Project description:The nematode Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been hampered by the fact that DNA binding motifs are known for only 71 (9%) of the estimated 763 high-confidence sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray (PBM) experiments on representatives of canonical TF families in the C. elegans TF repertoire, obtaining motifs for 129 distinct TFs. Moreover, we can infer motifs for 97 additional TFs that have DNA binding domains that are very similar to those already characterized, resulting in a total coverage of binding specificities for almost 40% of the C. elegans TF repertoire. These data highlight the diversification of binding motifs for the nuclear hormone receptor (NHR) and C2H2 zinc finger families, and reveal unexpected diversity of motifs for others, including the T-box and DM families. Enrichment of motifs in the promoters of functionally related genes is consistent with known biology in many cases, and also identifies putative new regulatory roles for poorly characterized TFs. The motifs are available at http:// http://cisbp.ccbr.utoronto.ca. Protein binding microarray (PBM) experiments were performed for a set of 129 diverse C. elegans transcription factors. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. Details of the PBM protocol are described in Berger et al., Nature Biotechnology 2006.
Project description:Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable binding of TFs with diverse tissue specificities, functions, and DNA binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly-phased nucleosome. The pluripotency factors OCT4- SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome internal sites, a preference that also guides binding genome-wide. By contrast, factors such as BANP, REST, or CTCF, engage throughout causing nucleosomal displacement.This reveals that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF-specific and influenced by nucleosome position in the cell.
Project description:Mammalian gene expression is controlled by transcription factors (TFs) that engage sequence motifs in a chromatinized genome, where nucleosomes can restrict DNA access. Yet, how nucleosomes affect individual TFs remains unclear. Here, we measure the ability of over one hundred TF motifs to recruit TFs in a defined chromosomal locus in mouse embryonic stem cells. This identifies a set sufficient to enable binding of TFs with diverse tissue specificities, functions, and DNA binding domains. These chromatin-competent factors are further classified when challenged to engage motifs within a highly-phased nucleosome. The pluripotency factors OCT4- SOX2 preferentially engage non-nucleosomal and entry-exit motifs, but not nucleosome internal sites, a preference that also guides binding genome-wide. By contrast, factors such as BANP, REST, or CTCF, engage throughout causing nucleosomal displacement.This reveals that TFs vary widely in their sensitivity to nucleosomes and that genome access is TF-specific and influenced by nucleosome position in the cell.
Project description:The nematode Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been hampered by the fact that DNA binding motifs are known for only 71 (9%) of the estimated 763 high-confidence sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray (PBM) experiments on representatives of canonical TF families in the C. elegans TF repertoire, obtaining motifs for 129 distinct TFs. Moreover, we can infer motifs for 97 additional TFs that have DNA binding domains that are very similar to those already characterized, resulting in a total coverage of binding specificities for almost 40% of the C. elegans TF repertoire. These data highlight the diversification of binding motifs for the nuclear hormone receptor (NHR) and C2H2 zinc finger families, and reveal unexpected diversity of motifs for others, including the T-box and DM families. Enrichment of motifs in the promoters of functionally related genes is consistent with known biology in many cases, and also identifies putative new regulatory roles for poorly characterized TFs. The motifs are available at http:// http://cisbp.ccbr.utoronto.ca.
Project description:A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3 and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Hydrogen deuterium exchange mass spectrometry was used to define two stages in the mode of binding of the CD20 epitope the antibody. In the early stage of the reaction, the antigen interacted with CDR3 (VH). In the equilibrium stages, stable binding occurred to CDR2 (VH) and CDR2 (VL), without any detectable CDR3 (VH) interactions. These data suggest that CDR3 (VH) functions as a transient antigen docking motif to nucleate the peptide into the antibody active site which resolves into antigen binding with the heavy and light chain CDR2 domains. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry to map the kinetic mode of antigen binding. These data support the further development of an engineered synthetic antibody for use as a canine lymphoma therapeutic that mimics the human anti-CD20 antibody therapeutic.
