HUB1, a novel Krüppel type zinc finger protein, represses the human T cell leukemia virus type I long terminal repeat-mediated expression.

We have shown that human T-cell leukemia virus type I (HTLV-I) gene expression is negatively regulated by the U5 repressive element (U5RE) of its long terminal repeat (LTR). To isolate factors binding to U5RE, we screened a cDNA expression library by south-western blotting with a U5RE probe. Screening 2 x10(6) clones gave a positive clone with a 3.8 kb insert encoding a novel 671 residue polypeptide, named HTLV-I U5RE binding protein 1 (HUB1), with five zinc finger domains and a Krüppel-associated box like domain; HUB1 may be related to a repressor belonging to the Krüppel type zinc finger protein. A 4.0 kb mRNA for HUB1 is ubiquitously expressed among all human tissues tested. HUB1 recognizes the TCCACCCC sequence as a core motif and exerts a strong repressive effect on HTLV-I LTR-mediated expression. A new repressive domain, named HUB1 repressive (HUR) domain, was identified, rather than the Krüppel-associated box like domain. The N-terminal region upstream of HUR domain seemed to be also indispensable to the repression. Thus, we propose that HUB1 is a new type repressor and plays an important role in the HTLV-I U5-mediated repression.     

Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.     

Remote homologue identification of Drosophila GAGA factor in mouse

GAGA factor (GAF) is involved in both gene activation and gene repression and plays a role in the modulation of chromatin structure. In Drosophila, Trithroax like (Trl) gene encodes the DNA binding protein called GAGA factor (GAF). Trl-GAF binds to GAGA sites through its C2H2 zinc finger domain and has an N-terminal BTB/POZ domain. Identification of Trl-GAF homologue in mouse helps in deeper understanding of the mechanism and function. Conventional alignment tools such as BLAST and FASTA cannot identify homologues in mouse genome as their sequence identity is below 30%. In the present study, various sequence and structure analyses were followed for the detection of remote homologues of Drosophila GAGA FACTOR in mouse to identify as Zbtb3. Through homology modeling and docking approach, the zinc finger region of mouse Zbtb3 showed conserved residues and favorable DNA binding sites with GAGA sites similar to that of Drosophila GAGA FACTOR.     

Evolutionary divergence in the broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain from the candidate tumor suppressor gene hypermethylated in cancer.

Hypermethylated in cancer, a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five Krüppel-like C2H2 zinc finger motifs and a N-terminal protein/protein interaction domain called broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain. Hypermethylated in cancer appears unique in the broad complex, tramtrack and bric à brac/poxviruses and zinc finger family since it contains a 13 amino acid insertion located in a loop between the conserved beta-strand beta5 and helix alpha5 which are involved in dimerization and scaffolding of the broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain. Cloning and sequencing of a murine hypermethylated in cancer gene suggests that this insertion has been acquired late in the evolution since it is present in two mammalian hypermethylated in cancer genes but absent in its zebrafish and avian counterparts. This is a unique example of a high divergence of the same broad complex, tramtrack and bric à brac/poxviruses and zinc finger domain in different species.     

Multiple genes encoding zinc finger domains are expressed in human T cells

Proteins containing zinc finger domains have been implicated in developmental control of gene expression in Drosophila, Xenopus, mouse, and humans. Multiple cDNAs encoding zinc (II) finger structures were isolated from human cell lines of T-cell origin to explore whether zinc finger genes participate in the differentiation of human hematopoietic cells. Initial restriction analysis, genomic Southern blotting, and partial sequence comparisons revealed at least 30 nonoverlapping cDNAs designated cKox(1-30) encoding zinc finger motifs. Analysis of cKox1 demonstrated that Kox1 is a single-copy gene that is expressed in a variety of hematopoietic and nonhaematopoietic cell lines. cKox1 encodes 11 zinc fingers that were shown to bind zinc when expressed as a beta-gal-Kox1 fusion protein. Further analysis of the predicted amino acid sequence revealed a heptad repeat of leucines NH2-terminal to the finger region, which suggests a potential domain for homo- or heterodimer protein formation. On the basis of screening results it was estimated that approximately 70 zinc finger genes are expressed in human T cells. Zinc finger motifs are probably present in a large family of proteins with quite diverse and distinct functions. However, comparisons of individual finger regions in cKox1 with finger regions of cKox2 to cKox30 showed that some zinc fingers are highly conserved in their putative alpha-helical DNA binding region, supporting the notion of a zinc finger-specific DNA recognition code.