- Top of page
- Experimental procedures
The zinc finger transcription factor GATA-3 is a master regulator of type 2 T-helper cell development. Interestingly, in GATA-3–/– mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA-3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA-3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA-3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH promoter residing at −61 to −39 bp. Surprisingly, this domain does not contain GATA-3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site-directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA-3. Furthermore, protein–protein interaction assays showed that GATA-3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA-3 may regulate TH gene transcription via a novel and distinct protein–protein interaction, and directly contributes to NA phenotype specification.
Vertebrate nervous system development is regulated by a complex regulatory network of extracellular signals and nuclear transcription factors controlling the process of cell fate specification of vast neural subtypes (Marquardt and Pfaff 2001; Goridis and Rohrer 2002). Among the various phenotypes of a particular neuron, neurotransmitter identity is an important feature because it determines the nature of the chemical neurotransmission a given neuron will mediate, and influences its specific connectivity with target cells.
As indicated by the group nomenclature of noradrenaline (NA)-producing cells (i.e. group A1–A7) in the CNS, the NA cell type is one of the earliest neurotransmitter systems to be defined by neuroscientists (Cooper et al. 1996). In the CNS, NA is mainly produced in the locus coeruleus (A4 and A6 cell groups) by sympathetic ganglia (SG) in the peripheral nervous system (PNS). The regulatory cascade controlling the NA neurotransmitter phenotype has been extensively studied, leading to the identification and functional characterization of critical signaling molecules and transcription factors. For example, bone morphogenic proteins (BMPs) are essential for sympathetic nervous system (SNS) development and induce the expression of the proneural gene, Mash1 (or Cash1) (Brunet and Pattyn 2002; Goridis and Rohrer 2002; Howard 2005). Thus, Mash1 is the first transcription factor shown to be essential for NA neuron development (Guillemot et al. 1993). Downstream of Mash1 lies the homeodomain transcription factor, Phox2a, which is a critical regulator of NA cell lineage development (Morin et al. 1997; Guo et al. 1999; Lo et al. 1999; Stanke et al. 1999). The closely related transcription factor, Phox2b, is also induced by BMPs independently of Mash1 and is another essential regulator of NA neuron development (Pattyn et al. 1999).
A potentially new player in NA cell fate specification is the zinc finger transcription factor, GATA-3. GATA-3 was originally isolated as a T cell-specific DNA binding factor. Extensive in vivo and in vitro studies demonstrated that GATA-3 is essential for the development of T cell lineage and is a master regulator for the differentiation of type 2 T helper (Th2) cells (Murphy and Reiner 2002). GATA-3–/– embryos die between days 11 and 12 post coitum (d.p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross fetal liver haematopoietic aberrations (Pandolfi et al. 1995). Interestingly, a re-evaluation of the GATA-3–/– mouse showed that embryonic lethality was partially averted by feeding catechol intermediates, suggesting that NA deficiency is a proximal cause of death (Lim et al. 2000). Consistent with this, GATA-3 null mutation led to reduced accumulation of TH and dopamine β-hydroxylase (d.b.h.) mRNAs in the SNS, whereas several other SNS genes were unaffected (Lim et al. 2000). Furthermore, Tsarovina et al. (2004) recently re-investigated the GATA-3–/– mouse and confirmed that TH and d.b.h. expression is affected in this mouse, albeit to a somewhat different degree. This study demonstrated a more global effect of GATA-3 on sympathetic development, including the reduced size of the sympathetic chain (Tsarovina et al. 2004). Together, these in vivo findings suggest significant roles for GATA-3 in the development of the SNS. However, important questions regarding the function of GATA-3 remain unanswered, e.g. does GATA-3 directly regulate the induction of the NA neurotransmitter phenotype, are NA-synthesizing genes, such as TH and d.b.h., immediate target genes of GATA-3 in SNS development, and by what mechanism does GATA-3 control its target genes. To address these questions, we performed a series of experiments. Here, we show that overexpression of GATA-3 resulted in an increase of NA neurons among avian NCSCs in vitro, supporting the notion that GATA-3 has a direct role in NA neurotransmitter phenotype specification. Furthermore, we found that GATA-3 directly transactivates the promoter function of the TH gene, strongly suggesting that TH may be an immediate target of GATA-3. Surprisingly, our promoter analysis and protein interaction assays demonstrated that GATA-3 regulates the TH gene promoter via a distinct protein–protein interaction with the known transcription factor, CREB. Taken together, our results suggest the possibility that GATA-3 controls TH gene transcription via a novel and distinct protein–protein interaction.
- Top of page
- Experimental procedures
Transcription factors belonging to the GATA family contain two C4-type zinc finger domains and bind to the DNA motif WGATAR (Patient and McGhee 2002). Among the six members of the GATA family, GATA-3 is best known as a master regulator of Th2 cell differentiation (Murphy and Reiner 2002). In the present study, we investigated the potential functional role of GATA-3 in the regulatory cascade of specification of the NA neurotransmitter identity during SNS development.
In the NA biosynthetic pathway, TH is the first and, presumably, rate-limiting step converting tyrosine to l-dopa. Thus, expression of TH is an essential feature of NA neurotransmitter identity. We hypothesized that GATA-3 may directly regulate NA phenotype specification, and that the TH gene is an immediate downstream target of GATA-3. Alternatively, GATA-3 can indirectly regulate TH gene expression by inducing proliferation of precursor cells and/or regulating other transcription factors, e.g. Phox2a and 2b, which are known to transactivate TH gene expression. Several lines of evidence described in this study support the first possibility. First, we found that GATA-3 was able to transactivate TH promoter prominently. Second, overexpression of GATA-3 increased the number of TH-expressing cells in NCSC culture. Third, in our clonal analysis, GATA-3 increased the number of TH-positive clones.
