ACTR/AIB1 functions as an E2F1 coactivator to promote breast cancer cell proliferation and antiestrogen resistance.

Overexpression or amplification of ACTR (also named AIB1, RAC3, p/CIP, TRAM-1, and SRC-3), a member of the p160 family of coactivators for nuclear hormone receptors, has been frequently detected in multiple types of human tumors, including breast cancer. However, its role in cancer cell proliferation and the underlying mechanism are unclear. Here, we show that overexpression of ACTR not only enhances estrogen-stimulated cell proliferation but also, more strikingly, completely negates the cell cycle arrest effect by tamoxifen and pure antiestrogens. Unexpectedly, we found that ACTR directly interacts, through its N-terminal domain, with E2F1 and is recruited to E2F target gene promoters. Elevation of ACTR in quiescent cells strongly stimulates the transcription of a subset of E2F-responsive genes that are associated with the G(1)/S transition. We also demonstrated, by adenovirus vector-mediated RNA interference, that ACTR is required for E2F1-mediated gene expression and the proliferation of estrogen receptor (ER)-negative breast cancer cells. Moreover, the ability of elevated ACTR to promote estrogen-independent cell proliferation depends on the function of E2F1 and the association between ACTR and E2F1, but not ER. Thus, our results reveal an essential role of ACTR in control of breast cancer cell proliferation and implicate the ACTR-E2F1 pathway as a novel mechanism in antiestrogen resistance.

The neutralizing effect of a polyclonal antibody raised against the N-terminal eighteen-aminoacid residues of birtoxin towards the whole venom of Parabuthus transvaalicus.

Scorpion venom is composed among other things of a large number of neurotoxic peptides affecting all major types of ion channels. The majority of the toxicity of the venom is attributed to the presence of these peptides. In our previous studies using a combination of HPLC and mass spectrometry, we showed that birtoxin like peptides are the major peptidic components of the venom of Parabuthus transvaalicus. These peptides are quite similar to each other differing by only few amino acid residues. In addition they all share a common N-terminus of eighteen amino acid residues. We hypothesize that neutralization of this domain will decrease the toxicity of the whole venom of P. transvaalicus. Polyclonal antibodies against the common N-terminal region of the peptides are generated. Here we show by bioassays that the polyclonal antibodies neutralize the venom of P. transvaalicus in a dose dependent manner and by mass spectrometry and western blotting that these peptides indeed react with the polyclonal antibodies. Previously antibodies generated against a single major toxic component of venom have proven to be an effective strategy for antivenin production. In the case of P. transvaalicus the generated antibody is against the majority of the peptidic fraction due to the presence of several highly similar and highly toxic components in this venom. We show that using the knowledge obtained through biochemical characterization studies it is possible to design very specific antibodies that will be useful for clinical applications against Parabuthus envenomation.

E2F in vivo binding specificity: comparison of consensus versus nonconsensus binding sites.

We have previously shown that most sites bound by E2F family members in vivo do not contain E2F consensus motifs. However, differences between in vivo target sites that contain or lack a consensus E2F motif have not been explored. To understand how E2F binding specificity is achieved in vivo, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using chromatin immunoprecipitation coupled with DNA microarray analysis (ChIP-chip) assays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are (1) the site must be in a core promoter and (2) the region must be utilized as a promoter in that cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including (1) indirect recruitment, (2) looping to the core promoter mediated by an E2F bound to a distal motif, and (3) assisted binding of E2F to a site that weakly resembles an E2F motif. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated fragments. Our findings suggest that in vivo (1) a consensus motif is not sufficient to recruit E2Fs, (2) E2Fs can bind to isolated regions that lack a consensus motif, and (3) binding can require regions other than the best match to the E2F motif.

Using ChIP-chip technology to reveal common principles of transcriptional repression in normal and cancer cells.

