| Nucleic Acids Research | Pages |
The corepressor N-CoR and its variants RIP13a and RIP13[Delta]1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70
Introduction
Materials And Methods
Primer sequences
Plasmids
Mammalian two hybrid assay
CAT assays
In vitro binding assays
Results
In vivo and in vitro interaction assays demonstrate that the repression and receptor interaction domains of N-CoR and the RIP13 variants interact specifically with TAFII32
N-CoR, TAFII32 and TFIIB can simultaneously interact with each other: the repression and receptor interaction domains of N-CoR interact specifically with TFIIB
N-CoR can simultaneously interact with TFIIB, TAFII32 and TAFII70
N-CoR, TAFII32 and TFIIB interact in a non-competitive manner
The RVR-corepressor complex also interacts with the general transcriptional machinery, and the physical association of TFIIB with N-CoR occurs in the presence of Sin3B and HDAc-1
The N-CoR repression domain inhibits the functionalinteraction between TFIIB and TAFII32 in vivo
Discussion
Acknowledgements
References
The corepressor N-CoR and its variants RIP13a and RIP13[Delta]1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70
ABSTRACT
INTRODUCTION
Cofactors function as bridges between DNA binding proteins and the basal transcriptional machinery. The identification ofcorepressors (i.e. N-CoR and SMRT) that interact with the thyroid hormone, retinoic acid receptors and vitamin D receptors (TR, RAR and VDR), has shed some light on the mechanism of repression (by classical nuclear receptors) in the absence of ligand (1-3). The C-terminal Receptor Interaction Domain (RID) of the corepressors interacts with the Ligand Binding Domain (LBD/DE) region of unliganded receptors. This interaction induces a series of protein-protein interactions that repress transcription. The corepressors contain two interaction domains that interact independently and synergistically with the nuclear receptors (4,5). These corepressors interact with Sin3 and recruit the histone deacetylases (HDAc-1 or RPD 3) that lead to hypo-acetylation of the histones. This de-acetylation leads to conformational changes that stabilise the nucleosome structure, and limit the accessibility of the chromatin to the transcriptional machinery (6-8). The N-CoR/Sin3/HDAc complex mediates transcriptional repression from a wide variety of other non-receptor mediated pathways including the Mad/Mxi mediated repression of myc activities and tumour suppresion, and the oncoproteins PLZF-RAR[alpha] (9) and LAZ3/BCL6 (10) that are involved in non-hodgkin lymphomas and acute promyelocytic leukemia, respectively. These receptor and non-receptor mediated pathways share common attributes; they are converted in response to environmental stimuli from the repressive to the operative condition and function in the management of differentiation and cell division.
N-CoR and the splice variants (RIP13a and RIP13[Delta]1) mediate transcriptional repression by the orphan nuclear receptors, Rev-erbA (5,11), RVR (5,11) and COUP-TF II (12). The C-terminal regions of N-CoR and RIP13 that encode the RIDs are almost identical. There are two distinct differences between RIP13a and N-CoR (4 and Fig.
Figure 1. Schematic presentation of regions and domains in N-CoR, RIP13a and RIP13[Delta]1 used in the mammalian two hybrid assay.The shaded regions indicated the position of ID-I and ID-II in the C-terminal RID of N-CoR and its variants, RIP13a and RIP13[Delta]1. Striped areas in the N-termini of RIP13 and RIP13[Delta]1 represent unique N-terminal regions. There are two distinct differences between RIP13a and N-CoR. The first 1016 amino acids of N-CoR that encode that encode repression domains I and II, are replaced by 10 unique amino acids at the N-terminal end of RIP13a. However, RIP13a contains seven copies of a repeated motif, G-s-l-s/t-q-G-t-P, that associated with repressor activity in SMRT. Another difference is the replacement of 48 amino acids of N-CoR (amino acids 1235-1282) with a serine in RIP13a (amino acid 228). There are several minor amino acid changes described in detail in Seol et al. (4). The N-CoR and RIP13a RIDs are completely identical. Specifically, ID-I corresponded to the-region