ABSTRACT
Pluripotent cells are promising tools in the arena of regenerative medicine. For many years, research efforts have been directed toward uncovering the underlying mechanisms that govern the pluripotent state and this involves identifying new pluripotency-associated factors. Zinc finger protein 553 (Zfp553) has been hypothesized to be one such factor because of its predominant expression in inner cell mass of the mouse early embryo. In this study, we have identified Zfp553 as a regulator of pluripotency. Zfp553 knockdown downregulates pluripotency markers and triggers differentiation in mouse embryonic stem cells (mESCs). Further investigation revealed that Zfp553 regulates pluripotency in mESCs through the transcriptional activation of Pou5f1 and Nanog. Microarray results revealed that depletion of Zfp553 downregulates many pluripotency genes, as well as genes associated with metabolism-related processes. ChIP-sequencing (ChIP-seq) depicted the genomic binding sites of Zfp553 in mESCs and its binding motif. In addition, we found that depletion of Zfp553 could impair somatic cell reprogramming, evidenced by reduced reprogramming efficiency and cell viability. Together, our preliminary findings provide novel insights to a newly identified pluripotency factor Zfp553 and its role in pluripotency regulation.
Subject(s)
Cell Proliferation/genetics , Cellular Reprogramming/genetics , DNA-Binding Proteins/physiology , Induced Pluripotent Stem Cells/metabolism , Nuclear Proteins/physiology , Animals , Cell Differentiation/genetics , Cells, Cultured , Embryonic Stem Cells/physiology , Energy Metabolism/genetics , Gene Expression Profiling , Gene Expression Regulation, Developmental , Induced Pluripotent Stem Cells/physiology , Mice , Microarray Analysis , Zinc FingersABSTRACT
The acquisition of pluripotent cells can be achieved by combined overexpression of transcription factors Oct4, Klf4, Sox2 and c-Myc in somatic cells. This cellular reprogramming process overcomes various barriers to re-activate pluripotency genes and re-acquire the highly dynamic pluripotent chromatin status. Many genetic and epigenetic factors are essentially involved in the reprogramming process. We previously reported that Patz1 is required for maintenance of ES cell identity. Here we report that Patz1 plays an inhibitory role in OKSM-induced reprogramming process since more iPS colonies can be induced from Patz1(+/-) MEFs than wild type MEFs; while the addition of Patz1 significantly repressed reprogramming efficiency. Patz1(+/-) MEFs can surpass the senescence barrier of Ink4a/Arf locus, thus enhancing iPS colonies formation. Moreover, Patz1(+/-) MEFs displayed higher levels of acetylated histone H3, H3K4me2, H3K4me3, H3K36me3 and lower levels of histone H3K9me3 and HP1α, indicating that heterozygous knockout of Patz1 results in a globally open chromatin which is more accessible for transcriptional activation. However, Patz1(-/-) MEFs gave the lowest reprogramming efficiency which may result from cell senescence trigged by up-regulated Ink4a/Arf locus. Together, we have demonstrated that the dosage of Patz1 modulates reprogramming process via significantly influencing cell senescence, proliferation and chromatin structure.
Subject(s)
Cellular Reprogramming/genetics , Kruppel-Like Transcription Factors/genetics , Animals , Cell Line , Cellular Reprogramming/physiology , Cellular Senescence/genetics , Cellular Senescence/physiology , Chromatin/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/physiology , Fibroblasts/metabolism , Fibroblasts/physiology , Histones/genetics , Kruppel-Like Factor 4 , Mice , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/physiology , Transcriptional Activation/geneticsABSTRACT
Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of blastocysts are pluripotent. Pluripotency is maintained by a transcriptional network in which Oct4 and Nanog are master regulators. Notably, several zinc finger transcription factors have important roles in this network. Patz1, a BTB/POZ-domain-containing zinc finger protein, is expressed at higher levels in the ICM relative to the trophectoderm. However, its function in pluripotency has been poorly studied. Here, we show that Patz1 is an important regulator of pluripotency in ESCs. Patz1 RNAi, chromatin immunoprecipitation (ChIP), and reporter assays indicate that Patz1 directly regulates Pou5f1 and Nanog. Global transcriptome changes upon Patz1 knockdown largely involve upregulation of apoptotic genes and downregulation of cell cycle and cellular metabolism genes. Patz1 ChIP sequencing further identified more than 5,000 binding sites of Patz1 in mouse genome, from which two binding motifs were extracted. Further, gene ontology analysis of genes associated with the binding sites displays enrichment for proximity to developmental genes. In addition, embryoid body assays suggest that Patz1 represses developmental genes. Together, these results propose that Patz1 is important for ESC pluripotency.
