The alternative macrophage relay: STAT6 passes the baton to EGR2.

Alternative polarization of macrophages induced by IL-4 is important for homeostasis and tissue repair. Downstream from IL-4 receptor signaling, STAT6 activation is transient, but induces stable transcriptional changes. These data suggest that STAT6 induces second messengers to carry out the alternative transcriptional program. In this issue of Genes & Development, Daniel and colleagues (pp. 1474-1492) identify EGR2 as a downstream regulator of STAT6 with broad functionality that further induces many transcription factors associated with alternative polarization. Identification of high EGR2 expression in a subset of mouse and human alveolar macrophages further highlights EGR2 as a conserved marker of alternatively activated macrophages.

The transcription factor EGR2 is the molecular linchpin connecting STAT6 activation to the late, stable epigenomic program of alternative macrophage polarization.

Macrophages polarize into functionally distinct subtypes while responding to microenvironmental cues. The identity of proximal transcription factors (TFs) downstream from the polarization signals are known, but their activity is typically transient, failing to explain the long-term, stable epigenomic programs developed. Here, we mapped the early and late epigenomic changes of interleukin-4 (IL-4)-induced alternative macrophage polarization. We identified the TF, early growth response 2 (EGR2), bridging the early transient and late stable gene expression program of polarization. EGR2 is a direct target of IL-4-activated STAT6, having broad action indispensable for 77% of the induced gene signature of alternative polarization, including its autoregulation and a robust, downstream TF cascade involving PPARG. Mechanistically, EGR2 binding results in chromatin opening and the recruitment of chromatin remodelers and RNA polymerase II. Egr2 induction is evolutionarily conserved during alternative polarization of mouse and human macrophages. In the context of tissue resident macrophages, Egr2 expression is most prominent in the lung of a variety of species. Thus, EGR2 is an example of an essential and evolutionarily conserved broad acting factor, linking transient polarization signals to stable epigenomic and transcriptional changes in macrophages.

A common molecular logic determines embryonic stem cell self-renewal and reprogramming.

During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.

Transcription factors regulate early T cell development via redeployment of other factors: Functional dynamics of constitutively required factors in cell fate decisions.

Establishment of cell lineage identity from multipotent progenitors is controlled by cooperative actions of lineage-specific and stably expressed transcription factors, combined with input from environmental signals. Lineage-specific master transcription factors activate and repress gene expression by recruiting consistently expressed transcription factors and chromatin modifiers to their target loci. Recent technical advances in genome-wide and multi-omics analysis have shed light on unexpected mechanisms that underlie more complicated actions of transcription factors in cell fate decisions. In this review, we discuss functional dynamics of stably expressed and continuously required factors, Notch and Runx family members, throughout developmental stages of early T cell development in the thymus. Pre- and post-commitment stage-specific transcription factors induce dynamic redeployment of Notch and Runx binding genomic regions. Thus, together with stage-specific transcription factors, shared transcription factors across distinct developmental stages regulate acquisition of T lineage identity.

Defining B Cell Chromatin: Lessons from EBF1.

Hematopoiesis is regulated by signals from the microenvironment, transcription factor networks, and changes of the epigenetic landscape. Transcription factors interact with and shape chromatin to allow for lineage- and cell type-specific changes in gene expression. During B lymphopoiesis, epigenetic regulation is observed in multilineage progenitors in which a specific chromatin context is established, at the onset of the B cell differentiation when early B cell factor 1 (EBF1) induces lineage-specific changes in chromatin, during V(D)J recombination and after antigen-driven activation of B cells and terminal differentiation. In this review, we discuss the epigenetic changes underlying B cell differentiation, focusing on the role of transcription factor EBF1 in B cell lineage priming.

Transcription factor network analysis based on single cell RNA-seq identifies that Trichostatin-a reverses docetaxel resistance in prostate Cancer.

