Genome-wide identification and characterization of functional neuronal activity-dependent enhancers.
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ABSTRACT:
SUBMITTER: Malik AN
PROVIDER: S-EPMC4297619 | biostudies-literature | 2014 Oct
REPOSITORIES: biostudies-literature
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Project description:Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in neurons. While ~12,000 putative activity-regulated enhancer sequences have previously been identified that are enriched for H3K4me1 and the histone acetyltransferase CBP, we find that this chromatin signature is not sufficient to distinguish which of these regulatory sequences are actively engaged in promoting activity-dependent transcription. We show here that a subset of H3K4me1/CBP positive enhancers that is enriched for H3K27 acetylation (H3K27ac) in vivo, and shows increased H3K27ac upon membrane depolarization of cortical neurons, function to regulate activity-dependent transcription. The function of many of these activity-regulated enhancers appears to be dependent on the binding of FOS, a protein that had previously been thought to interact primarily with the promoters of activity-regulated genes. Furthermore, many of these target genes in cortical neurons encode neuron specific proteins that regulate synaptic development and function. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function, and provide a resource of activity-dependent enhancers that may give insight into genetic variation that contributes to brain development and disease. Genome-wide maps of H3K27ac and AP1 transcription factors (CFOS, FOSB, JUNB) before and after neuronal activity in mouse cortical neurons.
2014-09-07 | E-GEOD-60192 | biostudies-arrayexpress
Project description:Experience-dependent gene transcription is required for nervous system development and function. However, the DNA regulatory elements that control this program of gene expression are not well defined. Here we characterize the enhancers that function across the genome to mediate activity-dependent transcription in neurons. While ~12,000 putative activity-regulated enhancer sequences have previously been identified that are enriched for H3K4me1 and the histone acetyltransferase CBP, we find that this chromatin signature is not sufficient to distinguish which of these regulatory sequences are actively engaged in promoting activity-dependent transcription. We show here that a subset of H3K4me1/CBP positive enhancers that is enriched for H3K27 acetylation (H3K27ac) in vivo, and shows increased H3K27ac upon membrane depolarization of cortical neurons, function to regulate activity-dependent transcription. The function of many of these activity-regulated enhancers appears to be dependent on the binding of FOS, a protein that had previously been thought to interact primarily with the promoters of activity-regulated genes. Furthermore, many of these target genes in cortical neurons encode neuron specific proteins that regulate synaptic development and function. These findings suggest that FOS functions at enhancers to control activity-dependent gene programs that are critical for nervous system function, and provide a resource of activity-dependent enhancers that may give insight into genetic variation that contributes to brain development and disease.
2014-09-07 | GSE60192 | GEO
Project description:BackgroundThe pig is an economically important livestock species and is a widely applied large animal model in medical research. Enhancers are critical regulatory elements that have fundamental functions in evolution, development and disease. Genome-wide quantification of functional enhancers in the pig is needed.ResultsWe performed self-transcribing active regulatory region sequencing (STARR-seq) in the porcine kidney epithelial PK15 and testicular ST cell lines, and reliably identified 2576 functional enhancers. Most of these enhancers were located in repetitive sequences and were enriched within silent and lowly expressed genes. Enhancers poorly overlapped with chromatin accessibility regions and were highly enriched in chromatin with the repressive histone modification H3K9me3, which is different from predicted pig enhancers detected using ChIP-seq for H3K27ac or/and H3K4me1 modified histones. This suggests that most pig enhancers identified with STARR-seq are endogenously repressed at the chromatin level and may function during cell type-specific development or at specific developmental stages. Additionally, the PPP3CA gene is associated with the loin muscle area trait and the QKI gene is associated with alkaline phosphatase activity that may be regulated by distal functional enhancers.ConclusionsIn summary, we generated the first functional enhancer map in PK15 and ST cells for the pig genome and highlight its potential roles in pig breeding.
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Project description:Enhancers are short non-coding DNA sequences outside of the target promoter regions that can be bound by specific proteins to increase a gene's transcriptional activity, which has a crucial role in the spatiotemporal and quantitative regulation of gene expression. However, enhancers do not have a specific sequence motifs or structures, and their scattered distribution in the genome makes the identification of enhancers from human cell lines particularly challenging. Here we present a novel, stacked multivariate fusion framework called SMFM, which enables a comprehensive identification and analysis of enhancers from regulatory DNA sequences as well as their interpretation. Specifically, to characterize the hierarchical relationships of enhancer sequences, multi-source biological information and dynamic semantic information are fused to represent regulatory DNA enhancer sequences. Then, we implement a deep learning-based sequence network to learn the feature representation of the enhancer sequences comprehensively and to extract the implicit relationships in the dynamic semantic information. Ultimately, an ensemble machine learning classifier is trained based on the refined multi-source features and dynamic implicit relations obtained from the deep learning-based sequence network. Benchmarking experiments demonstrated that SMFM significantly outperforms other existing methods using several evaluation metrics. In addition, an independent test set was used to validate the generalization performance of SMFM by comparing it to other state-of-the-art enhancer identification methods. Moreover, we performed motif analysis based on the contribution scores of different bases of enhancer sequences to the final identification results. Besides, we conducted interpretability analysis of the identified enhancer sequences based on attention weights of EnhancerBERT, a fine-tuned BERT model that provides new insights into exploring the gene semantic information likely to underlie the discovered enhancers in an interpretable manner. Finally, in a human placenta study with 4,562 active distal gene regulatory enhancers, SMFM successfully exposed tissue-related placental development and the differential mechanism, demonstrating the generalizability and stability of our proposed framework.
