Maria Barna

All Publications

• Decoding the Function of Expansion Segments in Ribosomes. Molecular cell Fujii, K., Susanto, T. T., Saurabh, S., Barna, M. 2018; 72 (6): 1013

  Abstract

  Expansion segments (ESs) are enigmatic insertions within the eukaryotic ribosome, the longest of which resemble tentacle-like extensions that vary in length and sequence across evolution, with a largely unknown function. By selectively engineering rRNA in yeast, we find that one of the largest ESs, ES27L, has an unexpected function in translation fidelity. Ribosomes harboring a deletion in the distal portion of ES27L have increased amino acid misincorporation, as well as readthrough and frameshifting errors. Byemploying quantitative mass spectrometry, we further find that ES27L acts as an RNA scaffold to facilitate binding of a conserved enzyme, methionine amino peptidase (MetAP). We show that MetAP unexpectedly controls the accuracy of ribosome decoding, which is coupled to an increase in its enzymatic function through its interaction with ES27L. These findings reveal that variable ESs of the ribosome serve important functional roles and act as platforms for the binding of proteins that modulate translation across evolution.

  View details for DOI 10.1016/j.molcel.2018.11.023

  View details for PubMedID 30576652

• The Discovery of Ribosome Heterogeneity and Its Implications for Gene Regulation and Organismal Life. Molecular cell Genuth, N. R., Barna, M. 2018; 71 (3): 364–74

  Abstract

  The ribosome has recently transitioned from being viewed as a passive, indiscriminate machine to a more dynamic macromolecular complex with specialized roles in the cell. Here, we discuss the historical milestones from the discovery of the ribosome itself to how this ancient machinery has gained newfound appreciation as a more regulatory participant in the central dogma of gene expression. The first emerging examples of direct changes in ribosome composition at the RNA and protein level, coupled with an increased awareness of the role individual ribosomal components play in the translation of specific mRNAs, is opening a new field of study centered on ribosome-mediated control of gene regulation. In this Perspective, we discuss our current understanding of the known functions for ribosome heterogeneity, including specialized translation of individual transcripts, and its implications for the regulation and expression of key gene regulatory networks. In addition, we suggest what the crucial next steps are to ascertain the extent of ribosome heterogeneity and specialization and its importance for regulation of the proteome within subcellular space, across different cell types, and during multi-cellular organismal development.

  View details for DOI 10.1016/j.molcel.2018.07.018

  View details for PubMedID 30075139

• The Mammalian Ribo-interactome Reveals Ribosome Functional Diversity and Heterogeneity. Cell Simsek, D., Tiu, G. C., Flynn, R. A., Byeon, G. W., Leppek, K., Xu, A. F., Chang, H. Y., Barna, M. 2017; 169 (6): 1051-1065 e18

  Abstract

  During eukaryotic evolution, ribosomes have considerably increased in size, forming a surface-exposed ribosomal RNA (rRNA) shell of unknown function, which may create an interface for yet uncharacterized interacting proteins. To investigate such protein interactions, we establish a ribosome affinity purification method that unexpectedly identifies hundreds of ribosome-associated proteins (RAPs) from categories including metabolism and cell cycle, as well as RNA- and protein-modifying enzymes that functionally diversify mammalian ribosomes. By further characterizing RAPs, we discover the presence of ufmylation, a metazoan-specific post-translational modification (PTM), on ribosomes and define its direct substrates. Moreover, we show that the metabolic enzyme, pyruvate kinase muscle (PKM), interacts with sub-pools of endoplasmic reticulum (ER)-associated ribosomes, exerting a non-canonical function as an RNA-binding protein in the translation of ER-destined mRNAs. Therefore, RAPs interconnect one of life's most ancient molecular machines with diverse cellular processes, providing an additional layer of regulatory potential to protein expression.

  View details for DOI 10.1016/j.cell.2017.05.022

  View details for PubMedID 28575669

• Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide. Molecular cell Shi, Z., Fujii, K., Kovary, K. M., Genuth, N. R., Röst, H. L., Teruel, M. N., Barna, M. 2017

  Abstract

  Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression.

  View details for PubMedID 28625553

• RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation. Nature Xue, S., Tian, S., Fujii, K., Kladwang, W., Das, R., Barna, M. 2015; 517 (7532): 33-38

  Abstract

  Emerging evidence suggests that the ribosome has a regulatory function in directing how the genome is translated in time and space. However, how this regulation is encoded in the messenger RNA sequence remains largely unknown. Here we uncover unique RNA regulons embedded in homeobox (Hox) 5' untranslated regions (UTRs) that confer ribosome-mediated control of gene expression. These structured RNA elements, resembling viral internal ribosome entry sites (IRESs), are found in subsets of Hox mRNAs. They facilitate ribosome recruitment and require the ribosomal protein RPL38 for their activity. Despite numerous layers of Hox gene regulation, these IRES elements are essential for converting Hox transcripts into proteins to pattern the mammalian body plan. This specialized mode of IRES-dependent translation is enabled by an additional regulatory element that we term the translation inhibitory element (TIE), which blocks cap-dependent translation of transcripts. Together, these data uncover a new paradigm for ribosome-mediated control of gene expression and organismal development.

