E2F-6, a member of the E2F family that can behave as a transcriptional repressor

Abstract

The E2F family of proteins is required to establish the correct cell-cycle-dependent transcription of genes that direct the process of cell division. All previously identified E2F proteins can act in a similar manner; depending on whether or not they are associated with the cell cycle inhibitors the retinoblastoma protein (pRB), p107, or p130, they can either repress or activate the transcription of E2F-responsive genes. We now report the cloning and characterization of another E2F family member, E2F-6, whose structure is reminiscent of the dominant inhibitors of other transcription factor families. The dimerization and DNA binding properties of E2F-6 are similar to those of the other E2F family members. However, it is not regulated by pRB, p107, or p130, and it is unable to activate transcription. Instead, it can act to repress the transcription of E2F responsive genes by countering the activity of the other E2F complexes via a pRB-, p107-, or p130-independent mechanism.

Figure 1

E2F-6 is another member of the E2F family. (A) Amino acid sequence comparison of human versus murine E2F-6. (B) Amino acid sequence comparison of the human E2F proteins. Domains responsible for DNA binding, dimerization (leucine zipper, marked box), and pocket protein binding are indicated. Conserved residues are denoted in boldface type.

Figure 2

Expression patterns of E2F-6. (A) Northern blot analysis of poly(A)⁺ RNA isolated from the indicated cell lines and tissues (human tissue blot from CLONTECH), screened with a probe derived from the full-length E2F-6 coding sequence. (B) Western blot analysis of extracts derived from C33-A cells transiently transfected with pCMV–HA–E2F-6 (200 ng) and from untransfected U2OS, C33-A, ML-1, and 293 cells (50 μg). The positions of HA–E2F-6 and endogenous E2F-6 proteins are indicated by arrows. The band running beneath HA–E2F-6 is a degradation product of this transfected protein (data not shown). The asterisk denotes a nonspecific band.

Figure 3

Dimerization and DNA binding properties of E2F-6 (A) C33-A cells were transiently transfected with expression vectors encoding E2F-1, E2F-4, or HA–E2F-6 in the presence or absence of DP (DP-1 or DP-2) or pocket proteins (pRB, p107) and immunoprecipitated with the indicated antibodies. (B) C33-A cells were transiently transfected with the identical combinations of expression vectors as in A. Gel shift assays were carried out on the indicated unlabeled cell extracts (1.5 μg of total protein per lane) by using the consensus E2F site from the adenoviral E2 promoter (TTTCGCGCCCTTT). Western blot assays were carried out on the same unlabeled cell extracts (300 ng of total protein per lane) by using monoclonal antibodies against E2F-1 (KH95), E2F-4 (LLF4–1), or the HA tag (12CA5). Gel retardation assays have also been conducted by using the additional E2F sites TTTCCCGCCTTT, TTTCCCGCCAAA, TTTCCCGCGTGT, or ATTCCCGCGCTTT with similar differences in affinity.

Figure 4

Effects of E2F-6 on transactivation of an E2F₄-CAT reporter C33-A cells were transiently transfected in duplicate with 4 μg of E2F₄-CAT, 2 μg of Rous sarcoma virus–luciferase (as an internal control), 14 μg of carrier DNA (pBKS+ and pCMV-neo-Bam) in the absence or presence of 3 μg of pCMV-HA-DP2 plus pCMV-E2F expression vectors as indicated. CAT and luciferase activity were determined 24 h after transfection. The values shown are the average of duplicate transfectants for representative experiments.