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Project description:Activity-regulated genes (ARGs) are important for neuronal functions like long-term memory and are well-characterized in mammals but poorly studied in other model organisms like Drosophila. Here we stimulated fly neurons with different paradigms and identified ARGs using high-throughput sequencing from brains as well as from sorted neurons: they included a narrow set of circadian neurons as well as dopaminergic neurons. Surprisingly, many ARGs are specific to the stimulation paradigm and very specific to neuron type. In addition and unlike mammalian immediate early genes (IEGs), fly ARGs do not have short gene lengths and are less enriched for transcription factor function. Chromatin assays using ATAC-sequencing show that the transcription start sites (TSS) of ARGs do not change with neural firing but are already accessible prior to stimulation. Lastly based on binding site enrichment in ARGs, we identified transcription factor mediators of firing and created neuronal activity reporters.
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Project description:Recent work has shown that RNA polymerase II-mediated transcription at distal cis-regulatory elements serves as a mark of highly active enhancers. Production of noncoding RNAs at enhancers, termed eRNAs, correlates with higher expression of genes that the enhancer interacts with; hence, eRNAs provide a new tool to model gene activity in normal and disease tissues. Moreover, this unique class of noncoding RNA has diverse roles in transcriptional regulation. Transcribed enhancers can be identified by a common signature of epigenetic marks by overlaying a series of genome-wide chromatin immunoprecipitation and RNA sequencing datasets. A computational approach to filter non-enhancer elements and other classes of noncoding RNAs is essential to not cloud downstream analysis. Here we present a protocol that combines wet and dry bench methods to accurately identify transcribed enhancers genome-wide as well as an experimental procedure to validate these datasets.
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Project description:Whole-genome sequencing is identifying growing numbers of non-coding variants in human disease studies, but the lack of accurate functional annotations prevents their interpretation. We describe the genome-wide landscape of distant-acting enhancers active in the developing and adult human heart, an organ whose impairment is a predominant cause of mortality and morbidity. Using integrative analysis of >35 epigenomic data sets from mouse and human pre- and postnatal hearts we created a comprehensive reference of >80,000 putative human heart enhancers. To illustrate the importance of enhancers in the regulation of genes involved in heart disease, we deleted the mouse orthologs of two human enhancers near cardiac myosin genes. In both cases, we observe in vivo expression changes and cardiac phenotypes consistent with human heart disease. Our study provides a comprehensive catalogue of human heart enhancers for use in clinical whole-genome sequencing studies and highlights the importance of enhancers for cardiac function.
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Project description:We investigated sequence features of enhancers separated from their target gene by at least one intermediate gene/exon (named tele-enhancers in this study) and enhancers residing inside their target gene locus. In this study, we used whole genome enhancer maps and gene expression profiles to establish a large panel of tele-enhancers. By contrasting tele-enhancers to proximal enhancers targeting heart genes, we observed that heart tele-enhancers use unique regulatory mechanisms based on the cardiac transcription factors SRF, TEAD, and NKX-2.5, whereas proximal heart enhancers rely on GATA4 instead. A functional analysis showed that tele-enhancers preferentially regulate house-keeping genes and genes with a metabolic role during heart development. In addition, tele-enhancers are significantly more conserved than their proximal counterparts. Similar trends have been observed for non-heart tissues and cell types, suggesting that our findings represent general characteristics of tele-enhancers.
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Project description:The limb is widely used as a model developmental system and changes to gene expression patterns in its signaling centers, notably the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER), are known to cause limb malformations and evolutionary differences in limb morphology. Although several genes that define these limb signaling centers have been described, the identification of regulatory elements that are active within these centers has been limited. By dissecting mouse E11.5 limbs that fluorescently mark the ZPA or AER, followed by fluorescence-activated cell sorting and low-cell H3K27ac ChIP-seq, we identified thousands of specific signaling-center enhancers. Our ChIP-seq datasets show strong correlation with ZPA- and AER-expressed genes, previously characterized functional ZPA and AER enhancers and enrichment for relevant biological terms related to limb development and malformation for the neighboring genes. Using transgenic assays, we show that several of these sequences function as ZPA and AER enhancers. Our results identify novel ZPA and AER enhancers that could be important regulators of genes involved in the establishment of these specialized regions and the patterning of tetrapod limbs.
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