Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription-factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC B-cell transformation.

Basaloid Follicular Hamartoma: An Additional Criterion of Nevoid Basal Cell Carcinoma Syndrome.

ABSTRACT: Basaloid follicular hamartoma (BFH) is a rare, benign follicular neoplasm which typically presents as brown to skin-colored papules on the face, scalp, and trunk. Histologically, BFH consists of cords and strands of basaloid cells forming cystic structures with scant stroma and should be distinguished from infundibulocystic basal cell carcinoma to avoid overly aggressive treatment. Although BFH has been found to be associated with distinct syndromes, including alopecia, myasthenia gravis, and cystic fibrosis, there is often clinical, histopathologic, and genetic overlap with nevoid basal cell carcinoma syndrome (NBCCS). In this article, we describe a case of a 13-year-old patient with NBCCS who presented with multiple BFHs and propose that it its inclusion into the diagnostic criteria for NBCCS be considered.

Improvement of Unilateral Breast Hypoplasia With Oral Spironolactone in a Patient With Becker Nevus Syndrome.

Becker nevus (BN) is a benign cutaneous smooth muscle hamartoma that presents with a hyperpigmented patch or plaque with or without hypertrichosis.1 BN may be associated with ipsilateral breast hypoplasia or other musculoskeletal abnormalities, an association which has been termed Becker nevus syndrome (BNS).

The benefit of poor mixing: kinetics of coacervation.

Complex coacervation has become a prominent area of research in the fields of food science, personal care, drug stabilization, and more. However, little has been reported on the kinetics of assembly of coacervation itself. Here, we describe a simple, low-cost way of looking at the kinetics of coacervation by creating poorly mixed samples. In particular, we examine how polymer chain length, the patterning and symmetry of charges on the oppositely charged polyelectrolytes, and the presence of salt and a zwitterionic buffer affect the kinetics of complex coacervation. Our results suggest an interesting relationship between the time for equilibration and the order of addition of polymers with asymmetric patterns of charge. Furthermore, we demonstrated that increasing polymer chain length resulted in a non-monotonic trend in the sample equilibration times as a result of opposing factors such as excluded volume and diffusion. We also observed differences in the rate of sample equilibration based on the presence of a neutral, zwitterionic buffer, as well as the presence and identity of added salt, consistent with previous reports of salt-specific effects on the rheology of complex coacervates. While not a replacement for more advanced characterization strategies, this turbidity-based method could serve as a screening tool to identify interesting and unique phenomena for further study.

Melanoma Ex Blue Nevus With GNA11 Mutation and BAP1 Loss: Case Report and Review of the Literature.

Cutaneous melanomas may demonstrate a variety of histopathological features and genetic abnormalities. Melanomas that arise in the setting of blue nevi, also known as "malignant blue nevus" or melanoma ex blue nevus (MBN), share a similar histopathological and mutational profile with uveal melanoma. Most uveal melanomas show characteristic GNA11 or GNAQ mutations; additional BAP1 mutation or loss is associated with the highest risk of metastasis and worst prognosis. However, the significance of BAP1 loss in melanomas ex blue nevus remains unclear. We present a case of MBN arising from the scalp of a 21-year-old woman. The diagnosis was established on histopathological findings demonstrating a markedly atypical melanocytic proliferation with increased mitotic activity, necrosis, and a focus of angiolymphatic invasion. Immunohistochemical analysis demonstrated the absence of BAP1 nuclear expression within tumor cells. Next generation sequencing detected GNA11 Q209L mutation and BAP1 loss (chromosome 3p region loss), supporting the diagnosis. We reviewed another 21 MBN cases with reported BAP1 status from the literature. MBN with BAP1 loss presented at a younger average age (41 vs. 61 years), demonstrated larger average lesion thickness (9.0 vs. 7.3 mm), and had a higher rate of metastasis (50% vs. 33%) compared with BAP1-retained MBN. BAP1 expression studies may assist in the diagnosis and management of MBN, but further research is needed.

ER71 specifies Flk-1+ hemangiogenic mesoderm by inhibiting cardiac mesoderm and Wnt signaling.

