Laszlo Jakoi

E2F4 and E2F5 Play an Essential Role in Pocket Protein–Mediated G1 Control

E2F transcription factors are major regulators of cell proliferation. The diversity of the E2F family suggests that individual members perform distinct functions in cell cycle control. E2F4 and E2F5 constitute a defined subset of the family. Until now, there has been little understanding of their individual biochemical and biological functions. Here, we report that simultaneous inactivation of E2F4 and E2F5 in mice results in neonatal lethality, suggesting that they perform overlapping functions during mouse development. Embryonic fibroblasts isolated from these mice proliferated normally and reentered from Go with normal kinetics compared to wild-type cells. However, they failed to arrest in G1 in response to p16INK4a. Thus, E2F4 and E2F5 are dispensable for cell cycle progression but necessary for pocket protein-mediated G1 arrest of cycling cells.

The accumulation of an E2F-p130 transcriptional repressor distinguishes a G0 cell state from a G1 cell state.

Previous studies have demonstrated cell cycle-dependent specificities in the interactions of E2F proteins with Rb family members. We now show that the formation of an E2F-p130 complex is unique to cells in a quiescent, G0 state. The E2F-p130 complex does not reform when cells reenter a proliferative state and cycle through G1. The presence of an E2F-p130 complex in quiescent cells coincides with the E2F-mediated repression of transcription of the E2F1 gene, and we show that the E2F sites in the E2F1 promoter are important as cells enter quiescence but play no apparent role in cycling cells. In addition, the decay of the E2F-p130 complex as cells reenter the cell cycle requires the action of G1 cyclin-dependent kinase activity. We conclude that the accumulation of the E2F-p130 complex in quiescent cells provides a negative control of certain key target genes and defines a functional distinction between these G0 cells and cells that exist transiently in G1.

Identification of a Novel E2F3 Product Suggests a Mechanism for Determining Specificity of Repression by Rb Proteins

ABSTRACT The tumor suppressor function of Rb is intimately related to its ability to interact with E2F and repress the transcription of E2F target genes. Here we describe a novel E2F product that specifically interacts with Rb in quiescent cells. This novel E2F, which we term E2F3b, is encoded by a unique mRNA transcribed from an intronic promoter within the E2F3 locus. The E2F3b RNA differs from the previously characterized E2F3 RNA, which we now term E2F3a, by the utilization of a unique coding exon. In contrast to the E2F3a product that is tightly regulated by cell growth, the E2F3b product is expressed equivalently in quiescent and proliferating cells. But, unlike the E2F4 and E2F5 proteins, which are also expressed in quiescent cells and form complexes with the p130 protein, the E2F3b protein associates with Rb and represents the predominant E2F-Rb complex in quiescent cells. Thus, the previously described specificity of Rb function as a transcriptional repressor in quiescent cells coincides with the association of Rb with this novel E2F product.

Interactions of the p107 and Rb proteins with E2F during the cell proliferation response

The E2F transcription factor is found in complexes with a variety of cellular proteins including the retinoblastoma tumor suppressor protein. Various assays have demonstrated a tight correlation between the functional capacity of Rb as a growth suppressor and its ability to bind to E2F. Moreover, only the underphosphorylated form of Rb, which appears to be the active species, interacts with E2F. Despite the fact that the majority of Rb becomes hyperphosphorylated at the end of G1, we now show that the E2F‐Rb interaction persists through the G1/S transition and into S phase. A distinct E2F complex does appear to be regulated in relation to the transition from G1 to S phase. We now demonstrate that this complex contains the Rb‐related p107 protein. Moreover, like the Rb protein, p107 inhibits E2F‐dependent transcription in a co‐transfection assay. This result, together with the observation that free, uncomplexed E2F accumulates as cells leave G1 and enter S phase, suggests that the p107 protein may regulate E2F‐dependent transcription during G1. In contrast, although Rb does regulate the transcriptional activity of E2F, this association does not coincide with the G1 to S phase transition.

Loss of E2F4 activity leads to abnormal development of multiple cellular lineages.

We have generated mice deficient in E2F4 activity, the major form of E2F in many cell types. Analysis of newborn pups deficient in E2F4 revealed abnormalities in hematopoietic lineage development as well as defects in the development of the gut epithelium. Specifically, we observed a deficiency of various mature hematopoietic cell types together with an increased number of immature cells in several lineages. This was associated with an increased frequency of apoptotic cells. We also found a substantial reduction in the thickness of the gut epithelium that normally gives rise to crypts as well as a reduction in the density of villi. These observations suggest a critical role for E2F4 activity in controlling the maturation of cells in a number of tissues.

FUNCTIONAL ANALYSIS OF E2F TRANSCRIPTION FACTOR

Publisher Summary This chapter discusses the functional analysis of E2F transcription factor. Originally identified as a transcriptional activity important for the regulation of the adenovirus E2 gene, the E2F (E2 factor) transcription factor has now been shown to play a critical role in the control of transcription during the G1 to S-phase transition. It is also evident that E2F activity is complex and is composed of a family of proteins that generates various heterodimeric complexes with specific DNA binding activity. A variety of studies have demonstrated specificities in the interaction of E2F family members with Rb family members during cell cycle progression. This chapter describes three aspects of functional assays for E2F activity: (1) the assay of E2F DNA binding activity in cell extracts by standard gel retardation, (2) transient transfection assays that measure the cell cycle-dependent expression of E2F controlled promoters, and (3) construction of recombinant adenovirus vectors that allow expression of E2F proteins to better explore the role of E2F in cellular growth control.

A combinatorial mechanism for determining the specificity of E2F activation and repression

Various studies have detailed the role of E2F proteins in both transcription activation and repression. Further study has shown that distinct promoter elements, but comprising the same E2F-recognition motif, confer positive or negative E2F control and that this reflects binding of either activator or repressor E2F proteins, respectively. We now show that the specificity of binding of an activator or repressor E2F protein is determined by adjacent sequences that bind a cooperating transcription factor. We propose that the functional E2F element is a module comprising not only the E2F-binding site but also the adjacent site for the cooperating transcription factor.

A method for semiautomatic differential analysis of urinary catecholamines.

Abstract A semiautomatic method for analysis of urinary catecholamines by the trihydroxyindole reaction utilizing an AutoAnalyzer is described in detail with modifications providing precision and ease of operation. Recovery of norepinephrine is 89 ± 8.9%; of epinephrine 76 ± 6.8%. Values for healthy ambulatory subjects are 27.5 ± 9.3 μg norepinephrine and 7.5 ± 2.2 μg epinephrine per 24 hr. Data regarding linearity, reproducibility, and repeatability are given.