Search

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes., The authors summarize the data produced by phase III of the Encyclopedia of DNA Elements (ENCODE) project, a resource for better understanding of the human and mouse genomes.

Background Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems. Results Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9. Conclusions These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type. Electronic supplementary material The online version of this article (10.1186/s13072-019-0275-8) contains supplementary material, which is available to authorized users.

Enhancers are short regulatory sequences bound by sequence-specific transcription factors and play a major role in the spatiotemporal specificity of gene expression patterns in development and disease. While it is now possible to identify enhancer regions genomewide in both cultured cells and primary tissues using epigenomic approaches, it has been more challenging to develop methods to understand the function of individual enhancers because enhancers are located far from the gene(s) that they regulate. However, it is essential to identify target genes of enhancers not only so that we can understand the role of enhancers in disease but also because this information will assist in the development of future therapeutic options. After reviewing models of enhancer function, we discuss recent methods for identifying target genes of enhancers. First, we describe chromatin structure-based approaches for directly mapping interactions between enhancers and promoters. Second, we describe the use of correlation-based approaches to link enhancer state with the activity of nearby promoters and/or gene expression. Third, we describe how to test the function of specific enhancers experimentally by perturbing enhancer–target relationships using high-throughput reporter assays and genome editing. Finally, we conclude by discussing as yet unanswered questions concerning how enhancers function, how target genes can be identified, and how to distinguish direct from indirect changes in gene expression mediated by individual enhancers.

DNA methylation, which occurs predominantly at CpG dinucleotides, is a potent epigenetic repressor of transcription. Because DNA methylation is reversible, there is much interest in understanding the mechanisms by which it can be regulated by DNA-binding transcription factors. We discuss several models that, by incorporating sequence motifs, CpG density, and methylation levels, attempt to link the binding of a transcription factor with the acquisition or loss of DNA methylation at promoters and distal regulatory elements. Additional in vivo genome-wide characterization of transcription factor binding patterns and high-resolution DNA methylation analyses are clearly required for stronger support of each model.

C2H2 zinc fingers are found in several key transcriptional regulators in the immune system. However, these proteins usually contain more fingers than are needed for sequence-specific DNA binding, which suggests that different fingers regulate different genes and functions. Here we found that mice lacking finger 1 or finger 4 of Ikaros exhibited distinct subsets of the hematological defects of Ikaros-null mice. Most notably, the two fingers controlled different stages of lymphopoiesis, and finger 4 was selectively required for tumor suppression. The distinct defects support the hypothesis that only a small number of genes that are targets of Ikaros are critical for each of its biological functions. The subcategorization of functions and target genes by mutagenesis of individual zinc fingers will facilitate efforts to understand how zinc-finger transcription factors regulate development, immunity and disease.

Variability in the quality of antibodies to histone post-translational modifications (PTMs) presents widely recognized hindrance in epigenetics research. Here, by using antibody engineering technologies we produced recombinant antibodies directed to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and their high specificity enabled us to identify positive and negative correlations among histone PTMs.

The self-renewal capacity ascribed to embryonic stem cells (ESC) is reminiscent of cancer cell proliferation, raising speculation that a common network of genes may regulate these traits. A search for general regulators of these traits yielded a set of microRNAs for which expression is highly enriched in human ESCs and liver cancer cells (HCC) but attenuated in differentiated quiescent hepatocytes. Here, we show that these microRNAs promote hESC self-renewal, as well as HCC proliferation, and when overexpressed in normally quiescent hepatocytes, induce proliferation and activate cancer signaling pathways. Proliferation in hepatocytes is mediated through translational repression of Pten, Tgfbr2, Klf11, and Cdkn1a, which collectively dysregulates the PI3K/AKT/mTOR and TGFβ tumor suppressor signaling pathways. Furthermore, aberrant expression of these miRNAs is observed in human liver tumor tissues and induces epithelial–mesenchymal transition in hepatocytes. These findings suggest that microRNAs that are essential in normal development as promoters of ESC self-renewal are frequently upregulated in human liver tumors and harbor neoplastic transformation potential when they escape silencing in quiescent human hepatocytes. Mol Cancer Res; 10(7); 979–91. ©2012 AACR.

Developmental and homeostatic remodeling of cellular organelles is mediated by a complex process termed autophagy. The cohort of proteins that constitute the autophagy machinery functions in a multistep biochemical pathway. Though components of the autophagy machinery are broadly expressed, autophagy can occur in specialized cellular contexts, and mechanisms underlying cell-type-specific autophagy are poorly understood. We demonstrate that the master regulator of hematopoiesis, GATA-1, directly activates transcription of genes encoding the essential autophagy component microtubule-associated protein 1 light chain 3B (LC3B) and its homologs (MAP1LC3A, GABARAP, GABARAPL1, and GATE-16). In addition, GATA-1 directly activates genes involved in the biogenesis/function of lysosomes, which mediate autophagic protein turnover. We demonstrate that GATA-1 utilizes the forkhead protein FoxO3 to activate select autophagy genes. GATA-1-dependent LC3B induction is tightly coupled to accumulation of the active form of LC3B and autophagosomes, which mediate mitochondrial clearance as a critical step in erythropoiesis. These results illustrate a novel mechanism by which a master regulator of development establishes a genetic network to instigate cell-type-specific autophagy.

