Plasmids (pLKO.1-puro) carrying pre-designed anti-NELF-A or anti-NELF-E short hairpin (designed by RNAi consortium or TRC) or scrambled hairpin sequences were obtained from Sigma. Self-inactivating HIV lentivirus particles were produced by transfecting 293T cells with the vector, envelope (pMD2.G; Addgene), and packaging plasmids (psPAX2; Addgene). For infection with recombinant lentiviruses, the viral supernatant was diluted in neuronal media and cells were infected at a multiplicity of infection ranging from 2 to 5. At seven days post infection, neurons were tested for NELF-A or NELF-E mRNA knockdown. Two anti-NELF-A hairpins (sh-nelfa: CCG GCC CAA TGT TCA AGA CAT CTT ACT CGA GTA AGA TGT CTT GAA CAT TGG GTT TTT G and sh-nelfa2: CCG GGA GGA ACA GAA CCC CAA TGT TCT CGA GAA CAT TGG GGT TCT GTT CCT CTT TTT G) and one anti-NELF-E hairpin (sh-nelfe: CCG GCT GGA TTC CTT GTG CCT CAT ACT CGA GTA TGA GGC ACA AGG AAT CCA GTT TTT G) were used in this study. For electrophysiological studies, NELF-A hairpin was subsequently cloned into a GFP containing lentiviral backbone (pLKO.3G; Addgene) using SphI and EcoRI restriction enzyme sites for co-expression of GFP along with the anti-NELF-A shRNA.
Cell culture and treatment
Cultures of cortical neurons were prepared from embryonic day 18 Sprague Dawley rats44
(Institution approval: ASP 01–21). Dissociated cortical neurons were plated in Neurobasal media (Invitrogen) supplemented with 25 mM glutamate (Sigma) and 0.5 mM L-Glutamine (Sigma) and either B27 (Invitrogen) or NS21 and maintained in a similar media without the glutamate. NS21 was prepared as previously described45
. Neurons were routinely used between 11–16 days in vitro
. In gene induction studies, cells were treated with 1–2
μM tetrodotoxin (TTX; Calbiochem) for 48 hours. To induce transcription, cells were washed twice with excess TTX-free warm media, and then incubated in fresh media at 34°C. The withdrawal time was recorded after the second wash.
Cultured neurons were fixed with 4% paraformaldehyde in PBS and permeabilized with pre-chilled absolute ethanol treatment for 5 minutes in −20°C. Fixed neurons were then immuno-stained with anti-Arc antibody (1:1000; Synaptic Systems) overnight at 4°C and then with anti-Rabbit Alexa 633 dye-conjugated secondary antibody (1:2500; Molecular Probes) at room temperature for 2 hours in blocking solutions.
Gene expression analysis using quantitative PCR (qPCR)
Total RNA was isolated from cortical cell cultures using the RNeasy kit with DNase treatment (Qiagen). To measure mature mRNA (qRT-PCR), cDNA was produced using MuLV reverse transcriptase with poly-dT and random hexamer primers. Gene expression was measured in quantitative real-time PCR (qPCR). To measure pre-mRNA (one-step qRT-PCR), primers that target the arc
introns (Supplementary Information
) served for cDNA synthesis and subsequent amplification (14 cycles) using the manufacturer’s protocol in the One-Step RT-PCR kit (Qiagen). The amplified product level was quantified by qPCR using the same primers against the arc
Gene expression analysis was conducted by the NIEHS Microarray Core using RNA obtained from three biological replicates. The Agilent Whole Rat Genome 4×44 multiplex format oligo arrays (014879, Agilent Technologies) were used following the Agilent 1-color microarray-based gene expression analysis protocol. Using 500ng of total RNA, Cy3 labeled cRNA was produced according to the manufacturer’s protocol. For each sample 1.65 μg of Cy3 labeled cRNAs were fragmented and hybridized for 17 hours in a rotating hybridization oven. Slides were washed and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v9.5), using the 1-color defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (version 7.2; Rosetta Biosoftware, Kirkland, WA).
