H3K27ac bookmarking promotes rapid post-mitotic activation of the pluripotent stem cell program without impacting 3D chromatin reorganization.

During self-renewal, cell-type-defining features are drastically perturbed in mitosis and must be faithfully reestablished upon G1 entry, a process that remains largely elusive. Here, we characterized at a genome-wide scale the dynamic transcriptional and architectural resetting of mouse pluripotent stem cells (PSCs) upon mitotic exit. We captured distinct waves of transcriptional reactivation with rapid induction of stem cell genes and transient activation of lineage-specific genes. Topological reorganization at different hierarchical levels also occurred in an asynchronous manner and showed partial coordination with transcriptional resetting. Globally, rapid transcriptional and architectural resetting associated with mitotic retention of H3K27 acetylation, supporting a bookmarking function. Indeed, mitotic depletion of H3K27ac impaired the early reactivation of bookmarked, stem-cell-associated genes. However, 3D chromatin reorganization remained largely unaffected, suggesting that these processes are driven by distinct forces upon mitotic exit. This study uncovers principles and mediators of PSC molecular resetting during self-renewal.

Promoter-proximal pausing of RNA polymerase II: a nexus of gene regulation.

Precise spatio-temporal control of gene activity is essential for organismal development, growth, and survival in a changing environment. Decisive steps in gene regulation involve the pausing of RNA polymerase II (Pol II) in early elongation, and the controlled release of paused polymerase into productive RNA synthesis. Here we describe the factors that enable pausing and the events that trigger Pol II release into the gene. We also discuss open questions in the field concerning the stability of paused Pol II, nucleosomes as obstacles to elongation, and potential roles of pausing in defining the precision and dynamics of gene expression.

An improved auxin-inducible degron system preserves native protein levels and enables rapid and specific protein depletion.

Rapid perturbation of protein function permits the ability to define primary molecular responses while avoiding downstream cumulative effects of protein dysregulation. The auxin-inducible degron (AID) system was developed as a tool to achieve rapid and inducible protein degradation in nonplant systems. However, tagging proteins at their endogenous loci results in chronic auxin-independent degradation by the proteasome. To correct this deficiency, we expressed the auxin response transcription factor (ARF) in an improved inducible degron system. ARF is absent from previously engineered AID systems but is a critical component of native auxin signaling. In plants, ARF directly interacts with AID in the absence of auxin, and we found that expression of the ARF PB1 (Phox and Bem1) domain suppresses constitutive degradation of AID-tagged proteins. Moreover, the rate of auxin-induced AID degradation is substantially faster in the ARF-AID system. To test the ARF-AID system in a quantitative and sensitive manner, we measured genome-wide changes in nascent transcription after rapidly depleting the ZNF143 transcription factor. Transcriptional profiling indicates that ZNF143 activates transcription in cis and regulates promoter-proximal paused RNA polymerase density. Rapidly inducible degradation systems that preserve the target protein's native expression levels and patterns will revolutionize the study of biological systems by enabling specific and temporally defined protein dysregulation.

A rapid, extensive, and transient transcriptional response to estrogen signaling in breast cancer cells.

We report the immediate effects of estrogen signaling on the transcriptome of breast cancer cells using global run-on and sequencing (GRO-seq). The data were analyzed using a new bioinformatic approach that allowed us to identify transcripts directly from the GRO-seq data. We found that estrogen signaling directly regulates a strikingly large fraction of the transcriptome in a rapid, robust, and unexpectedly transient manner. In addition to protein-coding genes, estrogen regulates the distribution and activity of all three RNA polymerases and virtually every class of noncoding RNA that has been described to date. We also identified a large number of previously undetected estrogen-regulated intergenic transcripts, many of which are found proximal to estrogen receptor binding sites. Collectively, our results provide the most comprehensive measurement of the primary and immediate estrogen effects to date and a resource for understanding rapid signal-dependent transcription in other systems.

Transcription factors GAF and HSF act at distinct regulatory steps to modulate stress-induced gene activation.

The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.

An AR-ERG transcriptional signature defined by long-range chromatin interactomes in prostate cancer cells.

The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR cis-regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention.

A bipartite boundary element restricts UBE3A imprinting to mature neurons.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele of UBE3A, a gene encoding an E3 ubiquitin ligase. UBE3A is only expressed from the maternally inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression of UBE3A is restricted to neurons by expression of UBE3A antisense transcript (UBE3A-ATS) from the paternally inherited allele, which silences the paternal allele of UBE3A in cis However, the mechanism restricting UBE3A-ATS expression and UBE3A imprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression of UBE3A-ATS in humans. Removal of this element led to up-regulation of UBE3A-ATS without repressing paternal UBE3A However, increasing expression of UBE3A-ATS in the absence of the boundary element resulted in full repression of paternal UBE3A, demonstrating that UBE3A imprinting requires both the loss of function from the boundary element as well as the up-regulation of UBE3A-ATS These results suggest that manipulation of the competition between UBE3A-ATS and UBE3A may provide a potential therapeutic approach for AS.

Signaling pathways differentially affect RNA polymerase II initiation, pausing, and elongation rate in cells.