In mammalian cells, multiple cellular processes, including gene silencing, cell growth and differentiation, pluripotency, neoplastic transformation, apoptosis, DNA repair, and maintenance of genomic integrity, converge on the evolutionarily conserved protein KAP1, which is thought to regulate the dynamic organization of chromatin structure via its ability to influence epigenetic patterns and chromatin compaction. In this minireview, we discuss how KAP1 might execute such pleiotropic effects, focusing on genomic targeting mechanisms, protein-protein interactions, specific post-translational modifications of both KAP1 and associated histones, and transcriptome analyses of cells deficient in KAP1.

GATA factors interact with simple DNA motifs (WGATAR) to regulate critical processes, including hematopoiesis, but very few WGATAR motifs are occupied in genomes. Given the rudimentary knowledge of mechanisms underlying this restriction, and how GATA factors establish genetic networks, we used ChIP-seq to define GATA-1 and GATA-2 occupancy genome-wide in erythroid cells. Coupled with genetic complementation analysis and transcriptional profiling, these studies revealed a rich collection of targets containing a characteristic binding motif of greater complexity than WGATAR. GATA factors occupied loci encoding multiple components of the Scl/TAL1 complex, a master regulator of hematopoiesis and leukemogenic target. Mechanistic analyses provided evidence for cross-regulatory and autoregulatory interactions among components of this complex, including GATA-2 induction of the hematopoietic corepressor ETO-2 and an ETO-2 negative autoregulatory loop. These results establish fundamental principles underlying GATA factor mechanisms in chromatin and illustrate a complex network of considerable importance for the control of hematopoiesis.

A crucial question in the field of gene regulation is whether the location at which a transcription factor binds influences its effectiveness or the mechanism by which it regulates transcription. Comprehensive transcription factor binding maps are needed to address these issues, and genome-wide mapping is now possible thanks to the technological advances of ChIP-chip and ChIP-seq. This Review discusses how recent genomic profiling of transcription factors gives insight into how binding specificity is achieved and what features of chromatin influence the ability of transcription factors to interact with the genome. It also suggests future experiments that may further our understanding of the causes and consequences of transcription factor-genome interactions.

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.

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.

Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE.

A single chromatin immunoprecipitation (ChIP) sample does not provide enough DNA for hybridization to a genomic tiling array. A commonly used technique for amplifying the DNA obtained from ChIP assays is ligation-mediated PCR (LM-PCR). However, using this amplification method, we could not identify Oct4 binding sites on genomic tiling arrays representing 1% of the human genome (ENCODE arrays). In contrast, hybridization of a pool of 10 ChIP samples to the arrays produced reproducible binding patterns and low background signals. However, the pooling method would greatly increase the number of ChIP reactions needed to analyze the entire human genome. Therefore, we have adapted the GenomePlex® whole genome amplification (WGA) method for use in ChIP-chip assays; detailed ChIP and amplification protocols used for these analyses are provided as supplementary material. When applied to ENCODE arrays, the products prepared using this new method resulted in an Oct4 binding pattern similar to that from the pooled Oct4 ChIP samples. Importantly, the signal-to-noise ratio using the GenomePlex WGA method is superior to the LM-PCR amplification method.

Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation.

The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (∼1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

Natural killer (NK) and CD1d-restricted Valpha14i natural killer T (NKT) cells play a critical early role in host defense. Here we show that mice with a targeted deletion of T-bet, a T-box transcription factor required for Th1 cell differentiation, have a profound, stem cell-intrinsic defect in their ability to generate mature NK and Valpha14i NKT cells. Both cell types fail to complete normal terminal maturation and are present in decreased numbers in peripheral lymphoid organs of T-bet(-/-) mice. T-bet expression is regulated during NK cell differentiation by NK-activating receptors and cytokines known to control NK development and effector function. Our results identify T-bet as a key factor in the terminal maturation and peripheral homeostasis of NK and Valpha14i NKT cells.

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.

The standard chromatin immunoprecipitation (ChIP) assay is used to examine the specific association of transcription factors with DNA in the context of living cells. Here we review two modifications to this protocol which are designed to identify novel target genes of transcription factors in mammalian cells. The main advantage to both of these approaches is that only DNA sequences directly bound by a factor within the context of a living cell will be identified. Therefore, artifacts associated with overexpression and/or alterations in signaling pathways are avoided. The first modification we describe, a ChIP cloning strategy, can be used to isolate any genomic fragment specifically associated with a particular factor. It requires no special equipment or reagents other than a high-affinity antibody to be used for immunoprecipitation of the factor of interest. However, it is most useful for the isolation of a small number of genomic targets. In contrast, the second modification, which combines ChIP with specialized CpG microarrays, is ideal for a more global analysis of target genes. Advantages, common problems, and detailed protocols for these two ChIP techniques are discussed.

The role of intermediate methylation states in DNA is unclear. Here, to comprehensively identify regions of intermediate methylation and their quantitative relationship with gene activity, we apply integrative and comparative epigenomics to 25 human primary cell and tissue samples. We report 18,452 intermediate methylation regions located near 36% of genes and enriched at enhancers, exons and DNase I hypersensitivity sites. Intermediate methylation regions average 57% methylation, are predominantly allele-independent and are conserved across individuals and between mouse and human, suggesting a conserved function. These regions have an intermediate level of active chromatin marks and their associated genes have intermediate transcriptional activity. Exonic intermediate methylation correlates with exon inclusion at a level between that of fully methylated and unmethylated exons, highlighting gene context-dependent functions. We conclude that intermediate DNA methylation is a conserved signature of gene regulation and exon usage., Many loci in the mammalian genome are intermediately methylated. Here, by comprehensively identifying these loci and quantifying their relationship with gene activity, the authors show that intermediate methylation is an evolutionarily conserved epigenomic signature of gene regulation.

