Search tips
Search criteria

Results 1-25 (64)

Clipboard (0)

Select a Filter Below

Year of Publication
more »
1.  Distant cis Regulatory Elements in Human Skeletal Muscle Differentiation 
Genomics  2011;98(6):401-411.
Identifying gene regulatory elements and their target genes in human cells remains a significant challenge. Despite increasing evidence of physical interactions between distant regulatory elements and gene promoters in mammalian cells, many studies consider only promoter-proximal regulatory regions. We identify putative cis-regulatory modules (CRMs) in human skeletal muscle differentiation by combining myogenic TF binding data before and after differentiation with histone modification data in myoblasts. CRMs that are distant (>20 kb) from muscle gene promoters are common and are more likely than proximal promoter regions to show differentiation-specific changes in myogenic TF binding. We find that two of these distant CRMs, known to activate transcription in differentiating myoblasts, interact physically with gene promoters (PDLIM3 and ACTA1) during differentiation. Our results highlight the importance of considering distal CRMs in investigations of mammalian gene regulation and support the hypothesis that distant CRM-promoter looping contacts are a general mechanism of gene regulation.
PMCID: PMC3229301  PMID: 21907276
cis-regulatory modules; DNA looping interactions; Transcriptional regulation; Transcription factors; DNA binding sites
2.  Modular Evolution of DNA-Binding Preference of a Tbrain Transcription Factor Provides a Mechanism for Modifying Gene Regulatory Networks 
Molecular Biology and Evolution  2014;31(10):2672-2688.
Gene regulatory networks (GRNs) describe the progression of transcriptional states that take a single-celled zygote to a multicellular organism. It is well documented that GRNs can evolve extensively through mutations to cis-regulatory modules (CRMs). Transcription factor proteins that bind these CRMs may also evolve to produce novelty. Coding changes are considered to be rarer, however, because transcription factors are multifunctional and hence are more constrained to evolve in ways that will not produce widespread detrimental effects. Recent technological advances have unearthed a surprising variation in DNA-binding abilities, such that individual transcription factors may recognize both a preferred primary motif and an additional secondary motif. This provides a source of modularity in function. Here, we demonstrate that orthologous transcription factors can also evolve a changed preference for a secondary binding motif, thereby offering an unexplored mechanism for GRN evolution. Using protein-binding microarray, surface plasmon resonance, and in vivo reporter assays, we demonstrate an important difference in DNA-binding preference between Tbrain protein orthologs in two species of echinoderms, the sea star, Patiria miniata, and the sea urchin, Strongylocentrotus purpuratus. Although both orthologs recognize the same primary motif, only the sea star Tbr also has a secondary binding motif. Our in vivo assays demonstrate that this difference may allow for greater evolutionary change in timing of regulatory control. This uncovers a layer of transcription factor binding divergence that could exist for many pairs of orthologs. We hypothesize that this divergence provides modularity that allows orthologous transcription factors to evolve novel roles in GRNs through modification of binding to secondary sites.
PMCID: PMC4166925  PMID: 25016582
evo-devo; gene regulatory networks; transcription factors; echinoderm; evolution; T-box
3.  Using a structural and logics systems approach to infer bHLH–DNA binding specificity determinants 
Nucleic Acids Research  2011;39(11):4553-4563.
Numerous efforts are underway to determine gene regulatory networks that describe physical relationships between transcription factors (TFs) and their target DNA sequences. Members of paralogous TF families typically recognize similar DNA sequences. Knowledge of the molecular determinants of protein–DNA recognition by paralogous TFs is of central importance for understanding how small differences in DNA specificities can dictate target gene selection. Previously, we determined the in vitro DNA binding specificities of 19 Caenorhabditis elegans basic helix-loop-helix (bHLH) dimers using protein binding microarrays. These TFs bind E-box (CANNTG) and E-box-like sequences. Here, we combine these data with logics, bHLH–DNA co-crystal structures and computational modeling to infer which bHLH monomer can interact with which CAN E-box half-site and we identify a critical residue in the protein that dictates this specificity. Validation experiments using mutant bHLH proteins provide support for our inferences. Our study provides insights into the mechanisms of DNA recognition by bHLH dimers as well as a blueprint for system-level studies of the DNA binding determinants of other TF families in different model organisms and humans.
