The genome as 3D-nucleic acid-[glyco-]protein nanomachine orchestrates translation of genetic [Me... more The genome as 3D-nucleic acid-[glyco-]protein nanomachine orchestrates translation of genetic [Mendelian] and epigenetic [non-Mendelian] information into phenotypical structural patterns. It is not restricted to transcription and translation of DNA scripts into proteins which matter only 2% of transcriptional output. By episcription, same genotypes [e.g. twins] deliver plethoras of not identical epigenetic phenotype variations. Functions of endogenous RNA isolated from total transcriptional output upon cell activation were investigated in vascular remodeling and tolerance phenomena. Structural codes [3D-episcripts] for epigenetic phenotyping were found escaping deciphering, so far. METHODS:
bioRxiv (Cold Spring Harbor Laboratory), May 22, 2018
Determining the biophysical principles that shape transcription factor (TF) binding in a cellspec... more Determining the biophysical principles that shape transcription factor (TF) binding in a cellspecific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes -autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding-site affinity are independent and additive. .
Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the... more Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation we used proteinbinding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis reveals a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the function of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. Our results provide new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression.
bioRxiv (Cold Spring Harbor Laboratory), Apr 6, 2024
Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that... more Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-κB as a primary regulator of acutely induced H3K27ac. Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data supports clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be .
A new method is introduced to structurally align interfaces observed in protein-DNA complexes. Th... more A new method is introduced to structurally align interfaces observed in protein-DNA complexes. The method is based on a procedure that describes the interfacial geometry in terms of the spatial relationships between individual amino acid-nucleotide pairs. An amino acid-amino acid similarity matrix, S, is defined that provides a quantitative measure of the geometric relationships of amino acids in different interfaces and the entire stretch of "local" DNA within some distance of each amino acid. S is used as a substitution matrix in a dynamic programming algorithm that aligns the interfacial amino acids of the two complexes. The quality of the alignment is determined by an interface alignment score, IAS, that provides a quantitative measure of the similarity in the docking geometry between two protein-DNA complexes. We have clustered a large set of protein-DNA complexes based on their IAS values. In general, proteins within a single family form identifiable clusters. Subgroup clustering is often observed within families offering a fine-grained description of docking geometries. Although proteins with similar folds tend to dock in similar ways, important differences are observed even for structural motifs that almost perfectly align. Relationships are observed between the interfaces formed in cognate and noncognate complexes involving the same proteins indicating a strong driving force to maintain certain contacts, even if this requires a distortion of the DNA. There are cases where inter-family similarities are greater than intrafamily similarities. Our method offers the possibility of comparing different protein-DNA interfaces in a detailed, objective and quantitative fashion. This offers the possibility of new approaches to the description of the determinants of molecular recognition and to the prediction of protein and DNA sequence combinations that are optimal for binding.
Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer... more Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer drivers. To predict driver non-coding variants (NCVs), we present a transcription factor (TF)-aware burden test based on a model of coherent TF function in promoters. We apply this test to NCVs from the Pan-Cancer Analysis of Whole Genomes cohort and predict 2555 driver NCVs in the promoters of 813 genes across 20 cancer types. These genes are enriched in cancer-related gene ontologies, essential genes, and genes associated with cancer prognosis. We find that 765 candidate driver NCVs alter transcriptional activity, 510 lead to differential binding of TF-cofactor regulatory complexes, and that they primarily impact the binding of ETS factors. Finally, we show that different NCVs within a promoter often affect transcriptional activity through shared mechanisms. Our integrated computational and experimental approach shows that cancer NCVs are widespread and that ETS factors are commonly disrupted. Cancer initiation and