Transcriptional misregulation is involved in the development of many diseases, especially neoplastic transformation. Distal regulatory elements, such as enhancers, play a major role in specifying cell-specific transcription patterns in both normal and diseased tissues, suggesting that enhancers may be prime targets for therapeutic intervention. By focusing on modulating gene regulation mediated by cell type-specific enhancers, there is hope that normal epigenetic patterning in an affected tissue could be restored with fewer side effects than observed with treatments employing relatively nonspecific inhibitors such as epigenetic drugs. New methods employing genomic nucleases and site-specific epigenetic regulators targeted to specific genomic regions, using either artificial DNA-binding proteins or RNA–DNA interactions, may allow precise genome engineering at enhancers. However, this field is still in its infancy and further refinements that increase specificity and efficiency are clearly required.

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.

Overexpression of c-Myc can lead to altered transcriptional regulation of cellular genes and to neoplastic transformation. Although DNA binding is clearly required, the mechanism by which recruitment of c-Myc to target promoters results in transcriptional activation is highly debated. Much of this controversy comes from the difficulty in clearly defining a true Myc target gene. We have previously determined that cad is a bona fide Myc target gene and thus now use the cad promoter as a model to study Myc function. Others have shown that Myc can interact indirectly with histone acetylases and have suggested that Myc mediates transcriptional activation by causing an increase in the levels of acetylated histones on target promoters. To directly test this model, we employed a chromatin immunoprecipitation assay to examine the levels of acetylated histones on the cad promoter. Although Myc was bound to the cad promoter in S phase but not in G(0) phase, we found high levels of acetylated histones on the promoter in both stages. We also examined acetylated histones on the cad promoter before and after differentiation of U937 cells. Although the levels of c-Myc bound to the cad promoter were greatly reduced after differentiation, we saw high levels of acetylated histones on the cad promoter both before and after differentiation. Finally, we found that a 30-fold change in binding of N-Myc to the telomerase promoter did not result in a concomitant change in histone acetylation. Thus, recruitment of a Myc family member to a target promoter does not necessarily influence the amount of acetylated histones at that promoter. Further investigations are in progress to define the role of Myc in transcriptional activation.

The E2F family of transcription factors plays an important role in the regulation of gene expression at the G1/S-phase transition of the mammalian cell cycle (see reference 16 for an extensive review of the E2F regulatory pathway). E2F binding sites are found in the promoters of genes whose products are required for nucleotide synthesis (e.g., dihydrofolate reductase [DHFR] and thymidine kinase [TK]), for DNA replication (e.g., DNA polymerase α and cdc6), and for cell cycle progression (e.g., cyclin E, cyclin D1, c-myc, b-myb, and cdc2). Transcription from each of these promoters increases during late G1 or early S phase, and this regulation is mediated by protein binding to one or more E2F binding sites (3, 11, 22, 30, 34, 43, 44). To date, eight members of the E2F family have been identified: six E2F proteins (E2F1 to -6) and two DP proteins (DP1 and DP2). The E2F and DP proteins bind to DNA as a heterodimer and can function as activators of transcription. Alternatively, E2F-DP heterodimers can also repress transcription when complexed with members of the Rb family of pocket proteins (pRb, p107, and p130) due to the ability of the pocket proteins to bind to and mask the E2F transactivation domain and to recruit histone deacetylases (6, 17, 25). Individual E2Fs preferentially bind to different pocket proteins. E2F1, E2F2, and E2F3, for example, bind to pRb, while E2F4 predominantly binds to p130 and p107 and E2F5 binds to p130. A popular model for how E2F family members regulate G1/S-phase-specific gene expression invokes a complex pattern of protein-protein and protein-DNA interactions that change as cells progress through the cell cycle (Fig. ​(Fig.1A).1A). As depicted, transcription from E2F site-containing promoters is thought to be repressed in G0 phase due to the binding of a trimolecular E2F-DP-pocket protein complex and recruitment of histone deacetylase activity by the pocket protein component (6, 17, 25). As cells progress through the cell cycle, various cyclin–cyclin-dependent kinase (cdk) complexes phosphorylate the pocket proteins, causing release of the hyperphosphorylated pocket protein and associated proteins from the DNA-bound E2F-DP heterodimer (1, 2, 7). Finally, traversal through S phase is thought to be accompanied by cyclin-cdk-mediated phosphorylation of the DP subunit of E2F1-3–DP complexes, resulting in release of the heterodimers from the promoter DNA (15, 21, 42). In some cells, E2F4-containing complexes are thought to be inactivated by relocation to the cytoplasm (28, 38). FIG. 1 E2F-mediated transcriptional regulation. (A) The current model of E2F-mediated transcriptional regulation is thought to involve binding of an E2F-pocket protein complex to promoters in G0 phase of the cell cycle and release of the pocket protein in late ... Although this model is attractive, it is largely based upon circumstantial data, and several important questions remain unanswered. For example, the transcriptional activity of complexes containing E2F4 and E2F5 may be shut off during mid- to late S phase by association with unphosphorylated p107 or p130, rather than by relocation to the cytoplasm (10). Additionally, it is not known if E2F target gene specificity exists or if all six E2Fs bind to and regulate every target gene or if Rb family members display target gene specificity. Determination of target gene specificity has been difficult due to the fact that most cells studied to date contain all of the E2Fs and pocket proteins. Thus, most analyses of E2F and pocket protein binding specificity have been performed using in vitro systems or by overexpression of an individual E2F or pocket protein in cells. In vitro systems, however, cannot recapitulate the complex environment of living cells, and altering the relative amounts of individual proteins through overexpression may abolish important protein-protein interactions. Therefore, we wished to determine which E2Fs and pocket proteins bind to and regulate expression of specific target genes in intact cells under physiological conditions. We felt that the use of an unperturbed in vivo system was of particular importance in the analysis of E2F target gene specificity since regulation occurs in the context of cell cycle progression which cannot be mimicked in vitro and which is often altered when individual E2F proteins are overexpressed. Toward this goal, we have used a formaldehyde cross-linking and immunoprecipitation system to monitor protein-DNA and protein-protein interactions on E2F target genes in living cells. Importantly, this procedure has allowed us to determine the in vivo patterns of E2F and pocket protein binding to specific E2F target genes as cells progress through a cell cycle. Our results indicate that, while some promoters fit the proposed model for E2F-mediated transcriptional regulation, others do not, necessitating individualization of the current model. Furthermore, our data suggest that cell cycle-regulated activity is a stochastic event, as cells have the ability to form several different E2F-pocket protein complexes on a given promoter at each stage of the cell cycle.

