Finally, we investigated the biological significance of the regulation of Tcf4 gene by Zac1. As
Tcf4 can act as a transcription factor, we tested whether the control of the expression of Tcf4 by
Zac1 can affect the transcription of Tcf4 target gene. We decided to focus on the paternally imprinted gene p57kip2 for 3 reasons.
1) It is co-regulated with Zac1 in the context of the Imprinted gene Network (Varrault et al., 2006).
2) p57kip2 plays an important role in differentiation and migration of RGC and IPC during neurogenesis (Tury et al., 2011a).
3) Zac1, Tcf4 and p57kip2 are known inducers of cell cycle arrest (Matsuoka et al., 2006; Herbst et al., 2009a).
p57kip2 is one of the three members of the the Cip/Kip family of cell cycle inhibitors. It is expressed in proliferating progenitors in the VZ and SVZ of the telencephalon, but also in cortical precursors and mature neurons. p57kip2 expression is strongly reduced in the adult brain, except in the developing cerebellum where it is transiently expressed during the postnatal cerebellar neurogenesis. p57kip2 has been identified as a functionally relevant target recruited by the E-proteins to induce cell cycle arrest in the human neuroblastoma cell line SK-N-SH (Rothschild et al., 2006). Hence, we first determined whether Tcf4 can regulate the expression of
p57kip2 in the murine C17.2 cell line. To address this, cells were transfected with Tcf4 expression vector and endogenous p57kip2gene expression was measured by qRT-PCR. Tcf4 overexpresssion induced p57kip2 by 1.5 fold confirming the role of Tcf4 as regulator of p57kip2gene expression in the mouse brain (Fig. 22.C). Previous studies showed that p57kip2might be directly regulated by
Zac1; hence, it is difficult to determine the relative contribution of both transcription factors to
p57kip2 gene regulation. However, in contrast to p57kip2, Zac1 gene expression was not affected by
Tcf4 overexpression (Fig. 22.C), we could therefore conclude that Tcf4 could regulate p57kip2
expression in a Zac1 independent manner. It is thus likely that both factors control the expression of the cell cycle regulator p57kip2. Additional Zac1 overexpression and knocking down approaches confirmed that the regulation of Zac1 expression mediates the expression of Tcf4 and of its target gene p57kip2 (Fig. 22.A-B-D).
59 Figure 22: Zac1 and Tcf4 regulate p57kip2expression in C17.2 cells. A). Knock down of Zac1 by transfection of 500 ng of a pool of shRNA targeted against Zac1 (shRNA-Zac1) for 24 hrs, resulted into a significant decrease of
Zac1 and Tcf4-B mRNA levels as measured by qRT-PCR. B) Immunoblot analysis of 50 µg of WCE from C17.2 cells transfected with 500 ng of scramble shRNA or shRNA-Zac1. The reduction of Tcf4 and Zac1 mRNAs after
Zac1-knockdown translates into their increased protein expression. C) C17.2 cells were transfected for 24 hrs with 100 ng of pCDEF3-Flag-Tcf4, and expression of Zac1 and p57kip2 was assessed by qRT-PCR. Overexpression of
Tcf4 increased p57kip2gene expression independently of Zac1. D) Knock down of Zac1 by transfection of 500 ng of a pool of shRNA targeted against Zac1 (shRNA-Zac1) for 24 hrs, resulted into a significant decrease of p57kip2 mRNA levels as measured by qRT-PCR. This indicates that regulation of Zac1 expression control Tcf4 and its target gene
p57kip2 expression. Bars represent the average of 3 independent experiments ran in duplicates; SD is shown. *p<0.05; **p<0.01 (t-test).
Finally, to verify these findings in vivo, we measured Tcf4 occupancy at p57kip2 promoter in two brain regions where they are both expressed (Alam et al., 2005; Ye et al., 2009): in the VZ and
B
C
A
60
SVZ of the lateral ventricles and in the developing cerebellum (Fig. 23) by ChIP. The two analyzed genomic regions on p57kip2promoter contain an E-box (E-protein binding motif) and
Tcf4 proximal promoter was used as negative control of binding as no E-Box motif could be identified in this region. We observed a strong enrichment of Tcf4 at p57kip2promoter in vivo, supporting a role for the proneural gene in the regulation of p57kip2gene transcription.
Figure 23: Tcf4 occupies p57kip2 promoter in neural progenitors. ChIP analysis of Tcf4 occupancy at p57kip2
promoter and Tcf4 proximal promoter in the regions surrounding the lateral ventricles of E15 mouse brain or in the developing murine cerebellum of P8. Brain punches from 6 animals for lateral ventricle were pooled and used for each immunoprecipitation (n=2) whereas 1 whole cerebellum was used for each immunoprecipitation (n=3). Values were first normalized to the Input, and then calculated as fold enrichment (Tcf4 antibody value divided by the IgG value) and plotted on a logarithmic scale. % of Input could not be used here as the value of the IgG differed grandly between the 3 primers.
Altogether, we could show that Zac1 is an important transcriptional activator during neurogenesis. Via the control of Tcf4, it might promote neuronal differentiation. By controlling
p57kip2 gene expression, Zac1 might also control the cell cycle exit of progenitor cells.