GATA family transcription factors bind to GATA-containing sequence motifs (Murphy and Reiner 2002). Interestingly, the TH subdomain responsive to GATA-3 does not contain any GATA-3 recognition motif. In the case of the TH proximal subdomain (at −61 to −39 bp), the CRE is the only known cis-regulatory motif, which is the binding site for the basic region-leucine zipper (bZip) transcription factor CREB. Mutation of this CRE motif almost completely abolished transactivation of the TH promoter by GATA-3, while it did not affect transactivation by another transcription factor, AP2α. These results suggest that GATA-3–CREB interaction may be involved in TH promoter regulation. Indeed, in vitro GST pull-down and in vivo Co-IP assays demonstrated that GATA-3 and CREB could physically interact with each other. Further protein interaction assays using a series of fragments delineated the regions of the proteins essential for their physical interaction. For GATA-3, the C-terminal half (aa 242–444) encompassing both fingers was sufficient while for CREB, the fragment (aa 270–327) encompassing bZip allowed the interaction. Taken together, we propose that GATA-3 is able to transactivate the TH gene via physical interaction with CREB. Despite prominent expression of CREB, forced expression of GATA-3 did not activate the TH promoter in TH-expressing cells. This observation can be explained by the assumption that GATA-3 is sufficiently expressed in the TH-expressing cells to maintain its activity. In line with this, Phox2a and Phox2b, NA-specific transcription factors, transactivated the d.b.h. promoter in d.b.h. non-expressing cell lines, but not in d.b.h.-expressing cell lines (Yang et al. 1998).
In developing embryos, neural crest cells originate from the roof plate of the neural tube and migrate between somites and the neural tube. At their final target sites, neural crest cells of the trunk region become different types of neurons or glia in response to environmental signals. To address further the in vivo function of GATA-3, we infected primary NCSC isolated from the trunk region of E2 quail embryos with replication competent RCAS-cGATA-3 or RCASBP(B) viruses. Whereas the total number of NCSC was not affected by GATA-3, the number of cells with the sympathoadrenergic phenotype was increased more than twofold as examined by TH expression. The increase in TH- positive cells could be explained by the increase in cell proliferation of NA progenitor cells or by fate changes of NCSCs by GATA-3. In our clonal analysis, the number of TH-expressing clones was increased from 8.3% to 17.0%. Thus, these results (i.e. increases of both TH-positive cells and clones) cannot be explained by simple up-regulation of TH gene expression by GATA-3 in TH-positive cells. Rather, our result strongly supports the hypothesis that GATA-3 directly controls the NA fate determination of NCSCs during sympathetic neuron development and that this process involves, at least in part, direct activation of TH gene transcription.
One interesting issue is the functional relationship between GATA-3 and GATA-2. Among the six GATA factors, GATA-3 and GATA-2 share the highest sequence homology, and only these two members are expressed in the nervous system in an overlapping manner. While a knockout study indicated a critical role of GATA-3 in mouse SNS development, a similar role was suggested for GATA-2 in chick embryo (Groves et al. 1995). In developing chick SNS, GATA-2 was expressed after Cash1 and Phox2 proteins, followed by SCG10 and TH (Groves et al. 1995). Moreover, Tsarovina et al. (2004) recently reported that GATA-2, but not GATA-3, is expressed in developing chick sympathetic precursors and controls SNS development, suggesting that GATA-2 in chick is the functional counterpart of GATA-3 in mouse in SNS development. Notably, GATA-3 is genetically downstream of Phox2b, because its expression is abolished in Phox2b knockout mice while Phox2b is induced in GATA-3 knockout mice (Lim et al. 2000). Though GATA-3 is critical for the development of SNS (Lim et al. 2000; Tsarovina et al. 2004), induction of NA phenotype by GATA-3 requires a certain cellular context. GATA-3 needs to work in concert with other factors, such as Phox2a/b and dHand, because the increase in TH-positive clones in NCSC culture was rather modest (2–2.5-fold).
In summary, our results show that GATA-3 can regulate the transcriptional activity of the TH gene via a novel and distinct protein–protein interaction. We propose that TH is an immediate target gene of GATA-3, and that GATA-3 directly participates in NA phenotype specification during SNS development. Although GATA-3 has been shown to interact with several proteins, such as FOG, ROG and Smad3 (Miaw et al. 2000; Blokzijl et al. 2002; Kurata et al. 2002), our study is the first to support the possibility that GATA-3 can regulate its target gene(s) via protein–protein interactions with a general transcription factor. In this context, it is noteworthy that another key transcription factor for dopamine neuron development, Nurr1, has been shown to enhance the transcriptional activity of the human dopamine transporter gene through a promoter region devoid of Nurr1 binding sites (Sacchetti et al. 2001). Thus, this potentially novel mechanism by which key fate-determining transcription factors (e.g. GATA-3 and Nurr1) control neurotransmitter identity warrants further investigation. In addition, as CREB is expressed in a vast number of tissues, it is of great interest to determine whether GATA-3 may regulate additional target genes via similar protein–protein interactions.