Zinc fingers 1 and 7 of yeast TFIIIA are essential for assembly of a functional transcription complex on the 5 S RNA gene

The binding of transcription factor (TF) IIIA to the internal control region of the 5 S RNA gene is the first step in the assembly of a DNA–TFIIIA–TFIIIC– TFIIIB transcription complex, which promotes accurate transcription by RNA polymerase III. With the use of mutations that are predicted to disrupt the folding of a zinc finger, we have examined the roles of zinc fingers 1 through 7 of yeast TFIIIA in the establishment of a functional transcription complex both in vitro and in vivo. Our data indicate that, in addition to their role in DNA binding, the first and seventh zinc fingers contribute other essential roles in the assembly of an active transcription complex. Alanine-scanning mutagenesis identified residues within zinc finger 1 that are not required for DNA binding but are required for incorporation of TFIIIC into the TFIIIA–DNA complex. Although disruption of zinc finger 2 or 3 had a deleterious effect on the activity of TFIIIA both in vitro and in vivo, we found that increasing the level of their in vivo expression allowed these mutant proteins to support cell viability. Disruption of zinc fingers 4, 5 or 6 had minimal effect on the DNA binding and TF activities of TFIIIA

An Omega-Based Bacterial One-Hybrid System for the Determination of Transcription Factor Specificity

From the yeast genome completed in 1996 to the 12 Drosophilagenomes published earlier this year; little more than a decade has provided an incredible amount of genomic data. Yet even with this mountain of genetic information the regulatory networks that control gene expression remain relatively undefined. In part, this is due to the enormous amount of non-coding DNA, over 98% of the human genome, which needs to be made sense of. It is also due to the large number of transcription factors, potentially 2,000 such factors in the human genome, which may contribute to any given network directly or indirectly. Certainly, one of the central limitations has been the paucity of transcription factor (TF) specificity data that would aid in the prediction of regulatory targets throughout a genome. The general lack of specificity data has hindered the prediction of regulatory targets for individual TFs as well as groups of factors that function within a common regulatory pathway. A large collection of factor specificities would allow for the combinatorial prediction of regulatory targets that considers all factors actively expressed in a given cell, under a given condition. Herein we describe substantial improvements to a previous bacterial one-hybrid system with increased sensitivity and dynamic range that make it amenable for the high-throughput analysis of sequence-specific TFs. Currently we have characterized 108 (14.3%) of the predicted TFs in Drosophilathat fall into a broad range of DNA-binding domain families, demonstrating the feasibility of characterizing a large number of TFs using this technology. To fully exploit our large database of binding specificities, we have created a GBrowse-based search tool that allows an end-user to examine the overrepresentation of binding sites for any number of individual factors as well as combinations of these factors in up to six Drosophila genomes (veda.cs.uiuc.edu/cgi-bin/gbrowse/gbrowse/Dmel4). We have used this tool to demonstrate that a collection of factor specificities within a common pathway will successfully predict previously validated cis-regulatory modules within a genome. Furthermore, within our database we provide a complete catalog of DNA-binding specificities for all 84 homeodomains in Drosophila. This catalog enabled us to propose and test a detailed set of recognition rules for homeodomains and use this information to predict the specificities of the majority of homeodomains in the human genome

Mapping Domains for ZIC3 Molecular Function

The zinc finger of the cerebellum (Zic) genes encode a family of transcriptional regulators critical for early embryogenesis. All ZIC proteins contain a zinc finger domain (ZFD) and other evolutionary conserved regions. They are pleiotropic in nature since they can influence gene expression directly by acting as transcription factors due to their ability to bind target DNA sequences, or indirectly as co-factors by interacting with protein partners. Little is known, however, about the structural components that allow ZIC proteins to perform these functions. Among ZIC family members the protein structure of ZIC3 is relatively well characterised, yet details regarding its transactivation domain remain unknown. During embryonic development ZIC3 is involved in maintaining pluripotency of embryonic stem cells, formation of the left-right (L-R) axis and arrangement of visceral organs. Mutations in Zic3 in humans and animal models cause congenital L-R axis defects. The work presented in this thesis maps structural domains required for ZIC3 molecular function. Characterisation of a novel allele of murine Zic3 revealed that removal of the ZFD and C-terminus renders the mutant protein functionally null and incapable of dominantly interfering with the function of other ZIC proteins. To further assess the transcription factor function of ZIC3, a new cell-based transactivation assay system using target ZIC3-DNA binding sequences was designed. This assay was used to identify regions within the ZFD and C-terminus vital for transactivation via ZIC3. In addition other evolutionary conserved domains were implicated in transactivation. This study provides a reliable and robust platform to investigate the transcription factor function of ZIC proteins and their variants

Investigation into the role of LSH ATPase in chromatin remodelling

Chromatin remodelling is a crucial nuclear process affecting replication, transcription and repair. Global reduction of DNA methylation is observed in Immunodeficiency-Centromeric Instability-Facial Anomaly (ICF) syndrome. Several proteins were found to be mutated in patients diagnosed with ICF, among them are LSH and CDCA7. LSH is a chromatin remodeller bearing homology to the members of Sf2 remodelling family. A point mutation in its ATPase lobe was identified in ICF. CDCA7 is a zinc finger protein that was recently found to be crucial for nucleosome remodelling activity of LSH. Several point mutations in its zinc finger domain were described in ICF patients. In vitro and in vivo studies have shown that LSH-/- phenotype demonstrates reduction of global DNA methylation, implying that chromatin remodelling LSH functions may be required for the efficient methyltransferase activity, linking this finding to ICF phenotype. Here, the LSH-mononucleosome interaction was explored in vitro using bioinformatics, biochemical, biophysical and structural techniques. LSH purification was further optimised, achieving near 100% purity, which is a useful improvement for any potential structural studies. LSH has been found to interact with the mononucleosome in vitro and no DNA linker was required for this interaction, indicating that LSH binds to the nuclesomal core through its ATPase domain. Qualitatively estimated Kd for this interaction was in nanomolar region, which did not translate into complex detection during size exclusion chromatography. CDCA7 was expressed in insect cell system and semi-purified, however, high nucleic acid presence in the final protein sample precluded any potential studies of CDCA7 interaction with chromatin. Homology and ab initio modelling for LSH and CDCA7, respectively, indicated that LSH is likely to bind the superhelical location 2 (SHL2), however, the exact location of CDCA7 and its interaction with LSH will have to be elucidated in further experimental work