The role of ZO-2 in the nucleus has started to be unraveled. Our work with reporter genes under the control of AP-1–regulated promoters revealed the participation of ZO-2 in the repression of gene transcription (Betanzos et al., 2004
). Because TJs seem to be involved in the regulation of cell proliferation, we analyzed whether ZO-2 was able to affect the transcription of cyclin D1, a key regulator of the cell cycle. Our results indicated that ZO-2 in a complex with c-Myc and HDAC1 repressed the transcription of the cyclin D1 gene. This repression seemed to be physiologically relevant, because cells overexpressing ZO-2 exhibited a diminished cell proliferation rate as determined through [3
H]thymidine incorporation and wound healing assays.
The relationship among TJ proteins, gene transcription, and cell proliferation was uncovered with the pioneering work of Balda and Matter. They revealed how ZO-1 inhibited G1/S phase transition by cytoplasmic sequestration of ZONAB, a transcription factor that forms a complex with CDK4 (Balda and Matter, 2000
; Balda et al., 2003
). Moreover, they showed that upon ZONAB overexpression cyclin D1 gene expression became up-regulated, whereas the expression of the SH3 domain of ZO-1, which inhibits ZONAB function, induced only a small inhibition of the cyclin D1 promoter (Sourisseau et al., 2006
). The latter observation is in accordance with our present findings in MDCK cells that revealed that in contrast to ZO-2, ZO-1 transfection exerted no direct effect on the activity of the human cyclin D1 promoter (B). These results demonstrated that each of these proteins exerted a particular function not shared by other ZO proteins.
Because ZO proteins do not have the characteristic features in their sequence that allow binding to DNA, they might form complexes with other proteins, such as transcription factors, to regulate gene transcription. The interaction between ZO-1 with ZONAB and ZO-2 with Jun, Fos, and C/EBP has confirmed this proposal and has allowed the exploration of regulatory regions of genes regulated by ZO protein expression.
Our work initially explored TRE(AP-1) and CRE sites of the human cyclin D1 promoter, located at positions −929 to −935 and −45 to −52, respectively. Because the mutation of just one of any of these sites generated a profound loss of promoter activity, it became extremely difficult to appreciate the impact ZO-2 overexpression had on them (A). However, we further pursued this point in a ChIP assay done by immunoprecipitating ZO-2 and performing PCR amplification with primers for the TRE site. Because no amplification was obtained, we suspect that ZO-2 is not exerting its inhibitory effect on cyclin D1 transcription through association to the TRE(AP-1) site (B). With regard to the CRE site, it should be pointed out that the construct containing the deletion of the E box and that showed no repression upon ZO-2 overexpression still has an intact CRE site (B). These results, therefore, suggested that this location is not involved in the inhibition of cyclin D1 transcription triggered by ZO-2 overexpression.
The deletion analysis performed around the E2F site highlighted the importance of the E box in the transcriptional regulation of cyclin D1 modulated by ZO-2 (). Structurally different factors of the basic-helix loop-helix (bHLH) type, like c-Myc and USF, or the zinc finger protein family, like Snail, recognize the CAC(A/G)TG sequence that characterizes E box elements (Blackwell et al., 1990
; Cole and McMahon, 1999
; Nieto, 2002
; Corre and Galibert, 2005
). We discarded the participation of Snail in the repression of cyclin D1 mediated by ZO-2, because Snail protein levels are so low in MDCK cells that they escape detection with commercial antibodies (Comijn et al., 2001
; Bolos et al., 2003
). However, to further address this issue, we performed ChIP assays in MDCK cells that overexpress Snail (generously provided by professor Roberto Montesano, Department of Cell Physiology and Metabolism, University of Geneva Medical Center), and we did not observe interaction of Snail with the E box region of the cyclin D1 promoter (data not shown). The participation of USF and/or E2F transcription factors in cyclin D1 down-regulation by ZO-2 is a matter of current work in our laboratory, because at least in the case of E2F, the electrophoretic mobility shift assays showed effective competition of this factor for complex 2 (A, left).
With regards to c-Myc, we demonstrated its ability to bind to the E box element of the cyclin D1 promoter, to repress the promoter activity either alone or in a cooperative manner with ZO-2 ( and ), and to inhibit cell proliferation (). Another tumor suppressor known to modulate the transcriptional activity regulated by c-Myc is, for example, Bin1, which interacts with the N-terminal region of c-Myc and inhibits malignant cell proliferation (Elliott et al., 1999
Besides cyclin D1 (Philipp et al., 1994
), other genes repressed in c-Myc–transformed cells included those that encode cell adhesion proteins, such as the β1 integrin subunit (Judware and Culp, 1995
); molecules of the immune system, such as major histocompatibility complex class I and lymphocyte function-associated antigen-1 (Bernards et al., 1986
; Versteeg et al., 1988
; Inghirami et al., 1990
); and cell cycle regulators, such as C/EBPα (Freytag and Geddes, 1992
) and c-Myc itself (Penn et al., 1990
). These findings suggested that gene repression contributes significantly to the phenotype of c-Myc transformed cells. Formation of Myc:Max heterodimers is essential for both Myc-dependent transcriptional activation and gene repression (Mao et al., 2003
). Other combinations of c-Myc heterodimers have been described that recruit remodeling enzymes, such as HDACs, and exert a repressing effect. Such is the case of MM-1, a novel c-Myc-binding protein that interferes with the E box-dependent transcription activity of c-Myc (Mori et al., 1998
). In HeLa cells, c-Myc repression is controlled by a complex containing c-Myc, MM-1, the corepressors mSin3 and TIF1β, and HDAC1 (Satou et al., 2001
). Here, we have described the formation of a complex consisting of ZO-2, c-Myc, and HDAC1 that participates in the negative regulation of cyclin D1; however, we cannot rule out the inclusion of other components in this complex.
In summary, we can conclude that ZO-2 inhibits the transcription of cyclin D1 by forming a complex that includes c-Myc and HDAC1, and this complex associates with the E box present in the cyclin D1 promoter (). The interaction of ZO-2 with this complex is important for understanding the role played by TJ proteins in the regulation of cell proliferation and tumorigenesis.