  View details for DOI 10.1038/nature14010

  View details for PubMedID 25409156

• Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning. Nature Sanders, T. A., Llagostera, E., Barna, M. 2013; 497 (7451): 628-632

  Abstract

  The ability of signalling proteins to traverse tissues containing tightly packed cells is of fundamental importance for cell specification and tissue development; however, how this is achieved at a cellular level remains poorly understood. For more than a century, the vertebrate limb bud has served as a model for studying cell signalling during embryonic development. Here we optimize single-cell real-time imaging to delineate the cellular mechanisms for how signalling proteins, such as sonic hedgehog (SHH), that possess membrane-bound covalent lipid modifications traverse long distances within the vertebrate limb bud in vivo. By directly imaging SHH ligand production under native regulatory control in chick (Gallus gallus) embryos, our findings show that SHH is unexpectedly produced in the form of a particle that remains associated with the cell via long cytoplasmic extensions that span several cell diameters. We show that these cellular extensions are a specialized class of actin-based filopodia with novel cytoskeletal features that have not been previously described. Notably, particles containing SHH travel along these extensions with a net anterograde movement within the field of SHH cell signalling. We further show that in SHH-responding cells, specific subsets of SHH co-receptors, including cell adhesion molecule downregulated by oncogenes (CDO) and brother of CDO (BOC), actively distribute and co-localize in specific micro-domains within filopodial extensions, far from the cell body. Stabilized interactions are formed between filopodia containing SHH ligand and those containing co-receptors over a long range. These results suggest that contact-mediated release propagated by specialized filopodia contributes to the delivery of SHH at a distance. Together, these studies identify an important mode of communication between cells that considerably extends our understanding of ligand movement and reception during vertebrate tissue patterning.

  View details for DOI 10.1038/nature12157

  View details for PubMedID 23624372

• Ribosome-Mediated Specificity in Hox mRNA Translation and Vertebrate Tissue Patterning CELL Kondrashov, N., Pusic, A., Stumpf, C. R., Shimizu, K., Hsieh, A. C., Xue, S., Ishijima, J., Shiroishi, T., Barna, M. 2011; 145 (3): 383-397

  Abstract

  Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than regulatory capacity in mRNA translation. Here we identify mutations of the Ribosomal Protein L38 (Rpl38) gene in mice exhibiting surprising tissue-specific patterning defects, including pronounced homeotic transformations of the axial skeleton. In Rpl38 mutant embryos, global protein synthesis is unchanged; however the translation of a select subset of Homeobox mRNAs is perturbed. Our data reveal that RPL38 facilitates 80S complex formation on these mRNAs as a regulatory component of the ribosome to confer transcript-specific translational control. We further show that Rpl38 expression is markedly enriched in regions of the embryo where loss-of-function phenotypes occur. Unexpectedly, a ribosomal protein (RP) expression screen reveals dynamic regulation of individual RPs within the vertebrate embryo. Collectively, these findings suggest that RP activity may be highly regulated to impart a new layer of specificity in the control of gene expression and mammalian development.

  View details for DOI 10.1016/j.cell.2011.03.028

  View details for Web of Science ID 000290022900008

  View details for PubMedID 21529712

• Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency NATURE Barna, M., Pusic, A., Zollo, O., Costa, M., Kondrashov, N., Rego, E., Rao, P. H., Ruggero, D. 2008; 456 (7224): 971-U79

  Abstract

  The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA. Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Emu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc-overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap-dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (also known as Cdc2l and PITSLRE), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Emu-Myc/+ mice. When accurate translational control is re-established in Emu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

  View details for DOI 10.1038/nature07449

  View details for Web of Science ID 000261768300050

  View details for PubMedID 19011615

• Translation control of the immune checkpoint in cancer and its therapeutic targeting. Nature medicine Xu, Y., Poggio, M., Jin, H. Y., Shi, Z., Forester, C. M., Wang, Y., Stumpf, C. R., Xue, L., Devericks, E., So, L., Nguyen, H. G., Griselin, A., Gordan, J. D., Umetsu, S. E., Reich, S. H., Worland, S. T., Asthana, S., Barna, M., Webster, K. R., Cunningham, J. T., Ruggero, D. 2019

