Julie Wells

Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation

In their article, A.S. Weinmann and P.J. Farnham (2002, Methods 26) described new techniques for isolating in vivo binding sites for any DNA-binding protein. In this article, we describe complementary methods for detailed in vivo characterizations of such identified protein-DNA interactions. First, we describe how formaldehyde crosslinking and chromatin immunoprecipitation (ChIP), in conjunction with transient transfections or the use of cell lines containing stably integrated constructs or episomes, can be employed to identify which specific nucleotides of a region of DNA are required for recruitment of a particular transcription factor. In contrast to in vivo footprinting, this method not only specifies which nucleotides are bound, but also identifies the protein(s) involved in binding. Next, we discuss the use of the ChIP assay to study how binding of a transcription factor is altered by passage through the cell cycle, by overexpression or deletion of another factor, or during tumorigenesis. Finally, a look toward the future suggests that the ChIP assay may be combined with Western blot analysis or mass spectrometry to identify additional proteins that interact with a transcription factor of interest.

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.

Mre11 Complex and DNA Replication: Linkage to E2F and Sites of DNA Synthesis

ABSTRACT We show that the Mre11 complex associates with E2F family members via the Nbs1 N terminus. This association and Nbs1 phosphorylation are correlated with S-phase checkpoint proficiency, whereas neither is sufficient individually for checkpoint activation. The Nbs1 E2F interaction occurred near the Epstein-Barr virus origin of replication as well as near a chromosomal replication origin in the c-myc promoter region and was restricted to S-phase cells. The Mre11 complex colocalized with PCNA at replication forks throughout S phase, both prior to and coincident with the appearance of nascent DNA. These data suggest that the Mre11 complex suppresses genomic instability through its influence on both the regulation and progression of DNA replication.

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.

Identification of novel pRb binding sites using CpG microarrays suggests that E2F recruits pRb to specific genomic sites during S phase

The retinoblastoma (Rb) tumor suppressor protein is an important regulator of cell proliferation and differentiation. Many studies have shown that pRb can negatively regulate the activity of the E2F family of transcription factors during G0 and G1 phases of the cell cycle, perhaps by serving as a bridge between the E2Fs and transcriptional repressors such as histone deacetylases and methylases. However, pRb has also been shown to localize to discrete DNA foci during S phase, a time at which pRb is thought to be dissociated from E2F. Numerous other DNA binding proteins have been shown to interact with pRb, suggesting that pRb may control progression through S phase by binding to sites in the genome distinct from E2F target gene promoters. To test this hypothesis, we have identified novel pRb binding sites within the human genome using an unbiased approach which relies upon a combination of chromatin immunoprecipitation and CpG microarray analysis. To provide the greatest opportunity of finding distinct sets of pRb binding sites, we examined pRb binding in chromatin obtained from human Raji cells synchronized in either G0/G1 phase or S phase. These experiments have allowed us to identify a large set of new genomic binding sites for the pRb protein. We found that some sites are occupied by pRb only during G0/G1 phase, as would be predicted from previous models of pRb function. We also identified sites to which pRb bound only during S phase and other sites which were bound constitutively by pRb. Surprisingly, we found that E2F1 was present at most of the CpG islands bound by pRb, independent of the phase of the cell cycle. Thus, although pRb has the potential to interact with numerous transcription factors, our data suggest that the majority of DNA-bound pRb is recruited to E2F target promoters during both G0/G1 and S phases.

The Chromatin Structure of the Dual c-myc Promoter P1/P2 Is Regulated by Separate Elements

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-mycchromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-mycpromoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

The tumor suppressor WTX shuttles to the nucleus and modulates WT1 activity

WTX encodes a tumor suppressor gene inactivated in Wilms tumor and recently implicated in WNT signaling through enhancement of cytoplasmic β-catenin (CTNNB1) degradation. Here, we report that WTX translocates to the nucleus, a property that is modified by an endogenous splicing variant and is modulated by a nuclear export inhibitor. WTX is present in distinct subnuclear structures and co-localizes with the paraspeckle marker p54NRB/NONO, suggesting a role in transcriptional regulation. Notably, WTX binds WT1, another Wilms tumor suppressor and stem cell marker that encodes a zinc-finger transcription factor, and enhances WT1-mediated transcription of Amphiregulin, an endogenous target gene. Together, these observations suggest a role for WTX in nuclear pathways implicated in the transcriptional regulation of cellular differentiation programs.

