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T Kouzarides

Publications and source records attributed to T Kouzarides.

At least 73 records · Page 4Linked to original sources
PubMed1995

An inhibitor domain in c-Fos regulates activation domains containing the HOB1 motif.

The c-Fos protein has three activation modules at its C-terminus, two of which contain motifs (HOB1 and HOB2) which are also present in the activation domains of c-Jun. Here we show the existence of two additional activation modules at the N-terminus of c-Fos, one of which contains a second HOB1 motif (HOB1-N). The N-terminus also contains an inhibitor domain (ID1) which silences HOB1 activity. GAL4 fusion experiments showed that ID1 can specifically silence HOB1-containing activation domains from c-Fos or c-Jun when linked in cis, but will not affect other distinct activation domains. The c-Fos related protein, FosB, also contains an inhibitor domain. Mutagenic and deletion analyses identify an inhibitor motif (IM1) conserved between c-Fos and FosB, which is required for inhibitor function. Mutagenesis of IM1 enhances the ability of c-Fos to activate an AP1 bearing promoter. Finally, squelching experiments suggest that c-Fos ID1 binds a limiting protein involved in inhibition. These results demonstrate the existence of a new class of inhibitor domain within transcriptional activators, which acts in a sequence specific manner to inhibit a subset of activation domains.

Amino Acid Sequence↗
PubMed1995

Transcriptional control by the retinoblastoma protein.

The retinoblastoma gene product is an abundant nuclear protein whose 'pocket domain' mediates numerous protein-protein interactions. A substantial proportion of the RB-interacting proteins are transcription factors suggesting that RB plays a fundamental role in the regulation of transcription. Via these interactions, RB can influence both the progression through the cell cycle and the expression of lineage specific products. In this review I discuss some of the likely mechanisms by which RB regulates cell proliferation and differentiation.

Animals↗
PubMed1995

Functions of pRb and p53: what's the connection?

The pRb and p53 proteins have tumour suppressor functions and both are regulators of transcription. Their mechanisms of transcriptional regulation are unrelated in many ways--in contrast, it would appear, to their biological functions. This review highlights their biological connections in the light of recent advances in understanding the mechanisms of pRb and p53 function.

Journal Article↗
PubMed1995

Regulation of transcription by E2F1/DP1.

The E2F1 transcription factor, in co-operation with DP1, controls the expression of several S-phase specific genes. This activity is most likely responsible for the oncogenic and S-phase inducing properties of E2F1, suggesting that this transcription factor plays a key role in regulating the cell cycle. The transcriptional activation functions of E2F1 are resident in a small C-terminal domain which can independently activate transcription. Here we review the protein-protein interactions which impinge upon and regulate this activation domain and put forward some models on their mechanism of action.

Animals↗
PubMed1994

Phosphorylation of the c-Fos and c-Jun HOB1 motif stimulates its activation capacity.

The c-Fos and c-Jun proteins bind an AP1 site and activate transcription synergistically. These two proteins have a common activation domain which has two co-operating motifs, HOB1 and HOB2. The HOB1 motif of c-Jun includes S73 which is required for Ha-Ras-induced super-activation and phosphorylation by MAP kinase-like enzymes. Since c-Fos HOB1 has a conserved Thr residue (T232) analogous to c-Jun S73 we have proposed that c-Fos HOB1 will be regulated in the same way as c-Jun HOB1. Here we show that the HOB1-containing activation domain of c-Fos is stimulated by Ha-Ras in vivo and phosphorylated by a MAP kinase family member in vitro and that mutating T232 to Ala abolishes both functions. Collectively these results suggest that phosphorylation of the HOB1 motif increases its activation capacity. To provide direct evidence for this we change the context of c-Fos T232 to a PKA recognition site, and show that HOB1 activity is now stimulated by the catalytic subunit of PKA. This 'PKA specificity' experiment represents a novel and powerful way to analyse phosphorylation events involved in a variety of biological functions.

Amino Acid Sequence↗
PubMed1994

A C-terminal domain in FosB, absent in FosB/SF and Fra-1, which is able to interact with the TATA binding protein, is required for altered cell growth.

Transcriptional regulation in eukaryotes is thought to occur through interactions between specific transcription factors and the general transcription machinery. We show that the regulatory protein FosB, but not FosB/SF or Fra-1, specifically and stably associates with the TATA box binding protein (TBP) and the multiprotein complex TFIID. The binding to TBP is specified by the last 55 C-terminal amino acids of FosB, requiring a small amino acid sequence, termed the 'TBP binding motif' (TBM). Deletion of the TBM affects transcriptional activity slightly, but it is adjacent to a proline-rich sequence which constitutes the major transactivation domain. However, both regions are required for the transformation of Rat-1A cells by FosB. Transfection experiments demonstrate that inhibition of transactivation due to excess levels of Gal4-FosB (squelching) can be partially relieved by the co-expression of TBP, which establishes that TFIID is a functional target of FosB. Since TBP binding is not exhibited by FosB/SF or Fra-1, we suggest that the activity mediated by the TBP interaction is one differentiating characteristic that distinguishes the FosB functions from those of FosB/SF and Fra-1.