  Abstract

  Cancer cells develop mechanisms to escape immunosurveillance, among which modulating the expression of immune suppressive messenger RNAs is most well-documented. However, how this is molecularly achieved remains largely unresolved. Here, we develop an in vivo mouse model of liver cancer to study oncogene cooperation in immunosurveillance. We show that MYC overexpression (MYCTg) synergizes with KRASG12D to induce an aggressive liver tumor leading to metastasis formation and reduced mouse survival compared with KRASG12D alone. Genome-wide ribosomal footprinting of MYCTg;KRASG12 tumors compared with KRASG12D revealed potential alterations in translation of mRNAs, including programmed-death-ligand 1 (PD-L1). Further analysis revealed that PD-L1 translation is repressed in KRASG12D tumors by functional, non-canonical upstream open reading frames in its 5' untranslated region, which is bypassed in MYCTg;KRASG12D tumors to evade immune attack. We show that this mechanism of PD-L1 translational upregulation was effectively targeted by a potent, clinical compound that inhibits eIF4E phosphorylation, eFT508, which reverses the aggressive and metastatic characteristics of MYCTg;KRASG12D tumors. Together, these studies reveal how immune-checkpoint proteins are manipulated by distinct oncogenes at the level of mRNA translation, which can be exploited for new immunotherapies.

  View details for DOI 10.1038/s41591-018-0321-2

  View details for PubMedID 30643286

• The p53 family members have distinct roles during mammalian embryonic development. Cell death and differentiation Van Nostrand, J. L., Bowen, M. E., Vogel, H., Barna, M., Attardi, L. D. 2017

  Abstract

  The p53 tumor suppressor is a member of a multi-protein family, including the p63 and p73 transcription factors. These proteins can bind to the same consensus sites in DNA and activate the same target genes, suggesting that there could be functional redundancy between them. Indeed, double mutant mice heterozygous for any two family member-encoding genes display enhanced cancer phenotypes relative to single heterozygous mutants. However, whether the family members play redundant roles during embryonic development has remained largely unexplored. Although p53(-/-); p73(-/-) mice are born and manifest phenotypes characteristic of each of the single mutants, the consequences of combined deficiency of p63 and either p53 or p73 have not been elucidated. To examine the functional overlap of p53 family members during development, we bred and analyzed compound mutant embryo phenotypes. We discovered that double knockout embryos and five allele knockout embryos only displayed obvious defects accounted for by loss of single p53 family members. Surprisingly, at mid-gestation (E11), we identified a single viable triple knockout embryo that appeared grossly normal. Together, these results suggest that the p53 family is not absolutely required for early embryogenesis and that p53 family members are largely non-redundant during early development.

  View details for DOI 10.1038/cdd.2016.128

  View details for PubMedID 28211873

• Pervasive translational regulation of the cell signalling circuitry underlies mammalian development NATURE COMMUNICATIONS Fujii, K., Shi, Z., Zhulyn, O., Denans, N., Barna, M. 2017; 8

  Abstract

  The degree and dynamics of translational control during mammalian development remain poorly understood. Here we monitored translation of the mammalian genome as cells become specified and organize into tissues in vivo. This identified unexpected and pervasive translational regulation of most of the core signalling circuitry including Shh, Wnt, Hippo, PI3K and MAPK pathways. We further identify and functionally characterize a complex landscape of upstream open reading frames (uORFs) across 5'-untranslated regions (UTRs) of key signalling components. Focusing on the Shh pathway, we demonstrate the importance of uORFs within the major SHH receptor, Ptch1, in control of cell signalling and neuronal differentiation. Finally, we show that the expression of hundreds of mRNAs underlying critical tissue-specific developmental processes is largely regulated at the translation but not transcript levels. Altogether, this work reveals a new layer of translational control to major signalling components and gene regulatory networks that diversifies gene expression spatially across developing tissues.

  View details for DOI 10.1038/ncomms14443

  View details for Web of Science ID 000393859100001

  View details for PubMedID 28195124

• Functional 5' UTR mRNA structures in eukaryotic translation regulation and how to find them. Nature reviews. Molecular cell biology Leppek, K., Das, R., Barna, M. 2017

  Abstract

  RNA molecules can fold into intricate shapes that can provide an additional layer of control of gene expression beyond that of their sequence. In this Review, we discuss the current mechanistic understanding of structures in 5' untranslated regions (UTRs) of eukaryotic mRNAs and the emerging methodologies used to explore them. These structures may regulate cap-dependent translation initiation through helicase-mediated remodelling of RNA structures and higher-order RNA interactions, as well as cap-independent translation initiation through internal ribosome entry sites (IRESs), mRNA modifications and other specialized translation pathways. We discuss known 5' UTR RNA structures and how new structure probing technologies coupled with prospective validation, particularly compensatory mutagenesis, are likely to identify classes of structured RNA elements that shape post-transcriptional control of gene expression and the development of multicellular organisms.

