Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analyses in neuronal cells reveal that USF transcription factors regulate lysosomal gene expression [Expression analysis]

ABSTRACT: The upstream stimulating factors (USFs) USF1 and USF2 are ubiquitously expressed transcription factors characterized by a conserved basic helix-loop-helix leucine zipper DNA-binding domain. They form homo- or heterodimers and recognize E-box motifs to modulate gene expression. They are known to regulate diverse cellular functions including the cell cycle, immune response and glucose-lipid metabolism, but their roles in neuronal cells remain to be clarified. Here, we performed chromatin immunoprecipitation of USF1 from mouse brain cortex preparations. Subsequent promoter array analysis (ChIP-chip) indicated that USF1 exclusively bound to the CACGTG E-box motifs in the proximal promoter regions. Importantly, functional annotation of the USF1-binding targets revealed an enrichment of genes related to lysosomal functions. Gene expression arrays using a neuronal cell line subsequently revealed that knockdown of USFs deregulated lysosomal gene expression. Altered expression was validated by quantitative RT-PCR, supporting the conclusion that USFs regulate lysosomal gene expression. Furthermore, USFs knockdown slightly increased LysoTracker staining, implying a role for USFs in modulating lysosomal homeostasis. Together, our comprehensive, genome-scale analyses identified lysosomal genes as targets of USFs in neuronal cells, suggesting a potential additional pathway of lysosomal regulation. Gene expression profiling of neuro2a cells knocking down USFs. To screen USFs-downstream genes, mouse neuro2a cells were transfected with vectors for USFs miR RNAi or an empty vector, and then subjected to microarray analysis using Affymetrix Mouse Gene 1.0 ST Array.

ORGANISM(S): Mus musculus

SUBMITTER: Tomoyuki Yamanaka

PROVIDER: E-GEOD-76615 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Voltage gated calcium channels play a central role in regulating the electrical and biochemical properties of neurons and muscle cells. Cells coordinate the expression of voltage gated calcium channels with the expression of other proteins that regulate membrane potential and calcium homeostasis. We report that the C-terminus of CaV 1.2, an L-type calcium channel (LTC) contains a C-terminal fragment that translocates to the nucleus and regulates transcription. This calcium channel associated transcription factor (CCAT) associates with transcriptional co-regulators such as p54nrb/NonO and binds to endogenous promoters. CCAT regulates the expression of gap junctions, sodium calcium exchangers, NMDA receptors, potassium channels and other proteins that regulate neuronal signaling. Electrical activity and developmental processes regulate the nuclear localization of CCAT, suggesting that the CCAT integrates information about the number of LTCs with information about the developmental history and electrical activity of a cell. These findings provide the first evidence that voltage gated calcium channels can directly activate transcription and suggest a novel mechanism linking voltage gated channels to the function and differentiation of excitable cells. Experiment Overall Design: The goal of these experiments was to identify the genes that are regulated by CCAT, a novel transcription factor derived from the C-terminus of CaV1.2. Neuro2A neuroblastoma cells were transfected with the last 503 AA of CaV1.2 which is full length CCAT (CCAT FL) or with the last 280 AA of CaV1.2, a form of CCAT that lacks the transcriptional activation domain (CCAT DTA). The mRNA from either CCAT FL or CCAT DTA expressing cells was hybridized to Agilent mouse genome microarrays along with mRNA from untransfected neuro2A cells (CCAT FL or DTA A series). Subsequent investigation revealed that transfection with the PA1 plasmid by itself increases the expression of some genes. To control for this effect we compared Neuro2A cells transfected with full length CCAT (in PA1) with Neuro2A cells transfectected with PA1 alone (CCAT FL B series). The microarray data was analyzed with the Rossetta Luminator gene expression data analysis system.

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Genome-wide analyses in neuronal cells reveal that USF transcription factors regulate lysosomal gene expression [Expression analysis]

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