Biographical Sketch:

Dr. Thimmapaya received his education in India. He obtained his B.Sc. degree in Biology from Mysore University, his M.Sc. degree in Biochemistry from Maharaja Sayajirao University Baroda, and his Ph.D. Degree in Biochemistry from the Indian Institute of Science. From 1975 to 1977 he worked with Dr. Sherman Weissman of the Department of Human Genetics at Yale University. In his laboratory he and his colleagues deduced the nucleotide sequence of Simian virus 40 (SV40) genome and mapped the protein coding genes that the virus encodes. From 1977 to 1980 he worked with Dr. Thomas Shenk in the Department of Microbiology at the University of Connecticut Health Center. In Dr. Shenk’s laboratory he continued the genetic analysis of the SV40 early region and began his work on human adenoviruses (Ad). He constructed Ad mutants in which the two viruses encoded small RNA polymerase III genes (also termed as Virus Associated RNAs I and II) are deleted. These studies showed that the VAI RNA is required for the translation of viral and cellular mRNAs at late times after adenovirus infection of human cells. Later, these studies led to our understanding of the role of a kinase that phosphorylates the translation initiation factor called eIF-2. Adenovirus VAI RNA binds to this enzyme and blocks its activity.

In December of 1980 Dr. Thimmapaya joined the Microbiology-Immunology Department of Northwestern University Medical School. For several years, his laboratory continued to work on the transcriptional and translational aspects of human adenovirus and simian virus 40. These studies included analysis of VAI RNA-PKR interactions, new translational regulation function in SV40, and the transcriptional regulation of the Ad early transcription region 2 (E2). He also briefly worked on certain transcriptional regulation aspects in Scizosachharomyces pombe and identified a novel protein that interacts with human E2F site in the early 1990’s.

During the last few years Thimmapaya’s laboratory has shifted focus and started to work on retinoblastoma (Rb) tumor suppressor protein and a class of transcriptional coactivators termed p300/CBP family proteins. His laboratory in collaboration with Dr. Sigmund Weitzman has recently started to work on the development of gene therapy strategies to treat metastatic breast cancer.

Research Description:

Dr. Thimmapaya’s laboratory currently focuses on the study of the roles played by p300 and CBP in (i) cell cycle regulation, (ii) transcriptional activation by Ets family transcription factors and (iii) breast epithelial cell differentiation and mammary gland development .

Research Abstract:

p300 and CBP proteins are two highly conserved members of a family of transcriptional coactivators that bridge the DNA bound sequence specific transcription factors and the basal transcription complex and increase transcriptional initiation signals. p300 was first identified as an adenovirus E1A binding protein; E1A is an adenovirus transforming protein. Both p300 and CBP are nuclear phosphoproteins that are about 2400 amino acids long and migrate at about 300 kDa proteins in SDS-polyacrylamide gels. CBP was first identified as a CREB binding protein that coactivates the CREB transcription factor. cAMP phosphorylates CREB which binds to CBP and stimulates cAMP responsive genes. p300/CBP contains three cysteine- and histidine rich (C/H) regions, one between a.a. 347 and 414 (C/H1), the second between a.a. 1163 and 1451 (C/H2), and the third between a.a. 1638 and 1806 (C/H3) . p300/CBP also contains a bromodomain between a.a.1070 and 1134. The bromodomain is characteristic of coactivators or adaptors that include yeast SNF2, its human homolog hbrm, and CBP. p300 and CBP interact with a number of sequence specific transcription factors. p300/CBP also binds to nuclear hormone receptors and sterioid receptor coactivator-1 (SRC-1) during hormone induced gene activation. Recent studies show that a p300-binding protein termed PCAF and p300 contain histone acetylation activity. p300/CBP can acetylate cellular transcription factors such as p53 and modulate its DNA binding activity. Protein-coding genes in chromatin are in a repressed state and they are not accessible for transcription factors unless the DNA-bound histones are acetylated. The properties of p300/CBP fit into a model in which these proteins facilitate the accessibility of chromatin to transcription factors by acetylation of histones as well as sequence specific transcription factors and thus positively regulating gene expression. p300/CBP proteins are present in limiting amounts in the cell and therefore the activities of these proteins must be controlled in the cell by factors that are yet to be identified. Mutations of CBP has recently been discovered to be responsible for cases of heritable Rubinstein-Taybi syndrome, an autosomal-dominant disorder which causes metal retardation and a predisposition to several forms of cancers. Further, transposition of CBP, which fuses the histone acetyl transferase domain of CBP to MOZ gene has been found in acute myeloid leukemia. The above two observations suggest that p300 and CBP may be tumor suppressor genes.