  View details for PubMedID 29165424

• An emerging role for the ribosome as a nexus for post-translational modifications. Current opinion in cell biology Simsek, D., Barna, M. 2017; 45: 92–101

  Abstract

  The ribosome is one of life's most ancient molecular machines that has historically been viewed as a backstage participant in gene regulation, translating the genetic code across all kingdoms of life in a rote-like fashion. However, recent studies suggest that intrinsic components of the ribosome can be regulated and diversified as a means to intricately control the expression of the cellular proteome. In this review, we discuss advances in the characterization of ribosome post-translational modifications (PTMs) from past to present. We specifically focus on emerging examples of ribosome phosphorylation and ubiquitylation, which are beginning to showcase that PTMs of the ribosome are versatile, may have functional consequences for translational control, and are intimately linked to human disease. We further highlight the key questions that remain to be addressed to gain a more complete picture of the array of ribosome PTMs and the upstream enzymes that control them, which may endow ribosomes with greater regulatory potential in gene regulation and control of cellular homeostasis.

  View details for DOI 10.1016/j.ceb.2017.02.010

  View details for PubMedID 28445788

• Translating the Genome in Time and Space: Specialized Ribosomes, RNA Regulons, and RNA-Binding Proteins. Annual review of cell and developmental biology Shi, Z., Barna, M. 2015; 31: 31-54

  Abstract

  A central question in cell and developmental biology is how the information encoded in the genome is differentially interpreted to generate a diverse array of cell types. A growing body of research on posttranscriptional gene regulation is revealing that both global protein synthesis rates and the translation of specific mRNAs are highly specialized in different cell types. How this exquisite translational regulation is achieved is the focus of this review. Two levels of regulation are discussed: the translation machinery and cis-acting elements within mRNAs. Recent evidence shows that the ribosome itself directs how the genome is translated in time and space and reveals surprising functional specificity in individual components of the core translation machinery. We are also just beginning to appreciate the rich regulatory information embedded in the untranslated regions of mRNAs, which direct the selective translation of transcripts. These hidden RNA regulons may interface with a myriad of RNA-binding proteins and specialized translation machinery to provide an additional layer of regulation to how transcripts are spatiotemporally expressed. Understanding this largely unexplored world of translational codes hardwired in the core translation machinery is an exciting new research frontier fundamental to our understanding of gene regulation, organismal development, and evolution. Expected final online publication date for the Annual Review of Cell and Developmental Biology Volume 31 is November 7, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

  View details for DOI 10.1146/annurev-cellbio-100814-125346

  View details for PubMedID 26443190

• Differential Requirements for eIF4E Dose in Normal Development and Cancer CELL Truitt, M. L., Conn, C. S., Shi, Z., Pang, X., Tokuyasu, T., Coady, A. M., Seo, Y., Barna, M., Ruggero, D. 2015; 162 (1): 59-71

  Abstract

  eIF4E, the major cap-binding protein, has long been considered limiting for translating the mammalian genome. However, the eIF4E dose requirement at an organismal level remains unexplored. By generating an Eif4e haploinsufficient mouse, we found that a 50% reduction in eIF4E expression, while compatible with normal development and global protein synthesis, significantly impeded cellular transformation. Genome-wide translational profiling uncovered a translational program induced by oncogenic transformation and revealed a critical role for the dose of eIF4E, specifically in translating a network of mRNAs enriched for a unique 5' UTR signature. In particular, we demonstrate that the dose of eIF4E is essential for translating mRNAs that regulate reactive oxygen species, fueling transformation and cancer cell survival in vivo. Our findings indicate eIF4E is maintained at levels in excess for normal development that are hijacked by cancer cells to drive a translational program supporting tumorigenesis.

  View details for DOI 10.1016/j.cell.2015.05.049

  View details for Web of Science ID 000357542300009

  View details for PubMedID 26095252

• The ribosome prophecy. Nature reviews. Molecular cell biology Barna, M. 2015; 16 (5): 268

  View details for DOI 10.1038/nrm3993

  View details for PubMedID 25907611

• Cis-regulatory RNA elements that regulate specialized ribosome activity. RNA biology Xue, S., Barna, M. 2015: 0

  Abstract

  Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5'UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.

