Year-end Sale % OFF 
Abstract
Stage-specific activator protein (SSAP) is the transcription factor responsible for the activation of the sea urchin late H1 gene at the mid-blastula stage of embryogenesis. SSAP contains an extremely potent transcription activation domain that functions 4-5-fold better than VP16 in a variety of mammalian cell lines. We used the two-hybrid screening technique to identify human cDNAs from an HL60 cell-derived cDNA library that encode proteins that interact with the transcription activation domain of SSAP. One of these cDNAs encodes ZFM1, a protein previously identified at the locus linked to multiple endocrine neoplasia type 1 (MEN1) and as presplicing factor SF1. Functional assays establish the ZFM1 protein as a transcriptional repressor. ZFM1 protein represses Gal4-GQC-mediated transcription, and this activity requires both a repression domain found in the N-terminal 137 amino acids of the protein, as well as a GQC interaction region. The physiological significance of repression mediated by ZFM1 comes from the ability of its specific repression domain to function when fused to Gal4 and tethered to promoters containing Gal4 binding sites. The activity is unique in that activated but not basal transcription levels are affected.
Highlights
Dergoes a posttranslational modification at about 12 h after fertilization, and dimerization coincides with the activation of late H1 gene (2)
Identification of ZFM1 as an Stage-specific activator protein (SSAP)-interacting Protein—To identify protein(s) that interact with the GQC activation domain, we employed a modified version of the yeast two-hybrid system
Gal4-GQC activates reporter genes 4 –5-fold better than Gal4-VP16 (4). It belongs to a class of transcription activation domain rich in glycine, glutamine, serine, threonine, and basic amino acids
Summary
Plasmids—To generate pBTM116-GQC as bait for two-hybrid screening, an EcoRI fragment derived from pSG424-GQC (4), which contains the entire GQC domain (amino acids 181– 404), was ligated into pBTM116 vector cut with EcoRI. To create pGAD424-ZFM1-E for yeast two-hybrid assays, two primers (5Ј-GGG GTC GAC CAG ACC ACA TGG CGA CCG GAG CGA AC-3Ј) and (5Ј-CCC GTC GAC TCA CTT GTC ATC GTC GTC CTT GTA GTC CCA ATG GGC GCG GAA AGT-3Ј) were used in a PCR reaction to amplify ZFM1-E from pBluescript SK phagemid DNA This PCR product was cut by SalI and ligated into the SalI site of pGAD424 to fuse ZFM1-E in frame with the Gal activation domain. To create pCR3.1-ZFM1-(1–320), this region of ZFM1-A was amplified by PCR using two primers (5Ј-GGG GTC GAC CCG CCA CCA TGG CGA CCG GAG CGA AC-3Ј and 5Ј-GAC GCG TCG ACT CAC TTG TCA TCG TCG TCC TTG TAG TCC AGT TCA GCC ATG AGG GA-3Ј) This PCR fragment is cloned in pCR3.1 and the expression of ZFM1-(1–320) with a C-terminal FLAG-tag is under the control of cytomegalovirus promoter in the resulting plasmid. The expression of transfected ZFM1 protein was detected using M2 antibody, which recognizes the FLAG-tag on its C terminus
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