BACKGROUND: Overcoming drug resistance is critical for increasing the survival rate of prostate cancer (PCa). Docetaxel is the first cytotoxic chemotherapeutical approved for treatment of PCa. However, 99% of PCa patients will develop resistance to docetaxel within 3 years. Understanding how resistance arises is important to increasing PCa survival. METHODS: In this study, we modeled docetaxel resistance using two PCa cell lines: DU145 and PC3. Using the Passing Attributes between Networks for Data Assimilation (PANDA) method to model transcription factor (TF) activity networks in both sensitive and resistant variants of the two cell lines. We identified edges and nodes shared by both PCa cell lines that composed a shared TF network that modeled changes which occur during acquisition of docetaxel resistance in PCa. We subjected the shared TF network to connectivity map analysis (CMAP) to identify potential drugs that could disrupt the resistant networks. We validated the candidate drug in combination with docetaxel to treat docetaxel-resistant PCa in both in vitro and in vivo models. RESULTS: In the final shared TF network, 10 TF nodes were identified as the main nodes for the development of docetaxel resistance. CMAP analysis of the shared TF network identified trichostatin A (TSA) as a candidate adjuvant to reverse docetaxel resistance. In cell lines, the addition of TSA to docetaxel enhanced cytotoxicity of docetaxel resistant PCa cells with an associated reduction of the IC50 of docetaxel on the resistant cells. In the PCa mouse model, combination of TSA and docetaxel reduced tumor growth and final weight greater than either drug alone or vehicle. CONCLUSIONS: We identified a shared TF activity network that drives docetaxel resistance in PCa. We also demonstrated a novel combination therapy to overcome this resistance. This study highlights the usage of novel application of single cell RNA-sequencing and subsequent network analyses that can reveal novel insights which have the potential to improve clinical outcomes.

Identification of small RNAs during cold acclimation in Arabidopsis thaliana.

BACKGROUND: Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. RESULT: We subjected A. thaliana plants to cold acclimation conditions (4 °C) and analyzed the sRNA transcriptomes after 3 h, 6 h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cis- and trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. CONCLUSION: In A. thaliana, miRNAs and sRNAs derived from cis- and trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.

RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection.

Brassica napus is one of the world's most valuable oilseeds and is under constant pressure by the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, the causal agent of white stem rot. Despite our growing understanding of host pathogen interactions at the molecular level, we have yet to fully understand the biological processes and underlying gene regulatory networks responsible for determining disease outcomes. Using global RNA sequencing, we profiled gene activity at the first point of infection on the leaf surface 24 hours after pathogen exposure in susceptible (B. napus cv. Westar) and tolerant (B. napus cv. Zhongyou 821) plants. We identified a family of ethylene response factors that may contribute to host tolerance to S. sclerotiorum by activating genes associated with fungal recognition, subcellular organization, and redox homeostasis. Physiological investigation of redox homeostasis was further studied by quantifying cellular levels of the glutathione and ascorbate redox pathway and the cycling enzymes associated with host tolerance to S. sclerotiorum. Functional characterization of an Arabidopsis redox mutant challenged with the fungus provides compelling evidence into the role of the ascorbate-glutathione redox hub in the maintenance and enhancement of plant tolerance against fungal pathogens.

Single-cell data revealed exhaustion of characteristic NK cell subpopulations and T cell subpopulations in hepatocellular carcinoma.

BACKGROUND: The treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) have been a major medical challenge. Unraveling the landscape of tumor immune infiltrating cells (TIICs) in the immune microenvironment of HCC is of great significance to probe the molecular mechanisms. METHODS: Based on single-cell data of HCC, the cell landscape was revealed from the perspective of TIICs. Special cell subpopulations were determined by the expression levels of marker genes. Differential expression analysis was conducted. The activity of each subpopulation was determined based on the highly expressed genes. CTLA4+ T-cell subpopulations affecting the prognosis of HCC were determined based on survival analysis. A single-cell regulatory network inference and clustering analysis was also performed to determine the transcription factor regulatory networks in the CTLA4+ T cell subpopulations. RESULTS: 10 cell types were identified and NK cells and T cells showed high abundance in tumor tissues. Two NK cells subpopulations were present, FGFBP2+ NK cells, B3GNT7+ NK cells. Four T cells subpopulations were present, LAG3+ T cells, CTLA4+ T cells, RCAN3+ T cells, and HPGDS+ Th2 cells. FGFBP2+ NK cells, and CTLA4+ T cells were the exhaustive subpopulation. High CTLA4+ T cells contributed to poor prognostic outcomes and promoted tumor progression. Finally, a network of transcription factors regulated by NR3C1, STAT1, and STAT3, which were activated, was present in CTLA4+ T cells. CONCLUSION: CTLA4+ T cell subsets in HCC exhibited functional exhaustion characteristics that probably inhibited T cell function through a transcription factor network dominated by NR3C1, STAT1, and STAT3.