  View details for DOI 10.1080/15476286.2015.1085149

  View details for PubMedID 26327194

• Specialized filopodia: at the 'tip' of morphogen transport and vertebrate tissue patterning. Current opinion in genetics & development Fairchild, C. L., Barna, M. 2014; 27C: 67–73

  Abstract

  For over a century, biologists have strived to unravel the mechanisms that establish how cells are informed of their position in the embryo and differentiate to give rise to complex organs and structures. However, the historical idea that one predominant mode of ligand transport, largely accounted for by free diffusion, can explain how all signaling molecules, known as morphogens, control tissue patterning has greatly hindered our ability to fully appreciate the complexities driving the delivery and reception of signaling molecules at a distance. In reality, a cell's shape, morphology, and location change continuously as development progresses. Thus, cellular context poses distinct challenges for morphogen transport in each unique cellular environment. Emerging studies reveal that some cells overcome such obstacles in an unexpected manner: via long, cellular projections, or specialized filopodia, that link distant cells and traffic signaling components. Here, we will review recent findings describing specialized filopodia and discuss the potential mechanisms and implications for filopodia-based long-range cell signaling and communication, particularly within the developing vertebrate embryo.

  View details for DOI 10.1016/j.gde.2014.03.013

  View details for PubMedID 24907447

• When the going gets tough: scientists' personal challenges. Cell Lengerke, C., Fernandez-Capetillo, O., Tolic-Norrelykke, I., Barna, M., Coleman, T., Zamboni, D. 2014; 159 (2): 225–26

  View details for PubMedID 25436265

• Tailor Made Protein Synthesis for HSCs. Cell stem cell Barna, M., Ruggero, D. 2014; 14 (4): 423–24

  Abstract

  Translation control is a prevalent form of gene expression regulation in developmental and stem cell biology. A recent paper by Signer et al. (2014) measures protein synthesis in the mouse hematopoietic compartment and reveals the importance of diminished protein production for maintaining hematopoietic stem cell function and restraining oncogenic potential.

  View details for DOI 10.1016/j.stem.2014.03.011

  View details for PubMedID 24702992

• Ribosomes take control PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Barna, M. 2013; 110 (1): 9-10

  View details for DOI 10.1073/pnas.1218764110

  View details for Web of Science ID 000313630300020

  View details for PubMedID 23243144

  View details for PubMedCentralID PMC3538271

• Specialized ribosomes: a new frontier in gene regulation and organismal biology NATURE REVIEWS MOLECULAR CELL BIOLOGY Xue, S., Barna, M. 2012; 13 (6): 355-369

  Abstract

  Historically, the ribosome has been viewed as a complex ribozyme with constitutive rather than intrinsic regulatory capacity in mRNA translation. However, emerging studies reveal that ribosome activity may be highly regulated. Heterogeneity in ribosome composition resulting from differential expression and post-translational modifications of ribosomal proteins, ribosomal RNA (rRNA) diversity and the activity of ribosome-associated factors may generate 'specialized ribosomes' that have a substantial impact on how the genomic template is translated into functional proteins. Moreover, constitutive components of the ribosome may also exert more specialized activities by virtue of their interactions with specific mRNA regulatory elements such as internal ribosome entry sites (IRESs) or upstream open reading frames (uORFs). Here we discuss the hypothesis that intrinsic regulation by the ribosome acts to selectively translate subsets of mRNAs harbouring unique cis-regulatory elements, thereby introducing an additional level of regulation in gene expression and the life of an organism.

  View details for DOI 10.1038/nrm3359

  View details for Web of Science ID 000304354100011

  View details for PubMedID 22617470

• Visualization of cartilage formation: Insight into cellular properties of skeletal progenitors and chondrodysplasia syndromes DEVELOPMENTAL CELL Barna, M., Niswander, L. 2007; 12 (6): 931-941

  Abstract

  The cellular events underlying skeletal morphogenesis and the formation of cartilage templates are largely unknown. We generated an imaging system to dynamically visualize limb mesenchymal cells undergoing successive phases in cartilage formation and to delineate the cellular function of key regulators of chondrogenesis found mutated in chondrodysplasia syndromes. We uncovered an unsuspected role for Sox9 in control of cell morphology, independent from its major downstream target ColIIa, critically required for the mesenchyme-to-chondrocyte transition. In contrast, Bmp signaling regulates a cellular program we term "compaction" in which mesenchymal cells acquire a cohesive cell behavior required to delineate the boundaries and size of cartilage elements. Moreover, we visualized labeled progenitor cells from different regions of the limb bud and identified unique cellular properties that may direct their contribution toward specific skeletal elements such as the humerus or digits. These findings shed light on the cellular basis for chondrodysplasia syndromes and formation of the vertebrate skeleton.

