Walter Schaffner

A rapid, sensitive, and specific method for the determination of protein in dilute solution

Abstract A protein assay is described in which the sample is precipitated with trichloroacetic acid in the presence of sodium dodecylsulfate, filtered off on a Millipore membrane and stained with Amidoschwarz 10B. The proteindye complex is eluted, and its absorbance determined at 630 nm. This assay is very reproducible, insensitive to variations in assay conditions, and linear from 3 to 30 μg of protein. It can be used on samples with a concentration as low as 0.75 μg/ml. There is no interference by commonly used reagents such as Tris, thiol reagents, EDTA, urea, sucrose, and many others. The color yield for a variety of proteins was determined and found to lie within ±15% of the value for bovine serum albumin which was used as standard. Of the proteins tested only insulin, which due to its low molecular weight was incompletely retained on the membrane in the filtration step, gave a low color yield, 50% of the standard.

A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus

A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and -524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors.

A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes

Transcriptional enhancers, originally discovered in viral genomes, are short, cis-acting, regulatory sequences that strongly stimulate transcription from promoters of nearby genes. We demonstrate the existence of an enhancer within a mouse immunoglobulin heavy chain gene. A DNA fragment located between the joining region and the switch recombination region in the intron upstream of the immunoglobulin mu constant region has been linked, in both orientations, to genes coding for rabbit beta-globin or SV40 T antigen. This element enhances the number of correct beta-globin gene transcripts by at least two orders of magnitude and also stimulates production of T antigen. It acts from several hundred to several thousand base pairs up or downstream of a promoter without amplifying template copy number. Of the various cell lines tested, the immunoglobulin gene enhancer functions only in lymphocyte-derived (myeloma) cells. We propose that this tissue-specific enhancer contributes to the activation of somatically rearranged immunoglobulin variable region genes and possibly to abnormal expression of other genes (e.g. c-myc) that become translocated to its domain of influence.

The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression.

We have described and cloned previously a factor (MTF‐1) that binds specifically to heavy metal‐responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF‐1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF‐1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF‐1. Moreover, due to the loss of MTF‐1, the endogenous metallothionein I and II genes are silent, indicating that MTF‐1 is required for both their basal and zinc‐induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal‐responsive promoters in null mutant cells. However, cotransfection of an MTF‐1 expression vector and metal‐responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF‐1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF‐1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.

Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter.

Metallothioneins (MTs) are small cysteine‐rich proteins whose structure is conserved from fungi to man. MTs strongly bind heavy metals, notably zinc, copper and cadmium. Upon exposure of cells to heavy metal and other adverse treatments, MT gene transcription is strongly enhanced. Metal induction is mediated by several copies of a 15 bp consensus sequence (metal‐responsive element, MRE) present in the promoter region of MT genes. We and others have demonstrated the presence of an MRE‐binding factor in HeLa cell nuclear extracts. We found that this factor, termed MTF‐1 (MRE‐binding transcription factor) is inactivated/reactivated in vitro by zinc withdrawal/addition. Here we report that the amounts of MTF‐1‐DNA complexes are elevated several‐fold in zinc‐treated cells, as measured by bandshift assay. We have also cloned the cDNA of mouse MTF‐1, a 72.5 kDa protein. MTF‐1 contains six zinc fingers and separate transcriptional activation domains with high contents of acidic and proline residues. Ectopic expression of MTF‐1 in primate or rodent cells strongly enhances transcription of a reporter gene that is driven by four consensus MREd sites, or by the complete mouse MT‐I promoter, even at normal zinc levels.

Enhancers and eukaryotic gene transcription

Abstract Enhancers were originally identified as long-range activators of gene transcription in higher eukaryotes and they were the first DNA sequences found to confer tissue specificity. These properties had set them apart from previously described upstream promoter elements of eukaryotic genes. More recent investigations suggest that enhancer and promoter elements overlap both physically and functionally. Our current view is that enhancers and upstream regulatory elements are composed of a modular arrangement of short sequence motifs each with specific function in conferring inducibility, tissue specificity, or a general enhancement of transcription. These motifs apparently are binding sites for nuclear proteins whose mechanism of action remains to be elucidated.

Different activation domains stimulate transcription from remote ('enhancer') and proximal ('promoter') positions.

