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, 29 (6), 755-66

TBL1 and TBLR1 Phosphorylation on Regulated Gene Promoters Overcomes Dual CtBP and NCoR/SMRT Transcriptional Repression Checkpoints

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TBL1 and TBLR1 Phosphorylation on Regulated Gene Promoters Overcomes Dual CtBP and NCoR/SMRT Transcriptional Repression Checkpoints

Valentina Perissi et al. Mol Cell.

Abstract

A key strategy to achieve regulated gene expression in higher eukaryotes is to prevent illegitimate signal-independent activation by imposing robust control on the dismissal of corepressors. Here, we report that many signaling pathways, including Notch, NF-kappaB, and nuclear receptor ligands, are subjected to a dual-repression "checkpoint" based on distinct corepressor complexes. Gene activation requires the release of both CtBP1/2- and NCoR/SMRT-dependent repression, through the coordinate action of two highly related exchange factors, the transducer beta-like proteins TBL1 and TBLR1, that license ubiquitylation and degradation of CtBP1/2 and NCoR/SMRT, respectively. Intriguingly, their function and differential specificity reside in only five specific Ser/Thr phosphorylation site differences, regulated by direct phosphorylation at the level of the promoter, as exemplified by the role of PKCdelta in TBLR1-dependent dismissal of NCoR. Thus, our data reveal a strategy of dual-factor repression checkpoints, in which dedicated exchange factors serve as sensors for signal-specific dismissal of distinct corepressors, with specificity imposed by upstream signaling pathways.

