Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach produces high-quality binding profiles using 1,000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.
The emerging genome-wide hairpin bisulfite sequencing (hairpin-BS-Seq) technique enables the determination of the methylation pattern for DNA double strands simultaneously. Compared with traditional bisulfite sequencing (BS-Seq) techniques, hairpin-BS-Seq can determine methylation fidelity and increase mapping efficiency. However, no computational tool has been designed for the analysis of hairpin-BS-Seq data yet. Here we present HBS-tools, a set of command line based tools for the preprocessing, mapping, methylation calling, and summarizing of genome-wide hairpin-BS-Seq data. It accepts paired-end hairpin-BS-Seq reads to recover the original (pre-bisulfite-converted) sequences using global alignment and then calls the methylation statuses for cytosines on both DNA strands after mapping the original sequences to the reference genome. After applying to hairpin-BS-Seq datasets, we found that HBS-tools have a reduced mapping time and improved mapping efficiency compared with state-of-the-art mapping tools. The HBS-tools source scripts, along with user guide and testing data, are freely available for download.
Genes that regulate hormone release are essential for maintaining metabolism and energy balance. Egr1 encodes a transcription factor that regulates hormone production and release, and a decreased in growth hormones has been reported in Egr1 knockout mice. A reduction in growth hormones has also been observed in Nestin-Cre mice, a model frequently used to study the nervous system. Currently, it is unknown how Egr1 loss or the Nestin-Cre driver disrupt pituitary gene expression. Here, we compared the growth curves and pituitary gene expression profiles of Nestin-Cre-mediated Egr1 conditional knockout (Egr1cKO) mice with those of their controls. Reduced body weight was observed in both the Nestin-Cre and Egr1cKO mice, and the loss of Egr1 had a slightly more severe impact on female mice than on male mice. RNA-seq data analyses revealed that the sex-related differences were amplified in the Nestin-Cre-mediated Egr1 conditional knockout mice. Additionally, in the male mice, the influence of Egr1cKO on pituitary gene expression may be overridden by the Nestin-Cre driver. Differentially expressed genes associated with the Nestin-Cre driver were significantly enriched for genes related to growth factor activity and binding. Altogether, our results demonstrate that Nestin-Cre and the loss of Egr1 in the neuronal cell lineage have distinct impacts on pituitary gene expression in a sex-specific manner.
The peripheral immune system is a major regulator of the pathophysiology associated with traumatic brain injury (TBI). While age-at-injury influences recovery from TBI, the differential effects on the peripheral immune response remain unknown. Here, we investigated the effects of TBI on gene expression changes in murine whole blood using RNAseq analysis, gene ontology and network topology-based key driver analysis. Genome-wide comparison of CCI-injured peripheral whole blood showed a significant increase in genes involved in proteolysis and oxidative-reduction processes in juvenile compared to adult. Conversely, a greater number of genes, involved in migration, cytokine-mediated signaling and adhesion, were found reduced in CCI-injured juvenile compared to CCI-injured adult immune cells. Key driver analysis also identified G-protein coupled and novel pattern recognition receptor (PRR), P2RY10, as a central regulator of these genes. Lastly, we found Dectin-1, a c-type lectin PRR to be reduced at the protein level in both naïve neutrophils and on infiltrating immune cells in the CCI-injured juvenile cortex. These findings demonstrate a distinct peripheral inflammatory profile in juvenile mice, which may impact the injury and repair response to brain trauma.
(Molecular Cell 62, 848–861; June 16, 2016) In the original version of the above article, a recent publication and its findings had not been acknowledged. The online and print versions have now been corrected, and the corrected sentence reads “… but the importance of loss of H3K9me2 is not clear (Walter et al., 2016).” The full citation to the reference has been added to the reference list: Walter, M., Teissandier, A., Pérez-Palacios, R., Bourc’his, D. (2016). An epigenetic switch ensures transposon repression upon dynamic loss of DNA methylation in embryonic stem cells. Elife 5. http://dx.doi.org/10.7554/eLife.11418 Impairment of DNA Methylation Maintenance Is the Main Cause of Global Demethylation in Naive Embryonic Stem Cellsvon Meyenn et al.Molecular CellMay 26, 2016In BriefGlobal demethylation during epigenetic reprogramming in preimplantation embryos, primordial germ cells, and naive embryonic stem cells is a consequence of impaired DNA methylation maintenance and synergistically regulated by a decrease in UHRF1 and H3K9me2 levels, while locus specific methylation maintenance is guided by specific recruitment of UHRF1 to H3K9me2/3. TET enzymes are dispensable for global DNA methylation reprogramming. Full-Text PDF Open Access
Global DNA methylation changes have been documented to occur during normal development, aging and disease progression. Genome-wide levels of methylation are commonly inferred from the average level of methylation of large sets of repetitive elements. Alu repeats, in particular, have been exploited in a variety of approaches to monitor genome-wide losses in CpG methylation. Alu elements encompass over 25% of the CpG dinucleotides in the human genome. However, due to the high frequency of deamination of methylated cytosines in repetitive elements occurring in the germline, the sensitivity to detect methylation changes based upon their methylation levels is greatly compromised. In addition, the number of repeats that were targeted in previous investigations for ascertainment of genome-wide levels of methylation could not be accurately estimated. In this study, we developed and implemented an algorithm to design highly specific primers altogether targeting 617,817 CpG sites in over 45,000 Alu elements. In combination with the pyrosequencing technology, these primers were shown to yield highly sensitive and reproducible results.
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