Biochemical study of Nolz-1 protein and its interacting proteins

• Main Authors: Shun-Yuan Lee, 李舜淵
• Other Authors: Fu-Chin Liu
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/10868189219254477978

碩士 === 國立陽明大學 === 神經科學研究所 === 97 === Nolz-1 is a nuclear protein enriched in the subventricular zone (SVZ) of lateral ganglionic eminence (LGE) in developing ventral telencephalon, and is one of the members of NET family (Noc, Nlz, Elbow, and Tlp-1). Previous studies have shown that Nolz-1 is a transcriptional repressor in ST14A and Neuro2A neural cell lines, but Nolz-1 is a transcriptional activator in non-neural HEK293T cells. Studies of Nolz-1 homologues in zebrafish, Nlz-1, and in Drosophalia, Elbow, also indicate that Nolz-1 is a transcriptional repressor in neurons. Nolz-1 could interact with Groucho (Grg), a transcriptional co-repressor, as other members of NET family do. By co-immunoprecipitation assay, I found that Nolz-1 interacted with Grg5 via the protein domains containing putative Groucho binding motif, FKPY, and the Buttonhead box. Furthermore, using reporter gene assay, I demonstrated that over-expression of Grg4 could convert Nolz-1 from transcriptional activator into repressor in HEK293T cells, suggesting the functional significance of the interaction between Nolz-1 and Grg4. The second part of my thesis is to study the interaction between Nolz-1 and β-catenin/Tcf-mediated Wnt signaling (canonical Wnt signal pathway). The investigation was carried out in P19 cells. By reporter gene assays, the promoter activities of Nolz-1 were analyzed, and these results showed that the putative Tcf/Lef1 response elements might be located in the promoter region between 200 bps to 2400 bps upstream from the Nolz-1 transcriptional start site. By immunoblotting assays, neither activation of Wnt signaling by constitutively active β-catenin or inhibition of Wnt signaling by DKK1, which is a secreted antagonist of the canonical Wnt signaling, did not change the protein levels of Nolz-1 in P19 cells. Further, knockdown of Nolz-1 leads to the increases of canonical Wnt signaling in neural induction of RA-aggregated P19 cells. These results suggested that Nolz-1 is not the downstream target gene of the canonical Wnt signal pathway in neural induction of P19 cells, but Nolz-1 could suppress canonical Wnt signaling activity. In addition, I hypothesized that Nolz-1 protein might interact with Tcf/Lef1 protein family, which are the transcription factors and effectors of canonical Wnt signaling, because both of them were associated with Groucho protein. Unfortunately, this hypothesis could not be verified by the co-immunoprecipitation assay of Nolz-1 and TCF7, LEF1, TCF7L1 or Tcf7l2 proteins. I studied the cell proliferation of P19 cells while knocking down Nolz-1 with Nolz-1 shRNAs. Knocking down Nolz-1 did not affect the expression levels of PCNA, a marker for cell proliferation, but the cell number assayed by cell viability test was increased. Taken together, Nolz-1 could regulate the canonical Wnt signaling activity in neural induction of P19 cells. The last part of my thesis is to identify other interacting proteins of Nolz-1 by proteomic approach using MALDI-TOF. One protein that I found is Btbd11. However, double immunostaining shows that Btbd11 is localized in cytoplasma whereas Nolz-1 is localized in the nucleus, and the results of in situ hybridization and RT-PCR indicate that Btbd11 is enriched in the insular cortices, posterior thalamus and the hindbrain region but not in the ventral telencephalon of E13.5 mouse embryo. Because the Nolz-1 and Btbd11 protein interaction could not be verified by co-immunoprecipitation assay, Btbd11 might be a false-positive protein identified by proteomic approach. In summary, my studies demonstrate that Nolz-1 is a transcriptional repressor, and it interacts with Groucho protein through the protein domains containing Buttonhead box and the FKPY Groucho binding motif. In addition, Nolz-1 could suppress the canonical Wnt signal pathway during neural induction of P19 clls, suggesting that Nolz-1 may control neural development by regulating canonical Wnt signal activity.