PMCID: PMC3113581  PMID: 21335608
4.  Bayesian hierarchical model of protein-binding microarray k-mer data reduces noise and identifies transcription factor subclasses and preferred k-mers 
Bioinformatics  2013;29(11):1390-1398.
Motivation: Sequence-specific transcription factors (TFs) regulate the expression of their target genes through interactions with specific DNA-binding sites in the genome. Data on TF-DNA binding specificities are essential for understanding how regulatory specificity is achieved.
Results: Numerous studies have used universal protein-binding microarray (PBM) technology to determine the in vitro binding specificities of hundreds of TFs for all possible 8 bp sequences (8mers). We have developed a Bayesian analysis of variance (ANOVA) model that decomposes these 8mer data into background noise, TF familywise effects and effects due to the particular TF. Adjusting for background noise improves PBM data quality and concordance with in vivo TF binding data. Moreover, our model provides simultaneous identification of TF subclasses and their shared sequence preferences, and also of 8mers bound preferentially by individual members of TF subclasses. Such results may aid in deciphering cis-regulatory codes and determinants of protein–DNA binding specificity.
Availability and implementation: Source code, compiled code and R and Python scripts are available from
Contact: or
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3661050  PMID: 23559638
5.  Analysis of the Sequence Specificities of DNA Binding Proteins with Protein Binding Microarrays 
Methods in enzymology  2006;410:279-299.
DNA binding proteins are important for various cellular processes, such as transcriptional regulation, recombination, replication, repair, and DNA modification. Of particular interest are transcription factors (TFs), since through interactions with their DNA binding sites, they modulate gene expression in a manner required for normal cellular growth and differentiation, and also for response to environmental stimuli. To date, the DNA binding specificities of most DNA binding proteins remain unknown, since earlier technologies aimed at characterizing DNA-protein interactions have been laborious and not highly scalable. Recently we developed a new DNA microarray-based technology, termed protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding site sequence specificities of TFs, or any DNA binding protein. The DNA binding site data from PBMs can be used to predict what genes are regulated by a given TF, what the functions are of a given TF and its predicted target genes, and how that TF may fit into the cell's transcriptional regulatory network.
PMCID: PMC2747587  PMID: 16938556
DNA microarrays; transcription factors; transcription factor binding sites; DNA binding proteins; protein-DNA binding; DNA regulatory motifs
6.  Design of Compact, Universal DNA Microarrays for Protein Binding Microarray Experiments 
Journal of Computational Biology  2008;15(7):655-665.
Our group has recently developed a compact, universal protein binding microarray (PBM) that can be used to determine the binding preferences of transcription factors (TFs). This design represents all possible sequence variants of a given length k (i.e., all k-mers) on a single array, allowing a complete characterization of the binding specificities of a given TF. Here, we present the mathematical foundations of this design based on de Bruijn sequences generated by linear feedback shift registers. We show that these sequences represent the maximum number of variants for any given set of array dimensions (i.e., number of spots and spot lengths), while also exhibiting desirable pseudo-randomness properties. Moreover, de Bruijn sequences can be selected that represent gapped sequence patterns, further increasing the coverage of the array. This design yields a powerful experimental platform that allows the binding preferences of TFs to be determined with unprecedented resolution.
PMCID: PMC3203512  PMID: 18651798
combinatorics; DNA arrays; genomics; linear algebra; strings
7.  DNA Microarray Technologies for Measuring Protein-DNA Interactions 
Current opinion in biotechnology  2006;17(4):422-430.
