Activator of CREM in Testis and its uses

Details Activator of CREM in Testis and its uses Activator of CREM in Testis and its uses

2000-03-10

2003-12-09

Saidha, Tekchand (Department: 1652)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S069100, C435S069700, C435S252300, C435S320100, C536S023500

Reexamination Certificate

active

06660841

ABSTRACT:

The present invention deals with a new protein activator of CREM in Testis named ACT, the cDNA encoding ACT and the use of said protein and cDNA for screening modulating compounds which are able to interact with ACT or its site of interaction on CREM to block or stimulate the CREM transcriptional activity in Testis. Such compounds may lead to control of male fertility.
Transcriptional activation by CREB and CREM requires serine phosphorylation within the activation domain (Ser133 in CREB; Ser117 in CREM) and consequent interaction with the co-activator CBP
1, 2
. The activator CREM is expressed at very high levels in male germ cells and is required for post-meiotic gene expression
2-4
. Using a two-hybrid screen, we have isolated a testis-derived cDNA encoding ACT (Activator of CREM in Testis), a novel LIM-only protein that specifically associates with CREM. ACT is expressed coordinately with CREM in a highly tissue- and developmentally-regulated manner. ACT powerfully stimulates CREM transcriptional activity in yeast and mammalian cells and it contains an intrinsic activation function. Strikingly, ACT bypasses the classical requirements for activation, namely Ser117-phosphorylation and interaction with CBP. Thus, ACT identifies a novel pathway of transcriptional activation by CREM and CREB. ACT may define a novel class of tissue-specific co-activators whose function could be specific of distinct cellular differentiation programs.
CREB and CREM are factors directly coupled to signaling pathways. Phosphorylation within the P-box, and consequent CBP interaction, turns them into powerful activators
1, 2
. The activation domain (AD) comprises also two glutamine-rich regions Q1 and Q2 interaction to the TBP-associated hTAF130 seems involved in activation
5
. While CREB is ubiquitous, CREM displays a remarkable tissue distribution and plays important roles in the nuclear response to physiological stimuli
2
. In particular, the activator CREM is hundred-fold more abundant in male germ cells than in any other tissue
4
. CREM controls the transcription of various post-meiotic genes
2,3
and its targeted inactivation in the mouse results in the complete block of spermatogenesis at early spermatids stage
6, 7
. Unexpected recent results show that the activator CREM is not phosphorylated at Ser117 in germ cells (in preparation). Thus, while the physiological role of CREM in germ cells is well established, the molecular mechanism by which it exerts its function remains elusive.
We wished to investigate how activation by CREM could occur in the absence of phosphorylation. A yeast two-hybrid was used to screen a murine testis cDNA expression library with the CREM AD (
FIG. 1
a
). The CREM AD fused to the GAL4 DBD (DNA-binding domain) is completely inactive (
FIGS. 1
b;
1
c
) for the lack of CBP and hTAF130 yeast homologs. One clone interacted with high affinity when tested for nutritional selection and &bgr;-galactosidase activity (
FIGS. 1
b;
1
c
). This encodes a novel 284aa protein (SEQ ID NO:1)
FIG. 1
d
) ACT (activator of CREM in testis), containing four complete and one N-terminal half LIM motif (
FIG. 1
d
). LIM domains are constituted by double zinc-finger structures, are present in various proteins and thought to mediate protein-protein interactions
8, 9
.
ACT displays a remarkable tissue-specific expression (
FIG. 2
a
). We have raised anti-ACT specific antibody which revealed a protein of the expected size (33 kDa). ACT protein colocalizes in purified spermatids with CREM
10
(
FIG. 2
b
). In situ analysis shows regulated expression during germ cell differentiation (
FIG. 2
c
), coordinated with CREM
10
. Immunohistochemistry demonstrates that ACT is nuclear and expressed in round and elongated spermatids (
FIG. 2
d
). Thus, CREM and ACT proteins are coexpressed in vivo.
ACT and CREM proteins efficiently associate (FIG.
3
