Biomedical subjects

P Raychaudhuri

Publications and source records attributed to P Raychaudhuri.

At least 37 records · Page 2Linked to original sources

PubMed1996

p21 Disrupts the interaction between cdk2 and the E2F-p130 complex.

In nonproliferating or growth-arrested cells, the transcription factor E2F remains bound to the retinoblastoma-related protein p130. Accumulation of this E2F-p130 complex correlates with an arrest of the cell cycle progression. Progression through G1 phase is associated with a cyclin-dependent binding of the cyclin-dependent kinase cdk2 to the E2F-p130 complex. By fractionating mouse L-cell extracts, we have obtained a partially purified preparation of the E2F-p130 complex that also contains cdk2. Incubation of this complex with recombinant p21 results in a disruption of the interaction between cdk2 and the E2F-p130 complex in extracts of a cell line that expresses a temperature-sensitive mutant of p53. Incubation at the permissive temperature (32 degrees C) results in an induction of p21 synthesis. An increase in the level of p21 in these cells correlates with a loss of cdk2 from the cdk2-containing E2F-p130 complex. We also show that the expression of a reporter gene containing E2F sites in the promoter region is reduced by the coexpression of p21. Since p21 is believed to be a mediator of p53, we speculated that the p21-mediated disruption of the cdk2-containing E2F-p130 complex plays a role in the growth suppression function of p53.

Animals↗

PubMed1995

HPV16 E7 oncoprotein induces expression of a 110 kDa heat shock protein.

Heat shock protein genes are induced by various kinds of stress. Besides stress, the heat shock family gene hsp70 has been shown to be induced by growth-stimulating agents such as the DNA virus oncoproteins and serum. Here, we report cloning of a novel cDNA that encodes a 100 kDa heat shock protein-related polypeptide as a human papillomavirus oncoprotein E7-inducible gene. E7 induces expression of this heat shock protein at the level of RNA synthesis. Moreover, the induction of this heat shock protein-mRNA was dependent on the conserved region 2 of the E7 protein, which is essential for binding to the proteins of the retinoblastoma family.

3T3 Cells↗

PubMed1995

Association of p107 with Sp1: genetically separable regions of p107 are involved in regulation of E2F- and Sp1-dependent transcription.

The retinoblastoma-related protein p107 has been shown to be a regulator of the transcription factor E2F. p107 associates with E2F via its pocket region and represses E2F-dependent transcription. In this study, we provide evidence for a novel interaction between p107 and the transcription factor Sp1. We show that p107 can be found endogenously associated with Sp1 in the extracts of several different cell lines. Moreover, in transient transfection assays, expression of p107 represses Sp1-dependent transcription. This repression of Sp1-dependent transcription does not require the DNA-binding domain of Sp1. Transcription driven by a chimeric protein containing the Ga14 DNA-binding domain and the Sp1 activation domains is inhibited by p107. Interestingly, unlike the repression of E2F-dependent transcription, the repression of Sp1-dependent transcription does not depend on an intact pocket region. We show that distinct regions of p107 are involved in the control of Sp1 and E2F.

Animals↗

PubMed1994

Activation of the c-fos gene by the HPV16 oncoproteins depends upon the cAMP-response element at -60.

The E6 and the E7 genes of the high-risk types of human papillomavirus (types 16 and 18) are associated with the induction or maintenance of malignant growth. The molecular mechanism by which these oncogenes contribute to the malignant phenotype is not clear. To study the effects of E7 on cellular processes, we constructed a stable cell line that inducibly expressed the E7 gene of HPV16. By using this cell line, we provide evidence that expression of E7 of HPV16 stimulates c-fos gene expression. Also, by doing transient transfection experiments, we show that the expression of either E6 or E7 induces transcription from the c-fos promoter. Analysis of a series of c-fos promoter mutants indicates that the activation by both E6 and E7 is dependent on the cyclic AMP response element. To further investigate the mechanism(s) of the activation of the c-fos gene and their relation to the oncogenic properties of E6 and E7, several mutants of the E6 and E7 genes were analyzed. The results of these studies indicate that the CR1 and CR2 regions in the E7 protein, and sequences distinct from the p53-binding region in the E6 protein, are critical for activation of the c-fos promoter.

3T3 Cells↗

PubMed1993

Association of the human papillomavirus type 16 E7 protein with the S-phase-specific E2F-cyclin A complex.

