Regulatory Nodes Research Articles

Neuronal programming by microbiota regulates intestinal physiology.

Neural control of the function of visceral organs is essential for homeostasis and health. Intestinal peristalsis is critical for digestive physiology and host defence, and is often dysregulated in gastrointestinal disorders1. Luminal factors, such as diet and microbiota, regulate neurogenic programs of gut motility2-5, but the underlying molecular mechanisms remain unclear. Here we show that the transcription factor aryl hydrocarbon receptor (AHR) functions as a biosensor in intestinal neural circuits, linking their functional output to the microbial environment of the gut lumen. Using nuclear RNA sequencing of mouse enteric neurons that represent distinct intestinal segments and microbiota states, we demonstrate that the intrinsic neural networks of the colon exhibit unique transcriptional profiles that are controlled by the combined effects of host genetic programs and microbial colonization. Microbiota-induced expression of AHR in neurons of the distal gastrointestinal tract enables these neurons to respond to the luminal environment and to induce expression of neuron-specific effector mechanisms. Neuron-specific deletion of Ahr, or constitutive overexpression of its negative feedback regulator CYP1A1, results in reduced peristaltic activity of the colon, similar to that observed in microbiota-depleted mice. Finally, expression of Ahr in the enteric neurons of mice treated with antibiotics partially restores intestinal motility. Together, our experiments identify AHR signalling in enteric neurons as a regulatory node that integrates the luminal environment with the physiological output of intestinal neural circuits to maintain gut homeostasis and health.

Nuclear Factor Kappa B Signaling Complexes in Acute Inflammation.

Significance: Nuclear factor kappa B (NF-κB) is a master regulator of the inflammatory response and represents a key regulatory node in the complex inflammatory signaling network. In addition, selective NF-κB transcriptional activity on specific target genes occurs through the control of redox-sensitive NF-κB interactions. Recent Advances: The selective NF-κB response is mediated by redox-modulated NF-κB complexes with ribosomal protein S3 (RPS3), Pirin (PIR). cAMP response element-binding (CREB)-binding protein (CBP)/p300, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), activator protein-1 (AP-1), signal transducer and activator of transcription 3 (STAT3), early growth response protein 1 (EGR-1), and SP-1. NF-κB is cooperatively coactivated with AP-1, STAT3, EGR-1, and SP-1 during the inflammatory process, whereas NF-κB complexes with CBP/p300 and PGC-1α regulate the expression of antioxidant genes. PGC-1α may act as selective repressor of phospho-p65 toward interleukin-6 (IL-6) in acute inflammation. p65 and nuclear factor erythroid 2-related factor 2 (NRF2) compete for binding to coactivator CBP/p300 playing opposite roles in the regulation of inflammatory genes. S-nitrosylation or tyrosine nitration favors the recruitment of specific NF-κB subunits to κB sites. Critical Issues: NF-κB is a redox-sensitive transcription factor that forms specific signaling complexes to regulate selectively the expression of target genes in acute inflammation. Protein-protein interactions with coregulatory proteins, other transcription factors, and chromatin-remodeling proteins provide transcriptional specificity to NF-κB. Furthermore, different NF-κB subunits may form distinct redox-sensitive homo- and heterodimers with distinct affinities for κB sites. Future Directions: Further research is required to elucidate the whole NF-κB interactome to fully characterize the complex NF-κB signaling network in redox signaling, inflammation, and cancer.

MicroRNA-184 is induced by store-operated calcium entry and regulates early keratinocyte differentiation.

Extracellular calcium (Ca2+ ) and store-operated Ca2+ entry (SOCE) govern homoeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report that exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1,25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+ -dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a fourfold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a threefold rise in levels of cyclin E and doubled the levels of γH2AX, a marker of DNA double-strand breaks. The p21 cyclin-dependent kinase inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to an SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

