GPS2 is a 327 amino acid nuclear protein with a predicted molecular mass of 37 kDa. Although the precise biological functions of GPS2 are largely unknown, several lines of evidence suggest its potential involvement in diverse cellular functions. It was initially isolated via its ability to suppress lethal G protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae
). Pheromone binds to its cognate receptor and triggers a Gβ,γ
-mediated kinase cascade that shares structural similarities with mammalian mitogen-activated protein kinase (MAPK) pathways. Consistent with its role in conserved signaling pathways, over-expression of GPS2 in mammalian cells strongly suppresses a RAS/MAPK-mediated signal and interferes with c-Jun N-terminal kinase 1 (JNK1) activation by serum factors or tumor necrosis factor α (TNFα) (21
). Studies have shown that GPS2 interacts with the human T cell lymphotrophic virus type I (HTLV-I) Tax oncoprotein, and suppresses its ability to activate JNK1 (21
). The N-CoR and histone deacetalyase 3 (HDAC3) complex participates in diverse repression pathways and contains about 10-12 associated proteins. GPS2 is one subunit of this complex (27
). GPS2 inhibition of JNK activation is mediated through an associated N-CoR-dependent corepressor function (28
). Furthermore, studies have also suggested a role for GPS2 in mediating cellular responses to DNA damage (29
), and GPS2 might function in concert with a hMSH4-hMSH5 heterocomplex during the process of homologous recombination (30
Our studies demonstrate the relationship between RFX4_v3 and GPS2. First, both RFX4_v3 and GPS2 are present throughout the brain and in differentiated neuronal cells, suggesting their interaction is physiologically relevant. Second, yeast two-hybrid screening and co-immunoprecipitation experiments in mammalian cells demonstrate the physical interaction between these two proteins. Third, GPS2 is able to enhance the activating abilities of RFX4_v3 on the Cx3cl1 promoter, suggesting that GPS2 is a co-activator for RFX4_v3-dependent transcriptional events.
Transcription cofactors may exert variable effects on transcriptional regulation depending on their interacting partners. As stated above, GPS2 can suppress
transcription by association with N-CoR and HDAC3 repressor complexes. However, GPS2 has also been reported to interact with several transcription factors and enhance
their transactivation. For example, GPS2 binds the papillomavirus E2 protein activation domain and is necessary for stimulation of E2 transcriptional activity (31
). GPS2 is also associated with the p53 tumor suppressor and facilitates the p53 response by augmenting p53-dependent transcription (29
). GPS2 can also stimulate the transcription function of a c-Jun AD-Gal4 DBD fusion protein (31
). The mechanisms of GPS2 functioning as a transcription co-activator are not well understood. GPS2 might activate E2-dependent transcription by directly interacting with both E2 and p300 (32
). Complex formation among E2, GPS2, and p300 may function by bringing p300 (with histone acetyltransferase activity) close to the transcription initiation sites. Histone acetylation of nearby nucleosomes is thought to remodel the chromatin structure and enhance access of the transcriptional machinery to DNA. Therefore, one possible mechanism by which GPS2 potentiates RFX4_v3 transactivation is that GPS2 might recruit other transcriptional activators to the promoters of RFX4_v3 target genes. Base on our ChIP results, another possible explanation for GPS2 co-activation of RFX4_v3 activities is that interaction of RFX4_v3 with GPS2 might enhance the binding of RFX4_v3 protein to the Cx3cl1
promoter. GPS2 alone was also shown to be sufficient to activate transcription from several artificial promoters (33
); however, it had no ability to either activate or repress the transcription of the Cx3cl1
gene by itself.
RFX4 proteins lack the Q/PQ and A regions, which play roles in transcriptional activation, and are believed to function as a transcriptional repressor (2
). However, our experiments indicate that RFX4_v3 can activate Cx3cl1
gene expression. The activation domain of RFX4_v3 protein has not been identified yet. It seems that the activation region is located within the first 574 amino acids, since RFX4_v3/1-574 can stimulate Cx3cl1
gene transcription to a similar extent as the full-length RFX4_v3 protein. Further carboxyl-terminal deletion of the RFX4_v3/1-574 protein to generate RFX4_v3/1-300 totally abolishes RFX4_v3 activation on the Cx3cl1
promoter, suggesting that the dimerization domain may be essential for RFX4_v3 activities since amino acids 301-574 mainly encode the dimerization domain. The carboxyl-terminal region of RFX4_v3 (amino acids 575-735) has no effect on RFX4_v3 transactivating abilities. However, this region was involved in the GPS2 potentiation of RFX4_v3 activation. There are several possible explanations for this phenomenon. RFX4_v3/1-574 alone was sufficient for GPS2 binding; however, additional binding between GPS2 and the RFX4_v3 carboxyl-terminal region could (a) enhance the interaction between two proteins, or (b) lead to conformational changes of either or both proteins. These changes might facilitate complex formation with other transcription factor(s), or alternatively increase the activating abilities of RFX4_v3 itself. Additional experiments will be needed to further examine these possibilities.
Besides the interaction with RFX4_v3, GPS2 has the ability to bind other mammalian RFX family members, including RFX2 and RFX3, but not RFX1. It has been suggested that the dimerization domain might be involved in the interaction. RFX4_v2 forms a heterodimer with RFX2 and RFX3, but not RFX1, and forms a homodimer with RFX4_v2 itself, theoretically through the dimerization domains (2
). It is interesting to note that both GPS2 and RFX4_v2 do not bind RFX1, although according to the sequence alignment, the dimerization domains of RFX1-RFX3 are closely related, and the dimerization domain of RFX4 is slightly different from the other three dimerization domains.
GPS2 is a relatively well-characterized transcriptional cofactor, therefore, we chose to focus our initial efforts on the interaction between GPS2 and RFX4_v3. However, eight other potential binding partners for RFX4_v3 were uncovered by the yeast two-hybrid screening and several may be worthy of further consideration. For example, calmodulin is associated with numerous neuronal functions including dendrite growth (34
) and synaptic plasticity (35
). There is a putative calmodulin binding site between amino acids 114-168 of the RFX4_v3 protein. This is consistent with our yeast two-hybrid results which show that the first 250 amino acids of RFX4_v3 could interact with calmodulin. Proteasome function is linked with neurodegenerative disorders (36
), and one proteasome subunit PMSD2 might interact with RFX4_v3. ZBTB1 contains both zinc finger and BTB domains. BTB domains from several zinc finger proteins have been shown to mediate transcriptional repression and to interact with components of histone deacetylase co-repressor complexes including N-CoR and SMRT (37
). NCDN, RICS, and FBF1 are believed to be cytosolic proteins, but it will be interesting to examine whether they could potentially translocate to the nucleus and interact with RFX4_v3 under certain circumstances. MLF2 and ZFP469 are novel proteins with no information on their cellular localization or physiological function. For each of these potential RFX4_v3 binding partners, examination of cellular localization, validation of physical interactions with RFX4_v3, and further investigation of the functional relevance of these interactions with respect to regulation of RFX4_v3-mediated gene transcription will provide for a better understanding of the role of RFX4_v3 in brain development.