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Genome-wide maps of CREB/CRTC binding in Drosophila mushroom body.

ABSTRACT: Purpose: Exploring the alteration in CREB/CRTC binding related to long-term memory Methods: The nuclei of Drosophila mushroom bodies were purified and the histone acetylation and CREB/CRTC binding were examined by ChIP-seq using specific antibodies. The sequencing was performed using Miseq, and aligned to dm3 using CLCbio. The sequence reads that passed quality filters were analyzed by peak calling using PICS and ERD equipped on Strand NGS software. This method using two algorisms excludes false-positive calling of peaks. The PICS and ERD were run using a default setting except for the following parameters; for PICS, 120 bp as an average fragment length, 10 bp as a minimum distance between forward and reverse reads, 200 bp as a minimum distance between forward and reverse reads, 100 bp as a window width, with 5% false discovery rate; for ERD, 1.5 as an enrichment factor, 100 bp as a window size, 10 bp as a window slide size, 100 bp as a minimum region size. The peaks obtained by ERD analysis were filtered by an enrichment factor of 2, and a density of reads at 0.12 for CREB and 0.2 for CRTC. The CREB and the CRTC binding sites were determined as the peak-called region in at least 2 samples out of the three replicates. The CREB and the CRTC binding sites located 200 bp vicinity to each other were defined as the CREB/CRTC binding sites. In parallel, the read counts were obtained in the 1 kb window covering entire genome and analyzed by DESeq2, to determine the region enriched with CREB/CRTC in a specific group of samples. The region with increased or decreased CREB/CRTC binding were determined if the region were defined by the CREB/CRTC binding sites in the above criteria. Results: Using an optimized data analysis workflow, the filtered reads amounted to 8-11million reads for CREB in each of 3 replicates, 8-17 million reads for CRTC in each of 3 replicates, and 4.4 million reads for input. Using anti-CREB antibody, we found 239 significant increases in CREB binding out of 4995 CREB/CRTC binding sites 1 day after training. Using anti-CRTC antibody, we found 4989 significant increases in CRTC binding out of 4995 CREB/CRTC binding sites 1 day after spaced training. Conclusions: Our study shows that, although CREB binding is mostly unchanged, CRTC binding is significantly increased after long-term memory formation in Drosophila mushroom bodies.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE81456 | GEO | 2017/02/09

SECONDARY ACCESSION(S): PRJNA321666

REPOSITORIES: GEO

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Project description:Purpose: Exploring the alteration in histone acetylation colocalized with CREB/CRTC binding, which are important for long-term memory maintenance Methods: The nuclei of Drosophila mushroom bodies were purified and the histone acetylation and CREB/CRTC binding were examined by ChIP-seq using specific antibodies. The sequencing was performed using SOLiD, and aligned to dm3 using Lifescope..The sequence reads that passed quality filters were analyzed by peak calling using PICS and ERD equipped on Strand NGS software. This method using two algorisms excludes false-positive calling of peaks. The PICS and ERD were run using a default setting except for the following parameters; for PICS, 120 bp as an average fragment length, 10 bp as a minimum distance between forward and reverse reads, 200 bp as a minimum distance between forward and reverse reads, 100 bp as a window width, with 5% false discovery rate; for ERD, 2 as an enrichment factor, 100 bp as a window size, 10 bp as a window slide size, 100 bp as a minimum region size. The peaks obtained by ERD analysis were filtered by an enrichment factor of 1.5, and a density of reads at 0.3 for H3K9Ac, 0.15 for H4K16Ac, 0.18 for CREB and 0.15 for CRTC. To determine the histone acetylation-increased sites, the peaks determined from 3 replicates of the trained samples were summed to analyze all peaks. Then the centers of the H3K9Ac and H4K16Ac peaks were extracted, and then used to create neighboring 400 bp windows. The mapped reads were counted in these 400 bp windows for each biological replicate of ChIP-seq analysis. The counted read numbers in individual regions were normalized by elav for H3K9Ac ChIP-seq, and gapdh2 for H4K16Ac ChIP. The normalized read numbers in individual windows obtained from the spaced trained flies and the naïve flies were analyzed by Student’s t test (p < 0.05). Standard deviations of the normalized read numbers in individual windows from 3 replicates of naïve flies were 0.07 in H3K9Ac-ChIP-seq and 0.10 in H4K16Ac-ChIP-seq. Significantly increased peaks showing a more than 1.2-fold increase (>2SD) were determined as sites with an increase in H3K9Ac or H4K16Ac. Results: Using an optimized data analysis workflow, the filtered reads amounted to 12-15 million reads for H3K9Ac in each of 3 replicates, 5-7 million reads for H4K16 in each of 3 replicates, 2.4 million reads for CREB, 2 million reads for CRTC, and 2.9 million reads for input. Using anti-H3K9Ac antibody, we found significant increases in H3K9Ac 1 day after training, and 75.0% of these mapped within 500bps of the transcriptional start sites (TSSs) of 1766 genes. Using anti-H4K16Ac antibody, we found significant increases in H4K16Ac 1 day after spaced training, and 61.6% of these mapped within 500bps of the TSSs of 1320 genes. Epigenetic changes in the vicinity of TSSs suggests that expression of these genes may be increased in LTM maintenance. We also examined CRTC and CREB binding 1 day after training. We identified 2390 CRTC binding sites, of which 79.6% of these were close to CREB binding sites. These CREB/CRTC binding sites mapped to 1394 genes, and were predominately located within 500bp from TSSs (55.4%). Of the 1394 CREB/CRTC target genes, 346 genes showed increases in H3K9Ac, 319 showed increases in H4K16Ac, and 135 showed increases in both. Conclusions: Our study represents the first detailed analysis of chromatin state related to memory in Drosophila mushroom bodied. The optimized data analysis workflows reported here should provide a framework for comparative investigations of tissue-specific epigenetic alterations.

