Transkriptiotekijöiden sitoutumiskohtien laskennallisesta määrittämisestä:ETS-faktorit ERG ja SPDEF

Tässä opinnäytetyössä tarkasteltiin ja sovellettiin laskennallisia menetelmiä transkriptiotekijöiden (TF) sitoutumiskohtien (TFBS) määrittämiseen. TF:t ovat geenien säätelyyn osallistuvia proteiineja, jotka tunnistavat spesifisen nukleotidijärjestyksen sisältävän DNA-jakson eli TFBS:n sitoutuen siihen. Saman TF:n eri TFBS:t eivät ole identtisiä, vaan ainoastaan samankaltaisia. Laskennallisten menetelmät perustuvat malleihin, joissa DNA:n rakenneosasten eli nukleotidien esiintyminen kussakin TFBS:n paikassa noudattaa jotakin todennäköisyysjakaumaa. Opinnäytetyö jakaantuu kirjallisuus- ja soveltavaan osaan. Ensimmäisessä luodaan yleiskatsaus tutkimusaiheen biologiseen taustaan, esitetään aiheen kannalta keskeisiä matemaattisia määritelmiä ja kaavoja sekä perehdytään erilaisiin TFBS-määrittelyihin, kuten konsensussekvenssi, paikkapainomatriisi (PWM) ja Markov-matriisimallit. TFBS-määrittelyn löytämiseksi kehitetyistä algoritmeista esitellään MEME-menetelmä. Toiseksi kuvataan pistemääräfunktio, jonka avulla etsitään uusia sitoutumiskohtia tunnetun TFBS-määrittelyn avulla ja jota hyödynnetään sovelletun osan MATCH-algoritmissa. Esimerkkinä laskennallisten menetelmien matemaattisesta pätevyydestä esitetään MEME-menetelmän käyttämän EM-(odotusarvo-maksimointi)-algoritmin suppenemistarkastelu todistuksineen. Tämän tarkastelun päälähde on ’Wu, C. (1983) On the Convergence Properties of the EM Algorithm. The Annals of Statistics, 11: 95–103.’ Tutkielman soveltavassa osassa tutkittiin MATCH-algoritmin käyttökelpoisuutta paikannettaessa ETS-transkriptiotekijöiden ERG ja SPDEF sitoutumiskohtia joukosta kromosomaalisia DNA-sekvenssejä, joiden tiedettiin sitovan kyseisiä transkriptiotekijöitä ihmisen eturauhassyöpäsoluissa CHIP-seq-analyysin perusteella (Wei et al.(2010), EMBO J., 29: 2147–2160.) MATCH-algoritmi perustuu oletukseen TFBS:n nukleotidien multinomisesta ja toisistaan riippumattomasta jakaantumisesta. Tri Gonghong Wei ystävällisesti auttoi algoritmissa käytettyjen, alunperin MEME-algoritmilla in vitro (koeputki) -sidontakokeiden tuloksista määritettyjen PWM-matriisien hankkimisessa. Datan analyysissä käytetyt skriptit ja funktiot laadittiin itse Matlab-ympäristössä. Olennaisena osana tähän kuului MATCH-algoritmin lisäksi permutaatioanalyysi, jonka avulla arvioitiin löydettyjen TFBS-kandidaattien tilastollista merkitsevyyttä. Tutkimuksessa analysoitiin 195 ERG-tekijän ja 193 SPDEF-tekijän sitomaa sekvenssiä analysoimalla DNA:n kumpikin juoste erikseeen. Tilastollisesti merkitseviä ERG-TFBS:iä löydettiin vain kaksi ja SPDEF-TFBS:iä viisi kappaletta kaikki eri sekvensseistä. Kun ERG-sekvenssien analyysissä käytettiin in vitro -sidontakokeista saadun PWM:n sijasta in vivo (solussa tapahtuva) -määritettyä edellisestä hieman poikkeavaa PWM:ää, tilastollisesti merkitseviä TFBS-kandidaatteja löydettiin 50 sekvenssistä yhteensä 58 kappaletta. ETS-transkriptiotekijöiden sitomat DNA-sekvenssit sisältävät ainoastaan viiden nukleotidin mittaisen (C/A)GGA(A/T) -ydinjakson, joka on yhteinen suurimmalle osalle näiden proteiinien sitoutumiskohtia. Koska käytetyssä mallissa oletettiin nukleotidien esiintymisen todennäköisyysjakauma riippumattomaksi ympäröivistä nukleotideista, on ymmärrettävää, että MATCH-algoritmi tuottaa tilastollisesti merkitseviä löydöksiä vain, jos kohdesekvenssi on