Starr: Simple Tiling ARRay analysis of Affymetrix ChIP-chip data

Background  

Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is an assay used for investigating DNA-protein-binding or post-translational chromatin/histone modifications. As with all high-throughput technologies, it requires thorough bioinformatic processing of the data for which there is no standard yet. The primary goal is to reliably identify and localize genomic regions that bind a specific protein. Further investigation compares binding profiles of functionally related proteins, or binding profiles of the same proteins in different genetic backgrounds or experimental conditions. Ultimately, the goal is to gain a mechanistic understanding of the effects of DNA binding events on gene expression.     

Affinity purification of specific DNA fragments using a lac repressor fusion protein

A new method for purification of specific DNA sequences using a solid phase technique has been developed based on a fusion between the Escherichia coli lac repressor gene (lacI) and the staphylococcal protein A gene (spa). The fusion protein, expressed in Escherichia coli, is active both in vivo and in vitro with respect to its three functional activities (DNA binding, IPTG induction, and IgG binding). The recombinant protein can be immobilized in a one-step procedure with high yield and purity using the specific interaction between protein A and the Fc-part of immunoglobulin G. The immobilized repressor can thereafter be used for affinity purification of specific DNA fragments containing the lac operator (lacO) sequence.     

Identification of centromeric and telomeric DNA‐binding proteins in rice

Centromeres and telomeres are DNA/protein complexes and essential functional components of eukaryotic chromosomes. Previous studies have shown that rice centromeres and telomeres are occupied by CentO (rice centromere satellite DNA) satellite and G‐rich telomere repeats, respectively. However, the protein components are not fully understood. DNA‐binding proteins associated with centromeric or telomeric DNAs will most likely be important for the understanding of centromere and telomere structure and functions. To capture DNA‐specific binding proteins, affinity pull‐down technique was applied in this study to isolate rice centromeric and telomeric DNA‐binding proteins. Fifty‐five proteins were identified for their binding affinity to rice CentO repeat, and 80 proteins were identified for their binding to telomere repeat. One CentO‐binding protein, Os02g0288200, was demonstrated to bind to CentO specifically by in vitro assay. A conserved domain, DUF573 with unknown functions was identified in this protein, and proven to be responsible for the specific binding to CentO in vitro. Four proteins identified as telomere DNA‐binding proteins in this study were reported by different groups previously. These results demonstrate that DNA affinity pull‐down technique is effective in the isolation of sequence‐specific binding proteins and will be applicable in future studies of centromere and telomere proteins.     

Mapping of sequence-specific chromatin proteins by a novel method: topoisomerase I on Tetrahymena ribosomal chromatin

DNA derived from the 5' spacers of the rRNA genes from Tetrahymena has unusual electrophoretic properties. These properties made it possible to devise a simple electrophoretic procedure for isolating specific rDNA spacer fragments from preparations of total nuclear DNA, enabling us to study DNA modifications at the level of unfractionated nuclei. We have employed the method to study the distribution of topoisomerase I binding sites on the r-chromatin (ribosomal chromatin) of Tetrahymena at the DNA sequence level. The presence of topoisomerase I in situ was detected by its ability to introduce single-strand cleavages into DNA. The positions of the cleavages were determined on DNA sequencing gels after isolation of the fragments. Topoisomerase I binding in r-chromatin is sequence specific and cleavage is confined to a 16 base-pair conserved sequence element previously determined to be a high-affinity binding site for topoisomerase I in vitro. The high degree of sequence specificity may be of important functional significance, as we find a similar sequence specificity with enzymes isolated from five evolutionarily distant species, indicating that preference for the 16 base-pair element is an intrinsic property of eukaryotic type I topoisomerases.     

Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains.

The mouse Pax-3 gene encodes a protein that is a member of the Pax family of DNA binding proteins. Pax-3 contains two DNA binding domains: a paired domain (PD) and a paired type homeodomain (HD). Both domains are separated by 53 amino acids and interact synergistically with a sequence harboring an ATTA motif (binding to the HD) and a GTTCC site (binding to the PD) separated by 5 base pairs. Here we show that the interaction of Pax-3 with these two binding sites is independent of their angular orientation. In addition, the protein spacer region between the HD and the PD can be shortened without changing the spatial flexibility of the two DNA binding domains which interact with DNA. Furthermore, by using circular permutation analysis we determined that binding of Pax-3 to a DNA fragment containing a specific binding site causes conformational changes in the DNA, as indicated by the different mobilities of the Pax-3-DNA complexes. The ability to change the conformation of the DNA was found to be an intrinsic property of the Pax-3 PD and of all Pax proteins that we tested so far. These in vitro studies suggest that interaction of Pax proteins with their specific sequences in vivo may result in an altered DNA conformation.     

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.     