Amino Acid Sequence↗
PubMed1994

Functional interaction between the HCMV IE2 transactivator and the retinoblastoma protein.

The 86 kDa immediate early IE2 protein of human cytomegalovirus (HCMV) can activate transcription of both viral and cellular genes and can repress transcription from its own promoter. Using two in vivo assays, we provide evidence of a functional interaction between IE2 and the retinoblastoma (RB) protein: IE2 alleviates RB-induced repression of a promoter bearing E2F binding sites and RB alleviates IE2-mediated repression of its own promoter. These functional effects are likely to be a result of a direct contact between IE2 and RB, which we can demonstrate both in vitro and in HCMV-infected cells. The interaction between IE2 and RB shows similar characteristics to the interaction between RB and E1A. First, binding to IE2 requires an intact RB pocket domain. Secondly, the binding is sensitive to the phosphorylation state of RB, because cyclin A-CDK-induced phosphorylation of RB diminishes IE2 binding. Thirdly, the IE2 domain required for RB binding is separate to the domains necessary for TBP and TFIIB binding. Our results demonstrate that large and small DNA viruses have a common interface with the host cell, namely the association with the RB tumour suppressor protein.

Adenovirus E1A Proteins↗
PubMed1994

Evidence for a protein domain superfamily shared by the cyclins, TFIIB and RB/p107.

Cyclins, TFIIB and RB play major roles in cell cycle and/or gene regulation. Earlier work has suggested common ancestry for the TFIIB repeats and RB pocket B which share 20% sequence identity. We now report that database searches with profiles based on a multiple alignment of cyclin core regions (the 'cyclin box') detect the TFIIB repeats with equivalent scores to divergent cyclins. Several features of the sequences support the notion of common ancestry: e.g. cyclins A/B, C and D share approximately 20-30% identity but each have approximately 15-20% identity with vertebrate TFIIB, showing that conserved cyclin features underlie the match. These results suggest the presence of a domain superfamily, which we term the TR domain, in nuclear regulatory proteins belonging to the TFIIB, cyclin and RB families, that has been duplicated many times during eukaryotic evolution. The TR domain appears to function in protein-protein interactions.

Amino Acid Sequence↗
PubMed1994

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

Adenovirus E1A Proteins↗
PubMed1993

The retinoblastoma protein binds E2F residues required for activation in vivo and TBP binding in vitro.

The retinoblastoma (RB) tumour suppressor protein is capable of repressing the activity of promoters containing DNA binding sites for the transcription factor E2F. Recently a protein which binds RB and possesses the DNA binding characteristics of E2F has been cloned. Here we show that the E2F activation domain is the target for RB-induced repression. RB can silence the 57 residue E2F activation domain but cannot effectively repress an E2F mutant which has reduced RB binding capacity. Extensive mutagenesis of E2F shows residues involved in RB binding are required for transcription activation. Mutations which affect both functions most dramatically lie within the minimal RB binding region. A further subset of sensitive residues lies within a new repeat motif E/DF XX L X P which flanks the minimum RB binding site. These data show that RB can mask E2F residues involved in the activation process, possibly by mimicking a component of the transcriptional machinery. Consistent with this model, we find that the TATA box binding protein TBP can bind to the E2F activation domain in vitro in a manner indistinguishable from that of RB.

Amino Acid Sequence↗
PubMed1993

c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro; definition of the minimal kinase recognition motif.

The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serine residue 249 (Ser-249) is required for phosphorylation to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, indicating that phosphorylation at other sites is required for this effect. Mutagenesis of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser-Gln, and that adjacent acidic residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, it confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.

Amino Acid Sequence↗
PubMed1993

The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB.

The retinoblastoma (RB) tumor suppressor protein and the TATA-box-binding protein TFIID form contacts with a number of viral transactivator proteins. One of these, the adenovirus E1A protein, can bind to both proteins. Here we present evidence that the cellular transcription factor PU.1 can bind to both RB and TFIID. Like E1A, PU.1 binds to the conserved C-terminal domain of TFIID and to the RB "pocket" domain. The PU.1 sequences required to bind either protein lie within a 75-amino acid region which functions as an independent activation domain in vivo. The ability of PU.1 to contact directly both RB and TFIID through the same 75-residue domain prompted us to look for sequence similarity between these two proteins. We find that the previously defined domain A of the RB pocket shows sequence similarity to the conserved C terminus of TFIID, whereas domain B shows sequence similarity to a second general transcription factor, TFIIB. The potential for RB to influence transcription by using TFIID- and TFIIB-related functions is discussed.