Molecular characterization of projection neuron subtypes in the mouse olfactory bulb.

Projection neurons (PNs) in the mammalian olfactory bulb (OB) receive input from the nose and project to diverse cortical and subcortical areas. Morphological and physiological studies have highlighted functional heterogeneity, yet no molecular markers have been described that delineate PN subtypes. Here, we used viral injections into olfactory cortex and fluorescent nucleus sorting to enrich PNs for high-throughput single nucleus and bulk RNA deep sequencing. Transcriptome analysis and RNA in situ hybridization identified distinct mitral and tufted cell populations with characteristic transcription factor network topology, cell adhesion, and excitability-related gene expression. Finally, we describe a new computational approach for integrating bulk and snRNA-seq data and provide evidence that different mitral cell populations preferentially project to different target regions. Together, we have identified potential molecular and gene regulatory mechanisms underlying PN diversity and provide new molecular entry points into studying the diverse functional roles of mitral and tufted cell subtypes.

Characterization of SALL2 Gene Isoforms and Targets Across Cell Types Reveals Highly Conserved Networks.

The SALL2 transcription factor, an evolutionarily conserved gene through vertebrates, is involved in normal development and neuronal differentiation. In disease, SALL2 is associated with eye, kidney, and brain disorders, but mainly is related to cancer. Some studies support a tumor suppressor role and others an oncogenic role for SALL2, which seems to depend on the cancer type. An additional consideration is tissue-dependent expression of different SALL2 isoforms. Human and mouse SALL2 gene loci contain two promoters, each controlling the expression of a different protein isoform (E1 and E1A). Also, several improvements on the human genome assembly and gene annotation through next-generation sequencing technologies reveal correction and annotation of additional isoforms, obscuring dissection of SALL2 isoform-specific transcriptional targets and functions. We here integrated current data of normal/tumor gene expression databases along with ChIP-seq binding profiles to analyze SALL2 isoforms expression distribution and infer isoform-specific SALL2 targets. We found that the canonical SALL2 E1 isoform is one of the lowest expressed, while the E1A isoform is highly predominant across cell types. To dissect SALL2 isoform-specific targets, we analyzed publicly available ChIP-seq data from Glioblastoma tumor-propagating cells and in-house ChIP-seq datasets performed in SALL2 wild-type and E1A isoform knockout HEK293 cells. Another available ChIP-seq data in HEK293 cells (ENCODE Consortium Phase III) overexpressing a non-canonical SALL2 isoform (short_E1A) was also analyzed. Regardless of cell type, our analysis indicates that the SALL2 long E1 and E1A isoforms, but not short_E1A, are mostly contributing to transcriptional control, and reveals a highly conserved network of brain-specific transcription factors (i.e., SALL3, POU3F2, and NPAS3). Our data integration identified a conserved molecular network in which SALL2 regulates genes associated with neural function, cell differentiation, development, and cell adhesion between others. Also, we identified PODXL as a gene that is likely regulated by SALL2 across tissues. Our study encourages the validation of publicly available ChIP-seq datasets to assess a specific gene/isoform's transcriptional targets. The knowledge of SALL2 isoforms expression and function in different tissue contexts is relevant to understanding its role in disease.

MicroRNAs: Tiny Regulators of Gene Expression with Pivotal Roles in Normal B-Cell Development and B-Cell Chronic Lymphocytic Leukemia.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs bearing regulatory potency. The implication of miRNAs in physiological cellular processes has been well documented so far. A typical process orchestrated by miRNAs is the normal B-cell development. A stage-specific expression pattern of miRNAs has been reported in the developmental procedure, as well as interactions with transcription factors that dictate B-cell development. Besides their involvement in normal hematopoiesis, miRNAs are severally implicated in hematological malignancies, a typical paradigm of which is B-cell chronic lymphocytic leukemia (B-CLL). B-CLL is a highly heterogeneous disease characterized by the accumulation of abnormal B cells in blood, bone marrow, lymph nodes, and spleen. Therefore, timely, specific, and sensitive assessment of the malignancy is vital. Several studies have attempted to highlight the remarkable significance of miRNAs as regulators of gene expression, biomarkers for diagnosis, prognosis, progression, and therapy response prediction, as well as molecules with potential therapeutic utility. This review seeks to outline the linkage between miRNA function in normal and malignant hematopoiesis by demonstrating the main benchmarks of the implication of miRNAs in the regulation of normal B-cell development, and to summarize the key findings about their value as regulators, biomarkers, or therapeutic targets in B-CLL.

