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Biomedical subjects

Peggy J Farnham

Publications and source records attributed to Peggy J Farnham.

At least 19 recordsLinked to original sources
PubMed2006

Locating mammalian transcription factor binding sites: a survey of computational and experimental techniques.

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.

Animals↗
PubMed2006

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.

Base Pairing↗
PubMed2006

Suz12 binds to silenced regions of the genome in a cell-type-specific manner.

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.

Animals↗
PubMed2006

Unbiased location analysis of E2F1-binding sites suggests a widespread role for E2F1 in the human genome.

The E2F family of transcription factors regulates basic cellular processes. Here, we take an unbiased approach towards identifying E2F1 target genes by examining localization of E2F1-binding sites using high-density oligonucleotide tiling arrays. To begin, we developed a statistically-based methodology for analysis of ChIP-chip data obtained from arrays that represent 30 Mb of the human genome. Using this methodology, we identified regions bound by E2F1, MYC, and RNA Polymerase II (POLR2A). We found a large number of binding sites for all three factors; extrapolation suggests there may be approximately 20,000-30,000 E2F1- and MYC-binding sites and approximately 12,000-17,000 active promoters in HeLa cells. In contrast to our results for MYC, we find that the majority of E2F1-binding sites (>80%) are located in core promoters and that 50% of the sites overlap transcription starts. Only a small fraction of E2F1 sites possess the canonical binding motif. Surprisingly, we found that approximately 30% of genes in the 30-Mb region possessed an E2F1 binding site in a core promoter and E2F1 was bound near to 83% of POLR2A-bound sites. To determine if these results were representative of the entire human genome, we performed ChIP-chip analyses of approximately 24,000 promoters and confirmed that greater than 20% of the promoters were bound by E2F1. Our results suggest that E2F1 is recruited to promoters via a method distinct from recognition of the known consensus site and point toward a new understanding of E2F1 as a factor that contributes to the regulation of a large fraction of human genes.

Binding Sites↗
PubMed2006

Comparison of sample preparation methods for ChIP-chip assays.

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.

Animals↗
PubMed2005

CpG Island microarray probe sequences derived from a physical library are representative of CpG Islands annotated on the human genome.

An effective tool for the global analysis of both DNA methylation status and protein-chromatin interactions is a microarray constructed with sequences containing regulatory elements. One type of array suited for this purpose takes advantage of the strong association between CpG Islands (CGIs) and gene regulatory regions. We have obtained 20,736 clones from a CGI Library and used these to construct CGI arrays. The utility of this library requires proper annotation and assessment of the clones, including CpG content, genomic origin and proximity to neighboring genes. Alignment of clone sequences to the human genome (UCSC hg17) identified 9595 distinct genomic loci; 64% were defined by a single clone while the remaining 36% were represented by multiple, redundant clones. Approximately 68% of the loci were located near a transcription start site. The distribution of these loci covered all 23 chromosomes, with 63% overlapping a bioinformatically identified CGI. The high representation of genomic CGI in this rich collection of clones supports the utilization of microarrays produced with this library for the study of global epigenetic mechanisms and protein-chromatin interactions. A browsable database is available on-line to facilitate exploration of the CGIs in this library and their association with annotated genes or promoter elements.

Base Sequence↗
PubMed2004

The use of transient chromatin immunoprecipitation assays to test models for E2F1-specific transcriptional activation.

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.

Animals↗
PubMed2004

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27.

Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27.

Carrier Proteins↗
PubMed2004

T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells.

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.

Animals↗
PubMed2004

Genomic approaches that aid in the identification of transcription factor target genes.

It is well-established that deregulation of the transcriptional activity of many different genes has been causatively linked to human diseases. In cancer, altered patterns of gene expression are often the result of the inappropriate expression of a specific transcriptional activator or repressor. Functional studies of cancer-specific transcription factors have relied upon the study of candidate target genes. More recently, gene expression profiling using DNA microarrays that contain tens of thousands of cDNAs corresponding to human mRNAs has allowed for a large-scale identification of genes that respond to increased or decreased levels of a particular transcription factor. However, such experiments do not distinguish direct versus indirect target genes. Coupling chromatin immunoprecipitation to micro-arrays that contain genomic regions (ChIP-chip) has provided investigators with the ability to identify, in a high-throughput manner, promoters directly bound by specific transcription factors. Clearly, knowledge gained from both types of arrays provides complementary information, allowing greater confidence that a transcription factor regulates a particular gene. In this review, we focus on Polycomb group (PcG) complexes as an example of transcriptional regulators that are implicated in various cellular processes but about which very little is known concerning their target gene specificity. We provide examples of how both expression arrays and ChIP-chip microarray-based assays can be used to identify target genes of a particular PcG complex and suggest improvements in the application of array technology for faster and more comprehensive identification of directly regulated target genes.

Animals↗
PubMed2003

E2F6 negatively regulates BRCA1 in human cancer cells without methylation of histone H3 on lysine 9.