We compared 12 different cell populations, including embryonic stem cells before and during differentiation into embryoid bodies as well as various types of normal and tumor cells to determine if pluripotent versus differentiated cell types use different mechanisms to establish their transcriptome. We first identified genes that were not expressed in the 12 different cell populations and then determined which of them were regulated by histone methylation, DNA methylation, at the step of productive elongation, or by the inability to establish a preinitiation complex. For these experiments, we performed chromatin immunoprecipitation using antibodies to H3me3K27, H3me3K9, 5-methyl-cytosine, and POLR2A. We found that (1) the percentage of low expressed genes bound by POLR2A, H3me3K27, H3me3K9, or 5-methyl-cytosine is similar in all 12 cell types, regardless of differentiation or neoplastic state; (2) a gene is generally repressed by only one mechanism; and (3) distinct classes of genes are repressed by certain mechanisms. We further characterized two transitioning cell populations, 3T3 cells progressing from G0/G1 into S phase and mES cells differentiating into embryoid bodies. We found that the transient regulation through the cell cycle was achieved predominantly by changes in the recruitment of the general transcriptional machinery or by post-POLR2A recruitment mechanisms. In contrast, changes in chromatin silencing were critical for the permanent changes in gene expression in cells undergoing differentiation.

A comprehensive ChIP-chip analysis of E2F1, E2F4, and E2F6 in normal and tumor cells reveals interchangeable roles of E2F family members.

Using ChIP-chip assays (employing ENCODE arrays and core promoter arrays), we examined the binding patterns of three members of the E2F family in five cell types. We determined that most E2F1, E2F4, and E2F6 binding sites are located within 2 kb of a transcription start site, in both normal and tumor cells. In fact, the majority of promoters that are active (as defined by TAF1 or POLR2A binding) in GM06990 B lymphocytes and Ntera2 carcinoma cells were also bound by an E2F. This very close relationship between E2F binding sites and binding sites for general transcription factors in both normal and tumor cells suggests that a chromatin-bound E2F may be a signpost for active transcription initiation complexes. In general, we found that several E2Fs bind to a given promoter and that there is only modest cell type specificity of the E2F family. Thus, it is difficult to assess the role of any particular E2F in transcriptional regulation, due to extreme redundancy of target promoters. However, Ntera2 carcinoma cells were exceptional in that a large set of promoters were bound by E2F6, but not by E2F1 or E2F4. It has been proposed that E2F6 contributes to gene silencing by recruiting enzymes involved in methylating histone H3. To test this hypothesis, we created Ntera2 cell lines harboring shRNAs to E2F6. We found that reduction of E2F6 only induced minimal alteration of the transcriptome of Ntera2 transcriptome. Our results support the concept of functional redundancy in the E2F family and suggest that E2F6 is not critical for histone methylation.

A computational genomics approach to identify cis-regulatory modules from chromatin immunoprecipitation microarray data--a case study using E2F1.

Advances in high-throughput technologies, such as ChIP-chip, and the completion of human and mouse genomic sequences now allow analysis of the mechanisms of gene regulation on a systems level. In this study, we have developed a computational genomics approach (termed ChIPModules), which begins with experimentally determined binding sites and integrates positional weight matrices constructed from transcription factor binding sites, a comparative genomics approach, and statistical learning methods to identify transcriptional regulatory modules. We began with E2F1 binding site information obtained from ChIP-chip analyses of ENCODE regions, from both HeLa and MCF7 cells. Our approach not only distinguished targets from nontargets with a high specificity, but it also identified five regulatory modules for E2F1. One of the identified modules predicted a colocalization of E2F1 and AP-2alpha on a set of target promoters with an intersite distance of <270 bp. We tested this prediction using ChIP-chip assays with arrays containing approximately 14,000 human promoters. We found that both E2F1 and AP-2alpha bind within the predicted distance to a large number of human promoters, demonstrating the strength of our sequence-based, unbiased, and universal protocol. Finally, we have used our ChIPModules approach to develop a database that includes thousands of computationally identified and/or experimentally verified E2F1 target promoters.