Cooperative, synergistic gene regulation by nuclear hormone receptors can increase sensitivity and amplify cellular responses to hormones. We investigated thyroid hormone (TH) and glucocorticoid (GC) synergy on the Krüppel-like factor 9 (Klf9) gene, which codes for a zinc finger transcription factor involved in development and homeostasis of diverse tissues. We identified regions of the Xenopus and mouse Klf9 genes 5-6 kb upstream of the transcription start sites that supported synergistic transactivation by TH plus GC. Within these regions, we found an orthologous sequence of approximately 180 bp that is highly conserved among tetrapods, but absent in other chordates, and possesses chromatin marks characteristic of an enhancer element. The Xenopus and mouse approximately 180-bp DNA element conferred synergistic transactivation by hormones in transient transfection assays, so we designate this the Klf9 synergy module (KSM). We identified binding sites within the mouse KSM for TH receptor, GC receptor, and nuclear factor κB. TH strongly increased recruitment of liganded GC receptor and serine 5 phosphorylated (initiating) RNA polymerase II to chromatin at the KSM, suggesting a mechanism for transcriptional synergy. The KSM is transcribed to generate long noncoding RNAs, which are also synergistically induced by combined hormone treatment, and the KSM interacts with the Klf9 promoter and a far upstream region through chromosomal looping. Our findings support that the KSM plays a central role in hormone regulation of vertebrate Klf9 genes, it evolved in the tetrapod lineage, and has been maintained by strong stabilizing selection.

A central feature of models of associative memory formation is the reliance on information convergence from pathways responsive to the conditioned stimulus (CS) and unconditioned stimulus (US). In particular, cells receiving coincident input are held to be critical for subsequent plasticity. Yet identification of neurons in the mammalian brain that respond to such coincident inputs during a learning event remains elusive. Here we use Arc cellular compartmental analysis of temporal gene transcription by fluorescence in situ hybridization (catFISH) to locate populations of neurons in the mammalian brain that respond to both the CS and US during training in a one-trial learning task, conditioned taste aversion (CTA). Individual neurons in the basolateral nucleus of the amygdala (BLA) responded to both the CS taste and US drug during conditioning. Coincident activation was not evident, however, when stimulus exposure was altered so as to be ineffective in promoting learning (backward conditioning, latent inhibition). Together, these data provide clear visualization of neurons in the mammalian brain receiving convergent information about the CS and US during acquisition of a learned association.Arc ͉ memory ͉ novelty ͉ plasticity ͉ taste aversion conditioning A central issue in behavioral neuroscience is how alterations in neural activity mediate the durable behavioral changes involved in learning. Current concepts of associative memory formation are based on the premise that plasticity relies on convergence of information from pathways responsive to the conditioned stimulus (CS) and the unconditioned stimulus (US) (1-3). Yet despite impressive progress in defining underlying neuronal circuitry and probable sites of association for several associative learning models (2, 4-6), identification of neuronal populations where convergent activation actually occurs during learning remains elusive. Electrophysiological studies have made inroads toward this goal, but are hampered by limited sampling ability, especially when the targets are convergent neurons, which are likely to be sparsely distributed during any single training trial. In those studies where convergent activation was recorded, animals were either anesthetized or had already reached asymptotic performance on a learning task (5-7).The imaging method known as cellular compartmental analysis of temporal gene transcription by fluorescence in situ hybridization (catFISH) can circumvent several of the technical limitations that have made it difficult to sample broad regions of the mammalian brain with high cellular and temporal resolution during a learning event (8). In particular, catFISH serves as a functional imager that allows investigators to distinguish neuronal populations activated by two distinct behavioral experiences. CatFISH utilizes Arc (or Arg 3.1), an immediate early gene (IEG) that is expressed in forebrain glutamatergic neurons after periods of enhanced activation (9, 10). The innovative features of catFISH rely on the time course of Arc mRNA locali...

Highlights d ataqv is a software package for ATAC-seq quality control (QC) and visualization d We show extensive variation in QC metrics for 2,009 public ATAC-seq datasets d Increased Tn5 dosage increases power to detect almost all regulatory genomic features d CTCF is a notable Tn5 dosage-insensitive factor

High-throughput reporter assays, such as self-transcribing active regulatory region sequencing (STARR-seq), allow for unbiased and quantitative assessment of enhancers at the genome-wide level. In order to cover the size of the human genome, recent advancements of STARR-seq technology have employed more complex genomic library and increased sequencing depths.These advances necessitate a reliable processing pipeline and peak-calling algorithm. Most studies of STARR-seq have relied on chromatin immunoprecipitation sequencing (ChIP-seq) processing pipeline to identify peak regions. However, here we highlight key differences in the processing of STARR-seq versus ChIP-seq data. STARR-seq uses transcribed RNA to measure enhancer activity, making determining the basal transcription rate important. Further, STARRseq coverage is non-uniform, overdispersed, and often confounded by sequencing biases such as GC content and mappability. We observed a correlation between RNA thermodynamic stability and STARR-seq RNA readout, suggesting that STARR-seq might be sensitive to RNA secondary structure and stability. Considering these findings, we developed a statistical framework for uniformly processing STARR-seq data: STARRPeaker. We applied our method to two whole human genome STARR-seq experiments; HepG2 and K562. Our method identifies highly reproducible and epigenetically active enhancers across replicates. Moreover, STARRPeaker outperforms other peak callers in terms of identifying known enhancers. Thus, our framework optimized for processing STARR-seq data accurately characterizes cell-typespecific enhancers, while addressing potential confounders.