The transcription factor E2F has been shown to be involved in the expression of several cell cycle-regulated genes, and the activity of this factor is controlled by cellular proteins such as pRB and p107. E2F is also a target of the DNA virus oncoproteins (adenovirus E1A, simian virus 40 T antigen, and human papillomavirus [HPV] E7) (see the review by J. R. Nevins [Science 258: 424-429, 1992]). These viral oncoproteins dissociate an inactive complex between E2F and the retinoblastoma tumor suppressor protein (pRB), and this dissociation of the E2F-pRB complex correlates with a stimulation of the E2F-dependent transcription. In the S phase of the cell cycle, E2F forms a complex with p107, cyclin A, and the cdk2 kinase (E2F-cyclin A complex). The cellular function of this S-phase-specific complex is unclear. The adenovirus E1A protein dissociates the E2F-cyclin A complex. The HPV type 16 (HPV-16) E7 protein, which possesses significant sequence homology with E1A, does not dissociate the E2F-cyclin A complex. We find that the HPV-16 E7 protein associates very efficiently with the E2F-cyclin A complex. This association is dependent on the sequences that are also necessary for the transforming activity of E7. Moreover, the E7 protein of a low-risk HPV (type 6b) is much less efficient in binding to the E2F-cyclin A complex compared with that of the high-risk type. We also find that the E2F-cyclin A complex remains endogenously associated with the E7 protein in extracts of Caski cells, which express high levels of HPV-16 E7 protein. Finally, we have extensively purified the E2F-cyclin A complex from mouse L-cell extracts and show that, in cell extracts, the E2F-cyclin A complex remains associated with other cellular proteins.

Animals↗

PubMed1992

Retinoblastoma-repression of E2F-dependent transcription depends on the ability of the retinoblastoma protein to interact with E2F and is abrogated by the adenovirus E1A oncoprotein.

The product of the retinoblastoma tumor suppressor gene interacts with the transcription factor E2F. Two distinct types of interactions can be detected between the retinoblastoma gene product (Rb) and E2F. The first type involves an Rb-binding protein, RBP60. The Rb/E2F complex formed in the presence of RBP60 is able to bind DNA and migrates with a distinct mobility in gel retardation assays. The second type of Rb/E2F complex is seen in the absence of RBP60. This second type of Rb/E2F complex does not form a band-shift complex in gel retardation assays and its formation results in an apparent inhibition or loss of the DNA binding activity of E2F. Using a series of Rb-mutants we show that these two types of Rb/E2F complexes depend on common domains of the Rb protein. The T/E1A-binding region as well as the carboxyl-terminus of the Rb protein are critical for these two types of Rb/E2F interactions. We also show that the retinoblastoma protein represses the E2F-dependent transcription, and this Rb-repression of the E2F-dependent transcription depends on the ability of Rb to interact with E2F. Moreover, the adenovirus E1A gene product, which binds Rb, counteracts the Rb-repression and restores E2F-dependent transcription.

3T3 Cells↗

PubMed1992

Identification of a 60-kilodalton Rb-binding protein, RBP60, that allows the Rb-E2F complex to bind DNA.

Several reports have indicated that the product of the retinoblastoma gene (Rb) complexes with the transcription factor E2F. We present evidence that the DNA-binding of the Rb-E2F complex involves another cellular factor. Addition of Rb to purified preparations of E2F does not generate an Rb-E2F complex that can bind DNA, and in fact, we see an inhibition of the DNA-binding ability of E2F. On the other hand, addition of Rb to cruder preparations of E2F results in the formation of an Rb-E2F complex (E2Fr) that can bind DNA and produces a distinct complex in gel retardation assays. We have identified and purified a 60-kDa protein that allows the Rb-E2F complex to bind DNA, and we show that this 60-kDa protein exerts its effect by directly interacting with Rb.

Animals↗

PubMed1991

The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F.

Recently, we identified an inhibitory protein, E2F-I, that blocks the DNA-binding activity of the transcription factor E2F. We also showed that the adenovirus E1A protein reverses this inhibitory activity of E2F-I, thereby restoring the DNA-binding activity of E2F. We have now further purified this inhibitory activity and show that the most purified preparation of E2F-I contains a 105 kd E1A-binding protein. This 105 kd E1A-binding protein cross-reacts with two different antibodies against the retinoblastoma (RB) gene product. Moreover, the RB gene product copurifies with E2F-I activity. Taken together, we conclude that the product of the RB gene is a part of E2F-I and is involved in the regulation of E2F activity.

Adenovirus Early Proteins↗

PubMed1991

Domains of the adenovirus E1A protein required for oncogenic activity are also required for dissociation of E2F transcription factor complexes.

Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of E1A mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.

Adenoviridae↗

PubMed1991

Analysis of trans activation by human papillomavirus type 16 E7 and adenovirus 12S E1A suggests a common mechanism.