To gain insight into the tumor suppressor properties of E2F1, we investigated growth inhibition by the E2F family of transcription factors using a tissue culture model system. We first show that exogenous E2F expression causes an 80% decrease in NIH3T3 colony formation and activated c-Ha-Ras-mediated focus formation. Inhibition of Ras-mediated transformation was dependent upon E2F DNA binding activity but did not require amino- or carboxy-terminal E2F1 protein interaction domains. Because E2F upregulation has been suggested to be associated with a neoplastic phenotype, it was possible that increased E2F activity would not be inhibitory to previously transformed cells. However, we found that exogenous E2F was also inhibitory to growth of NIH3T3 cells previously transformed by Ras or Neu. Further characterization revealed that exogenous E2F expression is inhibitory at very early times after transfection, causing dramatic losses in transfected cell populations. Interestingly, those few cells which do establish appear to be unaffected by the overexpressed E2F. Therefore, we propose that increased E2F activity may only be tolerated in a subset of cells which have acquired specific alterations that are dominant over E2F-mediated growth inhibition.

Previously, we have used a chromatin cross-linking and immunoprecipitation protocol for the analysis of Myc and USF binding to the cad promoter. The adaptation of this technique for the study of mammalian transcription factors was a big step forward in the analysis of transcription factor family member specificity, allowing for the first time a definitive knowledge of which factor binds to a promoter region under normal physiological conditions. However, due to limitations of the assay, our previous studies could not definitively prove that both Myc and USF bound to the exact same site on the cad promoter, nor could we directly correlate loss of in vivo binding of a particular factor with loss of transcriptional activity. Therefore, we have further modified the chromatin immunoprecipitation protocol to alleviate these problems. We have now shown that it is possible to coexamine growth-regulated transcriptional activity and promoter occupancy by using stably integrated promoter constructs. We show that both Myc and USF bind to the exact same E box on the cad promoter, suggesting that competition between these two factors for a single site occurs in living cells. We also find that cad promoter constructs that retain USF binding but lose Myc binding in vivo no longer display an increase in transcriptional activity in mid- to late G(1) phase of the cell cycle. Finally, we propose that cell cycle-regulated transcriptional activation of the cad promoter may be a stochastic, rather than a predetermined, process.

Activity of the dihydrofolate reductase (dhfr) promoter increases at the G1-S-phase boundary of the cell cycle. Mutations that abolish protein binding to an E2F element in the dhfr promoter also abolish the G1-S-phase increase in dhfr transcription, indicating that transcriptional regulation is mediated by the E2F family of proteins. To investigate the mechanism by which E2F regulates dhfr transcription, we moved the E2F element upstream and downstream of its natural position in the promoter. We found that the E2F element confers growth regulation to the dhfr promoter only when it is proximal to the transcription start site. Using a heterologous E2F element, we showed that position-dependent regulation is a property that is promoter specific, not E2F element specific. We demonstrated that E2F-mediated growth regulation of dhfr transcription requires activation of the dhfr promoter in S phase and that the C-terminal activation domains of E2F1, E2F4, and E2F5, when fused to the Gal4 DNA binding domain, are sufficient to specify position-dependent activation. To further investigate the role of activation in dhfr regulation, we tested other transactivation domains for their ability to activate the dhfr promoter. We found that the N-terminal transactivation domain of VP16 cannot activate the dhfr promoter. We propose that, unlike other E2F-regulated promoters, robust transcription from the dhfr promoter requires an E2F transactivation domain close to the transcription start site.

As cells approach S phase, many changes occur to create an environment conducive for DNA synthesis and commitment to cell division. The transcription rate of many genes encoding enzymes involved in DNA synthesis, including the dihydrofolate reductase (dhfr) gene, increases at the G1/S boundary of the cell cycle. Although a number of transcription factors interact to finely tune the levels of dhfr RNA produced, two families of transcription factors, Sp1 and E2F, play central roles in modulating dhfr levels. A region containing several Sp1-binding sites is required for both regulated and basal transcription levels. In contrast, the E2F-binding sites near the transcription start site are required only for regulated transcription. A model is presented for the regulation of the dhfr gene which may also pertain to other cell cycle-associated genes.

