XB-ART-40391Dev Growth Differ October 1, 2009; 51 (8): 699-706.
Interaction of ZFPIP with PBX1 is crucial for proper expression of neural genetic markers during Xenopus development.
ZFPIP/Zfp462 has been recently identified as a new vertebrate zinc finger encoding gene whose product interacts with Pbx1. Previous work indicates that ZFPIP is maternally expressed in Xenopus laevis oocytes and plays a key role during the cleavage phase of embryogenesis. This early expression is followed by a zygotic expression which overlaps with the neural Pbx1 expression pattern, suggesting an interaction between these two partners during Xenopus neurogenesis. In order to test the physiological interaction between ZFPIP and Pbx1, we carried out a dominant negative assay in which the Pbx1 interacting domain of ZFPIP (ZFPIPp) was overexpressed in Xenopus laevis embryos. We observed that ZFPIPp ectopic expression led to abnormal en2 and N-cam expression patterns, whereas krox-20 expression was not affected. Furthermore, we showed that while ZFPIPp alone was localized in the nucleus of Cos-7 cells, additional expression of Pbx1 induced a location of ZFPIPp at the perinuclear region of the cells. These overall data suggest that ZFPIP and Pbx1 could be partners and cooperate in the regulation of essential neural genes during Xenopus development.
PubMed ID: 19737294
Article link: Dev Growth Differ
Genes referenced: en2 egr2 ncam1 klf4 pbx1 znf462
Antibodies: Pbx1 Ab1 Pcna Ab3 ZNF462 Ab1
Article Images: [+] show captions
|Fig. 1. Expression patterns of ZFPIP (h) and Pbx1 ( ) in Xenopus. Quantitative reverse transcription–polymerase chain reaction (qRT–PCR) assays were carried out in order to follow ZFPIP and Pbx1 mRNAs dynamics during Xenopus laevis development (stage 1–33). For both genes, relative expression was primarily low, started to increase at stage 17 and kept on augmenting until stage 33 (a). Western blot analysis revealed overlapping expression of ZFPIP and Pbx1b proteins from stage 33–46. Proliferating Cell Nuclear antigen (PCNA) was used as an internal control (b).|
|Fig. 2. Interaction of xZFPIPp and mZFPIPp with Pbx1 in vitro. Xenopus laevis (Xl) peptide was compared to Mus musculus (Mm) ZFPIPp. The xZFPIPp peptide contained 660 AA and corresponded to the Pbx1 binding domain of the mouse ZFPIP protein (mZFPIPp) identified from the two hybrid screens. The identical or equivalent amino-acids between xZFPIPp and mZFPIPp proteins are indicated respectively by ‘‘:’’ and ‘‘.’’ (a). GST pull-down assays were carried out using bacterially expressed Pbx1-GST and in vitro translated xZFPIPp and mZFPIPp. The mouse protein corresponded to the band at approximately 32 kDa, whereas the xenopus protein band was about 35 kDa. While neither xZFPIPp nor mZFPIPp was retained by control GST proteins, they both interacted with Pbx1-GST (b).|
|Fig. 3. Injection of a dominant negative form of ZFPIP (ZFPIPp) perturbed neural markers. The xZFPIPp transcripts were injected in one blastomere of two cell-stage embryos. The correct injection of xZFPIPp mRNAs were checked by whole mount hybridization using xZFPIPp as riboprobes (a). The correct translation of the protein was checked by Western blots carried out with a-ZFPIP antibodies on extracts prepared from injected and non-injected stage 22 embryos (b). Whole mount hybridizations of late neurula embryos using N-cam, en2 or krox-20 riboprobes demonstrated an altered expression of N-cam and en2 expression in injected blastomeres. The side injected with xZFPIPp capped mRNAs and the non-injected side are visualized by b-galactosidase staining and are indicated respectively by * and ** (c).|
|Fig. 4. Co-expression of ZFPIPp- FLAG or N-ZFPIP-FLAG and Pbx1b-HA in Cos-7 cells. Immunocytochemistry assays were carried out using a-HA and a-FLAG antibodies on cells expressing independently or co-expressing ZFPIP proteins (ZFPIPp-FLAG or N-ZFPIPFLAG) and Pbx1b-HA. ZFPIPp-FLAG, N-ZFPIP-FLAG and Pbx1b-HAproteins were observed, respectively, in the nucleus and in the cytoplasm when they were expressed separately. In contrast, co-expression in the same cell type of ZFPIPp or N-ZFPIP and Pbx1b, led to cytoplasmic and perinuclear localization of ZFPIP ⁄ Pbx1 complexes.|
|Fig. 5. ZFPIP is contained within a syntenic region. Using AutoGRAPH software (see methods), ZFPIP region in Homo sapiens (chromosome 9), Mus musculus (chromosome 4) and Canis familiaris (chromosome 11) were compared. The ZFPIP gene belongs to a syntenic genomic region, which has not been rearranged in the dog, mouse and human lineages since their last common ancestor. ZFPIP is contiguous to another zing finger-containing gene referred to as KLF4 (AC: NC000070.5). Arrows indicate the location of the ZFPIP gene on the different genomes (from left to right: murin, human and canine genomes). The colored lines connecting orthologous genes show the conservation of gene order within conserved segments. Black lines between colored segments represent breakpoints.|