between amino acids 2218 and 2451 of N-CoR (1), and to amino acids 1164-1397 in RIP13a (ID-I in N-CoR, RIP13a and RIP13[Delta]1 are identical). ID-II corresponded to the region between amino acids 1848 and 2163 of N-CoR and to amino acids 794 and 1109 in RIP13a (ID-II in N-CoR and RIP13a are identical). ID-II[Delta]1 from RIP13[Delta]1 has an internal deletion of 120 amino acids, that lacks amino acids 805-925 from the RIP13a ID-II. Regions of proteins containing the RIDs of N-CoR/RIP13a and RIP13[Delta]1 and the N-terminal repression domains used in the mammalian two hybrid and GST pulldown assays that were linked to GAL4, VP16 or pSG5 are shown in black. Amino acids in the ID-II[Delta]1 and ID-I+II[Delta]1 constructs refer to the corresponding amino acids in RIP13[Delta]1. The C-terminal N-CoR and RIP13 RIDs are composed of interaction domains (IDs) I and II that function synergistically and can also independently interact with some nuclear receptors, albeit weakly (5,11,12). Although the RIDs from N-CoR and RIP13a are absolutely identical, the RIP13[Delta]1-RID has an internal deletion in ID II (see Fig. The corepressor complexes that mediate repression of transcription by the nuclear hormone receptors have been characterised in terms of the proteins that regulate chromatin architecture (6-8). Furthermore, notable progress in elucidating the central role of the corepressor complex (N-CoR/Sin3/HDAc) and protein-protein interactions involved in oncoprotein mediated transcriptional repression has been observed (9,10). However, the components of the basal transcriptional machinery involved/targeted in the repression of gene expression by the nuclear receptors and oncoproteins that interact with N-CoR and SMRT remains obscure. Clues to the involvement of the basal transcriptional machinery in N-CoR mediated inhibition may be gleaned from the fact that N-CoR is involved in the repression of GAL4VP16 mediated transactivation by the Rev-erb E-region, that involves a minimal region (~35 amino acids) that spans the ligand binding domain (LBD)-specific signature motif (F/WAKXXXXFXXLXXXDQXXLL), helix 3, loop 3-4, helix 4 and helix 5 of the Rev-erb family of orphan nuclear receptors (13-15). VP16 mediated trans-activation involves the direct interaction of TAFII32 (or its Drosophila homologue-TAFII40) and TFIIB with VP16 (16-18). Concurrently there is direct protein-protein interaction between TFIIB and TAFII32. Antibodies directed against TAFII32 block GAL4VP16 mediated trans-activation, suggesting that TAFII32 is a critical coactivator in the process of transcription (16-19). This is an obvious starting point in the search for targets of N-CoR mediated repression of transcription, since these factors are central and essential components of the transcription initiation complex. TAFII32 recruits TFIIB (16-18) and interacts very efficiently with TAFII70 (20,21) that efficiently binds TAFII250 in the TFIID complex during transcriptional activation (21). TAFII250 is histone acetyltransferase (22) and obviously any mechanism that affects a pathway of histone acetylation is an ideal target/pathway for corepressor action that leads to net deacteylation (16-26). The present study utilised mammalian two hybrid and direct in vitro binding assays to characterise the specific regions in N-CoR and its variants, RIP13a and RIP13[Delta]1, that interact with key targets in the process of transcriptional initiation. These experiments demonstrated that the N-terminal repressor region and the C-terminal receptor interaction domains of N-CoR made direct contact with three key components of the transcription initiation complex, TFIIB, TAFII32 and TAFII70. These studies suggested that N-CoR represses transcription via a mechanism that involves direct non-competitive interaction with the TAFs and TFIIB, and regulates the crucial interaction between TAFII32 and TFIIB. Interestingly, these are key components of the initiation process and a major rate limiting step in the initiation process. We suggest that the corepressor interactions with the basal transcriptional factors freezes them into a non-functional state or conformation that is not permissive to basal transcription.