Two distinct types of Flk-1(+) mesoderm, hemangiogenic and cardiogenic, are thought to contribute to blood, vessel, and cardiac cell lineages. However, our understanding of how Flk-1(+) mesoderm is specified is currently limited. In the present study, we investigated whether ER71, an Ets transcription factor essential for hematopoietic and endothelial cell lineage development, could modulate the hemangiogenic or cardiogenic outcome of the Flk-1(+) mesoderm. We show that Flk-1(+) mesoderm can be divided into Flk-1(+)PDGFRα(-) hemangiogenic and Flk-1(+)PDGFRα(+) cardiogenic mesoderm. ER71-deficient embryonic stem cells produced only the Flk-1(+)PDGFRα(+) cardiogenic mesoderm, which generated SMCs and cardiomyocytes. Enforced ER71 expression in the wild-type embryonic stem cells skewed toward the Flk-1(+)PDGFRα(-) mesoderm formation, which generated hematopoietic and endothelial cells. Whereas hematopoietic and endothelial cell genes were positively regulated by ER71, cardiac and Wnt signaling pathway genes were negatively regulated by ER71. We show that ER71 could inhibit Wnt signaling in VE-cadherin-independent as well as VE-cadherin-dependent VE-cadherin/ß-catenin/Flk-1 complex formation. Enforced ß-catenin could rescue cardiogenic mesoderm in the context of ER71 overexpression. In contrast, ER71-deficient Flk-1(+) mesoderm displayed enhanced Wnt signaling, which was reduced by ER71 re-introduction. We provide the molecular basis for the antagonistic relationship between hemangiogenic and cardiogenic mesoderm specification by ER71 and Wnt signaling.

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury.

BACKGROUND: The regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets. RESULTS: In this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery. CONCLUSIONS: This work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs.

MicroRNAs modulate Schwann cell response to nerve injury by reinforcing transcriptional silencing of dedifferentiation-related genes.

In the peripheral nervous system, Schwann cells (SCs) surrounding damaged axons undergo an injury response that is driven by an intricate transcriptional program and is critical for nerve regeneration. To examine whether these injury-induced changes in SCs are also regulated posttranscriptionally by miRNAs, we performed miRNA expression profiling of mouse sciatic nerve distal segment after crush injury. We also characterized the SC injury response in mice containing SCs with disrupted miRNA processing due to loss of Dicer. We identified 87 miRNAs that were expressed in mouse adult peripheral nerve, 48 of which were dynamically regulated after nerve injury. Most of these injury-regulated SC miRNAs were computationally predicted to inhibit drivers of SC dedifferentiation/proliferation and thereby re-enforce the transcriptional program driving SC remyelination. SCs deficient in miRNAs manifested a delay in the transition between the distinct differentiation states required to support peripheral nerve regeneration. Among the miRNAs expressed in adult mouse SCs, miR-34a and miR-140 were identified as functional regulators of SC dedifferentiation/proliferation and remyelination, respectively. We found that miR-34a interacted with positive regulators of dedifferentiation and proliferation such as Notch1 and Ccnd1 to control cell cycle dynamics in SCs. miR-140 targeted the transcription factor Egr2, a master regulator of myelination, and modulated myelination in DRG/SC cocultures. Together, these results demonstrate that SC miRNAs are important modulators of the SC regenerative response after nerve damage.

Regulation of the PMP22 gene through an intronic enhancer.

Successful myelination of the peripheral nervous system depends upon induction of major protein components of myelin, such as peripheral myelin protein 22 (PMP22). Myelin stability is also sensitive to levels of PMP22, as a 1.4 Mb duplication on human chromosome 17, resulting in three copies of PMP22, is the most common cause of the peripheral neuropathy Charcot-Marie-Tooth disease. The transcription factor Egr2/Krox20 is required for induction of high level expression of Pmp22 in Schwann cells but its activation elements have not yet been determined. Using chromatin immunoprecipitation analysis of the rat Pmp22 locus, we found a major peak of Egr2 binding within the large intron of the Pmp22 gene. Analysis of a 250 bp region within the largest intron showed that it is strongly activated by Egr2 expression in reporter assays. Moreover, this region contains conserved binding sites not only for Egr2 but also for Sox10, which is also required for Schwann cell development. Our analysis shows that Sox10 is required for optimal activity of the intronic site as well as PMP22 expression. Finally, mouse transgenic analysis revealed tissue-specific expression of this intronic sequence in peripheral nerve. Overall, these data show that Egr2 and Sox10 activity are directly involved in mediating the developmental induction of Pmp22 expression.

