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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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Transcriptional repression by CtBP2 and mutants and competition between HDAC and ZEB for RR-CtBP2 binding. A, Repression of E-cad promoter by CtBP2 and mutants. CtBP1/2 double knock-out cell line MEF90 cells were co-transfected with pE-cad-Luc, phRL-tk (for internal control), and various CtBP2 constructs. Dual luciferase assay was performed as described [11]. Lysates were examined for CtBP expression by western blot with the CtBP2 antibody (lower panel). B, Interaction of wt CtBP2 and RR-CtBP2 with endogenous ZEB and HDAC2 in the presence of GFP-ZEB101. HeLa cells were co-transfected with CtBP2 and GFP-ZEB101. Cell lysates were immunoprecipitated with the Flag antibody beads and precipitated proteins examined by western blots as indicated. Both CtBP proteins and GFP fusion proteins carry an HA tag and are recognized by the HA antibody. C, Competition between GFP-ZEB101 and HDAC2 for binding to RR-CtBP2. HeLa cells were co-transfected with CtBP2 and increasing amounts of GFP-ZEB101. Co-IP and western blot analyses were performed as in B.
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Figure 3: Transcriptional repression by CtBP2 and mutants and competition between HDAC and ZEB for RR-CtBP2 binding. A, Repression of E-cad promoter by CtBP2 and mutants. CtBP1/2 double knock-out cell line MEF90 cells were co-transfected with pE-cad-Luc, phRL-tk (for internal control), and various CtBP2 constructs. Dual luciferase assay was performed as described [11]. Lysates were examined for CtBP expression by western blot with the CtBP2 antibody (lower panel). B, Interaction of wt CtBP2 and RR-CtBP2 with endogenous ZEB and HDAC2 in the presence of GFP-ZEB101. HeLa cells were co-transfected with CtBP2 and GFP-ZEB101. Cell lysates were immunoprecipitated with the Flag antibody beads and precipitated proteins examined by western blots as indicated. Both CtBP proteins and GFP fusion proteins carry an HA tag and are recognized by the HA antibody. C, Competition between GFP-ZEB101 and HDAC2 for binding to RR-CtBP2. HeLa cells were co-transfected with CtBP2 and increasing amounts of GFP-ZEB101. Co-IP and western blot analyses were performed as in B.

Mentions: Transcriptional repression by CtBP1 and CtBP2 requires CtBP interaction with both DNA-targeting transcription repressors, such as ZEB [5,27-29], and enzymes involved in chromatin modification/remodeling, such as HDAC1/2 [23]. Since both RR-CtBP2 and GG-CtBP2 interacted with ZEB well (Fig. 2A), we examined the ability of these CtBP2 mutants to repress the E-cad promoter. Although several different repressors have been implicated in repression of the E-cad promoter, ZEB appears to play a dominant role [5,11,23]. CtBP2 wt or its mutants were co-transfected with the E-cad-Luc reporter into the CtBP1/2 double knock-out cell line, MEF90. Luciferase assay showed that GG-CtBP2 retained significant repression activity, while RR-CtBP2 had no repression activity (Fig. 3A). Thus, the ability of RR-CtBP2 to interact with ZEB (Fig. 2A) appears to be insufficient for repression of the E-cad promoter.


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)

Transcriptional repression by CtBP2 and mutants and competition between HDAC and ZEB for RR-CtBP2 binding. A, Repression of E-cad promoter by CtBP2 and mutants. CtBP1/2 double knock-out cell line MEF90 cells were co-transfected with pE-cad-Luc, phRL-tk (for internal control), and various CtBP2 constructs. Dual luciferase assay was performed as described [11]. Lysates were examined for CtBP expression by western blot with the CtBP2 antibody (lower panel). B, Interaction of wt CtBP2 and RR-CtBP2 with endogenous ZEB and HDAC2 in the presence of GFP-ZEB101. HeLa cells were co-transfected with CtBP2 and GFP-ZEB101. Cell lysates were immunoprecipitated with the Flag antibody beads and precipitated proteins examined by western blots as indicated. Both CtBP proteins and GFP fusion proteins carry an HA tag and are recognized by the HA antibody. C, Competition between GFP-ZEB101 and HDAC2 for binding to RR-CtBP2. HeLa cells were co-transfected with CtBP2 and increasing amounts of GFP-ZEB101. Co-IP and western blot analyses were performed as in B.
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Figure 3: Transcriptional repression by CtBP2 and mutants and competition between HDAC and ZEB for RR-CtBP2 binding. A, Repression of E-cad promoter by CtBP2 and mutants. CtBP1/2 double knock-out cell line MEF90 cells were co-transfected with pE-cad-Luc, phRL-tk (for internal control), and various CtBP2 constructs. Dual luciferase assay was performed as described [11]. Lysates were examined for CtBP expression by western blot with the CtBP2 antibody (lower panel). B, Interaction of wt CtBP2 and RR-CtBP2 with endogenous ZEB and HDAC2 in the presence of GFP-ZEB101. HeLa cells were co-transfected with CtBP2 and GFP-ZEB101. Cell lysates were immunoprecipitated with the Flag antibody beads and precipitated proteins examined by western blots as indicated. Both CtBP proteins and GFP fusion proteins carry an HA tag and are recognized by the HA antibody. C, Competition between GFP-ZEB101 and HDAC2 for binding to RR-CtBP2. HeLa cells were co-transfected with CtBP2 and increasing amounts of GFP-ZEB101. Co-IP and western blot analyses were performed as in B.
Mentions: Transcriptional repression by CtBP1 and CtBP2 requires CtBP interaction with both DNA-targeting transcription repressors, such as ZEB [5,27-29], and enzymes involved in chromatin modification/remodeling, such as HDAC1/2 [23]. Since both RR-CtBP2 and GG-CtBP2 interacted with ZEB well (Fig. 2A), we examined the ability of these CtBP2 mutants to repress the E-cad promoter. Although several different repressors have been implicated in repression of the E-cad promoter, ZEB appears to play a dominant role [5,11,23]. CtBP2 wt or its mutants were co-transfected with the E-cad-Luc reporter into the CtBP1/2 double knock-out cell line, MEF90. Luciferase assay showed that GG-CtBP2 retained significant repression activity, while RR-CtBP2 had no repression activity (Fig. 3A). Thus, the ability of RR-CtBP2 to interact with ZEB (Fig. 2A) appears to be insufficient for repression of the E-cad promoter.

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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