A validated regulatory network for Th17 cell specification.

Th17 cells have critical roles in mucosal defense and are major contributors to inflammatory disease. Their differentiation requires the nuclear hormone receptor RORγt working with multiple other essential transcription factors (TFs). We have used an iterative systems approach, combining genome-wide TF occupancy, expression profiling of TF mutants, and expression time series to delineate the Th17 global transcriptional regulatory network. We find that cooperatively bound BATF and IRF4 contribute to initial chromatin accessibility and, with STAT3, initiate a transcriptional program that is then globally tuned by the lineage-specifying TF RORγt, which plays a focal deterministic role at key loci. Integration of multiple data sets allowed inference of an accurate predictive model that we computationally and experimentally validated, identifying multiple new Th17 regulators, including Fosl2, a key determinant of cellular plasticity. This interconnected network can be used to investigate new therapeutic approaches to manipulate Th17 functions in the setting of inflammatory disease.

Liver cancer development driven by the AP-1/c-Jun~Fra-2 dimer through c-Myc.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.

Iron homeostasis proteins Grx4 and Fra2 control activity of the Schizosaccharomyces pombe iron repressor Fep1 by facilitating [2Fe-2S] cluster removal.

The Bol2 homolog Fra2 and monothiol glutaredoxin Grx4 together play essential roles in regulating iron homeostasis in Schizosaccharomyces pombe. In vivo studies indicate that Grx4 and Fra2 act as coinhibitory partners that inactivate the transcriptional repressor Fep1 in response to iron deficiency. In Saccharomyces cerevisiae, Bol2 is known to form a [2Fe-2S]-bridged heterodimer with the monothiol Grxs Grx3 and Grx4, with the cluster ligands provided by conserved residues in Grx3/4 and Bol2 as well as GSH. In this study, we characterized this analogous [2Fe-2S]-bridged Grx4-Fra2 complex in S. pombe by identifying the specific residues in Fra2 that act as ligands for the Fe-S cluster and are required to regulate Fep1 activity. We present spectroscopic and biochemical evidence confirming the formation of a [2Fe-2S]-bridged Grx4-Fra2 heterodimer with His66 and Cys29 from Fra2 serving as Fe-S cluster ligands in S. pombe. In vivo transcription and growth assays confirm that both His66 and Cys29 are required to fully mediate the response of Fep1 to low iron conditions. Furthermore, we analyzed the interaction between Fep1 and Grx4-Fra2 using CD spectroscopy to monitor changes in Fe-S cluster coordination chemistry. These experiments demonstrate unidirectional [2Fe-2S] cluster transfer from Fep1 to Grx4-Fra2 in the presence of GSH, revealing the Fe-S cluster dependent mechanism of Fep1 inactivation mediated by Grx4 and Fra2 in response to iron deficiency.

A systematic comparison of FOSL1, FOSL2 and BATF-mediated transcriptional regulation during early human Th17 differentiation.

Th17 cells are essential for protection against extracellular pathogens, but their aberrant activity can cause autoimmunity. Molecular mechanisms that dictate Th17 cell-differentiation have been extensively studied using mouse models. However, species-specific differences underscore the need to validate these findings in human. Here, we characterized the human-specific roles of three AP-1 transcription factors, FOSL1, FOSL2 and BATF, during early stages of Th17 differentiation. Our results demonstrate that FOSL1 and FOSL2 co-repress Th17 fate-specification, whereas BATF promotes the Th17 lineage. Strikingly, FOSL1 was found to play different roles in human and mouse. Genome-wide binding analysis indicated that FOSL1, FOSL2 and BATF share occupancy over regulatory regions of genes involved in Th17 lineage commitment. These AP-1 factors also share their protein interacting partners, which suggests mechanisms for their functional interplay. Our study further reveals that the genomic binding sites of FOSL1, FOSL2 and BATF harbour hundreds of autoimmune disease-linked SNPs. We show that many of these SNPs alter the ability of these transcription factors to bind DNA. Our findings thus provide critical insights into AP-1-mediated regulation of human Th17-fate and associated pathologies.

Chromatin accessibility uncovers KRAS-driven FOSL2 promoting pancreatic ductal adenocarcinoma progression through up-regulation of CCL28.

BACKGROUND: The epigenetic mechanisms involved in the progression of pancreatic ductal adenocarcinoma (PDAC) remain largely unexplored. This study aimed to identify key transcription factors (TFs) through multiomics sequencing to investigate the molecular mechanisms of TFs that play critical roles in PDAC. METHODS: To characterise the epigenetic landscape of genetically engineered mouse models (GEMMs) of PDAC with or without KRAS and/or TP53 mutations, we employed ATAC-seq, H3K27ac ChIP-seq, and RNA-seq. The effect of Fos-like antigen 2 (FOSL2) on survival was assessed using the Kaplan-Meier method and multivariate Cox regression analysis for PDAC patients. To study the potential targets of FOSL2, we performed Cleavage Under Targets and Tagmentation (CUT&Tag). To explore the functions and underlying mechanisms of FOSL2 in PDAC progression, we employed several assays, including CCK8, transwell migration and invasion, RT-qPCR, Western blotting analysis, IHC, ChIP-qPCR, dual-luciferase reporter, and xenograft models. RESULTS: Our findings indicated that epigenetic changes played a role in immunosuppressed signalling during PDAC progression. Moreover, we identified FOSL2 as a critical regulator that was up-regulated in PDAC and associated with poor prognosis in patients. FOSL2 promoted cell proliferation, migration, and invasion. Importantly, our research revealed that FOSL2 acted as a downstream target of the KRAS/MAPK pathway and recruited regulatory T (Treg) cells by transcriptionally activating C-C motif chemokine ligand 28 (CCL28). This discovery highlighted the role of an immunosuppressed regulatory axis involving KRAS/MAPK-FOSL2-CCL28-Treg cells in the development of PDAC. CONCLUSION: Our study uncovered that KRAS-driven FOSL2 promoted PDAC progression by transcriptionally activating CCL28, revealing an immunosuppressive role for FOSL2 in PDAC.

Long non-coding RNA MIR17HG impedes FOSL2-mediated transcription activation of HIC1 to maintain a pro-inflammatory phenotype of microglia during intracerebral haemorrhage.

Activation and polarization of microglia play decisive roles in the progression of intracerebral haemorrhage (ICH), and lactate exposure correlates with microglia polarization. This study explores molecules influencing lactate production and microglia phenotype alteration following ICH. A murine model of ICH was induced by intracerebral injection of collagenase. The mice experienced autonomous neurological function recovery, haematoma resolution and rapid lactate production, along with a gradual increase in angiogenesis activity, neuronal recovery and an M1-to-M2 phenotype change of microglia. Galloflavin, a lactate dehydrogenase antagonist, suppressed this phenotype change and the functional recovery in mice. FOS like 2 (FOSL2) was significantly upregulated in the brain tissues from day 7 post-ICH. Overexpression of FOSL2 induced an M1-to-M2 phenotype shift in microglia and accelerated lactate production in vivo and in haemoglobin-treated microglia in vitro. Long non-coding RNA MIR17HG impeded FOSL2-mediated transcription activation of hypermethylated in cancer 1 (HIC1). MIR17HG overexpression induced pro-inflammatory activation of microglia in mice, which was blocked by further HIC1 overexpression. Overall, this study demonstrates that MIR17HG maintains a pro-inflammatory phenotype of microglia during ICH progression by negating FOSL2-mediated transcription activation of HIC1. Specific inhibition of MIR17HG or upregulation of FOSL2 or HIC1 may favour inflammation inhibition and haematoma resolution in ICH.

Fra-1 regulates its target genes via binding to remote enhancers without exerting major control on chromatin architecture in triple negative breast cancers.

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.

Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2.

Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation.

Upregulation of microRNA-597 in myelodysplastic syndromes induces apoptosis through FOSL2 inhibition.

INTRODUCTION: Dysregulation of microRNAs (miRNAs) has been associated with the pathophysiology of myelodysplastic syndrome (MDS). Trisomy 8 is the most frequent chromosomal abnormalities in Korean patients with MDS. We investigated the dysregulation of miR-597-5p, located on chromosome 8, which is reported to induce cell death in numerous cancers. MATERIALS AND METHODS: We compared the expression profiles of miR-597-5p among 65 MDS patients and 11 controls, and analyzed the in vitro effects of miR-597 on leukemic cells using an acute myeloid leukemia cell line transfected with miR-597. RESULTS: We found that miR-597-5p levels were upregulated 4.05-fold in MDS patients compared to those in controls. In vitro study results demonstrated that transfection with a miR-597 mimic induced apoptosis through downregulation of FOS like 2 (FOSL2). CONCLUSION: These findings suggest that upregulation of miR-597 induces apoptosis and that miR-597 has a possible role in the pathophysiology of MDS.

Alternatively activated macrophages promote airway inflammation through JAK3-STAT5-Fra2 in asthma.

BACKGROUND: Fos-related antigen-2 (Fra-2) is a transcription factor belonging to the activator protein 1 (AP-1) family, which is associated with many chronic airway diseases such as asthma. Alternatively activated (M2) macrophages are associated with Fra2 in airway diseases such as pulmonary fibrosis. However, there is no specific study that explores the relationship between M2 macrophages and Fra2 in asthma. OBJECTIVE: We hypothesized that a potential mechanism of allergic asthma could be that Fra2 is highly expressed in M2 macrophages through JAK3-STAT5 and facilitates the production of downstream T-helper 2 (Th2) cytokines, thus promoting the pathogenesis of asthma. METHODS: Peripheral venous blood and airway tissue samples of patients with asthma and controls were obtained. Moreover, a C57BL/6 mouse model of asthma was established. Fra2 expression was detected using immunohistochemistry and immunofluorescence. Macrophages were obtained by flow sorting, and expression of the JAK3-STAT5-Fra2 signaling pathway was determined using PCR and western blotting. Enzyme-linked immunosorbent assay was used to determine M2 macrophage-associated Th2-type cytokine levels. RESULTS: Fra2 was highly expressed in patients with asthma and asthmatic mice. The JAK3-STAT5 was a signal pathway related to the high expression of Fra2 in M2 macrophages. Moreover, we found that Fra2 could affect the production of Th2 cytokines downstream of M2 macrophages, including interleukin 4 (IL-4) and IL-13. CONCLUSION: M2 macrophages could promote airway inflammation through JAK3-STAT5-Fra2 to induce allergic asthma. Our study offers a new insight to further understand the pathogenesis of asthma and also provides a new direction for targeted treatment.

Pulmonary fibrosis in Fra-2 transgenic mice is associated with decreased numbers of alveolar macrophages and increased susceptibility to pneumococcal pneumonia.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae, bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1ß, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae. Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

KLRD1, FOSL2 and LILRB3 as potential biomarkers for plaques progression in acute myocardial infarction and stable coronary artery disease.

BACKGROUND: Myocardial infarction (MI) contributes to high mortality and morbidity and can also accelerate atherosclerosis, thus inducing recurrent event due to status changing of coronary artery walls or plaques. The research aimed to investigate the differentially expressed genes (DEGs), which may be potential therapeutic targets for plaques progression in stable coronary artery disease (CAD) and ST-elevated MI (STEMI). METHODS: Two human datasets (GSE56885 and GSE59867) were analyzed by GEO2R and enrichment analysis was applied through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. To explore the seed genes, the protein-protein interaction (PPI) network was constructed and seed genes, as well as top30 ranking neighbours were screened out. To validate these findings, one human dataset GSE120521 was analyzed. Linear regression analysis and ROC curve were also performed to determine which seed genes above mentioned could be independent factors for plaques progression. Mice MI model and ELISA of seed genes were applied and ROC curve was also performed for in vivo validation. RESULTS: 169 DEGs and 573 DEGs were screened out in GSE56885 and GSE59867, respectively. Utilizing GO and KEGG analysis, these DEGs mainly enriched in immune system response and cytokines interaction. PPI network analysis was carried out and 19 seed genes were screened out. To validate these findings, GSE120521 was analyzed and three genes were demonstrated to be targets for plaques progression and stable CAD progression, including KLRD1, FOSL2 and LILRB3. KLRD1 and LILRB3 were demonstrated to be high-expressed at 1d after MI compared to SHAM group and FOSL2 expression was low-expressed at 1d and 1w. To investigate the diagnostic abilities of seed genes, ROC analysis was applied and the AUCs of KLRD1, FOSL2 and LILRB3, were 0.771, 0.938 and 0.972, respectively. CONCLUSION: This study provided the screened seed genes, KLRD1, FOSL2 and LILRB3, as credible molecular biomarkers for plaques status changing in CAD progression and MI recurrence. Other seed genes, such as FOS, SOCS3 and MCL1, may also be potential targets for treatment due to their special clinical value in cardiovascular diseases.

Circ_0091581 Promotes the Progression of Hepatocellular Carcinoma Through Targeting miR-591/FOSL2 Axis.

BACKGROUND: Circular RNAs (circRNAs) have shown crucial regulatory roles in cancer biology. We aimed to uncover the role and underlying mechanism of circ_0091581 in hepatocellular carcinoma (HCC) progression. METHODS: The abundance of circ_0091581, microRNA-591 (miR-591) and FOS like 2, AP-1 transcription factor subunit (FOSL2) was measured by quantitative real-time polymerase chain reaction. Cell viability, colony formation ability, and invasion ability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay, and transwell invasion assay. The migration ability was analyzed by transwell migration assay and wound healing assay. Flow cytometry was used to evaluate the cell cycle and apoptosis of HCC cells. The interaction between miR-591 and circ_0091581 or FOSL2 was predicted by Circular RNA Interactome database or TargetScan database and confirmed by dual-luciferase reporter assay and RNA immune co-precipitation assay. FOSL2 protein expression was measured by Western blot assay. Xenograft tumor assay was conducted to analyze the role of circ_0091581 in HCC tumor growth in vivo. RESULTS: Circ_0091581 was highly expressed in HCC tissue samples and cell lines in contrast to that in adjacent normal tissue samples and THLE-2 cell line. Circ_0091581 accelerated the viability, colony formation, metastasis, and cell cycle, while it impeded the apoptosis of HCC cells. MiR-591 bound to circ_0091581, and circ_0091581 knockdown-mediated effects in HCC cells were largely overturned by miR-591 silencing. FOSL2 was a target of miR-591, and FOSL2 overexpression largely reversed miR-591 accumulation-induced influences in HCC cells. FOSL2 protein expression was down-regulated by circ_0091581 silencing, and the addition of miR-591 inhibitor partly recovered the expression of FOSL2 in HCC cells. Circ_0091581 interference notably suppressed HCC tumor growth in vivo. CONCLUSION: Circ_0091581 acted as an oncogene to enhance the viability, colony formation, metastasis and cell cycle and inhibit the apoptosis of HCC cells through targeting miR-591/FOSL2 axis.

The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis.

BACKGROUND: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. METHODS AND RESULTS: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-ß. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-ß-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. CONCLUSION: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-ß-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

Unifying mechanism for different fibrotic diseases.

Fibrotic diseases are not well-understood. They represent a number of different diseases that are characterized by the development of severe organ fibrosis without any obvious cause, such as the devastating diseases idiopathic pulmonary fibrosis (IPF) and scleroderma. These diseases have a poor prognosis comparable with endstage cancer and are uncurable. Given the phenotypic differences, it was assumed that the different fibrotic diseases also have different pathomechanisms. Here, we demonstrate that many endstage fibrotic diseases, including IPF; scleroderma; myelofibrosis; kidney-, pancreas-, and heart-fibrosis; and nonalcoholic steatohepatosis converge in the activation of the AP1 transcription factor c-JUN in the pathologic fibroblasts. Expression of the related AP1 transcription factor FRA2 was restricted to pulmonary artery hypertension. Induction of c-Jun in mice was sufficient to induce severe fibrosis in multiple organs and steatohepatosis, which was dependent on sustained c-Jun expression. Single cell mass cytometry revealed that c-Jun activates multiple signaling pathways in mice, including pAkt and CD47, which were also induced in human disease. αCD47 antibody treatment and VEGF or PI3K inhibition reversed various organ c-Jun-mediated fibroses in vivo. These data suggest that c-JUN is a central molecular mediator of most fibrotic conditions.