MATERIALS AND METHODS
Primer sequences
| GMUQ296 | 5[prime]-GCGAATTCACCATGGTNAAA/GA/TCNAAG/AAAG/ACA-3[prime] |
| GMUQ297 | 5[prime]-GCGAATTCACCNCA/TA/GTCNG/CA/TNAA/GNGTT/CTCG/ATAT/CTG-3[prime] |
| GMUQ332 | 5[prime]-GCGGTCGACTAACCATGGCTGAGGAGAAGAAGCTG-3[prime] |
| GMUQ333 | 5[prime]-GCGGTCGACTTCACGGAGCAGGCTGAGGGG-3[prime] |
| GMUQ340 | 5[prime]-GCGGAATTCACCATGTCAAGTTCAGGTTATCCT-3[prime] |
| GMUQ341 | 5[prime]-GCGGAATTCCCACTCCCTGTTTGGACT-3[prime] |
| GMUQ390 | 5[prime]-GCGGAATTCACCATGTGTGCTTCCTGCTCT-3[prime] |
Plasmids
The expression plasmids pGAL0 (27), pNLVP16 (28) and pG5E1bCAT (29). pGAL0 contains the GAL4-DBD and pNLVP16 contains the acidic activation domain of VP16. All PCR amplifications were performed with Pfu DNA (Stratagene) or Pwo DNA polymerase (Boehringer Mannheim) according to the manufacturer's instructions. End-filling reactions were performed with Klenow DNA polymerase according to the manufacturer's instructions. All pBluescript and pBS clones and GAL and VP16 chimeras were sequenced by double stranded sequencing to verify identity and confirm the reading frame.
Full length RIP13a was amplified from pCDM8-RIP13a (4) using the two primers GMUQ390 and GMUQ297. The product was cleaved with EcoRI and ligated to pGAL0/EcoRI and pSG5/EcoRI to create GAL4-Rip13a and pSG5-Rip13a, respectively. To construct pSG5-RIP13[Delta]1, GAL4-RIP13[Delta]1 was cleaved with EcoRI and the resulting fragment was ligated to pSG5/EcoRI. To construct pSG5-N-CoR/RIP13a-RID, the RID was excised from GAL4-ID-I+II (14) with EcoRI and the resulting fragment was ligated to pSG5/EcoRI. N-CoR amino acids 1-1017 was amplified with Pwo DNA polymerase from GAL4-NCoR using the two primers GMUQ340 and GMUQ341. The product was cleaved with EcoRI and the resulting fragment was ligated to pGAL0/EcoRI and pSG5/EcoRI to form pGAL4-N-CoR amino acid 1-1017 and pSG5-N-CoR amino acids 1-1017, respectively. RIP13[Delta]1 amino acids 1-627 was excised from pSG5-RIP13[Delta]1 with EcoRI/NcoI and the resulting 1.8 kb fragment was end-filled and ligated to end-filled pNLVP16/NdeI to create VP16-RIP13[Delta]1 amino acids 1-627. VP16-R[Igr]P13[Delta]1 amino acids 1-627 was cleaved with SalI/XbaI and the resulting fragment was ligated to pGAL0/SalI/XbaI.
hTAFII-32 was cut from pBS-KS+-hTAF32 (an unpublished clone from the Tjian laboratory) with NdeI/EcoRI, end-filled and ligated to end-filled pNLVP16/XhoI. hTAFII-70 was amplified with Pfu DNA polymerase from phTAFII-70 (an unpublished clone from the Tjian laboratory) using the primers GMUQ332 and GMUQ333 and was ligated to pBluescript KS/EcoRV. Antisense pBluescript-TAFII-70 was cleaved with SalI and the resulting fragment was inserted into pGAL0/SalI and pNLVP16/SalI to create pGAL4-TAFII-70 and VP16-TAFII-70, respectively. TFIIB was excised from pGEX-KT-TFIIB with EcoRI end-filled and ligated to end-filled pGAL0/SalI or end-filled Vp16/SalI create pGAL4-TFIIB or VP16-TFIIB, respectively. GAL4-TBP and VP16-TBP were constructed by cleaving pT[beta]-hTBP (an unpublished clone from the Tjian laboratory) with NdeI and ligating the resulting fragment into pGAL0/NdeI and pNLVP16/NdeI, respectively. All other plasmids and primers have been described previously (5,11-15).
Mammalian two hybrid assay
Each well of a six well plate of JEG-3 cells (60-70% confluence) was co-transfected with 3 µg pG5E1bCAT reporter, 1 µg GAL chimeras and 1 µg VP16 chimeras in 1 ml of Dulbecco's Modified Eagle's Medium (DMEM) containing 5% charcoal-stripped foetal calf serum (FCS) by the DOTAP (Boehringer Mannheim) mediated procedure as described previously (30,31). After 24 h, the medium was replaced and cells were harvested for the assay of CAT activity 36-48 h after transfection. Each transfection was performed at least three times to overcome the variability inherent in transfections.
