Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection.

BackgroundFruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.MethodsA. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses.ResultsThe genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission.ConclusionsThese observations suggest that, as in mammals, innate immunity and mitochondrial responses are integrated in mosquitoes and that AsP38 MAPK-dependent signaling facilitates mosquito survival during parasite infection, a fact that may attest to the relatively longer evolutionary relationship of these parasites with their invertebrate compared to their vertebrate hosts. On a practical level, improved understanding of the balances and trade-offs between resistance and metabolism could be leveraged to generate fit, resistant mosquitoes for malaria control

Computational prediction of metabolism: sites, products, SAR, P450 enzyme dynamics, and mechanisms.

Metabolism of xenobiotics remains a central challenge for the discovery and development of drugs, cosmetics, nutritional supplements, and agrochemicals. Metabolic transformations are frequently related to the incidence of toxic effects that may result from the emergence of reactive species, the systemic accumulation of metabolites, or by induction of metabolic pathways. Experimental investigation of the metabolism of small organic molecules is particularly resource demanding; hence, computational methods are of considerable interest to complement experimental approaches. This review provides a broad overview of structure- and ligand-based computational methods for the prediction of xenobiotic metabolism. Current computational approaches to address xenobiotic metabolism are discussed from three major perspectives: (i) prediction of sites of metabolism (SOMs), (ii) elucidation of potential metabolites and their chemical structures, and (iii) prediction of direct and indirect effects of xenobiotics on metabolizing enzymes, where the focus is on the cytochrome P450 (CYP) superfamily of enzymes, the cardinal xenobiotics metabolizing enzymes. For each of these domains, a variety of approaches and their applications are systematically reviewed, including expert systems, data mining approaches, quantitative structure-activity relationships (QSARs), and machine learning-based methods, pharmacophore-based algorithms, shape-focused techniques, molecular interaction fields (MIFs), reactivity-focused techniques, protein-ligand docking, molecular dynamics (MD) simulations, and combinations of methods. Predictive metabolism is a developing area, and there is still enormous potential for improvement. However, it is clear that the combination of rapidly increasing amounts of available ligand- and structure-related experimental data (in particular, quantitative data) with novel and diverse simulation and modeling approaches is accelerating the development of effective tools for prediction of in vivo metabolism, which is reflected by the diverse and comprehensive data sources and methods for metabolism prediction reviewed here. This review attempts to survey the range and scope of computational methods applied to metabolism prediction and also to compare and contrast their applicability and performance.JK, MJW, JT, PJB, AB and RCG thank Unilever for funding

Selected Works in Bioinformatics

This book consists of nine chapters covering a variety of bioinformatics subjects, ranging from database resources for protein allergens, unravelling genetic determinants of complex disorders, characterization and prediction of regulatory motifs, computational methods for identifying the best classifiers and key disease genes in large-scale transcriptomic and proteomic experiments, functional characterization of inherently unfolded proteins/regions, protein interaction networks and flexible protein-protein docking. The computational algorithms are in general presented in a way that is accessible to advanced undergraduate students, graduate students and researchers in molecular biology and genetics. The book should also serve as stepping stones for mathematicians, biostatisticians, and computational scientists to cross their academic boundaries into the dynamic and ever-expanding field of bioinformatics

Strukturní studie vybraných komplexů signálních proteinů.

