Characterization of MLE RNA helicase, a subunit of the Dosage Compensation Complex (DCC) in Drosophila melanogaster

2. Summary In Drosophila melanogaster the transcriptional activity of the male X chromosome is upregulated to compensate for the reduced dosage of X-linked genes as compared to the two X chromosomes in females. This process is mediated by the Dosage Compensation Complex (DCC), a ribonucleoprotein complex consisting of five proteins (MSL1, MSL2, MSL3, MOF and MLE) and two non-coding RNAs (roX1 and roX2). The DCC preferentially localizes on the X chromosomes in males where it doubles its transcription rate. Two enzymes are associated with the DCC: the acetyltransferase MOF, specific for the lysine 16 of H4 (H4-K16), and the DNA/RNA helicase MLE. Genetic experiments demonstrated that both activities are required for dosage compensation in male flies. However, the weak association of MLE to the DCC has complicated its biochemical analysis and, so far, the involvement of MLE RNA helicase in dosage compensation has only been demonstrated genetically. Using different in vivo and in vitro approaches the physical and functional interactions of MLE with the other MSL proteins and with the roX RNAs was addressed. Monoclonal antibodies, specifically recognizing MLE, were raised in rats, offering a new tool for MLE characterization. By coexpression of the DCC subunits in SF9 cells, a recombinant complex containing MSL1-2-3, MOF, MLE and the roX2 RNA was reconstituted and purified. A specific integration of roX2 into the DCC could be observed only in the absence of MLE. Non specific RNA binding properties seemed instead associated to MLE RNA helicase. Moreover, the purified MSL complex did not affect the ATPase activity of MLE in the presence or absence of roX2 RNA. In vitro, MLE showed a preferential association with MSL1 and MSL2 and MLE interaction with both MSL proteins were not RNA mediated. In view of these results we suggest that binding to roX2 is not the main determinant for MLE integration into the DCC complex and protein-protein interactions might instead contribute to its proper recruitment to the X chromosome. MLE is a member of the DEAD-box RNA helicase family and it shares with the other members the same domain organization. In addition to a central ATPase/helicase domain, two predicted N-terminal double strand (ds) RNA-binding motifs (dsRBM1 and dsRBM2) and a predicted C-terminal single strand (ss) RNA/DNA-binding domain (RGG-box) are also present in MLE protein. These domains have been extensively characterized in RHA, human ortholog of MLE, and their RNA binding properties confirmed. However, it is not known how MLE binds RNA and how the different RNA binding modules contribute to stimulate its enzymatic activities. A preferential binding of MLE to dsRNA compared to ssRNA was shown by binding assays. In addition, changes in the affinity of MLE for both ssRNA and dsRNA were observed in the presence of different nucleotides. Deletion mutants of MLE were produced and purified from insect cells in order to address the contribution of the different RNA binding domains to MLE enzymatic activities. By transient expression in Drosophila cells of the same deletion mutants fused to GFP, the effects of individual domains on MLE recruitment to the X chromosome were also determined. The results show that unlike RHA, the dsRB1 and the RGG domains are dispensable for MLE RNA binding and unwinding, whereas dsRB2 seems to play the major role in coordinating both activities. However, the enzymatic activities alone are not sufficient to properly target MLE to the X chromosome. These results provide new data on the functional properties of MLE RNA helicase that may help to elucidate its molecular mechanisms of action.1. Zusammenfassung In der Taufliege