  View details for DOI 10.1016/j.devcel.2007.04.016

  View details for Web of Science ID 000247119700013

  View details for PubMedID 17543865

• Gli3 and Plzf cooperate in proximal limb patterning at early stages of limb development NATURE Barna, M., Pandolfi, P. P., Niswander, L. 2005; 436 (7048): 277-281

  Abstract

  The vertebrate limb initially develops as a bud of mesenchymal cells that subsequently aggregate in a proximal to distal (P-D) sequence to give rise to cartilage condensations that prefigure all limb skeletal components. Of the three cardinal limb axes, the mechanisms that lead to establishment and patterning of skeletal elements along the P-D axis are the least understood. Here we identify a genetic interaction between Gli3 (GLI-Kruppel family member 3) and Plzf (promyelocytic leukaemia zinc finger, also known as Zbtb16 and Zfp145), which is required specifically at very early stages of limb development for all proximal cartilage condensations in the hindlimb (femur, tibia, fibula). Notably, distal condensations comprising the foot are relatively unperturbed in Gli3(-/-);Plzf(-/-) mouse embryos. We demonstrate that the cooperative activity of Gli3 and Plzf establishes the correct temporal and spatial distribution of chondrocyte progenitors in the proximal limb-bud independently of known P-D patterning markers and overall limb-bud size. Moreover, the limb defects in Gli3(-/-);Plzf(-/-) embryos correlate with the transient death of a specific subset of proximal mesenchymal cells that express bone morphogenetic protein receptor, type 1B (Bmpr1b) at the onset of limb development. These findings suggest that the development of proximal and distal skeletal elements is distinctly regulated early during limb-bud formation. The initial division of the vertebrate limb into two distinct molecular domains is consistent with fossil evidence indicating that the upper and lower extremities of the limb have different evolutionary origins.

  View details for DOI 10.1038/nature03801

  View details for Web of Science ID 000230459500045

  View details for PubMedID 16015334

• Essential role of Plzf in maintenance of spermatogonial stem cells NATURE GENETICS Costoya, J. A., Hobbs, R. M., Barna, M., Cattoretti, G., Manova, K., Sukhwani, M., Orwig, K. E., Wolgemuth, D. J., Pandolfi, P. P. 2004; 36 (6): 653-659

  Abstract

  Little is known of the molecular mechanisms whereby spermatogonia, mitotic germ cells of the testis, self-renew and differentiate into sperm. Here we show that Zfp145, encoding the transcriptional repressor Plzf, has a crucial role in spermatogenesis. Zfp145 expression was restricted to gonocytes and undifferentiated spermatogonia and was absent in tubules of W/W(v) mutants that lack these cells. Mice lacking Zfp145 underwent a progressive loss of spermatogonia with age, associated with increases in apoptosis and subsequent loss of tubule structure but without overt differentiation defects or loss of the supporting Sertoli cells. Spermatogonial transplantation experiments revealed a depletion of spermatogonial stem cells in the adult. Microarray analysis of isolated spermatogonia from Zfp145-null mice before testis degeneration showed alterations in the expression profile of genes associated with spermatogenesis. These results identify Plzf as a spermatogonia-specific transcription factor in the testis that is required to regulate self-renewal and maintenance of the stem cell pool.

  View details for DOI 10.1038/ng1367

  View details for Web of Science ID 000221763700026

  View details for PubMedID 15156143

• Plzf mediates transcriptional repression of HoxD gene expression through chromatin remodeling DEVELOPMENTAL CELL Barna, M., Merghoub, T., Costoya, J. A., Ruggero, D., Branford, M., Bergia, A., Samori, B., Pandolfi, P. P. 2002; 3 (4): 499-510

  Abstract

  The molecular mechanisms that regulate coordinated and colinear activation of Hox gene expression in space and time remain poorly understood. Here we demonstrate that Plzf regulates the spatial expression of the AbdB HoxD gene complex by binding to regulatory elements required for restricted Hox gene expression and can recruit histone deacetylases to these sites. We show by scanning forced microscopy that Plzf, via homodimerization, can form DNA loops and bridge distant Plzf binding sites located within HoxD gene regulatory elements. Furthermore, we demonstrate that Plzf physically interacts with Polycomb proteins on DNA. We propose a model by which the balance between activating morphogenic signals and transcriptional repressors such as Plzf establishes proper Hox gene expression boundaries in the limb bud.

  View details for Web of Science ID 000178629300008

  View details for PubMedID 12408802

• Plzf regulates limb and axial skeletal patterning NATURE GENETICS Barna, M., Hawe, N., Niswander, L., Pandolfi, P. P. 2000; 25 (2): 166-172

  Abstract

  The promyelocytic leukaemia zinc finger (Plzf) protein (encoded by the gene Zfp145) belongs to the POZ/zinc-finger family of transcription factors. Here we generate Zfp145-/- mice and show that Plzf is essential for patterning of the limb and axial skeleton. Plzf inactivation results in patterning defects affecting all skeletal structures of the limb, including homeotic transformations of anterior skeletal elements into posterior structures. We demonstrate that Plzf acts as a growth-inhibitory and pro-apoptotic factor in the limb bud. The expression of members of the abdominal b (Abdb) Hox gene complex, as well as genes encoding bone morphogenetic proteins (Bmps), is altered in the developing limb of Zfp145-/- mice. Plzf regulates the expression of these genes in the absence of aberrant polarizing activity and independently of known patterning genes. Zfp145-/- mice also exhibit anterior-directed homeotic transformation throughout the axial skeleton with associated alterations in Hox gene expression. Plzf is therefore a mediator of anterior-to-posterior (AP) patterning in both the axial and appendicular skeleton and acts as a regulator of Hox gene expression.

  View details for Web of Science ID 000087459200014

  View details for PubMedID 10835630

• Interleukin-12 promotes recovery from viral encephalitis VIRAL IMMUNOLOGY Komatsu, T., Barna, M., Reiss, C. S. 1997; 10 (1): 35-47

  Abstract

  Infusion of interleukin-12 (IL-12) enhances recovery from lethal experimental vesicular stomatitis virus (VSV) infection of the central nervous system (CNS). Interleukin-12 treatment resulted in: 1) increased survival frequency; 2) faster recovery from weight loss; 3) substantially decreased VSV titers in brain homogenates and diminished immunohistochemical detection of VSV antigens in tissue sections; 4) earlier and increased CNS expression of types 1, 2, and 3 nitric oxide synthase (NOS) and both major histocompatibility complex (MHC) class I and class II antigens; 5) earlier and increased blood and CNS levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These results suggest that IL-12 enhances recovery from VSV infection of the CNS.

  View details for Web of Science ID A1997YA74900005

  View details for PubMedID 9095530

• Activation of type III nitric oxide synthase in astrocytes following a neurotropic viral infection VIROLOGY Barna, M., Komatsu, T., Reiss, C. S. 1996; 223 (2): 331-343

  Abstract

  Type III nitric oxide synthase (type III NOS), also known as endothelial cell nitric oxide synthase (eNOS or ecNOS or NOS-3), is a constitutively expressed, calcium- and calmodulin-dependent, isoform of NOS. Its expression has been localized to endothelial cells and a subset of neurons in the brain. We report here that resident astrocytes of the central nervous system (CNS) of mice express type III NOS. Following an experimental neurotropic viral infection, the expression of type III NOS on reactive astrocytes increases substantially, predominantly in virally infected regions of the brain. This upregulation of type III NOS expression is also evident following cytokine treatment in vitro. The intraperitoneal (i.p.) administration of IL-12, a potent activator of IFN-gamma and TNF-alpha production, results in a substantial increase in type III NOS immunoreactivity in astrocytes. Cytokine-mediated activation of type III NOS is observed in vitro following exposure of a C6 glioma cells, which constitutively express type III NOS, to IL-12, IFN-gamma, and TNF-alpha treatment. We conclude that astrocytes of the murine CNS express type III NOS, which may be positively regulated by a number of cytokines following viral infection. Type III NOS expression by astrocytes represents a novel source of nitric oxide in the brain. It may be important in regulating perfusion and maintaining the blood-brain barrier. Given the intimate association of astrocytes with endothelial cells and neurons, increased activity of type III NOS following viral infection may be beneficial in inhibition of viral infection in neighboring cells.

  View details for Web of Science ID A1996VJ81000007

  View details for PubMedID 8806568

• Sex differences in susceptibility to viral infection of the central nervous system JOURNAL OF NEUROIMMUNOLOGY Barna, M., Komatsu, T., Bi, Z. B., Reiss, C. S. 1996; 67 (1): 31-39

  Abstract

  We have characterized striking differences in recovery of male and female BALB/c and BALB/c-H-2dm2 (dm2) mice from an experimental neurotropic viral infection of the central nervous system (CNS). Following intranasal inoculation of vesicular stomatitis virus (VSV), assays of tissue homogenates from female mice produced lower viral titers. There was also a significant reduction in the spread of virus from the rostral to caudal end of the brain in female mice. Enhanced recovery by female mice of both strains in response to this viral insult correlates with increased levels of Nitric Oxide Synthase (NOS) types I, II, and III expression, an increased prevalence of reactive astrocytes, earlier and enhanced levels of expression of Major Histocompatibility Complex (MHC) class II molecules on astrocytes, endothelial and microglial cells, and increased T cell infiltration in the female BALB/c mouse. Taken together, these findings document sexual dimorphism in CNS immunity, and may provide an understanding of some of the mechanisms underlying many sex-biased diseases.