progression are often associated with environmentally induced or spontaneous mutations, and inherited genomic variants that increase cancer risk 1-3. Large scale projects such as the Cancer Genome Atlas (TCGA) and the International Genome Consortium (ICGC) have identified millions of somatic variants in tumors 4-6. However, in most cases, it is not known whether these mutations affect any cellular function, confer growth advantage, or are causally implicated in cancer development 7. The difficulty in annotating variants is that only a few cancer driver mutations are needed to initiate tumor growth, development, and metastasis and these mutations must be distinguished from thousands of passenger mutations that do not alter fitness 7. Even though more than 90% of somatic variants are in non-coding regions, few non-coding cancer drivers have been identified 6,8,9 , highlighting the need for approaches to identify and validate non-coding variants (NCVs) in cancer. Mutational burden tests have been used to predict driver NCVs. These tests are based on determining an increased mutational frequency in DNA regions of interest (e.g., cis-regulatory elements (CREs)) compared to a background mutational frequency 10-18. Methods have employed a range of different parameters to estimate the background mutational frequency in CREs, including cancer-specific mutational signatures, sequence conservation, functional annotations, mutational frequencies in neighboring regions or other "similar" genomic regions, replication timing, and expression levels 9,19. Despite these varied approaches to estimate mutational burden and the increasing number of sequenced tumor samples, studies have only identified~100 driver NCVs. For example, burden tests within specific cancer types have identified NCVs in the promoters of TERT, FOXA1, HES1, SDHD, and PLEKHS1 20-22. Further, a global analysis of 2568 cancer whole genome samples from the Pan-Cancer Analysis of Whole Genomes (PCAWG) identified driver NCVs in the promoters of TERT, HES1 and seven additional genes 9. A more recent analysis of 3949 tumors from PCAWG and the Hartwig Medical Foundation identified driver NCVs in the promoters and enhancers of 52 genes 19. Additionally, driver NCVs have been identified in the super-enhancers of BCL6, BCL2, CXCR4 in diffuse large B-cell lymphomas 23. Whether this somewhat limited number of
Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As... more Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to 41300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel 'recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4-Met28-Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNAbinding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. Molecular Systems Biology 7: 555;
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Dec 10, 2012
The genome-wide identification of pairs of interacting proteins is an important step in the eluci... more The genome-wide identification of pairs of interacting proteins is an important step in the elucidation of cell regulatory mechanisms 1,2. Much of our current knowledge derives from highthroughput techniques such as yeast two hybrid and affinity purification 3 , as well as from manual curation of experiments on individual systems 4. A variety of computational approaches based, for example, on sequence homology, gene co-expression, and phylogenetic profiles have also been developed for the genome-wide inference of protein-protein interactions (PPIs) 5,6. Yet, comparative studies suggest that the development of accurate and complete repertoires of PPIs is still in its early stages 7-9. Here we show that three-dimensional structural information can be used to predict PPIs with an accuracy and coverage that are superior to predictions based on nonstructural evidence. Moreover, an algorithm, PrePPI, that combines structural information with Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
A major challenge in obtaining a full molecular description of evolutionary adaptation is to char... more A major challenge in obtaining a full molecular description of evolutionary adaptation is to characterize how transcription factor (TF) DNA-binding specificity can change. To identify mechanisms of TF diversification, we performed detailed comparisons of yeast C2H2 ZF proteins with identical canonical recognition residues that are expected to bind the same DNA sequences. Unexpectedly, we found that ZF proteins can adapt to recognize new binding sites in a modular fashion whereby binding to common core sites remains unaffected. We identified two distinct mechanisms, conserved across multiple Ascomycota species, by which this molecular adaptation occurred. Our results suggest a route for TF evolution that alleviates negative pleiotropic effects by modularly gaining new binding sites. These findings expand our current understanding of ZF DNA binding and provide evidence for paralogous ZFs utilizing alternate modes of DNA binding to recognize unique sets of noncanonical binding sites.
