Faculty Bibliography

In the absence of serum, nerve growth factor (NGF) promotes the survival and differentiation of the PC12 pheochromocytoma cell line. In the presence of serum, NGF acts primarily as a differentiation factor and negative regulator of cell cycling. To investigate NGF control of cell cycling, we have analyzed the regulation of cyclin dependent kinases during PC12 cell differentiation. NGF treatment leads to a reduction in the steady-state protein levels of p33cdk2 and p34cdc2, two key regulators of cell cycle progression. The decrease in p33cdk2 and p34cdc2 coincides with a decrease in the enzymatic activity of cyclinA-p34cdc2, cyclinB-p34cdc2, cyclinE-p33cdk2, and cyclinA-p33cdk2 kinases. The decline in p33cdk2 and p34cdc2 kinase activity in response to NGF is accelerated in cells that over-express the p140trk NGF receptor, suggesting that the timing of the down- regulation is dependent on the level of p140trk and the strength of the NGF signal. The level of cyclin A, a regulatory subunit of p33cdk2 and p34cdc2, is relatively constant during PC12 differentiation. Nevertheless, the DNA binding activity of the cyclinA-associated transcription factor E2F/DP decreases. Thus, NGF down-regulates the activity of cyclin dependent kinases and cyclin-transcription factor complexes during PC12 differentiation

We show that c-Myc, in addition to activating transcription through E-box Myc binding sites (Ems), also represses transcription by a mechanism dependent on initiator (Inr) elements of the basal promoters of susceptible genes. Repression was first observed as a component of c-Myc biphasic regulation of the adenovirus-2 major late promoter (MLP), which contains both Inr and Ems sequences. Two differentiation-specific genes containing Inr, the C/EBP alpha and albumin genes, are repressed through their basal promoters by c-Myc, but are activated by the related B-HLH-LZ factor, USF. Repression requires both the B-HLH-LZ and Myc box II (MBII) domains. Significantly, a MBII deletion mutant which is deficient in repression, but transactivates normally, fails to cooperate with an activated ras gene to transform primary fibroblasts. Thus Myc-dependent transactivation is insufficient for Ras cooperation and the novel transcription repression function is implicated in Ras cooperation as well as the suppression of Inr-dependent genes

The amino-terminal region of the adenovirus type 5 E1a protein including conserved regions (CRs) 1 and 2 binds the 105-kDa retinoblastoma protein and a second, 300-kDa, cellular protein. We show that mutant viruses with deletions of CR1 which release the binding of either p105 or p300 still activate early promoters and infect cells productively. However, mutations which disrupt binding of both proteins disrupt early promoter activity and block the viral life cycle. Ela CR3, which has an established role in early promoter activation, can act in trans to the amino-terminal functions. This suggests that the amino terminus provides distinct, redundant functions related to p300 and Rb binding that synergize with CR3 to transactivate early genes

Growth of human adenoviruses is severely restricted in monkey cells. We examined the synthesis of mRNAs from the Ad2 late transcription unit (LTU) in abortively infected monkey cells at late times in infection. All L2, L3, and L5 mRNAs were absent or drastically reduced in abortive infections. Most L1 and L4 mRNAs were also greatly decreased in abortive infections; however, a single large messenger was produced from each of the L1 and L4 families, at levels approaching those found in productive infections. The pattern of i-leader containing mRNAs was also changed in abortive infections. These defects could be corrected in monkey cells by the presence of SV40 T antigen or an altered adenoviral DNA binding protein. These defects in late gene expression in abortive infections could not be attributed to differences in transcription along the LTU or levels of DNA replication. In abortively infected cells, nuclear levels of L5 mRNA were decreased 2 to 6 fold, while cytoplasmic levels were decreased over 200-fold. These findings imply a general defect in processing of viral mRNAs, most likely due to defective splicing and/or transport, during abortive infections

We have studied the activation of the serum albumin promoter by transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) in the HepG2 hepatoma cell line. We find that three distinct mechanisms determine the ability of C/EBP alpha to activate this promoter in a cell-type-specific and cooperative manner. First, the trans-activating function of C/EBP alpha is generated through cooperation between three separate domains of the protein that we have named trans-activation elements (TE-I through TE-III). The TEs have little or no ability to activate transcription by themselves, but any two can cooperate to do so, both in the C/EBP alpha protein and when linked to the GAL4 DNA-binding domain. Second, TE-III was found to contain a negative regulatory subdomain, the function of which was alleviated when C/EBP alpha was bound in the environment of the albumin promoter. This formed the basis for cooperative activation of this promoter by C/EBP alpha. Finally, we demonstrate that the leucine zipper of C/EBP alpha participates in determining the cell type specificity of albumin promoter activation, as it exerts a strong negative effect on albumin promoter activation in the nonhepatic HeLa cell line but not in HepG2 cells. These findings shed new light on the mode of action of C/EBP alpha and show a novel function for leucine zipper in cell-type-specific gene expression

Nerve growth factor induces the neuronal-like differentiation of PC12 cells, and epidermal growth factor promotes PC12 viability and is weakly mitogenic. Despite these differences, both growth factors induce indistinguishable patterns of transient delayed transcription of the tyrosine hydroxylase (TH) gene and the expression of proteins encoded by Fos gene family members. Thus, TH expression is sensitive to signaling pathways common to these two growth factors. We show that c-fos and fosB successively occupy an AP-1 site-like element of the TH promoter after nerve growth factor treatment. Furthermore, under conditions of transient transfection, Fos family proteins may synergize with c-jun to transrepress TH gene transcription through the TH-fat-specific element. We show that the target of repression is the AP-1 site-like element that lies within the TH-fat-specific element. We demonstrate that this site is also a major positive acting site for TH control. These results suggest a model in which the long term effect of c-fos family protein expression is to limit the expression of the TH gene. We consider the novel properties of this element in providing temporal and cell type-specific regulation of TH transcription