Hans-Jürgen Thiesen

Transition metals modulate DNA-protein interactions of SP1 zinc finger domains with its cognate target site

The metal free apoprotein of recombinant human transcription factor SP1 was used in metal reconstitution experiments to study the importance of zinc in facilitating DNA binding of zinc finger proteins. Our functional analysis indicates that several transition metals are capable of modulating DNA-protein interactions of zinc finger domains with their cognate DNA target sites. Excess or deficiency of divalent zinc, or the presence of transition metals, such as divalent cadmium, cobalt, copper, manganese and nickel impair DNA binding of zinc reconstituted SP1. In addition, functionally active SP1 protein can be obtained by metal reconstitutions in absence of zinc(II) by presence of cadmium(II)- and cobalt(II)-, to lesser extents by presence of nickel(II)- or manganese(II)chloride. This study indicates that zinc might play a functional role in regulating DNA protein interactions of zinc finger proteins in vivo. It is postulated that fluctuating divalent zinc alone or transition metals bound to cellular components might form mixed-ligand complexes that alter the zinc finger protein conformation and impair DNA binding.

Amino acid substitutions in the SP1 zinc finger domain alter the DNA binding affinity to cognate SP1 target site

Non conserved amino acids, which are located in the postulated alpha-helical region of the third zinc finger in human transcription factor SP1, have been replaced by amino acids, which occur at the analogous zinc finger position in human protein Kox 15. This helical domain was mutated from SDHLSKH to SSHLIOH (SP1-M3). Aspartic acid (D), serine (S) and lysine (K) were substituted by serine (S), isoleucine (I) and glutamine (Q). The DNA binding of the mutated SP1-M3 protein to the SP1 cognate target site GGG GCG GGG was significantly impaired, indicating that the amino acids, aspartic acid, serine and lysine play a pivotal role in DNA recognition. The mutated SP1 finger cannot imitate the function of the wild type SP1 finger in interacting with the cognate SP1 target site. This structure-function analysis indicates that the third SP1 zinc finger participates in sequence-specific DNA recognition. Thus, the affinity of zinc finger domains can be altered by substituting a limited number of amino acids. This observation is consistent with the notion that zinc finger domains are positioned in the major groove of the DNA and wrap around the DNA. Structure-function analysis of this kind might lead to the description of a zinc finger specific recognition code.

A cluster of expressed zinc finger protein genes in the pericentromeric region of human chromosome 10

Three members of the human zinc finger Krüppel family, ZNF11/KOX2, ZNF22/KOX15, and ZNF25/KOX19, have been regionally localized to the pericentromeric region of chromosome 10 by in situ chromosomal hybridization and somatic cell hybrid analysis. ZNF25/KOX19 is located centromeric to a breakpoint in chromosome band 10q11.2 in the chromosome region 10p11.2-q11.2, whereas ZNF22/KOX15 maps distal to it in band 10q11.2. Sequences hybridizing to the KOX2 probe are found at two loci, ZNF11A and ZNF11B, that map proximal and distal to the 10q11.2 breakpoint, respectively. The two ZNF11 loci probably represent two related sequences in 10p11.2-q11.2. This cluster of ZNF/KOX genes is of particular interest since the loci for multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes have been assigned to this region by linkage analysis.

Chromosomal localization of two human zinc finger protein genes, ZNF24 (KOX17) and ZNF29 (KOX26), to 18q12 and 17p13–p12, respectively

Two members of the zinc finger Krüppel family, ZNF24 (KOX17) and ZNF29 (KOX26), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization to human chromosomes 18q12 and 17p13-p12, respectively. The mapping of ZNF29 together with the previously reported localization of ZFP3 suggests that a zinc finger gene complex is located on human chromosome 17p. ZNF29 maps centromeric to the human p53 tumor antigen gene (TP53). In the analogous murine position, the two mouse zinc finger genes Zfp2 and Zfp3 have recently been assigned to the distal region of mouse chromosome 11, the murine homolog of human chromosome 17. Both human zinc finger genes ZNF24 and ZNF29 are in chromosomal regions that have been noted to be deleted in neoplasms of the lung and of the central nervous system at chromosome 17p and in colorectal neoplasia at chromosomes 17p and 18q.

