eRNA profiling uncovers the enhancer landscape of oesophageal adenocarcinoma and reveals new deregulated pathways.

Cancer is driven by both genetic and epigenetic changes that impact on gene expression profiles and the resulting tumourigenic phenotype. Enhancers are transcriptional regulatory elements that are key to our understanding of how this rewiring of gene expression is achieved in cancer cells. Here, we have harnessed the power of RNA-seq data from hundreds of patients with oesophageal adenocarcinoma (OAC) or its precursor state Barrett's oesophagus coupled with open chromatin maps to identify potential enhancer RNAs and their associated enhancer regions in this cancer. We identify ~1000 OAC-specific enhancers and use these data to uncover new cellular pathways that are operational in OAC. Among these are enhancers for JUP, MYBL2, and CCNE1, and we show that their activity is required for cancer cell viability. We also demonstrate the clinical utility of our dataset for identifying disease stage and patient prognosis. Our data therefore identify an important set of regulatory elements that enhance our molecular understanding of OAC and point to potential new therapeutic directions.

Oncogenic ERRB2 signals through the AP-1 transcription factor to control mesenchymal-like properties of oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) is a deadly disease with poor survival statistics and few targeted therapies available. One of the most common molecular aberrations in OAC is amplification or activation of the gene encoding the receptor tyrosine kinase ERBB2, and ERBB2 is targeted in the clinic for this subset of patients. However, the downstream consequences of these ERBB2 activating events are not well understood. Here we used a combination of phosphoproteomics, open chromatin profiling and transcriptome analysis on cell line models and patient-derived datasets to interrogate the molecular pathways operating downstream from ERBB2. Integrated analysis of these data sets converge on a model where dysregulated ERBB2 signalling is mediated at the transcriptional level by the transcription factor AP-1. AP-1 in turn controls cell behaviour by acting on cohorts of genes that regulate cell migration and adhesion, features often associated with EMT. Our study therefore provides a valuable resource for the cancer cell signalling community and reveals novel molecular determinants underlying the dysregulated behaviour of OAC cells.

Complexities in the role of acetylation dynamics in modifying inducible gene activation parameters.

High levels of histone acetylation are associated with the regulatory elements of active genes, suggesting a link between acetylation and gene activation. We revisited this model, in the context of EGF-inducible gene expression and found that rather than a simple unifying model, there are two broad classes of genes; one in which high lysine acetylation activity is required for efficient gene activation, and a second group where the opposite occurs and high acetylation activity is inhibitory. We examined the latter class in more detail using EGR2 as a model gene and found that lysine acetylation levels are critical for several activation parameters, including the timing of expression onset, and overall amplitudes of the transcriptional response. In contrast, DUSP1 responds in the canonical manner and its transcriptional activity is promoted by acetylation. Single cell approaches demonstrate heterogenous activation kinetics of a given gene in response to EGF stimulation. Acetylation levels modify these heterogenous patterns and influence both allele activation frequencies and overall expression profile parameters. Our data therefore point to a complex interplay between acetylation equilibria and target gene induction where acetylation level thresholds are an important determinant of transcriptional induction dynamics that are sensed in a gene-specific manner.

PEGS: An efficient tool for gene set enrichment within defined sets of genomic intervals.

Many biological studies of transcriptional control mechanisms produce lists of genes and non-coding genomic intervals from corresponding gene expression and epigenomic assays. In higher organisms, such as eukaryotes, genes may be regulated by distal elements, with these elements lying 10s-100s of kilobases away from a gene transcription start site. To gain insight into these distal regulatory mechanisms, it is important to determine comparative enrichment of genes of interest in relation to genomic regions of interest, and to be able to do so at a range of distances. Existing bioinformatics tools can annotate genomic regions to nearest known genes, or look for transcription factor binding sites in relation to gene transcription start sites. Here, we present PEGS ( Peak set Enrichment in Gene Sets). This tool efficiently provides an exploratory analysis by calculating enrichment of multiple gene sets, associated with multiple non-coding elements (peak sets), at multiple genomic distances, and within topologically associated domains. We apply PEGS to gene sets derived from gene expression studies, and genomic intervals from corresponding ChIP-seq and ATAC-seq experiments to derive biologically meaningful results. We also demonstrate an extended application to tissue-specific gene sets and publicly available GWAS data, to find enrichment of sleep trait associated SNPs in relation to tissue-specific gene expression profiles.

ZIC3 Controls the Transition from Naive to Primed Pluripotency.

