
5 TTTATGTATTTGCGCGGTTTTAAG 3
MYC3 – 5 TTCAAAATAGAATTTCTGGGAAAGGT 3
5 GCGGCCATGATCACTGATT 3 ;
MYC4 - 5 GGTTTTCCTTTTATGCCCTTG 3
5 CTATTAACCATTTGAACCCGAAATC 3
MYC3 UTR - 5 AGGGGTTAGAGTTTACGAGTGA 3
5 CCAAATCAAATCGCGCGGAA 3
All statistical tests were performed with Graphpad Prism 6
using unpaired 2-tailed t-test with 95% confidence interval.
In all figures error bars represent SEM and according to the
Graphpad classification of significance points *(P = 0.01–
0.05), **P = 0.001–0.01, ***P = 0.0001–0.001 and ****P
Psi is required for cell and tissue growth
Knockdown of Psi in the dorsal compartment of the
larval wing imaginal disc with the serrate-GAL4 (ser-GAL4)
driver results in a curled adult wing phenotype (Figure 1A).
Similar ‘wings up’ phenotypes have been reported
previously following manipulation of growth regulators in the
dorsal wing compartment of the larval wing disc, which
develops into the top layer of the adult wing (
growth of the top sheet of the wing bilayer causes wing
cupping and bending due to torsional strain associated with a
comparatively smaller top sheet juxtaposing a larger
bottom sheet. We observed this phenotype using 2 alternate
and non-overlapping UAS-Psi RNAi lines (Figure 1A), and
confirmed depletion of Psi protein in the dorsal
compartment using our Psi antibody (Figure 1B). Quantification of
a defined area of the adult wing revealed a significant
reduction in wing size (Figure 1C, P < 0.0001 for Psi RNAi 1 and
Psi RNAi 2 compared with control, and see Supplementary
Table S2). As each wing-blade cell protrudes a single hair,
counting hair numbers within a fixed area provides a
measure of cell size, i.e. increased wing hairs indicate reduced
cell size. In line with Psi being essential for cell growth we
observed a significant increase in wing hairs (Figure 1D, P
< 0.0001 for Psi RNAi 1 compared with control within the
central region posterior of wing vein L5, and see
Supplementary Table S3). Psi depletion therefore resulted in
impaired wing size, primarily as a consequence of reduced cell
Psi interacts with RNA Pol II machinery including the transcriptional Mediator complex
The large scale DPiM Co-IP mass spectrometry study
generated a significant metazoan protein map; and included
rigorous statistical analysis and follow up validation of
interaction networks (
). The top 65 Psi interactors
identified following in silico mining of these data are listed in
Figure 2A (see also Supplementary Table S1) and the
percentage of hits comprising major functional classes
summarized in Figure 2B. Strikingly, 41 (or 63%) of these
Psiinteractors fell into ontology classes associated with RNA
Pol II-dependent transcription. In accordance with
previously reported RNA-binding roles for Psi (
), the majority
of the remaining Psi interactors were implicated in RNA
processing (18%) and/or mRNA translation (6%) (Figure
2A and B). These data suggest that in addition to
previously reported roles in mRNA processing, Psi might
interact with the RNA Pol II machinery to regulate
transcription. To further interrogate potential transcriptional roles,
the 41 interactors with designated functions in RNA Pol
II transcription were divided into sub-classes. Strikingly,
23 of the 41 transcriptional class interactors (56%)
comprised subunits of the Mediator (MED) complex (Figure
2C and D). The remaining 18 interactors were either part
of the chromatin-remodeling machinery (32%) or gene
specific transcriptional regulators (12%) (Figure 2C and D).
As the DPiM studies (
) that detected Psi in
complex with most MED subunits were performed in vitro
using overexpressed tagged protein in Drosophila S2
culture cells, we first confirmed that key MED subunits could
be detected in complex with Psi in vivo, by conducting
Co-immunoprecipitation (Co-IP) from wild type 3rd
instar larval imaginal disc lysates (Figure 2E and F). In
general, the MED complex can behave as either an activator
or inhibitor of RNA Pol II-dependent transcription. The
‘small’ or core MED complex is required for activation of
RNA Pol II transcription. The ‘large’ complex has been
predominantly characterised as a transcriptional repressor
and is comprised of an additional 4 proteins; the kinase
module comprising the Cyclin dependent kinase complex
(CDK8/CycC) and 2 additional MED subunits (MED12
and MED13) (
). As the DPiM detected Psi in complex
with both core subunits of the MED complex and the
CDK8/CycC module, we conducted Co-IP with either
antiCDK8 or anti-MED17 (a gift from Michael Marr) and
Western blot with an anti-Psi antibody. The 97 kDa band
for Psi was detected in association with the kinase
module subunit of the MED complex, i.e. the CDK8 subunit
(Figure 2E) and using the MED17 antibody we were able
to IP endogenous Psi from wild type larvae lysates (Figure
2F, representative blots from at least 3 biological replicates).
