trans Repression of the Human Metallothionein IIA Gene Promoter by PZ120, a Novel 120-Kilodalton Zinc Finger Protein

Abstract

Metallothioneins are small, highly conserved, cysteine-rich proteins that bind a variety of metal ions. They are found in virtually all eukaryotic organisms and are regulated primarily at the transcriptional level. In humans, the predominant metallothionein gene is hMTIIA, which accounts for 50% of all metallothioneins expressed in cultured human cells. The hMTIIA promoter is quite complex. In addition to cis-acting DNA sequences that serve as binding sites for trans-acting factors such as Sp1, AP1, AP2, AP4, and the glucocorticoid receptor, the hMTIIA promoter contains eight consensus metal response element sequences. We report here the cloning of a novel zinc finger protein with a molecular mass of 120 kDa (PZ120) that interacts specifically with the hMTIIA transcription initiation site. The PZ120 protein is ubiquitously expressed in most tissues and possesses a conserved poxvirus and zinc finger (POZ) motif previously found in several zinc finger transcription factors. Intriguingly, we found that a region of PZ120 outside of the zinc finger domain can bind specifically to the hMTIIA DNA. Using transient-transfection analysis, we found that PZ120 repressed transcription of the hMTIIA promoter. These results suggest that the hMTIIA gene is regulated by an additional negative regulator that has not been previously described.Metallothioneins (MTs) were initially discovered by biochemists searching for tissue constituents responsible for the natural accumulation of cadmium. They have been reported to occur throughout the animal kingdom, as well as in plants, eukaryotic microorganisms, and prokaryotes (reviewed in references 25 and 35). In humans, MTs have been isolated from the liver (57), cultured cells (41, 59), and the brain (growth inhibitory factor) (75). They are present in four distinguishable forms known as hMTI, hMTII, hMTIII, and hMTIV (reviewed in references 2, 25, 37, and 55).Different tissues and cell types synthesize different MTs in various levels. However, the hMTIIA gene is responsible for the majority of MTs expressed in most tissues in humans (41). This high level of hMTIIA basal constitutive expression coupled with the gene's remarkable inducibility makes hMTIIA easily assayable and, therefore, provides an ideal model for studying transcriptional regulation in eukaryotic cells. Although there are reports that hMTIIA may be regulated by gene amplification (47), DNA methylation (33), or posttranscriptional events (60), there is no question that the principal mechanism of regulation lies at the level of transcriptional initiation (44).The interest in transcriptional regulation of the hMTIIA gene centers on three key issues. First, shortly after the cloning of the hMTIIA gene, there was an explosive increase of interest in the identification of the cis-acting DNA sequences in the hMTIIA promoter that are responsible for basal and induced transcription. Second, many laboratories have intensely pursued the identification of trans-acting proteins that interact with these cis-acting DNA sequences. Fina...

The Pu.1 target gene Zbtb11 regulates neutrophil development through its integrase-like HHCC zinc finger

“…Furthermore, deletion mutants lacking the carboxyl-terminal zinc finger domains were able to rescue (Fig. 5c), consistent with a prior study demonstrating their dispensability for repression of the metallothionein promoter19. Of note, deletion of the N terminus did not interfere with functional rescue by Zbtb11 in this bioassay, suggesting that the steric consequences of point mutation of these four key residues are more detrimental to whole protein function than complete absence of this domain.…”

“…However, this question will only be resolved by biophysical data. Since removal of the C-terminal zinc fingers remarkably does not appear to impede function, either in our in vivo bioassay or a single previous in vitro study19, the newly identified HHCC zinc finger could serve to preserve Zbtb11 function in this context. It is thought that in viruses this zinc finger recognizes viral DNA42 and we have shown a requirement for the intact HHCC domain for recognition of cellular DNA through the transrepression and autorepression functions of Zbtb11.…”

“…Previous observations have correlated high ZBTB11 expression with human myeloid lineage cells and several subtypes of acute myeloid leukaemia1718. Originally identified as a regulator of metallothionein 2A 18 years ago19, little else is known about Zbtb11 and its biological function.…”

Metallothionein: the multipurpose protein

“…However, there are possibly other pathways of metal induction, because protein kinase C inhibitors have been found to inhibit Zn and Cd induction of MT in Chinese hamster cells [84]. Moreover, MREs can interact with a variety of nuclear proteins that either activate or inhibit transcription [17,85,86]. This may partly explain why Cd induction of MT-1 is inhibited by the administration of 17-β-estradiol and progesterone to ovariectomised mice [87].…”

Characteristics, functions, and applications of metallothionein in aquatic vertebrates

“…Moreover, MREs can interact with various nuclear proteins that either activate or inhibit transcription (Tang et al, 1999;Miles et al, 2000;Ogra et al, 2001). There is evidence that several MREs could respond directly to hypoxia and oxidants, possibly via MTF-1, although these conditions may also separate Zn from protein ligands which could then activate MTF-1 (Murphy et al, 1999).…”