Investigation of transcriptome mechanism associated with osteoporosis explored by microarray analysis.

Microarray data of osteoporosis (OP) were analyzed based on prediction of transcription factors (TFs) or their targets as well as influences of TFs or TF network to uncover key TFs in OP. The microarray data E-GEOD-35956 was downloaded from the GPL570 platform. Differentially expressed genes (DEGs) with logarithm of fold change (|logFC|) >2 and P-value <0.05 were identified between OP samples and normal controls. TF genes were screened from the DEGs based on ITFP, Marbach 2016, TRRUST databases. TF targets were enriched from DEGs using Fisher's exact test. TF targets were selected based on their impact factors. TF targets were chosen from TF network analysis. Finally, key TFs were identified by based on TFs coverage. A total of 300 DEGs were obtained. There were no TF genes screened from the DEGs. In total 165, 87 and 178 TF targets were screened from DEGs respectively based on Fisher's exact test, influence of TFs or TF network analysis. According to the optimal TF set with TFs having maximum coverage of DEGs, 178 TF targets was the most. Thus, the optimal sets of TFs were FOXO1, KLF16, RXRA, RARA, HNF4A, CEBPB, ESR1, SOX8, ZNF219, and SP1. Altogether, these results suggested identified crucial TFs in OP might play a significant role in OP development, showing these key TFs probably would aid in unveiling the underlying molecular mechanisms and may be therapeutic targets, diagnostic or prognostic biomarkers for OP.

Genome-Scale Transcriptional Regulatory Network Models of Psychiatric and Neurodegenerative Disorders.

Transcriptional regulatory changes in the developing and adult brain are prominent features of brain diseases, but the involvement of specific transcription factors (TFs) remains poorly understood. We integrated brain-specific DNase footprinting and TF-gene co-expression to reconstruct a transcriptional regulatory network (TRN) model for the human brain. We identified key regulator TFs whose predicted target genes were enriched for differentially expressed genes in the prefrontal cortex of individuals with psychiatric and neurodegenerative diseases. Many of these TFs were further implicated in the same diseases through disruption of their binding sites by disease-associated SNPs and associations of TF loci with disease risk. Using primary human neural stem cells, we validated network predictions that link the TF POU3F2 to schizophrenia and bipolar disorder via both cis- and trans-acting mechanisms. Our models of brain-specific TF binding sites and target genes provide a resource for network analysis of brain diseases.

Arabidopsis thaliana WRKY25 Transcription Factor Mediates Oxidative Stress Tolerance and Regulates Senescence in a Redox-Dependent Manner.

Senescence is the last developmental step in plant life and is accompanied by a massive change in gene expression implying a strong participation of transcriptional regulators. In the past decade, the WRKY53 transcription factor was disclosed to be a central node of a complex regulatory network of leaf senescence and to underlie a tight multi-layer control of expression, activity and protein stability. Here, we identify WRKY25 as a redox-sensitive up-stream regulatory factor of WRKY53 expression. Under non-oxidizing conditions, WRKY25 binds to a specific W-box in the WRKY53 promoter and acts as a positive regulator of WRKY53 expression in a transient expression system using Arabidopsis protoplasts, whereas oxidizing conditions dampened the action of WRKY25. However, overexpression of WRKY25 did not accelerate senescence but increased lifespan of Arabidopsis plants, whereas the knock-out of the gene resulted in the opposite phenotype, indicating a more complex regulatory function of WRKY25 within the WRKY subnetwork of senescence regulation. In addition, overexpression of WRKY25 mediated higher tolerance to oxidative stress and the intracellular H2O2 level is lower in WRKY25 overexpressing plants and higher in wrky25 mutants compared to wildtype plants suggesting that WRKY25 is also involved in controlling intracellular redox conditions. Consistently, WRKY25 overexpressers had higher and wrky mutants lower H2O2 scavenging capacity. Like already shown for WRKY53, MEKK1 positively influenced the activation potential of WRKY25 on the WRKY53 promoter. Taken together, WRKY53, WRKY25, MEKK1 and H2O2 interplay with each other in a complex network. As H2O2 signaling molecule participates in many stress responses, WRKK25 acts most likely as integrators of environmental signals into senescence regulation.