The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation

For the study of DNA-membrane interaction and the regulation of replication initiation we have determined the total nucleotide sequence of pUB110. As previously reported, this plasmid replicates in B. subtilis at a copy number of 30-50 per cell, with a majority of plasmids (60-80%) bound to the membrane (type-I binding). The type-I membrane binding is apparently necessary for pUB110 initiation of replication in vivo, but the membrane binding site is not known. Furthermore, four areas of the plasmid specifically bind to Bacillus subtilis membrane in an in vitro binding reaction (type II binding). These two types of membrane binding of pUB110 are different in that the in vivo binding (type-I) requires one (dnaBI) of the host initiation genes and is high-salt resistant, whereas the in vitro binding (type-II) does not require the dnaBI gene product and is high-salt sensitive. 7-mer double-strand sequence, TCAGCAA/AGTCGTT, or one-base derivatives of this sequence are frequently (17 of 23 of the 7-mer sequences) found in or close to the type-II binding areas. One of them is found at a restriction enzyme recognition site of a binding area that destroys the type-II membrane binding. These sequences may or may not have significance in type-II membrane binding. In addition to the neomycin resistance gene, the sequence data indicate two sizable open reading frames, ORF alpha and ORF beta, and two small ORF, gamma, and delta. All of these reading frames are in the same direction, which coincides with the direction of the replication. The open reading frame alpha (ORF alpha) corresponding to 334 amino acids close to the replication origin may be essential for the initiation of replication of PUB110. The putative protein alpha corresponding to this open reading frame contains a consensus sequence of the DNA binding sites which are found in a number of known DNA-binding proteins. The consensus DNA binding site of protein alpha is flanked by two hydrophobic areas. These two observations suggest that the corresponding protein may have both an affinity to a specific site in pUB110, and an affinity to the membrane.     

Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.

We have developed a simple procedure for rapid determination of a DNA sequence recognized by a DNA binding protein based on immobilization of the protein on nitrocellulose filters. The procedure consists of the following steps: A recombinant protein with a functional DNA binding domain is expressed in E. coli. The protein is purified to homogeneity, immobilized on nitrocellulose paper, and exposed to a pool of double stranded oligonucleotides carrying in the central part a 20 bp random sequence, which is flanked by conserved sequences with restriction endonuclease recognition sites for analytical and subcloning purposes and sequences complementary to polymerase chain reaction primers. Oligonucleotides retained by the DNA-binding protein are liberated by increasing the ionic strength and used in a new binding process after amplification by the polymerase chain reaction technique. Finally the amplified product is cloned for determination of the DNA sequence selected by the DNA-binding protein. Murine Zn-finger and basic helix-loop-helix DNA binding proteins were used to demonstrate the efficiency of the method. We show that the yield of oligonucleotides binding to the protein was increased by several consecutive rounds of filter binding and amplification, and that the protein extracted a specific sequence from the pool of random oligonucleotides.     

The optimal binding sequence of the Hox11 protein contains a predicted recognition core motif.

HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. We determined the DNA binding specificity of the Hox11 protein by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contained a core TAAGTG motif that is consistent with a prediction based on the residues at specific positions that potentially make DNA base contacts and models of homeodomain-DNA interaction proposed from studies with other homeodomains. The specific interaction between Hox11 and the selected optimal binding sequence was further confirmed by band-shift and DNA competition assays. Given that the Hox11 homeodomain shares low homology with other well studied homeodomains, the presence of a predictable recognition core motif in its optimal binding sequence supports the notion that different homeodomains interact with DNA in a similar manner, through highly conserved residues at specific positions that allow contact with DNA.     

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.     

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.     

Mechanism for specificity by HMG-1 in enhanceosome assembly

Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.     

Purification of nuclear factor I by DNA recognition site affinity chromatography

Nuclear factor I (NF-I) is a cellular protein that enhances the initiation of adenovirus DNA replication in vitro. The enhancement of initiation correlates with the ability of NF-I to bind a specific nucleotide sequence within the viral origin of replication. We have developed a method for the purification of NF-I which is based upon the high affinity interaction between the protein and its recognition site. This approach may be generally applicable to the purification of other site-specific DNA binding proteins. The essential feature of the method is a two-step column chromatographic procedure in which proteins are first fractionated on an affinity matrix consisting of nonspecific (Escherichia coli) DNA and then on a matrix that is highly enriched in the specific DNA sequence that is recognized by NF-I. During the first step NF-I coelutes with proteins that have similar general affinity for DNA. During the second step NF-I elutes at a much higher ionic strength than the contaminating nonspecific DNA binding proteins. The DNA recognition site affinity matrix used in the second step is prepared from a plasmid (pKB67-88) that contains 88 copies of the NF-I binding site. This plasmid was constructed by means of a novel cloning strategy that generates concatenated NF-I binding sites arranged exclusively in a direct head to tail configuration. Using this purification scheme, we have obtained a 2400-fold purification of NF-I from crude HeLa nuclear extract with a 57% recovery of specific DNA binding activity. Throughout the purification procedure NF-I retained the ability to enhance the efficiency of initiation of adenovirus DNA replication in vitro. Electrophoresis of the purified fraction on sodium dodecyl sulfate-polyacrylamide gels revealed a population of related polypeptides that ranged in apparent molecular weight from 66,000 to 52,000. The native molecular weight of NF-I deduced from gel filtration and glycerol sedimentation studies is 55,000 and the frictional ratio is 1.3. These results suggest that NF-I exists as a globular monomer in solution.