CAT assays
Cells were harvested and CAT activity measured as described previously (32). Aliquots of cell extracts were incubated at 37°C, with 0.1-0.4 µCi of 14C-chloramphenicol (ICN) in the presence of 5 mM acetyl CoA in 0.25 M Tris-HCl, pH 7.8. After a 1-4 h incubation period, 1 ml ethyl acetate was used to extract the chloramphenicol and its acetylated forms. Extracted materials were analysed on Silica gel thin layer chromatography plates as described previously (32). Quantitation of all CAT assays was performed with an AMBIS [beta]-scanner.
In vitro binding assays
GST and GST-fusion proteins were expressed in Escherichia coli (BL21) and purified using glutathione agarose affinity chromato-graphy as described previously (31). The GST-fusion proteins were analysed on 10% SDS-PAGE for integrity and to normalise the amount of each protein. The Promega TNT-coupled transcription-translation system was used to produce 35S-methionine labelled N-CoR, TFIIB, TAFII32 and TAFII70 proteins that were visualised by SDS-PAGE. In vitro binding assays were performed with glutathione agarose beads (Sigma) coated with ~500 ng of GST-fusion protein and 2-30 µl of 35S-methionine-labelled protein in 200 µl of binding buffer containing 100 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.5% Nonidet P-40, 5 µg ethidium bromide and 100 µg BSA. The reaction was allowed to proceed for 1-2 h at 4°C with rocking. The affinity beads were then collected by centrifugation and washed five times with 1 ml of binding buffer without ethidium bromide and BSA. The beads were resuspended in 20 µl SDS-PAGE sample buffer and boiled for 5 min. The eluted proteins were fractionated by SDS-PAGE, the gel was treated with Amersham Amplify fluor, dried at 70°C and autoradiographed (11).
RESULTS
In vivo and in vitro interaction assays demonstrate that the repression and receptor interaction domains of N-CoR and the RIP13 variants interact specifically with TAFII32
In an initial investigation to address whether the corepressors interact with the basal transcriptional machinery that is required for initiation, we examined the ability of the corepressors to interact with TFIIB, TAFII32 and TAFII70 in vivo and in vitro. We tested these components of the basal transcriptional machinery since N-CoR mediates the repression of GAL4VP16 mediated transactivation by the orphan nuclear receptors, and it has been demonstrated that the VP16 acidic activation domain interacts directly with (i) TFIIB and TBP and (ii) TAFII32 and TAFII70. Furthermore, TAFII32 is recruited into the TFIID complex by TAFII70 (16-19,25,26).
Recruitment of TAFII32 is critical to the initiation of transcription. Hence, we examined the ability of the N-CoR/RIP13a and RIP13[Delta]1-RIDs (Fig.
Figure 2. TAFII32 interacts with the N-terminal and C-terminal regions of N-CoR and its variants: analysis of the interaction of the N-CoR and its variants with TAFII32 by the mammalian two hybrid assay. JEG-3 cells were co-transfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± standard deviation and were derived from three independent transfections. VP16 or the indicated VP16-TAFII32 (+) were co-transfected with GAL4 chimeras of (A) the receptor interaction domains (RIDs) and N-terminal regions of N-CoR and the RIP13 variants and (B) the indicated GAL4 chimeras of the independent interaction domains I and II were co-transfected with VP16 or VP16-TAFII32. Fold activation is expressed relative to CAT activity obtained after co-transfection of GAL4 DBD and the VP16 vector alone arbitrarily set to 1.0 (A and B). As discussed earlier, the N-CoR/RIP13a and RIP13[Delta]1 RIDs are each composed of two interaction domains (I and II) that can independently interact with some nuclear receptors, albeit weakly. ID-I corresponded to the region between amino acids 2218 and 2451 of N-CoR (1), and to amino acids 1164-1397 in RIP13a (ID-I in N-CoR, RIP13a and RIP13[Delta]1 are identical). ID-II corresponded to the region between amino acids 1848 and 2163 of N-CoR and to amino acids 794 and 1109 in RIP13a (ID-II in N-CoR and RIP13a are identical). ID-II[Delta]1 from RIP13[Delta]1 has an internal deletion of 120 amino acids, that lacks amino acids 805-925 from the RIP13a ID-II. We therefore examined the ability of ID-I and ID-II and ID-II[Delta]1 from N-CoR, RIP13a and R[Igr]P13[Delta]1 to independently interact with TAFII32. As demonstrated above, both RIDs interacted very efficiently with TAFII32. ID-I poorly interacted with TAFII32 (<2-fold); however, ID-II very strongly interacted with TAFII32 (~130-fold). In contrast, ID-II[Delta]1 weakly interacted with TAFII32 (~10-fold) (Fig. The demonstration of interaction between TAFII32 and the corepressors in the in vivo mammalian two hybrid