Embryonic stem cells (ESCs) must transition through a series of intermediate cell states before becoming terminally differentiated. Here, we investigated the early events in this transition by determining the changes in the open chromatin landscape as naive mouse ESCs transition to epiblast-like cells (EpiLCs). Motif enrichment analysis of the newly opening regions coupled with expression analysis identified ZIC3 as a potential regulator of this cell fate transition. Chromatin binding and genome-wide transcriptional profiling following Zic3 depletion confirmed ZIC3 as an important regulatory transcription factor, and among its targets are genes encoding a number of transcription factors. Among these is GRHL2, which acts through enhancer switching to maintain the expression of a subset of genes from the ESC state. Our data therefore place ZIC3 upstream of a set of pro-differentiation transcriptional regulators and provide an important advance in our understanding of the regulatory factors governing the early steps in ESC differentiation.

Jun-Mediated Changes in Cell Adhesion Contribute to Mouse Embryonic Stem Cell Exit from Ground State Pluripotency.

Embryonic stem cells (ESC) are able to give rise to any somatic cell type. A lot is known about how ESC pluripotency is maintained, but comparatively less is known about how differentiation is promoted. Cell fate decisions are regulated by interactions between signaling and transcriptional networks. Recent studies have shown that the overexpression or downregulation of the transcription factor Jun can affect the ESC fate. Here we have focussed on the role of the Jun in the exit of mouse ESCs from ground state pluripotency and the onset of early differentiation. Transcriptomic analysis of differentiating ESCs reveals that Jun is required to upregulate a programme of genes associated with cell adhesion as ESCs exit the pluripotent ground state. Several of these Jun-regulated genes are shown to be required for efficient adhesion. Importantly this adhesion is required for the timely regulated exit of ESCs from ground state pluripotency and the onset of early differentiation events. Stem Cells 2016;34:1213-1224.

Otx2 and Oct4 drive early enhancer activation during embryonic stem cell transition from naive pluripotency.

Embryonic stem cells (ESCs) are unique in that they have the capacity to differentiate into all of the cell types in the body. We know a lot about the complex transcriptional control circuits that maintain the naive pluripotent state under self-renewing conditions but comparatively less about how cells exit from this state in response to differentiation stimuli. Here, we examined the role of Otx2 in this process in mouse ESCs and demonstrate that it plays a leading role in remodeling the gene regulatory networks as cells exit from ground state pluripotency. Otx2 drives enhancer activation through affecting chromatin marks and the activity of associated genes. Mechanistically, Oct4 is required for Otx2 expression, and reciprocally, Otx2 is required for efficient Oct4 recruitment to many enhancer regions. Therefore, the Oct4-Otx2 regulatory axis actively establishes a new regulatory chromatin landscape during the early events that accompany exit from ground state pluripotency.

PARP1 orchestrates variant histone exchange in signal-mediated transcriptional activation.

Transcriptional activation is accompanied by multiple molecular events that remodel the local chromatin environment in promoter regions. These molecular events are often orchestrated in response to the activation of signalling pathways, as exemplified by the response of immediate early genes such as FOS to ERK MAP kinase signalling. Here, we demonstrate that inducible NFI recruitment permits PARP1 binding to the FOS promoter by a mutually reinforcing loop. PARP1 and its poly(ADP-ribosyl)ation activity are required for maintaining FOS activation kinetics. We also show that the histone variant H2A.Z associates with the FOS promoter and acts in a transcription-suppressive manner. However, in response to ERK pathway signalling, H2A.Z is replaced by H2A; PARP1 activity is required to promote this exchange. Thus, our work has revealed an additional facet of PARP1 function in promoting dynamic remodelling of promoter-associated nucleosomes to allow transcriptional activation in response to cellular signalling.

MAP kinase signalling cascades and transcriptional regulation.

The MAP kinase (MAPK) signalling pathways play fundamental roles in a wide range of cellular processes and are often deregulated in disease states. One major mode of action for these pathways is in controlling gene expression, in particular through regulating transcription. In this review, we discuss recent significant advances in this area. In particular we focus on the mechanisms by which MAPKs are targeted to the nucleus and chromatin, and once there, how they impact on chromatin structure and subsequent gene regulation. We also discuss how systems biology approaches have contributed to our understanding of MAPK signaling networks, and also how the MAPK pathways intersect with other regulatory pathways in the nucleus. Finally, we summarise progress in studying the physiological functions of key MAPK transcriptional targets.