Schopnost proteinů vázat jiné molekuly v reakci na různé podněty ve svém mikrookolí je základem rozsáhlých regulačních sítí, které koordinují následnou činnost buněk. Správná funkce těchto signálních drah závisí převážně na nekovalentních interakcích, které často ovlivňují strukturu proteinů a proteinových komplexů. Pochopení molekulárního mechanismu funkce proteinu v buněčné signalizaci je proto často závislé na znalosti jeho trojdimenzionální struktury. V této disertační práci představuji studie, které vedly na molekulární úrovni k pochopení několika protein-proteinových a ligand-proteinových interakcí podílejících se na buněčné signalizaci. Použila jsem nukleární magnetickou rezonanci (NMR), malo- úhlový rozptyl rentgenového záření (SAXS) a další biofyzikální metody pro určení molekulární podstaty inhibice čtyř signálních proteinů: vápník/kalmodulin (Ca2+ /CaM)- dependentní proteinkinasy kinasy 2 (CaMKK2); proteasy kaspasa-2; forkhead transkripčního faktoru FOXO3 a proteinkinasy ASK1. Konkrétněji byla zkoumána role proteinu 14-3-3 a CaM v regulaci CaMKK2 aktivity. Dále byl detailně studován mechanizmus, jakým protein 14-3-3 ovlivňuje schopnost oligomerizace a jaderné lokalizace kaspasy-2 a také byla objasněna podstata modulace transkripční aktivity FOXO transkripčních faktorů díky zkoumání...The ability of proteins to bind other molecules in response to various stimuli in their microenvironment serves as a platform for extensive regulatory networks coordinating downstream cell actions. The correct function of these signaling pathways depends mostly on noncovalent interactions often affecting the structure of proteins and protein complexes. Understanding the molecular mechanism of a protein function in cell signaling therefore often depends on our knowledge of a three-dimensional structure. In this doctoral thesis, I present the work that led to the understanding of several protein-protein and protein-ligand interactions implicated in cell signaling at the molecular level. I applied nuclear magnetic resonance spectroscopy, small angle X-ray scattering and other biophysical methods to determine the molecular basis of inhibition of four signaling proteins: Calcium/Calmodulin (Ca2+ /CaM)-dependent protein kinase kinase 2 (CaMKK2); protease Caspase-2; Forkhead transcription factor FOXO3, and Apoptosis signal-regulating protein kinase 1 (ASK1). In particular, I investigated the distinct roles of 14-3-3 and Ca2+ /CaM in the regulation of CaMKK2 activity. I also studied in detail the mechanism how 14-3-3 interferes with the caspase-2 oligomerization and its nuclear localization as well as...Department of Physical and Macromolecular ChemistryKatedra fyzikální a makromol. chemieFaculty of SciencePřírodovědecká fakult

Antimicrobial Detection Illuminated: Developing Bioluminescent Antibiotic Biosensors Based on Bacterial Gene Regulatory Elements

