Genome-wide RNA pol II initiation and pausing in neural progenitors of the rat.

BACKGROUND: Global RNA sequencing technologies have revealed widespread RNA polymerase II (Pol II) transcription outside of gene promoters. Small 5'-capped RNA sequencing (Start-seq) originally developed for the detection of promoter-proximal Pol II pausing has helped improve annotation of Transcription Start Sites (TSSs) of genes as well as identification of non-genic regulatory elements. However, apart from the most well studied genomes of human and mouse, mammalian transcription has not been profiled with sufficiently high precision. RESULTS: We prepared and sequenced Start-seq libraries from rat (Rattus norgevicus) primary neural progenitor cells. Over 48 million uniquely mappable reads from two independent biological replicates allowed us to define the TSSs of 7365 known genes in the rn6 genome, reannotating 2503 TSSs by more than 5 base pairs, characterize promoter-associated antisense transcription, and profile Pol II pausing. By combining TSS data with polyA-selected RNA sequencing, we also identified thousands of potential new genes producing stable RNA as well as non-genic transcripts representing possible regulatory elements. CONCLUSIONS: Our study has produced the first Start-seq dataset for the rat. Apart from profiling transcription initiation, our data reaffirm the prevalence of Pol II pausing across the rat genome and indicate conservation of pausing mechanisms across metazoan genomes. We suggest that pausing location, at least in mammals, is constrained by a distance from initiation of transcription, whether it occurs at or outside of a gene promoter. Abundant antisense transcription initiation around protein coding genes indicates that Pol II recruited to the vicinity of a promoter is distributed to available start sites of transcription at either DNA strand. Transcriptome profiling of neural progenitors presented here will facilitate further studies of other rat cell types as well as other organisms.

Persistent 6-OH-BDE-47 exposure impairs functional neuronal maturation and alters expression of neurodevelopmentally-relevant chromatin remodelers.

Polybrominated diphenyl ethers (PBDEs) are a pervasive class of brominated flame retardants that are present in the environment at particularly high levels, especially in the United States. Their environmental stability, propensity for bioaccumulation, and known potential for neurotoxicity has evoked interest regarding their effects on the developing nervous system. Exposure to PBDEs has been strongly associated with neurodevelopmental disorders. However, the details of their mechanistic roles in such disorders are incompletely understood. Here, we report the effects of one of the most prevalent congeners, BDE-47, and its hydroxylated metabolites on the maturation and function of embryonic rat cortical neurons. Prolonged exposure to 6OH-BDE-47 produces the strongest effects amongst the parent BDE-47 congener and its tested hydroxylated metabolites. These effects include: i) disruption of transcriptional responses to neuronal activity, ii) dysregulation of multiple genes associated with neurodevelopmental disorders, and intriguingly, iii) altered expression of several subunits of the developmentally-relevant BAF (Brg1-associated factors) chromatin remodeling complex, including the key subunit BAF170. Taken together, our data indicate that persistent exposure to 6OH-BDE-47 may interfere with neurodevelopmental chromatin remodeling mechanisms and gene transcription programs, which in turn are likely to interfere with downstream processes such as synapse development and overall functional maturity of neurons. Results from this study have identified a novel aspect of 6OH-BDE-47 toxicity and open new avenues to explore the effects of a ubiquitous environmental toxin on epigenetic regulation of neuronal maturation and function.

Different Neuronal Activity Patterns Induce Different Gene Expression Programs.

A vast number of different neuronal activity patterns could each induce a different set of activity-regulated genes. Mapping this coupling between activity pattern and gene induction would allow inference of a neuron's activity-pattern history from its gene expression and improve our understanding of activity-pattern-dependent synaptic plasticity. In genome-scale experiments comparing brief and sustained activity patterns, we reveal that activity-duration history can be inferred from gene expression profiles. Brief activity selectively induces a small subset of the activity-regulated gene program that corresponds to the first of three temporal waves of genes induced by sustained activity. Induction of these first-wave genes is mechanistically distinct from that of the later waves because it requires MAPK/ERK signaling but does not require de novo translation. Thus, the same mechanisms that establish the multi-wave temporal structure of gene induction also enable different gene sets to be induced by different activity durations.

Histone Hypervariants H2A.Z.1 and H2A.Z.2 Play Independent and Context-Specific Roles in Neuronal Activity-Induced Transcription of Arc/Arg3.1 and Other Immediate Early Genes.

The histone variant H2A.Z is an essential and conserved regulator of eukaryotic gene transcription. However, the exact role of this histone in the transcriptional process remains perplexing. In vertebrates, H2A.Z has two hypervariants, H2A.Z.1 and H2A.Z.2, that have almost identical sequences except for three amino acid residues. Due to such similarity, functional specificity of these hypervariants in neurobiological processes, if any, remain largely unknown. In this study with dissociated rat cortical neurons, we asked if H2A.Z hypervariants have distinct functions in regulating basal and activity-induced gene transcription. Hypervariant-specific RNAi and microarray analyses revealed that H2A.Z.1 and H2A.Z.2 regulate basal expression of largely nonoverlapping gene sets, including genes that code for several synaptic proteins. In response to neuronal activity, rapid transcription of our model gene Arc is impaired by depletion of H2A.Z.2, but not H2A.Z.1. This impairment is partially rescued by codepletion of the H2A.Z chaperone, ANP32E. In contrast, under a different context (after 48 h of tetrodotoxin, TTX), rapid transcription of Arc is impaired by depletion of either hypervariant. Such context-dependent roles of H2A.Z hypervariants, as revealed by our multiplexed gene expression assays, are also evident with several other immediate early genes, where regulatory roles of these hypervariants vary from gene to gene under different conditions. Together, our data suggest that H2A.Z hypervariants have context-specific roles that complement each other to mediate activity-induced neuronal gene transcription.