A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

MAP Kinase: SUMO pathway interactions.

The convergence and coordinated cross talk of different signalling pathways forms a regulatory network which determines the biological outcome to environmental cues. The MAPK pathways are one of the important routes by which extracellular signals are transduced into intracellular responses. Through protein phosphorylation mechanisms, they can play a pivotal role in regulating other posttranslational modifications such as protein acetylation and ubiquitination. In addition, protein sumoylation has emerged as an important pathway which also functions through post-translational modification. The SUMO pathway modulates a diverse range of cellular processes including signal transduction, chromosome integrity, and transcription. Interestingly, recent studies have provided links between the SUMO and MAPK signalling pathways which converge to modulate transcription factor activity. This was first demonstrated by the observation that the activation of the ERK pathway caused de-sumoylation of the transcription factor, Elk-1. Furthermore, a growing number of links are now being made between the MAPK pathway and protein sumoylation. Given the nature of protein sumoylation in diverse biological functions, it is not surprising that the effect of MAPK pathways on sumoylation varies between different proteins. Here, we describe protocols that can be used in studying the cross talk between the MAPK and SUMO pathways, particularly at the level of gene regulation.

The SUMO E3 ligase activity of Pc2 is coordinated through a SUMO interaction motif.

Protein modification by SUMO conjugation has emerged to be an important regulatory event. Recently, the mechanisms through which SUMO elicits its effects on target proteins have been elucidated. One of these is the noncovalent association between SUMO and coregulatory proteins via SUMO interaction motifs (SIMs). We therefore searched for additional binding proteins to elucidate how SUMO acts as a signal to potentiate novel noncovalent interactions with SUMO-binding proteins. We identified an E3 ligase, Pc2, as a SUMO-binding protein with two functionally distinct SIMs. Here, we focus on the role of SIM2 and demonstrate that it is crucial for many of the documented Pc2 functions, which converge on determining its E3 ligase activity. One role of SUMO binding in this context is the subnuclear partitioning of the active form of Ubc9 (SUMO approximately Ubc9) by Pc2. The significance of the SIM2-dependent functions of Pc2 is demonstrated in the control of the precise expression of lineage-specific genes during embryonic stem cell differentiation.

A role for non-covalent SUMO interaction motifs in Pc2/CBX4 E3 activity.

BACKGROUND: Modification of proteins by the small ubiquitin like modifier (SUMO) is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs) that are generally short hydrophobic peptide motifs have been identified. METHODOLOGY/PRINCIPAL FINDINGS: Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci. CONCLUSIONS/SIGNIFICANCE: This work provides the first clear evidence for a role for SIMs in SUMO E3 activity.

MAP kinase-mediated c-fos regulation relies on a histone acetylation relay switch.

Gene activation is often associated with high levels of histone acetylation. Enhanced acetylation levels can promote the recruitment of further chromatin modifying complexes or the basal transcription machinery. Here, we have studied MAP kinase-mediated upregulation of c-fos and uncover a role for histone acetylation in promoting the recruitment of a second transcription factor, NFI. MAP kinase signaling to Elk-1 enhances the net histone acetylase activity associated with the c-fos promoter, which leads to changes in the acetylation state and structure of a promoter-proximal nucleosome, which allows NFI binding. Binding of NFI provides a permissive state for the recruitment of basal machinery and subsequent promoter activation. Our results provide insights into how MAP kinase signaling promotes inducible gene expression; phosphorylation of recipient transcription factors (primary effectors) triggers a HAT relay switch, which facilitates the recruitment of additional transcription factors (secondary effectors) through alteration of the local nucleosomal structure.

Rev7/MAD2B links c-Jun N-terminal protein kinase pathway signaling to activation of the transcription factor Elk-1.

The mitogen-activated protein (MAP) kinases represent one of the most important classes of signaling cascades that are used by eukaryotic cells to sense extracellular signals. One of the major responses to these cascades is a change in cellular gene expression profiles mediated through the direct targeting of transcriptional regulators, such as the transcription factor Elk-1. Here we have identified human Rev7 (hRev7)/MAD2B/MAD2L2 as an interaction partner for Elk-1 and demonstrate that hRev7 acts to promote Elk-1 phosphorylation by the c-Jun N-terminal protein kinase (JNK) MAP kinases. As phosphorylation of Elk-1 potentiates the activity of its transcriptional activation domain, hRev7 therefore contributes to the upregulation of Elk-1 target genes, such as egr-1, following exposure of cells to stress conditions caused by DNA-damaging agents. Thus, given its previous roles in permitting DNA damage bypass during replication and regulating cell cycle progression, our data linking hRev7 to gene expression changes suggest that hRev7 has a widespread role in coordinating the cellular response to DNA damage.