Thyroid hormone (T3) is essential for proper neurological development. The hormone, bound to its receptors, regulates gene transcription in part by modulating posttranslational modifications of histones. Methylation of DNA, which is established by the de novo DNA methyltransferase (DNMT)3a and DNMT3b, and maintained by DNMT1 is another epigenetic modification influencing gene transcription. The expression of Dnmt3a, but not other Dnmt genes, increases in mouse brain in parallel with the postnatal rise in plasma [T3]. We found that treatment of the mouse neuroblastoma cell line Neuro2a[TRβ1] with T3 caused rapid induction of Dnmt3a mRNA, which was resistant to protein synthesis inhibition, supporting that it is a direct T3-response gene. Injection of T3 into postnatal day 6 mice increased Dnmt3a mRNA in the brain by 1 hour. Analysis of two chromatin immunoprecipitation-sequencing datasets, and targeted analyses using chromatin immunoprecipitation, transfection-reporter assays, and in vitro DNA binding identified 2 functional T3-response elements (TREs) at the mouse Dnmt3a locus located +30.3 and +49.3 kb from the transcription start site. Thyroid hormone receptors associated with both of these regions in mouse brain chromatin, but with only 1 (+30.3 kb) in Neuro2a[TRβ1] cells. Deletion of the +30.3-kb TRE using CRISPR/Cas9 genome editing eliminated or strongly reduced the Dnmt3a mRNA response to T3. Bioinformatics analysis showed that both TREs are highly conserved among eutherian mammals. Thyroid regulation of Dnmt3a may be an evolutionarily conserved mechanism for modulating global changes in DNA methylation during postnatal neurological development.

Interactions between transcription factors and chromatin are fundamental to genome organization and regulation and, ultimately, cell state. Here, we use information theory to measure signatures of organized chromatin resulting from transcription factor-chromatin interactions encoded in the patterns of the accessible genome, which we term chromatin information enrichment (CIE). We calculate CIE for hundreds of transcription factor motifs across human samples and identify two classes: low and high CIE. The 10–20% of common and tissue-specific high CIE transcription factor motifs, associate with higher protein–DNA residence time, including different binding site subclasses of the same transcription factor, increased nucleosome phasing, specific protein domains, and the genetic control of both chromatin accessibility and gene expression. These results show that variations in the information encoded in chromatin architecture reflect functional biological variation, with implications for cell state dynamics and memory.

STARR-seq technology has employed progressively more complex genomic libraries and increased sequencing depths. An issue with the increased complexity and depth is that the coverage in STARR-seq experiments is non-uniform, overdispersed, and often confounded by sequencing biases, such as GC content. Furthermore, STARR-seq readout is confounded by RNA secondary structure and thermodynamic stability. To address these potential confounders, we developed a negative binomial regression framework for uniformly processing STARR-seq data, called STARRPeaker. Moreover, to aid our effort, we generated whole-genome STARR-seq data from the HepG2 and K562 human cell lines and applied STARRPeaker to comprehensively and unbiasedly call enhancers in them.

Thyroid hormone is essential for normal development in vertebrates. In amphibians, T3 controls metamorphosis by inducing tissue-specific gene regulation programs. A hallmark of T3 action is the modification of chromatin structure, which underlies changes in gene transcription. We found that mRNA for the de novo DNA methyltransferase (DNMT) dnmt3a, but not dnmt1, increased in the brain of Xenopus tadpoles during metamorphosis in parallel with plasma [T3]. Addition of T to the rearing water caused a time-dependent increase in dnmt3a mRNA in tadpole brain, tail, and hind limb. By analyzing data from a genome-wide analysis of T3 receptor (TR) binding in tadpole tail, we identified several putative T3 response elements (TREs) within the dnmt3a locus. Using in vitro DNA binding, transient transfection-reporter, and chromatin immunoprecipitation assays for TRs, we identified two functional TREs at -7.1 kb and +5.1 kb relative to the dnmt3a transcription start site. Sequence alignment showed that these TREs are conserved between two related frog species, X. laevis and X. tropicalis, but not with amniotes. Our previous findings showed that this gene is directly regulated by liganded TRs in mouse brain, and whereas the two mouse TREs are conserved among Eutherian mammals, they are not conserved in Xenopus species. Thus, although T3 regulation of dnmt3a may be an ancient pathway in vertebrates, the genomic sites responsible for hormone regulation may have diverged or arisen by convergent evolution. We hypothesize that direct T regulation of dnmt3a may be an important mechanism for modulating global changes in DNA methylation.