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

DNA motifs are short sequences varying from 6 to 25 bp and can be highly variable and degenerated. One major approach for predicting transcription factor (TF) binding is using position weight matrix (PWM) to represent information content of regulatory sites; however, when used as the sole means of identifying binding sites suffers from the limited amount of training data available and a high rate of false-positive predictions. ChIPMotifs program is a de novo motif finding tool developed for ChIP-based high-throughput data, and W-ChIPMotifs is a Web application tool for ChIPMotifs. It composes various ab initio motif discovery tools such as MEME, MaMF, Weeder and optimizes the significance of the detected motifs by using bootstrap re-sampling error estimation and a Fisher test. Using these techniques, we determined a PWM for OCT4 which is similar to canonical OCT4 consensus sequence. In a separate study, we also use de novo motif discovery to suggest that ZNF263 binds to a 24-nt site that differs from the motif predicted by the zinc finger code in several positions.

The self-renewal capacity ascribed to hESCs is paralleled in cancer cell proliferation, suggesting that a common network of genes may facilitate the promotion of these traits. However, the molecular mechanisms that are involved in regulating the silencing of these genes as stem cells differentiate into quiescent cellular lineages remain poorly understood. Here, we show that a differentiated cell specific miR-122 exemplifies this regulatory attribute by suppressing the translation of a gene, Pkm2, which is commonly enriched in hESCs and liver cancer cells (HCCs), and facilitates self-renewal and proliferation. Through a series of gene expression analysis, we show that miR-122 expression is highly elevated in quiescent human primary hepatocytes (hPHs) but lost or attenuated in hESCs and HCCs, while an opposing expression pattern is observed for Pkm2. Depleting hESCs and HCCs of Pkm2, or overexpressing miR-122, leads to a common deficiency in self-renewal and proliferation. Likewise, during the differentiation process of hESCs into hepatocytes, a reciprocal expression pattern is observed between miR-122 and Pkm2. An examination of the genomic region upstream of miR-122 uncovered hyper-methylation in hESCs and HCCs, while the same region is de-methylated and occupied by a transcription initiating protein, RNA polymerase II (RNAPII), in hPHs. These findings indicate that one possible mechanism by which hESC self-renewal is modulated in quiescent hepatic derivatives of hESCs is through the regulatory activity of a differentiated cell-specific miR-122, and that a failure to properly turn “on” this miRNA is observed in uncontrollably proliferating HCCs.

Understanding the mechanisms underlying ErbB3 overexpression in breast cancer will facilitate the rational design of therapies to disrupt ErbB2-ErbB3 oncogenic function. Although ErbB3 overexpression is frequently observed in breast cancer, the factors mediating its aberrant expression are poorly understood. In particular, the ErbB3 gene is not significantly amplified, raising the question as to how ErbB3 overexpression is achieved. In this study we showed that the ZNF217 transcription factor, amplified at 20q13 in 20% of breast tumors, regulates ErbB3 expression. Analysis of a panel of human breast cancer cell lines (n50) and primary human breast tumors (n15) showed a strong positive correlation between ZNF217 and ErbB3 expression. Ectopic expression of ZNF217 in human mammary epithelial cells induced ErbB3 expression, whereas ZNF217 silencing in breast cancer cells resulted in decreased ErbB3 expression. Although ZNF217 has previously been linked with transcriptional repression because of its close association with C-terminal-binding protein (CtBP)1/2 repressor complexes, our results show that ZNF217 also activates gene expression. We showed that ZNF217 recruitment to the ErbB3 promoter is CtBP1/2-independent and that ZNF217 and CtBP1/2 have opposite roles in regulating ErbB3 expression. In addition, we identify ErbB3 as one of the mechanisms by which ZNF217 augments PI-3K/Akt signaling. © 2010 Macmillan Publishers Limited All rights reserved.

krox 20 is an inducible immediate early response gene. To determine if krox 20 has a physiological role in the adult central nervous system (CNS), this study sought to demonstrate the presence of krox 20 in adult rat brain. RNA analysis showed the presence of krox 20 transcripts in the CNS, including the cortex. Polyclonal antibodies to a Krox 20 fusion protein demonstrated 79 and 55 kDa antigens in nuclear CNS homogenates. Neither RNA nor protein analysis was able to demonstrate an induction of krox 20 by a seizure at times when other immediate early response genes are known to be induced. Immunohistochemical analysis revealed staining at several levels throughout the nervous system. This staining was predominantly nuclear, consistent with the role of krox 20 as a transcription factor. These data show that krox 20 is present in the adult CNS, yet differs in response to stimuli as compared to other related transcription factors with a zinc finger motif, such as NGFI-A and NGFI-C.

