Stephanie M. Bartley

Use of Chromatin Immunoprecipitation To Clone Novel E2F Target Promoters

ABSTRACT We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles.

Target Gene Specificity of E2F and Pocket Protein Family Members in Living Cells

ABSTRACT E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G0 phase of the cell cycle and release of the pocket protein in late G1, followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G0 and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

The identification of E2F1-specific target genes

The E2F family of transcriptional regulators consists of six different members. Analysis of E2F-regulated promoters by using cultured cells suggests that E2Fs may have redundant functions. However, animal studies have shown that loss of individual E2Fs can have distinct biological consequences. Such seemingly conflicting results could be due to a difference in E2F-mediated regulation in cell culture vs. animals. Alternatively, there may be genes that are specifically regulated by an individual E2F which have not yet been identified. To investigate this possibility further, we have analyzed gene expression in E2F1 nullizygous mice. We found that loss of E2F1 did not cause changes in expression of known E2F target genes, suggesting that perhaps E2F1-specific promoters are distinct from known E2F target promoters. Therefore, we used oligonucleotide microarrays to identify mRNAs whose expression is altered on loss of E2F1. We demonstrate by chromatin immunoprecipitation that several of the promoters that drive expression of the deregulated mRNAs selectively recruit E2F1, but not other E2Fs, and this recruitment is via an element distinct from a consensus E2F binding site. To our knowledge, these are as yet undocumented examples of promoters being occupied in asynchronously growing cells by a single E2F family member. Interestingly, the E2F1-specific target genes that we identified encode proteins having functions quite different from the function of known E2F target genes. Thus, whereas E2F1 may share redundant functions in cell growth control with other E2F family members, it may also play an important biological role distinct from the other E2Fs.

A method for identification of vocal fold lamina propria fibroblasts in culture.

Objective Vocal fold biology research is emerging as a vital area of study in laryngology. One impediment is the lack of both commercially available vocal fold lamina propria fibroblasts and a constitutively expressed specific marker for fibroblasts. We present an in vitro technique that allows for identification of fibroblasts by ruling out the possibility of the cells belonging to other lineages that are found in vocal fold tissue. Study Design An in vitro study. Methods Two primary vocal fold fibroblast cell lines and one immortalized vocal fold fibroblast cell line were cultured. Immunohistologic staining for α-actinin, cytokeratin 19, and von Willebrand factor was completed for the three fibroblast lines in addition to skeletal, endothelial, and epithelial cell lines. Cell type was differentiated by positive staining for α-actinin, cytokeratin 19, and von Willebrand factor. Results Fibroblast cultures did not express α-actinin, cytokeratin 19, and von Willebrand factor, whereas skeletal muscle, endothelial, and epithelial cultured cells expressed each respectively. Conclusions This simple rule-out methodology for fibroblast confirmation is an important step when establishing cell culture, and it establishes sound internal validity particularly in the early stages of this emerging area of study.

No effect of loss of E2F1 on liver regeneration or hepatocarcinogenesis in C57BL/6J or C3H/HeJ mice

The E2F family of transcription factors regulates the expression of genes needed for DNA synthesis and cell‐cycle control. However, the individual contributions of the different E2F family members in regulating proliferation in various tissues have not been well characterized. Mouse liver is an excellent system for investigating proliferation because its growth state can be experimentally manipulated. As observed in cell culture systems, E2F1 protein is present at low levels in the quiescent liver, with an increase in expression during proliferation. Therefore, we expected that E2F1 may play an important role in cell‐growth control during periods of robust proliferation. Using E2F1‐nullizygous mice, we performed partial hepatectomies to investigate the role of E2F1 in the synchronous proliferation of adult hepatocytes. We found that E2F1 deficiency resulted in only minor changes in gene expression and that the timing of liver regeneration was not altered in E2F1 nullizygous mice. E2F1 has displayed properties of both a tumor suppressor and an oncogene in different model systems. Therefore, we investigated the role of E2F1 in rapidly growing liver tumor cells in strains of mice that have high (C3H/HeJ) and low (C57BL/6J) rates of hepatocarcinogenesis. We observed no significant differences in the number of liver tumors that developed after diethylnitrosamine treatment of wild type versus E2F1‐nullizygous mice. We suggest that abundant levels of E2F4 in the mouse liver compensate for loss of E2F1. Mol. Carcinog. 25:295–303, 1999.

Vocal fold myofibroblast profile of scarring

Vocal fold fibroblasts (VFF) are responsible for extracellular matrix synthesis supporting lamina propria in normal and diseased conditions. When tissue is injured, VFF become activated and differentiate into myofibroblasts to facilitate wound healing response. We investigated if vocal fold myofibroblasts can be utilized as surrogate cells for scarred VFF.