  View details for Web of Science ID A1996VA91900005

  View details for PubMedID 8707928

• Interleukin-12: Promotes enhanced recovery from viral infection of neurons in the central nervous system Conference on Interleukin-12 - Cellular and Molecular Immunology of an Important Regulatory Cytokine Reiss, C. S., Komatsu, T., Barna, M., Bi, Z. B. NEW YORK ACAD SCIENCES. 1996: 257–265

  View details for Web of Science ID 000070968300027

  View details for PubMedID 8958937

• Host immune response to vesicular stomatitis virus infection of the central nervous system in C57BL/6 mice VIRAL IMMUNOLOGY CHRISTIAN, A. Y., Barna, M., Bi, Z. B., Reiss, C. S. 1996; 9 (3): 195-205

  Abstract

  In this report, the kinetics of cellular inflammatory changes in the brain of vesicular stomatitis virus (VSV)-infected C57BL/6 (B6) mice was determined. The behavior and survival rate of infected B6 were carefully monitored each day. Infectious viral titers and VSV antigen distribution were determined at several time points during the course of infection. Strong activation of both astrocytes and microglia was observed after VSV infection. Induction of type II nitric oxide synthase (iNOS) was detected in activated microglia in the olfactory bulb (OB) starting at day 4 postinfection. Induced expression of major histocompatibility complex (MHC) molecules and rapid infiltration of both T cells and natural killer (NK) cells were detected in the VSV-infected CNS. Collectively, these data indicate that the response to CNS infection in B6 mice, which is often primarily Th1 in characteristics, is comparable to BALB/c mice, a strain that often shows a Th2-dominated immune response.

  View details for Web of Science ID A1996VK05900007

  View details for PubMedID 8890478

• IL-12 PROMOTES ENHANCED RECOVERY FROM VESICULAR STOMATITIS-VIRUS INFECTION OF THE CENTRAL-NERVOUS-SYSTEM JOURNAL OF IMMUNOLOGY Bi, Z. B., QUANDT, P., Komatsu, T., Barna, M., Reiss, C. S. 1995; 155 (12): 5684-5689

  Abstract

  To investigate the role of a cytokine in host defense against the vesicular stomatitis virus (VSV) infection of the central nervous system (CNS), IL-12 was injected i.p. into groups of 10 BALB/c mice on days -1, 0, 1, 2, and 3 postinfection. Four days postinfection, mice were examined. IL-12 strongly enhanced immunity to VSV infection in the CNS as demonstrated by 1) decreased VSV titers in brain homogenate of IL-12-injected mice compared with those of controls; 2) increased expression of inducible nitric oxide synthase in the CNS; 3) enhanced expression of both MHC class I and class II Ags in the CNS; 4) increased T cell infiltration in the CNS, especially in the olfactory bulb; and 5) diminished VSV-induced apoptosis in olfactory bulb. No detrimental effect was observed even with the 200 ng/mouse dose of IL-12. Protective effects of IL-12 were dose dependent. Collectively, these results demonstrate that exogenously added IL-12, even when injected peripherally, significantly enhances recovery from VSV infection of the CNS.

  View details for Web of Science ID A1995TJ63100030

  View details for PubMedID 7499854

• VESICULAR STOMATITIS-VIRUS INFECTION OF THE CENTRAL-NERVOUS-SYSTEM ACTIVATES BOTH INNATE AND ACQUIRED-IMMUNITY JOURNAL OF VIROLOGY Bi, Z. B., Barna, M., Komatsu, T., Reiss, C. S. 1995; 69 (10): 6466-6472

  Abstract

  Vesicular stomatitis virus (VSV) causes acute infection of the central nervous system (CNS) when intranasally applied. We have examined cellular inflammatory changes in the CNS following VSV infection. As early as 1 day postinfection (p.i.), astrocytes were activated in the olfactory bulb (OB). This was followed by activation of microglia, first observed in the OB at day 3 p.i. Expression of inducible nitric oxide synthase was observed in activated microglia in the OB at day 3 p.i., and increased inducible nitric oxide synthase expression coincided with decreased virus titers in tissue homogenates. Expression of major histocompatibility complex (MHC) class I molecules on astrocytes and microglial, endothelial, and ependymal cells was also rapidly induced and followed by induced expression of MHC class II molecules on astrocytes and microglial and endothelial cells. Consistent with the pattern of viral dissemination, MHC molecules were expressed temporally from the rostral-to-caudal direction. Infiltration of CD8+ cells was observed as early as 1 day p.i. in the OB. CD4+ cells were detected in the OB at day 4 p.i. Increasing T-cell infiltration coincided with decreased virus titers. In contrast, B-cell infiltration of the CNS was not detected until day 14 p.i., after the virus was cleared and mice were showing behavioral signs of recovery. Breakdown of the blood-brain barrier was detected beginning at day 6 p.i., was most severe at day 8 p.i., and was followed by full recovery. Collectively, these data show that both innate immunity (production of nitric oxide) and acquired immunity (expression of MHC molecules and T-cell infiltration) are activated following VSV infection in the CNS.

  View details for Web of Science ID A1995RU78400061

  View details for PubMedID 7545248