assay strongly suggests these proteins may interact by a direct mechanism. However, this does not eliminate the possibility of an indirect mechanism in which additional factor(s) mediate the interaction. We tested this hypothesis using a biochemical approach, the in vitro GST pulldown assay. Glutathione agarose immobilised GST-RIP13[Delta]1-RID and the GST-N-CoR/RIP13a-RID were tested for direct interaction with in vitro 35S-radiolabelled full length native TAFII32. GST-RIP13[Delta]1- and N-CoR/RIP13a-RIDs both showed a direct interaction with full length TAFII32 (Fig. Figure 3. N-CoR directly interacts with TAFII32 invitro. Interaction of TAFII32 and corepressors in vitro. (A) TAFII32 was radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone, GST-RIP13[Delta]1-RID and GST-N-CoR/RIP13a-RID. Inputs of the radiolabelled 35S-methionine protein are also shown. (B) N-CoR, N-CoR amino acids 1-1017 and the N-CoR/RIP13a-RID were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TAFII32. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. In conclusion, these in vivo and in vitro studies demonstrate that the repression region and receptor interaction domain of the corepressors are involved in the interaction with TAFII32. There is a quantitative (but not qualitative) discrepancy between the in vivo and in vitro assays with respect to the strength of the interactions between TAFII32, and the repression and RID regions of N-CoR. Whether this is reflects the true situation, or assay anomalies, has not been resolved.
N-CoR, TAFII32 and TFIIB can simultaneously interact with each other: the repression and receptor interaction domains of N-CoR interact specifically with TFIIB
TAFII32 interacts with TAFII70 and TFIIB during the formation of the transcriptional initiation complex (18,23), which we verified by the in vivo mammalian two hybrid assay and in vitro GST-pulldown assay (Fig.
Figure 4. N-CoR directly interacts with TFIIB and TAFII32. Analysis of the interaction of the N-CoR family of co-repressors with factors known to be involved in transcriptional activation by the mammalian two hybrid assay. JEG-3 cells were co-transfected with the indicated plasmids (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Results shown are mean ± standard deviation and were derived from three independent transfections. (A) Interaction of factors known to be involved in transcriptional activation with TAFII32. GAL4-DBD or GAL4-TAFII70-TFIIB or -TBP were co-transfected with VP16 or the VP16-TAFII-32 chimera. (B) TAFII70 and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TAFII32. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes in all gels contain ~10% input. (C)N-CoR and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TAFII32. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes in all gels contain ~10% input. (D) N-CoR and TAFII32 were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TFIIB. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. We examined the ability of glutathione agarose immobilised GST-TAFII32 and GST-TFIIB for direct interaction with in vitro 35S-radiolabelled full length native N-CoR and TFIIB or TAFII32, respectively (Fig. We then examined the ability of in vitro 35S-radiolabelled full length/native N-CoR, the repression region (amino acids 1-1017) and the receptor interaction domains to interact with the immobilised GST-TFIIB fusion protein. Full length N-CoR very efficiently interacted with TFIIB in vitro. The amino acids between 1 and 1017 encoding the repression domain also interacted very efficiently with TFIIB (Fig. Figure 5. TFIIB interacts with the N-terminal and C-terminal regions of N-CoR: interaction of TFIIB and corepressors in vitro. (A) N-CoR, N-CoR amino acids 1-1017 and the N-CoR/RIP13a-RID were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TFIIB. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. (B) TAFII32 and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-N-CoR/RIP13a-RID. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. These studies confirmed the interaction between N-CoR and TFIIB and suggested that the corepressor, TFIIB and TAFII32 were directly involved in the repression of transcription.
N-CoR can simultaneously interact with TFIIB, TAFII32 and TAFII70
Since N-CoR had been independently shown to interact with TAFII32 and TFIIB, and that TAFII32 could directly interact with TFIIB and TAFII70, we investigated whether N-CoR could simultaneously interact with TAFII32 and TAFII70 in the GST-pulldown assay. Hence, we examined the ability of glutathione agarose immobilised GST-N-CoR/RIP13a-RID to interact with a mixture of in vitro 35S-radiolabelled full length native TAFII32 and TFIIB, and TAFII32 and TAFII70 (Fig.