Congenital diaphragmatic hernia candidate genes derived from embryonic transcriptomes

Congenital diaphragmatic hernia (CDH) is a common (1 in 3,000 live births) major congenital malformation that results in significant morbidity and mortality. The discovery of CDH loci using standard genetic approaches has been hindered by its genetic heterogeneity. We hypothesized that gene expression profiling of developing embryonic diaphragms would help identify genes likely to be associated with diaphragm defects. We generated a time series of whole-transcriptome expression profiles from laser captured embryonic mouse diaphragms at embryonic day (E)11.5 and E12.5 when experimental perturbations lead to CDH phenotypes, and E16.5 when the diaphragm is fully formed. Gene sets defining biologically relevant pathways and temporal expression trends were identified by using a series of bioinformatic algorithms. These developmental sets were then compared with a manually curated list of genes previously shown to cause diaphragm defects in humans and in mouse models. Our integrative filtering strategy identified 27 candidates for CDH. We examined the diaphragms of knockout mice for one of the candidate genes, pre–B-cell leukemia transcription factor 1 (Pbx1), and identified a range of previously undetected diaphragmatic defects. Our study demonstrates the utility of genetic characterization of normal development as an integral part of a disease gene identification and prioritization strategy for CDH, an approach that can be extended to other diseases and developmental anomalies.

SUMO-1 Modification of the Wilms’ Tumor Suppressor WT1

SUMO-1 conjugation modulates numerous cellular functions, including the subnuclear localization of its target proteins. The WT1 tumor suppressor encodes a four-zinc finger protein with distinct splicing isoforms. WT1(−KTS), encoding uninterrupted zinc fingers, functions as a transcription factor and has a diffusely nuclear distribution; WT1(+KTS), with an insertion of three amino acids (KTS) between zinc fingers three and four, localizes to discrete nuclear speckles, the function of which is unknown. Because the SUMO-1 E2-conjugating enzyme, Ubc9, interacts with WT1, we tested whether sumoylation modulates the cellular localization of WT1. We find here that both WT1 isoforms are directly sumoylated on lysine residues 73 and 177. Although RNA interference-mediated Ubc9 depletion effectively suppresses WT1 nuclear speckles, a SUMO-1–deficient WT1(+KTS)(K73, 177R) double mutant retains localization to speckles. Thus, direct sumoylation of WT1 is not responsible for its cellular localization, and other sumoylated proteins may target WT1 to these nuclear structures. Identification of other components of WT1-associated speckles is likely to provide clues to their function.

Ovol2 Suppresses Cell Cycling and Terminal Differentiation of Keratinocytes by Directly Repressing c-Myc and Notch1

Ovol2 belongs to the Ovo family of evolutionarily conserved zinc finger transcription factors that act downstream of key developmental signaling pathways including Wg/Wnt and BMP/TGF-β. We previously reported Ovol2 expression in the basal layer of epidermis, where epidermal stem/progenitor cells reside. In this work, we use HaCaT human keratinocytes to investigate the cellular and molecular functions of Ovol2. We show that depletion of Ovol2 leads to transient cell expansion but a loss of cells with long term proliferation potential. Mathematical modeling and experimental findings suggest that both faster cycling and precocious withdrawal from the cell cycle underlie this phenotype. Ovol2 depletion also accelerates extracellular signal-induced terminal differentiation in two- and three-dimensional culture models. By chromatin immunoprecipitation, luciferase reporter, and functional rescue assays, we demonstrate that Ovol2 directly represses two critical downstream targets, c-Myc and Notch1, thereby suppressing keratinocyte transient proliferation and terminal differentiation, respectively. These findings shed light on how an epidermal cell maintains a proliferation-competent and differentiation-resistant state.