![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 2045-2051
Departments of Medicine and Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD 21205
The regulation of tissue- and temporal-specific eukaryotic gene expression and the activation of genes in response to extracellular inducers are two fundamental processes that attract many a molecular biologist. The development of methods for cloning individual genes has provided the opportunity to study the mechanisms underlying these processes at a molecular level. What has been learned is that eukaryotic promoters consist of defined short stretches of DNA sequences, which are recognized by a variety of specific DNA-binding proteins that regulate transcription. The current challenges include an understanding of 1) which specific cis-acting DNA sequence elements and which trans-acting factors (transcription factors) are required for the expression of a given gene, 2) how a given set of DNA-protein interactions regulates the expression of a tissue-specific gene, and 3) how these interactions are integrated into the overall regulation of gene expression during development.
This article will address the current methodologies used to identify and characterize trans-acting eukaryotic transcription factors. In addition, some of the common classes of eukaryotic transcription factors studied to date and their functions in gene regulation will also be described. For a detailed description of methodologies, readers are referred to a number of recent publications (Hames and Higgins 1993 The principal strategy in identifying and characterizing transcription factors is based on their ability to recognize and interact with specific DNA sequences present in the promoters of eukaryotic genes. The detection of sequence-specific DNA binding activities from crude cell extracts is usually the first step that leads to the eventual purification and cloning of various transcription factors. Two techniques are often used in assessing DNA-protein interactions: the electrophoretic mobility shift assay (EMSA)3 (Fried and Crothers 1981 Electrophoretic mobility shift assay (EMSA)
DNase I protection (footprinting) assay
Other commonly employed DNA binding assays
(i) Methylation interference assay.
This assay is based on the fact that methylation of specific guanine or adenine residues within the target DNA sequence inhibits the binding of a transcription factor to that site (Fig. 3). A singly end-labeled DNA probe is first partially methylated with dimethyl sulfate (DMS) and incubated with the nuclear extract of interest. The protein-DNA complex is then separated from the free DNA using EMSA. Both protein-bound and free DNA are eluted from the gel, cleaved at the site of modification with piperidine and resolved by denaturing polyacrylamide gel electrophoresis. If methylation occurs at a particular guanine or adenine residue that is critical for the DNA-protein interaction, the binding of the protein to that DNA will be inhibited, resulting in the recovery of that DNA only from the free DNA fraction. The presence of particular guanine and adenine bands in the free DNA fraction and their concomitant absence from the bound DNA fraction are indicative of those nucleotides being the contact points of the protein.
(ii) UV crosslinking.
Irradiation of DNA with ultraviolet light produces pyrimidine free radicals that are chemically active and can form covalent bonds such as thymidine dimers. This reactive property of UV-irradiated DNA can be used to link transcription factors to their respective recognition sites. When a protein-DNA complex is irradiated with UV light, it causes the formation of covalent bonds between pyrimidines and certain amino acid residues in the transcription factor that are in close proximity to the DNA. The labeling of a transcription factor in this fashion allows for the easy and rapid determination of its approximate molecular weight in a denaturing polyacrylamide gel even in crude extracts. Frequently, halogenated analogues of thymidine (for example, bromodeoxyuridine or BrdU) are incorporated into the DNA enzymatically to enhance the crosslinking between protein and DNA.
(iii) Southwestern blotting.
As the name implies, Southwestern blotting is a variation of the Western blotting technique. Cell extracts containing the DNA-binding protein of interest are resolved by denaturing polyacrylamide gel electrophoresis followed by electrophoretic transfer to a nitrocellulose membrane. The membrane is then probed with a radioactively labeled DNA fragment bearing the recognition site, preferably in the form of tandem repeats. The protein that interacts with the probe can be visualized by autoradiography after nonspecifically bound DNA is first washed away from the membrane.