Ever since World War II, antibiotics have been medicine’s number one asset in fighting microbial infection, one of the leading causes of death worldwide. Misuse of antibiotics has, however, led to rapid spread of antibiotic resistance among bacteria and ensuing development of multiple resistant pathogens. Therefore, antibiotics are rapidly losing their antimicrobial value, which can be seen a failure of society to protect one of its valuable resources.The use of antibiotics in food production animals is strictly controlled by the European Union. Veterinary use is regulated to prevent spreading of resistance due to unwarranted use and to prevent antibiotic residues in food products. EU legislation establishes maximum residue limits (MRLs) of veterinary medicinal products in foodstuffs of animal origin, and enforces countries to establish and execute a national monitoring plan of animal products to implement food control measures. Among samples selected for monitoring, suspect noncompliant samples are screened for and then subjected to confirmatory analysis to establish the identity and concentration of the contaminant. Screening methods for antibiotic residues are typically based on microbiological growth inhibition, whereas physico-chemical methods are used for confirmatory analysis.In this study, antibiotic whole-cell biosensor assays were examined as a novel screening method. Utilizing a tetracycline-specific bioluminescent whole-cell biosensor, a screening method for tetracycline residues in poultry meat was developed. Assay sensitization to meet the EU MRLs was achieved by improving tetracycline accumulation into the biosensor cells with a combination of membrane-permeabilizing agent polymyxin B and chelating agent EDTA. The result was a rapid, simple and cost-effective high-throughput screening method that could detect all four veterinary relevant tetracyclines and their 4-epimer metabolites in poultry meat with sensitivity below the MRLs. The study also provided proof of antimicrobial activity of tetracycline 4-epimer metabolites, a quality previously thought absent from 4-epidoxycycline.Nisin is a lantibiotic, a peptide antibiotic produced by lactococci. The industrial use of nisin as a food preservative (E234) and maximum allowed levels set by the EU warrant developing methods for nisin quantification in foods. In this study, a bioluminescent whole-cell biosensor for nisin was constructed and utilized in determining nisin concentrations in milk. The developed assay was rapid and simple to perform, and required no sample pretreatment except dilution. Sensitivity of the assay was in the sub-picogram per ml level, exceeding the performance of all previously published methods. The assay was also used in determining nisin-production efficiency by quantifying nisin in growth medium of a nisin-producing Lactococcus strain. Simultaneously, nisin producers could be distinguished from non-producers. This idea was expanded in a follow-up study, which utilized the nisin biosensor in screening for nisin producers in raw milk. Screening was based on simple overlay of raw milk cultures and identification of nisin producers by a bioluminescent zone surrounding the nisinogenic colony. The seven identified nisinogenic colonies were divided in three groups by genetic fingerprinting,and characterized as nisin variant Z producing Lactococcus lactis subsp. lactis. In addition, four nisin A producers were identified in a panel of 91 dairy lactococcal strains. Specificity studies showed that only nisin and not other bacteriocin peptides induced bioluminescence in the sensor strain. Also, all nisin-gene harboring colonies induced bioluminescence, with the exception of one lactococcal strain shown to carry a nonfunctional nisin gene.The development of novel inducible whole-cell biosensors for different groups of antimicrobials can be limited by the lack of regulatory elements specifically responsive for these substances. In this study, we characterized DNA and ligand binding of the macrolide antibiotic-responsive repressor protein, MphR(E). The protein was modified by rational design of mutations to improve DNA affinity and dimerization. DNA and ligand binding as well as macrolide-induced dissociation from DNA were studied by fluorescence anisotropy and mass spectrometry. Mutants with improved DNA affinity and retained ligand binding and dissociation characteristics were identified. One mutant surprisingly formed a covalent dimer through disulfide bridge formation. This was shown to improve DNA affinity, but ligand binding and induction was impaired. Ligand binding spectrum of MphR(E) was shown to cover macrolides with a 14-membered lactone ring structure, but macrolides with a 16-membered ring or lincosamides showed no binding. MphR(E) and its mutants showed interesting novel characteristics that could benefit biosensor design.In conclusion, this study shows the applicability of whole-cell biosensors in developing simple, robust and cost-effective screening methods for antimicrobials in food products. These methods show high sensitivity and specificity towards the target analyte, and can be used in semi-quantitative to quantative analysis. In addition to residue monitoring, whole-cell biosensors can be used for producer identification. The identified nisin producers can find use as protective starter cultures in fermented food production. The modified repressor MphR(E) shows promise as an improved regulator of reporter gene production in whole-cell biosensor applications, and is an example of purposeful effort to develop regulatory elements for novel biosensor designs.Antibiootit ovat olleet tärkein bakteeri-infektioiden hoitokeino aina toisesta maailmansodasta lähtien. Bakteeri-infektiot ovat maailmanlaajuisten kuolemansyytilastojen kärkisijoilla. Antibioottien