An extended consensus motif enhances the specificity of substrate modification by SUMO.

Protein modification by SUMO conjugation is an important regulatory event. Sumoylation usually takes place on a lysine residue embedded in the core consensus motif psiKxE. However, this motif confers limited specificity on the sumoylation process. Here, we have probed the roles of clusters of acidic residues located downstream from the core SUMO modification sites in proteins such as the transcription factor Elk-1. We demonstrate that these are functionally important in SUMO-dependent transcriptional repression of Elk-1 transcriptional activity. Mechanistically, the acidic residues are important in enhancing the efficiency of Elk-1 sumoylation by Ubc9. Similar mechanisms operate in other transcription factors and phosphorylation sites can functionally substitute for acidic residues. Thus, an extended sumoylation motif, termed the NDSM (negatively charged amino acid-dependent sumoylation motif), helps define functional SUMO targets. We demonstrate that this extended motif can be used to correctly predict novel targets for SUMO modification.

PIASxalpha differentially regulates the amplitudes of transcriptional responses following activation of the ERK and p38 MAPK pathways.

Activation of the MAP kinase pathways leads to changes in gene expression profiles through direct targeting of transcription factors and their coregulators. Here we identify PIASxalpha as a key regulator that determines the differential response of the transcription factor Elk-1 to the ERK and the stress-activated p38 MAP kinase pathways. While PIASxalpha functions as a coactivator to facilitate SUMO and HDAC-2 removal from Elk-1 in response to ERK pathway activation, PIASxalpha acts in the opposite manner to inhibit HDAC-2 and SUMO loss following stress-activated MAP kinase pathway signaling. Thus, PIASxalpha either enhances or dampens down the activation of Elk-1 target genes, depending on the pathway activated. p38 MAP kinase-mediated PIASxalpha phosphorylation allows it to switch between these two alternative modes of operation. Thus, PIASxalpha acts as a key signal integrator that permits different responses from the same transcription factor, depending on the signaling pathway that is activated.

Convergence of the SUMO and MAPK pathways on the ETS-domain transcription factor Elk-1.

The ETS-domain transcription factor Elk-1 is regulated by phosphorylation in response to activation of the MAPK (mitogen-activated protein kinase) pathways. This phosphorylation triggers a series of molecular events that convert Elk-1 from a transcriptionally silent state into a highly active state and then back to a basal level. At the same time, activation of the ERK (extracellular-signal-regulated kinase) MAPK pathway leads to loss of modification of Elk-1 by SUMO (small ubiquitin-related modifier). As SUMO imparts repressive properties on Elk-1, ERK-mediated SUMO loss leads to de-repression at the same time as the ERK pathway promotes activation of Elk-1. Thus a two-step mechanism is employed to convert Elk-1 into its fully activated state. Here, the molecular events underlying these changes in Elk-1 status, and the role of PIASxalpha [protein inhibitor of activated STAT (signal transducer and activator of transcription) xalpha] as a co-activator that facilitates this process, are discussed.

PIASx acts as an Elk-1 coactivator by facilitating derepression.

The ETS-domain transcription factor Elk-1 is a MAP kinase-inducible transcriptional activator protein. However, in the basal state, its activity is repressed by SUMO-dependent histone deacetylase (HDAC) recruitment. Relief of this repression accompanies the activation process. Here, we demonstrate that PIASx(alpha) acts to facilitate this derepression process. Members of the PIAS family of proteins can act as E3 enzymes that enhance the sumoylation status of a variety of substrates. However, PIASx-mediated coactivation of Elk-1 occurs in an E3 activity-independent manner. PIASx(alpha) binds to Elk-1 in vivo and enhances its transcriptional activity. The coactivating properties of PIASx(alpha) require Elk-1 to be modified with SUMO and the integrity of the SUMO binding motif in PIASx(alpha). PIASx(alpha) activates Elk-1 through alterations in the HAT/HDAC activities associated with Elk-1. In particular, PIASx(alpha) facilitates the loss of the repressive HDAC-2 from sumoylated Elk-1, a key event in the activation of Elk-1 in response to signalling through the ERK MAP kinase pathway. Our data therefore reveal a novel coactivator function for PIASx(alpha) through reversing SUMO-mediated repression of transcription factor activity.