To characterize the biochemical properties of transcription factors, it is often necessary to study them in pure (cloned) forms. Several different approaches have recently been developed to achieve the cloning of cDNAs encoding various transcription factors. They generally fall into two major categories: protein purification by conventional biochemical methods, followed by peptide sequencing or antibody generation for cDNA library screening, and expression cloning of transcription factors based on their sequence-specific DNA recognition.
Biochemical purification of transcription factors
INTRODUCTION
Introduction
References
, Latchman 1993, Revzin 1993). Readers are also encouraged to consult a number of recently published excellent textbooks on the molecular mechanisms of transcriptional regulation (Conaway and Conaway 1994, Goodburn 1996, Latchman 1995, McKnight and Yamamoto 1992, Wingender 1993).
DNA BINDING ASSAYS USED TO STUDY TRANSCRIPTION FACTORS
, Garner and Revzin 1981) and the DNase I protection (footprinting) assay (Brenowitz et al. 1986, Galas and Schmitz 1978). Variations of these two methods include methylation interference (Brunelle and Schleif 1987), ultraviolet (UV) crosslinking (Chodosh et al. 1986), and Southwestern blotting (Kwast-Welfeld et al. 1993). All of these techniques are described below.
View larger version (19K):
[in a new window]
Fig 1.
EMSA. (A) The DNA probe is a double-stranded synthetic oligonucleotide containing the binding site of interest (gray area) that has been end-labeled with 32P (asterisk). The probe is incubated with a nuclear extract containing the protein (stippled circles) that binds to the specific DNA sequence. The reaction mixture is then resolved by nondenaturing polyacrylamide gel electrophoresis, and the locations of labeled probe are visualized with autoradiography. In the left lane in which the DNA runs alone without any added nuclear extract, the probe shows up as a single, fast-migrating band. In the right lane the binding of the protein to the specific sequence within the DNA results in the formation of a slower migrating DNA-protein complex. (B) The specificity of the interaction between the protein and DNA in a complex is determined by competition experiments using specific or nonspecific unlabeled DNA. Lane 1 is probe alone and lanes 2-7 include added nuclear extracts. A single DNA-protein complex is formed as seen in lane 2. Lanes 3 and 4 contain increasing amounts of an unlabeled specific competitor DNA, whereas lanes 5 and 6 contain increasing amounts of an unlabeled nonspecific competitor DNA. In lane 7 an antibody directed against the specific DNA-binding protein is included, which results in the formation of an even slower migrating DNA-protein-antibody complex in a process referred to as supershift.
View larger version (35K):
[in a new window]
Fig 2.
DNase I protection (footprinting) assay. A double-stranded DNA fragment labeled at one end only (asterisk) is the probe and is incubated with a nuclear extract containing a specific binding protein. The mixture is digested with a diluted solution of DNase I (thin hollow arrows) for a short duration before being resolved on a denaturing DNA sequencing gel. In the absence of any nuclear extract, the digested DNA runs as a ladder without any interruption (left lane). The binding of a protein (striped circle) to a specific region (gray box) of the probe will protect that region from being digested by DNase I, resulting in a void or protected area referred to as a footprint.
View larger version (28K):
[in a new window]
Fig 3.
Methylation interference assay. An end-labeled (asterisk) double-stranded DNA fragment is treated with a diluted solution of DMS so that on the average each molecule of DNA is methylated (Me) at one site. Following incubation of the partially methylated probe with a nuclear extract, EMSA is performed. Both the free probe and the bound probe (representing the DNA-protein complex) are extracted from the polyacrylamide gel, treated with piperidine (which cleaves at methylated bases) and resolved on a denaturing DNA sequencing gel. The absence of cleavage in certain regions of the bound DNA indicates that these particular residues are involved in contacting the protein and that their methylation interferes with the formation of the complex.