Figures

Figure 1
Figure 1. TBL1 and TBLR1 are required for dismissal of distinct corepressors
(A) Single-cell nuclear microinjection of purified IgGs against either TBL1 or TBLR1 inhibited transcriptional activation of a T3RE-dependent and of an ERE-dependent LacZ reporter in Rat1 cells. Depletion of TBLR1, but not TBL1, could be rescued by downregulating NCoR and SMRT by specific siRNAs. (B) A LacZ reporter driven by a 1.2kb fragment of the pS2 promoter was used in MCF7 to screen for other corepressors removal by siRNAs. Downregulation of CtBP1and CtBP2 fully rescued estrogen-induced activation in absence of TBL1. (C) αTBL1 IgGs microinjection blocked activation of LacZ reporters driven by an ERE response element, a PPARγ-responsive fragment of the AOX3 promoter, a TR-responsive fragment of the Dio1 promoter and a 3xp65 response element. All were rescued by removal of CtBP1 and CtBP2 by specific siRNAs microinjection. (D) Analysis of GREB1 mRNA expression in transiently transfected MCF7 cells by RT-PCR at 4h after induction. Expression of the GAPDH gene was used for normalization. Validation of the siRNA used here is shown in Supplemental Figure S1.
Figure 2
Figure 2. CtBP1/2 and the NCoR complex are both recruited to ER- and Notch-target genes for repression
(A) ChIP analysis of the occupancy of the pS2 promoter upon basal growth conditions in U2OS cells showed specific recruitment of CtBP, NCoR, TBLR1 and TBL1 as indicated by qPCR performed on the chromatin immunoprecipitated with each specific antibody compared to the chromatin immunoprecipitated with non-specific IgGs. qPCR of an unrelated genomic region was used as negative control. Results illustrated are representative of three or more biological experiments. (B) Similar ChIP analysis was performed on the pS2 promoter upon estrogen stimulation in MCF7 cells. A peak of recruitment of both CtBP and NCoR could be observed 30’ after ligand stimulation, together with ligand-dependent recruitment of ERα and Rip140, followed by their rapid dismissal at 45’ (C) Single-cell nuclear microinjection of purified IgGs against TBL1 inhibited transcriptional activation of an ERE-dependent LacZ reporter upon E2 in MCF7 cells carrying the endogenous Rip140 or in cells where overexpression of wild-type Rip140 substituted for endogenous Rip140. When a mutant Rip140, unable to bind CtBP, was used to rescue for the loss of endogenous Rip140 the basal repression in absence of stimulation could not be restored and activation became TBL1-independent. (D) ChIP analysis in undifferentiated C2C12 cells (GM) showed enrichment of the Hes1 promoter in the sonicated chromatin fraction immunoprecipitated by CtBP, NCoR and HDAC3 specific antibodies. On the contrary, occupancy of the Hes1 promoter by the Trimethyl-Lys4-histone 3 mark is decreased upon induction of differentiation (DM). Corresponding expression of the Hes1 gene during differentiation is shown in Supplemental Figure S2. (E) The relative induction of Hes1 expression in C2C12 myoblasts upon NICD transient transfection was measured by RT-PCR and normalized for 18S expression. Co-transfection of specific siRNAs against TBL1 and TBLR1 showed that both are required for Notch-mediated transcriptional activation. (F) Single cell microinection assay in C2C12 cells showing that transcriptional activation of a LacZ reporter driven by the Notch-responsive Hes1 promoter requires NICD, CBF, SRC1, CBP, TBL1 and TBLR1.
Figure 3
Figure 3. TBL1 function in mediating CtBP degradation
(A) Endogenous co-immunoprecipitation of CtBP with TBL1 and Rip140 performed in estradiol-stimulated MCF-7 cells. Co-immunoprecipitation of CtBP and Rip140 was used as positive control. (B) GST pull down analysis revealing direct interaction between CtBP C’terminus and TBL1 WD40 domain. (C) Direct interaction between TBLR1 and CtBP in a GST pull down assay. In vivo interaction between CtBP and TBL1 was also observed in 293 cells, while co-immunoprecipitation between CtBP and TBLR1 could not be recorded. Immunoprecipitation of NCoR (see Supplemental Figure S3-A) was performed in parallel as positive control. (D) Immunoblot of U2OS whole cell extracts with α-CtBP antibody revealed that TBL1 downregulation by siRNA transfection stabilized CtBP protein level. (E) Immunoblot analysis of extracts from U2OS treated with 50J/cm3 UV showed that CtBP protein degradation is impaired in cells transfected with specific siRNAs against TBL1 or HIPK2. Changes in CtBP protein level is better appreciated on the lower exposure or on the slower-running bands representing post-transcriptionally modified CtBP, as confimed by specific siRNA downregulation (see Supplemental Figure S3-B). Quantification of the CtBP bands from the shorter exposed panel was done using the GeneTools software on a GeneGenius Byo Imaging System and is represented in the bar graph below. (F) Immunoblot with αFLAG of 293 cell extracts upon transient transfection with Flag-CtBP and HA-HIPK2 confirming that CtBP protein level is downregulated by HIPK2 overexpression in a TBL1-dependent manner.
Figure 4
Figure 4. TBL1X and TBLR1 are specifically regulated by phosphorylation
(A) Schematic representation of murine TBL1 and TBLR1 showing the chromosomal locations of the genes, the position of F-box and WD-40 domains in the translated proteins, the homology between TBL1 and TBLR1 and the localization of five putative phosphorylation sites that are uniquely present on each protein. (B) Immunoblot of whole cell extracts from U2OS cells showing multiple specific bands identified by αTBL1 antibody and removed by siRNA transient transfection. The higher molecular weight bands indicate phosphorylated forms of TBL1 as shown by their depletion with CIP alkaline phosphatase. (C) Immunoblot with αTBLR1 antibody comparing nuclear extracts (NE) and whole cell extracts (WCE) from U2OS cells identified higher molecular weight bands enriched in the nuclear fraction. Quantification of the relative intensity of the bands from the longer exposed blot for the NE and the shorter exposed blot for the WCE is shown below. (D) Immunoblot with α-MonoP-TBLR1 and α-TriP-TBLR1 antibodies on U2OS nuclear extracts confirmed that TBLR1 is phosphorylated in vivo in the nuclear compartment. The antibodies specificity is confirmed by TBLR1 and TBL1 siRNAs; αHDAC2 blotting is used as loading control. (E) Depletion of the immunoprecipitated phospho-TBLR1 from nuclear extracts with CIP phosphatase and treatment of cells with Rottlerin confirmed in vivo phosphorylation by PKC. (F) Immunoblot of nuclear extracts from U2OS transiently transfected with siRNAs against PKCα or PKCδ confirmed that PKCδ phosphorylates TBLR1 at Ser 123, as measured by western blot with the α-MonoP-TBLR1. Specificity of the siRNAs is confirmed by immunoblotting for PKCα and PKCδ (G) ChIP analysis of the occupancy of the pS2 promoter by ERα, Rip140, NCoR and Ser123-phospho-TBLR1 in MCF7 cells treated with estrogen for the indicated times. (H) Occupancy of the RARβ promoter in 293 cells showing transient recruitment of PKCα, PKCδ and GSK3 kinases and dismissal of NCoR 5’ after treatment with retinoic acid. (I) Immunoblot with α-MonoP-TBLR1 antibody showing enrichment of nuclear poly-ubiquitylated phospho-TBLR1 when proteasome activity is blocked by MG132. (J) Poly-ubiquitylated phospho-TBLR1 is immunoprecipitated by endogenous TBL1 and specifically depleted by TBLR1 siRNA transfection in 293 cells. The immunoprecipitation with TBL1 is increased by MG132 while no interaction between poly-ubiquitylated phosphoTBLR1 and HDAC3 is detected. Immunoprecipitation of NCoR is shown as positive control.
Figure 5
Figure 5. TBL1 and TBLR1 identities and functions are determined by five specific phosphorylation sites
(A) Inhibition of T3R, ER and RAR dependent transcriptional activity by αTBL1 or αTBLR1 was rescued by co-injecting expression vectors for either TBL1 or TBLR1, while activation could not be rescued when the specific phosphorylation sites, selectively present in the two factors, were mutated. (B) Schematic representation of the results of the experiments shown in (A) and others not shown: activation by ER, T3R and PPARγ is blocked by αTBL1 microinjection and rescued by overexpression of TBL1 wild-type but not by the mutated form. Similar results for TBLR1 rescue on ER-, T3R-, PPARγ- and RAR-mediated transcription. The results obtained are marked as positive when at least 60% of the original activity is rescued. (C) Activation of a LacZ reporter driven by the RARE response element is inhibited by TBLR1 siRNA microinjection and can be rescued by wild-type TBLR1 or phosphorylation mimicking mutants (Glu), but not by mutations deleting each of the two TBLR1-specific phosphorylation sites. (D) Selective requirement of TBLR1 for RAR-mediated transcription can be specifically changed by swapping the five specific phosphorylation sites that distinguish TBL1 and TBLR1. (E) Model. While TBLR1 specifically functions to mediate the dismissal of the NCoR/SMRT/HDAC3 corepressor complex, TBL1 function is key to promoting the ubiquitylation and degradation of the corepressor CtBP based on direct interaction. The specificity between TBL1 and TBLR1 functions is regulated upon activation via local phosphorylation, with TBLR1 being specifically phosphorylated by PKCδ at Ser123.

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