RNA polymerase II (Pol II) transcribes hundreds of kilobases of DNA, limiting the production of mRNAs and lncRNAs. We used global run-on sequencing (GRO-seq) to measure the rates of transcription by Pol II following gene activation. Elongation rates vary as much as 4-fold at different genomic loci and in response to two distinct cellular signaling pathways (i.e., 17ß-estradiol [E2] and TNF-α). The rates are slowest near the promoter and increase during the first ~15 kb transcribed. Gene body elongation rates correlate with Pol II density, resulting in systematically higher rates of transcript production at genes with higher Pol II density. Pol II dynamics following short inductions indicate that E2 stimulates gene expression by increasing Pol II initiation, whereas TNF-α reduces Pol II residence time at pause sites. Collectively, our results identify previously uncharacterized variation in the rate of transcription and highlight elongation as an important, variable, and regulated rate-limiting step during transcription.

Extensive polymerase pausing during Drosophila axis patterning enables high-level and pliable transcription.

Cascades of zygotic gene expression pattern the anterior-posterior (AP) and dorsal-ventral (DV) axes of the early Drosophila embryo. Here, we used the global run-on sequencing assay (GRO-seq) to map the genome-wide RNA polymerase distribution during early Drosophila embryogenesis, thus providing insights into how genes are regulated. We identify widespread promoter-proximal pausing yet show that the presence of paused polymerase does not necessarily equate to direct regulation through pause release to productive elongation. Our data reveal that a subset of early Zelda-activated genes is regulated at the level of polymerase recruitment, whereas other Zelda target and axis patterning genes are predominantly regulated through pause release. In contrast to other signaling pathways, we found that bone morphogenetic protein (BMP) target genes are collectively more highly paused than BMP pathway components and show that BMP target gene expression requires the pause-inducing negative elongation factor (NELF) complex. Our data also suggest that polymerase pausing allows plasticity in gene activation throughout embryogenesis, as transiently repressed and transcriptionally silenced genes maintain and lose promoter polymerases, respectively. Finally, we provide evidence that the major effect of pausing is on the levels, rather than timing, of transcription. These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints.

The RNA exosome contributes to gene expression regulation during stem cell differentiation.

Gene expression programs change during cellular transitions. It is well established that a network of transcription factors and chromatin modifiers regulate RNA levels during embryonic stem cell (ESC) differentiation, but the full impact of post-transcriptional processes remains elusive. While cytoplasmic RNA turnover mechanisms have been implicated in differentiation, the contribution of nuclear RNA decay has not been investigated. Here, we differentiate mouse ESCs, depleted for the ribonucleolytic RNA exosome, into embryoid bodies to determine to which degree RNA abundance in the two states can be attributed to changes in transcription versus RNA decay by the exosome. As a general observation, we find that exosome depletion mainly leads to the stabilization of RNAs from lowly transcribed loci, including several protein-coding genes. Depletion of the nuclear exosome cofactor RBM7 leads to similar effects. In particular, transcripts that are differentially expressed between states tend to be more exosome sensitive in the state where expression is low. We conclude that the RNA exosome contributes to down-regulation of transcripts with disparate expression, often in conjunction with transcriptional down-regulation.

The polycomb group mutant esc leads to augmented levels of paused Pol II in the Drosophila embryo.

Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutants.

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells.

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, "bivalent" ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5' proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation.

Identification of active transcriptional regulatory elements from GRO-seq data.

Modifications to the global run-on and sequencing (GRO-seq) protocol that enrich for 5'-capped RNAs can be used to reveal active transcriptional regulatory elements (TREs) with high accuracy. Here, we introduce discriminative regulatory-element detection from GRO-seq (dREG), a sensitive machine learning method that uses support vector regression to identify active TREs from GRO-seq data without requiring cap-based enrichment (https://github.com/Danko-Lab/dREG/). This approach allows TREs to be assayed together with gene expression levels and other transcriptional features in a single experiment. Predicted TREs are more enriched for several marks of transcriptional activation­including expression quantitative trait loci, disease-associated polymorphisms, acetylated histone 3 lysine 27 (H3K27ac) and transcription factor binding­than those identified by alternative functional assays. Using dREG, we surveyed TREs in eight human cell types and provide new insights into global patterns of TRE function.

X chromosome dosage compensation via enhanced transcriptional elongation in Drosophila.

The evolution of sex chromosomes has resulted in numerous species in which females inherit two X chromosomes but males have a single X, thus requiring dosage compensation. MSL (Male-specific lethal) complex increases transcription on the single X chromosome of Drosophila males to equalize expression of X-linked genes between the sexes. The biochemical mechanisms used for dosage compensation must function over a wide dynamic range of transcription levels and differential expression patterns. It has been proposed that the MSL complex regulates transcriptional elongation to control dosage compensation, a model subsequently supported by mapping of the MSL complex and MSL-dependent histone 4 lysine 16 acetylation to the bodies of X-linked genes in males, with a bias towards 3' ends. However, experimental analysis of MSL function at the mechanistic level has been challenging owing to the small magnitude of the chromosome-wide effect and the lack of an in vitro system for biochemical analysis. Here we use global run-on sequencing (GRO-seq) to examine the specific effect of the MSL complex on RNA Polymerase II (RNAP II) on a genome-wide level. Results indicate that the MSL complex enhances transcription by facilitating the progression of RNAP II across the bodies of active X-linked genes. Improving transcriptional output downstream of typical gene-specific controls may explain how dosage compensation can be imposed on the diverse set of genes along an entire chromosome.