The AP-1 transcription factor component Fosl2 potentiates the rate of myocardial differentiation from the zebrafish second heart field.

The vertebrate heart forms through successive phases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heart field (FHF) progenitors assemble the linear heart tube. Thereafter, second heart field (SHF) progenitors differentiate into cardiomyocytes that are accreted to the poles of the heart tube over a well-defined developmental window. Although heart tube elongation deficiencies lead to life-threatening congenital heart defects, the variables controlling the initiation, rate and duration of myocardial accretion remain obscure. Here, we demonstrate that the AP-1 transcription factor, Fos-like antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. fosl2 mutants initiate accretion appropriately, but cardiomyocyte production is sluggish, resulting in a ventricular deficit coupled with an accumulation of SHF progenitors. Surprisingly, mutant embryos eventually correct the myocardial deficit by extending the accretion window. Overexpression of Fosl2 also compromises production of SHF-derived ventricular cardiomyocytes, a phenotype that is consistent with precocious depletion of the progenitor pool. Our data implicate Fosl2 in promoting the progenitor to cardiomyocyte transition and uncover the existence of regulatory mechanisms to ensure appropriate SHF-mediated cardiomyocyte contribution irrespective of embryonic stage.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer.

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

BRD4 localization to lineage-specific enhancers is associated with a distinct transcription factor repertoire.

Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.

Fra-2 is a novel candidate drug target expressed in the podocytes of lupus nephritis.

Lupus nephritis (LN) is a common and devastating complication caused by systemic lupus erythematosus. In this study, we evaluated the expression and mechanism of Fos-related antigen 2 (Fra-2) in LN. The results showed that Fra-2 was significantly increased in kidney biopsies of LN patients compared with healthy controls and other kidney disease in glomerular podocytes. The MRL/lpr mouse strain is a murine model of lupus, and it was used to study the mechanisms of Fra-2 in LN. The results showed that Fra-2 was expressed in the glomerular podocytes. We investigated the effects of inflammatory stimuli on Fra-2 protein expression in the glomerular podocytes, and found that interferon gamma was most effective at increasing Fra-2 protein expression. Knockdown of Fra-2 using siRNA enhanced the protein expression of nephrin. Therefore, Fra-2 may be a specific drug target for podocyte injury in LN.

Regulation of Hspb7 by MEF2 and AP-1: implications for Hspb7 in muscle atrophy.

Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) transcription complexes have been individually implicated in myogenesis, but their genetic interaction has not previously been addressed. Using MEF2A, c-Jun and Fra-1 chromatin immunoprecipitation sequencing (ChIP-seq) data and predicted AP-1 consensus motifs, we identified putative common MEF2 and AP-1 target genes, several of which are implicated in regulating the actin cytoskeleton. Because muscle atrophy results in remodelling or degradation of the actin cytoskeleton, we characterized the expression of putative MEF2 and AP-1 target genes (Dstn, Flnc, Hspb7, Lmod3 and Plekhh2) under atrophic conditions using dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock protein b7 (Hspb7) was induced by Dex treatment and further analyses revealed that loss of MEF2A using siRNA prevented Dex-regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun expression reduced Dex-mediated upregulation of Hspb7 whereas AP-1 depletion enhanced Hspb7 expression. In vivo, expression of Hspb7 and other autophagy-related genes was upregulated in response to atrophic conditions in mice. Manipulation of Hspb7 levels in mice also impacted gross muscle mass. Collectively, these data indicate that MEF2 and AP-1 confer antagonistic regulation of Hspb7 gene expression in skeletal muscle, with implications for autophagy and muscle atrophy.