DNA binding proteins play key roles in many cellular processes, including transcriptional regulation and replication. Microarray-based technologies permit high-throughput identification of binding sites and functional roles of these proteins. In particular, microarray readout either of chromatin immunoprecipitation (‘ChIP-chip’) or of DNA adenine methyltransferase fusion proteins (‘DamID’) enables the identification of in vivo genomic target sites of proteins. A complementary approach, in vitro binding of proteins directly to double-stranded DNA microarrays (‘protein binding microarrays’), permits rapid characterization of their DNA binding site sequence specificities. Recent advances in DNA microarray synthesis technologies have permitted the definition of proteins’ DNA binding sites at much higher resolution and coverage, and further advances in these and emerging technologies will further increase the efficiencies of these exciting new approaches.
PMCID: PMC2727741  PMID: 16839757
8.  Discovery of DNA regulatory elements with bacteria 
Nature biotechnology  2005;23(8):942-944.
PMCID: PMC2720157  PMID: 16082362
9.  Protein Binding Microarrays (PBMs) for the Rapid, High-Throughput Characterization of the Sequence Specificities of DNA Binding Proteins 
DNA binding proteins play a number of key roles in cells, in processes including transcriptional regulation, recombination, genome rearrangements, and DNA replication, repair, and modification. Of particular interest are the interactions between transcription factors and their DNA binding sites, as they are an integral part of the transcriptional regulatory networks that control gene expression. Despite their importance, the DNA binding specificities of most DNA binding proteins remain unknown, as earlier technologies aimed at characterizing DNA-protein interactions have been time-consuming and not highly scalable. We have developed a new DNA microarray-based technology, termed protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding site sequence specificities of transcription factors in a single day. The resulting DNA binding site data can be used in a number of ways, including for the prediction of the genes regulated by a given transcription factor, annotation of transcription factor function, and functional annotation of the predicted target genes.
PMCID: PMC2690637  PMID: 16888363
DNA microarrays; transcription factors; DNA binding proteins; protein-DNA binding; DNA regulatory motifs
10.  Genomic regions flanking E-box binding sites influence DNA binding specificity of bHLH transcription factors through DNA shape 
Cell reports  2013;3(4):1093-1104.
DNA sequence is a major determinant of the binding specificity of transcription factors (TFs) for their genomic targets. However, eukaryotic cells often express, at the same time, TFs with highly similar DNA binding motifs but distinct in vivo targets. Currently, it is not well understood how TFs with seemingly identical DNA motifs achieve unique specificities in vivo. Here, we used custom protein binding microarrays to analyze TF specificity for putative binding sites in their genomic sequence context. Using yeast TFs Cbf1 and Tye7 as our case study, we found that binding sites of these bHLH TFs (i.e., E-boxes) are bound differently in vitro and in vivo, depending on their genomic context. Computational analyses suggest that nucleotides outside E-box binding sites contribute to specificity by influencing the 3D structure of DNA binding sites. Thus, local shape of target sites might play a widespread role in achieving regulatory specificity within TF families.
PMCID: PMC3640701  PMID: 23562153
transcription factors; bHLH; DNA binding sites; protein binding microarrays; DNA shape; support vector regression
11.  Machine learning classification of cell-specific cardiac enhancers uncovers developmental subnetworks regulating progenitor cell division and cell fate specification 
Development (Cambridge, England)  2014;141(4):878-888.
The Drosophila heart is composed of two distinct cell types, the contractile cardial cells (CCs) and the surrounding non-muscle pericardial cells (PCs), development of which is regulated by a network of conserved signaling molecules and transcription factors (TFs). Here, we used machine learning with array-based chromatin immunoprecipitation (ChIP) data and TF sequence motifs to computationally classify cell type-specific cardiac enhancers. Extensive testing of predicted enhancers at single-cell resolution revealed the added value of ChIP data for modeling cell type-specific activities. Furthermore, clustering the top-scoring classifier sequence features identified novel cardiac and cell type-specific regulatory motifs. For example, we found that the Myb motif learned by the classifier is crucial for CC activity, and the Myb TF acts in concert with two forkhead domain TFs and Polo kinase to regulate cardiac progenitor cell divisions. In addition, differential motif enrichment and cis-trans genetic studies revealed that the Notch signaling pathway TF Suppressor of Hairless [Su(H)] discriminates PC from CC enhancer activities. Collectively, these studies elucidate molecular pathways used in the regulatory decisions for proliferation and differentiation of cardiac progenitor cells, implicate Su(H) in regulating cell fate decisions of these progenitors, and document the utility of enhancer modeling in uncovering developmental regulatory subnetworks.