Chromatin Immunoprecipitation (ChIP)
Cells were cross-linked with formaldehyde after treatment. In TTX samples, cells were washed with 2 μM TTX in PBS. Cross-linked cell lysates were sonicated for fifteen cycles (bursts and intervals of 30 seconds each) with the Bioruptor (Diagenode). Sonicated samples were pre-cleared for 1.5 hours with 50% A/G bead slurry (SantaCruz Biotechnology), then immunoprecipitated overnight at 4 degrees with 5 μg anti-Rpb1 (8WG16; Abcam), anti-Rpb1 pSer5 (Abcam), anti-H3 (Abcam), anti-tri methyl H3K4 (Millipore), anti-acetylated H3K9 (Millipore), anti-NELF-A or anti-NELF-E (Sigma) antibodies. The anti-NELF-A (rat) antibody was custom made (Yenzyme) with a twenty amino acid synthetic peptide (QIKLSEHTEDLPKADGQGS-amide) corresponding to 481–499 amino acids of rat NELF-A (WHSC2) as the immunogen. Immunoprecipitation with H5 (anti-Rpb1 pS2-CTD IgM; Covance) antibody was carried out overnight at 4°C and then anti-mouse IgM was added for an additional day. Antigen-antibody complexes were immunoprecipitated with 50% A/G bead slurry, washed once with low salt buffer, three times with high salt buffer for most IPs, once with LiCl buffer and finally once with Tris-EDTA. As a deviation, NELF-A imunoprecipitation was done in 200mM NaCl and two and four high salt washes were performed for the H5 and NELF-A immunocomplexes respectively. Samples were reverse cross-linked and chromatin DNA was eluted using the QIAquick Nucleotide Removal Kit (Qiagen). Eluted chromatin was quantified by qPCR. Data for ChIP with H5 was obtained by subtracting anti-mouse IgM ChIP values from the corresponding H5 values. For ChIP with brain samples, isolated minced cortices (with hippocampus) were passed through 19 and 22 gauge needles in ice-cold PBS containing 1% Formaldehyde. These cross-linked samples were then processed as above.
Chromatin Immunoprecipitation-sequencing (ChIP-seq)
ChIP was performed with Pol II M01 antibody and the immunoprecipitated DNA fragments were used to prepare ChIP-Seq libraries. These libraries were constructed using the standard protocol from Illumina with NEBNext DNA Sample Prep Reagent Set 1 and 2 (New England Biolabs; E600L; E6020L) and single-end adapters from Illumina (Genomic DNA Sample Prep Oligo Only Kit; FC-102-1003) with two modifications to the protocol. Platinum® Pfx DNA Polymerase (Invitrogen; 11708-039) was used for library amplification and Agencourt AmPure Reagent (Beckman Coulter Genomics; A29152) was used to clean up the amplification reaction. Libraries were quantitated by qPCR (Power SYBR® Green PCR Master Mix; 4368702; Life Technologies, Inc.) with following primers:
Flow cells were prepared with Illumina’s Standard Cluster Generation Kit V 2 (FC-103-2001) on an Illumina Cluster Station and run on a Genome AnalyzerIIx within one week using 36-Cycle Sequencing Kits V4 at National Intramural Sequencing Center (NISC), NIH.
Short Read alignment and data analysis
Reads provided by NISC were aligned with Bowtie 0.12.346
to the R. norvegicus
rn4 index. Biological replicates were in good agreement and were combined, resulting in a data set of 21,229,004 unique hits. ChIP-seq hit locations were centered by moving the forward and reverse strand hit location 75 nt in the appropriate direction because the average fragment size was ~150bp and placed in 25 nt bins for visualization using the UCSC genome browser. R. norvegicus
Ensembl Genes 58 data were used to define TSS and gene bodies. Promoter proximal regions were defined as the physical genomic region +/− 200 nt from the annotated TSS. Gene bodies were defined as the physical region from +501 to the end of the gene. To define the significant number of reads in bound promoter proximal regions, we used the Fisher’s exact test implemented in R. We compared 28,117 promoter proximal regions to 11,426 size matched intergenic regions that are at least 5,000 nt from the start or end of an annotated gene. The p
-values from the Fisher’s exact tests were corrected using the Benjamini-Hochberg method to control for false discovery, and a corrected P
-value < 0.01 was deemed significant. Promoter proximal stalling index
was defined as the ratio of the number of reads in a promoter proximal region to a gene body normalized by the search size. Genes with gene bodies less than 401 nt were excluded from Stalling index analyses.
arc intron-enriched riboprobes and fluorescent in situ hybridization (FISH)
The plasmid containing the arc
gene sequence spanning both the introns (and the exon in between) has been previously described8
and was a kind gift from J. Guzowski. Riboprobes were generated from linearized plasmids by in vitro
transcription using fluorescein-labeled dUTPs. FISH was subsequently performed with this riboprobe as detailed previously by Guzowski et al.