Systemic oxidative stress and antioxidant capacity in cancer patients

Various factors impact the outcome of patients with the diagnosis of cancer. Common treatment modalities of cancer, including surgery, radiation therapy and cytostatic agents, all lead to a systemic inflammatory reaction. In particular, this reaction is of physiological importance as it is crucial for patient recovery. However, in some patients, a self-perpetuating inflammatory response develops due to the presence of unfavorable risk factors, several of which are still unknown, but might lead to a worse disease prognosis. Inflammation has been intimately associated with oxidative stress, that is characterized by an imbalance between pro-oxidants, also termed reactive species, and antioxidants. Systemically, oxidative stress can be quantified by measuring thiols (R-SH, sulfhydryl compounds), which are considered to be regulatory nodes within the extracellular antioxidant network. Most importantly, thiol measurements in serum or plasma form a robust and powerful read-out of the in vivo reduction-oxidation (redox) status as thiols are highly redox-active and thus readily oxidized by circulating reactive species. Therefore, systemic quantification of thiols might be a valuable addition to the clinically available diagnostic and prognostic armamentarium as it is able to reliably capture the overall balance between oxidants and the antioxidant capacity of patients. In this review, we summarized the currently available literature on thiol levels as amendable markers for oxidative stress in patients with lung, prostate and colorectal cancer. Total thiols, native (free) thiols and disulfide levels are significantly altered in these patients compared to healthy individuals. In general, these findings indicate that the extracellular antioxidant capacity is severely affected in patients with these types of cancer. Moreover, lower thiol levels are associated with a lowered overall survival. Future research should focus on exploration of the clinical significance of thiols in cancer.

ID1 Mediates Escape from TGFβ Tumor Suppression in Pancreatic Cancer.

TGFβ is an important tumor suppressor in pancreatic ductal adenocarcinoma (PDA), yet inactivation of TGFβ pathway components occurs in only half of PDA cases. TGFβ cooperates with oncogenic RAS signaling to trigger epithelial-to-mesenchymal transition (EMT) in premalignant pancreatic epithelial progenitors, which is coupled to apoptosis owing to an imbalance of SOX4 and KLF5 transcription factors. We report that PDAs that develop with the TGFβ pathway intact avert this apoptotic effect via ID1. ID1 family members are expressed in PDA progenitor cells and encode components of a set of core transcriptional regulators shared by PDAs. PDA progression selects against TGFβ-mediated repression of ID1. The sustained expression of ID1 uncouples EMT from apoptosis in PDA progenitors. AKT signaling and mechanisms linked to low-frequency genetic events converge on ID1 to preserve its expression in PDA. Our results identify ID1 as a crucial node and potential therapeutic target in PDA. SIGNIFICANCE: Half of PDAs escape TGFβ-induced tumor suppression without inactivating the TGFβ pathway. We report that ID1 expression is selected for in PDAs and that ID1 uncouples TGFβ-induced EMT from apoptosis. ID1 thus emerges as a crucial regulatory node and a target of interest in PDA.This article is highlighted in the In This Issue feature, p. 1.

STCH4/REIL2 Confers Cold Stress Tolerance in Arabidopsis by Promoting rRNA Processing and CBF Protein Translation

Plants respond to cold stress by inducing the expression of transcription factors that regulate downstream genes to confer tolerance to freezing. We screened an Arabidopsis transfer DNA (T-DNA) insertion library and identified a cold-hypersensitive mutant, which we named stch4 (sensitive to chilling 4). STCH4/REIL2 encodes a ribosomal biogenesis factor that is upregulated upon cold stress. Overexpression of STCH4 confers chilling and freezing tolerance in Arabidopsis. The stch4 mutation reduces CBF protein levels and thus delayed the induction of C-repeat-binding factor (CBF) regulon genes. Ribosomal RNAprocessing is reduced in stch4 mutants, especially under cold stress. STCH4 associates with multiple ribosomal proteins, and these interactions are modulated by cold stress. These results suggest that the ribosome is a regulatory node for cold stress responses and that STCH4 promotes an altered ribosomal composition and functions in low temperatures to facilitate the translation of proteins important for plant growth and survival under cold stress.

Full-length tran sequencing provides insight into lignan and lignin metabolism relation

Flax (Linum usitatissimum L.) is an important economic crop worldwide. The lignin content of flax stems directly determines the quality of flax fiber, and excessively high lignin content in the fiber leads to pollutant discharge by the textile industry. Flax seeds also contain lignans, a group of compounds that are structurally similar to lignin and highly beneficial for human health. The biosynthesis of lignan and lignin occurs in the same metabolic pathway. To elucidate the metabolic relationship between these two compounds and the regulatory nodes of their metabolic processes, third generation (Iso-Seq) and second generation (Illumina) sequencing technologies were used in this study to obtain the transcriptomes of a pair of flax cultivars with significant differences in lignan content. The results showed that the differentially expressed genes (DEGs) are significantly enriched in the lignin and lignan biosynthesis pathways. Ten genes with significant differences in expression (|Log2 FC|>4) were selected for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression levels of these genes all were significantly different in Shuangya 4 and NEW at different stages of flax seed and stem development. We speculated that these genes were most likely closely related to the lignan and lignin metabolism. We also found that the lignan and lignin content are significantly negatively correlated with each other during flax seed and stem development. The findings of this study provide a scientific basis for the application of biotechnological methods to the development of new high-quality flax cultivars with high lignan and low lignin content.