PMCID: PMC3912831  PMID: 24496624
Machine learning; Gene regulation; Transcription factors; Progenitor specification; Cell division; Organogenesis; Drosophila
12.  5′RNA-Seq identifies Fhl1 as a genetic modifier in cardiomyopathy  
The Journal of Clinical Investigation  2014;124(3):1364-1370.
The transcriptome is subject to multiple changes during pathogenesis, including the use of alternate 5′ start-sites that can affect transcription levels and output. Current RNA sequencing techniques can assess mRNA levels, but do not robustly detect changes in 5′ start-site use. Here, we developed a transcriptome sequencing strategy that detects genome-wide changes in start-site usage (5′RNA-Seq) and applied this methodology to identify regulatory events that occur in hypertrophic cardiomyopathy (HCM). Compared with transcripts from WT mice, 92 genes had altered start-site usage in a mouse model of HCM, including four-and-a-half LIM domains protein 1 (Fhl1). HCM-induced altered transcriptional regulation of Fhl1 resulted in robust myocyte expression of a distinct protein isoform, a response that was conserved in humans with genetic or acquired cardiomyopathies. Genetic ablation of Fhl1 in HCM mice was deleterious, which suggests that Fhl1 transcriptional changes provide salutary effects on stressed myocytes in this disease. Because Fhl1 is a chromosome X–encoded gene, stress-induced changes in its transcription may contribute to gender differences in the clinical severity of HCM. Our findings indicate that 5′RNA-Seq has the potential to identify genome-wide changes in 5′ start-site usage that are associated with pathogenic phenotypes.
PMCID: PMC3934171  PMID: 24509080
13.  Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos 
Nature methods  2013;10(8):774-780.
Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses, and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here, we have developed ‘enhancer-FACS-Seq’ (eFS) technology for highly parallel identification of active, tissue-specific enhancers in Drosophila embryos. Analysis of enhancers identified by eFS to be active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, novel mesodermal transcription factors (TFs). Naïve Bayes classifiers using TF binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded within them.
PMCID: PMC3733245  PMID: 23852450
14.  Nucleotides of transcription factor binding sites exert interdependent effects on the binding affinities of transcription factors 
Nucleic Acids Research  2002;30(5):1255-1261.
We can determine the effects of many possible sequence variations in transcription factor binding sites using microarray binding experiments. Analysis of wild-type and mutant Zif268 (Egr1) zinc fingers bound to microarrays containing all possible central 3 bp triplet binding sites indicates that the nucleotides of transcription factor binding sites cannot be treated independently. This indicates that the current practice of characterizing transcription factor binding sites by mutating individual positions of binding sites one base pair at a time does not provide a true picture of the sequence specificity. Similarly, current bioinformatic practices using either just a consensus sequence, or even mononucleotide frequency weight matrices to provide more complete descriptions of transcription factor binding sites, are not accurate in depicting the true binding site specificities, since these methods rely upon the assumption that the nucleotides of binding sites exert independent effects on binding affinity. Our results stress the importance of complete reference tables of all possible binding sites for comparing protein binding preferences for various DNA sequences. We also show results suggesting that microarray binding data using particular subsets of all possible binding sites can be used to extrapolate the relative binding affinities of all possible full-length binding sites, given a known binding site for use as a starting sequence for site preference refinement.
PMCID: PMC101241  PMID: 11861919
15.  Evaluation of methods for modeling transcription-factor sequence specificity 
Nature biotechnology  2013;31(2):126-134.