hyvin lähellä PWM-matriisin määräämää konsensussekvenssiä. Tällöin menetetään TFBS- kandidaatit, joissa ydinjakson ulkopuolisten nukleotidien yhteisesiintyminen puhtaasti sattumalta on epätodennäköistä, vaikka erillisinä tapahtumina, riippumattomuusoletuksen vallitessa, esiintyminen ei poikkea tilastollisesti merkitsevästi taustasta. Täten tämä tutkimus vahvistaa käsitystä, että uusia TFBS-kandidaatteja etsittäessä olisi syytä käyttää malleja, jotka sallivat riippuvuuden TFBS:n eri nukleotidipaikkojen välillä.The computational methods used in the analysis of transcription factor binding sites were reviewed and utilized in this Pro gradu thesis. Transcription factors (TF) are proteins that regulate the activity of genes. They bind specific DNA sequences, hereafter transcription factor binding sites (TFBS), which share the similar but usually not identical sequences of DNA building block nucleotides. Computational methods are based on models, where the presence of certain nucleotide at the specific position of the TFBS obeys some probability distribution. The thesis consists of a literature review and an applied study. First, a general review of biological background and key mathematical definitions and formulas are given. Secondly, different TFBS definitions like a consensus sequence, a position weight matrix (PWM) and markovian matrix models are presented. MEME is described as an example of an algorithm for extracting matrix form TFBS definition from DNA sequences which are known to bind a specific transcription factor. The solidity of the mathematical basis of MEME is illustrated by showing the proof for the convergence of the EM-algorithm used in MEME. Finally the principles of using a scoring function in the search of novel TFBS are presented. In the applied part of this thesis, the MATCH algorithm is used in the search for human ERG and SPDEF transcription factor binding sites in chromosomal DNA. The analysed sequences come from CHIP-seq analysis (Wei et al.(2010), EMBO J., 29: 2147–2160.) which represent real binding events in human cells. The MATCH algorithm is based on the assumption of independent and multinomial distribution of nucleotides in each TFBS position. Dr Gonghong Wei kindly helped to access the ERG and SPDEF specific PWMs used in this study. These PWMs have been produced by the MEME analysis of in vitro binding data. All the scripts and functions used in the data analysis were written in Matlab environment. In addition to the MATCH algorithm, the permutation analysis was compiled and used to estimate the statistical significance of found TFBS candidates. Altogether 195 ERG specific and 193 SPDEF specific sequences were analysed (both strands). Only two ERG TFBSs and five SPDEF TFBSs were found with statistical significance. When the in vitro PWM was replaced with an in vivo PWM which originated from CHIP-seq analysis, the analysis of ERG specific sequences provided 58 TFBS from 50 different sequences. The poor efficiency of the MATCH algorithm is obviously a consequence from the pre-assumptions of the algorithm. In contrast to the independent distribution of nucleotides postulated in the MATCH algorithm, the binding specificities of ETS-factors are likely dependent on the co-presence of multiple nucleotides. Therefore, models which take account the dependence between the nucleotide positions in the TFBS, should be preferred in the future analysis

