Changes in regeneration-responsive enhancers shape regenerative capacities in vertebrates.

Vertebrates vary in their ability to regenerate, and the genetic mechanisms underlying such disparity remain elusive. Comparative epigenomic profiling and single-cell sequencing of two related teleost fish uncovered species-specific and evolutionarily conserved genomic responses to regeneration. The conserved response revealed several regeneration-responsive enhancers (RREs), including an element upstream to inhibin beta A (inhba), a known effector of vertebrate regeneration. This element activated expression in regenerating transgenic fish, and its genomic deletion perturbed caudal fin regeneration and abrogated cardiac regeneration altogether. The enhancer is present in mammals, shares functionally essential activator protein 1 (AP-1)-binding motifs, and responds to injury, but it cannot rescue regeneration in fish. This work suggests that changes in AP-1-enriched RREs are likely a crucial source of loss of regenerative capacities in vertebrates.

Coupling Computational and Intracellular Screening and Selection Toward Co-compatible cJun and cFos Antagonists.

Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.

Activation of Oncogenic Super-Enhancers Is Coupled with DNA Repair by RAD51.

DNA double-strand breaks (DSBs) are deleterious and tumorigenic but could also be essential for DNA-based processes. Yet the landscape of physiological DSBs and their role and repair are still elusive. Here, we mapped DSBs at high resolution in cancer and non-tumorigenic cells and found a transcription-coupled repair mechanism at oncogenic super-enhancers. At these super-enhancers the transcription factor TEAD4, together with various transcription factors and co-factors, co-localizes with the repair factor RAD51 of the homologous recombination pathway. Depletion of TEAD4 or RAD51 increases DSBs at RAD51/TEAD4 common binding sites within super-enhancers and decreases expression of related genes, which are mostly oncogenes. Co-localization of RAD51 with transcription factors at super-enhancers occurs in various cell types, suggesting a broad phenomenon. Together, our findings uncover a coupling between transcription and repair mechanisms at oncogenic super-enhancers, to control the hyper-transcription of multiple cancer drivers.

Insight into the molecular function and transcriptional regulation of activator protein 1 (AP-1) components c-Jun/c-Fos ortholog in red lip mullet (Liza haematocheila).

The transcription factor, activator protein-1 (AP-1), is a dimeric protein and a downstream member of the mitogen-activated protein kinase (MAPK) signaling pathway. It regulates a wide array of functions including, cell proliferation, survival, differentiation, response to UV-irradiation, immune responses, and inflammatory conditions. AP-1 belongs to the basic leucine zipper (bZIP) protein family, which consists of members from Jun, Fos, Maf, and ATF subfamilies. In the present study, c-Jun and c-Fos homologs were identified from a transcriptome database of Liza haematocheila and designated as Lhc-Jun and Lhc-Fos. In both sequences, the signature bZIP domain was identified and also the DNA binding sites, dimerization sites, as well as the phosphorylation sites, were found to be highly conserved through evolution. Tissue distribution analysis revealed that both Lhc-Jun and Lhc-Fos transcripts were ubiquitously expressed in all examined tissues of healthy mullets. In order to determine the transcriptional modulations of Lhc-Jun and Lhc-Fos, challenge experiments were carried out using LPS, poly I:C, and L. garvieae. The qRT-PCR analysis revealed significant upregulation of Lhc-Jun and Lhc-Fos in blood, gill, liver, and spleen. This is the first study that explores the correlation between UV-irradiation and AP-1 ortholog expression in teleosts. Also, this is the first time that the functional characterization of the teleost c-Fos ortholog has been carried out. Sub-cellular localization of Lhc-Jun and Lhc-Fos was observed in the nucleus. AP-1-Luc reporter assays revealed significant higher luciferase activities in both Lhc-Jun and Lhc-Fos proteins compared to mock controls. These results strongly suggest that Lhc-Jun and Lhc-Fos might play a significant role in Liza haematocheila immunity by regulating AP-1 promoter sequences in immune and stress-related genes.

The AP-1 transcriptional complex: Local switch or remote command?

The ubiquitous family of AP-1 dimeric transcription complexes is involved in virtually all cellular and physiological functions. It is paramount for cells to reprogram gene expression in response to cues of many sorts and is involved in many tumorigenic processes. How AP-1 controls gene transcription has largely remained elusive till recently. The advent of the "omics" technologies permitting genome-wide studies of transcription factors has however changed and improved our view of AP-1 mechanistical actions. If these studies confirm that AP-1 can sometimes act as a local transcriptional switch operating in the vicinity of transcription start sites (TSS), they strikingly indicate that AP-1 principally operates as a remote command binding to distal enhancers, placing chromatin architecture dynamics at the heart of its transcriptional actions. They also unveil novel constraints operating on AP-1, as well as novel mechanisms used to regulate gene expression via transcription-pioneering-, chromatin-remodeling- and chromatin accessibility maintenance effects.

Structural basis for effects of CpA modifications on C/EBPß binding of DNA.

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPß binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPß DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPß. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

Activator Protein-1: redox switch controlling structure and DNA-binding.

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the 'OFF' state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.

Tethering not required: the glucocorticoid receptor binds directly to activator protein-1 recognition motifs to repress inflammatory genes.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls the expression of extensive gene networks, driving both up- and down-regulation. GR utilizes multiple DNA-binding-dependent and -independent mechanisms to achieve context-specific transcriptional outcomes. The DNA-binding-independent mechanism involves tethering of GR to the pro-inflammatory transcription factor activator protein-1 (AP-1) through protein-protein interactions. This mechanism has served as the predominant model of GR-mediated transrepression of inflammatory genes. However, ChIP-seq data have consistently shown GR to occupy AP-1 response elements (TREs), even in the absence of AP-1. Therefore, the current model is insufficient to explain GR action at these sites. Here, we show that GR regulates a subset of inflammatory genes in a DNA-binding-dependent manner. Using structural biology and biochemical approaches, we show that GR binds directly to TREs via sequence-specific contacts to a GR-binding sequence (GBS) half-site found embedded within the TRE motif. Furthermore, we show that GR-mediated transrepression observed at TRE sites to be DNA-binding-dependent. This represents a paradigm shift in the field, showing that GR uses multiple mechanisms to suppress inflammatory gene expression. This work further expands our understanding of this complex multifaceted transcription factor.

Methyl-dependent and spatial-specific DNA recognition by the orthologous transcription factors human AP-1 and Epstein-Barr virus Zta.

T: Activator protein 1 (AP-1) is a transcription factor that recognizes two versions of a 7-base pair response element, either 5΄- GAG CA-3΄ or 5΄- GAG CA-3΄ (where M = 5-methylcytosine). These two elements share the feature that 5-methylcytosine and thymine both have a methyl group in the same position, 5-carbon of the pyrimidine, so each of them has two methyl groups at nucleotide positions 1 and 5 from the 5΄ end, resulting in four methyl groups symmetrically positioned in duplex DNA. Epstein-Barr Virus Zta is a key transcriptional regulator of the viral lytic cycle that is homologous to AP-1. Zta recognizes several methylated Zta-response elements, including meZRE1 (5΄- GAG C A-3΄) and meZRE2 (5΄- GAG G A-3΄), where a methylated cytosine occupies one of the inner thymine residues corresponding to the AP-1 element, resulting in the four spatially equivalent methyl groups. Here, we study how AP-1 and Zta recognize these methyl groups within their cognate response elements. These methyl groups are in van der Waals contact with a conserved di-alanine in AP-1 dimer (Ala265 and Ala266 in Jun), or with the corresponding Zta residues Ala185 and Ser186 (via its side chain carbon Cß atom). Furthermore, the two ZRE elements differ at base pair 6 (C:G versus G:C), forming a pseudo-symmetric sequence (meZRE1) or an asymmetric sequence (meZRE2). In vitro DNA binding assays suggest that Zta has high affinity for all four sequences examined, whereas AP-1 has considerably reduced affinity for the asymmetric sequence (meZRE2). We ascribe this difference to Zta Ser186 (a unique residue for Zta) whose side chain hydroxyl oxygen atom interacts with the two half sites differently, whereas the corresponding Ala266 of AP-1 Jun protein lacks such flexibility. Our analyses demonstrate a novel mechanism of 5mC/T recognition in a methylation-dependent, spatial and sequence-specific approach by basic leucine-zipper transcriptional factors.

Baicalein reduces angiogenesis in the inflammatory microenvironment via inhibiting the expression of AP-1.

Increasing clinical and experimental studies have demonstrated that refractory chronic inflammation will result in malignant tumor and anti-angiogenic therapy may be an effective way to thwart the progression. Baicalein, one of the major active flavanoids found in Scutellaria baicalensis Georgi, has been exhibited potent anti-inflammation and anti-tumor effects by reducing angiogenesis. However, the exact mechanism of baicalein on endothelial cells in inflammatory microenvironment was not clear yet. Here, we investigated the anti-angiogenic effect of baicalein by incubating human umbilical vein endothelial cells (HUVECs) with THP-1 conditioned medium in vitro. The tube formation of HUVECs and microvessel outgrowth of rat aorta were attenuated, as well as the number of newly formed blood vessels in chicken chorioallantoic membrane (CAM) was reduced by baicalein. This anti-angiogenic effect was mainly on account of the inhibited motility, migration and invasion of HUVECs. In addition, mechanistic studies showed that baicalein could bind to AP-1 directly and the expression of c-Jun and c-Fos in HUVECs was reduced, accompanied by their increased proteasomal degradation. Besides, baicalein suppressed the nuclear translation, heterodimer formation and DNA binding affinity of c-Jun and c-Fos. What's more, the anti-angiogenic effect of baicalein was further confirmed by matrigel plug assay in vivo. Taken together, our study demonstrated that baicalein could exert its anti-angiogenic effect in the inflammation microenvironment via inhibiting the transcriptional activity of AP-1, which suggested that baicalein might be an alternative treatment against refractory chronic inflammation.

Integrated microfluidic approach for quantitative high-throughput measurements of transcription factor binding affinities.

Protein binding to DNA is a fundamental process in gene regulation. Methodologies such as ChIP-Seq and mapping of DNase I hypersensitive sites provide global information on this regulation in vivo In vitro methodologies provide valuable complementary information on protein-DNA specificities. However, current methods still do not measure absolute binding affinities. There is a real need for large-scale quantitative protein-DNA affinity measurements. We developed QPID, a microfluidic application for measuring protein-DNA affinities. A single run is equivalent to 4096 gel-shift experiments. Using QPID, we characterized the different affinities of ATF1, c-Jun, c-Fos and AP-1 to the CRE consensus motif and CRE half-site in two different genomic sequences on a single device. We discovered that binding of ATF1, but not of AP-1, to the CRE half-site is highly affected by its genomic context. This effect was highly correlated with ATF1 ChIP-seq and PBM experiments. Next, we characterized the affinities of ATF1 and ATF3 to 128 genomic CRE and CRE half-site sequences. Our affinity measurements explained that in vivo binding differences between ATF1 and ATF3 to CRE and CRE half-sites are partially mediated by differences in the minor groove width. We believe that QPID would become a central tool for quantitative characterization of biophysical aspects affecting protein-DNA binding.

Identification and characterization a novel transcription factor activator protein-1 in the sea cucumber Apostichopus japonicus.

The transcription factor activator protein-1 (AP-1) is an important gene expression regulator with typical Jun and region-leucine zipper (bZIP) domains and can respond to a plethora of physiological and pathological stimulus. In this study, we identified a novel AP-1 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjAP-1). The full-length of AjAP-1 was of 2944 bp including a 5' untranslated region (UTR) of 201 bp, a 3' UTR of 1753 bp and a putative open reading frame of 990 bp encoding a polypeptide of 329 amino acid residues. Two representative domains of Jun and bZIP as well as two nuclear localization signals (NLSs) were also detected in deduced amino acid of AjAP-1. Spatial distribution expression indicated that AjAP-1 was ubiquitously expressed in all examined tissues with predominant expression in the body wall, moderate in the tube feet, respiratory tree and colemocytes and slightly weak in the intestine and longitudinal muscle. Time-course expression analysis in intestine and coelomocytes revealed that AjAP-1 both reached its peak expression at 4 h after Vibrio splendidus challenge with a 2.6 and 8.2-fold increase compared to their control groups, respectively. Taken together, all these results suggested that AjAP-1 was a novel immune factor and might be involved in the processes of anti-bacteria response in sea cucumber.

Imaging Fos-Jun transcription factor mobility and interaction in live cells by single plane illumination-fluorescence cross correlation spectroscopy.

We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.

Small molecule inhibitors targeting activator protein 1 (AP-1).

Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases.

Hepatitis C virus core+1/ARF protein decreases hepcidin transcription through an AP1 binding site.

Chronic viral hepatitis C is characterized by iron accumulation in the liver, and hepcidin regulates iron absorption. Hepatitis C virus (HCV) core+1/ARFP is a novel protein produced by a second functional ORF within the core gene. Here, using reporter assays and HCV bicistronic replicons, we show that, similarly to core, core+1/ARFP decreases hepcidin expression in hepatoma cells. The activator protein 1 (AP1) binding site of the human hepcidin promoter, shown here to be relevant to basal promoter activity and to the repression by core, is essential for the downregulation by core+1/ARFP while the previously described C/EBP (CCAAT/enhancer binding protein) and STAT (signal transducer and activator of transcription) sites are not. Consistently, expression of the AP1 components c-jun and c-fos obliterated the repressive effect of core and core+1/ARFP. In conclusion, we provide evidence that core+1/ARFP downregulates AP1-mediated transcription, providing new insights into the biological role of core+1/ARFP, as well as the transcriptional modulation of hepcidin, the main regulator of iron metabolism.

Transcriptional regulation of the mouse CD11c promoter by AP-1 complex with JunD and Fra2 in dendritic cells.

CD11c, a member of the ß(2) integrin family of adhesion molecule, is expressed on the surface of myeloid lineages and activated lymphoid cells and forms a heterodimeric receptor with CD18. We analyzed the mouse CD11c promoter structure to elucidate the transcriptional regulation in dendritic cells (DCs). By reporter assay, the -84/-65 region was identified to be essential for activity of the mouse CD11c promoter in the mouse bone marrow-derived (BM) DCs and monocyte cell line RAW264.7. An electrophoretic mobility shift assay using a number of antibodies against transcription factors revealed that the target region was recognized by a complex including JunD and Fra2, which are transcription factors belonging to the AP-1 family. The direct interaction of JunD and Fra2 with the CD11c promoter was further confirmed by a chromatin immunoprecipitation assay using CD11c-positive cells purified from BMDCs. Finally, mouse JunD and/or Fra2 siRNA was introduced into BMDCs to evaluate the involvement of these factors against CD11c transcription and found that Fra2 siRNA reduced cell surface expression level of CD11c. These results indicate that AP-1 composed with JunD and Fra2 protein plays a primary role in enhancing the transcription level of the CD11c gene in DC.

Regulation and control of AP-1 binding activity in embryotoxicity.

The electrophoretic mobility shift assay (EMSA) is a sensitive relatively straightforward methodology used to detect sequence-specific DNA-protein interactions. It is the fundamental procedure of several variants that allow qualitative and quantitative assessments of protein-nucleic acid complexes. Classically, nuclear proteins and DNA are combined and the resulting mixture is electrophoretically separated in polyacrylamide or agarose gel under native conditions. The distribution within the gel is generally detected with autoradiography of the ³²P-labeled DNA. The underlying principle is that nucleic acid with protein bound to it will migrate more slowly through a gel matrix than the free nucleic acid. In this chapter, a representative protocol is described that addresses specific challenges of using whole embryos as the nuclear protein source, and the most common and informative EMSA variant, the "supershift," is also presented. The important points are underscored and approaches for troubleshooting are explained. References are provided for alternative methods and extensions of the basic protocol.

RAS/ERK pathway transcriptional regulation through ETS/AP-1 binding sites.

The RAS/RAF/MEK/ERK signaling pathway is activated by mutation in many cancers. Neighboring ETS and AP-1 DNA binding sequences can act as response elements for transcriptional activation by this pathway. ERK phosphorylation of an ETS transcription factor is one mechanism of activating the RAS/ERK gene expression program that can promote cancer cell phenotypes such as proliferation, invasion, and metastasis. Recent genome-wide mapping of ETS proteins over-expressed by chromosomal rearrangement in prostate cancer reveals a second mechanism for activation of this gene expression program. An oncogenic subset of ETS transcription factors can activate RAS/ERK target genes even in the absence of RAS/ERK pathway activation by binding ETS/AP-1 sequences. Thus, regulation of cancer cell invasion and metastasis via ETS/AP-1 sequence elements depends on which ETS protein is bound, and the status of the RAS/ERK pathway. This commentary will focus on what is known about the selectivity of ETS/AP-1 sequences for different ETS transcription factors and the transcriptional consequences of ETS protein selection.

Midkine upregulates MICA/B expression in human gastric cancer cells and decreases natural killer cell cytotoxicity.

Midkine (MK) is a heparin-binding growth factor overexpressed in various human cancers. In the current study, a positive correlation was observed between MK expression and MICA/B serum levels of gastric cancer patients. In addition, MK transfection significantly increased MICA/B expression in gastric cancer cells. The soluble MICA/B expression was also elevated. Furthermore, MK transfection inhibited CD107a and Granzyme B expression, thereby suppressing the natural killer (NK) cell cytotoxicity in vitro. The phosphorylation of p38 MAPK and its promotion of CHOP expression were also observed after MK treatment and transfection. CHOP was indirectly bound to the MICA/B promoter region by interacting with AP-1, leading to MICA/B transcription. Overall, the current study shows that MK expression in tumor cells indirectly suppresses NK cytotoxicity by inducing MICA/B expression and suppressing NKG2D expression.

Binding site specificity and factor redundancy in activator protein-1-driven human papillomavirus chromatin-dependent transcription.

Activator protein-1 (AP-1) regulates diverse gene responses triggered by environmental cues and virus-induced cellular stress. Although many signaling events leading to AP-1 activation have been described, the fundamental features underlying binding site selection and factor recruitment of dimeric AP-1 complexes to their target genes remain mostly uncharacterized. Using recombinant full-length human AP-1 dimers formed between c-Jun and Fos family members (c-Fos, FosB, Fra-1, Fra-2) for DNA binding and transcriptional analysis, we found that each of these AP-1 complex exhibits differential activity for distinct non-consensus AP-1 sites present in human papillomavirus (HPV), and each AP-1 complex is capable of activating transcription from in vitro-reconstituted HPV chromatin in a p300- and acetyl-CoA-dependent manner. Transcription from HPV chromatin requires AP-1-dependent and contact-driven recruitment of p300. Acetylation of dimeric AP-1 complexes by p300 enhances AP-1 binding to DNA. Using a human C-33A cervical cancer-derived cell line harboring the episomal HPV type 11 genome, we illustrate binding site selectivity recognized by c-Jun, JunB, JunD, and various Fos family members in a combinatorial and unique pattern, highlighting the diversity and importance of non-canonical binding site recognition by various AP-1 family proteins.