Binding of proteins to particular DNA sites across the genome is a primary determinant of specifi... more Binding of proteins to particular DNA sites across the genome is a primary determinant of specificity in genome maintenance and gene regulation. DNAbinding specificity is encoded at multiple levels, from the detailed biophysical interactions between proteins and DNA, to the assembly of multi-protein complexes. At each level, variation in the mechanisms used to achieve specificity has led to difficulties in constructing and applying simple models of DNA binding. We review the complexities in protein-DNA binding found at multiple levels and discuss how they confound the idea of simple recognition codes. We discuss the impact of new highthroughput technologies for the characterization of protein-DNA binding, and how these technologies are uncovering new complexities in protein-DNA recognition. Finally, we review the concept of multi-protein recognition codes in which new DNAbinding specificities are achieved by the assembly of multi-protein complexes. Side-chain flexibility and water molecules Protein side-chains and water molecules in the binding interface create a flexible network of interactions that can readily adopt new conformations in response to changes in DNA or protein sequence (2-5). Two examples illustrate the relevant complexities. Comparing protein-DNA crystal structures for wild-type and mutant zinc finger protein Zif268/Egr1, it was demonstrated that a single amino acid mutation (Zif268 D20A) could lead to altered protein side-chain conformations and DNAbinding preferences (6). Further, the side-chain rearrangement occurred without a change in the protein docking geometry, but resulted in a new, positioned water molecule in the binding interface for one of the DNA sequences. Structural comparison of two Cre recombinase variants in complex with different DNA sequences revealed that both DNA and protein differences affect the contacts made in the binding interface (7). In complex with the same LoxM7 DNA sequence, two Cre variants, which differ at only three residue positions, had distinctly different conformations for the common sidechains mediating base contacts. Further, comparison of one of the variants bound to a different LoxP DNA site revealed that recognition of the two DNA sequences, LoxM7 and LoxP, led to altered side-chain conformations and different side-chain and water-mediated contacts. These studies highlight the complex interplay between side-chains, water molecules and DNA bases in the binding interface. DNA structure and indirect readout 'Direct readout' (also known as 'base readout') refers to the situation where proteins discriminate different bases in a DNA sequence via direct (or water-mediated)
How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate ... more How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate quantitative changes in spatiotemporally restricted expression patterns during animal development is not well understood. Pax6 is a dosage-sensitive gene essential for eye development. Here, we identify the Prep1 (pKnox1) transcription factor as a critical dose-dependent upstream regulator of Pax6 expression during lens formation. We show that Prep1 activates the Pax6 lens enhancer by binding to two phylogenetically conserved lower-affinity DNA-binding sites. Finally, we describe a mechanism whereby Pax6 levels are determined by transcriptional synergy of Prep1 bound to the two sites, while timing of enhancer activation is determined by binding site affinity.
bioRxiv (Cold Spring Harbor Laboratory), Mar 15, 2021
These results represent the first functional characterization of the biologically important trans... more These results represent the first functional characterization of the biologically important transcription factor NF-κB in a taxonomic kingdom other than Animalia. As such, they provide information on the evolutionary origins and basal diversification of NF-κB outside of metazoans. These results suggest that NF-κB plays life stage-specific roles in Capsaspora, the closest unicellular ancestor to all metazoans. Finally, the analysis of three NF-κB proteins in a single choanoflagellate indicates that choanoflagellates have subclasses of NF-κBs, which can form heterodimers, suggesting that NF-κB subunit expansion and diversification has occurred at least twice in evolution. .
A paradigm in transcriptional regulation is that a graded increase in transcription factor (TF) c... more A paradigm in transcriptional regulation is that a graded increase in transcription factor (TF) concentration is ''digitally'' translated into an on/off transcriptional response by means of cooperative TF binding to adjacent DNA binding sites. Such paradigm stems from the analysis of TFs operating in developmental processes, notably embryonic segmentation, that require the definition of sharp borders separating different body regions. Here we show that NF-kB, a key TF responsible for the expression of genes implicated in the inflammatory and immune responses, is an ''analogical'' transcriptional regulator. We demonstrate that increasing doses of inflammatory stimuli lead to gradually increasing concentrations of NF-kB in the cell nucleus, which in turn are translated into gradually increasing levels of transcriptional activity of NF-kB target genes. Differently to what observed in developmental systems, we show that the number of NF-kB binding sites in 304a Monday, March 2, 2009 CORE Metadata, citation and similar papers at core.ac.uk
The genome as 3D-nucleic acid-[glyco-]protein nanomachine orchestrates translation of genetic [Me... more The genome as 3D-nucleic acid-[glyco-]protein nanomachine orchestrates translation of genetic [Mendelian] and epigenetic [non-Mendelian] information into phenotypical structural patterns. It is not restricted to transcription and translation of DNA scripts into proteins which matter only 2% of transcriptional output. By episcription, same genotypes [e.g. twins] deliver plethoras of not identical epigenetic phenotype variations. Functions of endogenous RNA isolated from total transcriptional output upon cell activation were investigated in vascular remodeling and tolerance phenomena. Structural codes [3D-episcripts] for epigenetic phenotyping were found escaping deciphering, so far. METHODS:
bioRxiv (Cold Spring Harbor Laboratory), May 22, 2018
Determining the biophysical principles that shape transcription factor (TF) binding in a cellspec... more Determining the biophysical principles that shape transcription factor (TF) binding in a cellspecific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes -autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding-site affinity are independent and additive. .
Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the... more Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation we used proteinbinding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis reveals a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the function of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. Our results provide new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression.
bioRxiv (Cold Spring Harbor Laboratory), Apr 6, 2024
Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that... more Transcription factor (TF)-cofactor (COF) interactions define dynamic, cell-specific networks that govern gene expression; however, these networks are understudied due to a lack of methods for high-throughput profiling of DNA-bound TF-COF complexes. Here we describe the Cofactor Recruitment (CoRec) method for rapid profiling of cell-specific TF-COF complexes. We define a lysine acetyltransferase (KAT)-TF network in resting and stimulated T cells. We find promiscuous recruitment of KATs for many TFs and that 35% of KAT-TF interactions are condition specific. KAT-TF interactions identify NF-κB as a primary regulator of acutely induced H3K27ac. Finally, we find that heterotypic clustering of CBP/P300-recruiting TFs is a strong predictor of total promoter H3K27ac. Our data supports clustering of TF sites that broadly recruit KATs as a mechanism for widespread co-occurring histone acetylation marks. CoRec can be .
A new method is introduced to structurally align interfaces observed in protein-DNA complexes. Th... more A new method is introduced to structurally align interfaces observed in protein-DNA complexes. The method is based on a procedure that describes the interfacial geometry in terms of the spatial relationships between individual amino acid-nucleotide pairs. An amino acid-amino acid similarity matrix, S, is defined that provides a quantitative measure of the geometric relationships of amino acids in different interfaces and the entire stretch of "local" DNA within some distance of each amino acid. S is used as a substitution matrix in a dynamic programming algorithm that aligns the interfacial amino acids of the two complexes. The quality of the alignment is determined by an interface alignment score, IAS, that provides a quantitative measure of the similarity in the docking geometry between two protein-DNA complexes. We have clustered a large set of protein-DNA complexes based on their IAS values. In general, proteins within a single family form identifiable clusters. Subgroup clustering is often observed within families offering a fine-grained description of docking geometries. Although proteins with similar folds tend to dock in similar ways, important differences are observed even for structural motifs that almost perfectly align. Relationships are observed between the interfaces formed in cognate and noncognate complexes involving the same proteins indicating a strong driving force to maintain certain contacts, even if this requires a distortion of the DNA. There are cases where inter-family similarities are greater than intrafamily similarities. Our method offers the possibility of comparing different protein-DNA interfaces in a detailed, objective and quantitative fashion. This offers the possibility of new approaches to the description of the determinants of molecular recognition and to the prediction of protein and DNA sequence combinations that are optimal for binding.
Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer... more Although >90% of somatic mutations reside in non-coding regions, few have been reported as cancer drivers. To predict driver non-coding variants (NCVs), we present a transcription factor (TF)-aware burden test based on a model of coherent TF function in promoters. We apply this test to NCVs from the Pan-Cancer Analysis of Whole Genomes cohort and predict 2555 driver NCVs in the promoters of 813 genes across 20 cancer types. These genes are enriched in cancer-related gene ontologies, essential genes, and genes associated with cancer prognosis. We find that 765 candidate driver NCVs alter transcriptional activity, 510 lead to differential binding of TF-cofactor regulatory complexes, and that they primarily impact the binding of ETS factors. Finally, we show that different NCVs within a promoter often affect transcriptional activity through shared mechanisms. Our integrated computational and experimental approach shows that cancer NCVs are widespread and that ETS factors are commonly disrupted. Cancer initiation and progression are often associated with environmentally induced or spontaneous mutations, and inherited genomic variants that increase cancer risk 1-3. Large scale projects such as the Cancer Genome Atlas (TCGA) and the International Genome Consortium (ICGC) have identified millions of somatic variants in tumors 4-6. However, in most cases, it is not known whether these mutations affect any cellular function, confer growth advantage, or are causally implicated in cancer development 7. The difficulty in annotating variants is that only a few cancer driver mutations are needed to initiate tumor growth, development, and metastasis and these mutations must be distinguished from thousands of passenger mutations