A TFAP2C Gene Signature Is Predictive of Outcome in HER2-Positive Breast Cancer.

The AP-2γ transcription factor, encoded by the TFAP2C gene, regulates the expression of estrogen receptor-alpha (ERα) and other genes associated with hormone response in luminal breast cancer. Little is known about the role of AP-2γ in other breast cancer subtypes. A subset of HER2+ breast cancers with amplification of the TFAP2C gene locus becomes addicted to AP-2γ. Herein, we sought to define AP-2γ gene targets in HER2+ breast cancer and identify genes accounting for physiologic effects of growth and invasiveness regulated by AP-2γ. Comparing HER2+ cell lines that demonstrated differential response to growth and invasiveness with knockdown of TFAP2C, we identified a set of 68 differentially expressed target genes. CDH5 and CDKN1A were among the genes differentially regulated by AP-2γ and that contributed to growth and invasiveness. Pathway analysis implicated the MAPK13/p38δ and retinoic acid regulatory nodes, which were confirmed to display divergent responses in different HER2+ cancer lines. To confirm the clinical relevance of the genes identified, the AP-2γ gene signature was found to be highly predictive of outcome in patients with HER2+ breast cancer. We conclude that AP-2γ regulates a set of genes in HER2+ breast cancer that drive cancer growth and invasiveness. The AP-2γ gene signature predicts outcome of patients with HER2+ breast cancer and pathway analysis predicts that subsets of patients will respond to drugs that target the MAPK or retinoic acid pathways. IMPLICATIONS: A set of genes regulated by AP-2γ in HER2+ breast cancer that drive proliferation and invasion were identified and provided a gene signature that is predictive of outcome in HER2+ breast cancer.

Dynamic Interactions Between LH and Testosterone in Healthy Community-Dwelling Men: Impact of Age and Body Composition.

Aging is associated with diminished testosterone (Te) secretion, which may be attributed to Leydig cell dysfunction, decreased pituitary stimulation, and altered Te feedback. To study all regulatory nodes-gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and Leydig cell-in the same cohort of healthy men. This was a placebo-controlled, blinded, prospectively randomized cross-over study in 40 men, age range 19 to 73 years, and body mass index (BMI) range 20 to 34.3 kg/m2. A submaximal dose of the GnRH antagonist ganirelix was used to assess outflow of GnRH, by calculating the difference between LH output during the control arm and ganirelix arm. Ketoconazole (a steroidogenic inhibitor) was used to estimate feedback, by the difference in LH output during the ketoconazole and control arm. High-dose ganirelix and repeated LH infusions were used to measure testicular responsivity. Blood sampling was performed at 10-minute intervals. There were age-related, but not body composition-related decreases in estimated GnRH secretion, the feedback strength of Te on LH, and Leydig cell responsivity to LH, accompanied by changes in approximate entropy. Bioavailable Te levels were negatively related to both age and computed tomography (CT)-estimated abdominal visceral mass (AVF), without interaction between these variables. The LH response to a submaximal dose of GnRH was independent of age and AVF. Advancing age is associated with (1) attenuated bioavailable Te secretion caused by diminished GnRH outflow and not by decreased GnRH responsivity of the gonadotrope, (2) diminished testicular responsivity to infused LH pulses, and (3) partial compensation by diminished Te feedback on central gonadotropic regulation.

Integrated Pan-Cancer Map of EBV-Associated Neoplasms Reveals Functional Host-Virus Interactions.