(i). Role of p300/CBP in cell cycle progression

Cell division of a eukaryotic cell takes place in four successive phases. After the nuclear and cytoplasmic division (M phase), the daughter cells begin interphase of a new cycle. Interphase starts with the G1 phase in which biosynthetic activities are resumed at high rate, S phase in which DNA replication begins and ends, G2 phase which separate the S phase the next M phase. Progression through the cell cycle is a tightly regulated preocess, responding to a plethora of positive and negative regulators of growth. These include cyclins, cyclin dependent kinases (Cdk), Cdk inhibitors such as p21, p27, p16, and p57, and retinoblastoma (Rb) protein. Previous studies have suggested that p300/CBP may have a role in maintaining the G0 or G1 state in normal cells. We are investigating the role of p300 in cell cycle progression by depleting the p300 levels in cells using adenovirus vectors expressing p300-specific antisense sequences and by conditional overexpression of p300.

In order to study the role of p300 in the cell cycle, we have created adenovirus vectors which express antisense versions of the 5' end of p300 mRNA. Expression of these vectors in MCF10 immortalized human breast cells reduces the amount of p300 present in the cell. When these antisense constructs are expressed in MCF10 cells synchronized in early G1, the cells exit G1 early, within a few hours of serum (growth) stimulation. As the cell cycle progresses, these cells appear to congregate in S and G2 and never cycle back into G1. Via Westerns and kinase assays, we have examined the amounts and activities of the G1 and S phase cyclins. Cyclin A (S phase cyclin) protein and kinase levels and cyclin E (G1 phase cyclin) kinase levels are greatly elevated in cells which express p300 antisense mRNA, starting at about 26 hours after infection with the antisense producing viruses. Cyclin E protein levels and cyclin D1 protein and kinase levels do not change, however, indicating that the cells do not undergo a normal cell cycle. None of the known inhibitors of cyclin A and cyclin E, p21/Cip1, p27/Kip1, or p57/Kip2, seem to be involved in the changes in cyclins. Cdk-2 levels also change slightly, and it is possible that changes in the kinases or phosphatase that regulate cdk-2 are responsible for the changes in cyclin activity seen. These results indicate that p300 is involved in the regulation of early cell cycle events and in the repression of cyclin A and cyclin E activity in G0/G1 phase cells.

(ii): Role of p300/CBP in the transcriptional activation activity of Ets family transcription factors:

The Ets family of transcription factors include a large number of proteins which perform diverse functions in the cell including serum stimulation of the c-fos promoter (Elk-1/SAP-1), activation of HSV immediate early promoters (GABP a and b), regulation of immunoglobulin light chain enhancers (Pu.1/Spi-1, erythroid differentiation, and Drosophila development . Constitutively active mutant Ets proteins (v-Ets-1 and -2) are involved in cellular transformation. A characteristic feature of this class of proteins is a highly conserved 85 amino acid (a.a.) DNA binding domain termed the Ets domain which contains a helix-turn-helix motif. The Ets domain binds to a GGAA purine-rich core sequence found in the promoters and enhancers of viral and cellular genes.

Ets-1 and -2 are ubiquitous proteins which shares significant homology. The transactivation and the DNA binding (Ets) domains in both Ets-1 and 2 map to N and the C-terminal regions, respectively. The homologous regions include the Ets domain which is 95% conserved between Ets-1 and 2 and the Pointed domain located in the transactivation domain. The Pointed domain consists of approximately 100 a.a. region that is conserved within a subgroup of Ets factors including Drosophila Ets factor pointed P2 and Ets-1 and 2. The Pointed domain contains a MAP kinase site and pointed P2 is a target of Ras/MAP kinase signaling pathways in Drosophila. Ets-1 and -2 are targets of the Ras signaling pathway and Ras mediated activation of Ets-1 and 2 transactivation activity requires phosphorylation of Ets-2 threonine 72 and the corresponding Ets-1 threonine 38, which are also conserved in pointed P2 and yan (a Drosophila repressor). The conserved MAP kinase site (threonine 72) in Ets-2 is phosphorylated by MAP kinase.