Human DNA polymerase ε:expression, phosphorylation and protein-protein interactions

Abstract DNA replication is a process in which a cell duplicates its genome before cell division, and must proceed accurately and in organized manner to guarantee maintenance of the integrity of the genetic information. DNA polymerases are enzymes that catalyse the synthesis of the new DNA strand by utilizing the parental strand as a template. In addition to chromosomal replication, DNA synthesis and therefore DNA polymerases are also needed in other processes like DNA repair and DNA recombination. The DNA polymerase is an essential DNA polymerase in eukaryotes and is required for chromosomal DNA replication. It has also been implicated in DNA repair, recombination, and in transcriptional and cell cycle control. The regulation of the human enzyme was explored by analysing its expression, phosphorylation and protein-protein interactions. Expression of both the A and B subunits of the human DNA polymerase ε was strongly growth-regulated. After serum-stimulation of quiescent fibroblasts, the steady-state mRNA levels were up-regulated at least 5-fold. In actively cycling cells, however, the steady-state mRNA and protein levels fluctuated less than 2-fold, being highest in G1/S phase. The promoter of the B subunit gene was analysed in detail. The 75 bp core promoter was essentially dependent on the Sp1 transcription factor. Furthermore, mitogenic control of the promoter required an intact E2F binding element, and binding of E2F2, E2F4 and p107 was demonstrated in vitro. A down-regulation element, located immediately downstream from the core promoter, bound E2F1, NF-1 and pRb transcription factors. A model of the promoter function is presented. Topoisomerase IIβ binding protein 1 (TopBP1) was found to be associated with human DNA polymerase ε. TopBP1 contains eight BRCT domains and is homologous to Saccharomyces cerevisiae Dpb11, Schizosaccharomyces pombe Cut5, Drosophila melanogaster Mus101 and the human Breast Cancer susceptibility protein 1 (BRCA1). TopBP1 is a phosphoprotein, whose expression is induced at the G1/S border and is required for chromosomal DNA replication. It co-localizes in S phase with BRCA1 into discrete foci, which do not represent sites of ongoing DNA replication. However, if DNA is damaged or replication is blocked in S phase cells, TopBP1 and BRCA1 re-localize into proliferating cell nuclear antigen (PCNA) containing foci that represent stalled replication forks. Finally, phosphorylation of DNA polymerase ε was described and at least three immunologically distinct and differentially phosphorylated forms were shown to exist. Phosphorylation is on serine and threonine residues and shows a cell cycle dependent fluctuation, but is not affected by DNA damage or by inhibition of DNA replication. BRCA1 co-immunoprecipitates with a hypophosphorylated form of DNA polymerase ε. In contrast, TopBP1 was shown to be associated with a hyperphosphorylated form

BP180/Collagen XVII:a molecular view

Abstract BP180 is a type II collagenous transmembrane protein and is best known as the major autoantigen in the blistering skin disease bullous pemphigoid (BP). The BP180 trimer is a central component in type I hemidesmosomes (HD), which cause the adhesion between epidermal keratinocytes and the basal lamina, but BP180 is also expressed in several non-HD locations, where its functions are poorly characterized. The immunological roles of intact and proteolytically processed BP180, relevant in BP, have been subject to intensive research, but novel functions in cell proliferation, differentiation, and aging have also recently been described. To better understand the multiple physiological functions of BP180, the focus should return to the protein itself. Here, we comprehensively review the properties of the BP180 molecule, present new data on the biochemical features of its intracellular domain, and discuss their significance with regard to BP180 folding and protein–protein interactions

The intracellular domain of BP180/collagen XVII is intrinsically disordered and partially folds in an anionic membrane lipid-mimicking environment

Abstract The trimeric transmembrane collagen BP180, also known as collagen XVII, is an essential component of hemidesmosomes at the dermal–epidermal junction and connects the cytoplasmic keratin network to the extracellular basement membrane. Dysfunction of BP180 caused by mutations in patients with junctional epidermolysis bullosa or autoantibodies in those with bullous pemphigoid leads to severe skin blistering. The extracellular collagenous domain of BP180 participates in the protein’s triple-helical folding, but the structure and functional importance of the intracellular domain (ICD) of BP180 are largely unknown. In the present study, we purified and characterized human BP180 ICD. When expressed in Escherichia coli as glutathione-S-transferase or 6 × histidine tagged fusion protein, the BP180 ICD was found to exist as a monomer. Analysis of the secondary structure content by circular dichroism spectroscopy revealed that the domain is intrinsically disordered. This finding aligned with that of a bioinformatic analysis, which predicted a disordered structure. Interestingly, both anionic detergent micelles and lipid vesicles induced partial folding of the BP180 ICD, suggesting that in its natural environment, the domain’s folding and unfolding may be regulated by interaction with the cell membrane or accompanying proteins. We hypothesize that the intrinsically disordered structure of the ICD of BP180 contributes to the mechanism that allows the remodeling of hemidesmosome assembly