Thus Psi could be detected in association with endogenous
MED subunits from both the kinase module and the core
MED complex in vivo, however, further studies are required
to determine whether Psi directly interacts with these
Psi-dependent growth is sensitive to MED abundance
To determine the importance of the Psi-MED interaction
to development, we tested whether either transcriptional
core or kinase MED subunits were capable of modifying
the Psi ‘wings up’ phenotype. We predicted suppressors of
Psi-dependent growth would increase the size of the dorsal
sheet to bend wings down, i.e. to become more like wild type
(i.e. through loss of an inhibitor or increased abundance of
an activator), while enhancers (i.e. loss of an activator or
increased abundance of an inhibitor) of Psi-driven growth
would result in further wing bending. Interestingly, genetic
manipulation of core and kinase module subunits of MED
differentially modified the Psi RNAi phenotype (Figure 3).
Co-knockdown of CDK8 or CycC suppressed the bending
of the Psi adult wings and significantly increased
compartment size (Figure 3A and B P < 0.0001 for Psi RNAi +
CDK8 RNAi and Psi RNAi + CycC RNAi compared with
Psi RNAi alone, see Supplementary Tables S4 and S5),
consistent with the large MED module normally inhibiting
Although there was no obvious wing bending following
MED18 knockdown alone we observed a significant
reduction in compartment size (Figure 3C and D, P < 0.0074). As
the MED complex is required for modulating all RNA Pol
II-dependent transcription, the reduced wing phenotype
associated with depletion of the MED18 subunit alone may
reflect this more general role. Co-knockdown of MED18
and Psi did not significantly further reduce compartment
size, however, MED18 overexpression suppressed the Psi
wings up phenotype (Figure 3C and D for Psi RNAi +
MED18 RNAi compared with Psi RNAi alone, and see
Supplementary Table S6). Further analyses for additional
components of the core MED complex revealed that the
impaired growth associated with Psi knockdown in the
wing was suppressed by overexpression of MED4, MED7,
MED17, MED19, MED20, MED30 and MED31 (Figure
3D and see Supplementary Table S6). Together with the
ability to Co-IP Psi with antibodies to the MED complex
these data demonstrate both core and kinase module
subunits of MED physically interact with Psi, and that
Psidependent growth is sensitive to MED abundance.
Psi is required for maintaining endogenous levels of dMYC
mRNA and impaired growth in Psi knockdown depends on
Given mammalian FBP1 has been implicated in controlling
activated MYC transcription in vitro (
) and the
observation that MYC is a potent driver of cell and tissue growth
), we tested whether the decreased cell and tissue
growth associated with the Psi ‘wings up’ phenotype might
be sensitive to abundance of Drosophila MYC, dMYC.
In accordance with the reduced compartment size
following Psi knockdown in the wing being dMYC-dependent,
dMYC overexpression suppressed the ‘wings up’
phenotype. Furthermore co-knockdown of dMYC using RNAi
enhanced the Psi RNAi phenotype (Figure 4A and B, and
see Supplementary Table S7). To test whether impaired
wing growth was associated with decreased dMYC
abundance, we ubiquitously depleted Psi and measured dMYC
mRNA in wing imaginal discs by qPCR. Psi mRNA
abundance was not decreased following dMYC RNAi, however,
following a significant reduction in Psi mRNA (Figure 4C,
P < 0.0001 for Psi RNAi 1 and 2 compared with control,
Supplementary Table S8), dMYC mRNA was significantly
decreased (Figure 4D, P = 0.0006 for Psi RNAi 1 and P =
0.0066 for Psi RNAi 2 compared with control,
Supplementary Table S9). Taken together these data suggest that Psi
is required for maintaining dMYC mRNA at endogenous
levels and, thus, for cell growth during Drosophila wing
dMYC-dependent wing growth is sensitive to
As observed for Psi RNAi (Figure 1A), depletion of dMYC
in the dorsal wing compartment (i.e. Ser-GAL4 driven
UAS-dMYC RNAi) resulted in a ‘wings up’ phenotype
(Figure 4A). Given the observation that Psi-dependent
growth was modulated by MED and dMYC (Figures 3 and
4, respectively), we hypothesised that dMYC-dependent
growth might be sensitive to MED activity/abundance.
