RNA packaging motor: From structure to quantum mechanical modelling and sequential-stochastic mechanism

The bacteriophages of the Cystoviridae family package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate, i.e. the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work

Chromatin interaction maps identify Wnt responsive cis-regulatory elements coordinating Paupar-Pax6 expression in neuronal cells

Central nervous system-expressed long non-coding RNAs (lncRNAs) are often located in the genome close to protein coding genes involved in transcriptional control. Such lncRNA-protein coding gene pairs are frequently temporally and spatially co-expressed in the nervous system and are predicted to act together to regulate neuronal development and function. Although some of these lncRNAs also bind and modulate the activity of the encoded transcription factors, the regulatory mechanisms controlling co-expression of neighbouring lncRNA-protein coding genes remain unclear. Here, we used high resolution NG Capture-C to map the cis-regulatory interaction landscape of the key neuro-developmental Paupar-Pax6 lncRNA-mRNA locus. The results define chromatin architecture changes associated with high Paupar-Pax6 expression in neurons and identify both promoter selective as well as shared cis-regulatory-promoter interactions involved in regulating Paupar-Pax6 co-expression. We discovered that the TCF7L2 transcription factor, a regulator of chromatin architecture and major effector of the Wnt signalling pathway, binds to a subset of these candidate cis-regulatory elements to coordinate Paupar and Pax6 co-expression. We describe distinct roles for Paupar in Pax6 expression control and show that the Paupar DNA locus contains a TCF7L2 bound transcriptional silencer whilst the Paupar transcript can act as an activator of Pax6. Our work provides important insights into the chromatin interactions, signalling pathways and transcription factors controlling co-expression of adjacent lncRNAs and protein coding genes in the brain

RNA packaging motor: From structure to quantum mechanical modelling and sequential-stochastic mechanism

The bacteriophages of the Cystoviridae family package their single stranded RNA genomic precursors into empty capsid (procapsids) using a hexameric packaging ATPase motor (P4). This molecular motor shares sequence and structural similarity with RecA-like hexameric helicases. A concerted structural, mutational and kinetic analysis helped to define the mechanical reaction coordinate, i.e. the conformational changes associated with RNA translocation. The results also allowed us to propose a possible scheme of coupling between ATP hydrolysis and translocation which requires the cooperative action of three consecutive subunits. Here, we first test this model by preparing hexamers with defined proportions of wild type and mutant subunits and measuring their activity. Then, we develop a stochastic kinetic model which accounts for the catalytic cooperativity of the P4 hexamer. Finally, we use the available structural information to construct a quantum-chemical model of the chemical reaction coordinate and obtain a detailed description of the electron density changes during ATP hydrolysis. The model explains the results of the mutational analyses and yields new insights into the role of several conserved residues within the ATP binding pocket. These hypotheses will guide future experimental work

A gain-of-function single nucleotide variant creates a new promoter which acts as an orientation-dependent enhancer-blocker

Many single nucleotide variants (SNVs) associated with human traits and genetic diseases are thought to alter the activity of existing regulatory elements. Some SNVs may also create entirely new regulatory elements which change gene expression, but the mechanism by which they do so is largely unknown. Here we show that a single base change in an otherwise unremarkable region of the human α-globin cluster creates an entirely new promoter and an associated unidirectional transcript. This SNV downregulates α-globin expression causing α-thalassaemia. Of note, the new promoter lying between the α-globin genes and their associated super-enhancer disrupts their interaction in an orientation-dependent manner. Together these observations show how both the order and orientation of the fundamental elements of the genome determine patterns of gene expression and support the concept that active genes may act to disrupt enhancer-promoter interactions in mammals as in Drosophila. Finally, these findings should prompt others to fully evaluate SNVs lying outside of known regulatory elements as causing changes in gene expression by creating new regulatory elements

DNA methylation of intragenic CpG islands depends on their transcriptional activity during differentiation and disease

