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Interaction of ZEB and histone deacetylase with the PLDLS-binding cleft region of monomeric C-terminal binding protein 2.

Zhao LJ, Kuppuswamy M, Vijayalingam S, Chinnadurai G - BMC Mol. Biol. (2009)

Bottom Line: CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs.These results suggest a competition between the canonical PLDLS-motif factors such as E1A and non-PLDLS factor HDAC for interaction with CtBP.Our results are consistent with a model that the CtBP2 dimer may interact with a PLDLS-containing repressor through one monomer and recruit HDAC and other chromatin modifying enzymes through the second monomer in the CtBP2 dimer.

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Affiliation: Institute for Molecular Virology, Saint Louis University Health Sciences Center, St, Louis, Missouri 63104, USA. zhaol@slu.edu

ABSTRACT

Background: Proteins of the C-terminal binding protein (CtBP) family, CtBP1 and CtBP2 are closely related transcriptional regulators that are coded by two different gene loci in the vertebrate genomes. They perform redundant and unique functions during animal development. CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs. The N-terminal region of CtBP1/2 forms a hydrophobic cleft and is involved in interaction with both PLDLS-containing factors and non-PLDLS factors. CtBP proteins function as dimers to mediate transcriptional repression and dimerization is modulated by specific binding to NAD/NADH.

Results: In this study, we have investigated the role of dimerization of CtBP2 in recruitment of PLDLS-motif cofactors and non-PLDLS cofactors. Our results indicate that mutations in CtBP2 that interfere with dimerization abolish CtBP2 interaction with most cellular factors, except the PLDLS-motif factor zinc-finger E-box binding homeobox (ZEB) and the non-PLDLS factor HDAC2. Unlike most PLDLS-containing CtBP-binding proteins, ZEB contains three PLDLS-like motifs and all three contribute to the interaction with the CtBP2 monomer. Despite the ability to interact with ZEB and HDAC, the CtBP2 monomer fails to mediate ZEB-dependent transcriptional repression. The lack of repression activity of the CtBP2 monomer is correlated with the competition between ZEB and HDAC for interaction with the CtBP2 monomer.

Conclusion: These results suggest a competition between the canonical PLDLS-motif factors such as E1A and non-PLDLS factor HDAC for interaction with CtBP. They also indicate that the affinity for the CtBP monomer may be determined by the number as well as amino acid sequence compositions of the PLDLS-like motifs. Our results are consistent with a model that the CtBP2 dimer may interact with a PLDLS-containing repressor through one monomer and recruit HDAC and other chromatin modifying enzymes through the second monomer in the CtBP2 dimer.

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HDAC-dependent transcriptional repression by Gal4-RR-CtBP2. A, Transcriptional repression by Gal4-ZEB101. Gal4 fusion constructs were co-transfected into MCF7 cells with pG5-Luc and phRL-tk. Dual luciferase assay was performed as in Fig. 3A. DBD: Gal4 DNA binding domain. B, Failure of RR-CtBP2 to repress transcription through Gal4-ZEB101. CtBP1/2 double knockout cells were co-transfected with pG5-Luc, phRL-tk, Gal4-ZEB101 and CtBP2 or RR-CtBP2. Dual luciferase assay was performed as in A. C, HDAC-dependent transcriptional repression by Gal4-CtBP2 and Gal4-RR-CtBP2. MEF90 cells were co-transfected with pG5-Luc, phRL-tk, and Gal4-CtBP2 or Gal4-RR-CtBP2. HDAC inhibitor TSA was added at 0.2 ng/ml during transfection. Dual luciferase assay was carried out as in A.
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Figure 6: HDAC-dependent transcriptional repression by Gal4-RR-CtBP2. A, Transcriptional repression by Gal4-ZEB101. Gal4 fusion constructs were co-transfected into MCF7 cells with pG5-Luc and phRL-tk. Dual luciferase assay was performed as in Fig. 3A. DBD: Gal4 DNA binding domain. B, Failure of RR-CtBP2 to repress transcription through Gal4-ZEB101. CtBP1/2 double knockout cells were co-transfected with pG5-Luc, phRL-tk, Gal4-ZEB101 and CtBP2 or RR-CtBP2. Dual luciferase assay was performed as in A. C, HDAC-dependent transcriptional repression by Gal4-CtBP2 and Gal4-RR-CtBP2. MEF90 cells were co-transfected with pG5-Luc, phRL-tk, and Gal4-CtBP2 or Gal4-RR-CtBP2. HDAC inhibitor TSA was added at 0.2 ng/ml during transfection. Dual luciferase assay was carried out as in A.

Mentions: To examine the functional correlation between CtBP2 interaction with GFP-ZEB101 and CtBP2 transcriptional repression, ZEB101 was expressed as a Gal4-ZEB101 fusion protein. Co-transfection of Gal4-ZEB101 with the luciferase reporter, G5-MLP-Luc, into MCF7 cells resulted in transcriptional repression (Fig. 6A). Similarly, Gal4-ZEB-DLm2 was also active in repression, consistent with the ability of GST-ZEB-DLm2 to interact with both the CtBP2 monomer and CtBP2 dimer (Fig. 4). In contrast, Gal4-ZEB-PLm1-3 did not repress transcription, possibly due to its defective interaction with CtBP.


Interaction of ZEB and histone deacetylase with the PLDLS-binding cleft region of monomeric C-terminal binding protein 2.