Complete characterization of the microRNAome in a patient with acute myeloid leukemia.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression and have been implicated in the pathogenesis of cancer. In this study, we applied next generation sequencing techniques to comprehensively assess miRNA expression, identify genetic variants of miRNA genes, and screen for alterations in miRNA binding sites in a patient with acute myeloid leukemia. RNA sequencing of leukemic myeloblasts or CD34(+) cells pooled from healthy donors showed that 472 miRNAs were expressed, including 7 novel miRNAs, some of which displayed differential expression. Sequencing of all known miRNA genes revealed several novel germline polymorphisms but no acquired mutations in the leukemia genome. Analysis of the sequence of the 3'-untranslated regions (UTRs) of all coding genes identified a single somatic mutation in the 3'-UTR of TNFAIP2, a known target of the PML-RARα oncogene. This mutation resulted in translational repression of a reporter gene in a Dicer-dependent fashion. This study represents the first complete characterization of the "miRNAome" in a primary human cancer and suggests that generation of miRNA binding sites in the UTR regions of genes is another potential mechanism by which somatic mutations can affect gene expression.

Enhancing the Photoelectrochemical Hydrogen Evolution Reaction through Nanoscrolling of Two-Dimensional Material Heterojunctions.

The clean production of hydrogen from water using sunlight has emerged as a sustainable alternative toward large-scale energy generation and storage. However, designing photoactive semiconductors that are suitable for both light harvesting and water splitting is a pivotal challenge. Atomically thin transition metal dichalcogenides (TMD) are considered as promising photocatalysts because of their wide range of available electronic properties and compositional variability. However, trade-offs between carrier transport efficiency, light absorption, and electrochemical reactivity have limited their prospects. We here combine two approaches that synergistically enhance the efficiency of photocarrier generation and electrocatalytic efficiency of two-dimensional (2D) TMDs. The arrangement of monolayer WS2 and MoS2 into a heterojunction and subsequent nanostructuring into a nanoscroll (NS) yields significant modifications of fundamental properties from its constituents. Spectroscopic characterization and ab initio simulation demonstrate the beneficial effects of straining and wall interactions on the band structure of such a heterojunction-NS that enhance the electrochemical reaction rate by an order of magnitude compared to planar heterojunctions. Phototrapping in this NS further increases the light-matter interaction and yields superior photocatalytic performance compared to previously reported 2D material catalysts and is comparable to noble-metal catalyst systems in the photoelectrochemical hydrogen evolution reaction (PEC-HER) process. Our approach highlights the potential of morphologically varied TMD-based catalysts for PEC-HER.

CCAAT/enhancer-binding protein ß and NF-κB mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to IL-1ß.

A large set of chemokines is highly up-regulated in human chondrocytes in response to IL-1ß (Sandell, L. J., Xing, X., Franz, C., Davies, S., Chang, L. W., and Patra, D. (2008) Osteoarthr. Cartil. 16, 1560-1571). To investigate the mechanism of transcriptional regulation, deletion constructs of selected chemokine gene promoters, the human CCL3 (MIP-1α) and CCL4 (MIP-1ß), were transfected into human chondrocytes with or without IL-1ß. The results show that an IL-1ß-responsive element is located between bp -300 and -140 of the CCL3 promoter and between bp -222 and -100 of the CCL4 promoter. Because both of these elements contain CCAAT/enhancer-binding protein ß (C/EBPß) motifs, the function of C/EBPß was examined. IL-1ß stimulated the expression of C/EBPß, and the direct binding of C/EBPß to the C/EBPß motif was confirmed by EMSA and ChIP analyses. The -300 bp CCL3 promoter and -222 bp CCL4 promoter were strongly up-regulated by co-transfection with the C/EBPß expression vector. Mutation of the C/EBPß motif and reduction of C/EBPß expression by siRNA decreased the up-regulation. Additionally, another cytokine-related transcription factor, NF-κB, was also shown to be involved in the up-regulation of chemokines in response to IL-1ß, and the binding site was identified. The regulation of C/EBPß and NF-κB was confirmed by the inhibition by C/EBPß and NF-κB and by transfection with C/EBPß and NF-κB expression vectors in the presence or absence of IL-1ß. Taken together, our results suggest that C/EBPß and NF-κB are both involved in the IL-1ß-responsive up-regulation of chemokine genes in human chondrocytes. Time course experiments indicated that C/EBPß gradually and steadily induces chemokine up-regulation, whereas NF-κB activity was highest at the early stage of chemokine up-regulation.

