E1A Interacts with Two Opposing Transcriptional Pathways To Induce Quiescent Cells into S Phase

Abstract

Despite data suggesting that the adenovirus E1A protein of 243 amino acids creates an S-phase environment in quiescent cells by overcoming the nucleosomal repression of E2F-regulated genes, the precise mechanisms underlying E1A's ability in this process have not yet been defined at the biochemical level. In this study, we show by kinetic analysis that E1A, as opposed to an E1A mutant failing to bind p130, can temporally eliminate corepressor complexes consisting of p130-E2F4 and HDAC1/2-mSin3B from the promoters of E2F-regulated genes in quiescent cells. Once the complexes are removed, the di-methylation of H3K9 at these promoters becomes dramatically diminished, and this in turn allows for the acetylation of H3K9/14 and the recruitment of activating E2F family members, which is then followed by the transcriptional activity of the E2F-regulated genes. Remarkably, although an E1A mutant that can no longer bind to a histone acetyltransferase (PCAF) is as capable as wild-type E1A in eliminating corepressor complexes and methyl groups from the promoters of these genes, it cannot mediate the acetylation of H3K9/14 or induce their transcription. These findings suggest that corepressors as well as coactivators are acted upon by E1A to derepress E2F-regulated genes in quiescent cells. Thus, our results highlight for the first time a functional relationship between E1A and two transcriptional pathways of differing functions for transitioning cells out of quiescence and into S phase.Human adenoviruses normally infect quiescent or terminally differentiated cells. Central to this infectivity is the action of the small-size adenovirus E1A protein of 243 amino acid (aa) residues (243R), which creates a condition favorable for viral replication (1). As such, this protein is principally responsible for transitioning cells out of quiescence and into S phase or for reactivating DNA synthesis in terminally differentiated muscle cells (3,17,25). Earlier studies have shown that the activities of E1A in this context are largely dependent upon its ability to physically associate with members of the retinoblastoma family of proteins, e.g., pRb and p130 (6,10,17). Both of these proteins are widely known for their ability to regulate the E2F family of transcription factors (E2F1 to E2F5), which play pivotal roles in regulating the expression of genes involved in cell cycle reentry and DNA synthesis (2). In general, the functions of the E2Fs serve broad roles, with E2F1 to -3 acting as transcriptional activators and E2F4 to -5 as transcriptional repressors. The remaining E2Fs (E2F6 to -8) can also act as transcriptional repressors, but in an Rb-independent manner (29). In cycling cells, pRb is believed to inhibit the activating function of E2F1 by recruiting chromatin-modifying complexes with histone deacetylase (HDAC) or histone methyltransferase activity to E2F-regulated genes (8). However, such recruitment may be important only for repressing key E2F promoters under specific conditions (e.g., Ras-induced senescence) since chromatin imm...

The Chaperone-assisted E3 Ligase C Terminus of Hsc70-interacting Protein (CHIP) Targets PTEN for Proteasomal Degradation

“…For ICC, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton-X-100, and blocked with 2.5% BSA in PBS before incubation with primary antibodies overnight at 4°C, followed by secondary antibodies for 1 h at room temperature under dark conditions according to established protocols (48). The images were captured on an Olympus BX61 motorized fluorescence microscope using Image ProPlus software and a Nikon A1 Laser scanning confocal microscope.…”

Adenovirus E1A Directly Targets the E2F/DP-1 Complex

“…The region of E1A that is responsible for binding to pRb and inhibiting its interaction with E2Fs is the CR2 domain, with a portion of the CR1 domain stabilizing the interaction and displacing E2Fs (2). E1A 12S overcomes the repression of E2F-regulated genes to force quiescent cells to enter the cell cycle by eliminating corepressor complexes consisting of p130-E2F4 and HDAC1/ 2-mSin3B from the promoters of E2F-regulated genes (29). Once these repressive complexes are removed, the promoters become occupied by activating E2F family members, which stimulate transcription (29).…”

“…E1A 12S overcomes the repression of E2F-regulated genes to force quiescent cells to enter the cell cycle by eliminating corepressor complexes consisting of p130-E2F4 and HDAC1/ 2-mSin3B from the promoters of E2F-regulated genes (29). Once these repressive complexes are removed, the promoters become occupied by activating E2F family members, which stimulate transcription (29). In summary, E1A interacts with several pRb family members in order to inhibit their interaction with E2Fs and induce cell cycle entry, as well as to activate a variety of cellular and viral genes.…”

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

“…24 The E1A protein from adenoviruses also displays this property. 26,27 A potential co-infection with an adenovirus was investigated by PCR analysis using cerebellumextracted DNA from the 4 cats with extracerebellar FPV protein expression, but no evidence of an adenoviral co-infection was found. Herpes viruses interfere with the E2F transcription factors through different mechanisms, either by retinoblastoma protein phosphorylation and E2F dependent genes expression, or by the production of viral retinoblastoma-inactivating proteins.…”

“…[24][25][26][27] Nested PCRs using either pan herpes (215-235 bp products from the viral DNA polymerase) or pan adeno primers (324 bp product from the viral DNA polymerase gene) were conducted on cats 7, 10-12 ( Fig. 2).…”