Drosophila melanogaster wird die transkriptionelle Aktivität des männlichen X Chromosoms erhöht, um die (im Vergleich zu weiblichen Zellen) verminderte Gen-Dosis X-chromosomaler Gene auszugleichen. Dieser Prozess wird durch den Dosis-Kompensationskomplex (DCC) vermittelt, einen Ribonucleoproteinkomplex, der aus fünf Proteinen (MSL1, MSL2, MSL3, MOF und MLE) sowie zwei nicht-kodierenden RNAs (roX1 und roX2) besteht. DCC bindet bevorzugt an das X Chromosom, wo er die Transkriptionsrate verdoppelt. Zwei Enzyme sind Teil des DCC: die Acetyltransferase MOF, die spezifisch das Lysin 16 des Histon H4 (H4K16) acetyliert, sowie die RNA/DNA-helicase MLE. Beide Aktivitäten sind für die korrekte Dosiskompensation in männlichen Fliegen verantwortlich. Allerdings erschwert die schwache Assoziation von MLE mit den übrigen MSL Proteinen die biochemische Analyse der Wirkung von MLE. Bislang konnte der Beitrag von MLE zur Dosiskompensation nur genetisch gezeigt werden. In dieser Arbeit werden mithilfe von verschiedenen experimentellen Ansätzen in vivo und in vitro die physischen und funktionalen Wechselwirkungen von MLE mit den übrigen MSL Proteinen, sowie mit den roX RNAs untersucht. Monoklonale Antikörper gegen MLE wurden in Ratten induziert, ein wichtiges neues Werkzeug zur Charakterisierung von MLE. Durch Co-Expression von DCC Untereinheiten in sf9 Zellen konnte ein rekombinanter Komplex rekonstituiert und gereinigt werden. Ein präferentieller Einbau von roX RNA konnte beobachtet werden, allerdings nur in Abwesenheit von MLE. In vitro konnte nur die unspezifische Bindung von MLE an RNA charakterisiert werden. Der gereinigte DCC hatte keinen Einfluss auf die ATPase Aktivität von MLE, unabhängig von der Anwesenheit von RNA. In vitro konnte eine spezifische Assoziation von MLE mit MSL1 und MSL2 beobachtet werden, die direkt und nicht durch RNA vermittelt war. Angesichts dieser Ergebnisse erscheint eine wichtige Rolle der roX2 RNA bei der Koppelung von MLE an den DCC unwahrscheinlich; vielmehr tragen Protein-Protein Wechselwirkungen zur Rekrutierung von MLE an das X Chromosom wesentlich bei. MLE gehört zur Familie der DEAD-box RNA Helicasen, mit denen es eine ähnliche Domänen-Organisation verbindet. Neben der zentralen ATPase/Helicase Domäne, werden für MLE zwei N-terminale Doppelstrang-RNA (dsRNA) Bindungsmotive (dsRBM1 und dsRBM2), sowie eine C-terminale Einzelstrang RNA/DNA Bindungsdomäne (RGG Box) aus Sequenzvergleichen vorhergesagt. Diese Domänen sind bislang im Kontext von RHA (RNA Helicase A), dem menschlichen Orthologen zu MLE, charakterisiert und ihre RNA Bindungseigenschaften bestätigt worden. Allerdings ist nicht bekannt, wie MLE an die RNA bindet und wie die verschiedenen RNA-bindenden Module zur RNA Stimulierung der ATPase beitragen. Hier konnte eine bevorzugte Bindung von MLE an doppelsträngige RNA (im Vergleich zu einzelsträngiger) in Bindungsassays beschrieben werden. Die Affinität der Helicase für RNA wird zudem durch Adenin-Nucleotide moduliert. Um den Beitrag einzelner Domänen zu den MLE Funktionen zu bestimmen, wurden eine Reihe von Deletionsmutanten von MLE in Insektenzellen hergestellt und gereinigt. Durch transiente Expression von entsprechenden MLE Derivaten als Fusionen mit GFP (Green Fluorescent Protein) wurde der Einfluss der Domänen auf die Rekrutierung von MLE and das X-Chromosom bestimmt. Im Gegensatz zu den beschriebenen Daten zu RHA erwiesen sich die RB1 und RGG als verzichtbar zur RNA Bindung und deren Entwindung, während dsRB2 eine wichtige Rolle zukommt. Zur korrekten Zielsteuerung von MLE an das X Chromosom ist allerdings die Funktionalität des Enzyms als ATPase alleine nicht ausreichend. Die hier vorgestellte Struktur-Funktionsanalyse leistet einen wichtigen Beitrag zur Beschreibung des Wirkmechanismus der RNA Helicase MLE