huolimaton käyttö on kuitenkin johtanut antibioottiresistenssin nopeaan leviämiseen bakteerien keskuudessa sekä useille antibiooteille resistenttien patogeenien kehittymiseen. Tämän vuoksi antibiootit menettävät nopeasti antimikrobiaalista voimakkuuttaan, mitä voidaan pitää yhteiskunnan kyvyttömyytenä suojella arvokasta pääomaansa. Euroopan Unioni valvoo antibioottien käyttöä eläimissä, joita hyödynnetään elintarvikkeiden tuotannossa. Eläinlääketieteellistä käyttöä säädellään resistenssin leviämisen ja ruoassa esiintyvien antibioottijäämien estämiseksi. EU:n lainsäädäntö osoittaa enimmäisjäämärajat eläinlääkinnällisille aineille eläinperäisissä elintarvikkeissa ja velvoittaa jäsenvaltiot laatimaan ja toteuttamaan eläimistä saatavien elintarvikkeiden kansallisen vierasainevalvontaohjelman. Valvontaan valittujen näytteiden joukosta seulotaan näytteet, joiden epäillään sisältävän jäämiä, joiden luonne ja pitoisuus varmistetaan lisäanalyysillä. Seulonnassa käytetään tavallisesti mikrobiologiseen kasvuinhibitioon perustuvia menetelmiä, ja varmistukseen fysikaalis-kemiallisia analyysejä. Tässä tutkimuksessa tarkasteltiin kokosolubioantureilla tehtävien antibioottimääritysten soveltuvuutta antibioottijäämien seulontamenetelmäksi. Tetrasykliinispesifistä bioluminoivaa kokosolubiosensoria käyttäen kehitettiin seulontamenetelmä tetrasykliinijäämien tunnistamiseksi kananlihasta. Määritys saatiin herkistettyä EU:n enimmäisjäämärajojen tasolle helpottamalla tetrasykliinien pääsyä bioanturisoluun solukalvon läpäisykykyä lisäävällä polymyksiini B:llä sekä kahdenarvoisia kationeja kelatoivalla EDTA:lla. Tuloksena oli nopea, yksinkertainen ja kustannustehokas seulontamenetelmä, jolla oli korkea suoritusteho. Menetelmä kykeni havaitsemaan kaikki neljä eläinlääketieteessä käytettävää tetrasykliiniä sekä niiden 4-epimeeri aineenvaihduntatuotteet kananlihassa enimmäisjäämärajat alittavissa pitoisuuksissa. Tutkimus myös tuotti todisteita tetrasykliinien 4-epimeerien antimikrobiaalisesta aktiivisuudesta, joka aiemmin arveltiin puuttuvan 4-epidoksisykliiniltä. Nisiini on laktokokkien tuottama lantibiootti eli peptidiantibiootti. Nisiinin käyttö elintarviketeollisuudessa säilöntäaineena (E234) sekä EU:n nisiinille asettama sallittu enimmäismäärä elintarvikkeissa luovat tarpeen nisiinin määritysmenetelmille. Tässä tutkimuksessa rakennettiin bioluminoiva nisiinispesifinen kokosolubioanturi, jota käytettiin määrittämään nisiinipitoisuuksia maidossa. Kehitetty määritys oli nopea ja yksinkertainen, eikä vaatinut laimennusta monimutkaisempaa näytteen esikäsittelyä. Määrityksen herkkyys oli alle pg/ml mittaluokassa, ja se oli herkin koskaan julkaistu nisiinimääritys. Määritystä käytettiin myös nisiinintuotannon tehokkuuden arvioinnissa mittaamalla nisiinipitoisuus sitä tuottavan Lactococcus lactis -kannan kasvumediumista. Samanaikaisesti nisiinintuottaja voitiin erottaa nisiiniä tuottamattomista kannoista. Tätä ajatusta tarkasteltiin laajemmin jatkotutkimuksessa,jossa nisiinibioanturia käytettiin seulomaan nisiinintuottajakantoja raakamaidosta. Yksinkertainen seulontamenetelmä perustui raakamaitobakteerien viljelmien peittämiseen ohuella bioanturikerroksella, jolloin nisiinintuottajapesäkkeiden ympärille muodostui biolumine-senssivyöhyke. Seitsemän tunnistettua nisiinintuottajapesäkettä jakautuivat geneettisen sormen-jäljen perusteella kolmeen ryhmään, ja ne olivat kaikki Lactococcus lactis subsp. lactis -alalajiin kuuluvia nisiini Z -variantin tuottajia. Lisäksi 91 laktokokkikannan paneelista tunnistettin kolme nisiini A -variantin tuottajaa. Spesifisyystutkimukset osoittivat, että vain nisiini indusoi bioluminesenssin bioanturibakteerissa eivätkä muut bakteriosiinipeptidit. Lisäksi kaikki nisiinigeeniä kantavat pesäkkeet indusoivat bioluminesenssin. Poikkeuksena oli yksi laktokokkikanta, jonka todettiin kantavan toimimatonta nisiinigeeniä. Tietyille antibioottiryhmille spesifisen vasteen antavien säätelyelementtien puute saattaa vaikeuttaa kokosolubioanturien kehittämistä. Tässä tutkimuksessa karakterisoitiin makrolidispesifisen repressoriproteiini MphR(E):n DNA- ja ligandinsitomisominaisuuksia. Proteiinia muokattiin rationaalisen mutaatiosuunnittelun keinoin tarkoituksena tuottaa DNA- ja dimerisoitumisominaisuuksiltaan parempia mutantteja. DNA- ja ligandisitoutumista sekä makrolidiligandien indusoimaa dissosiaatiota DNA:sta tutkittiin fluoresenssianisotropialla ja massaspektrometrialla. Tutkimuksessa löydettiin mutantteja, joilla oli villityypin proteiinia parempi DNA-affiniteetti sekä ennallaan säilynyt kyky sitoa ligandeja ja indusoitua sitomisen vaikutuksesta. Yksi mutanteista muodosti rikkisillan avulla kovalenttisen dimeerin vastoin odotuksia. Kovalentti dimerisaatio paransi DNA-affiniteettia, mutta haittasi ligandin sitomista sekä induktiota. MphR(E):n ligandikirjo kattoi 14-jäsenisen laktonirenkaan makrolidit, mutta ei 16-jäsenisen renkaan makrolideja eikä linkosamideja. MphR(E) ja sen mutantit osoittivat mielenkiintoisia uusia ominaisuuksia, jotka voivat hyödyttää bioantureiden suunnittelua. Johtopäätöksenä voidaan sanoa, että tämä tutkimus osoittaa kokosolubioanturien soveltuvan yksinkertaisten, luotettavien ja kustannustehokkaiden seulontamenetelmien kehittämiseen antibioottijäämien osoittamiseen elintarvikkeista. Nämä menetelmät osoittavat suurta herkkyyttä ja spesifisyyttä analyyttimolekyyliä kohtaan, ja niitä voidaan käyttää semikvantitatiiviseen sekä kvantitatiiviseen analysiin. Jäämien havainnoinnin lisäksi bioantureita voidaan käyttää antimikrobiaalisten aineiden tuottajien tunnistamiseen. MphR(E)-mutantit ovat lupaavia paranneltua säätelykykyä osoittavia reportterigeenin tuoton repressoreita käytettäväksi bioanturisovelluksissa. Ne ovat myös esimerkki rationaalisesta säätelyelementtien parantelusta uusien bioanturisovellusten kehittämiseksi