PURIFICATION AND CLONING OF TRANSCRIPTION FACTORS
, Rosenfeld and Kelly 1986). To construct the column, multiple tandem repeats of a double-stranded oligonucleotide bearing the high affinity-binding sequence for the transcription factor are covalently attached to a matrix support such as cyanogen bromide-activated Sepharose beads. After passing the proteins through this column, the transcription factor will bind to the matrix, which can then be eluted with a salt gradient. The procedure can be repeated to increase the protein purity. Many transcription factors can be purified up to 1000-fold by two sequential DNA affinity chromatographic steps.
Expression cloning of transcription factors
Two recently described techniques can be used in expression cloning, which is based primarily on the sequence-specific interaction of a transcription factor with its high affinity-binding site. One involves the screening of an expression cDNA library with a radiolabeled oligonucleotide probe containing the recognition site for the transcription factor (Singh et al. 1989, Vinson et al. 1989). The other is a genetic selection using the yeast one-hybrid system (Wang and Reed 1993). Both will be briefly discussed below.
(i) In situ detection of transcription factors.
The principal of this technique is similar to that of Southwestern blotting. An expression cDNA library is constructed using bacteriophage vectors, such as 
gt11, in which the cloned cDNA is expressed as a fusion protein with 
-galactosidase upon addition of an inducer of the lac operon. The induced proteins are transferred to nitrocellulose membranes followed by an optional step of denaturation and stepwise renaturation. The membranes are then probed with radiolabeled tandem repeats of an oligonucleotide bearing the high affinity-binding sequence. After washing the membranes to remove any nonspecifically attached probes, autoradiography is performed to identify the cDNA clones that bind to the probe. The authenticity of these clones can subsequently be verified by binding crude extracts prepared from lysogens of the recombinant phage to the radiolabeled probe using EMSA or DNase I footprinting assay.
(ii) The yeast one-hybrid selection system.
The yeast one-hybrid system stems from the important observation that most eukaryotic transcription factors are modular by nature, composed of a target-specific DNA-binding domain and a target-independent transcription activation domain. In this procedure (Fig. 4) candidates of cDNA clones encoding the transcription factor of interest (TF X) are expressed in the yeast as fusion proteins that contain a target-independent activation domain (AD) of the potent yeast transcription factor, GAL4. A reporter construct is generated that consists of multiple copies of the target sequence (X) adjacent to a low activity promoter (Pmin) directing HIS3 gene expression. The presence of this reporter allows sufficient growth for the selection of a stably integrated yeast strain when it is introduced into a his3
parent yeast strain. The further introduction of a GAL4-candidate fusion protein capable of binding to the target sequence present in the reporter will strongly activate transcription and increase levels of HIS3. This allows rapid growth and subsequent positive identification of candidate clones in media that lack histidine.
|
| |
COMMON CLASSES OF TRANSCRIPTION FACTORS AND THEIR FUNCTIONS |
|---|
Transcription factors are often classified based on the structural motifs that constitute their DNA-binding domains. In most cases the protein makes a large number of contacts with the DNA, involving hydrogen bonds, ionic bonds and hydrophobic interactions. Although each individual contact is weak, the 20 or so contacts that typically form at the protein-DNA interface ensure that the interaction is both specific and strong. In this section several major classes of eukaryotic transcription factors (Fig. 5) and their functions will be briefly reviewed. Readers are reminded that transcription of eukaryotic genes requires the participation of many additional regulatory proteins other than the sequence-specific DNA-binding transcription factors addressed below. Examples of these important proteins involved in transcriptional regulation include the TATA-binding protein (TBP), TBP-associated factors (TAF) and the recently identified family of transcription coactivators such as p300/CBP. Several recent articles provide excellent reviews of these topics (Glass et al. 1997, Goodrich and Tjian 1994, Sauer and Tjian 1997, Shikama et al. 1997, Verrijzer and Tjian 1996).
|
The helix-turn-helix motif (including the homeodomain proteins)
The first DNA-binding protein motif to be recognized was the helix-turn-helix. It is constructed from two
helices connected by a short chain of amino acids, which constitutes the turn. The more carboxyl-terminal helix is called the recognition helix because it fits into the major groove of DNA. As is the case with many sequence-specific DNA binding proteins, helix-turn-helix proteins bind as symmetrical dimers to a DNA composed of two symmetrically arranged half sites with similar sequences. Examples of this class of proteins include the bacterial tryptophan repressor and the phage repressor.