Diagnosis of anal human papillomavirus infection: polymerase chain reaction or cytology?

Summary Objectives To investigate the relationship between human immunodeficiency virus (HIV)-positive and HIV-negative patients engaging in promiscuous behaviors and anal human papillomavirus (HPV) infection diagnosed by polymerase chain reaction (PCR) and cytology. Methods Fifty-six HIV-positive patients and 49 HIV-negative patients who engaged in sexually promiscuous behavior were enrolled in the study. We performed cytological exams using the Pap smear and PCR for HPV-DNA detection, with identification of oncogenic strains. The 2001 Bethesda System terminology was used for the cytological exams. We also evaluated the immunologic status of the HIV-infected patients. Results PCR positivity for HPV-DNA was higher in the group of HIV-positive patients than in the group of HIV-negative patients with a statistically significant difference. In contrast we did not find any statistically significant difference by cytological exam. Oncogenic strains were equally distributed in the two groups. Conclusions Our results indicate the importance of the cytological exam for anal HPV screening in the population at high risk of sexually transmitted disease and that HPV-DNA PCR can be used only as adjunct test

Histone H1 variant-specific lysine methylation by G9a/KMT1C and Glp1/KMT1D

BACKGROUND: The linker histone H1 has a key role in establishing and maintaining higher order chromatin structure and in regulating gene expression. Mammals express up to 11 different H1 variants, with H1.2 and H1.4 being the predominant ones in most somatic cells. Like core histones, H1 has high levels of covalent modifications; however, the full set of modifications and their biological role are largely unknown. RESULTS: In this study, we used a candidate screen to identify enzymes that methylate H1 and to map their corresponding methylation sites. We found that the histone lysine methyltransferases G9a/KMT1C and Glp1/KMT1D methylate H1.2 in vitro and in vivo, and we mapped this novel site to lysine 187 (H1.2K187) in the C-terminus of H1. This H1.2K187 methylation is variant-specific. The main target for methylation by G9a in H1.2, H1.3, H1.5 and H1.0 is in the C-terminus, whereas H1.4 is preferentially methylated at K26 (H1.4K26me) in the N-terminus. We found that the readout of these marks is different; H1.4K26me can recruit HP1, but H1.2K187me cannot. Likewise, JMJD2D/KDM4 only reverses H1.4K26 methylation, clearly distinguishing these two methylation sites. Further, in contrast to C-terminal H1 phosphorylation, H1.2K187 methylation level is steady throughout the cell cycle. CONCLUSIONS: We have characterised a novel methylation site in the C-terminus of H1 that is the target of G9a/Glp1 both in vitro and in vivo. To our knowledge, this is the first demonstration of variant-specific histone methylation by the same methyltransferases, but with differing downstream readers, thereby supporting the hypothesis of H1 variants having specific functions

Methylation of H2AR29 is a novel repressive PRMT6 target

<p>Abstract</p> <p>Background</p> <p>Covalent histone modifications are central to all DNA-dependent processes. Modifications of histones H3 and H4 are becoming well characterised, but knowledge of how H2A modifications regulate chromatin dynamics and gene expression is still very limited.</p> <p>Results</p> <p>To understand the function of H2A modifications, we performed a systematic analysis of the histone H2A methylation status. We identified and functionally characterised two new methylation sites in H2A: R11 (H2AR11) and R29 (H2AR29). Using an unbiased biochemical approach in combination with candidate assays we showed that protein arginine methyltransferase (PRMT) 1 and PRMT6 are unique in their ability to catalyse these modifications. Importantly we found that H2AR29me2 is specifically enriched at genes repressed by PRMT6, implicating H2AR29me2 in transcriptional repression.</p> <p>Conclusions</p> <p>Our data establishes R11 and R29 as new arginine methylation sites in H2A. We identified the specific modifying enzymes involved, and uncovered a novel functional role of H2AR29me2 in gene silencing <it>in vivo</it>. Thus this work reveals novel insights into the function of H2A methylation and in the mechanisms of PRMT6-mediated transcriptional repression.</p

Multimodal epigenetic changes and altered NEUROD1 chromatin binding in the mouse hippocampus underlie FOXG1 syndrome

Forkhead box G1 (FOXG1) has important functions in neuronal differentiation and balances excitatory/inhibitory network activity. Thus far, molecular processes underlying FOXG1 function are largely unexplored. Here, we present a multiomics data set exploring how FOXG1 impacts neuronal maturation at the chromatin level in the mouse hippocampus. At a genome-wide level, FOXG1 i) both represses and activates transcription, ii) binds mainly to enhancer regions, iii) reconfigures the epigenetic landscape through bidirectional alteration of H3K27ac, H3K4me3, and chromatin accessibility, and iv) operates synergistically with NEUROD1. Interestingly, we could not detect a clear hierarchy of FOXG1 and NEUROD1, but instead, provide the evidence that they act in a highly cooperative manner to control neuronal maturation. Genes affected by the chromatin alterations impact synaptogenesis and axonogenesis. Inhibition of histone deacetylases partially rescues transcriptional alterations upon FOXG1 reduction. This integrated multiomics view of changes upon FOXG1 reduction reveals an unprecedented multimodality of FOXG1 functions converging on neuronal maturation. It fuels therapeutic options based on epigenetic drugs to alleviate, at least in part, neuronal dysfunction

Galaxy Formation and Symbiotic Evolution with the Inter-Galactic Medium in the Age of ELT-ANDES

High-resolution absorption spectroscopy toward bright background sources has had a paramount role in understanding early galaxy formation, the evolution of the intergalactic medium and the reionisation of the Universe. However, these studies are now approaching the boundaries of what can be achieved at ground-based 8-10m class telescopes. The identification of primeval systems at the highest redshifts, within the reionisation epoch and even into the dark ages, and of the products of the first generation of stars and the chemical enrichment of the early Universe, requires observing very faint targets with a signal-to-noise ratio high enough to detect very faint spectral signatures. In this paper, we describe the giant leap forward that will be enabled by ANDES, the high-resolution spectrograph for the ELT, in these key science fields, together with a brief, non-exhaustive overview of other extragalactic research topics that will be pursued by this instrument, and its synergistic use with other facilities that will become available in the early 2030s.Comment: 40 pages, 7 figures; submitted to Experimental Astronomy on behalf of the ANDES Science Tea

Structure-function analysis of the RNA helicase maleless

Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity