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A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17β-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA–dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.After acute agonist stimulation, phase-separated complexes form at estrogen-receptor-bound enhancer sites and coalesce into condensates with cooperative enhancer activity. Chronic stimulation causes the condensates to mature into a less dynamic, gel-like state.

MicroRNAs (miRNAs) are a class of endogenous small noncoding RNAs that participate in a majority of biological processes via regulating target gene expression. The post-transcriptional repression through miRNA seed region binding to 3′ UTR of target mRNA is considered as the canonical mode of miRNA-mediated gene regulation. However, emerging evidence suggests that other regulatory modes exist beyond the canonical mechanism. In particular, the function of intranuclear miRNA in gene transcriptional regulation is gradually revealed, with evidence showing their contribution to gene silencing or activating. Therefore, miRNA-mediated regulation of gene transcription not only expands our understanding of the molecular mechanism underlying miRNA regulatory function, but also provides new evidence to explain its ability in the sophisticated regulation of many bioprocesses. In this review, mechanisms of miRNA-mediated gene transcriptional and post-transcriptional regulation are summarized, and the synergistic effects among these actions which form a regulatory network of a miRNA on its target are particularly elaborated. With these discussions, we aim to emphasize the importance of miRNA regulatory network on target gene regulation and further highlight the potential application of the network mode in the achievement of a more effective and stable modulation of the target gene expression.

Quercetin 3-O-β-D-glucuronide (Q-3-G), the glucuronide conjugate of quercetin, has been reported as having anti-inflammatory properties in the lipopolysaccharide-stimulated macrophages, as well as anticancer and antioxidant properties. Unlike quercetin, which has been extensively described to possess a wide range of pharmacological activities including skin protective effects, the pharmacological benefits and mechanisms Q-3-G in the skin remained to be elucidated. This study focused on characterizing the skin protective properties, including anti-inflammatory and antioxidant properties, of Q-3-G against UVB-induced or H2O2-induced oxidative stress, the hydration effects, and antimelanogenesis activities using human keratinocytes (HaCaT) and melanoma (B16F10) cells. Q-3-G down-regulated the expression of the pro-inflammatory gene and cytokine such as cyclooxygenase-2 (COX-2) and tumor necrosis factor (TNF)-α in H2O2 or UVB-irradiated HaCaT cells. We also showed that Q-3-G exhibits an antioxidant effect using free radical scavenging assays, flow cytometry, and an increased expression of nuclear factor erythroid 2- related factor 2 (Nrf2). Q-3-G reduced melanin production in α-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. The hydration effects and mechanisms of Q-3-G were examined by evaluating the moisturizing factor-related genes, such as transglutaminase-1 (TGM-1), filaggrin (FLG), and hyaluronic acid synthase (HAS)-1. In addition, Q-3-G increased the phosphorylation of c-Jun, Jun N-terminal kinase (JNK), Mitogen-activated protein kinase (MAPK) kinase 4 (MKK4), and TAK1, involved in the MAPKs/AP-1 pathway, and the phosphorylation of IκBα, IκB kinase (IKK)-α, Akt, and Src, involved in the NF-κB pathway. Taken together, we have demonstrated that Q-3-G exerts anti-inflammatory, antioxidant, moisturizing, and antimelanogenesis properties in human keratinocytes and melanoma cells through NF-κB and AP-1 pathways.

Modification of the CRISPR/Cas9 genome editing system by the addition of the light inducible proteins CRY2 and CIBI1 enables blue light–mediated transcription of endogenous genes in mammalian cells. Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR-Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes.

The influence of the histone variant H2A.Z on transcription remains a long-standing conundrum. Here, by analyzing the actin-related protein6 mutant, which is impaired in H2A.Z deposition, and by H2A.Z profiling in stress conditions, we investigated the impact of this histone variant on gene expression in Arabidopsis thaliana. We demonstrate that the arp6 mutant exhibits anomalies in response to osmotic stress. Indeed, stress-responsive genes are overrepresented among those hyperactive in arp6. In wild-type plants, these genes exhibit high levels of H2A.Z in the gene body. Furthermore, we observed that in droughtresponsive genes, levels of H2A.Z in the gene body correlate with transcript levels. H2A.Z occupancy, but not distribution, changes in parallel with transcriptional changes. In particular, we observed H2A.Z loss upon transcriptional activation and H2A.Z gain upon repression. These data suggest that H2A.Z has a repressive role in transcription and counteracts unwanted expression in noninductive conditions. However, reduced activity of some genes in arp6 is associated with distinct behavior of H2A.Z at their +1 nucleosome, which exemplifies the requirement of this histone for transcription. Our data support a model where H2A.Z in gene bodies has a strong repressive effect on transcription, whereas in +1 nucleosomes, it is important for maintaining the activity of some genes.

Lymphatic dissemination from the primary tumor is a major mechanism by which breast cancer cells access the systemic circulation, resulting in distant metastasis and mortality. Numerous studies link activation of hypoxia-inducible factor 1 (HIF-1) with tumor angiogenesis, metastasis, and patient mortality. However, the role of HIF-1 in lymphatic dissemination is poorly understood. In this study, we show that HIF-1 promotes lymphatic metastasis of breast cancer by direct transactivation of the gene encoding platelet-derived growth factor B (PDGF-B), which has proliferative and chemotactic effects on lymphatic endothelial cells. Lymphangiogenesis and lymphatic metastasis in mice bearing human breast cancer orthografts were blocked by administration of the HIF-1 inhibitor digoxin or the tyrosine kinase inhibitor imatinib. Immunohistochemical analysis of human breast cancer biopsies demonstrated colocalization of HIF-1α and PDGF-B, which were correlated with lymphatic vessel area and histological grade. Taken together, these data provide experimental support for breast cancer clinical trials targeting HIF-1 and PDGF-B.

