| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 101 === Nuclear respiratory factor-1 (NRF-1) is a major transcription factor in the human genome and plays a key role in neurite outgrowth in human neuroblastoma IMR-32 cells. Neurite outgrowth is a critical process in neuronal development, and many genes are involved in this well-regulated process. However, how NRF-1 regulates neurite outgrowth is still unclear. In this study, we used bioinformatic tools to search for genes downstream from NRF-1 and hypothesized that these genes mediate NRF-1 function in neurite outgrowth in neurons.
In the first part of this study, we hypothesized that synapsin I is downstream of NRF-1 and mediates its function in neurite outgrowth. Synapsin I protein is a well-known phosphoprotein in neuronal terminals and has been implicated in neuronal differentiation. Human synapsin I gene promoter has a putative NRF-1 response element (NRE), but it is not known whether this NRE is functional. Gel electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP), site-directed mutagenesis, and promoter studies indicated that NRF-1 is a positive regulator of synapsin I promoter. Exogenous NRF-1 overexpression increased synapsin I protein levels in IMR-32 and HEK293T cells. Down-regulating synapsin I expression markedly decreased the percentage of neurite-bearing cells and the average length of the longest neurites in IMR-32 cells that stably or transiently overexpressed NRF-1. We confirmed that the human synapsin I gene is positively regulated by NRF-1 and mediates the function of NRF-1 in neurite outgrowth.
In the second part, genome-wide analyses have identified that 916 genes in the human genome are potential NRF-1-regulated genes, including 691 annotated and 225 hypothetical genes. Fifteen annotated genes from different biological processes, cell cycle-related genes- MAPRE3, NPDC1, SUV39H2, SKA3, transport-related genes- RAB3IP, TRAPPC3, signal transduction-related genes- SMAD5, PIP5K1A, USP10, SPRY4, transcription-related genes- GTF2F2, NR1D1, and regulation of GTPase activity-related genes- RHOA, RAPGEF6, SMAP1, were selected for biological confirmation. EMSA and ChIP confirmed that all NREs of these fifteen genes are critical for NRF-1 binding. Quantitative RT-PCR demonstrated that mRNA levels of 12 of these genes are regulated by NRF-1. Overexpression or knockdown of candidate genes demonstrated that MAPRE3, NPDC1, SMAD5, USP10, SPRY4, GTF2F2, SKA3, RAPGEF6 positively regulated, and RHOA and SMAP1 negatively regulated neurite outgrowth. In this part, we have confirmed that 12 annotated genes are regulated by NRF-1 and 10 of them mediate NRF-1 function in neurite outgrowth in IMR-32 cells.
In the third part, we focused on three hypothetical genes, FAM134C, C3orf10, and ENOX1, and determine whether these hypothetical genes mediate NRF-1 function in neurite outgrowth in IMR-32 cells and primary hippocampal neurons. We found that NRF-1 positively regulated FAM134C and ENOX1, but negatively regulated C3orf10 in IMR-32 cells and primary rat cortical neurons. FAM134C positively regulates and C3orf10 negatively regulates neurite outgrowth, but ENOX1 plays no role in neurite outgrowth regulation. FAM134C and C3orf10 mediates NRF-1-enhanced neurite outgrowth. In primary rat hippocampal neurons, Fam134c is predominantly expressed in the axon hillock and C3orf10 is ubiquitously expressed in all neurites and cell bodies at different developmental stages, suggesting their roles in axonal and dendritic outgrowth. Fam134c positively regulates axonal length, but C3orf10 negatively regulates the number of axonal collaterals and dendrites. In the third part, we annotated FAM134C, C3orf10, and ENOX1 as NRF-1-regulated genes, which have differential effects on neurite outgrowth in neuroblastoma cells as well as neurons. Overall, we found that NRF-1 regulates neurite outgrowth through cell cycle-, transport-, signal transduction-, transcription-, regulation of GTPase activity-related genes and hypothetical genes, which suggest that NRF-1 regulates neuronal differentiation through a variety of biological processes.
Keywords: nuclear respiratory factor-1; neuroblastoma cells; primary hippocampal neurons; neurite outgrowth; axonal and dendritic outgrowth; bioinformatics.
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