We reported previously that the lymphocyte‐derived octamer transcription factor 2A (Oct‐2A or OTF‐2A) activated both natural immunoglobulin promoters and synthetic promoters which contain the ‘octamer’ site, but was unable by itself to stimulate transcription from a remote enhancer position. Here we examine a larger set of transcription factors with respect to their proximal versus remote activation. Since a transcription factor may contain more than one activation domain, we have chosen to study the potential of individual activation domains in the context of fusion proteins that contain the DNA binding domain of GALA. We have identified at least two distinct functional classes of transcriptional activation domains. ‘Proximal’ activation domains, exemplified by glutamine‐rich domains of Oct‐1, Oct‐2A and Sp1, stimulate transcription only from a position close to the TATA box, usually in response to a remote enhancer. ‘General’ activation domains, derived from VP16, GAL4, p65 (NF‐chi B), TFE3, ITF‐1 and ITF‐2, can activate transcription from remote as well as proximal positions. These domains contain many acidic amino acids and/or other features such as clusters of serine and threonine. The proline‐rich activation domains of AP‐2 and CTF/NF1 may represent a third class with considerable promoter activity and low but significant enhancer activity. Furthermore, activation domains of both the acidic and glutamine‐rich types seem to have a modular structure, since duplicated subdomains can substitute for the entire domain.

Embryonic lethality and liver degeneration in mice lacking the metal-responsive transcriptional activator MTF-1.

We have shown previously that the heavy metal‐responsive transcriptional activator MTF‐1 regulates the basal and heavy metal‐induced expression of metallothioneins. To investigate the physiological function of MTF‐1, we generated null mutant mice by targeted gene disruption. Embryos lacking MTF‐1 die in utero at approximately day 14 of gestation. They show impaired development of hepatocytes and, at later stages, liver decay and generalized edema. MTF‐1−/− embryos fail to transcribe metallothionein I and II genes, and also show diminished transcripts of the gene which encodes the heavy‐chain subunit of the γ‐glutamylcysteine synthetase, a key enzyme for glutathione biosynthesis. Metallothionein and glutathione are involved in heavy metal homeostasis and detoxification processes, such as scavenging reactive oxygen intermediates. Accordingly, primary mouse embryo fibroblasts lacking MTF‐1 show increased susceptibility to the cytotoxic effects of cadmium or hydrogen peroxide. Thus, MTF‐1 may help to control metal homeostasis and probably cellular redox state, especially during liver development. We also note that the MTF‐1 null mutant phenotype bears some similarity to those of two other regulators of cellular stress response, namely c‐Jun and NF‐κB (p65/RelA).

Thionein (apometallothionein) can modulate DNA-binding and transcription activation by zinc finger containing factor-Spl

A number of transcription factors contain so‐called zinc finger domains for the interaction with their cognate DNA sequence. It has been shown that removal of the zinc ions complexed in these zinc fingers abrogates DNA binding and transcription activation. Therefore we wanted to test the hypothesis that the activity of transcription factors could be regulated by physiological chelators of zinc. A prominent candidate for such a chelator is the Cys‐rich protein thionein (apometallothionein) that is inducible by heavy metal loads, and by other environmental stimuli. Here we show with DNA binding and in vitro transcriptions assays that thionein indeed can inactive the zinc finger‐containing Spl in a reversible manner. By contrast, transcription factor Oct‐l, which binds DNA via a homeo‐domain, i.e. a helix‐turn‐helix motif not involving zinc ions, is refractory to thionein action. We propose that modulation of intracellular thionein concentration is used for the coordinated regulation of a large subset of genes whose transcription depends on zinc finger proteins.

A zinc-responsive factor interacts with a metal-regulated enhancer element (MRE) of the mouse metallothionein-I gene.

Heavy metal ions are effective inducers of metallothionein gene transcription. The metal response is dependent on short DNA motifs, so‐called MREs (metal responsive elements) that occur in multiple copies in the promoter region of these genes. We have analysed an MRE of the mouse metallothionein‐I gene (MREd) and we demonstrate that this can function over long distances as a bona fide metal ion‐inducible enhancer. The transcription factor Sp1 and a zinc‐inducible factor, designated MTF‐1, bind to the MREd enhancer in vitro. The combined use of MREd mutants in a transient assay in HeLa cells and a competition band shift assay show that the zinc‐inducible formation of the MTF‐1/DNA complex in vitro correlates with zinc‐inducible transcription in vivo. A chemical methylation interference assay revealed remarkably similar but non‐identical guanine interference patterns for the MTF‐1 and Sp1 complexes, which may mean that MTF‐1 is related to the Sp1 factor.