It is unclear whether bidirectional non-coding RNAs transcribed from enhancer elements (eRNAs) have any functional role; here, eRNA transcription is shown to be functionally important during the activation of genes by the oestrogen receptor in human breast cancer cells. Regulatory role for eRNAs Bidirectional non-coding RNAs are transcribed from enhancer elements, but it is unclear whether these enhancer-derived RNAs (eRNAs) have a functional role or are merely a reflection of enhancer activity. Two manuscripts in this issue of Nature examine this question in the context of the positive and negative transcriptional functions of different nuclear receptors. Wenbo Li et al . provide evidence for the functional importance of eRNA transcription during the activation of genes by the oestrogen receptor in breast cancer cell lines; and Michael Lam et al . show that the repressive functions of Rev-Erb nuclear receptors in macrophages are linked to their ability to inhibit the transcription of eRNAs. Taken together these studies provide evidence for a role for eRNAs in contributing to enhancer functions. The functional importance of gene enhancers in regulated gene expression is well established 1 , 2 , 3 . In addition to widespread transcription of long non-coding RNAs (lncRNAs) in mammalian cells 4 , 5 , 6 , bidirectional ncRNAs are transcribed on enhancers, and are thus referred to as enhancer RNAs (eRNAs) 7 , 8 , 9 . However, it has remained unclear whether these eRNAs are functional or merely a reflection of enhancer activation. Here we report that in human breast cancer cells 17β-oestradiol (E2)-bound oestrogen receptor α (ER-α) causes a global increase in eRNA transcription on enhancers adjacent to E2-upregulated coding genes. These induced eRNAs, as functional transcripts, seem to exert important roles for the observed ligand-dependent induction of target coding genes, increasing the strength of specific enhancer–promoter looping initiated by ER-α binding. Cohesin, present on many ER-α-regulated enhancers even before ligand treatment, apparently contributes to E2-dependent gene activation, at least in part by stabilizing E2/ER-α/eRNA-induced enhancer–promoter looping. Our data indicate that eRNAs are likely to have important functions in many regulated programs of gene transcription.

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. The CRISPR-Cas9 system, a powerful tool for genome editing, has been engineered to activate endogenous gene transcription specifically and potently on a genome-wide scale and applied to a large-scale gain-of-function screen for studying melanoma drug resistance. CRISPR-Cas9 used for gene-expression regulation The CRISPR-Cas9 system has emerged as a powerful tool for genome editing and transcriptional regulation of specific genes. Feng Zhang and colleagues have successfully modified the system to specifically and potently activate endogenous gene transcription on a genome-wide scale, such that it can be used for large-scale functional genomics screens. Application to a genome-wide screen of melanoma cells for genes which when overexpressed can confer resistance to a BRAF inhibitor demonstrates the feasibility of such screens, and also led to the discovery of potential new resistance mechanisms.

Summary Although starch degradation has been well studied in model systems such as Arabidopsis leaves and cereal seeds, this process in starchy fruits during ripening, especially in bananas, is largely unknown. In this study, 38 genes encoding starch degradation‐related proteins were identified and characterized from banana fruit. Expression analysis revealed that 27 candidate genes were significantly induced during banana fruit ripening, with concomitant conversion of starch‐to‐sugars. Furthermore, iTRAQ‐based proteomics experiments identified 18 starch degradation‐associated enzymes bound to the surface of starch granules, of which 10 were markedly up‐regulated during ripening. More importantly, a novel bHLH transcription factor, MabHLH6, was identified based on a yeast one‐hybrid screening using MaGWD1 promoter as a bait. Transcript and protein levels of MabHLH6 were also increased during fruit ripening. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transient expression experiments confirmed that MabHLH6 activates the promoters of 11 starch degradation‐related genes, including MaGWD1, MaLSF2, MaBAM1, MaBAM2, MaBAM8, MaBAM10, MaAMY3, MaAMY3C, MaISA2, MaISA3 and MapGlcT2‐2 by recognizing their E‐box (CANNTG) motifs present in the promoters. Collectively, these findings suggest that starch degradation during banana fruit ripening may be attributed to the complex actions of numerous enzymes related to starch breakdown at transcriptional and translational levels, and that MabHLH6 may act as a positive regulator of this process via direct activation of a series of starch degradation‐related genes.

Intrinsically disordered regions (IDRs) play important roles in proteins that regulate gene expression. A prominent example is the intracellular domain of the Notch receptor (NICD), which regulates the transcription of Notch-responsive genes. The NICD sequence includes an intrinsically disordered RAM region and a conserved ankyrin (ANK) domain. The 111-residue RAM region mediates bivalent interactions of NICD with the transcription factor CSL. Although the sequence of RAM is poorly conserved, the linear patterning of oppositely charged residues shows minimal variation. The conformational properties of polyampholytic IDRs are governed as much by linear charge patterning as by overall charge content. Here, we used sequence design to assess how changing the charge patterning within RAM affects its conformational properties, the affinity of NICD to CSL, and Notch transcriptional activity. Increased segregation of oppositely charged residues leads to linear decreases in the global dimensions of RAM and decreases the affinity of a construct including a C-terminal ANK domain (RAMANK) for CSL. Increasing charge segregation from WT RAM sharply decreases transcriptional activation for all permutants. Activation also decreases for some, but not all, permutants with low charge segregation, although there is considerable variation. Our results suggest that the RAM linker is more than a passive tether, contributing local and/or long-range sequence features that modulate interactions within NICD and with downstream components of the Notch pathway. We propose that sequence features within IDRs have evolved to ensure an optimal balance of sequence-encoded conformational properties, interaction strengths, and cellular activities.