Epstein-Barr virus (EBV) is a complex oncogenic symbiont. The molecular mechanisms governing EBV carcinogenesis remain elusive and the functional interactions between virus and host cells are incompletely defined. Here we present a comprehensive map of the host cell-pathogen interactome in EBV-associated cancers. We systematically analyzed RNA sequencing from >1,000 patients with 15 different cancer types, comparing virus and host factors of EBV+ to EBV- tissues. EBV preferentially integrated at highly accessible regions of the cancer genome, with significant enrichment in super-enhancer architecture. Twelve EBV transcripts, including LMP1 and LMP2, correlated inversely with EBV reactivation signature. Overexpression of these genes significantly suppressed viral reactivation, consistent with a "virostatic" function. In cancer samples, hundreds of novel frequent missense and nonsense variations in virostatic genes were identified, and variant genes failed to regulate their viral and cellular targets in cancer. For example, one-third of patients with EBV+ NK/T-cell lymphoma carried two novel nonsense variants (Q322X, G342X) of LMP1 and both variant proteins failed to restrict viral reactivation, confirming loss of virostatic function. Host cell transcriptional changes in response to EBV infection classified tumors into two molecular subtypes based on patterns of IFN signature genes and immune checkpoint markers, such as PD-L1 and IDO1. Overall, these findings uncover novel points of interaction between a common oncovirus and the human genome and identify novel regulatory nodes and druggable targets for individualized EBV and cancer-specific therapies. SIGNIFICANCE: This study provides a comprehensive map of the host cell-pathogen interactome in EBV+ malignancies.See related commentary by Mbulaiteye and Prokunina-Olsson, p. 5917.

Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.

Regulation of Erythroid Heme Synthesis By the Mitochondrial Clpx Unfoldase

Differentiating erythroid cells synthesize large quantities of heme for hemoglobinization. While the transcriptional regulation and enzymatic mechanisms of the heme synthetic enzymes are well characterized, we lack mechanistic understanding of how their protein stability, cofactor incorporation and functional interactions with mitochondrial housekeeping proteins are regulated. These mechanisms can rapidly alter the rate of heme synthesis in response to external stimuli and metabolic requirements, and are critical for heme regulation within a tissue-specific and developmental context. CLPX, a mitochondrial protein unfoldase best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (the CLPXP ATP-dependent protease) plays a central role in regulation of mitochondrial protein turnover, is one such heme regulatory protein. CLPX activates yeast ALAS, which catalyzes the committed step of the heme synthesis pathway, by facilitating the incorporation of its cofactor, PLP, and is required for erythroid heme synthesis in zebrafish (Kardon et al. Cell 2015). Paradoxically, it regulates the turnover of ALAS1 and ALAS2 protein in vertebrate cell lines and appears to regulate the heme synthesis downstream of ALAS (Kubota et al. JBC 2016, Yien et al. PNAS 2017). However, it is not known if vertebrate ALAS was activated by ALAS, or if the requirement for CLPX in vertebrate heme synthesis was caused its regulation of ALAS activity (Figure A). To dissect the roles of CLPX in erythroid heme synthesis, we knocked out Clpxand Clpp in murine erythroleukemia (MEL) cells and assayed the activity, stability, and steady state levels of the heme synthesis enzymes, ALAS2 and FECH, which colocalize with ALAS in the mitochondrial matrix. Consistent with previous observations, Clpx -/- MEL cells had a heme defect, while Clpp -/-cells did not (Figure B). However, in contrast to previous observations in the yeast model, CLPX is not required for ALAS activation in erythroid cells, but plays a key role in regulating ALAS2 turnover in concert with the CLPP peptidase (Figure C). During erythroid differentiation, CLPP protein levels are decreased, stabilizing ALAS2 protein (Figure D). Although differentiating Clpx -/-and Clpp -/- MEL cells did not demonstrate any changes in ALAS2 turnover, likely because steady-state levels of CLPP protein were already decreased (Figure E), we observed an increase in steady-state ALAS2 protein levels and a dramatic increase in ALAS2 enzyme activity. In vitro mitochondrial iron transport/heme synthesis assays revealed a heme defect in Clpx -/-MEL cells, suggesting that CLPX plays a role in mitochondrial iron metabolism. Collectively, these data suggest a complex, differentiation-stage specific regulation of heme synthesis by the CLPXP proteolytic complex (Figure F). As Clpx -/- mouse embryos die by about E9.5 (mousephenotype.org), we dissected the in vivo role of Clpxin erythropoiesis by analyzing the phenotypes of clpxa and clpxb mutant zebrafish obtained from ZIRC. To accomplish this, we crossed clpxa and clpxb mutant zebrafish into Tg(lcr:GFP) zebrafish line in which erythroid cells are fluorescently labeled with GFP (Ganis et al Dev Biol 2012). We observed that clpxa mutant zebrafish had an early erythropoietic defect at 24 hpf that resolved at 48hpf; this developmental defect was not observed in clpxbmutant zebrafish. Benzidine staining of heme in mutant zebrafish revealed that while clpxa was dispensable for erythroid heme synthesis, clpxb was required for erythroid hemoglobinization (Figure G). Lastly, clpxbzebrafish mutants continued to be developmentally delayed and did not survive past 5 dpf. Collectively, our observations in cell lines and in the zebrafish model demonstrate that Clpx is essential for the maintenance of differentiated erythroid cells, as well as for the differentiation of the erythroid lineage. The control of heme synthesis and erythroid development by CLPX reveals how mitochondrial physiology and heme synthesis are interdependent. Our results reveal an important regulatory node where the mitochondrial protein quality control machinery intersects with key steps in heme synthesis. Further, our studies provide important genetic tools for dissecting these regulatory components in isolation as well as within the in vivocontext of erythropoiesis. Figure Disclosures No relevant conflicts of interest to declare.