The zinc finger motifs
As the name zinc finger implies, this family of proteins contains a domain that utilizes zinc as an important component of the DNA-binding region. Amino acid residues such as cysteine and histidine tetrahedrally coordinate the zinc atom. Together they form a finger-like projection that is in close contact with the DNA. A common type of zinc finger protein is exemplified by the Xenopus transcription factor TFIIIA that is involved in the transcription of ribosomal genes. The finger is a simple structure that consists of an helix and a sheet held together by the zinc (Fig. 5C). This type of zinc finger is often found in a cluster with additional fingers, arranged one after the other so that the helix of each can contact the major groove of DNA, forming nearly a continuous stretch of helix along the groove (Fig. 5D).
helices are packed together with two zinc atoms. Like helix-turn-helix proteins, these proteins form dimers and allow one of the two helices of each subunit to interact with the major groove of the DNA.
The leucine zipper motif
The leucine zipper proteins bind to DNA as dimers. Although in many other proteins the dimerization and the DNA-binding domains are distinct, the leucine zipper motif combines both functions. Two helices, one from each monomer, are joined together to form a short coiled-coil. The helices are held together by interactions between hydrophobic amino acid side chains (usually leucines). Just beyond the dimerization interface, the two helices separate to form a Y-shaped structure, allowing the side chains (often basic residues) to contact the major groove of DNA (Fig. 5E).
The helix-loop-helix (HLH) motif
An HLH motif consists of a short helix connected by a loop to a second longer helix. The flexibility of the loop allows the two helices to pack against each other. This motif is involved in both dimer formation and DNA contact (Fig. 5F). As with the leucine zipper proteins, HLH proteins are capable of forming either homodimer or heterodimer. An important example of the HLH protein is myo D1, a regulatory protein that is essential for the formation of muscle cells.
| |
SUMMARY |
|---|
Eukaryotic transcription factors are modular proteins that utilize distinct domains for transcriptional activation (or repression) and DNA binding. The highly specific interaction between a given transcription factor and its cognate binding sequence forms the basis for the biochemical characterization and eventual purification of these important regulatory proteins. Commonly used techniques in the assessment of DNA binding of a transcription factor include EMSA and DNase I protection (footprinting) assay. Transcription factors are often purified and cloned based on their specific binding sequences. Finally, several important classes of structural motifs present in many eukaryotic transcription factors are presented. The understanding of the structure and function relationship between transcription factors and the genes that they regulate should provide the basis for understanding the overall molecular mechanisms controlling gene expression.
| |
FOOTNOTES |
|---|
Manuscript received 1 June 1998. Initial reviews completed . Revision accepted 3 August 1998.
| |
LITERATURE CITED |
|---|
Introduction
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  Z. Qin, F. Ren, X. Xu, Y. Ren, H. Li, Y. Wang, Y. Zhai, and Z. Chang ZNF536, a Novel Zinc Finger Protein Specifically Expressed in the Brain, Negatively Regulates Neuron Differentiation by Repressing Retinoic Acid-Induced Gene Transcription Mol. Cell. Biol., July 1, 2009; 29(13): 3633 - 3643. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. Geiman, H. Ton-That, J. M. Johnson, and V. W. Yang Transactivation and growth suppression by the gut-enriched Kruppel-like factor (Kruppel-like factor 4) are dependent on acidic amino acid residues and protein-protein interaction Nucleic Acids Res., March 1, 2000; 28(5): 1106 - 1113. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