Project description:Low energy states delay aging in multiple species, yet mechanisms coordinating energetics and longevity across tissues remain poorly defined. The conserved energy sensor AMP-activated protein kinase (AMPK) and its corresponding phosphatase calcineurin modulate longevity via the ‘CREB regulated transcriptional coactivator (CRTC)-1 in C. elegans. We show that CRTC-1 specifically uncouples AMPK/calcineurin mediated effects on lifespan from pleiotropic side effects by reprogramming mitochondrial and metabolic function. Strikingly, this pro-longevity metabolic state is regulated cell-nonautonomously by CRTC-1 in the nervous system. CRTC-1/CREB act antagonistically with the functional PPARα ortholog, NHR-49 to promote distinct peripheral metabolic programs. Neuronal CRTC-1 drives mitochondrial fragmentation in distal tissues and suppresses the effect of AMPK on systemic mitochondrial metabolism and longevity via a cell-nonautonomous catecholamine signal. These results demonstrate that transcriptional control of neuronal signals can override enzymatic regulation of metabolism in peripheral tissues. Central perception of energetic state therefore represents a target to promote healthy aging. Experiment was performed with three biological replicates. Gravid adults grown at 20¡C on 100 mm NG plates seeded with OP50-1 E. coli were collected and treated with hypochlorite to release eggs. Eggs were incubated overnight in M9 media to obtain L1 synchronized populations. One thousand L1 larvae were grown on a 100 mm NG plate seeded with OP50-1 E. coli. Worms were harvested for RNA extraction when L4 larval stage was reached. Animals were collected and washed extensively with M9 media to remove bacteria. Worms were then snap frozen in liquid nitrogen. RNA was extracted by five freeze/thaw cycles in Qiazol then purified by RNeasy mini kit (Qiagen). RNA quality was checked using an Agilent Technologies 2100 Bioanalyzer. All samples had an RNA integrity number of 10. cDNA libraries were prepared from 4 ugs of total RNA using the TruSeq RNA Sample Preparation v2 kit (Illumina). 50-cycle paired-end sequencing was performed on an Illumina HiSeq 2000 by the Harvard Biopolymer Core. Read quality was evaluated with FASTQC. Adapter sequences and poor quality bases (<20) were trimmed and filtered with CUTADAPT, resulting in a median of 44 million reads per replicate. These were aligned to the C. elegans genome (ce6, WS238) using TopHat version 2.0.8 (Kim et al., 2013), with a median 35 million reads mapped in proper pairs. The number of reads mapping to each gene was counted with htseq-count. Genes with less than 1 Count Per Million Reads (CPM) were discarded from further analysis. Counts were normalized for sequencing depth and RNA composition across all samples with edgeR (Robinson et al., 2010). Genes were tested for differential expression between each mutant strain and wild-type using edgeR’s glm method. For each comparison, genes with less than 5 CPM were filtered and those with at least 50% change and False Discovery Rate (FDR) of 1% or less were considered differentially expressed.

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Genome-wide maps of CREB/CRTC binding in Drosophila mushroom body.

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