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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.

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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Roles of PLDLS-like motifs of ZEB in interaction with CtBP2 and RR-CtBP2. A, Diagram of constructs. Numbers denote the amino acid residue number in the full-length ZEB1. B, Interaction of CtBP2 and RR-CtBP2 with GST-ZEB101 and GST-E1A. GST-ZEB101, GST-E1A, and GST-E1A-C* (with a DL→AS mutation in PLDLS motif) were charged to glutathione beads and then incubated with purified H6-CtBP2 or H6-RR-CtBP2. Bound CtBP2 was examined by western blot with the CtBP2 antibody and GST-fusion proteins visualized by Coomassie blue. Input (lanes 1 and 2) represents 5% of input proteins. C, Interaction of CtBP2 and RR-CtBP2 with mutants of GST-ZEB101. Conditions were the same as in B, except that Input (lane 1) represent 10% of input proteins. Bottom panel: Coomassie blue-stained GST-fusion proteins for the binding assays involving H6-CtBP2 (top panel). GST-fusion proteins for the binding of H6-RR-CtBP2 were the same (not shown).
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Figure 4: Roles of PLDLS-like motifs of ZEB in interaction with CtBP2 and RR-CtBP2. A, Diagram of constructs. Numbers denote the amino acid residue number in the full-length ZEB1. B, Interaction of CtBP2 and RR-CtBP2 with GST-ZEB101 and GST-E1A. GST-ZEB101, GST-E1A, and GST-E1A-C* (with a DL→AS mutation in PLDLS motif) were charged to glutathione beads and then incubated with purified H6-CtBP2 or H6-RR-CtBP2. Bound CtBP2 was examined by western blot with the CtBP2 antibody and GST-fusion proteins visualized by Coomassie blue. Input (lanes 1 and 2) represents 5% of input proteins. C, Interaction of CtBP2 and RR-CtBP2 with mutants of GST-ZEB101. Conditions were the same as in B, except that Input (lane 1) represent 10% of input proteins. Bottom panel: Coomassie blue-stained GST-fusion proteins for the binding assays involving H6-CtBP2 (top panel). GST-fusion proteins for the binding of H6-RR-CtBP2 were the same (not shown).

Mentions: The near normal interaction between RR-CtBP2 and ZEB was in sharp contrast to the defective interaction between RR-CtBP2 and E1A (Fig. 2A and Fig. 3B). Within the CtBP-interaction domain of ZEB, there are three PLDLS-like motifs (Fig. 4A) - a central PLDLS motif, and a PLNLS motif on both sides. The middle motif is identical to the canonical CtBP-binding motif of E1A [31]. To directly examine the roles of the individual PLDLS-like motifs in interaction with wt CtBP2 and RR-CtBP2, ZEB101 region was expressed as a GST-ZEB101 fusion protein. Mutants of GST-ZEB101 were constructed as shown in Fig. 4A, to incorporate mutations in the PLDLS-like motifs. The DLm2 mutant contains DL→AS mutations in the central PLDLS motif, the PLm1/3 mutant contains PL→AS mutations in the two PLNLS motifs, and the PLm1-3 mutant contains mutations in all three motifs.


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)

Roles of PLDLS-like motifs of ZEB in interaction with CtBP2 and RR-CtBP2. A, Diagram of constructs. Numbers denote the amino acid residue number in the full-length ZEB1. B, Interaction of CtBP2 and RR-CtBP2 with GST-ZEB101 and GST-E1A. GST-ZEB101, GST-E1A, and GST-E1A-C* (with a DL→AS mutation in PLDLS motif) were charged to glutathione beads and then incubated with purified H6-CtBP2 or H6-RR-CtBP2. Bound CtBP2 was examined by western blot with the CtBP2 antibody and GST-fusion proteins visualized by Coomassie blue. Input (lanes 1 and 2) represents 5% of input proteins. C, Interaction of CtBP2 and RR-CtBP2 with mutants of GST-ZEB101. Conditions were the same as in B, except that Input (lane 1) represent 10% of input proteins. Bottom panel: Coomassie blue-stained GST-fusion proteins for the binding assays involving H6-CtBP2 (top panel). GST-fusion proteins for the binding of H6-RR-CtBP2 were the same (not shown).
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Figure 4: Roles of PLDLS-like motifs of ZEB in interaction with CtBP2 and RR-CtBP2. A, Diagram of constructs. Numbers denote the amino acid residue number in the full-length ZEB1. B, Interaction of CtBP2 and RR-CtBP2 with GST-ZEB101 and GST-E1A. GST-ZEB101, GST-E1A, and GST-E1A-C* (with a DL→AS mutation in PLDLS motif) were charged to glutathione beads and then incubated with purified H6-CtBP2 or H6-RR-CtBP2. Bound CtBP2 was examined by western blot with the CtBP2 antibody and GST-fusion proteins visualized by Coomassie blue. Input (lanes 1 and 2) represents 5% of input proteins. C, Interaction of CtBP2 and RR-CtBP2 with mutants of GST-ZEB101. Conditions were the same as in B, except that Input (lane 1) represent 10% of input proteins. Bottom panel: Coomassie blue-stained GST-fusion proteins for the binding assays involving H6-CtBP2 (top panel). GST-fusion proteins for the binding of H6-RR-CtBP2 were the same (not shown).
Mentions: The near normal interaction between RR-CtBP2 and ZEB was in sharp contrast to the defective interaction between RR-CtBP2 and E1A (Fig. 2A and Fig. 3B). Within the CtBP-interaction domain of ZEB, there are three PLDLS-like motifs (Fig. 4A) - a central PLDLS motif, and a PLNLS motif on both sides. The middle motif is identical to the canonical CtBP-binding motif of E1A [31]. To directly examine the roles of the individual PLDLS-like motifs in interaction with wt CtBP2 and RR-CtBP2, ZEB101 region was expressed as a GST-ZEB101 fusion protein. Mutants of GST-ZEB101 were constructed as shown in Fig. 4A, to incorporate mutations in the PLDLS-like motifs. The DLm2 mutant contains DL→AS mutations in the central PLDLS motif, the PLm1/3 mutant contains PL→AS mutations in the two PLNLS motifs, and the PLm1-3 mutant contains mutations in all three motifs.

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.

Show MeSH