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.2016 Oct 20:9:99.
doi: 10.3389/fnmol.2016.00099. eCollection 2016.

Dynamic Regulation of the Adenosine Kinase Gene during Early Postnatal Brain Development and Maturation

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Dynamic Regulation of the Adenosine Kinase Gene during Early Postnatal Brain Development and Maturation

Katharina Kiese et al. Front Mol Neurosci..

Abstract

The ubiquitous metabolic intermediary and nucleoside adenosine is a "master regulator" in all living systems. Under baseline conditions adenosine kinase (ADK) is the primary enzyme for the metabolic clearance of adenosine. By regulating the availability of adenosine, ADK is a critical upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. ADK protein exists in the two isoforms nuclear ADK-L, and cytoplasmic ADK-S, which are subject to dynamic expression changes during brain development and in response to brain injury; however, gene expression changes of theAdk gene as well as regulatory mechanisms that direct the cell-type and isoform specific expression of ADK have never been investigated. Here we analyzed potential gene regulatory mechanisms that may influenceAdk expression including DNA promoter methylation, histone modifications and transcription factor binding. Our data suggest binding of transcription factor SP1 to theAdk promoter influences the regulation ofAdk expression.

Keywords: adenosine kinase; brain; development; epigenetic; rat; specificity protein 1.

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Figures

Figure 1
Figure 1
Adk gene expression in early postnatal brain development. (A) Adk gene expression of long and short Adk isoforms separately as well as total Adk expression in rat hippocampal neurons and(B) in rat hippocampal glial cells at postnatal days 4 and 14 (P4 and P14, respectively) is shown. Adk gene expression was calculated relative to internal reference gene GAPDH. Neuronal and glial Adk gene expression at P4 was normalized to respective expression at P14, which was always set at 100%. Adk-L - long Adk isoform; Adk-S - short Adk isoform.*indicates significance.
Figure 2
Figure 2
Impact of epigenetic chromatin modifications on Adk-L promoter activity in P4 and P14 rat hippocampal neurons. (A) Promoter region of the long Adk isoform was analyzed from pos. −735 to + 470 relative to the TSS.(B) Activating histone modifications were identified at the Adk-L promoter in both P4 and P14 hippocampal neurons, including acetylation of H3 and H4, as well as H3 phosphoacetylation and lysine 4 trimethylation, with a relative sparing of the TSS. There was no evidence for repressive H3K27 trimethylation. Upper right corner of each diagram with insets showing enrichment of positive controls for each antibody presented as percent of the total input chromatin (% input). Adk-L promoter, promoter regulating long Adk isoform expression; ChIP, chromatin immunoprecipitation; H3K9ac, acetylation of lysine (K) 9 of histone H3; H4ac, pan-acetylation of histone H4; H3S10phK14ac, phosphoacetylation of histone H3 targeting Serine (S) 10 and Lysine (K) 14; H3K4me3, trimethylation of lysine (K) 4 of histone H3; H3K27me3, trimethylation of lysine (K) 27 of histone H3; TSS, transcriptional start site.
Figure 3
Figure 3
Impact of epigenetic chromatin modifications on Adk-S promoter activity in P4 and P14 rat hippocampal neurons. (A) Promoter region of the short Adk isoform was analyzed from pos. −1043 to +1084 relative to the TSS.(B) Enrichment of activating histone modifications at the Adk-S promoter in both P4 and P14 hippocampal neurons, including acetylation of H3 and H4, as well as H3 phosphoacetylation and lysine 4 trimethylation, particularly upstream of the TSS was identified. No evidence for repressive H3K27 trimethylation. Adk-S promoter, promoter regulating short Adk isoform expression; ChIP, chromatin immunoprecipitation; H3K9ac, acetylation of lysine (K) 9 of histone H3; H4ac, pan-acetylation of histone H4; H3S10phK14ac, phosphoacetylation of histone H3 targeting Serine (S) 10 and Lysine (K) 14; H3K4me3, trimethylation of lysine (K) 4 of histone H3; H3K27me3, trimethylation of lysine (K) 27 of histone H3; Hist1H4B, Histone cluster 1, H4B; MyoD, Myogenic differentiation 1; TSS, transcriptional start site.
Figure 4
Figure 4
DNA promoter methylation. CpG methylation was analyzed using bisulfite sequencing following amplification and subcloning of the region of interest. A minimum of three clones was analyzed per sample. Promoter regions were amplified from pos. −588 to +298 (long isoform) and pos. −391 to +293 (short isoform) relative to TSS.
Figure 5
Figure 5
Identification of transcription factor binding. (A) Upper panel: SP1 binding sites (indicated in red) at the amplified region of Adk 1d primers (indicated in blue) around the TSS (pos. +1, bold). Lower panel: Binding of SP1 to the promoter region of the long Adk isoform are shown relative to a positive control primer in P4 and P14 rat hippocampal neurons. The analyzed promoter region spanned over 1 kb between position −735 and +470 relative to the TSS. There is significant enrichment of SP1 binding around the TSS of the Adk-L promoter in P14 rat hippocampal neurons, which may be implicated in the suppression of the long Adk isoform. Inset displays enrichment of positive control for Sp1 antibody presented as percent of the total input chromatin (% input).(B) Luciferase assay was performed to analyze the binding of SP1 to the Adk promoter and its impact on gene expression. Therefore, the Adk-L promoter including 1.000 bp upstream the TSS was subcloned in front of luciferase 2 (luc2) gene in the pGL4.10 vector and transfected into HEK293 cells. Transfected cells were incubated with increasing concentrations of recombinant SP1 for 3 h and then used for luciferase activity measurements.(C) We identified a concentration dependent negative regulation of the Adk-L promoter and luc2 gene through transcription factor SP1. Adk, Adenosine kinase; Adk-L promoter, promoter regulating expression of the long Adk isoform; Ap3m1, adaptor-related protein complex 3, mu 1 subunit; ChIP, chromatin immunoprecipitation; CpG, cytosine guanine dinucleotide; DHFR, Dihydrofolate reductase; kb, kilobase; SP1, specificity protein 1;TSS, transcriptional start site.*indicates significance.
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