Discovery Of Human Scan-nf Protein Family Genes And Their Functions

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse including DNA recognition, RNA packaging, transcriptional activation, protein folding and assembly, regulation of apoptosis, and lipid binding. The C₂H₂ type, which is a kind of important transcriptional factors, is the most common one in this family. There are some tandem zinc-finger motifs with varied numbers in C₂H₂ zinc-finger proteins. The N-terminus usually has some highly conserved domains, such as POZ, KRAB and SCAN domains.Among them, the SCAN domain is a highly conserver motif that is vertebrate-specific. The SCAN-ZFPs constitute a subfamily of C2H2 zinc finger proteins. Although some genes of the SCAN family have been implicated in the transcriptional regulation of growth factors, genes involved in lipid metabolism, as well as other genes involved in cell survival and differentiation, the function of most members of the SCAN family is unknown.In this study, we utilize a bioinformatics approach with experiments to define the structures and gene locations of the 54 members of the human SCAN family. The genes encoding SCAN domains are clustered in the human genomes, such as 3p21, 6p21.3, 7q22, 15q25, 16p13.3, 17p11.2, 18q12, 19q13.4, and Xq26. We also found that many genes of the SCAN family were capable of generating isoforms. And one type of the isoforms doesn't have the zinc fingers, we named them nf (no zinc finger), and fu (full length with zinc finger) to the isoforms with zinc fingers.To study the function of the nf isoforms, we cloned several members of the SCAN family, including fu and nf isoforms. Then we confirm their interactions through co-IP and GST-pull down. Our results showed that the fu isoforms can interact with the nf isoforms, and this interaction was mediated by the SCAN domain. It is known that the C₂H₂ zinc finger proteins need binding to specific DNA segments via their zinc fingers and interact with other cellular factors to control the transcription of target genes. Thus, if the isoforms without zinc fingers can interact with the ones with zinc fingers, the multi types of transcripts will make the mechanism of transcription regulation more complicated and variegated. So our results suggested that the nf isofrms may be very important to the complexity of the transcription regulation. And we know that the transcriptional factors need to enter the nuleusbefore regulating the transcriptional activity of their target genes. So we detected the subcellular localization of the isoforms. And we found that the nf isofroms we studied can be transferred to the nucleus by the fu isforms through the interaction meditated by the SCAN domain.Since the C₂H₂ zinc finger proteins are important transcription factors, we used the dual-luciferase reporter system to study the function on transcription regulation of the SCAN family members. The results showed that the ZNF191fu and ZNF434fu functioned as transcription repressors while the ZNF396fu can only weakly suppress the transcription activity of the reporter, and the ZNF397fu and ZNF447fu showed no signanificant capability of transcription regulation. Besides that, we also studied the function on transcription regulation of the nf isoforms, and we found that they showed different capability of transcription regulation to the fu isoforms. The ZNF191nf and ZNF434nf showed weaker regulation ability than their fu isoforms, while the ZNF447nf can significantly activate the transcription activity of the reporter gene. And ZNF397nf function as transcription repressors. The different transcriptional regulation activity of the nf isoforms and their interaction with the fu isoforms would contribute to the complexity of the transcription regulation.To further examine the biological function of interaction between different isoforms we also analyze the potential role of ZNF191 and ZNF434 on signaling pathway. We found that ZNF191fu can significantly enhance the HSE-luciferase activity, while ZNF434fu can enhance the AP1 (PMA)-luciferase activity, then we analyze whether the nf isoforms have impact on the fu isoforms. And we found the nf isoforms of ZNF191 and ZNF434 can inhibit the activity of their fu isoforms respectively.In this study, we also identified a novel gene encoding a SCAN family member named ZNF445. The ZNF445 mRNA consists of 9105 nucleotides and has a 1031-amino acid open reading frame. It is mapped to chromosome 3p21.32. The predicted 119-kDa protein contains a SCAN domain at the N-terminus, followed by a well-conserved Kriippel-associated box (KRAB) domain. At the C-terminus of the protein, there are 14 C₂H₂ (Cys2-His2) zinc finger motifs. Northern blot analysis indicates that a 9.1 kb transcript specific for ZNF445 is expressed in uterus, thymus, small intestine, colon, pancreas, peripheral blood leukocyte, and especially at a higher level in the testis and skeletal muscle in human adult tissues. Reporter gene assays showed that ZNF445 is a transcriptional repressor, and ZNF445 protein was located inthe nucleus when overexpressed in cultured cells. Overexpression of ZNF445 in the HEK 293T cells activates the transcriptional activities of AP1 and SRE. Since AP1 and SRE are important nuclear effectors of the MAPK pathway, we examine whether this activation was related to the MAPK pathway. We found that the activation of AP1 and SRE by ZNF445 can be supressed by PD98059, an inhibitor of MEK. This suggested that the ZNF445 affected AP1 and SRE through the MAPK pathyway. Then we studied the potential impact on MAPK pathway of ZNF445. The results showed that expression of ZNF445 can increase Elk-1, ERK1/2, MEK and Raf-1 phosphorylation and this impact is dose-dependent. In addition, deletion studies showed that the SCAN domain of ZNF445 may be involved in this activation. These results clearly indicate that ZNF445 is a member of the SCAN family of zinc finger transcription factor and may function in MAPK pathway through Raf-1/MEK/ERK /Elk-1 signals. And we also study the function of ZNF445 on cell growth and proliferation by the experiment of cloning efficiency in L02. The result showed that ZNF445 can significantly increase the cloning efficiency when compared with the control.In summary, we found a kind of isoforms when we study the human SCAN family. This kind isoforms don't contain zinc fingers so called nf isoforms. And they can interact with the fu isoforms which contain the zinc fingers through the SCAN domain. This interaction between them can affect the biological function of the fu isoforms. This finding is important to futher study the transcriptional regulation function of the zinc finger proteins, and may be one important answer to the problem - how can such a small group of transcription factors regulate so many genes expression. And in the study of ZNF445, one member of the SCAN family, we confirmed that the protein can affect the MAPK pathway, and found its biological function. We also found the SCAN domain of ZNF445 may be involved in this function. This is an important supplementary to the study of the SCAN family, and threw some light on the question about the cell growth and proliferation.