Figure 6. N-CoR directly interacts with TAFII32, TAFII70 and TFIIB. (A) Interaction of TFIIB, TAFs 32 and 70 with N-CoR in vitro. TAFII32, TAFII70 and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-N-CoR/RIP13a-RID. (B) N-CoR, N-CoR amino acids 1-1017, TAFII70 and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for simultaneous interaction with GST-alone, and GST-TAFII32. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. Since TAFII32 had been independently shown to interact with N-CoR, TFIIB and TAFII70, we investigated whether these proteins could simultaneously interact with TAFII32 in the GST-pulldown assay. The previous experiments had demonstrated that TAFII32 could independently interact with all these proteins that were part of the basal transcription machinery. Hence, we examined the ability of glutathione agarose immobilised GST-TAFII32 to interact with a mixture of in vitro 35S-radiolabelled full length native N-CoR (or the repression domain, amino acids 1-1017), TFIIB and TAFII70 (Fig. These studies added weight to the suggestion that N-CoR directly interacted with TFIIB and two TAFs that are critical components of the transcriptional initiation process. Interestingly, the TAFs normally function as essential coactivators, but may have been targeted by the corepressor to be frozen in a non-functional state during transcriptional repression.
N-CoR, TAFII32 and TFIIB interact in a non-competitive manner
We then embarked on experiments designed to analyse whether competitive binding between these interacting proteins was an issue with respect to the formation of a ternary complex. We examined the ability of increasing amounts of N-CoR (1, 2 and 4 µl) to affect the efficiency of interaction between GST-TAFII32 and radiolabelled TFIIB (Fig.
Figure 7. N-CoR, TAFII32 and TFIIB interact in a non-competitive manner. (A) N-CoR and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TAFII32. We examined the effect of increasing amounts of N-CoR (1, 2 and 4 µl) on the efficiency of the interaction between GST-TAFII32 and TFIIB. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. (B) N-CoR and TAFII32 were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-TFIIB. We examined the affect of increasing amounts of N-CoR (1, 2 and 4 µl) on the efficiency of the interaction between GST-TFIIB and TAFII32. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~10% input. Based on these observations and the previous experiments, we suggest a structure/model involving multiple contacts among these components that secures them in a non-productive complex and/or blocks the functional assembly of an initiation complex.
The RVR-corepressor complex also interacts with the general transcriptional machinery, and the physical association of TFIIB with N-CoR occurs in the presence of Sin3B and HDAc-1
We subsequently investigated whether the corepressor, N-CoR, could still interact with TFIIB and the TAFs when it was bound or anchored to a nuclear receptor. Since we had demonstrated that N-CoR could interact with TFIIB, TAFII32 and TAFII70, we investigated whether N-CoR could maintain these contacts to the generalized transcription machinery when bound to the orphan nuclear receptor, RVR, in the GST-pulldown assay. Hence, we examined the ability of glutathione agarose immobilised GST-RVR to interact with a mixture of in vitro 35S-radiolabelled full length native N-CoR, TFIIB, TAFII32 and TAFII70 (Fig.
Repression of transcription by the nuclear receptors and the Max associated Mad/Mxi1 family involves the formation of multiprotein complexes that are comprised of N-CoR/SMRT, Sin3A/B and the histone deacteylases. We decided to examine whether the corepressor, N-CoR, anchored to the orphan nuclear receptor, RVR/Rev-erb[beta], could interact with (i) Sin 3B, that functions to recruit proteins that condense chromatin structure and (ii) HDAc-1, a histone deacteylase that is involved in nucleosome condensation, in the presence of TFIIB. Hence, we investigated the ability of glutathione agarose immobilised RVR to interact with N-CoR, TFIIB, Sin3B and HDAc-1. We observed that Sin3B and HDAc-1 could still bind to N-CoR, in the presence of TFIIB (Fig.
These studies suggested that repression of transcription by the orphan nuclear receptor RVR/Rev-erb[beta] and the the corepressor N-CoR probably involves intimate contacts with the generalised transcriptional machinery and the proteins (Sin3 and HDAc-1) involved in nucleosomal condensation.