Genomic analyses often involve scanning for potential transcription-factor (TF) binding sites using models of the sequence specificity of DNA binding proteins. Many approaches have been developed to model and learn a protein’s binding specificity, but these methods have not been systematically compared. Here we applied 26 such approaches to in vitro protein binding microarray data for 66 mouse TFs belonging to various families. For 9 TFs, we also scored the resulting motif models on in vivo data, and found that the best in vitro–derived motifs performed similarly to motifs derived from in vivo data. Our results indicate that simple models based on mononucleotide position weight matrices learned by the best methods perform similarly to more complex models for most TFs examined, but fall short in specific cases (<10%). In addition, the best-performing motifs typically have relatively low information content, consistent with widespread degeneracy in eukaryotic TF sequence preferences.
PMCID: PMC3687085  PMID: 23354101
16.  Contribution of Distinct Homeodomain DNA Binding Specificities to Drosophila Embryonic Mesodermal Cell-Specific Gene Expression Programs 
PLoS ONE  2013;8(7):e69385.
Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.
PMCID: PMC3724861  PMID: 23922708
17.  LOESS correction for length variation in gene set-based genomic sequence analysis 
Bioinformatics  2012;28(11):1446-1454.
Motivation: Sequence analysis algorithms are often applied to sets of DNA, RNA or protein sequences to identify common or distinguishing features. Controlling for sequence length variation is critical to properly score sequence features and identify true biological signals rather than length-dependent artifacts.
Results: Several cis-regulatory module discovery algorithms exhibit a substantial dependence between DNA sequence score and sequence length. Our newly developed LOESS method is flexible in capturing diverse score-length relationships and is more effective in correcting DNA sequence scores for length-dependent artifacts, compared with four other approaches. Application of this method to genes co-expressed during Drosophila melanogaster embryonic mesoderm development or neural development scored by the Lever motif analysis algorithm resulted in successful recovery of their biologically validated cis-regulatory codes. The LOESS length-correction method is broadly applicable, and may be useful not only for more accurate inference of cis-regulatory codes, but also for detection of other types of patterns in biological sequences.
Availability: Source code and compiled code are available from
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3356840  PMID: 22492312
18.  Dual transcriptional activator and repressor roles of TBX20 regulate adult cardiac structure and function 
Human Molecular Genetics  2012;21(10):2194-2204.
The ongoing requirement in adult heart for transcription factors with key roles in cardiac development is not well understood. We recently demonstrated that TBX20, a transcriptional regulator required for cardiac development, has key roles in the maintenance of functional and structural phenotypes in adult mouse heart. Conditional ablation of Tbx20 in adult cardiomyocytes leads to a rapid onset and progression of heart failure, with prominent conduction and contractility phenotypes that lead to death. Here we describe a more comprehensive molecular characterization of the functions of TBX20 in adult mouse heart. Coupling genome-wide chromatin immunoprecipitation and transcriptome analyses (RNA-Seq), we identified a subset of genes that change expression in Tbx20 adult cardiomyocyte-specific knockout hearts which are direct downstream targets of TBX20. This analysis revealed a dual role for TBX20 as both a transcriptional activator and a repressor, and that each of these functions regulates genes with very specialized and distinct molecular roles. We also show how TBX20 binds to its targets genome-wide in a context-dependent manner, using various cohorts of co-factors to either promote or repress distinct genetic programs within adult heart. Our integrative approach has uncovered several novel aspects of TBX20 and T-box protein function within adult heart.
Sequencing data accession number ( GSE30943.
PMCID: PMC3335310  PMID: 22328084
19.  iSyTE: Integrated Systems Tool for Eye Gene Discovery 
Based on mouse lens gene expression profiling, a systems tool was developed for identification of genes associated with human congenital cataract. iSyTE ranked 88% of known isolated congenital cataract–associated genes within the top two of all candidates in the originally mapped genomic intervals.
To facilitate the identification of genes associated with cataract and other ocular defects, the authors developed and validated a computational tool termed iSyTE (integrated Systems Tool for Eye gene discovery; iSyTE uses a mouse embryonic lens gene expression data set as a bioinformatics filter to select candidate genes from human or mouse genomic regions implicated in disease and to prioritize them for further mutational and functional analyses.
Microarray gene expression profiles were obtained for microdissected embryonic mouse lens at three key developmental time points in the transition from the embryonic day (E)10.5 stage of lens placode invagination to E12.5 lens primary fiber cell differentiation. Differentially regulated genes were identified by in silico comparison of lens gene expression profiles with those of whole embryo body (WB) lacking ocular tissue.
Gene set analysis demonstrated that this strategy effectively removes highly expressed but nonspecific housekeeping genes from lens tissue expression profiles, allowing identification of less highly expressed lens disease–associated genes. Among 24 previously mapped human genomic intervals containing genes associated with isolated congenital cataract, the mutant gene is ranked within the top two iSyTE-selected candidates in approximately 88% of cases. Finally, in situ hybridization confirmed lens expression of several novel iSyTE-identified genes.
iSyTE is a publicly available Web resource that can be used to prioritize candidate genes within mapped genomic intervals associated with congenital cataract for further investigation. Extension of this approach to other ocular tissue components will facilitate eye disease gene discovery.
PMCID: PMC3339920  PMID: 22323457
20.  DNA Sequence Preferences of Transcriptional Activators Correlate More Strongly than Repressors with Nucleosomes 
Molecular Cell  2012;47(2):183-192.
Transcription factors (TFs) and histone octamers are two abundant classes of DNA binding proteins that coordinate the transcriptional program in cells. Detailed studies of individual TFs have shown that TFs bind to nucleosome-occluded DNA sequences and induce nucleosome disruption/repositioning, while recent global studies suggest this is not the only mechanism used by all TFs. We have analyzed to what extent the intrinsic DNA binding preferences of TFs and histones play a role in determining nucleosome occupancy, in addition to nonintrinsic factors such as the enzymatic activity of chromatin remodelers. The majority of TFs in budding yeast have an intrinsic sequence preference overlapping with nucleosomal histones. TFs with intrinsic DNA binding properties highly correlated with those of histones tend to be associated with gene activation and might compete with histones to bind to genomic DNA. Consistent with this, we show that activators induce more nucleosome disruption upon transcriptional activation than repressors.
Graphical Abstract
► We studied DNA-binding preferences for yeast nucleosomes and 137 TFs ► The majority of yeast TFs have intrinsic binding sequences similar to histones ► Activators have more similar intrinsic binding sequences than repressors to histones ► Activators compete more effectively with histones, disrupting nucleosomes formation
PMCID: PMC3566590  PMID: 22841002
21.  Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding 
Nature Immunology  2011;13(1):95-102.
The unique DNA-binding properties of distinct NF-κB dimers are known to influence the selective regulation of NF-κB target genes. To gain a stronger appreciation for these dimer-specific differences, we have combined protein-binding microarrays (PBM) and surface plasmon resonance (SPR) to evaluate DNA sites recognized by eight different NF-κB dimers. We observed three distinct binding-specificity classes and provide insight into mechanisms by which dimers might regulate distinct sets of genes. We identified many new non-traditional κB site sequences and highlight an under-appreciated plasticity of NF-κB dimers in recognizing κB sites with a single consensus half-site. This study provides a database that will be of broad utility in efforts to identify NF-κB target sites and uncover gene regulatory circuitry.
PMCID: PMC3242931  PMID: 22101729
22.  Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-κB family DNA binding 
Nature immunology  2011;13(1):95-102.
The unique DNA-binding properties of distinct NF-κB dimers are known to influence the selective regulation of NF-κB target genes. To gain a stronger appreciation for these dimer-specific differences, we have combined protein-binding microarrays (PBM) and surface plasmon resonance (SPR) to evaluate DNA sites recognized by eight different NF-κB dimers. We observed three distinct binding-specificity classes and provide insight into mechanisms by which dimers might regulate distinct sets of genes. We identified many new non-traditional κB site sequences and highlight an under-appreciated plasticity of NF-κB dimers in recognizing κB sites with a single consensus half-site. This study provides a database that will be of broad utility in efforts to identify NF-κB target sites and uncover gene regulatory circuitry.
PMCID: PMC3242931  PMID: 22101729
23.  An Evolutionarily Conserved Enhancer Regulates Bmp4 Expression in Developing Incisor and Limb Bud 
PLoS ONE  2012;7(6):e38568.
To elucidate the transcriptional regulation of Bmp4 expression during organogenesis, we used phylogenetic footprinting and transgenic reporter analyses to identify Bmp4 cis-regulatory modules (CRMs). These analyses identified a regulatory region located ∼46 kb upstream of the mouse Bmp4 transcription start site that had previously been shown to direct expression in lateral plate mesoderm. We refined this regulatory region to a 396-bp minimal enhancer, and show that it recapitulates features of endogenous Bmp4 expression in developing mandibular arch ectoderm and incisor epithelium during the initiation-stage of tooth development. In addition, this enhancer directs expression in the apical ectodermal ridge (AER) of the developing limb and in anterior and posterior limb mesenchyme. Transcript profiling of E11.5 mouse incisor dental lamina, together with protein binding microarray (PBM) analyses, allowed identification of a conserved DNA binding motif in the Bmp4 enhancer for Pitx homeoproteins, which are also expressed in the developing mandibular and incisor epithelium. In vitro electrophoretic mobility shift assays (EMSA) and in vivo transgenic reporter mutational analyses revealed that this site supports Pitx binding and that the site is necessary to recapitulate aspects of endogenous Bmp4 expression in developing craniofacial and limb tissues. Finally, Pitx2 chromatin immunoprecipitation (ChIP) demonstrated direct binding of Pitx2 to this Bmp4 enhancer site in a dental epithelial cell line. These results establish a direct molecular regulatory link between Pitx family members and Bmp4 gene expression in developing incisor epithelium.
PMCID: PMC3373496  PMID: 22701669
25.  Differential regulation of mesodermal gene expression by Drosophila cell type-specific Forkhead transcription factors 
Development (Cambridge, England)  2012;139(8):1457-1466.
A common theme in developmental biology is the repeated use of the same gene in diverse spatial and temporal domains, a process that generally involves transcriptional regulation mediated by multiple separate enhancers, each with its own arrangement of transcription factor (TF)-binding sites and associated activities. Here, by contrast, we show that the expression of the Drosophila Nidogen (Ndg) gene at different embryonic stages and in four mesodermal cell types is governed by the binding of multiple cell-specific Forkhead (Fkh) TFs – including Biniou (Bin), Checkpoint suppressor homologue (CHES-1-like) and Jumeau (Jumu) – to three functionally distinguishable Fkh-binding sites in the same enhancer. Whereas Bin activates the Ndg enhancer in the late visceral musculature, CHES-1-like cooperates with Jumu to repress this enhancer in the heart. CHES-1-like also represses the Ndg enhancer in a subset of somatic myoblasts prior to their fusion to form multinucleated myotubes. Moreover, different combinations of Fkh sites, corresponding to two different sequence specificities, mediate the particular functions of each TF. A genome-wide scan for the occurrence of both classes of Fkh domain recognition sites in association with binding sites for known cardiac TFs showed an enrichment of combinations containing the two Fkh motifs in putative enhancers found within the noncoding regions of genes having heart expression. Collectively, our results establish that different cell-specific members of a TF family regulate the activity of a single enhancer in distinct spatiotemporal domains, and demonstrate how individual binding motifs for a TF class can differentially influence gene expression.
PMCID: PMC3308180  PMID: 22378636
Transcription factors; Transcription factor binding sites; Forkhead proteins; Transcriptional regulation; Enhancers; Mesoderm

Results 1-25 (64)