Christine Dozier

Involvement of poly (ADP-Ribose)-polymerase in the Pax-6 gene regulation in neuroretina

The quail Pax-6 gene is expressed from two promoters named P0 and P1. P0 promoter is under the control of a neuroretina-specific enhancer (EP). This enhancer activates the P0 promoter specifically in neuroretina cells and in a developmental stage-dependent manner. The EP enhancer binds efficiently, as revealed by southwestern experiments, to a 110 kDa protein present in neuroretina cells but not in Quail Embryos Cells and Retinal Pigmented Epithelium which do not express the P0-initiated mRNAs. To study the role of p110 in Pax-6 regulation, we have purified the p110 from neuroretina cells extracts. Based on the peptide sequence of the purified protein, we have identified the p110 as the poly(ADP-ribose) polymerase (PARP). Using bandshift experiments and footprinting studies, we present evidence that PARP is a component of protein complexes bound to the EP enhancer that increases the on rate of the protein complex formation to DNA. Using PARP inhibitors (3AB and 6.5 Hphe), we show that these products are able to inhibit EP enhancer activity in neuroretina cells. Finally, we demonstrate that these inhibitors are able to decrease the expression of the P0-initiated mRNA in the MC29-infected RPE cells which, in contrast to the RPE cells, accumulated the PARP in response to v-myc expression. Our results suggest that PARP is involved in the Pax-6 regulation.

Evidence that POU factor Brn-3B regulates expression of Pax-6 in neuroretina cells.

The Pax-6 gene encodes a transcriptional master regulator involved in the development of the eye. The quail Pax-6 gene is expressed in the neuroretina from two promoters, P0 and P1, and is regulated by an intragenic neuroretina-specific enhancer (EP enhancer). The activity of this enhancer is restricted to the P0 promoter, which is activated at the onset of neuronal differentiation. In this article, we show that the POU domain transcription factor Brn-3b, which is expressed in various regions of the brain including retina and sensory neurons, is one of the factors interacting with the EP enhancer. Brn-3b strongly activates the EP enhancer in neuroretina cells but not in other cell types. Interestingly, this activation appears to be specific for Brn-3b, as the closely related POU factors Brn-3a and Brn-3c do not show activation of the EP enhancer. Our results identify the Pax-6 gene as a new potential downstream effector of the POU transcription factor Brn-3b.

Induction of proliferation of neuroretina cells by long terminal repeat activation of the carboxy-terminal part of c-mil.

Expression of the P100gag-mil protein of avian retrovirus MH2 in cultured chicken embryo neuroretina cells was previously shown to result in the proliferation of normally quiescent cell populations. We show here that long terminal repeat activation of the carboxy terminus of the c-mil gene is sufficient to induce neuroretina cell proliferation.

Prolifération cellulaire et coopération des oncogènes v-mil et v-myc

Abstract Retroviruses which possess the property to recombine with genetic material from the cell, have cloned and activated some oncogenes and hence are a privileged source for the study of these genes. Cellular oncogene activation can occur following two non mutually exclusive ways: (i) by over-expression of their products; (ii) by modifications of their products through mutations. Retroviruses can combine these two ways of activation leading to the over-expression of a modified product. In this paper, we present results obtained in the study of MH2, a retrovirus containing two oncogenes. We have shown that the two oncogenes of MH2 (v-mil and v-myc) cooperate in vitro to transform neuroretina cells from chicken embryos. These cells which normally do not grow in a defined medium, are induced to proliferate and become transformed upon infection by MH2. Our data enabled us to show that in MH2 v-mil was responsible for the induction of proliferation and v-myc for the transformation of the proliferating cells. Using in vitro constructs we located two regions in the protein encoded by v-mil which are important for its mitogenic property. We have also cloned the cellular counterpart of v-mil and the study of its biological activity on neuroretina cells enabled us to propose a mechanism of activation of the cellular gene by truncation of its 5′ part.

Retroviruses with Two Oncogenes

Less than a decade ago the first retroviral oncogene was discovered [1] and shown to derive from a normal cellular gene [2]. To date over 20 different retroviral oncogenes have been found, all derived from cellular counterparts. How a single oncogene can cause cancer is still not fully understood. Natural cancers are known to involve multi-step molecular changes, raising a paradox, as compared to cancers induced by retroviral oncogenes. To turn a cellular gene into an active retroviral oncogene, an overex-pression due to the very potent retroviral promoter may suffice in a few cases, whereas in most cases several modifications are probably required. These modifications might involve enhanced transcription, truncation of the gene, specific mutations, deletions or insertions (Fig. 1) as well as the viral route of transmission.