The N-CoR repression domain inhibits the functionalinteraction between TFIIB and TAFII32 in vivo
To further understand the consequence of these interactions between the corepressors and the general transcription factors in vivo, we investigated the effect of the expression of the corepressor receptor interaction domain and repressor region on the ability of TAFII32 to interact with TFIIB in the in vivo mammalian two hybrid assays. As observed in Figure
Figure 8. RVR forms a complex with N-CoR, the general transcriptional machinery and the Sin3B-HDAc-1 complex. N-CoR, TAFII70, TAFII32, Sin3B and HDAc-1 and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-RVR (Rev-erb[beta]) in vitro. (A) N-CoR, TFIIB and the TAFs were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-RVR. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~5-10% input. (B) N-CoR, Sin3B, HDAc-1 and TFIIB were radiolabelled with 35S-methionine by in vitro transcription/translation and tested for interaction with GST-alone and GST-RVR. Inputs of the radiolabelled 35S-methionine protein are also shown. The input lanes contain ~5-10% input. Figure 9. The N-CoR repression domain inhibits the functionalinteraction between TFIIB and TAFII32 in vivo. (A) JEG-3 cells were co-transfected with GAL4-TFIIB (1 µg) and VP16-TAFII-32 (1 µg) and with the indicated pSG5 plasmids (1 µg) (+) in the presence of pG5E1bCAT reporter plasmid and assayed for CAT activity. Percent conversion of 14C-chloramphenicol to its acetylated forms was calculated. Results shown are mean ± standard deviation and were derived from three independent transfections. (B) A cartoon that highlights the dual role of N-CoR in the repression of gene expression. N-CoR is involved in mediating nucleosome condensation and inhibiting the basal transcriptional machinery. This suggested that N-CoR binding to TAFII32 and TFIIB may repress/regulate transcription by controlling the functional/productive interaction between these key interactions. This interaction is central to the formation of the transcriptional initiation complex. TAFII32 normally interacts with activators/coactivators to mediate the signal to the basal transcription machinery. The targeting of TAFII32 indicates that repressors/corepressor(s) may function by recruiting the TAFs and TFIIB in a non-productive manner that blocks the subsequent formation of an active transcription complex.
DISCUSSION
Sequence specific binding proteins regulate transcription by protein-protein interactions with the basal transcriptional machinery, which includes TFIID a complex containing TBP and the TBP-associated factors (TAFs) that function as co-activators. Site specific regulatory protein interactions with the TFIID multiprotein complex have been proposed to function in the productive recruitment of TFIIB and/or a conformational change in the TFIID/TFIIB promoter-protein complex that facilitates the binding of other components of the transcription complex (19,21).
Nuclear receptor mediated repression of transcription is mediated by the cofactor(s) N-CoR, SMRT and RIP13, that function as corepressors (1-4). Here we present in vivo and in vitro studies demonstrating that N-CoR directly interacts with TAFII32, TAFII70 and TFIIB. Our results suggest that a model for N-CoR mediated repression must explain interactions between these components of the basal transcriptional machinery and the corepressor. Although we have demonstrated that N-CoR can directly interact with these central components of the basal transcription apparatus and that the corepressor can simultaneously pulldown TFIIB, TAFII32 and TAFII70, the presence of a ternary complex has not been identified directly. However, we have analysed the distinct interactions of each pair of potential partners. Furthermore, our investigation did not identify any indication for competitive binding between these components of the inhibitory complex. Hence we propose a structure/model involving multiple contacts among these components that locks them in a non-productive network and/or blocks the functional assembly of an initiation complex. This suggestion is supported by our observation that N-CoR inhibits the functional interaction or the transmission of the signal between TFIIB and TAFII32 in the mammalian two hybrid assay, even in the presence of VP16.
Analogously, Tjian and colleagues (16) demonstrated VP16 mediated transactivation involves direct non-competitive inter-actions between VP16, TFIIB and TAFII32/40 resulting in a conformational change in the TFIID/TFIIB complex that is permissive for complex assembly and subsequent initiation.
Interestingly, our study suggested that the N-terminal region of N-CoR encoding the repression domains very efficiently interacted with TFIIB; this is in agreement with the observations from Privalsky and colleagues who have shown that TFIIB interacted in a functional and efficient manner with the silencing domains in SMRT (M.Privalsky, personal communication; 39).