We have shown that Ets-1 and -2 recruit p300/CBP during the transcriptional activation of the human stromelysin promoter which contains palindromic Ets binding sites (Stromelysin 1 (MMP3) is a major MMP of connective tissues and can degrade most components of the extracellular matrix including laminin, fibronectins, cartilage proteoglycans and basement). p300/CBP and Ets-1 or -2 also cooperate to transactivate a synthetic reporter construct containing Ets binding sites. Using a variety of in vitro and in vivo approaches, we show that Ets-1 and -2 interact with the N- and the C-terminal regions of p300/CBP. We also show that binding of p300 to Ets-2 and transcriptional coactivation of Ets-2 by p300 are not affected by mutation of this Ets-2 MAP kinase phosphorylation site. Thus, our results indicate that p300/CBP plays an important role in regulating the activity of Ets-1 and Ets-2.

(iii) Role of p300/CBP in the epithelial cell differentiation and mammary gland development:

By using the p300 binding protein E1A as a tool, it has been shown that p300 is involved in the differentiation of muscle, neuronal and embryonal carcinoma (F9) cells. More direct studies have shown that p300/CBP binds to and collaborate with bHLH proteins in muscle and B-cell differentiation. Thus p300/CBP may be an important component of differentiation pathways of many cell types and may cooperate with transcription factors in the transcriptional activation of differentiation specific genes. The above observations prompted us to examine the role of p300/CBP in breast epithelial cell differentiation and duct morphogenesis.

In contrast to most other tissues, mammary tissue reaches its full growth potential following the onset of pregnancy and during lactation. Epithelial cells of the mammary gland undergo a normal cycle of proliferation, differentiation, and apoptosis during pregnancy, lactation, and involution following weaning. Hormonal control of mammary gland development is well studied. However, the role of intracellular factors, extracellular matrix, and the molecular mechanisms involved in the mammary gland development are incompletely understood.

We hypothesize that p300/CBP has an essential role in breast epithelial cell differentiation and morphogenesis. Thus, we propose to study the role of p300/CBP in the growth, differentiation and morphogenesis of mammary tissue in vivo using a transgenic mouse model. CBP specific antisense sequences and a dominant negative mutant of p300 will be targeted to breast tissue of transgenic mice using breast tissue specific promoters, and the resulting perturbations in the mammary gland development will be investigated. In addition, the role of p300/CBP in breast epithelial cell differentiation/duct morphogenesis will be examined in vitro using a Matrigel extracellular matrix system. p300 specific antisense sequences and a dominant negative mutant of p300 will be introduced into primary breast cells or an immortalized breast epithelial cell line (MCF10A) in culture using replication defective adenovirus vectors, and the effects of these mutants on differentiation and duct morphogenesis on Matrigel will be investigated. These studies will advance our knowledge of the mammary gland development, including the identification of possible pathways leading to differentiation and development of breast tissue and clues to the molecular basis of abnormalities associated with mammary gland development that may be responsible for the malignant conversion of breast epithelial cells.

Publications:

Rajan, P., Swaminathan, S., Zhu, J., Cole, C. N., Barber, G., Tevethia, M. J., and Thimmapaya, B. A novel translational regulation function for the simian virus 40 large-T antigen. J. Virol. 69:785-795, 1995.

Somasundaram, K., Jayaraman, G., Williams T., Moran, E., Frisch, S. and Thimmapaya, B. Repression of a Matrix metalloprotease gene by E1A correlates with its ability to bind to cell type-specific transcription factor AP-2. Proc. Natl. Acad. Sci. U.S.A , 93: 3088-3093, 1996.

Swaminathan, S. Rajan, P., Savinova, O., Jagus, R., and Thimmapaya, B. Simian virus 40 large-T bypasses the translational block imposed by the phoshorylation of eIF-2 alpha . Virology, 219: 321-323, 1996.

Swaminathan, S. and Thimmapaya, B. Transactivation of adenovirus E2 early promoter by E1A and E4 in vivo. J. Mol. Biol. 258: 736-746, 1996.

Porras, A., Bennett, J., How, A., Karen, T., Bouck, N., Henglein, B., Swaminathan, S., Thimmapaya, B., and Rundell, K. A novel simian virus 40 early-region domain mediates transactivation of the cyclin A promoter by small-t antigen and is required for transformation in small-t dependent assays. J. Virol. 70:6902-6908, 1996.

Zagaria, A., Mungre, S., Lovis, R., Birrer, M., Ness, S., Thimmapaya, B., and Pope, R. Tumor necrosis factor alpha gene regulation: Enhancement of C/EBP-induced activation by c-Jun. Mol. Cell. Biol. 18:2815-2824, 1998.

Buchmann, A., Swaminathan, S., and Thimmapaya, B. Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein. Mol. Cell. Biol.. 18:4505-4576, 1998.