We therefore tested whether impaired wing growth
associated with dMYC RNAi was modified by the core or
kinase module MED subunits. As observed for Psi
knockdown (Figure 3), manipulation of core and kinase module
subunits of MED differentially modified the dMYC RNAi
wings up phenotype (Figure 5). Co-knockdown of CDK8
or CycC suppressed adult wing bending associated with
dMYC RNAi and significantly increased compartment size
(Figure 5A and B, P < 0.0001 for dMYC RNAi compared
with dMYC RNAi + CDK8 RNAi and dMYC RNAi +
CycC RNAi, Supplementary Table S10), consistent with the
CDK8/CycC module normally acting as a negative
regulator of dMYC-dependent growth. Although MED18
coknockdown did not significantly further reduce
compartment size (as also observed for MED18 co-knockdown with
Psi, Figure 3C and D), MED18 or MED 17 overexpression
suppressed the dMYC RNAi small wing phenotype (Figure
5A and B). Thus dMYC-dependent tissue growth was
sensitive to MED abundance; wing growth was restored
following depletion of kinase module CDK8/CycC subunits
or via overexpression of core MED subunits.
Psi regulates transcription in vitro
To determine whether Psi might regulate dMYC by
behaving as a transcriptional activator in vitro we transfected
HeLa cells with GAL4 DNA binding domain-Psi fusion
constructs and a reporter plasmid (pLGB2) containing a
single DNA binding site for the Gal4 transcriptional
activator 5 of the minimal TATAA box sequence (13 bp from
E1B, for loading of RNA Pol II and the basic
transcriptional machinery) and upstream of a luciferase reporter
(Figure 6A). Strikingly, Psi activates more strongly than
control transactivators (E1A and VP16) for this minimal
promoter (Figure 6C, see Supplementary Table S11).
Alignment of the mammalian FBP-KH domain protein FBP1
with Psi revealed regions of homology in addition to the KH
domains, i.e. the N-terminus (N-box) and C-terminus
(tyrosine diad YM-motifs) (Figure 6B). To determine whether
particular regions of Psi might be important for
transactivation we therefore conducted a deletion series spanning both
C-terminal and N-terminal domains (Figure 6B). Deletion
of the C terminal YM2 domain, alone ( 1-610, orange
bar in 6B and D) or together with the YM1 domain (
1591, green bar in 6B and D), abolished the transactivation
capacity of Psi (Figure 6D). Interestingly, N-terminal
constructs containing even a single C-terminal Y motif were
capable of activating transcription ( 659-610, pink bar in
6B and D). The strongest luciferase activity was detected for
N-terminal constructs containing both the YM1 or YM2
repeat domains ( 659–676, indigo bar in Figure 6B and
D). Together these data suggest that full-length Psi has
potent capacity as a transcriptional activator, which requires
the C terminal YM1 and YM2 repeat domains. However,
the presence of the N-terminal -helix and KH-containing
ssDNA/RNA binding domains diminished transactivation
associated with the C-terminal domain (Figure 6B and D,
e.g. compare Psi full length with 659–676).
The interaction between FBP and the active MYC
promoter will be dynamic and encompass both ssDNA and
protein interfaces of the transcriptional machinery,
including the multi-subunit mediator and general transcription
factor complex (TFIIH). The observation that C-terminal
constructs transactivate more strongly than full length
Psi might suggest the N-terminus normally represses
Psidependent transcription, either directly by masking the
activation module and/or via interaction with a second
transcriptional repressor protein. In light of this possibility,
FBP1 has been shown to interact directly with the RNA
recognition motif (RRM) containing protein FUSE
Interacting Repressor (FIR), which also binds ssDNA to
repress transcription (
). In humans the N-terminal
domain in FBP1 is required for the interaction with FIR in
vitro (8), which is supported by NMR studies
demonstrating the second RNA recognition motif (RRM2) of FIR
interacts with the N-terminal -helix (or N-box) of FBP (
Cellular studies have demonstrated that this interaction
between FIR and the FBP1 N-box is essential for FIR
recruitment to FUSE DNA and thus transcriptional repression
). However, further studies are required to determine the
likely cause(s) of the variation in transcriptional capacity
associated with the different Psi deletion constructs.