Many studies have demonstrated that TAFII32/40 and TAFII60/70 directly interact with each other, bind TBP and are key mediators in the transmittance of the signals from the activators and the initiation complex (16-19). Furthermore, TAFII32/40 functions in TFIIB recruitment (16-19) and TAFII60/70 interacts with TAFII230/250 (20,23), a histone acetyltransferase that is involved in the transcriptional access to chromatin. The process of transcriptional initiation and activation has been viewed in two alternate models; one suggests that activators induce the step-wise formation of a multiprotein complex, whereas the alternate idea is that the complex is preassembled and that the activator recruits the complex to the DNA and produces functional conformational changes (19). Analysis has been complicated by data that suggests the components are involved in preinitiation/basal transcription and activation. These data for example have shown that TFIIB mutants are competent in basal transcription, but do not function in activation; furthermore, antibodies to TAFII32/40 inhibit activation but not basal transcription (16,17 and references therein). Our studies in this context suggest that N-CoR forms a complex with TAFII32, TAFII70 and TFIIB that is non-functional and may prevent the incorporation of these components into the initiation complex and/or produce conformational changes that are detrimental to transcription. The interaction of N-CoR with TAFII70 is very interesting; TAFII70 strongly binds TAFII250, a histone acetyltransferase. These observations, in light of the corepressor interactions with Sin3 and the histone deacetylases, suggest that the corepressor multiprotein complex may actively induce deacetylation and passively repress acetylation to assure minimum leakage. This hypothesis is supported by our observation that the orphan nuclear receptor/corepressor complex (RVR-N-CoR) also interacts with TFIIB and the TAFs, and that the physical association of TFIIB with N-CoR can occur in the context of interactions with Sin3B and HDAc-1 (Fig.
The strong interactions of the corepressor with TFIIB correlate with the many reports of nuclear receptor interactions with TFIIB, although these reports have been very contradictory spanning the entire spectrum of positive and negative effects (24,33-35). Specifically, it has been reported that in the absence of ligand, the thyroid hormone receptor (TR) LBD interacts with TFIIB, and that this interaction may freeze TFIIB in a non-functional conformation that is relieved by hormone binding which facilitates the assembly of functional initiation complex. Concomitant with these events is the reduction of non-productive interactions between TR and the initiation factors (24,33-35). Another report has suggested that TFIIB interacts with the AB region of TR, and that the interaction has decreased affinity in the presence of T3, although the association is still stable (36). Many other reports have demonstrated that TFIIB mediates vitamin D and retinoid receptor dependent gene expression, via mechanisms that involve direct binding (37,38).
TFIIB appears to be a key target in the activation and repression of gene expression. Furthermore, the cofactor SRC-1 (40), a coactivator for the steroid receptor superfamily, encodes histone acetyltransferase activity, interacts with p300 and PCAF and also makes strong contacts with TFIIB (41). SRC-1 is a member of the a gene family that includes SRC-1, TIF-2/GRIP-1/SRC-2 and RAC-3/ACTR/AIBI/SRC-3 (which is amplified in breast cancer; 40 and references therein). Our studies and the other reports suggest interaction with TFIIB is a crucial rate limiting step in the positive and negative regulation of transcription, and may be a key target in the control of cell division and differentiation. Furthermore, TFIIB and the TAFs are obviously key targets in the repression of transcription during oncoprotein (PLZF-RAR[alpha], LAZ3/BCL6 and Mad/Mxi-1) mediated repression (6-10).
We conclude that N-CoR mediated repression of transcription by the nuclear receptors involves direct interactions with central components of the preinitiation complex that locks them into a non-functional state or conformation that passively leads to reduced acetylation. This process occurs in a background of active deacetylation induced by the corepressor mediated Sin3 and histone deacetylase interactions. We speculate that the resolution of the corepressor structures will provide valuable insights into the mechanics of the process and provide clues as to which other members of the initiation complex will be likely targets of corepressor action.
ACKNOWLEDGEMENTS
We thank Dr R.Tjian for the gifts of plasmids encoding human TAFII32 and TAFII70, Drs L.Alland and R.DePinho for the Sin3B expression vector and Dr C.Hassig for the HDAc-1 cDNA clone. This investigation was supported by the National Health and Medical Research Council (NHMRC) of Australia. G.E.O.M. is an NHMRC Supported Senior Research Fellow. The Centre for Molecular and Cellular Biology is the recipient of an Australia Research Council (ARC) special research grant.
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