that do not alter fitness 7. Even though more than 90% of somatic variants are in non-coding regions, few non-coding cancer drivers have been identified 6,8,9 , highlighting the need for approaches to identify and validate non-coding variants (NCVs) in cancer. Mutational burden tests have been used to predict driver NCVs. These tests are based on determining an increased mutational frequency in DNA regions of interest (e.g., cis-regulatory elements (CREs)) compared to a background mutational frequency 10-18. Methods have employed a range of different parameters to estimate the background mutational frequency in CREs, including cancer-specific mutational signatures, sequence conservation, functional annotations, mutational frequencies in neighboring regions or other "similar" genomic regions, replication timing, and expression levels 9,19. Despite these varied approaches to estimate mutational burden and the increasing number of sequenced tumor samples, studies have only identified~100 driver NCVs. For example, burden tests within specific cancer types have identified NCVs in the promoters of TERT, FOXA1, HES1, SDHD, and PLEKHS1 20-22. Further, a global analysis of 2568 cancer whole genome samples from the Pan-Cancer Analysis of Whole Genomes (PCAWG) identified driver NCVs in the promoters of TERT, HES1 and seven additional genes 9. A more recent analysis of 3949 tumors from PCAWG and the Hartwig Medical Foundation identified driver NCVs in the promoters and enhancers of 52 genes 19. Additionally, driver NCVs have been identified in the super-enhancers of BCL6, BCL2, CXCR4 in diffuse large B-cell lymphomas 23. Whether this somewhat limited number of
Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As... more Recruitment of cofactors to specific DNA sites is integral for specificity in gene regulation. As a model system, we examined how targeting and transcriptional control of the sulfur metabolism genes in Saccharomyces cerevisiae is governed by recruitment of the transcriptional co-activator Met4. We developed genome-scale approaches to measure transcription factor (TF) DNA-binding affinities and cofactor recruitment to 41300 genomic binding site sequences. We report that genes responding to the TF Cbf1 and cofactor Met28 contain a novel 'recruitment motif' (RYAAT), adjacent to Cbf1 binding sites, which enhances the binding of a Met4-Met28-Cbf1 regulatory complex, and that abrogation of this motif significantly reduces gene induction under low-sulfur conditions. Furthermore, we show that correct recognition of this composite motif requires both non-DNAbinding cofactors Met4 and Met28. Finally, we demonstrate that the presence of an RYAAT motif next to a Cbf1 site, rather than Cbf1 binding affinity, specifies Cbf1-dependent sulfur metabolism genes. Our results highlight the need to examine TF/cofactor complexes, as novel specificity can result from cofactors that lack intrinsic DNA-binding specificity. Molecular Systems Biology 7: 555;
Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature, Dec 10, 2012
The genome-wide identification of pairs of interacting proteins is an important step in the eluci... more The genome-wide identification of pairs of interacting proteins is an important step in the elucidation of cell regulatory mechanisms 1,2. Much of our current knowledge derives from highthroughput techniques such as yeast two hybrid and affinity purification 3 , as well as from manual curation of experiments on individual systems 4. A variety of computational approaches based, for example, on sequence homology, gene co-expression, and phylogenetic profiles have also been developed for the genome-wide inference of protein-protein interactions (PPIs) 5,6. Yet, comparative studies suggest that the development of accurate and complete repertoires of PPIs is still in its early stages 7-9. Here we show that three-dimensional structural information can be used to predict PPIs with an accuracy and coverage that are superior to predictions based on nonstructural evidence. Moreover, an algorithm, PrePPI, that combines structural information with Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
A major challenge in obtaining a full molecular description of evolutionary adaptation is to char... more A major challenge in obtaining a full molecular description of evolutionary adaptation is to characterize how transcription factor (TF) DNA-binding specificity can change. To identify mechanisms of TF diversification, we performed detailed comparisons of yeast C2H2 ZF proteins with identical canonical recognition residues that are expected to bind the same DNA sequences. Unexpectedly, we found that ZF proteins can adapt to recognize new binding sites in a modular fashion whereby binding to common core sites remains unaffected. We identified two distinct mechanisms, conserved across multiple Ascomycota species, by which this molecular adaptation occurred. Our results suggest a route for TF evolution that alleviates negative pleiotropic effects by modularly gaining new binding sites. These findings expand our current understanding of ZF DNA binding and provide evidence for paralogous ZFs utilizing alternate modes of DNA binding to recognize unique sets of noncanonical binding sites.