The human papillomavirus E7 gene product is an oncoprotein with properties similar to those of the adenovirus E1A proteins. The human papillomavirus E7 proteins possess substantial amino acid sequence similarity to portions of conserved regions 1 and 2 of E1A, and the human papillomavirus type 16 E7 protein trans-activates the adenovirus E2 early promoter. Analysis of point mutations in the E2 promoter indicated that the E2F recognition sites were critical to E7 stimulation. In contrast to the activation of the E2 promoter, E7 could not trans-activate various other E1A-inducible promoters. Although the promoter specificity for E7 differs from that of 13S E1A trans activation, it is very similar to activation by the E1A 12S product. Moreover, analysis of the E7 protein has suggested that amino acid sequences critical for trans activation include those shared with E1A within conserved region 2. Biochemical studies demonstrate that the E7 protein, like the 12S E1A product, can alter the interaction of cellular factors with the E2F transcription factor. We therefore conclude that E7 trans activation is functionally related to that mediated by the 12S E1A product.

Adenovirus Early Proteins↗

PubMed1990

Adenovirus E1A proteins can dissociate heteromeric complexes involving the E2F transcription factor: a novel mechanism for E1A trans-activation.

Adenovirus infection activates the E2F transcription factor, in part through the formation of a heteromeric protein complex involving a 19 kd E4 gene product that then allows cooperative and stable promoter binding. We now find that cellular factors are complexed to E2F in extracts of several uninfected cell lines. E1A proteins can dissociate these complexes, releasing free E2F. This activity of E1A is independent of conserved domain 3 but is dependent on conserved domain 2 sequence. The E1A-mediated dissociation of the complexes allows the E4 protein to interact with E2F, generating a stable DNA-protein complex with the E2 promoter and a stimulation of transcription. These experiments demonstrate a function for E1A in mediating a dissociation of transcription factor complexes, allowing new interactions to form and thus changing the transcriptional specificity.

Adenovirus Early Proteins↗

PubMed1990

Activation of the E2F transcription factor in adenovirus-infected cells involves E1A-dependent stimulation of DNA-binding activity and induction of cooperative binding mediated by an E4 gene product.

Previous experiments have demonstrated that the DNA-binding activity of the E2F transcription factor is increased upon adenovirus infection and that both the E1A and E4 genes are required for activation. In this study, we demonstrated that this enhanced binding of E2F to the E2 promoter is the result of two events. (i) There is stimulation of the DNA-binding activity of the E2F factor; this stimulation is E1A dependent but independent of E4. (ii) There is also induction of a stabilized interaction between E2F molecules bound to adjacent promoter sites; induction of stable E2F binding requires E4 gene function. This two-step activation process was also demonstrated in vitro. A heat-stable fraction from extracts of adenovirus-infected cells, which contains the 19-kilodalton E4 protein, was capable of stimulating stable E2F binding in an ATP-independent manner and appeared to involve direct interaction of the E4 protein with E2F. An extract from virus-infected cells devoid of the E4 19-kilodalton protein stimulated E2F DNA binding without forming the stable complex. This reaction required ATP. We conclude that activation of E2F during adenovirus infection is a two-step process involving a change in both the DNA-binding activity of the factor and the capacity to stabilize the interaction through protein-protein contacts.

Adenovirus Early Proteins↗

PubMed1990

E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.

Activating Transcription Factors↗

PubMed1989

Phosphorylation-dependent activation of the adenovirus-inducible E2F transcription factor in a cell-free system.

Adenovirus infection induces a large increase in the DNA binding activity of a cellular transcription factor that is utilized by the viral E2 promoter and termed E2F. Using cell-free extracts, we have developed an assay for the in vitro activation of DNA binding activity of E2F. E2F activity is undetectable in HeLa extracts but upon incubation with a fraction from adenovirus-infected cells, there is an ATP-dependent increase in E2F DNA binding activity. This increase does not occur using an equivalent fraction from dl312 (E1A-)-infected cells. Incubation of E2F with phosphatase inactivates E2F binding activity. Incubation of the phosphatase-inactivated E2F with an infected cell fraction restores E2F activity as does incubation with a known protein kinase. In contrast, incubation with an extract from mock-infected cells does not restore activity. We conclude that the DNA binding activity of E2F is regulated by phosphorylation in an E1A-dependent manner.

Adenovirus Early Proteins↗

PubMed1989

DNA-binding activity of the adenovirus-induced E4F transcription factor is regulated by phosphorylation.