Paused Pol II captures enhancer activity and acts as a potent insulator.

Enhancers act over many kilobase pairs to activate target promoters, but their activity is constrained by insulator elements that prevent indiscriminate activation of nearby genes. In the July 1, 2009, issue of Genes & Development, Chopra and colleagues (pp. 1505-1509) report that promoters containing a stalled Pol II are activated by enhancers, but these promoters also serve as insulators that block enhancers from reaching more distal genes. This new class of insulators provide critical clues to regulatory mechanisms.

Centromeric transposable elements and epigenetic status drive karyotypic variation in the eastern hoolock gibbon.

Great apes have maintained a stable karyotype with few large-scale rearrangements; in contrast, gibbons have undergone a high rate of chromosomal rearrangements coincident with rapid centromere turnover. Here we characterize assembled centromeres in the Eastern hoolock gibbon, Hoolock leuconedys (HLE), finding a diverse group of transposable elements (TEs) that differ from the canonical alpha satellites found across centromeres of other apes. We find that HLE centromeres contain a CpG methylation centromere dip region, providing evidence this epigenetic feature is conserved in the absence of satellite arrays; nevertheless, we report a variety of atypical centromeric features, including protein-coding genes and mismatched replication timing. Further, large structural variations define HLE centromeres and distinguish them from other gibbons. Combined with differentially methylated TEs, topologically associated domain boundaries, and segmental duplications at chromosomal breakpoints, we propose that a "perfect storm" of multiple genomic attributes with propensities for chromosome instability shaped gibbon centromere evolution.

Bacillus subtilis RapA phosphatase domain interaction with its substrate, phosphorylated Spo0F, and its inhibitor, the PhrA peptide.

Rap proteins in Bacillus subtilis regulate the phosphorylation level or the DNA-binding activity of response regulators such as Spo0F, involved in sporulation initiation, or ComA, regulating competence development. Rap proteins can be inhibited by specific peptides generated by the export-import processing pathway of the Phr proteins. Rap proteins have a modular organization comprising an amino-terminal alpha-helical domain connected to a domain formed by six tetratricopeptide repeats (TPR). In this study, the molecular basis for the specificity of the RapA phosphatase for its substrate, phosphorylated Spo0F (Spo0F∼P), and its inhibitor pentapeptide, PhrA, was analyzed in part by generating chimeric proteins with RapC, which targets the DNA-binding domain of ComA, rather than Spo0F∼P, and is inhibited by the PhrC pentapeptide. In vivo analysis of sporulation efficiency or competence-induced gene expression, as well as in vitro biochemical assays, allowed the identification of the amino-terminal 60 amino acids as sufficient to determine Rap specificity for its substrate and the central TPR3 to TPR5 (TPR3-5) repeats as providing binding specificity toward the Phr peptide inhibitor. The results allowed the prediction and testing of key residues in RapA that are essential for PhrA binding and specificity, thus demonstrating how the widespread structural fold of the TPR is highly versatile, using a common interaction mechanism for a variety of functions in eukaryotic and prokaryotic organisms.

From telomere to telomere: The transcriptional and epigenetic state of human repeat elements.

Mobile elements and repetitive genomic regions are sources of lineage-specific genomic innovation and uniquely fingerprint individual genomes. Comprehensive analyses of such repeat elements, including those found in more complex regions of the genome, require a complete, linear genome assembly. We present a de novo repeat discovery and annotation of the T2T-CHM13 human reference genome. We identified previously unknown satellite arrays, expanded the catalog of variants and families for repeats and mobile elements, characterized classes of complex composite repeats, and located retroelement transduction events. We detected nascent transcription and delineated CpG methylation profiles to define the structure of transcriptionally active retroelements in humans, including those in centromeres. These data expand our insight into the diversity, distribution, and evolution of repetitive regions that have shaped the human genome.

Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme.

The human genome encodes a variety of poorly understood RNA species that remain challenging to identify using existing genomic tools. We developed chromatin run-on and sequencing (ChRO-seq) to map the location of RNA polymerase for almost any input sample, including samples with degraded RNA that are intractable to RNA sequencing. We used ChRO-seq to map nascent transcription in primary human glioblastoma (GBM) brain tumors. Enhancers identified in primary GBMs resemble open chromatin in the normal human brain. Rare enhancers that are activated in malignant tissue drive regulatory programs similar to the developing nervous system. We identified enhancers that regulate groups of genes that are characteristic of each known GBM subtype and transcription factors that drive them. Finally we discovered a core group of transcription factors that control the expression of genes associated with clinical outcomes. This study characterizes the transcriptional landscape of GBM and introduces ChRO-seq as a method to map regulatory programs that contribute to complex diseases.