Binding of proteins to particular DNA sites across the genome is a primary determinant of specifi... more Binding of proteins to particular DNA sites across the genome is a primary determinant of specificity in genome maintenance and gene regulation. DNAbinding specificity is encoded at multiple levels, from the detailed biophysical interactions between proteins and DNA, to the assembly of multi-protein complexes. At each level, variation in the mechanisms used to achieve specificity has led to difficulties in constructing and applying simple models of DNA binding. We review the complexities in protein-DNA binding found at multiple levels and discuss how they confound the idea of simple recognition codes. We discuss the impact of new highthroughput technologies for the characterization of protein-DNA binding, and how these technologies are uncovering new complexities in protein-DNA recognition. Finally, we review the concept of multi-protein recognition codes in which new DNAbinding specificities are achieved by the assembly of multi-protein complexes. Side-chain flexibility and water molecules Protein side-chains and water molecules in the binding interface create a flexible network of interactions that can readily adopt new conformations in response to changes in DNA or protein sequence (2-5). Two examples illustrate the relevant complexities. Comparing protein-DNA crystal structures for wild-type and mutant zinc finger protein Zif268/Egr1, it was demonstrated that a single amino acid mutation (Zif268 D20A) could lead to altered protein side-chain conformations and DNAbinding preferences (6). Further, the side-chain rearrangement occurred without a change in the protein docking geometry, but resulted in a new, positioned water molecule in the binding interface for one of the DNA sequences. Structural comparison of two Cre recombinase variants in complex with different DNA sequences revealed that both DNA and protein differences affect the contacts made in the binding interface (7). In complex with the same LoxM7 DNA sequence, two Cre variants, which differ at only three residue positions, had distinctly different conformations for the common sidechains mediating base contacts. Further, comparison of one of the variants bound to a different LoxP DNA site revealed that recognition of the two DNA sequences, LoxM7 and LoxP, led to altered side-chain conformations and different side-chain and water-mediated contacts. These studies highlight the complex interplay between side-chains, water molecules and DNA bases in the binding interface. DNA structure and indirect readout 'Direct readout' (also known as 'base readout') refers to the situation where proteins discriminate different bases in a DNA sequence via direct (or water-mediated)
How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate ... more How transcription factors interpret the cis-regulatory logic encoded within enhancers to mediate quantitative changes in spatiotemporally restricted expression patterns during animal development is not well understood. Pax6 is a dosage-sensitive gene essential for eye development. Here, we identify the Prep1 (pKnox1) transcription factor as a critical dose-dependent upstream regulator of Pax6 expression during lens formation. We show that Prep1 activates the Pax6 lens enhancer by binding to two phylogenetically conserved lower-affinity DNA-binding sites. Finally, we describe a mechanism whereby Pax6 levels are determined by transcriptional synergy of Prep1 bound to the two sites, while timing of enhancer activation is determined by binding site affinity.
bioRxiv (Cold Spring Harbor Laboratory), Mar 15, 2021
These results represent the first functional characterization of the biologically important trans... more These results represent the first functional characterization of the biologically important transcription factor NF-κB in a taxonomic kingdom other than Animalia. As such, they provide information on the evolutionary origins and basal diversification of NF-κB outside of metazoans. These results suggest that NF-κB plays life stage-specific roles in Capsaspora, the closest unicellular ancestor to all metazoans. Finally, the analysis of three NF-κB proteins in a single choanoflagellate indicates that choanoflagellates have subclasses of NF-κBs, which can form heterodimers, suggesting that NF-κB subunit expansion and diversification has occurred at least twice in evolution. .
A paradigm in transcriptional regulation is that a graded increase in transcription factor (TF) c... more A paradigm in transcriptional regulation is that a graded increase in transcription factor (TF) concentration is ''digitally'' translated into an on/off transcriptional response by means of cooperative TF binding to adjacent DNA binding sites. Such paradigm stems from the analysis of TFs operating in developmental processes, notably embryonic segmentation, that require the definition of sharp borders separating different body regions. Here we show that NF-kB, a key TF responsible for the expression of genes implicated in the inflammatory and immune responses, is an ''analogical'' transcriptional regulator. We demonstrate that increasing doses of inflammatory stimuli lead to gradually increasing concentrations of NF-kB in the cell nucleus, which in turn are translated into gradually increasing levels of transcriptional activity of NF-kB target genes. Differently to what observed in developmental systems, we show that the number of NF-kB binding sites in 304a Monday, March 2, 2009 CORE Metadata, citation and similar papers at core.ac.uk
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