Psi depletion reduced Ser 5 and Ser2 RNA Pol II abundance
on the dMYC promoter
To investigate the capacity of Psi to alter dMYC
transcription in vivo we examined RNA Pol II activity on the dMYC
promoter. The transcription cycle in all eukaryotes
generally occurs as follows: (i) chromatin remodeling proteins
and gene specific transcription factors (TFs) engage, (ii)
the Mediator complex, the general transcription initiation
factors (GTFs, e.g. TFIIH), and the hypophosphorylated
RNA Pol II holoenzyme are recruited to form the
preinitiation complex (PIC) (
) and (iii) initiation of
transcription and release of Pol II allows elongation. Activation
of Pol II requires phosphorylation of the carboxyl-terminal
domain (CTD) of the largest RNA Pol II subunit at (i)
Ser5 for initiation and promoter escape and (ii) Ser-2 for
transcript elongation (
Ex vivo studies have shown that in response to serum
stimulation of previously starved human fibroblasts, FBP1
physically interacts with single-stranded DNA upstream of
the MYC transcriptional start site, which precedes release of
paused RNA Pol II and activation of MYC expression (
We therefore examined enrichment for Ser 5 RNA Pol II on
the dMYC promoter in larval imaginal tissues following
depletion of Psi. ChIP for control tissue revealed a peak for
Ser 5 Pol II proximal to the dMYC TSS (Figure 7A and B,
see Supplementary Table S12). In comparison, Psi
knockdown resulted in a significant decrease in Ser 5 RNA Pol II
enrichment proximal to the dMYC TSS (MYC2 amplicon,
Figure 7B, see Supplementary Table S12). Thus, Psi was
required to maintain the pool of Ser 5 phosphorylated RNA
Pol II on the dMYC promoter.
The significant decrease in dMYC mRNA in wing discs
from these animals is consistent with the reduced Ser 5
RNA Pol II, and would suggest the decrease in RNA Pol
II is not due to precocious Pol II escape and increased
elongation. Indeed, we also observed a significant decrease in
elongating (Ser 2 phosphorylated RNA Pol II) in Psi
depleted larval tissues (Figure 7C, see Supplementary Table
S12). Together the reduction in Ser 5 and Ser 2 RNA Pol
II, and decrease in dMYC mRNA abundance are
consistent with either diminished loading and/or
phosphorylation of RNA Pol II, either at the initiation/promoter
escape step alone (and thus leading to a follow-on reduction
in elongation), or also at the elongation step. However, the
observation that Psi activates from a single DNA binding
site within the minimal promoter (Figure 6, as observed for
)), compared with most G4-activators that require
tandem sites, suggests that Psi is unlikely to act at the level
of Pol II recruitment, but downstream of PIC assembly.
The capacity to integrate extracellular growth and
developmental signals is fundamental to coordinated growth of
organs and tissues in multicellular animals. Although the
MED complex is required for most (if not all) RNA Pol
II dependent transcription, the CDK8 module in
particular has been noted for its capacity to sense developmental
and environmental cues to activate specific transcriptional
). In Drosophila, MED responds to
specific developmental networks to control patterning of the
wing imaginal disc (
). Consistent with specific roles
in regulating growth networks, CDK8 has also been
implicated as a negative regulator of tissue growth in mice
(45). Our observation that Psi contains potent
transcriptional activation capacity suggest Psi helps activate
endogenous RNA Pol II activity on dMYC to modulate dMYC
transcription and control tissue growth during
development. Together with the impaired growth phenotype
associated with Psi depletion being suppressed following either
co-depletion of subunits from the transcriptionally
repressive CDK8/CycC kinase module or overexpression of core
subunits, our findings suggests Psi/dMYC-dependent tissue
growth depends on MED abundance and activity.
Thus, in Drosophila Psi and MED integrate growth
signals to maintain developmentally regulated MYC
transcription, cell and tissue growth. Mammalian signaling networks
are well known to co-ordinate transcriptional up- or
downregulation of many growth and cell cycle genes,
particularly MYC. Ex vivo studies from the early 80s
demonstrated that serum stimulation of mammalian tissue
culture cells results in rapid activation of MYC transcription
). Subsequently, extensive studies revealed that torsional
stress and strain on dsDNA in the MYC promoter
following initiation of MYC transcription results in melting of
the double stranded FUSE (the Far Upstream Sequence
Element/FUSE −1.7 kb upstream of the major MYC
transcription start site) into ssDNA (
maximal activation of MYC transcription correlates with
dissociation of RNA Pol II from the MYC TSS and
recruitment of FBP1 (
), which has been extensively characterized
for specificity in binding the single stranded FUSE
). The rapid release of RNA Pol II prior to
the peak in MYC mRNA levels was associated with
maximal enrichment for FBP1, consistent with FBP1 promoting
RNA Pol II release to hyperactivate MYC transcription (
Based on these observations, and our current study in
Drosophila, we propose the following model for the action
of Psi/FBP1 in activated MYC transcription (Figure 7D).