E2F6 contains a DNA binding domain that is very similar to that of the other members of the E2F family of transcriptional regulators. However, E2F6 cannot bind to all promoters that contain consensus E2F-binding sites. Therefore, we used a combination of chromatin immunoprecipitation and genomic microarrays to identify promoters bound by E2F6 in human cells. Although most of the identified promoters were bound by multiple E2F family members, one promoter was bound only by E2F6. To determine which of the newly identified promoters were regulated by E2F6, we reduced the level of E2F6 by using RNA interference technology. We found that mRNA transcribed from promoters bound by E2F6 was increased after reduction of the amount of E2F6 protein in the cell. Interestingly, many of the E2F6-regulated genes encoded functions involved in tumor suppression and the maintenance of chromatin structure. Specifically, our results suggest that E2F6 represses transcription of the brca1, ctip, art27, hp1alpha, and the rbap48 genes. E2F6 has been postulated to mediate transcriptional repression by recruiting a histone H3 methyltransferase to the DNA. However, we found that the E2F6-regulated promoters did not contain histone H3 methylated at lysine 9. To determine the mechanism by which E2F6 regulates transcription, we performed chromatin immunoprecipitation before and after the introduction of small inhibitory ribonucleic acids specific to E2F6. We found that depletion of E2F6 resulted in the recruitment of E2F1 to the target promoters. In summary, we have identified 48 endogenous target genes of E2F6 and have shown that E2F6 can repress target promoters in a manner that does not require histone H3 methylation at lysine 9.

BRCA1 Protein↗
PubMed2003

Analysis of Myc bound loci identified by CpG island arrays shows that Max is essential for Myc-dependent repression.

The c-myc proto-oncogene encodes a transcription factor, c-Myc, which is deregulated and/or overexpressed in many human cancers. Despite c-Myc's importance, the identity of Myc-regulated genes and the mechanism by which Myc regulates these genes remain unclear. By combining chromatin immunoprecipitation with CpG island arrays, we identified 177 human genomic loci that are bound by Myc in vivo. Analyzing a cohort of known and novel Myc target genes showed that Myc-associated protein X, Max, also bound to these regulatory regions. Indeed, Max is bound to these loci in the presence or absence of Myc. The Myc:Max interaction is essential for Myc-dependent transcriptional activation; however, we show that Max bound targets also include Myc-repressed genes. Moreover, we show that the interaction between Myc and Max is essential for gene repression to occur. Taken together, the identification and analysis of Myc bound target genes supports a model whereby Max plays an essential and universal role in the mechanism of Myc-dependent transcriptional regulation.

CpG Islands↗
PubMed2003

Identification and characterization of CRG-L2, a new marker for liver tumor development.

Liver cancer is very common worldwide and the rates of hepatocellular carcinoma (HCC) have increased by over 70% in the last 2 decades in the US. Late diagnosis, because of the lack of clinical symptoms, and decreased hepatic function, because of underlying hepatic disease, lead to the extremely high mortality rates associated with HCC. Clearly, the identification of markers that are expressed early in the development of HCC and that are easily detected in high-risk patients would aid in early diagnosis and increased survival. We present the cloning and characterization of a novel gene, CRG-L2 (Cancer related gene-Liver 2), which displays high expression in murine and human hepatocellular carcinomas. Using in situ hybridization, we show that CRG-L2 mRNA levels are increased early during the development of liver tumors in C3H/HeJ mice, and that in normal tissues CRG-L2 mRNA is restricted to the murine testis and human placenta. Its restricted expression in normal tissues and unique early upregulation during tumor development make CRG-L2 an excellent candidate as a new clinical marker of HCC.

Base Sequence↗
PubMed2003

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 G(0) and G(1) 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 G(0)/G(1) 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 G(0)/G(1) 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 G(0)/G(1) and S phases.

Binding Sites↗
PubMed2003

Identification of the polycomb group protein SU(Z)12 as a potential molecular target for human cancer therapy.

We have previously identified SU(Z)12 as an E2F target gene. Because many E2F target genes encode proteins that are critical for the control of cell proliferation, we have further characterized the regulation and expression of SU(Z)12. To understand the molecular mechanisms responsible for expression of SU(Z)12 mRNA, we have analyzed the promoter region. We found that the SU(Z)12 gene is controlled by dual promoters, one of which functions bidirectionally. In addition to the E2F binding site, we have identified two binding sites for T cell factor (TCF)/beta-catenin complexes. Using gel mobility shift assays, we demonstrated that both TCF sites can be bound by TCF4. TCF/beta-catenin complexes have been shown to be a critical regulator of gene expression in tumors of the colon, breast, and liver. Accordingly, we have used chromatin immunoprecipitation assays to confirm that TCF4/beta-catenin complexes are bound to the SU(Z)12 promoter in colon cancer cells but not in HeLa cells. We next adapted the chromatin immunoprecipitation assay for use with primary colon tumor samples, and, using matched pairs of normal and tumor tissue obtained from several different colon cancer patients, we demonstrate that levels of beta-catenin bound to the SU(Z)12 promoter are increased in colon tumors. Finally, we show that the SU(Z)12 mRNA is up-regulated in a number of different human tumors, including tumors of the colon, breast, and liver. Recent studies have found that SU(Z)12 is a component of the Drosophila ESC-E(Z) and the human EED-EZH2 Polycomb chromatin remodeling complexes. Therefore, we suggest that SU(Z)12, which may modulate the tumor phenotype by changing gene expression profiles, may be a logical target for the design of a new antitumor agent

Animals↗
PubMed2002

Myc recruits P-TEFb to mediate the final step in the transcriptional activation of the cad promoter.

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.

3T3 Cells↗