The human genome contains ∼30,000 CpG islands (CGIs). While CGIs associated with promoters nearly always remain unmethylated, many of the ∼9,000 CGIs lying within gene bodies become methylated during development and differentiation. Both promoter and intragenic CGIs may also become abnormally methylated as a result of genome rearrangements and in malignancy. The epigenetic mechanisms by which some CGIs become methylated but others, in the same cell, remain unmethylated in these situations are poorly understood. Analyzing specific loci and using a genome-wide analysis, we show that transcription running across CGIs, associated with specific chromatin modifications, is required for DNA methyltransferase 3B (DNMT3B)-mediated DNA methylation of many naturally occurring intragenic CGIs. Importantly, we also show that a subgroup of intragenic CGIs is not sensitive to this process of transcription-mediated methylation and that this correlates with their individual intrinsic capacity to initiate transcription in vivo. We propose a general model of how transcription could act as a primary determinant of the patterns of CGI methylation in normal development and differentiation, and in human disease.</p

Ihmisen kyynelnesteen rasvakalvon ominaisuuksista - Näkökulmia molekyylidynamiikka-simulaatioista

The outer layer of human tear film – the tear film lipid layer – covers the surface of the eye. This layer is the interface between the tear film and the air surrounding the eye. Abnormal composition of the tear film and its lipid layer may have quite severe consequences for health in terms of the so-called "dry eyes syndrome". People with dry eyes experience uncomfortable feelings, which bear a resemblance to having "sand in the eyes", and they have also excessive tear production and blurry vision. In addition to the abnormal lipid composition, also abnormal secretion of the tears themselves can lead to the development of dry eyes. Untreated dry eyes can lead to inflammation of the eye surface, and even scarring of the eye surface tissue. Dry eyes are treated with eye drops, which are selected to match the symptoms of the patient – some people need more lipids to the layer, while some others lack the aqueous tears. In this thesis, the tear film lipid layer was investigated by the means of molecular dynamics simulations. First, it was investigated how lipids in a native composition organize themselves to the interface between air and water. The systems considered were exposed to varying interfacial areas to see what happens to the interfacial region during the early phases of closing and opening the eyes. Second, consequences of abnormal lipid compositions typical to blepharitis patients were explored. Further, these considerations were complemented with simulations of lipid layers with quite diverse compositions to elucidate the roles of certain specific lipids in the function of tear film lipid layers. It was found that the lipids form a layer to the very interface of air and water. In the normal tear film composition, phospholipids formed the main body of the interfacial layer, while cholesteryl esters were observed to penetrate into it, promoting tighter packing of the interfacial layer. When the system was exposed to decreasing interfacial area to investigate what happens when eyes are being blinked, triglycerides helped the layer to stay intact even at very small interfacial areas. Meanwhile, with lipid layers characteristic to blepharitis patients, the ability to form a continuous lipid layer to the surface was impaired. Should the tear film lipid layer really be a monolayer-like also in reality, the results suggest that each lipid type in the layer has a specific role to maintain the function of the layer: phospholipids form the interface, cholesteryl esters make it stiffer, and triglycerides enable easier adjustment to changes in interfacial area. The results presented in the thesis pave the way for new avenues in clinical research of dry eyes, and aid the developing of more suitable eye drops for the treatment of dry eye patients.Ihmisen silmän pinnalla on ohut kerros kyynelnestettä, kosteuttamassa ja suojaamassa silmän pintaa. Kyynelnestekalvon uloin kerros koostuu rasvamolekyyleistä. Tämä pintakerros on ns. kyynelnesteen rasvakalvo. Häiriöt kyynelnesteen ja sen rasvakalvon koostumuksessa voivat johtaa ns. "kuivasilmäisyyteen". Kuivasilmäisyyden oireita voivat olla tunne, että on "hiekkaa silmissä", sumentunut näkö, tai vuotavat silmät. Hoitamattomana kuivasilmäisyys johtaa silmän pinnan tulehdukseen ja pahimmillaan edelleen silmän pinnan arpeutumiseen. Kuivasilmäisyyttä hoidetaan silmätipoilla, joiden koostumus valitaan potilaan oireiden mukaan – joidenkin ihmisten kyynelnesteestä puuttuu vettä, toiset tarvitsevat lisää rasvamolekyylejä. Tässä väitöstutkimuksessa silmän pinnan rasvakalvoa tutkittiin molekyylidynamiikka-simulaatioilla. Ensin tutkittiin rasvakalvoja, jotka vastasivat koostumukseltaan silmän pinnan rasvakalvon normaalia tai lähes normaalia koostumusta. Sitten simulaatioilla tutkittiin silmäluomen tulehduksesta (blepariitti) kärsivien potilaiden rasvakalvon rakennetta. Simulaatiotuloksia tarkasteltaessa nähtiin rasvojen asettuvan ilman ja veden rajapinnalle monokerrokseksi (yhden rasvamolekyylikerroksen paksuiseksi kerrokseksi). Fosfolipidit muodostivat kalvon perusrakenteen, ja kolesteroliesterit asettuivat niiden väliin, järjestäen rasvamolekyylit tiukemmin pakatuksi kalvorakenteeksi. Kun systeemiä simuloitiin eri rajapinnan pinta-aloilla, huomattiin, että triglyseridit auttoivat kalvoa säilyttämään muotonsa silloinkin, kun ilman ja veden välisen rajapinnan pinta-ala oli hyvin pieni. Tämä saattaa olla triglyseridien rooli silmän pintakalvossa – auttaa kalvoa mukautumaan silmän räpäyttämisessä tapahtuviin pinta-alan muutoksiin. Blepariittipotilaiden rasvakalvo ei simulaatioissa muodostanut vastaavan kaltaista säännöllistä monokerrosrakennetta. Jos silmän pinnan rasvakalvo todellisuudessakin on yhden lipidikerroksen vahvuinen, tuloksia voitaisiin suoraan soveltaa lääketieteellisessä tutkimuksessa. Toivonkin, että väitöskirjassa esitetyt tulokset voivat auttaa kuivasilmäisyyden tutkimuksessa, ja hoitomuotojen kuten silmätippojen ominaisuuksien kehittämisessä