PARP-1 activation regulates the DNA damage response to DNA double-strand breaks

Les cassures double-brin de l'ADN, lorsque incorrectement réparées, peuvent avoir des conséquences fatales telles que des délétions et des réarrangements chromosomiques, favorisant la carcinogenèse. La poly(ADP-ribosyl)ation réalisée par la protéine poly(ADP-ribose) polymérase-1 (PARP-1) est l'une des premières modifications post-traductionnelles qui se produisent en réponse aux dommages à l'ADN. La PARP-1 utilise la nicotinamide pour générer un polymère chargé négativement, nommé poly(ADP-ribose) polymère (PAR), lequel est attaché en majorité à la PARP-1 elle-même ainsi qu'à d'autres protéines cibles. Le PAR a récemment été reconnu comme un signal de recrutement pour certaines protéines de réparation aux sites de dommages à l'ADN, mais un débat est en cours quant au rôle précis de la PARP-1 et du PAR dans la réponse aux dommages de l'ADN. Au cours de mon projet de doctorat, nous avons pu confirmer que les protéines qui se retrouvent en complexe avec le PAR immédiatement après les dommages à l'ADN sont principalement des facteurs de réparation. Étonnamment, les complexes protéiques associés au PAR pendant la période de récupération suite aux dommages sont enrichis en facteurs de liaison à l'ARN. Toutefois, la protéine liant l'ARN la plus abondante que nous avons détectée dans l'interactome du PAR, soit NONO, ne suit pas cette dernière cinétique puisqu'elle est fortement enrichie immédiatement après les dommages à l'ADN. Notre étude subséquente de NONO dans la réponse aux cassures double-brin de l'ADN a étonnamment révélé une implication directe de celle-ci par le mécanismede réparation de jonction des extrémités non-homologues. En plus, nous avons constaté que NONO se lie fortement et spécifiquement au PAR via son motif 1 de la reconnaissance de l'ARN, soulignant la compétition entre les PAR et l'ARN pour le même site de liaison. Fait intéressant, le recrutement in vivo de NONO aux sites de dommages de l'ADN dépend entièrement du PAR et nécessite le motif 1 de la reconnaissance de l'ARN. En conclusion, nos résultats établissent NONO comme une nouvelle protéine impliquée dans la réponse aux cassures double-brin de l'ADN et plus généralement démontrent un autre niveau de complexité supplémentaire dans l'interdépendance de la biologie de l'ARN et la réparation de l'ADN.DNA double-strand breaks are potentially lethal lesions, which if not repaired correctly, can have harmful consequences such as carcinogenesis promoted by chromosome deletions and rearrangements. Poly(ADP-ribosyl)ation carried out by poly(ADP-ribose) polymerase 1 (PARP-1) is one of the first posttranslational modifications occurring in response to DNA damage. In brief, PARP-1 uses nicotinamide to generate a negatively charged polymer called poly(ADP-ribose) polymer (PAR), that can be attached to acceptor proteins, which is to a large extent PARP-1 itself. PAR has recently been recognized as a recruitment signal for key DNA repair proteins to sites of DNA damage but the precise role of PARP-1 and its catalytic product PAR in the DNA damage response are still a matter of ongoing debate. Throughout my doctoral work, we confirmed that the proteins in complex with PAR promptly after DNA damage are mostly DNA repair proteins, whereas during the period of recovery from DNA damage, the PAR interactome is highly enriched with RNA processing factors. Interestingly, one of the most abundant RNA-binding proteins detected in the PAR interactome, namely NONO, did not follow these kinetics as it was highly enriched immediately after DNA damage in the DNA repair protein complexes centered on PAR. Our subsequent investigation of NONO in the DNA damage response to double-strand breaks strikingly revealed a direct implication for NONO in repair by nonhomologous end joining (NHEJ). Moreover, we found that NONO strongly and specifically binds to PAR through its RNA-recognition motif 1 (RRM1), highlighting competition between PAR and RNA for the same binding site. Remarkably, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR and requires the RRM1 motif. In conclusion, our results establish NONO as a new protein implicated in the DNA damage response to double-strand break and in broader terms add another layer of complexity to the cross-talk between RNA-biology and DNA repair