Zhao LJ, Kuppuswamy M, Vijayalingam S, Chinnadurai G - BMC Mol. Biol. (2009)

HDAC-dependent transcriptional repression by Gal4-RR-CtBP2. A, Transcriptional repression by Gal4-ZEB101. Gal4 fusion constructs were co-transfected into MCF7 cells with pG5-Luc and phRL-tk. Dual luciferase assay was performed as in Fig. 3A. DBD: Gal4 DNA binding domain. B, Failure of RR-CtBP2 to repress transcription through Gal4-ZEB101. CtBP1/2 double knockout cells were co-transfected with pG5-Luc, phRL-tk, Gal4-ZEB101 and CtBP2 or RR-CtBP2. Dual luciferase assay was performed as in A. C, HDAC-dependent transcriptional repression by Gal4-CtBP2 and Gal4-RR-CtBP2. MEF90 cells were co-transfected with pG5-Luc, phRL-tk, and Gal4-CtBP2 or Gal4-RR-CtBP2. HDAC inhibitor TSA was added at 0.2 ng/ml during transfection. Dual luciferase assay was carried out as in A.
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Figure 6: HDAC-dependent transcriptional repression by Gal4-RR-CtBP2. A, Transcriptional repression by Gal4-ZEB101. Gal4 fusion constructs were co-transfected into MCF7 cells with pG5-Luc and phRL-tk. Dual luciferase assay was performed as in Fig. 3A. DBD: Gal4 DNA binding domain. B, Failure of RR-CtBP2 to repress transcription through Gal4-ZEB101. CtBP1/2 double knockout cells were co-transfected with pG5-Luc, phRL-tk, Gal4-ZEB101 and CtBP2 or RR-CtBP2. Dual luciferase assay was performed as in A. C, HDAC-dependent transcriptional repression by Gal4-CtBP2 and Gal4-RR-CtBP2. MEF90 cells were co-transfected with pG5-Luc, phRL-tk, and Gal4-CtBP2 or Gal4-RR-CtBP2. HDAC inhibitor TSA was added at 0.2 ng/ml during transfection. Dual luciferase assay was carried out as in A.
Mentions: To examine the functional correlation between CtBP2 interaction with GFP-ZEB101 and CtBP2 transcriptional repression, ZEB101 was expressed as a Gal4-ZEB101 fusion protein. Co-transfection of Gal4-ZEB101 with the luciferase reporter, G5-MLP-Luc, into MCF7 cells resulted in transcriptional repression (Fig. 6A). Similarly, Gal4-ZEB-DLm2 was also active in repression, consistent with the ability of GST-ZEB-DLm2 to interact with both the CtBP2 monomer and CtBP2 dimer (Fig. 4). In contrast, Gal4-ZEB-PLm1-3 did not repress transcription, possibly due to its defective interaction with CtBP.

Bottom Line: CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs.These results suggest a competition between the canonical PLDLS-motif factors such as E1A and non-PLDLS factor HDAC for interaction with CtBP.Our results are consistent with a model that the CtBP2 dimer may interact with a PLDLS-containing repressor through one monomer and recruit HDAC and other chromatin modifying enzymes through the second monomer in the CtBP2 dimer.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Molecular Virology, Saint Louis University Health Sciences Center, St, Louis, Missouri 63104, USA. zhaol@slu.edu

ABSTRACT

Background: Proteins of the C-terminal binding protein (CtBP) family, CtBP1 and CtBP2 are closely related transcriptional regulators that are coded by two different gene loci in the vertebrate genomes. They perform redundant and unique functions during animal development. CtBP proteins mediate their transcriptional function through interaction with various DNA-binding repressors that contain PLDLS-like motifs and chromatin modifying enzymes, such as class I histone deacetylases (HDAC) that do not contain such motifs. The N-terminal region of CtBP1/2 forms a hydrophobic cleft and is involved in interaction with both PLDLS-containing factors and non-PLDLS factors. CtBP proteins function as dimers to mediate transcriptional repression and dimerization is modulated by specific binding to NAD/NADH.

Results: In this study, we have investigated the role of dimerization of CtBP2 in recruitment of PLDLS-motif cofactors and non-PLDLS cofactors. Our results indicate that mutations in CtBP2 that interfere with dimerization abolish CtBP2 interaction with most cellular factors, except the PLDLS-motif factor zinc-finger E-box binding homeobox (ZEB) and the non-PLDLS factor HDAC2. Unlike most PLDLS-containing CtBP-binding proteins, ZEB contains three PLDLS-like motifs and all three contribute to the interaction with the CtBP2 monomer. Despite the ability to interact with ZEB and HDAC, the CtBP2 monomer fails to mediate ZEB-dependent transcriptional repression. The lack of repression activity of the CtBP2 monomer is correlated with the competition between ZEB and HDAC for interaction with the CtBP2 monomer.

Conclusion: These results suggest a competition between the canonical PLDLS-motif factors such as E1A and non-PLDLS factor HDAC for interaction with CtBP. They also indicate that the affinity for the CtBP monomer may be determined by the number as well as amino acid sequence compositions of the PLDLS-like motifs. Our results are consistent with a model that the CtBP2 dimer may interact with a PLDLS-containing repressor through one monomer and recruit HDAC and other chromatin modifying enzymes through the second monomer in the CtBP2 dimer.

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