Characterization of HRG22, a human homologue of the putative tumor suppressor gene HIC1.

Database searches identified on chromosome 22q11.2, a region subject to translocations, an homologue of the HIC1 (hypermethylated in cancer) candidate tumor suppressor gene located at 17p13.3. This gene was termed HRG22 for HIC1-related gene on chromosome 22. We have characterized a new HRG22 upstream coding exon and defined the complete coding sequence of the human and zebrafish HRG22 genes. Alignment of the HRG22 and HIC1 proteins from various species revealed high sequence homology in their N-terminal BTB/POZ and five C-terminal C(2)H(2) zinc finger domains and highlighted a conserved GLDLSKK/R peptide in their middle region. The full-length HRG22 and HIC1 proteins colocalize onto nuclear dots and share several functional properties since their BTB/POZ domains heterodimerize and are autonomous transcriptional repression domain insensitive to Trichostatin A, a histone deacetylase (HDAC) inhibitor. Thus, HIC1 and HRG22 define a subgroup of BTB/POZ domains unable to recruit repressing complexes containing an HDAC activity.

Identification in the human candidate tumor suppressor gene HIC-1 of a new major alternative TATA-less promoter positively regulated by p53.

HIC-1 (hypermethylated in cancer 1), a BTB/POZ transcriptional repressor, was isolated as a candidate tumor suppressor gene located at 17p13.3, a region hypermethylated or subject to allelic loss in many human cancers and in the Miller-Dieker syndrome. The human HIC-1 gene is composed of two exons, a short 5'-untranslated exon and a large second coding exon. Recently, two murine HIC-1 isoforms generated by alternative splicing have been described. To determine whether such isoforms also exist in human, we have further analyzed the human HIC-1 locus. Here, we describe and extensively characterize a novel alternative noncoding upstream exon, exon 1b, associated with a major GC-rich promoter. We demonstrate using functional assays that the murine exon 1b previously described as coding from computer analyses of genomic sequences is in fact a noncoding exon highly homologous to its human counterpart. In addition, we report that the human untranslated exon is presumably a coding exon, renamed exon 1a, both in mice and humans. Both types of transcripts are detected in various normal human tissues with a predominance for exon 1b containing transcripts and are up-regulated by TP53, confirming that HIC-1 is a TP53 target gene. Thus, HIC-1 function in the cell is controlled by a complex interplay of transcriptional and translational regulation, which could be differently affected in many human cancers.

Recruitment of SMRT/N-CoR-mSin3A-HDAC-repressing complexes is not a general mechanism for BTB/POZ transcriptional repressors: the case of HIC-1 and gammaFBP-B.

Hypermethylated in cancer (HIC-1), a new candidate tumor suppressor gene located in 17p13.3, encodes a protein with five C(2)H(2) zinc fingers and an N-terminal broad complex, tramtrack, and bric à brac/poxviruses and zinc-finger (BTB/POZ) domain found in actin binding proteins or transcriptional regulators involved in chromatin modeling. In the human B cell lymphoma (BCL-6) and promyelocityc leukemia (PLZF) oncoproteins, this domain mediates transcriptional repression through its ability to recruit a silencing mediator of retinoid and thyroid hormone receptor (SMRT)/nuclear receptor corepressor (N-CoR)-mSin3A-histone deacetylase (HDAC) complex, a mechanism shared with numerous transcription factors. HIC-1 appears unique because it contains a 13-aa insertion acquired late in evolution, because it is not found in its avian homologue, gammaF1-binding protein isoform B (gammaFBP-B), a transcriptional repressor of the gammaF-crystallin gene. This insertion, located in a conserved region involved in the dimerization and scaffolding of the BTB/POZ domain, mainly affects slightly the ability of the HIC-1 and gammaFBP-B BTB/POZ domains to homo- and heterodimerize in vivo, as shown by mammalian two-hybrid experiments. Both the HIC-1 and gammaFBP-B BTB/POZ domains behave as autonomous transcriptional repression domains. However, in striking contrast with BCL-6 and PLZF, both HIC-1 and gammaFBP-B similarly fail to interact with members of the HDAC complexes (SMRT/N-CoR, mSin3A or HDAC-1) in vivo and in vitro. In addition, a general and specific inhibitor of HDACs, trichostatin A, did not alleviate the HIC-1- and gammaFBP-B-mediated transcriptional repression, as previously shown for BCL-6. Taken together, our studies show that the recruitment onto target promoters of an HDAC complex is not a general property of transcriptional repressors containing a conserved BTB/POZ domain.

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.

The carboxy-terminal end of the candidate tumor suppressor gene HIC-1 is phylogenetically conserved.

HIC-1 (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 BTB/POZ. These two domains appeared as the only conserved regions found between human HIC-1 and its avian homologue, gammaFBP-B, isolated as a transcriptional repressor of the gammaF-Crystallin gene. We have recloned the HIC-1 gene and found four nucleotide differences within the 3' part of its published coding sequence. The corrected HIC-1 C-terminal end exhibits now significant homology (70%) to the chicken gammaFBP-B C-terminal end and appears thus as a third phylogenetically conserved domain that may serve an important, yet unknown function in HIC-1.