(2006). Briefly, fixed neurons were hybridized with the riboprobe overnight at 56°C and FISH signals were then detected with anti-Fluorescein–HRP conjugate (Jackson) and amplified using cyanine-3 tyramide signal amplification kit (PerkinElmer).
Whole-cell patch-clamp recordings were performed on dissociated cortical neurons co-expressing GFP and shRNA. Neurons were perfused at 1 ml/min at room temperature in ACSF (in mM): 124 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, 17 D-glucose, 0.5 Picrotoxin and 0.001 TTX. Patch electrodes (3–7 MΩ) were filled with (in mM): 120 K-gluconate, 10 KCl, 3 MgCl2, 0.5 EGTA, 40 HEPES, 2Na2-ATP, 0.3 Na-GTP, with pH adjusted to 7.2 by NaOH. Cells were voltage clamped at −60 mV and mEPSCs were recorded over 3 minute periods. mEPSCs were detected and analyzed using the Mini Analysis program (Version 6.0.3, Axon Instruments). For multi-electrode arrays, cortical neurons were prepared and plated onto a square grid of 64 planar electrodes (MED64; MED Sciences, Japan). Recordings were acquired with an 64-channel integrated amplifier and MED64 Conductor software (Sampling rate 20kHz, LCF 100Hz). Spontaneous activity was recorded at 34°C both at the end of the TTX treatment and immediately following washout of TTX, achieved by washing 2 times.
Co-immunoprecipitation and Western blotting
For Co-IPs, nuclear material was extracted using the Nuclear Complex Co-IP Kit (Active Motif). Extracted material was pre-cleared for one hour with A/G beads (Santa Cruz Biotechnology) and then incubated with primary antibody overnight. As negative control, normal IgG was used that showed negligible signal. The following day, A/G beads were added to the IPs for one hour. The beads were washed with IP wash buffer prepared from the CoIP Kit and were prepared for Western blotting by adding LDS Sample Buffer (NuPAGE) and boiling. To extract nuclear proteins, neurons were washed with PBS + 5 mM MgCl2 and then lysed with plasma membrane lysis buffer (10mM HEPES pH 7.5, 60 mM KCl, 1.5 mM MgCl2, 0.1% NP40, and 0.5 mM DTT). Nuclei were collected by centrifugation, resuspended in nuclear extraction buffer (20mM HEPES pH 7.5, 100 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.1 mM EGTA, and 20% glycerol) and disrupted by brief sonication. Proteins were resolved by gel electrophoresis and transferred to a nitrocellulose membrane using the iBlot gel transfer apparatus (Invitrogen). Immunoblots were incubated with primary antibody overnight. Blots were visualized with an Odyssey infrared scanner (Li-COR Biosciences) after immunolabeling primary antibodies with infrared fluorophore-tagged secondary antibody (Molecular Probes). Images were analyzed using the Odyssey 2.1 software.
Novel environment studies
Sixteen to twenty one day-old rat pups were handled daily for several days prior to experimentation (Institution approval: ASP 2009–0023). The novel environment was a square plexiglass platform with high walls on a temperature regulated bed. Toys were included in this environment along with cotton swabs dipped in artificial vanilla, lemon and almond extracts. The control rat was decapitated immediately upon removal from the cage in the adjacent procedure room whereas the experimental rat was allowed to explore the environment and was decapitated immediately at the designated time. The brain was split into two hemispheres and one was frozen immediately in methyl-butane pre-chilled in a dry ice and methanol slurry for FISH. The other hemisphere was utilized to obtain tissue samples for PCR. Samples and sections were taken from the primary somatosensory area of the cortex. Similar findings have been observed in the hippocampus8
ChIP data are reported as means and S.Ds. All remaining data are expressed as mean and S.E.Ms. Statistical comparison of datasets were performed with two-tailed Student’s t test with Bonferroni corrections (for multiple comparisons of ChIP data), Kruskal-Wallis non-parametric test (comparison of Pol II Stalling indices) and two way ANOVA (for multiple comparisons in NELF-A knockdown studies). Statistics were performed with Sigmastat 3.5 software.