We examined the ability of purified RNA polymerase (RNAP) II lacking the carboxy-terminal heptapeptide repeat domain (CTD), called RNAP IIB, to transcribe a variety of promoters in HeLa extracts in which endogenous RNAP II activity was inhibited with anti-CTD monoclonal antibodies. Not all promoters were efficiently transcribed by RNAP IIB, and transcription did not correlate with the in vitro strength of the promoter or with the presence of a consensus TATA box. This was best illustrated by the GC-rich, non-TATA box promoters of the bidirectional dihydrofolate reductase (DHFR)-REP-encoding locus. Whereas the REP promoter was transcribed by RNAP IIB, the DHFR promoter remained inactive after addition of RNAP IIB to the antibody-inhibited reactions. However, both promoters were efficiently transcribed when purified RNAP with an intact CTD was added. We analyzed a series of promoter deletions to identify which cis elements determine the requirement for the CTD of RNAP II. All of the promoter deletions of both DHFR and REP retained the characteristics of their respective full-length promoters, suggesting that the information necessary to specify the requirement for the CTD is contained within approximately 65 bp near the initiation site. Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro. Since the synthetic minimal promoter of DHFR and the smallest REP promoter deletion are both activated by Sp1, the differential response in this assay does not result from upstream activators. However, the sequences around the start sites of DHFR and REP are not similar and our data suggest that they bind different proteins. Therefore, we propose that specific initiator elements are important for determination of the requirement of some promoters for the CTD.

The transcription rate of the dihydrofolate reductase (DHFR) gene increases at the G1/S boundary of the proliferative cell cycle. Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene. Mutation of a protein binding site that spans sequences from -16 to +11 in the DHFR promoter resulted in loss of the transcriptional increase at the G1/S boundary. Purification of an activity from HeLa nuclear extract that binds to this region enriched for a 180-kDa polypeptide (HIP1). Using this HIP1 preparation, we have identified specific positions within the binding site that are critical for efficient protein-DNA interactions. An analysis of association and dissociation rates suggests that bound HIP1 protein can exchange rapidly with free protein. This rapid exchange may facilitate the burst of transcriptional activity from the DHFR promoter at the G1/S boundary.

The Krox-20 gene is rapidly and transiently induced when quiescent 3T3 cells are stimulated to reenter the proliferative cycle. We identified the major serum-responsive transcription initiation site and found that it differs from the initiation sites previously identified for the Krox-20 gene. Transcripts from the major serum-responsive initiation site increased at least 40-fold in serum-stimulated cells compared with logarithmically growing cells.

The murine dihydrofolate reductase gene is regulated by a bidirectional promoter that lacks a TATA box. To identify the DNA sequences required for dihydrofolate reductase transcription, the activities of various templates were determined by in vitro transcription analysis. Our data indicate that sequences both upstream and downstream of the transcription initiation site modulate the activity of the dihydrofolate reductase promoter. We have focused on two regions downstream of the transcription initiation site that are important in determining the overall efficiency of the promoter. Region 1, which included exon 1 and part of intron 1, could stimulate transcription when placed in either orientation in the normal downstream position and when inserted upstream of the transcription start site. This region could also stimulate transcription in trans when the enhancer was physically separate from the promoter. Deletion of region 2, spanning 46 nucleotides of the 5' untranslated region, reduced transcriptional activity by fivefold. DNase I footprinting reactions identified protein-binding sites in both downstream stimulatory regions. Protein bound to two sites in region 1, both of which contain an inverted CCAAT box. The protein-binding site in the 5' untranslated region has extensive homology to binding sites in promoters that both lack (simian virus 40 late) and contain (adenovirus type 2 major late promoter and c-myc) TATA boxes.

Fields such as genomics and systems biology are built on the synergism between computational and experimental techniques. This type of synergism is especially important in accomplishing goals like identifying all functional transcription factor binding sites in vertebrate genomes. Precise detection of these elements is a prerequisite to deciphering the complex regulatory networks that direct tissue specific and lineage specific patterns of gene expression. This review summarizes approaches for in silico, in vitro, and in vivo identification of transcription factor binding sites. A variety of techniques useful for localized- and high-throughput analyses are discussed here, with emphasis on aspects of data generation and verification.

The E2F family of transcription factors regulates the expression of genes involved in cell cycle progression, DNA synthesis, repair, and recombination, and a variety of other cellular processes. Although E2F proteins are often redundant in function, specificity of binding and activity can occur. For example, E2F1, but not other E2F family members, was shown previously to bind the murine carboxylesterase promoter in chromatin immunoprecipitation studies. This promoter region lacks a consensus E2F binding site, suggesting that E2F1 may be recruited to the DNA in a unique fashion. To further investigate this E2F1-specific binding, we have employed a "transient chromatin immunoprecipitation" approach. Using various deletions and mutations of the promoter region, we localized the E2F1-specific binding site and demonstrated that it was required for E2F1-mediated transcription of the carboxylesterase promoter. The identified site was similar to the 8-bp consensus E2F site but differed from the consensus at a crucial position. To address whether E2F1 directly bound to this non-consensus site, we demonstrated that the DNA binding domain of E2F1 is necessary for E2F1-mediated activation of the carboxylesterase promoter. Interestingly, a "UP" mutation of the site, making it more similar to the consensus element, did not improve the ability of E2F1 to bind the promoter. Rather, E2F1 could no longer bind to the carboxylesterase promoter that contained the consensus E2F site. We propose a model in which E2F1-specific regulation of the carboxylesterase promoter requires both E2F1/DNA interactions and protein-protein interaction between E2F1 and a factor that binds adjacent to the non-consensus site.

