William S. Dynan

Specific Regions of Contact between Human T-cell Leukemia Virus Type I Tax Protein and DNA Identified by Photocross-linking

The human T-cell leukemia virus type I Tax protein forms a ternary complex on DNA in association with a host factor, the cyclic AMP response element-binding protein (CREB). An understanding of the precise geometry of this complex has been elusive. We have used photocross-linking to investigate Tax-DNA contacts. Our data show that Tax contacts the DNA at two symmetric positions 14 nucleotides apart on either side of the Tax responsive element. The presence of symmetric, widely separated regions of contact suggests that at least two molecules of Tax are present in the complex. Mapping the contacts onto a three-dimensional model of the CREB-DNA binary complex shows that they lie on the same face of the DNA near the regions where the N termini of the CREB bZIP domains enter the major groove. This location correlates well with previous evidence that CREB amino acid residues immediately N-terminal to the bZIP domain are crucial for the formation of the ternary complex. The limited number of cross-links observed suggests that contacts are primarily with the phosphate backbone and does not support the idea that a major structural element of the Tax protein inserts into the major or minor grooves of the DNA.

Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

ABSTRACT Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominant-negative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.

Understanding the molecular mechanism by which methylation influences gene expression

CpG dinucleotides that are found in the DNA of higher eukaryotes often contain cytosine that has been modified by methylation at the C5 position. The frequency with which 5-methyl cytosine is found at a particular site varies between cell types. Many studies have shown that the transcriptional control regions of genes that are expressed in a given cell type tend to be undermethylated, relative to the same sequences in other cell types. These observations support the idea that methylation may be involved in tissue-specific regulation of gene expression (recently reviewed in Refs 1, 2).

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

In vitro activation of transcription by the human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I (HTLV-I) regulatory protein Tax activates transcription of the proviral long terminal repeats and a number of cellular promoters. We have developed an in vitro system to characterize the mechanism by which Tax interacts with the host cell transcription machinery. Tax was purified from cells infected with a baculovirus expression vector. Addition of these Tax preparations to nuclear extracts from uninfected human T lymphocytes activated transcription of the HTLV-I long terminal repeat approximately 10-fold. Transcription-stimulatory activity copurified with the immunoreactive 40-kDa Tax polypeptide on gel filtration chromatography, and, as expected, the effect of recombinant Tax was diminished in HTLV-I-infected T-lymphocyte extracts containing endogenous Tax. Tax-mediated transactivation in vivo has been previously shown to require 21-bp-repeat Tax-responsive elements (TxREs) in the promoter DNA. Stimulation of transcription in vitro was also strongly dependent on these sequences. To investigate the mechanism of Tax transactivation, cellular proteins that bind the 21-bp-repeat TxREs were prepared by DNA affinity chromatography. Recombinant Tax markedly increased the formation of a specific host protein-DNA complex detected in an electrophoretic mobility shift assay. These data suggest that Tax activates transcription through a direct interaction with cellular proteins that bind to the 21-bp-repeat TxREs.

Autoantibodies to DNA-dependent protein kinase. Probes for the catalytic subunit.

DNA-dependent protein kinase (DNA-PK) is an important nuclear enzyme which consists of a catalytic subunit known as DNA-PKcs and a regulatory component identified as the Ku autoantigen. In the present study, we surveyed 312 patients in a search for this specificity. 10 sera immunoprecipitated a large polypeptide which exactly comigrated with DNA-PKcs in SDS-PAGE. Immunoblot analysis demonstrated that this polypeptide was recognizable by a rabbit antiserum specific for DNA-PKcs. Although the patient sera did not bind to biochemically purified DNA-PKcs in immunoblots or ELISA, they were able to deplete DNA-PK catalytic activity from extracts of HeLa cells in a dose-dependent manner. We conclude that these antibodies should be useful probes for studies which aim to define the role of DNA-PK in cells. Since six sera simultaneously contained antibodies to the Ku protein, these studies suggest that relatively intact forms of DNA-PK complex act as autoantigenic particles in selected patients.