High-Throughput Genotyping of CRISPR/Cas Edited Cells in 96-Well Plates

The emergence in recent years of DNA editing technologies&mdash;Zinc finger nucleases (ZFNs), transcription activator-like effector (TALE) guided nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/Cas family enzymes, and Base-Editors&mdash;have greatly increased our ability to generate hundreds of edited cells carrying an array of alleles, including single-nucleotide substitutions. However, the infrequency of homology-dependent repair (HDR) in generating these substitutions in general requires the screening of large numbers of edited cells to isolate the sequence change of interest. Here we present a high-throughput method for the amplification and barcoding of edited loci in a 96-well plate format. After barcoding, plates are indexed as pools which permits multiplexed sequencing of hundreds of clones simultaneously. This protocol works at high success rate with more than 94% of clones successfully genotyped following analysis

Single-allele chromatin interactions identify regulatory hubs in dynamic compartmentalized domains

The promoters of mammalian genes are commonly regulated by multiple distal enhancers, which physically interact within discrete chromatin domains. How such domains form and how the regulatory elements within them interact in single cells is not understood. To address this we developed Tri-C, a new chromosome conformation capture (3C) approach, to characterize concurrent chromatin interactions at individual alleles. Analysis by Tri-C identifies heterogeneous patterns of single-allele interactions between CTCF boundary elements, indicating that the formation of chromatin domains likely results from a dynamic process. Within these domains, we observe specific higher-order structures that involve simultaneous interactions between multiple enhancers and promoters. Such regulatory hubs provide a structural basis for understanding how multiple cis-regulatory elements act together to establish robust regulation of gene expression