Screening Of Interactive Proteins Of ZBTB8A And Initial Exploration Of ZBTB6

The ZBTB proteins which are important member of the zinc finger proteinfamily that function as transcription factors play important roles in various cellularfunctions, such as transcriptional regulation, cell proliferation, differentiation andapoptosis. Firstly, we cloned a gene, named as ZBTB8A （zinc finger and BTB domaincontaining8A）（NM₀01040441） which is a member of human ZBTB family, fromgastric cancer cell AGS cDNA by RT-PCR. Its function remains unknown. Preliminarystudies have shown that ZBTB8A gene has a full open reading frame （ORF） of1326bp,which was localized on chromosome1p35.1. The ZBTB8A gene encodes a protein with441amino acids. The molecular weight is predicted to be50kDa. ZBTB8A proteincontains an N-terminal BTB/POZ domain and two typical C2H2zinc-finger motifs thatextend to the end of the protein sequence. Comparing BTB/POZ domains derived fromZBTB8A with other known proteins, we found that the BTB/POZ domain of ZBTB8Ais highly homologous to the BTB/POZ domain of other known proteins. ZBTB8A waslocalized in the nucleus, forming dot-like structures.The interactions between proteins exist in each cell of the life and they control lotsof activities in cells. These interactions are the basis of life and each activitiy of life iscarried out by such interactions. Studying the roles of such proteins is helpful for us todiscover some basic problems about life. To fish the novel proteins interacting withZBTB8A, we screened fetal brain cDNA library by yeast two-hybrid system.Thirty-four yeast positive clones were obtained. We chose one of candidate proteins forfurther characterization: SMARCB1（SWI/SNF related, matrix associated, actindependent regulator of chromatin, subfamily b, member1）, also known as INI1（integrase interactor1protein） or BAF47（BRG1-associated factor47）.The recombinant plasmids of pCMV-Myc-SMARCB1, pCMV-GST-ZBTB8A,pCMV-GST-ZBTB8A-BTB and pCMV-GST-ZBTB8A-ZNF were constructed successfully. The expressions of these proteins have been confirmed in293T cells.SMARCB1can interact with ZBTB8A by mammalian pull-down assay. But the BTB orZNF motif of ZBTB8A can not interact with SMARCB1. It suggested that ZBTB8Aneeded to maintain its structural integrity in interaction with SMARCB1.We were also concerned about another ZBTB family zinc finger protein ZBTB6（zinc finger and BTB domain containing6）, also known as ZBTB26or ZID （zinc fingerprotein with interaction domain）. To verify the subcelluar location of ZBTB6protein,we constructed the plasmid pEGFP-ZBTB6which expressed the fused proteinEGFP-ZBTB6. The results indicated that ZBTB6was localized in the nucleus, formingdot-like structures. Further study showed that the zinc finger domain was required fornuclear localization, and the BTB domain was required for spots like localization of theZBTB6protein.Four ZBTB6interference vectors were designed and synthesized for further study.Moreover, we identified effective ZBTB6interference expression vector by semi-quantitative PCR. All of the related work will be helpful for further research of ZBTB6biological functions.