). Purified full-length ACT fused to glutathione-S-transferase (GST) was tested for binding to different CREM isoforms. The presence of the sole P-box is sufficient for binding ACT (
FIG. 3
a
). Consistent with the yeast two-hybrid assay, ACT does not bind to the repressor ICER
11
, indicating that the DBD is dispensable for interaction. To map the region involved in CREM association, we generated ACT truncations in each LIM motif. Mutants lacking the fourth LIM domain (GST-ACT 1-221) or the N-terminal half LIM domain (GST-ACT 38-284) interact with CREM, while deletion of the two C-terminal LIM domains (GST-ACT 1-162) impairs association (
FIG. 3
b
). Thus, the third LIM domain appears critical for CREM-ACT interaction. CREM-ACT association is also revealed by co-immunoprecipitation after expression in mammalian cells (
FIG. 3
c
). Extracts from cells transfected with CREM and Myc-tagged ACT expression vectors, individually or in combination, were immunoprecipitated using anti-CREM antibodies. Western analysis (
FIG. 3
c
) revealed that ACT associates with CREM in immunoprecipitated complexes. No ACT protein was co-immunoprecipitated by anti-CREM antibodies when ACT and CREM were expressed separately. Conversely, CREM-ACT association was detected when the Myc-tagged ACT protein was immunoprecipitated from co-transfected cells, using an anti-Myc antibody (
FIG. 3
c
). Finally, the CREM-ACT association is also revealed by co-immunoprecipitation of native proteins from a testis extract (
FIG. 3
d
).
Sequence analysis of the ACT cDNA from the two-hybrid screening held a surprise: the coding sequence was not in frame with the GAL4 AD within the yeast expression vector. Thus ACT could be translated from its own AUG and therefore have intrinsic coactivator properties in yeast. To test this, the GAL4-AD was deleted generating ACT(delGal4-AD) (
FIG. 4
a
). Strikingly, ACT alone powerfully activates CREM-dependent transcription. Thus ACT bypasses the requirement of CBP for activation, as CBP is lacking in yeast. A fusion of ACT in frame with GAL4 AD (ACT/Gal4-AD) elicits only a two-fold higher activity than ACT, indicating that ACT, per se, has an efficient activation potential. To assess whether ACT has an intrinsic AD we fused it to the GAL4 DBD (ACT/Gal4-DBD). This fusion protein efficiently induces activation (
FIG. 4
a
). Thus, ACT bears an autonomous AD as its recruitment to DNA is sufficient to elicit activation.
The CREM gene generates both activators and repressors
2,12
. Is ACT able to modify the repressors function? In a two-hybrid assay ACT-mediated co-activation takes place only in the presence of CREM activator isoforms, as the P-box in combination with at least one glutamine-rich domain is required (CREM&tgr;1 and CREM&tgr;2,
FIG. 4
b
).
ACT is a powerful activator in mammalian cells. Using a GAL4-based reporter
13
CREM-mediated activation was enhanced by ACT in a dose-dependent manner. Strikingly, ACT functions in absence of co-expressed PKA catalytic subunit (
FIG. 4
c
). Analogous results were obtained with full-length CREM and CREB on a CRE-driven somatostatin reporter and the testis-specific angiotensin converting enzyme (ACE) and calspermin promoters
14,15
(
FIG. 4
d,e;
not shown). ACT displays inherent activating function also in mammalian cells, as ACT fused to the GAL4 DBD strongly stimulates transcription from the G4CAT reporter (
FIG. 4
f
).
We wished to test ACT function on CREB, as it is known that GAL4 DBD fusion proteins containing the glutamine-rich activation domains are inactive in yeast
16
. We show that ACT significantly activates CREB-mediated transcription both in yeast and in mammalian cells (
FIG. 4
g;
and not shown).
Phosphorylation at Ser133 in CREB and Ser117 in CREM is critical for activation and CBP interaction
17-21
. As ACT bypasses the CBP requirement (FIG.
4
), we wondered whether a Ser>Ala substitution may prevent ACT function. The Ser>Ala117 mutation did not affect the CREM-ACT physical association tested by co-immunoprecipitation after transfection in mammalian cells (
FIG. 5
a
). Strikingly, the Ser>Ala117 mutation did not decrease the stimulating ACT function, both in yeast and mammalian cells (

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