Mouse Zinc Finger Protein Research Articles

Zfp322a Regulates Mouse ES Cell Pluripotency and Enhances Reprogramming Efficiency

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts are characterised by their ability to self-renew and their potential to differentiate into many different cell types. Recent studies have shown that zinc finger proteins are crucial for maintaining pluripotent ES cells. Mouse zinc finger protein 322a (Zfp322a) is expressed in the ICM of early mouse embryos. However, little is known regarding the role of Zfp322a in the pluripotency maintenance of mouse ES cells. Here, we report that Zfp322a is required for mES cell identity since depletion of Zfp322a directs mES cells towards differentiation. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays revealed that Zfp322a binds to Pou5f1 and Nanog promoters and regulates their transcription. These data along with the results obtained from our ChIP-seq experiment showed that Zfp322a is an essential component of mES cell transcription regulatory network. Targets which are directly regulated by Zfp322a were identified by correlating the gene expression profile of Zfp322a RNAi-treated mES cells with the ChIP-seq results. These experiments revealed that Zfp322a inhibits mES cell differentiation by suppressing MAPK pathway. Additionally, Zfp322a is found to be a novel reprogramming factor that can replace Sox2 in the classical Yamanaka's factors (OSKM). It can be even used in combination with Yamanaka's factors and that addition leads to a higher reprogramming efficiency and to acceleration of the onset of the reprogramming process. Together, our results demonstrate that Zfp322a is a novel essential component of the transcription factor network which maintains the identity of mouse ES cells.

Activation of transcriptional activity of HSE by a novel mouse zinc finger protein ZNFD specifically expressed in testis

Zinc finger proteins (ZFPs) that contain multiple cysteine and/or histidine residues perform important roles in various cellular functions, including transcriptional regulation, cell proliferation, differentiation, and apoptosis. The Cys-Cys-His-His (C(2)H(2)) type of ZFPs are the well-defined members of this super family and are the largest and most complex proteins in eukaryotic genomes. In this study, we identified a novel C(2)H(2) type of zinc finger gene ZNFD from mice which has a 1,002 bp open reading frame and encodes a protein with 333 amino acid residues. The predicted 37.4 kDa protein contains a C(2)H(2) zinc finger domain. ZNFD gene is located on chromosome 18qD1. RT-PCR analysis revealed that the ZNFD gene was specifically expressed in mouse testis but not in other tissues. Subcellular localization analysis demonstrated that ZNFD was localized in the nucleus. Reporter gene assays showed that overexpression of ZNFD in the COS7 cells activates the transcriptional activities of heat shock element (HSE). Overall, these results suggest that ZNFD is a member of the zinc finger transcription factor family and it participates in the transcriptional regulation of HSE. Many heat shock proteins regulated by HSE are involved in testicular development. Therefore, our results suggest that ZNFD may probably participate in the development of mouse testis and function as a transcription activator in HSE-mediated gene expression and signaling pathways.

Cloning and Expression Analysis of a Novel Mouse Zinc Finger Protein Gene Znf313 Abundantly Expressed in Testis

We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a C(3)HC(4) ring finger domain and three C(2)H(2) domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.

The p53-induced mouse zinc finger protein wig-1 binds double-stranded RNA with high affinity

The p53-induced mouse wig-1 gene encodes a Cys2His2-type zinc finger protein of unknown function. The zinc fingers in wig-1 are connected by long (56-75) amino acid linkers. This distribution of zinc finger domains resembles that of the previously described double-stranded (ds)RNA-binding proteins dsRBP-ZFa and JAZ. Ectopically expressed FLAG-tagged mouse wig-1 protein localized to nuclei and in some cells to nucleoli, whereas GFP-tagged mouse wig-1 localized primarily to nucleoli. Electrophoretic mobility shift assay using a recombinant GST-wig-1 fusion protein showed that wig-1 preferentially binds dsRNA rather than single-stranded RNA or dsDNA. A set of deletion/truncation mutants of wig-1 was tested to determine the dsRNA-binding domain(s) or region(s) in wig-1 that is involved in the stabilization of wig-1-dsRNA complexes in vitro. This revealed that the first zinc finger in wig-1 is essential for binding to dsRNA, whereas zinc fingers 2 and 3 are dispensable. wig-1 protein expressed in mammalian cells also showed a high affinity for dsRNA. wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA binding plays a role in the p53-dependent stress response.

Cloning and expression of the human single copy homologue of the mouse zinc finger protein zfr

A human homologue of the murine zinc finger protein zfr is transcriptionally induced in the Epstein–Barr virus-positive Burkitt lymphoma cell line Raji upon treatment with the granulocyte/macrophage lineage ganglioside IV 3NeuAc-nLcOse 4Cer. The gene was cloned by a rapid amplification of cDNA ends approach based on a cDNA clone. The resulting hzfr sequence is 3393 base pairs in length coding for a protein of 1057 amino acids. Sequence alignments between hzfr and zfr reveal an identity of 92% on the nucleotide level and an identity of 96.4% on the amino acid level, respectively. Based on Southern blot data hzfr can be addressed as a single copy gene. Tissue-specific expression was determined by semi-quantitative PCR of normalized cDNA populations from various human tissues with glyceraldehyde-3-phosphate dehydrogenase as an internal control. Highest levels of transcripts were found in brain. hzfr transcripts could not be detected in skeletal muscle.