The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages

SUMMARYWhile transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein’s ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.

Promising strategy for improving calycosin-7-O-β-D-glucoside production in Astragalus membranaceus adventitious root cultures

Culture duration, elicitation, and precursor feeding can significantly affect secondary metabolite production in adventitious root cultures. Hydrogen peroxide (H2O2) can act as a diffusible signal to induce the production of secondary metabolites. L-phenylalanine (L-Phe) is the precursor in the biosynthesis of the bioactive compound calycosin-7-O-β-D-glucoside (CG). To improve the accumulation of CG in Astragalus membranaceus adventitious roots (AMARs), the present study investigated the effects of culture duration and H2O2 and L-Phe treatment on the accumulation of CG in AMARs. The CG content fluctuated during AMAR culture, with the highest productivity observed after 35 d of suspension culture. Furthermore, the H2O2 treatment efficiently enhanced CG content in a concentration- and time-dependent manner, and under 20 μM H2O2, the CG reached its maximum level after 12 h of treatment. However, the addition of L-Phe to H2O2 treatment remarkably improved the CG accumulation, with a maximum CG level observed after 120 h of treatment with 200 μM H2O2 and 300 μM L-Phe, which was 8.6-fold greater than the cultures treated with 200 μM H2O2 alone. The effectiveness of these parameters was confirmed by measuring the expression of four major CG-biosynthesis genes, and both the chalcone reductase and isoflavone synthase genes were identified as key regulatory nodes in the CG biosynthetic pathway. The improvement strategy was also applied using a large-scale bioreactor system. Both the CG content and antioxidant activity of the H2O2-elicited and L-Phe-fed bioreactor culture were greater than those of the untreated bioreactor culture and field-grown plants. Thus, the simultaneous application of H2O2 elicitation and L-Phe-feeding in 35-d-old AMAR cultures is a promising strategy to economically improve the industrial production of CG.

The signaling proteins GPR158 and RGS7 modulate excitability of L2/3 pyramidal neurons and control A-type potassium channel in the prelimbic cortex

Stress profoundly affects physiological properties of neurons across brain circuits and thereby increases the risk for depression. However, the molecular and cellular mechanisms mediating these effects are poorly understood. In this study, we report that chronic physical restraint stress in mice decreases excitability specifically in layer 2/3 of pyramidal neurons within the prelimbic subarea of the prefrontal cortex (PFC) accompanied by the induction of depressive-like behavioral states. We found that a complex between G protein-coupled receptor (GPCR) 158 (GPR158) and regulator of G protein signaling 7 (RGS7), a regulatory GPCR signaling node recently discovered to be a key modulator of affective behaviors, plays a key role in controlling stress-induced changes in excitability in this neuronal population. Deletion of GPR158 or RGS7 enhanced excitability of layer 2/3 PFC neurons and prevented the impact of stress. Investigation of the underlying molecular mechanisms revealed that the A-type potassium channel Kv4.2 subunit is a molecular target of the GPR158-RGS7 complex. We further report that GPR158 physically associates with Kv4.2 channel and promotes its function by suppressing inhibitory modulation by cAMP-protein kinase A (PKA)-mediated phosphorylation. Taken together, our observations reveal a critical mechanism that adjusts neuronal excitability in L2/3 pyramidal neurons of the PFC and may thereby modulate the effects of stress on depression.

A systems biology pipeline identifies regulatory networks for stem cell engineering.