DNase I Footprinting as an Assay for Mammalian Gene Regulatory Proteins

The DNase I footprinting method was first described by Galas and Schmitz (1). The technique remains unsurpassed as a way of gaining direct and immediate information about the location of a protein binding site in the DNA sequence. In addition, the footprinting method provides a way of studying specific binding in the presence of a high nonspecific background, a common situation when working with rare proteins that have been only partially purified. So effective is the footprinting method in discriminating between specific and nonspecific binding that it is often possible to visualize specific protein-DNA interactions with entirely unfractionated protein extracts from mammalian nuclei.

Identification of a human T-cell leukemia virus type I tax peptide in contact with DNA.

The human T-cell leukemia virus Tax protein directs binding of a host factor, cAMP response element binding protein, to an extended recognition sequence in the proviral promoter. Prior cross-linking experiments have revealed that Tax makes restricted contact with this DNA at two symmetric positions, 14 nucleotides apart on opposite strands of the DNA. Tax lacks a conventional DNA binding domain, and the sequences in Tax that are in contact with DNA have not been previously identified. Analysis of cross-linked peptides now shows that the contact occurs between Tax residues 89 and 110, corresponding to a protease-sensitive linker joining two protein structural domains. The linker assumes a protease-resistant conformation in the cross-linked complex. Point mutations within the linker prevent cross-linking and interfere with Tax function. These data suggest that entry of Tax into the ternary complex may be coupled to folding of an unstructured protein domain, which then makes base-specific contacts with DNA.

A downstream-element-binding factor facilitates assembly of a functional preinitiation complex at the simian virus 40 major late promoter.

Recent work has shown that many promoters recognized by eucaryotic RNA polymerase II contain essential sequences located downstream of the transcriptional initiation site. We show here that the activity of a promoter element centered 28 base pairs downstream of the simian virus 40 major late initiation site appears to be mediated by a DNA-binding protein, which was isolated by affinity chromatography from HeLa cell nuclear extracts. In the absence of the other components of the transcriptional machinery, the protein bound specifically but weakly to its recognition sequence, with a Kd of approximately 10(-8) M. Analysis of kinetic data showed that mutation of the downstream element decreased the number of functional preinitiation complexes assembled at the promoter without significantly altering the time required for half the complexes to assemble. This suggests that in the absence of the downstream activating protein, preinitiation complexes are at least partially assembled but are not transcriptionally competent.

[17] In Vitro binding and transcription assays using the human T-cell leukemia virus type I tax protein

Publisher Summary Human T-cell leukemia virus type I (HTLV-I) encodes a 40 kDa regulatory protein, Tax, which interacts with the host cell transcriptional machinery to increase the expression of viral and cellular RNAs. Because the Tax protein plays a role in both productive viral infection and viral oncogenesis, the mechanism by which Tax interacts with the transcriptional machinery is of considerable interest. For better understanding of the way Tax activates transcription, a Tax-responsive in vitro transcription system is developed. In this system, purified recombinant Tax increases the transcription of the HTLV-I long terminal repeats (LTR) promoter approximately 10-fold. This Tax-mediated transactivation shows a strong dependence on the in vivo -defined TxREs, suggesting that it occurs by the same mechanism in vitro as in vivo . Purified recombinant Tax increases the binding of affinity-purified host cell proteins to the TxREs. It is suggested that this increased binding drives the increase in transcription. Recent work has shown that Tax affects the DNA binding activity of a number of different proteins. Purified Tax enhances the DNA binding of several proteins in the activating transcription factors (ATF)/ cAMP response element-binding (CREB) family. A quantitative treatment is developed to describe this phenomenon. Tax has also been reported to bind in ternary complexes containing DNA and CREB. Tax also enhances the binding of other transcription factors that may be relevant for the effect of Tax in vivo , including NF- k B, serum response factor (SRF), and c-jun. This ability to influence the DNA binding activity of a number of different transcription factors suggests that in vitro systems may be useful for studying the effects of Tax on a wide range of promoters. This chapter describes methods for establishing in vitro systems for studying the biochemical activities of the Tax protein.