Amino Acid Sequence↗
PubMed1993

Influence of the v-Myb transactivation domain on the oncoprotein's transformation specificity.

The v-myb-containing viruses AMV and E26 induce the proliferation of myelomonocytic cells. The E26 Myb protein, by virtue of its fusion to Ets, is also able to transform multipotent haematopoietic cells (MEPs). We have examined the biological effects of substituting the v-Myb transactivation domain with the strong acidic activator domain from the C-terminus of the HSV-1 VP16 protein. In the absence of Ets, deletion of the transactivation domain destroyed the ability of v-Myb to stimulate transcription and to transform cells, whilst the substitution of the VP16 transactivation domain into v-Myb resulted in a greatly enhanced transactivation potential and altered TATA box binding protein (TBP) binding properties. In spite of these functional differences, the v-Myb VP16 protein regained the ability to transform myeloid cells with the same characteristics as wild type v-Myb. A construct encoding v-Myb VP16 fused to v-Ets was still capable of inducing leukaemia and of transforming both myeloid cells and MEPs in vitro, although the latter cells exhibited an altered phenotype. Our results demonstrate that the transformation of myeloid cells by v-Myb is largely independent of the type and potency of the transactivation domain it contains, whereas transformation of MEPs by the Myb-Ets fusion protein has more stringent transactivation requirements of Myb.

Acetyltransferases↗
PubMed1993

Transcriptional regulation by the retinoblastoma protein.

The retinoblastoma protein (RB) plays a key role in the control of cell proliferation and mediates the terminal differentiation of certain cell types. Increasing evidence suggests that RB functions by contacting and modifying the behaviour of transcription factors. RB can form complexes with E2F and MyoD in vivo, and complexes with a number of other transcription factors have also been demonstrated in vitro. The interaction of regulatory transcription factors with RB may be explained by sequence similarity between RB and two general transcription factors: TBP and TFIIB. Here I review the evidence for a role of RB in the regulation of transcription and highlight some of the likely mechanisms of RB function.

Journal Article↗
PubMed1993

The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation.

The 86K immediate early (IE) 2 protein of human cytomegalovirus trans-activates a number of homologous and heterologous promoters, including the cellular promoter for the 70K heat-shock protein (hsp70), and the human immunodeficiency virus long terminal repeat. We have previously shown that IE2 trans-activates these two promoters in a TATA-dependent manner, and that IE2 is able to form a direct contact with TATA-box binding protein (TBP) in vitro. We now show that IE2 binds to the basic repeat region of TBP. In addition IE2 can contact a second general transcription factor, TFIIB. We have mapped the TBP- and TFIIB-binding regions within IE2 and show that these regions overlap, and also lie within parts of the protein previously identified as being required for the trans-activation and autoregulation functions of IE2.

Base Sequence↗
PubMed1992

Conserved motifs in Fos and Jun define a new class of activation domain.

Fos and Jun form a tight heterodimeric complex that activates transcription by AP1 sites. We have recognized that two adjacent regions of the Jun A1 activation domain are conserved in the Fos protein, and we refer to these two homologous regions as homology box 1 (HOB1) and homology box 2 (HOB2). Using GAL4 chimeras, we show that the HOB1/HOB2 region of Fos and Jun is an independent activation domain in which HOB1 and HOB2 act cooperatively to activate transcription. This cooperativity is retained after the replacement of Fos HOB1 or HOB2 with the equivalent domain of Jun or when duplicated HOB1/HOB1 and HOB2/HOB2 combinations are generated. In the Fos protein, HOB1 or HOB2 can also cooperate with a distinct domain at the carboxyl terminus of the protein. Using the HOB2 consensus sequence as a guide, we identified a HOB2-containing activation domain in the CCAAT/enhancer binding protein (C/EBP) protein. This HOB2 motif can cooperate with as yet undefined sequences in C/EBP and will function even when linked to Jun HOB1. Thus, HOB1 and HOB2 represent inert "cooperating modules" that are combined to generate a functional activation domain. Each of these modules has the potential to cooperate with both distinct and identical domains. The presence of HOB-like modules in three different transcription factors indicates that the HOB motifs characterize a new class of activation domain. These motifs can be used now to identify other transcription factors with such modular characteristics.

Amino Acid Sequence↗
PubMed1992

The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID.

We have asked how the human cytomegalovirus major immediate-early 1 (IE1) and 2 (IE2) proteins act to transactivate heterologous cellular and viral promoters. Here we show that transactivation of the human immunodeficiency virus long terminal repeat and the 70,000-molecular-weight heat shock protein (hsp70) promoter by IE1 is TATA box independent and that the IE1 protein does not interact directly with the TATA box-binding factor TFIID. Conversely, transactivation of these promoters by IE2 is TATA box dependent and a direct interaction between IE2 and TFIID occurs, suggesting that IE2 transactivation is mediated through interaction with TFIID.

Cells, Cultured↗