The c-Myc protein is up-regulated in many different types of cancer, suggesting that a detailed understanding of Myc function is an important goal. Our previous studies have focused on determining the mechanism by which Myc activates transcription using the target gene cad as an experimental model. Previously, we found that Myc activates cad transcription at a post-RNA polymerase II recruitment step and that the Myc transactivation domain interacts with a number of cdk-cyclin complexes. We now extend these studies to determine the role of these cyclin-cdk complexes in Myc-mediated transactivation. We have found that cyclin T1 binding to Myc localizes to the highly conserved Myc Box I, whereas cdk8 binding localizes to the amino-terminal 41 amino acids of the Myc transactivation domain. We showed that recruitment of cdk8 is sufficient for activation of a synthetic promoter construct. In contrast, the ability of Myc to activate transcription of the cad promoter correlates with binding of cyclin T1. Furthermore, recruitment of cyclin T1 to the cad promoter via a Gal4 fusion protein or through protein-protein interaction with the HIV-1 Tat protein can also activate cad transcription. These results suggest that Myc activates transcription by stimulating elongation and that P-TEFb is a key mediator of this process.

The c-Myc protein is a site-specific DNA-binding transcription factor that is up-regulated in a number of different cancers. We have previously shown that binding of Myc correlates with increased transcription of the cad promoter. We have now further investigated the mechanism by which Myc mediates transcriptional activation of the cad gene. Using a chromatin immunoprecipitation assay, we found high levels of RNA polymerase II bound to the cad promoter in quiescent NIH 3T3 cells and in differentiated U937 cells, even though the promoter is inactive. However, chromatin immunoprecipitation with an antibody that recognizes the hyperphosphorylated form of the RNA polymerase II carboxyl-terminal domain (CTD) revealed that phosphorylation of the CTD does correlate with c-Myc binding and cad transcription. We have also found that the c-Myc transactivation domain interacts with cdk9 and cyclin T1, components of the CTD kinase P-TEFb. Furthermore, activator bypass experiments have shown that direct recruitment of cyclin T1 to the cad promoter can substitute for c-Myc to activate the promoter. In summary, our results suggest that c-Myc activates transcription of cad by stimulating promoter clearance and elongation, perhaps via recruitment of P-TEFb.

The processes that take place during development and differentiation are directed through coordinated regulation of expression of a large number of genes. One such gene regulatory network provides cell cycle control in eukaryotic organisms. In this work, we have studied the structural features of the 5' regulatory regions of cell cycle-related genes. We developed a new method for identifying composite substructures (modules) in regulatory regions of genes consisting of a binding site for a key transcription factor and additional contextual motifs: potential targets for other transcription factors that may synergistically regulate gene transcription. Applying this method to cell cycle-related promoters, we created a program for context-specific identification of binding sites for transcription factors of the E2F family which are key regulators of the cell cycle. We found that E2F composite modules are found at a high frequency and in close proximity to the start of transcription in cell cycle-related promoters in comparison with other promoters. Using this information, we then searched for E2F sites in genomic sequences with the goal of identifying new genes which play important roles in controlling cell proliferation, differentiation and apoptosis. Using a chromatin immunoprecipitation assay, we then experimentally verified the binding of E2F in vivo to the promoters predicted by the computer-assisted methods. Our identification of new E2F target genes provides new insight into gene regulatory networks and provides a framework for continued analysis of the role of contextual promoter features in transcriptional regulation. The tools described are available at http://compel.bionet.nsc.ru/FunSite/SiteScan.html.

Liver cancer is the fifth most common cancer worldwide and unlike certain other cancers, such as colon cancer, a mutational model has not yet been developed. We have performed gene expression profiling of normal and neoplastic livers in C3H/HeJ mice treated with diethylnitrosamine. Using oligonucleotide microarrays, we compared gene expression in liver tumors to three different states of the normal liver: quiescent adult, regenerating adult, and newborn. Although each comparison revealed hundreds of differentially expressed genes, only 22 genes were found to be deregulated in the tumors in all three comparisons. Three of these genes were examined in human hepatocellular carcinomas and were found to be upregulated. As a second method of analysis, we used Representational Difference Analysis (RDA) to clone mRNA fragments differentially expressed in liver tumors versus regenerating livers. We cloned several novel mRNAs that are differentially regulated in murine liver tumors. Here we report the sequence of a novel cDNA whose expression is upregulated in both murine and human hepatocellular carcinomas. Our results suggest that DEN-treated mice provide an excellent model for human hepatocellular carcinomas.

Uncertainty as to which member of a family of DNA-binding transcription factors regulates a specific promoter in intact cells is a problem common to many investigators. Determining target gene specificity requires both an analysis of protein binding to the endogenous promoter as well as a characterization of the functional consequences of transcription factor binding. By using a formaldehyde crosslinking procedure and Gal4 fusion proteins, we have analyzed the timing and functional consequences of binding of Myc and upstream stimulatory factor (USF)1 to endogenous cellular genes. We demonstrate that the endogenous cad promoter can be immunoprecipitated with antibodies against Myc and USF1. We further demonstrate that although both Myc and USF1 can bind to cad, the cad promoter can respond only to the Myc transactivation domain. We also show that the amount of Myc bound to the cad promoter fluctuates in a growth-dependent manner. Thus, our data analyzing both DNA binding and promoter activity in intact cells suggest that cad is a Myc target gene. In addition, we show that Myc binding can occur at many sites in vivo but that the position of the binding site determines the functional consequences of this binding. Our data indicate that a post-DNA-binding mechanism determines Myc target gene specificity. Importantly, we have demonstrated the feasibility of analyzing the binding of site-specific transcription factors in vivo to single copy mammalian genes.