Neurogenin and NeuroD direct transcriptional targets and their regulatory enhancers.

Proneural basic helix-loop-helix proteins are key regulators of neurogenesis but their 'proneural' function is not well understood, partly because primary targets have not been systematically defined. Here, we identified direct transcriptional targets of the bHLH proteins Neurogenin and NeuroD and found that primary roles of these transcription factors are to induce regulators of transcription, signal transduction, and cytoskeletal rearrangement for neuronal differentiation and migration. We determined targets induced in both Xenopus and mouse, which represent evolutionarily conserved core mediators of Neurogenin and NeuroD activities. We defined consensus sequences for Neurogenin and NeuroD binding and identified responsive enhancers in seven shared target genes. These enhancers commonly contained clustered, conserved consensus-binding sites and drove neural-restricted transgene expression in Xenopus embryos. We then used this enhancer signature in a genome-wide computational approach to predict additional Neurogenin/NeuroD target genes involved in neurogenesis. Taken together, these data demonstrate that Neurogenin and NeuroD preferentially recognize neurogenesis-related targets through an enhancer signature of clustered consensus-binding sites and regulate neurogenesis by activating a core set of transcription factors, which build a robust network controlling neurogenesis.

Resistin induces expression of proinflammatory cytokines and chemokines in human articular chondrocytes via transcription and messenger RNA stabilization.

OBJECTIVE: To elucidate the effects of resistin on human articular chondrocytes and to generate a picture of their regulation at the transcriptional and posttranscriptional levels. METHODS: Human articular chondrocytes were cultured with resistin. Changes in gene expression were analyzed at various doses and times. Cells were also treated with the transcription inhibitor actinomycin D after resistin treatment or with the NF-kappaB inhibitor IKK-NBD before resistin treatment. Gene expression was tested by quantitative real-time polymerase chain reaction. Computational analysis for transcription factor binding motifs was performed on the promoter regions of differentially expressed genes. TC-28 chondrocytes were transfected with CCL3 and CCL4 promoter constructs, pNF-kappaB reporter, and NF-kappaB and CCAAT/enhancer binding protein beta (C/EBPbeta) expression vectors with or without resistin. RESULTS: Resistin-treated human articular chondrocytes increased the expression of cytokines and chemokines. Levels of messenger RNA (mRNA) for matrix metalloproteinase 1 (MMP-1), MMP-13, and ADAMTS-4 also increased, while type II collagen alpha1 (COL2A1) and aggrecan were down-regulated. The cytokine and chemokine genes could be categorized into 3 groups according to the pattern of mRNA expression over a 24-hour time course. One pattern suggested rapid regulation by mRNA stability. The second and third patterns were consistent with transcriptional regulation. Computational analysis suggested the transcription factors NF-kappaB and C/EBPbeta were involved in the resistin-induced up-regulation. This prediction was confirmed by the cotransfection of NF-kappaB and C/EBPbeta and the IKK-NBD inhibition. CONCLUSION: Resistin has diverse effects on gene expression in human chondrocytes, affecting chemokines, cytokines, and matrix genes. Messenger RNA stabilization and transcriptional up-regulation are involved in resistin-induced gene expression in human chondrocytes.

Locus-wide identification of Egr2/Krox20 regulatory targets in myelin genes.

Myelination of peripheral nerves by Schwann cells depends upon a gene regulatory network controlled by early growth response Egr2/Krox20, which is specifically required for Schwann cells to initiate and maintain myelination. To elucidate the mechanism by which Egr2 regulates gene expression during myelination, we have performed chromatin immunoprecipitation analysis on myelinating rat sciatic nerve in vivo. The resulting samples were applied to a tiled microarray consisting of a broad spectrum of genes that are activated or repressed in Egr2-deficient mice. The results show extensive binding within myelin-associated genes, as well as some genes that become repressed in myelinating Schwann cells. Many of the Egr2 peaks coincide with regions of open chromatin, which is a marker of enhancer regions. In addition, further analysis showed that there is substantial colocalization of Egr2 binding with Sox10, a transcription factor required for Schwann cell specification and other stages of Schwann cell development. Finally, we have found that Egr2 binds to promoters of several lipid biosynthetic genes, which is consistent with their dramatic up-regulation during the formation of lipid-rich myelin. Overall, this analysis provides a locus-wide profile of Egr2 binding patterns in major myelin-associated genes using myelinating peripheral nerve.

