November 1, 2014;
Carboxy terminus of GATA4 transcription factor is required for its cardiogenic activity and interaction with CDK4.
-6 transcription factors regulate numerous aspects of development and homeostasis in multiple tissues of mesodermal and endodermal origin. In the heart
, the best studied of these factors, GATA4
, has multiple distinct roles in cardiac specification, differentiation, morphogenesis, hypertrophy and survival. To improve understanding of how GATA4
achieves its numerous roles in the heart
, here we have focused on the carboxy-terminal domain and the residues required for interaction with cofactors FOG2 and Tbx5
. We present evidence that the carboxy terminal region composed of amino acids 362-400 is essential for mediating cardiogenesis in Xenopus pluripotent explants and embryos. In contrast, the same region is not required for endoderm
-inducing activity of GATA4
. Further evidence is presented that the carboxy terminal cardiogenic region of GATA4
does not operate as a generic transcriptional activator. Potential mechanism of action of the carboxy terminal end of GATA4
is provided by the results showing physical and functional interaction with CDK4
, including the enhancement of cardiogenic activity of GATA4
. These results establish CDK4
as a GATA4
partner in cardiogenesis. The interactions of GATA4
with its other well described cofactors Tbx5
and FOG2 are known to be involved in heart
morphogenesis, but their requirement for cardiac differentiation is unknown. We report that the mutations that disrupt interactions of GATA4
and FOG2, G295S and V217G, respectively, do not impair cardiogenic activity of GATA4
. These findings add support to the view that distinct roles of GATA4
in the heart
are mediated by different determinants of the protein. Finally, we show that the rat GATA4
likely induces cardiogenesis cell autonomously or directly as it does not require activity of endodermal transcription factor Sox17
, a GATA4
target gene that induces cardiogenesis non-cell autonomously.
[+] show captions
Fig. 1 – Carboxy-terminal domain (362–440) of GATA4 is required for cardiogenesis. (A) Schematic representation of GATA4
structure showing transcriptional activation domains (TAD), Zn fingers (Zn), nuclear localisation signal (nls) and the
position of mutations tested in this study. Right – sequence of the C-terminal region of GATA4 (rat, NP_653331; Xenopus
NP_001085355) and GATA5 (rat NP_001019487; Xenopus NP_001081962), showing poor conservation in these cardiogenic
factors. (B) Deletion of C-terminal 78 amino acids abolishes cardiogenic activity of GATA4. Animal caps injected with wt or
1–361 mutant rGATA4 were tested for expression of cardiomyocyte-specific genes myl7 and myh6 (synonyms: Myosin Light
Chain 2 and Myosin Heavy Chain α, respectively) at st.34 and for levels of GATA4 protein at st. 10 (lower panel). Note that
the effect of this deletion is negligible on endothelial marker aplnr (apelin receptor; synonym- msr) and is less pronounced
on hba3 (haemoglobin alpha 3; synonym- αglobin), a blood marker. odc1- loading control (expression of ornithine
decarboxylase 1). (C) Nuclear localisation of 1–361 rGATA4 was determined by immunohistochemistry at st. 7–8. (D) Lack of
activity of 1–361 in vivo. Embryos injected with 1–361 rGATA4 at 8–16 cell-stage in anterior ectodermal precursors were
analysed for myl7 expression by whole mount hybridisation. myl7 expression was only observed in the heart (h) in all
embryos examined (n = 50, from 2 independent experiments). In contrast, cardiogenic GATA4 versions readily induce
ectopic myl7 expression (Figs 3 and 5). (E) Non-cardiogenic 1–361 GATA4 mutant retains gene expression-inducing activity
in animal cap explants. At st. 10 the mutant 1–361 induces endogenous gata4 and (F) at st. 34 pan-endodermal marker
alpha-2 macroglobulin (a2m; synonym – endodermin) is induced (as well as by N-terminal deletions 153–440 and 201–440
non-cardiogenic GATA4 (Gallagher et al., 2012)).
Fig. 2 – CDK4 interacts with C-terminal GATA4 in vitro and enhances cardiogenic activity of GATA4 (A) In vitro translated
35S radio-labelled CDK4 protein was incubated with GST-N-terminal GATA4, GST-C-terminal GATA4 or GST proteins on
glutathione sepharose beads. The bound proteins were resolved by SDS/PAGE and revealed by autoradiography. Note that
CDK4 binds C-terminal GATA4 (1st lane). The experiment is one representative of two. (B) Fold synergy of wild type GATA4
(1–440) or C-terminal deletion (1–361) with CDK4 on GATA-dependent promoter. The C-terminal region is required for
synergy. 10 ng of GATA4 and 100 ng of CDK4 expression vectors were used. The experiment was performed twice.
* p < 0.005 of GATA4 1–440/CDK4 fold synergy vs GATA4 1–440 using Student’s t test. (C) NIH3T3 cells were transiently
transfected with GATA-luc and increasing doses of the indicated GATA4 expression vector (5, 25, 50 and 100 ng) with our
without treatment with 3 M CDK4 inhibitor. The cells were treated the following day after transfection and kept for 18 hrs.