Regulated Histone H3 Proteolysis During Mouse Embryonic Stem Cell Differentiation

The association of genomic DNA with histone proteins in the three-dimensional structure known as chromatin is the central framework for “epigenetics,†which is defined as inherited phenotypes governed by differences that cannot be explained by changes in DNA sequence. In recent years, studies have shown that regulated changes in the chemical and physical properties of chromatin often lead to dynamic changes in many cellular processes, including development and differentiation, by affecting the accessibility of the genomic information stored in the DNA. The cell uses many different mechanisms to regulate chromatin in order to establish, maintain, and propagate patterns of gene expression that are necessary for proper development and differentiation. Many of these mechanisms involve the histone component of chromatin, both through chemical and structural changes of the histone proteins themselves and via complex interactions with other non-histone chromatin proteins. Here, in my thesis work, I describe how a few of these chromatin regulatory mechanisms are used during mammalian differentiation, specifically focusing on those involving histone H3. First, in Chapter 2, I describe how certain non-histone chromatin proteins that are key to development specifically interact with modified histones using biochemical, biophysical and structural approaches. Next, in Chapter 3, I describe how chromatin undergoes specific, dramatic changes as cells lose their capacity for self-renewal and proceed toward a specific lineage using a mouse embryonic stem cell model of differentiation and early embryonic development. These changes involve both the post-translational modification of histone H3 and the incorporation of different H3 variant proteins into the chromatin fiber. Finally, I describe the observation that differentiating mouse embryonic stem cells proteolytically cleave histone H3 and identify a protease that accomplishes this cleavage

The Role of Nodal in the Regulation of Bi-Potential Trophoblast Progenitor Cells

The human placenta develops from highly proliferative and phenotypically plastic cells called trophoblasts. Bi-potential trophoblast stem cells differentiate into the villous pathway to form the syncytiotrophoblast layer and the extravillous trophoblast (EVT). The HTR-8/SVneo cell line is widely used to study trophoblast biology. These cells variably express villous-specific or EVT-specific genes depending on conditions. Such phenotypic plasticity is indicative of a bi-potential cytotrophoblast progenitor. Preliminary work has shown that similar to progenitors in situ, a subpopulation of HTR-8/SVneo cells expresses a6b4 integrin. This a6b4high subset exhibits enhanced clonogenicity and differentiation capacity. This cell line also expresses Nodal, a stem cell-associated factor that sustains the pluripotency of embryonic stem cells and is re-expressed in certain cancers. I hypothesized that the a6b4high subset within HTR-8/SVneo is enriched with bi-potential cytotrophoblast progenitor-like cells that are maintained by Nodal signaling. Our results revealed that the a6b4high subset expresses greater amounts of Nodal protein relative to the a6b4low subset. To investigate the role of Nodal in regulating trophoblast progenitors, stable Nodal knock-down and over-expressing cells were analyzed. It was found that Nodal is required for clonogenicity, maintenance of enhanced a6b4 expression and endovascular differentiation along the EVT pathway. These results suggest that the a6b4high population may represent a villous cytotrophoblast progenitor cell in which Nodal regulates clonogenicity and capacity for differentiation along the EVT pathway