Project description:A comparative canine-human therapeutics model is being developed in B-cell lymphoma through the generation of a hybridoma cell that produces a murine monoclonal antibody specific for canine CD20. The hybridoma cell produces two light chains, light chain-3 and light chain-7. However, the contribution of either light chain to the authentic full-length hybridoma derived IgG is undefined. Mass spectrometry was used to identify only one of the two light chains, light chain-7, as predominating in the full-length IgG. Gene synthesis created a recombinant murine-canine chimeric monoclonal antibody expressing light chain-7 that reconstituted the IgG binding to CD20. Hydrogen deuterium exchange mass spectrometry was used to define two stages in the mode of binding of the CD20 epitope the antibody. In the early stage of the reaction, the antigen interacted with CDR3 (VH). In the equilibrium stages, stable binding occurred to CDR2 (VH) and CDR2 (VL), without any detectable CDR3 (VH) interactions. These data suggest that CDR3 (VH) functions as a transient antigen docking motif to nucleate the peptide into the antibody active site which resolves into antigen binding with the heavy and light chain CDR2 domains. These approaches define a methodology for fine mapping of CDR contacts using nested enzymatic reactions and hydrogen deuterium exchange mass spectrometry to map the kinetic mode of antigen binding. These data support the further development of an engineered synthetic antibody for use as a canine lymphoma therapeutic that mimics the human anti-CD20 antibody therapeutic.
Project description:RNA-protein interactions are pivotal to proper gene regulation. Many RNA-binding proteins possess multiple RNA-binding domains; however, how these domains interplay to specify and regulate RNA targets remains poorly understood. Here, we investigate three multi-domain proteins, Musashi-1, Musashi-2, and Unkempt, three factors which share a high degree of RNA specificity. We use a combination of massively parallel in vitro assays with random or naturally derived RNA sequences and find that individual domains within a protein can have differing affinities, specificities, and spacing preferences. Further, we emphasize that while all three proteins have overlapping motif specificities, non-overlapping sequences may allow for target discrimination. We carry out large scale competition assays between these proteins and determine how individual protein specificities and affinities influence competitive binding. Integration of in vivo binding and regulation with in vitro specificities shows that target selection involves a combination of the protein intrinsic specificities described here, but cellular context is critical to drive these proteins to motifs in specific transcript regions. Finally, evolutionarily conserved RNA regions display evidence of binding multiple RBPs in vivo, and these RNA regions recapitulate this trend with the highest affinity in vitro. We highlight the importance of understanding features of complex RNA-protein interactions and how protein-target discrimination can be established.
Project description:RNA binding proteins are key regulators of gene expression, yet only a small fraction of these proteins has been functionally characterized. Here, we report the first large-scale analysis of the RNA motifs recognised by RNA binding proteins, encompassing 205 distinct proteins from 24 diverse eukaryotes. The sequence specificities of RBPs display deep evolutionary conservation, such that the recognition preferences for a large fraction of metazoan RNA binding proteins can be inferred from the sequences of their binding domains. The motifs we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA binding proteins in both normal physiology and in human disease. These data provide an unprecedented global view of RBPs and their targets and constitute an invaluable resource for defining post-transcriptional regulatory mechanisms in eukaryotes. Here, we analyze the RNA-binding preferences of 205 distinct RNA-binding proteins from 24 different eukaryotes, using RNAcompete. In this assay, purified GST-tagged RBPs are incubated with an excess of RNA pool and bound RNA from individual pulldowns are directly labeled, hybridized to a custom Agilent 244K microarray, and analyzed computationally to identify RNA-binding motifs.
Project description:Expansions of CD4+ T cells utilizing TRAV12-1 and TRBV2 were found exclusively in HLA-DR3+ LS patients. These TCRs shared CDR3 homology, suggesting that these T cells are responsible for recognizing sarcoidosis-associated antigen(s) and are involved in the local alveolitis.
Project description:Expansions of CD4+ T cells utilizing TRAV12-1 and TRBV2 were found exclusively in HLA-DR3+ LS patients. These TCRs shared CDR3 homology, suggesting that these T cells are responsible for recognizing sarcoidosis-associated antigen(s) and are involved in the local alveolitis.