HZAC encodes a zinc finger protein with antiproliferative properties and maps to a chromosomal region frequently lost in cancer.

We previously reported the identification of mZac, a novel mouse zinc finger protein that shared with p53 the ability to regulate concomitantly apoptosis and cell cycle progression. We describe here the isolation, chromosomal localization, and functional in vitro characterization of its human homolog. hZAC is a widely expressed zinc finger protein that reveals transactivation and DNA-binding activity. hZAC inhibits tumor cell growth through induction of apoptotic cell death and G1 arrest. Thus hZAC, like its mouse counterpart, displays antiproliferative properties through pathways known to be central to the activity of p53. We mapped hZAC on chromosome 6q24-q25, a region frequently deleted in many solid tumors. Indeed, allelic loss at 6q24-q25 has been shown in breast and ovary cancers, melanomas, astrocytomas, and renal cell carcinomas. Furthermore, Abdollahi et al. [Abdollahi, A., Godwin, A. K., Miller, P. D., Getts, L. A., Schultz, D. C., Tagushi, T., Testa, J. R. & Hamilton, T. C. (1997) Cancer Res. 57, 2029-2034] recently isolated ZAC through its loss of expression in a surface epithelial ovary tumor model and accordingly named it Lot for "lost on transformation." In view of these observations, the functional properties we report here provide further arguments to consider hZAC as a tumor suppressor gene candidate.

Multiplex differential display identifies a novel zinc-finger protein repressed during withdrawal from cocaine

Multiplex differential display (MDD), a modification of differential display reverse transcriptase polymerase chain reaction (DD-PCR), was used to identify cocaine-dependent regulation of previously known and unknown gene products. Direct comparison of the MDD amplification profiles of duplicate, total RNA samples from the caudate putamen (CPu) of either vehicle or cocaine treated Sprague–Dawley rats indicated that the relative induction of a 240 bp (8G247) product, likely to represent c- fos mRNA, closely paralleled changes in c- fos mRNA as measured by Northern blot analysis. MDD and Northern blot analysis also revealed substantial repression of another PCR product (8G226) at 1 h and 1 day after repeated administration of cocaine. At 2 days after cocaine exposure, the level of 8G226 had returned to control levels. The DNA sequence of 8G226 exhibited near identity with a mouse zinc-finger protein (PZf) and is thus likely to represent a transcriptional regulator. Interestingly, the repression of 8G226 immediately after cocaine treatment is in direct contrast to the cocaine-dependent increase in expression documented for NGFI-A, another zinc-finger protein which also functions as a transcriptional regulator. Detailed characterization of the prolonged reduction in the expression of 8G226 may lead to the identification of additional regulatory pathways that produce changes in cellular response after repeated cocaine exposure.

The ubiquitous transactivator Zfp-38 is upregulated during spermatogenesis with differential transcription

We describe the complete nucleotide sequence of a full length cDNA clone encoding a new mouse zinc finger protein gene, Zfp-38 and localize it on chromosome 5 by the interspecific backcross analysis. The N-terminal domain of the Zfp-38 protein (64 kDa) contains 358 amino acids and the C-terminal domain of 197 residues encodes 7 zinc fingers. We also present evidence that Zfp-38 is a strong transcriptional activator. The transactivation domain was localized in the non finger region and a fusion protein containing 112 amino acid residues from this region of the Zfp-38 and the DNA binding domain of the yeast Gal 4 protein, very efficiently transactivated the expression of a reported CAT plasmid, harboring the Gal4 target site. By in situ hybridization and northern blotting technique, the Zfp-38 transcript can be detected at a highly elevated level during spermatogenesis. Its expression accompanies the progression from pachytene spermatocytes to round spermatids. The undifferentiated spermatogonia or the haploid elongated spermatid and the spermatozoa do not show detectable level of the transcript. Interestingly, other tissues express low levels of a slightly shorter transcript with a different 5′ end as determined by RNase protection. The presence of both a transcriptional activating domain and 7 DNA binding zinc fingers, coupled with the cell type(s) specific expression pattern, suggests that Zfp-38 has the potential to regulate transcription during spermatogenesis.

Expression of a mouse zinc finger protein gene in both spermatocytes and oocytes during meiosis

In order to identify genes regulating meiosis, a mouse spermatocyte cDNA library was screened for sequences encoding proteins with C 2H 2-type zinc finger motifs which are typically expressed by the Drosophila Krüppel gene. Three new cDNAs were isolated, and they were designated CTfin33, CTfin51, and CTfin92. Among them, CTfin51 was selected for further study. The deduced amino acid sequence revealed seven zinc finger motifs in its C-terminal region. Northern blot and in situ hybridization showed CTfin51 mRNA expression in spermatocytes after the pachytene stage and in early stage round spermatids of prepuberal and adult males. Immunocytochemical staining with an antiserum against β-gal- CTfin51 fusion protein was localized within nuclei of spermatocytes and spermatids. Oocyte nuclei after the pachytene stage also were immunoreactive for CTfin51 protein. Immunoblots revealed a band at M r 75,000 in protein extracts from the testis and the ovary. These results suggest that the CTfin51 gene encodes a DNA-binding regulatory protein functionally associated with meiosis in both male and female gametogenesis.