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HGNC Approved Gene Symbol: NFE4
Cytogenetic location: 7q22.1 Genomic coordinates (GRCh38) : 7:102,973,430-102,988,853 (from NCBI)
Transcription factor p22-NFE4 plays a role in regulation of the human beta-globin (141900) locus through activation of the gamma-globin (142200) genes and competitive silencing of beta-globin gene expression. Along with the ubiquitous transcription factor CP2 (TFCP2; 189889), it forms part of the stage selector protein (SSP) complex. The p14-NFE4 isoform functions as a repressor of gamma- and epsilon (142100)-globin gene expression (Zhao et al., 2004).
By yeast 2-hybrid screen of a myelogenous leukemia (K562) cell cDNA library using the protein dimerization domain of CP2 as bait, followed by 5-prime RACE, Zhou et al. (2000) cloned NFE4. The full-length 179-amino acid protein has a calculated molecular mass of 22 kD. An internal ATG codon at position 101 was predicted to result in a 14-kD isoform. Western blot analysis detected both the 22-kD and 14-kD isoforms. Interaction of NFE4 with CP2 was confirmed by retention of in vitro transcribed/translated NFE4 on GST-CP2 beads. Coimmunoprecipitation showed that CP2 and NFE4 formed a physiologic complex in vivo, and electromobility shift assay (EMSA) showed that NFE4 is a component of the SSP-SSE (stage selector element) complex. RT-PCR analysis detected NFE4 expression in bone marrow, fetal liver, and cord blood.
By Northern blot analysis and 5-prime RACE, Zhao et al. (2006) determined that p14-NFE4 was derived from the full-length NFE4 transcript by translation initiation at the methionine at codon 101. By in vitro transcription/translation of the NFE4 cDNA truncated at codon 101, they showed that the truncated cDNA gave rise to a 14-kD product that could be visualized with anti-NFE4 antisera and comigrated with the 14-kD species previously observed in crude K562 nuclear extract.
By transducing K562 cells with NFE4, Zhou et al. (2000) enforced expression of NFE4 and observed induction of fetal and embryonic globin gene expression. They confirmed 5- to 10-fold upregulation of gamma-globin gene expression by Northern blot analysis and observed comparable effects on epsilon-globin gene expression. Activation of beta-globin gene expression was not observed. They transduced CD34+ cells derived from human cord blood with NFE4 and observed a 2-fold increase in gamma-globin gene expression.
Zhou et al. (2004) enforced expression of the p22-NFE4 protein in transgenic mice carrying the human beta-globin locus and observed that the ratio of gamma-globin to beta-globin expression was increased in embryonic day (E) 12.5 to E14.5 fetal liver. This increase in the gamma:beta ratio resulted in a delay in the fetal-to-adult hemoglobin switch. At E12.5, the 6-fold increase in the ratio was due to reduction in beta-globin gene expression. At E14.5, the 4-fold increase in the ratio was attributed to enhanced gamma-globin gene expression. Zhou et al. (2004) hypothesized that elevated p22-NFE4 levels maintained the fetal gene in the active state, rather than repressing adult globin gene expression, by facilitating competition of the fetal genes for the locus control region (LCR; see 152424).
Zhao et al. (2004) determined that NFE4 interacts with PCAF (602303) and is acetylated on lysine-43 in the N-terminal domain. Acetylation of NFE4 prolonged the protein half-life by preventing ubiquitin-mediated degradation in a PCAF-dependent manner. Stabilization of NFE4 by blocking ubiquitination did not increase transcription, suggesting that acetylation is required for NFE4-mediated gamma-globin gene activation. GST pull-down assays showed that nonacetylated NFE4 bound greater amounts of HDAC1 (601241), a member of a family of deacetylases involved in transcriptional repression and gene silencing; however, acetylated and nonacetylated NFE4 bound CP2 equally.
p14-NFE4 Function
Zhao et al. (2006) transduced K562 cells with p14-NFE4 and observed a reduction in gamma- and epsilon-globin gene expression but no change in beta-globin gene expression as determined by Northern blot analysis. The authors transduced CD34+ progenitors with p14-NFE4 and also observed reduction in gamma-globin gene expression by Northern blot analysis. Coimmunoprecipitation experiments using extracts from transduced 293T cells showed that p14-NFE4 binds CP2 but does not bind p22-NFE4. Quantitative chromatin immunoprecipitation (ChIP) assays showed that overexpression of p14-NFE4 in K562 cells repressed binding of CP2 to the SSE. The authors also observed loss of p45-NFE2 (601490) recruitment, resulting in reduction of RNA polymerase II (see 180660) and TBP (600075) binding to the gamma-globin gene promoter. Mutant (E110L and D137G) p14-NFE4 was unable to bind CP2, resulting in gamma-globin gene expression. Zhao et al. (2006) concluded that the dominant-negative role of p14-NFE4 results through sequestration of CP2 with subsequent loss of CP2 interaction with the gamma-globin promoter.
Gross (2008) mapped the NFE4 gene to chromosome 7q22.1 based on an alignment of the NFE4 sequence (GenBank AY258907) with the genomic sequence (build 36.1).
Gross, M. B. Personal Communication. Baltimore, Md. 6/26/2008.
Zhao, Q., Cumming, H., Cerruti, L., Cunningham, J. M., Jane, S. M. Site-specific acetylation of the fetal globin activator NF-E4 prevents its ubiquitination and regulates its interaction with the histone deacetylase, HDAC1. J. Biol. Chem. 279: 41477-41486, 2004. [PubMed: 15273251] [Full Text: https://doi.org/10.1074/jbc.M405129200]
Zhao, Q., Zhou, W., Rank, G., Sutton, R., Wang, X., Cumming, H., Cerruti, L., Cunningham, J. M., Jane, S. M. Repression of human gamma-globin gene expression by a short isoform of the NF-E4 protein is associated with loss of NF-E2 and RNA polymerase II recruitment to the promoter. Blood 107: 2138-2145, 2006. [PubMed: 16263792] [Full Text: https://doi.org/10.1182/blood-2005-06-2497]
Zhou, W., Clouston, D. R., Wang, X., Cerruti, L., Cunningham, J. M., Jane, S. M. Induction of human fetal globin gene expression by a novel erythroid factor, NF-E4. Molec. Cell. Biol. 20: 7662-7672, 2000. [PubMed: 11003662] [Full Text: https://doi.org/10.1128/MCB.20.20.7662-7672.2000]
Zhou, W., Zhao, Q., Sutton, R., Cumming, H., Wang, X., Cerruti, L., Hall, M., Wu, R., Cunningham, J. M., Jane, S. M. The role of p22 NF-E4 in human globin gene switching. J. Biol. Chem. 279: 26227-26232, 2004. [PubMed: 15084587] [Full Text: https://doi.org/10.1074/jbc.M402191200]