Chromosomal localization of 9 KOX zinc finger genes: physical linkages suggest clustering of KOX genes on chromosomes 12, 16, and 19

Nine KOX zinc finger genes were localized on four human chromosomes by in situ hybridization of cDNA probes to metaphase chromosomes. KOX1 (ZNF10), KOX11 (ZNF18), and KOX12 (ZNF19) were mapped to chromosome bands 12q24.33, 17p13-p12, and 16q22-q23, respectively. Six other KOX genes were localized on chromosome 19: KOX6 (ZNF14) and KOX13 (ZNF20) to 19p13.3-p13.2, KOX5 (ZNF13) and KOX22 (ZNF27) to 19q13.2-qter, and KOX24 (ZNF28) and KOX28 (ZNF30) to 19q13.4. Pulsed field gel electrophoresis experiments showed that the pairs of KOX genes found on the chromosome bands 12q24.33, 16q22-q23, 19p13.3-p13.2, or 19q13.3-qter lie within 200–300 kb DNA fragments. This suggests the existence of KOX gene clusters on these chromosomal bands.

A new Cys2/His2 zinc finger gene, rKr2, is expressed in differentiated rat oligodendrocytes and encodes a protein with a functional repressor domain

Abstract: The function of the vertebrate nervous system is dependent on the proper myelination of its fiber tracts. Myelin of the CNS is produced by oligodendrocytes. To identify gene regulatory proteins expressed in this particular glial cell type, we isolated cDNAs coding for Cys2/His2 zinc finger proteins from a rat oligodendrocyte cDNA library. One clone, named rKr2 (rKr for rat Krüppel‐type protein), encodes a protein with 19 carboxy‐terminal zinc finger domains and an amino‐terminal Krüppel‐associated box domain. This amino‐terminal domain of the rKr2 protein behaved as a strong transcriptional repressor module when fused to the DNA binding domain of yeast GAL4 and tested on an appropriate reporter construct. High levels of rKr2 mRNA in adult rat tissues were found only in the CNS and testis; in the CNS, the message was predominantly expressed in differentiated oligodendrocytes. The modular structure of the rKr2 protein (carboxy‐terminal DNA binding domain, amino‐terminal repressor module) and its expression pattern suggest that it acts as a sequence‐specific transcriptional repressor in the myelin‐producing glial cells of the CNS.

Two human genes encoding zinc finger proteins, ZNF12 (KOX 3) and ZNF 26 (KOX 20), map to chromosomes 7p22-p21 and 12q24.33, respectively

SummaryTwo members of the human zinc finger Krüppel family, ZNF 12 (KOX 3) and ZNF 26 (KOX 20), have been localized by somatic cell hybrid analysis and in situ chromosomal hybridization. The presence of individual human zinc finger genes in mouse-human hybrid DNAs was correlated with the presence of specific human chromosomes or regions of chromosomes in the corresponding cell hybrids. Analysis of such mouse-human hybrid DNAs allowed the assignment of the ZNF 12 (KOX 3) gene to chromosome region 7p. The ZNF 26 (KOX 20) gene segregated with chromosome region 12q13-qter. The zinc finger genes ZNF 12 (KOX 3) and ZNF 26 (KOX 20) were localized by in situ chromosomal hybridization to human chromosome regions 7p22-21 and 12q24.33, respectively. These genes and the previously mapped ZNF 24 (KOX 17) and ZNF 29 (KOX 26) genes, are found near fragile sites.

[27] Use of PCR to determine genomic DNA target sites for zinc finger protein expressed in mouse cerebellum

Summary Randomized oligonucleotide approaches as well as genomic DNA fragments have been used in conjunction with the polymerase chain reaction to identify DNA target sites and potential DNA target genes. All techniques currently used incorporate principles developed and discussed in manuscripts by Oliphant et al. (1989) , by Kinzler and Vogelstein (1989) , and by Thiesen and Bach (1990). Besides others, DNA binding sites have been identified specific for GLI-1 protein ( Kinzler and Vogelstein, 1990 ), specific for retinoic acid receptors ( Costa-Giomi et al., 1992 ), specific for EVI 1 ( Delwel et al., 1993 ; Matsugi et al., 1993 ), and specific for the chicken CdxA homeobox gene ( Margalit et al., 1993 ). However, the determination of functional target genes for DNA binding proteins remains one of the challenges in the near future in order to decipher the function of hundreds of zinc finger genes present in the mammalian genome.