MED will first integrate the activity of MYC enhancers,
stimulated by growth or developmental signals, with the
general transcription factors (GTFs) and RNA Pol II to
form the PIC. In a signaling environment conducive to
MYC transcription, MED will recruit TFIIH and stabilise
the PIC (
). MED will also bring TFIIH kinase
activity into close proximity with the CTD of RNA Pol II
(49), consistent with MED stimulating TFIIH-dependent
CTD phosphorylation and RNA Pol II promoter clearance
The increased RNA Pol II activity will result in
conformational changes in the MYC promoter, including
supercoiling to cause torsional stress and generation of the
singlestranded FUSE, which binds FBP1/Psi. FBP1 can also
directly interact with the XPB helicase subunit of TFIIH, and
this interaction is required for formation of the promoter
loop between RNA Pol II and FUSE (
). Psi is found in
complex with the kinase module subunits of MED, thus
we predict Psi/FBP1 will first interact with the preactivated
MYC promoter, i.e. with the large MED complex still in
residence. Structural changes in ssDNA following Psi/FBP1
loading will modulate promoter architecture further to
facilitate exit of the CDK8 module, thus maximising
MEDdriven RNA Pol II activity and MYC transcription. At this
stage we cannot make conclusions on whether Psi/FBP1
interacts directly with a given MED subunit, or indirectly via
the TFIIH complex, however, we predict the transition to a
maximally activated MYC promoter will be dependent on
Subsequently, FIR/Hfp will be recruited to the MYC
promoter via binding to ssDNA, FBP1 and TFIIH to
facilitate FBP1 exit, inactivation of RNA Pol II and return of
MYC transcription to basal levels. Moreover, RNA
interference studies suggest FIR is required for repression of MYC
) and is dysregulated in cancers with
associated elevation of MYC (
). Our previous Drosophila
studies suggested the RRM protein with most similarity to FIR,
Half Pint (Hfp), is essential for developmentally driven
downregulation of dMYC transcription in vivo (
Moreover, as observed for FIR (
), Hfp interacts with
the XPB helicase component of the general transcription
factor TFIIH to maintain a pool of engaged RNA Pol II
on the MYC promoter, consistent with poised RNA Pol II
being required to attenuate MYC transcription (36).
Our analysis of the Drosophila protein interaction map
revealed the XPB/Haywire subunit of TFIIH was not one
of the 3488 bait proteins in the DPiM screen; nor was
XPB/Haywire detected as one of the 4927 prey proteins
with 0.8% (or less) false discovery. This was despite known
interactors (including other TFIIH complex subunits such
as CycH, Cdk7 and MAT1) being included as bait in the
DPiM screen. Moreover, CycH, and Cdk7 only detected
other CAK subunits (MAT1 and Cdk7 or CycH), but did
not detect any of the core subunits (e.g. dXPB/Haywire).
MAT1 as bait detected Cdk7, CycH and XPD from the
core, but no other subunits. Thus, the DPiM is not
saturating, in particular the core TFIIH subunits appear to be
under represented, and further studies are required to
establish whether Psi interacts with XBP/TFIIH.
Our studies demonstrate nuanced mechanisms of MYC
transcriptional regulation, requiring interaction between
the MED complex and ssDNA binding proteins are
essential for normal tissue growth. Further studies are required to
determine whether human FBP1 also interacts with MED
as we predict this interaction will also modulate expression
of the MYC oncogene. As even subtle increases in MYC
expression (>2-fold) can promote the cell and tissue
overgrowth fundamental to cancer initiation and progression,
these observations will have implications for human disease
Supplementary Data are available at NAR Online.
The authors thank Michael Marr for the MED17 and
MED26 antibodies. We thank Bloomington and VDRC
stock centres for Drosophila strains and the DSHB for
Author contributions: L.G, J.E.A.L., O.Z., N.C.M., Z.N.,
G.P. T.W., R.L. and L.M.P. conceived experiments, designed
the experiments, performed the experiments, analysed the
data and assisted with drafting the manuscript. R.D.H. and
D.L.L. conceived experiments, contributed reagents and
assisted with drafting the manuscript. L.M.Q. conceived
experiments, designed experiments, analysed data and drafted
Project Grants and a Senior Research Fellowship from the
National Health and Medical Research Council of
Australia (to L.Q and R.H.); NIH (to D.L.); Cancer Council
of Victoria (to L.Q.). Funding for open access charge:
Cancer Council Victoria.
Conflict of interest statement. None declared.
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