Previous experiments have identified E4F, an inducible cellular factor that binds to sequences in the adenovirus E4 promoter that are critical for E1A-dependent transcriptional activation. The E4F factor has been purified and shown to stimulate transcription in vitro from the E4 promoter. Analysis of the affinity-purified factor identifies a single polypeptide of 50 kD that has E4F-specific binding activity. E4F binding activity is also regulated during F9 cell differentiation and can be activated in differentiated F9 cells by viral infection. Furthermore, the activation process appears to involve a phosphorylation event, because treatment of E4F with alkaline phosphatase abolishes activity and incubation of the phosphatase-inactivated factor with an extract from virus infected cells restores activity.

Adenoviridae↗

PubMed1989

The adenovirus E4 gene, in addition to the E1A gene, is important for trans-activation of E2 transcription and for E2F activation.

Previous experiments have demonstrated that adenovirus infection of human and mouse cells leads to an E1A-dependent activation of the DNA-binding capacity of a cellular transcription factor termed E2F. E2F binds to two sites in the adenovirus E2 early promoter which have been shown to be critical for E1A-dependent E2 early transcription, and the E2F-binding sites can confer E1A-induced transcription to a heterologous promoter. In addition, under a variety of circumstances, the increase in E2F-binding activity coincides with the activation of E2 transcription. We now find that, in addition to the E1A gene, another early viral gene, the E4 gene, is necessary for the activation of E2F-binding activity. Extracts prepared from human 293 cells, which express the E1A and E1B genes, had low levels of E2F activity, whereas infection of 293 cells with the E1A mutant dl312 increased E2F activity. This increase did not occur when 293 cells were infected with dl366, an E4 deletion mutant, nor was there an increase in E2F activity in HeLa cells infected with either dl366 or dl312; however, a coinfection with the two mutants yielded the normal wild-type increase in E2F. Furthermore, infection of HeLa cells with a high multiplicity of dl312, conditions that allow E4 gene expression in the absence of E1A, did not yield an increase in E2F activity. Thus, it appears that both the E1A gene and the E4 gene are directly involved in E2F activation. Measurements of E2 RNA production in a dl366 infection as compared with a wild-type or dl312 infection demonstrate that the E4 gene is essential for full E2 transcription. Furthermore, transfection assays of the E2 promoter demonstrate that, although E1A alone can trans-activate the E2 promoter, it is not as effective as the combination of E1A and E4 in the induction of the E2 promoter. We therefore conclude that the activation of the E2F factor leading to the activation of E2 transcription requires the combined action of both the E1A 289-amino-acid protein and an E4 product.

Adenoviridae↗

PubMed1989

The adenovirus-inducible factor E2F stimulates transcription after specific DNA binding.

The promoter-specific factor E2F interacts with critical regulatory sequences within the adenovirus E2 promoter. In addition, the level of active factor increases markedly during a virus infection, dependent on E1A function and coincident with the trans activation of E2 transcription. We have purified the E2F factor through a combination of standard biochemical procedures and DNA affinity chromatography. The purified factor was a single polypeptide of 54,000 molecular weight, as determined by UV crosslinking and renaturation of gel-fractionated protein. Addition of affinity-purified factor to an in vitro transcription system resulted in stimulation of transcription from a promoter containing two E2F-binding sites but not promoters lacking binding sites. We thus conclude that E2F is indeed capable of stimulating transcription once it has bound to the promoter.

Adenovirus Early Proteins↗

PubMed1988

Transactivation by the adenovirus E1A gene.

The 289aa product of the adenovirus E1A gene mediates the transcriptional activation of the set of early viral genes as well as several cellular genes. The E1A protein is not a DNA binding protein but, rather, acts indirectly to achieve the activation. The process of viral gene activation involves the use of cellular transcription factors, and in at least one case, in vivo assays have demonstrated a stimulation of stable promoter complex formation as a function of the E1A gene product. Analysis of transcription factors in nuclear extracts has identified a cellular factor, termed E2F, with specificity for the viral E2 promoter. The concentration of this factor increases as a result of the action of E1A. This increase in DNA binding activity does not require protein synthesis, thus indicating an E1A-mediated modification of a pre-existing factor. The E2F factor has been purified to homogeneity and is a polypeptide of 54,000 molecular weight. Analysis of an additional viral promoter, the E4 promoter, has identified a protein that interacts with sequences critical for transcription. This factor, termed E4F, is also increased as a function of the E1A product. The E4F factor has also been purified to homogeneity and has a molecular weight of 50,000. Therefore, the coordinate control of transcription by the E1A gene product involves the activation of multiple promoter specific factors.

Adenoviridae↗