An integrative approach identifies dysregulated long non-coding RNAs as microRNA decoys during nevus to melanoma transformation.

Mounting evidence supports a role for dysregulated long non-coding RNAs (lncRNA) in the development of many cancers. A recently discovered function of lncRNAs is to act as microRNA (miR) decoys or competing endogenous RNAs, which sequester specific miRs and relieve negative regulation of mRNA expression by miRs. Although a large number of non-coding RNAs are thought to function as competing endogenous RNAs, miR-sequestering lncRNAs involved in nevus to melanoma transformation remain largely unknown. In this study, we applied a bioinformatics approach to a unique dataset of benign melanocytic nevi and primary melanomas of the skin in order to fill this research gap. We modified a previously published miR target prediction algorithm, RNAhybrid, and improved its search efficiency. We reported the presence of many lncRNAs and miRs deregulated when transitioning from a senescence-like state of nevi to melanoma. We provided evidence of a relatively new and understudied mechanism of gene regulation during this process and identified for the first time lncRNAs (n = 122) that may potentially function as miR decoys as well as their target miRs during nevus to melanoma transformation. The knowledge presented here can be employed for developing biomarkers for diagnostic and risk stratification purposes.

Analysis of peripheral nerve expression profiles identifies a novel myelin glycoprotein, MP11.

The myelin sheath insulates axons and allows for rapid salutatory conduction in the nervous system of all vertebrates. The formation of peripheral myelin requires expression of the transcription factor Egr2, which is responsible for inducing such essential myelin-associated genes as Mpz, Mbp, Pmp22, and Mag. Using microarray analysis to compare gene expression patterns in peripheral nerve during development, during remyelination after nerve injury, and in a congenital hypomyelinating mouse model, we identified an evolutionarily conserved novel component of myelin called Mp11 (myelin protein of 11 kDa). The Mp11 genomic locus contains multiple conserved Egr binding sites, and Mp11 induction is regulated by the expression of Egr2. Similar to other Egr2-dependent genes, it is induced during developmental myelination and remyelination after nerve injury. Mp11 is a glycoprotein expressed preferentially in the myelin of the peripheral nervous system versus CNS and is specifically localized to the Schmidt-Lanterman incisures and paranodes of peripheral nerve. The Mp11 protein contains no identifiable similarity to other known protein domains or motifs. However, like other myelin genes, strict Mp11 expression levels are a requirement for the in vitro myelination of DRG neurons, indicating that this previously uncharacterized gene product is a critical component of peripheral nervous system myelin.

PAP: a comprehensive workbench for mammalian transcriptional regulatory sequence analysis.

Given the recent explosion of publications that employ microarray technology to monitor genome-wide expression and that correlate these expression changes to biological processes or to disease states, the determination of the transcriptional regulation of these co-expressed genes is the next major step toward deciphering the genetic network governing the pathway or disease under study. Although computational approaches have been proposed for this purpose, there is no integrated and user-friendly software application that allows experimental biologists to tackle this problem in higher eukaryotes. We have previously reported a systematic, statistical model of mammalian transcriptional regulatory sequence analysis. We have now made crucial extensions to this model and have developed a comprehensive, user-friendly web application suite termed the Promoter Analysis Pipeline (PAP). PAP is available at: http://bioinformatics.wustl.edu/webTools/portalModule/PromoterSearch.do.

Designing Electrostatic Interactions via Polyelectrolyte Monomer Sequence.

Charged polymers are ubiquitous in biological systems because electrostatic interactions can drive complicated structure formation and respond to environmental parameters such as ionic strength and pH. In these systems, function emerges from sophisticated molecular design; for example, intrinsically disordered proteins leverage specific sequences of monomeric charges to control the formation and function of intracellular compartments known as membraneless organelles. The role of a charged monomer sequence in dictating the strength of electrostatic interactions remains poorly understood despite extensive evidence that sequence is a powerful tool biology uses to tune soft materials. In this article, we use a combination of theory, experiment, and simulation to establish the physical principles governing sequence-driven control of electrostatic interactions. We predict how arbitrary sequences of charge give rise to drastic changes in electrostatic interactions and correspondingly phase behavior. We generalize a transfer matrix formalism that describes a phase separation phenomenon known as "complex coacervation" and provide a theoretical framework to predict the phase behavior of charge sequences. This work thus provides insights into both how charge sequence is used in biology and how it could be used to engineer properties of synthetic polymer systems.