Charting Developmental Dissolution of Pluripotency.

The formation of tissues and organs during metazoan development begs fundamental questions of cellular plasticity: How can the very same genome program have diverse cell types? How do cell identity programs unfold during development in space and time? How can defects in these mechanisms cause disease and also provide opportunities for therapeutic intervention? And ultimately, can developmental programs be exploited for bioengineering tissues and organs? Understanding principle designs of cellular identity and developmental progression is crucial for providing answers. Here, I will discuss how the capture of embryonic pluripotency in murine embryonic stem cells (ESCs) in vitro has allowed fundamental insights into the molecular underpinnings of a developmental cell state and how its ordered disassembly during differentiation prepares for lineage specification.

Genome-Wide Insights into the Development and Function of Thermogenic Adipocytes.

Brown and brown-like adipocytes are specialized adipocytes with a high capacity to convert metabolic energy to heat. This function is not only eminent in supporting organismal thermogenesis, but may also have potential in the fight against obesity. The latter has spurred a massive interest in understanding the development and regulation of these thermogenic adipocytes. Here, we review how genome-wide studies based on next-generation sequencing have provided insight into how the chromatin and transcriptional landscapes are established in thermogenic adipocytes and how thermogenic signals can change the genomic programming of white adipocytes. Furthermore, we discuss how the integration of genomic data can be used to discover novel transcriptional pathways that may be modulated as part of therapeutic strategies for the treatment of obesity.

Transcriptional control of sexual development in Cryptococcus neoformans.

Developmental processes are essential for the normal life cycles of many pathogenic fungi, and they can facilitate survival in challenging environments, including the human host. Sexual development of the human fungal pathogen Cryptococcus neoformans not only produces infectious particles (spores) but has also enabled the evolution of new disease-related traits such as drug resistance. Transcription factor networks are essential to the development and pathogenesis of C. neoformans, and a variety of sequence-specific DNA-binding proteins control both key developmental transitions and virulence by regulating the expression of their target genes. In this review we discuss the roles of known transcription factors that harbor important connections to both development and virulence. Recent studies of these transcription factors have identified a common theme in which metabolic, stress, and other responses that are required for sexual development appear to have been co-opted for survival in the human host, thus facilitating pathogenesis. Future work elucidating the connection between development and pathogenesis will provide vital insights into the evolution of complex traits in eukaryotes as well as mechanisms that may be used to combat fungal pathogens.

Quantitative iTRAQ LC-MS/MS Proteomics Reveals Transcription Factor Crosstalk and Regulatory Networks in Hypopharyngeal Squamous Cell Carcinoma.

To date, no effective therapeutic treatments have been developed for hypopharyngeal squamous cell carcinoma (HPSCC), a disease that has a five-year survival rate of approximately 31% because of its late diagnosis and aggressive nature. Despite recent improvements in diagnostic methods, there are no effective measures to prevent or detect HPSCC in an early stage. The goal of the current study was to identify molecular biomarkers and networks that can facilitate the speedy identification of HPSCC patients who could benefit from individualized treatment. Isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed with two-dimensional liquid chromatography-tandem mass spectrometry to identify quantitatively the differentially expressed proteins among three types of HPSCC disease stages. The iTRAQ results were evaluated by literature searches and western blot analysis. For example, FUBP1, one of 412 proteins with significantly altered expression profiles, was confirmed to have elevated expression in fresh HPSCC tissues. Integrin-mediated cell matrix adhesion and actin filament-inducing cytoskeleton remodeling were the cellular events that were the most relevant to HPSCC tumorigenesis and the metastatic process. The construction of transcriptional regulation networks led to the identification of key transcriptional regulators of tumor development and lymph node metastasis of HPSCC, including Sp1, c-Myc and p53. Additionally, our study indicated that the interactions among Sp1, c-Myc and p53 may play vital roles in the carcinogenesis and metastasis of HPSCC.