A major challenge for stem cell engineering is achieving a holistic understanding of the molecular networks and biological processes governing cell differentiation. To address this challenge, we describe a computational approach that combines gene expression analysis, previous knowledge from proteomic pathway informatics and cell signaling models to delineate key transitional states of differentiating cells at high resolution. Our network models connect sparse gene signatures with corresponding, yet disparate, biological processes to uncover molecular mechanisms governing cell fate transitions. This approach builds on our earlier CellNet and recent trajectory-defining algorithms, as illustrated by our analysis of hematopoietic specification along the erythroid lineage, which reveals a role for the EGF receptor family member, ErbB4, as an important mediator of blood development. We experimentally validate this prediction and perturb the pathway to improve erythroid maturation from human pluripotent stem cells. These results exploit an integrative systems perspective to identify new regulatory processes and nodes useful in cell engineering.

SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

Identification of microRNA-target modules from rice variety Pusa Basmati-1 under high temperature and salt stress.

High temperature and salinity stress are major factors limiting the growth and productivity of rice crop on a global scale. It is therefore an essential prerequisite to understand the molecular genetic regulation of plant responses to dual stresses. MicroRNAs (miRs) are recognized as key controllers of gene expression which act mainly at the post-transcriptional level to regulate various aspects of plant development. The present study attempts to investigate the miR circuits that are modulated in response to high temperature and salinity stress in rice. To gain insights into the pathway, preliminary miR profiles were generated using the next-generation sequencing (NGS) datasets. The identified molecules were filtered on the basis of fold differential regulation under high temperature, and time kinetics of their expression under the two individual stresses was followed to capture the regulatory windows. The analysis revealed the involvement of common miR regulatory nodes in response to two different abiotic stresses, thereby broadening our perspective about the stress-mediated regulatory mechanisms operative in rice.

RNA-binding proteins: The next step in translating skeletal muscle adaptations?

The decline of skeletal muscle mass during illness, injury, disuse, and aging is associated with poor health outcomes. Therefore, it is important to pursue a greater understanding of the mechanisms that dictate skeletal muscle adaptation. In this review, we propose that RNA-binding proteins (RBPs) comprise a critical regulatory node in the orchestration of adaptive responses in skeletal muscle. While RBPs have broadly pleiotropic molecular functions, our discussion is constrained at the outset by observations from hibernating animals, which suggest that RBP regulation of RNA stability and its impact on translational reprogramming is a key component of skeletal muscle response to anabolic and catabolic stimuli. We discuss the limited data available on the expression and functions of RBPs in adult skeletal muscle in response to disuse, aging, and exercise. A model is proposed in which dynamic changes in RBPs play a central role in muscle adaptive processes through their differential effects on mRNA stability. While limited, the currently available data suggest that understanding how adaptive (and maladaptive) changes in the expression of RBPs regulate mRNA stability in skeletal muscle could be an informative and productive research area for finding new strategies to limit atrophy and promote hypertrophy.

Selectively hampered activation of lymph node-resident dendritic cells precedes profound T cell suppression and metastatic spread in the breast cancer sentinel lymph node

BackgroundImmune regulated pathways influence both breast cancer (BrC) development and response to (neo)adjuvant chemotherapy. The sentinel lymph node (SLN), as the first metastatic site, is also the first site where BrC-induced suppression of immune effector subsets occurs. Since intricate knowledge of the phenotypic and functional status of these immune effector subsets is lacking, we set out to map the immune landscape of BrC SLN.MethodsViable LN cells from BrC SLN (n = 58) were used for detailed flowcytometry-assisted mapping of the immune landscape of BrC SLN in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n = 17). Findings were related to clinicopathological characteristics.ResultsOur data show that BrC-induced immune suppression in tumor-involved SLN, as evidenced by increased Treg and MDSC rates as well as by a generalized state of T cell anergy, coincides with hampered activation of LN-resident (LNR) dendritic cell (DC) subsets rather than of migratory DC subsets. Importantly, suppression of these LN-resident DC subsets preceded profoundly disabled T cell effector functions in tumor-involved SLN. Furthermore, we provide evidence that the suppressed state of LNR-cDC is not only related to nodal involvement but is also related to high-risk breast cancer subtypes that lack expression of hormone receptors and may be a negative predictor of disease-free survival.ConclusionThese data thus provide new insights in the mechanisms underlying loco-regional immune suppression induced by BrC and how these relate to clinical outcome. They identify the LNR-cDC subset as a pivotal regulatory node in cellular immune suppressive pathways and therefore as a promising therapeutic target to combat immune suppression and secure the induction of effective antitumor immunity, e.g. in combination with neo-adjuvant chemotherapy.