Isolation and Characterization of a Novel Zinc-finger Protein with Transcriptional Repressor Activity

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The SCAN domain is a conserved region of 84 residues found predominantly in zinc finger DNA-binding proteins in vertebrates. The SCAN domain appears to control the association of SCAN domain containing proteins into noncovalent complexes and may be the primary mechanism underlying partner choice in the oligomerization of these transcription factors. Here we have overexpressed, purified, and characterized the isolated SCAN domain (amino acids 37-132) from ZNF174. Both size exclusion chromatography and equilibrium sedimentation analysis demonstrate that the ZNF174 SCAN domain forms a homodimer. Circular dichroism shows that the isolated SCAN domain dimer has ϳ42% ␣-helix. Thermal denaturation experiments indicate that the SCAN domain undergoes a single reversible unfolding transition with a T m of over 70°C. The midpoint of the equilibrium unfolding transition increases with increasing protein concentration, consistent with a two-state unfolding transition in which folded dimer is in equilibrium with unfolded monomer. These findings demonstrate that the isolated SCAN domain forms a stable dimer and support a model in which the SCAN domain is capable of mediating the selective dimerization of a large family of vertebrate-specific, zinc finger-containing transcription factors.Transcription factors are composed of modular elements that include a DNA-binding domain and one or more separable effector domains that activate or repress transcription. Other modules within these factors regulate subcellular localization and gene expression by mediating selective association of the transcription factors with each other, or with other cellular components. Identification of these domains often provides a conceptual framework for understanding the function of the transcription factor.The SCAN 1 domain is a highly conserved vertebrate-specific protein domain found in ϳ60 genes in the human genome (1, 2). Only a handful of these gene products have been characterized thus far, and they appear to control a wide range of biological processes including development, cell differentiation, and lipid metabolism (reviewed in Refs. 3 and 4). The name for the SCAN domain was derived from the first letters of the names of four proteins initially found to contain this domain (SRE-ZBP, CTfin51, AW-1 (ZNF174), and Number 18 cDNA or ZNF197) (5). Alternatively, this domain has been referred to as LeR for leucine-rich domain (6). The SCAN domain consists of an 84-residue, leucine-rich region and is predicted to contain a high degree of ␣-helix. It is located at the N terminus when it is part of a zinc finger-containing transcription factor. The primary amino acid sequence of the domain does not resemble any of the other zinc finger-associated domains, such as the Kruppelassociated box (KRAB) or the poxvirus and zinc finger (POZ) domain, which is also known as the BTB (Broad-complex, Tramtrack, and Bric-a-brac) domain (7-9). Members of the SCAN domain family are broadly expressed and appear to function as either activators or repressors of transcri...

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The SCAN Domain of ZNF174 Is a Dimer

Stone¹,

Maki²,

Blacklow

et al. 2002

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“…These conserved repression domains include the BTB/POZ, WRPW, SNAG, SCAN, and Kruppel-associated box (KRAB) 1 (12)(13)(14)(15)(16). We have focused on the KRAB domain as a model system for the analysis of repression modules (17)(18)(19) (Fig.…”

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confidence: 99%

Biochemical Analysis of the Kruppel-associated Box (KRAB) Transcriptional Repression Domain

Peng¹,

Begg²,

Harper

et al. 2000

100

The Kruppel-associated box (KRAB) domain is a 75-amino acid transcriptional repressor module commonly found in eukaryotic zinc finger proteins. KRAB-mediated gene silencing requires binding to the RING-B boxcoiled-coil domain of the corepressor KAP-1. Little is known about the biochemical properties of the KRAB domain or the KRAB⅐KAP-1 complex. Using purified components, a combination of biochemical and biophysical analyses has revealed that the KRAB domain from the KOX1 protein is predominantly a monomer and that the KAP-1 protein is predominantly a trimer in solution. The analyses of electrophoretic mobility shift assays, GST association assays, and plasmon resonance interaction data have indicated that the KRAB binding to KAP-1 is direct, highly specific, and high affinity. The optical biosensor data for the complex was fitted to a model of a one-binding step interaction with fast association and slow dissociation rates, with a calculated K d of 142 nM. The fitted R max indicated three molecules of KAP-1 binding to one molecule of the KRAB domain, a stoichiometry that is consistent with quantitative SDSpolyacrylamide gel electrophoresis analysis of the complex. These structural and dynamic parameters of the KRAB/KAP-1 interaction have implications for identifying downstream effectors of KAP-1 silencing and the de novo design of new repression domains.

“…subfamilies based on the number of zinc finger motifs, sequence homology between the zinc-fingers, and the presence of specific repressor and activation domains (3)(4)(5)(6).…”

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confidence: 99%

Identification of Glis1, a Novel Gli-related, Krüppel-like Zinc Finger Protein Containing Transactivation and Repressor Functions

Kim

Lewandoski

Perantoni

et al. 2002

In this study, we describe the identification and characterization of a novel Krü ppel-like protein named Glisimilar 1 (Glis1). The Glis1 gene encodes an 84.3-kDa proline-rich protein. Its five tandem zinc finger motifs exhibit highest homology with those of members of the Gli and Zic subfamilies of Krü ppel-like proteins. Glis1 was mapped to mouse chromosome 4C6. Northern blot analysis showed that expression of the 3.3-kb Glis1 mRNA is most abundant in placenta and adult kidney and expressed at lower levels in testis. Whole mount in situ hybridization on mouse embryos demonstrated that Glis1 is expressed in a temporal and spatial manner during development; expression was most prominent in several defined structures of mesodermal lineage, including craniofacial regions, branchial arches, somites, vibrissal and hair follicles, limb buds, and myotomes. Confocal microscopic analysis showed that Glis1 is localized to the nucleus. The zinc finger region plays an important role in the nuclear localization of Glis1. Electrophoretic mobility shift assays demonstrated that Glis1 is able to bind oligonucleotides containing the Glibinding site consensus sequence GACCACCCAC. Although monohybrid analysis showed that in several cell types Glis1 was unable to induce transcription of a reporter, deletion mutant analysis revealed the presence of a strong activation function at the carboxyl terminus of Glis1. The activation through this activation function was totally suppressed by a repressor domain at its amino terminus. Constitutively active Ca 2؉ -dependent calmodulin kinase IV enhanced Glis1-mediated transcriptional activation about 4-fold and may be mediated by phosphorylation/activation of a co-activator. Our results suggest that Glis1 may play a critical role in the control of gene expression during specific stages of embryonic development.