The E2F family of transcription factors consists of two subgroups termed E2F and DP. E2F is required for cell proliferation, and is necessary for fruit fly development. E2F activity is a target for regulation by the retinoblastoma gene family, which includes pRB, p107 and p130. Mutant RB − − , RB − − : p 107 − − and p 107 − − : p 130 − − mice develop abnormally, probably as a result of dysregulation in the activity of E2F, indicating the importance of E2F in mammalian development. To investigate the role of E2F in murine development, we have examined the patterns of expression of E2F-1 through E2F-5, and DP-1 in the developing nervous system by in situ hybridization. E2F-1, E2F-2 and E2F-5 are first detected in the 9.5 days post-coitus (dpc) forebrain. Expression of these E2F forms extends caudally thereafter and includes the developing brain and the upper half of the 10.5 dpc spinal cord. By 11.5 dpc, these E2F factors are expressed throughout the central nervous system. In 12.5 dpc embryos, E2F-1, E2F-2 and E2F-5 are highly expressed in proliferating, undifferentiated neuronal precursors. As neurons differentiate and migrate to the outer marginal zones in the nervous system, expression of these E2F members is extinguished. In the developing retina, another neuronal tissue, E2F-1 expression is also confined to the proliferating, undifferentiated retinoblastic layer. In contrast, E2F-3 expression is up-regulated as retinoblasts differentiate into the ganglion cell layer. In non-neuronal tissues, high E2F-4 transcript levels are present in regions corresponding to proliferative chondrocytes, whereas E2F-2 and E2F-4 transcripts are very abundant in the thymic cortex, which contains immature thymocytes. We conclude that individual E2F forms are differentially regulated during the development of distinct tissues, and especially during neuronal development.

Previous studies have indicated that the presence of an E2F site is not sufficient for G1/S phase transcriptional regulation. For example, the E2F sites in the E2F1 promoter are necessary, but not sufficient, to mediate differential promoter activity in G0 and S phase. We have now utilized the E2F1 minimal promoter to test several hypotheses that could account for these observations. To test the hypothesis that G1/S phase regulation is achieved via E2F-mediated repression of a strong promoter, a variety of transactivation domains were brought to the E2F1 minimal promoter. Although many of these factors caused increased promoter activity, growth regulation was not observed, suggesting that a general repression model is incorrect. However, constructs having CCAAT or YY1 sites or certain GC boxes cloned upstream of the E2F1 minimal promoter displayed E2F site-dependent regulation. Further analysis of the promoter activity suggested that E2F requires cooperation with another factor to activate transcription in S phase. However, we found that the requirement for E2F to cooperate with additional factors to achieve growth regulation could be relieved by bringing the E2F1 activation domain to the promoter via a Gal4 DNA binding domain. Our results suggest a model that explains why some, but not all, promoters that contain E2F sites display growth regulation.

Carbamoyl-phosphate synthase/aspartate carbamoyltransferase/dihydroorotase, which is encoded by the cad gene, is required for the first three rate-limiting steps of de novo pyrimidine biosynthesis. It has been previously demonstrated that cad transcription increases at the G1/S-phase boundary, as quiescent cells reenter the proliferative cell cycle. The growth-responsive element has been mapped to an E box at +65 in the hamster cad promoter. Using an in vivo UV cross-linking and immunoprecipitation assay, we show that Myc, Max, and upstream stimulatory factor (USF) bind to the chromosomal cad promoter. To determine whether binding of Myc-Max or USF is critical for cad growth regulation, we analyzed promoter constructs which contain mutations in the nucleotides flanking the E box. We demonstrate that altering nucleotides which flank the cad E box to sequences which decrease Myc-Max binding in vitro correlates with a loss of cad G1/S-phase transcriptional activation. This result supports the conclusion that binding of Myc-Max, but not USF, is essential for cad regulation. Our investigations demonstrate that the endogenous cad E box can be bound by more than one transcription factor, but growth-induced cad expression is achieved only by Myc.

C3H/HeJ(C3H) mice are approximately 50-fold more susceptible to liver-tumor induction than C57BL/6J(B6) mice. This difference in susceptibility is a consequence of allelic differences in hepatocarcinogen sensitivity(Hcs) genes that control the growth of preneoplastic lesions in the liver. We have shown previously that these two strains differ in their responses to partial hepatectomy, which acts as a promoter of hepatocarcinogenesis in B6 mice but not in C3H mice. To determine whether there are also strain-specific differences in normal regulation of hepatic growth, we compared liver regeneration in C3H and B6 mice at the levels of DNA synthesis and gene expression. Partial hepatectomy induced a cascade of controlled events resulting in the regeneration of the liver to its original mass 11 d after surgery. We observed a two-fold greater level of DNA synthesis in C3H mice relative to B6 mice during the first peak of DNA synthesis, which occurred 35 h after hepatectomy in both strains. While the c-fos transcript was readily induced in both strains, there was a reduction in the expression of the late response genes E2F1 and dihydrofolate reductase in the livers of B6 mice when compared with the expression of these transcripts in the livers of C3H mice. The differential regulation of E2F1 between B6 and C3H mice may indicate that the Hcs genes and E2F1 function in the same signal-transduction pathway of normal growth control.© 1995 Wiley-Liss, Inc