Global analysis of Drosophila Cys2-His2 zinc finger proteins reveals a multitude of novel recognition motifs and binding determinants

Cys2-His2 zinc finger proteins (ZFPs) are the largest group of transcription factors in higher metazoans. A complete characterization of these ZFPs and their associated target sequences is pivotal to fully annotate transcriptional regulatory networks in metazoan genomes. As a first step in this process, we have characterized the DNA-binding specificities of 129 zinc finger sets from Drosophila using a bacterial one-hybrid system. This data set contains the DNA-binding specificities for at least one encoded ZFP from 70 unique genes and 23 alternate splice isoforms representing the largest set of characterized ZFPs from any organism described to date. These recognition motifs can be used to predict genomic binding sites for these factors within the fruit fly genome. Subsets of fingers from these ZFPs were characterized to define their orientation and register on their recognition sequences, thereby allowing us to define the recognition diversity within this finger set. We find that the characterized fingers can specify 47 of the 64 possible DNA triplets. To confirm the utility of our finger recognition models, we employed subsets of Drosophila fingers in combination with an existing archive of artificial zinc finger modules to create ZFPs with novel DNA-binding specificity. These hybrids of natural and artificial fingers can be used to create functional zinc finger nucleases for editing vertebrate genomes

FlyFactorSurvey: a database of Drosophila transcription factor binding specificities determined using the bacterial one-hybrid system

FlyFactorSurvey (http://pgfe.umassmed.edu/TFDBS/) is a database of DNA binding specificities for Drosophila transcription factors (TFs) primarily determined using the bacterial one-hybrid system. The database provides community access to over 400 recognition motifs and position weight matrices for over 200 TFs, including many unpublished motifs. Search tools and flat file downloads are provided to retrieve binding site information (as sequences, matrices and sequence logos) for individual TFs, groups of TFs or for all TFs with characterized binding specificities. Linked analysis tools allow users to identify motifs within our database that share similarity to a query matrix or to view the distribution of occurrences of an individual motif throughout the Drosophila genome. Together, this database and its associated tools provide computational and experimental biologists with resources to predict interactions between Drosophila TFs and target cis-regulatory sequences

An improved predictive recognition model for Cys2-His2 zinc finger proteins

Cys2-His2 zinc finger proteins (ZFPs) are the largest family of transcription factors in higher metazoans. They also represent the most diverse family with regards to the composition of their recognition sequences. Although there are a number of ZFPs with characterized DNA-binding preferences, the specificity of the vast majority of ZFPs is unknown and cannot be directly inferred by homology due to the diversity of recognition residues present within individual fingers. Given the large number of unique zinc fingers and assemblies present across eukaryotes, a comprehensive predictive recognition model that could accurately estimate the DNA-binding specificity of any ZFP based on its amino acid sequence would have great utility. Toward this goal, we have used the DNA-binding specificities of 678 two-finger modules from both natural and artificial sources to construct a random forest-based predictive model for ZFP recognition. We find that our recognition model outperforms previously described determinant-based recognition models for ZFPs, and can successfully estimate the specificity of naturally occurring ZFPs with previously defined specificities

Studies of initiation and elongation in T7 RNA polymerase

During transcription initiation, competition between the initiation and elongation processes is thought to lead to the production of abortive transcripts (2–8 nucleotides of RNA) by the RNA polymerases. Our results show that A-16C and T-14G promoter mutations would lead to earlier promoter escape events relative to A-15C/G and wild type promoters as previously reported. This indicates that even for the A-15C/G promoter, interactions between the polymerase and bases at positions −16 and −14 are still important. The only physiological regulator of transcription known in the T7 phage is the phage protein T7 lysozyme. Several models have been proposed about its mechanism of action. By running transcription assays on crosslinked promoter-polymerase complexes in the presence and absence of lysozyme, in addition to partial tryptic digests on promoter complexes stalled at different translocation positions, we have shown that lysozyme largely inhibits the initiation complex to elongation complex transition by causing the increased dissociation of the already unstable initiation complex. In transcription, double stranded DNA is locally melted (forming a bubble ), containing RNA annealed to the template strand. Thus, there is an energy cost to maintain the open bubble, but an energy gain from formation of the hybrid. We have independently varied the DNA-DNA and RNA-DNA duplex stabilities by employing 5-brominated pyrimidines, which stabilize duplexes by 4.3° per substitution. Our results show that increasing hybrid stability stabilizes the elongation complex more than increasing DNA-DNA stability (favoring collapse of the bubble) destabilizes the complex. The recent solving of the crystal structures of T7 RNA polymerase initiation complexes involved in de novo RNA synthesis, has revealed a novel NTP binding site (the D-site), distinct from previously known P- and N-sites. The apparent inflexibility of the D- and P-sites in the initiation complex has inspired the hypothesis that initiation of transcription with GTP is by a lock-and-key mechanism. By using a 5-propynyl-dC base in place of dC at the +1 and +2 sites, which would still allow for initiation with GTP we observe a decrease in initiation efficiency. This indicates that initiation of transcription in the T7 RNA polymerase system is by a lock-and-key mechanism

Cas9 effector-mediated regulation of transcription and differentiation in human pluripotent stem cells

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks, we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells, we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes, which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions, which can then be employed to direct cellular differentiation down desired pathways