Note that 1–361 GATA4 activation is not inhibited by CDK4. Experiment was performed twice. # p < 0.001 of GATA4 1–440
with CDK4 inhibitor vs. GATA4 1–440 using two-way ANOVA. (D) CDK4 enhances cardiogenic activity of GATA4. Animal cap
explants injected with indicated capped mRNAs (400 and 100 pg/embryo of CDK4 and CyclinD2 (CycD2), respectively;
300 pg/embryo of rGATA4 (suboptimal dose)) were analysed for expression of cardiomyocyte marker myl7 at st. 34.
Fig. 3 – Deletion of 15 or 40 C-terminal amino acids does not block cardiogenic activity of GATA4. (A) Expression of myh6
and odc1 in st. 34 animal caps expressing wt, 1–400, 1–425 and 1–425 mPKC (S419A; S420A) versions of rGATA4.Western
blot analysis (lower panel) of protein levels at st. 10. (B) Nuclear localisation of 1–400, 1–425 and 1–425 rGATA4. (C) Embryos
injected in anterior ectodermal blastomeres at 8–16 cell stage with 1–400, 1–425 and 1–425 were analysed for myl7
expression at st. 34. myl7 was detected in the heart (h) and in ectopic locations (arrows) of representative embryos (out of
40, 4–7 embryos showed ectopic cardiac expression).
Fig. 4 – Non-cardiogenic GATA4 mutants lacking N- or
C-terminus can be partially rescued by a heterologous
transcriptional activator. (A) 1–361 and 201–440 VP fusions
are stronger transcriptional activators than the wt rGATA4.
Embryos were injected at 1- or 2-cell stage with a firefly
luciferase reporter under the control of two GATA sites,
Renilla luciferase plasmid driven by the thymidine kinase
(TK) promoter and indicated mRNAs. Control-DNA alone
sample. Animal caps were excised at st. 8.5 and collected
for analysis 3 hours later. GATA-luc activity represents
relative fold activation (firefly luciferase activity normalised
by Renilla luciferase activity, with control (no mRNA)
sample set at 1). In three separate experiments 201–
440 + VP and 1–361 + VP were found to be considerably
more active than the wt rGATA4, with variable fold
differences but consistent trend of 201–440 + VP and
1–361 + VP showing 5–10 times greater activity than the wt
rGATA4. (B) VP16 minimal activation domain (VP) weakly
rescues cardiogenic activity of 1–361 and 201–440, as
assessed by expression of myl7 and myh6. (C) In contrast,
the ability of 1–361 and 201–440 VP fusions to induce the
expression of endothelial marker aplnr and a blood marker
hba3 is comparable to the wt rGATA4.
Fig. 5 – Cardiogenic activity of GATA4 does not require residues which are essential for interaction with FOG2 or Tbx5.
(A) E215D, V217G and G295S mutations do not affect cardiogenesis induced by GATA4 in animal pole explants, as assessed
by expression of myh6 at st. 34. Bottom panel –Western blot analysis of rGATA4 variants in animal caps collected at st. 10.
(B) Nuclear localisation of E215, V217G and G295S GATA4 mutants. (C) Ectopic myl7 expression (arrows) induced by
indicated mutants in representative embryos (out of 50, 4–10 embryos showed ectopic myl7 expression). (D) In addition to
being able to efficiently induce cardiac differentiation markers myh6 and myl7, V217G and G295S mutants are
indistinguishable from the wt rGATA4 in their ability to induce liver (nr1h5; synonym-for1), endothelium (aplnr) and blood
(hba3) markers. (E) Model of the C-terminal Zn finger of GATA4 with G295S mutation. The key side chains, Ser295 and
Arg282, are indicated. The model shows the clash that would occur with the mutation in the absence of backbone or side
chain adaptations to resolve it.
Fig. 6 – Non-cell autonomous induction of cardiogenesis in animal cap explants by Sox17. Animal caps derived from
cardiac actin–GFP (CA-GFP) embryos injected with 200 pg of Sox17 mRNA or uninjected (AC) were conjugated and cultured
until st. 34. Different animal caps in each conjugate were distinguished by the presence of rhodamine-dextran in one of
them. (A–D) Bright field images of representative examples of indicated types of explants. (A’–D’) Corresponding red and
green fluorescence images (merged), showing contribution of rhodamine-dextran injected animal caps and the presence or
absence of striated muscles (CA-GFP; Latinkic et al., 2003). CA-GFP activity was only detected in naïve, uninjected part of
Sox17+AC conjugates, indicating non-cell autonomous cardiac induction. The number of conjugates showing the
represented phenotype and the total number of conjugates are shown in the lower right corner (A–D). (E) Activity of Sox17
is shown by induction of endoderm marker a2m in st. 10 animal cap explants. (F) Inhibition of Sox17 function by
Sox17::EnR does not block cardiogenesis by rGATA4. rGATA4 (400 pg/embryo) and Sox17::EnR (200 pg per embryo) were
uniformly injected at 1- or 2-cell stage and RT-PCR was performed at st. 34 for myl7 and odc1 loading control.