The Parkinson’s-disease-associated receptor GPR37 undergoes metalloproteinase-mediated N-terminal cleavage and ectodomain shedding

Abstract The G-protein-coupled receptor 37 ( GPR37) has been implicated in the juvenile form of Parkinson’s disease, in dopamine signalling and in the survival of dopaminergic cells in animal models. The structure and function of the receptor, however, have remained enigmatic. Here, we demonstrate that although GPR37 matures and is exported from the endoplasmic reticulum in a normal manner upon heterologous expression in HEK293 and SH-SY5Y cells, its long extracellular N-terminus is subject to metalloproteinase-mediated limited proteolysis between E167 and Q168. The proteolytic processing is a rapid and efficient process that occurs constitutively. Moreover, the GPR37 ectodomain is released from cells by shedding, a phenomenon rarely described for GPCRs. Immunofluorescence microscopy further established that although full-length receptors are present in the secretory pathway until the trans-Golgi network, GPR37 is expressed at the cell surface predominantly in the N-terminally truncated form. This notion was verified by flow cytometry and cell surface biotinylation assays. These new findings on the GPR37 N-terminal limited proteolysis may help us to understand the role of this GPCR in the pathophysiology of Parkinson’s disease and in neuronal function in general

Gliptin-associated bullous pemphigoid and the expression of dipeptidyl peptidase-4/CD26 in bullous pemphigoid

Abstract Dipeptidyl peptidase-4 inhibitors (DPP-4i or gliptins) increase the risk of developing bullous pemphigoid (BP). To clarify, whether gliptin-associated BP has special features, we analyzed the clinical, histopathological and immunological features of 27 BP patients, 10 of which previously used gliptin medication. Compared to those who had not previously received gliptins, subjects who had, showed higher BP180-NC16A ELISA (enzyme-linked immunosorbent assay) values, fewer neurological co-morbidities and shorter time to remission, but differences were not statistically significant. The HLA-DQB1*03:01 allele was more commonly present among the BP patients than the control population, but was not more common in those with gliptin history. To determine the effect of gliptins on the expression of the DPP-4/CD-26 protein we performed immunohistochemistry, which showed that the skin expression of DPP-4/CD-26 was increased in BP patients, but not affected by prior gliptin treatment. We conclude that DPP-4i medication is common among BP patients and prior gliptin treatment may be associated with some specific features

Absence of NC14A domain of COLXVII/BP180 in mice results in IL-17‒associated skin inflammation

Abstract The deletion of exon 18 from Col17a1 in transgenic ΔNC14A mice results in the absence of the NC14A domain. NC14A corresponds to the human NC16A domain, the immunodominant epitope in bullous pemphigoid. Before the age of 1 year, 84% of ΔNC14A mice have developed severe itch and skin erosion. Further characterization of mice with mutated CoLXVII (Bp180) revealed acanthosis; subepidermal blistering; and inflammatory cell infiltrates, especially neutrophils, eosinophils, and mast cells in the lesional skin. Direct immunofluorescence analysis detected linear complement C3, IgG, and/or IgA deposition in the dermo‒epidermal junction of symptomatic ΔNC14A mice. Elevated gene expression of IL-17‒associated cytokines was detected in the lesional skin. An increased proportion of dendritic cells, myeloid-derived suppressor cells, and NK cells and a decrease of T cells were found in both the spleen and lymph nodes of symptomatic ΔNC14A mice. The proportions of B cells and regulatory T cells were increased in lymph nodes. An 8-week treatment with an anti‒IL-17A decreased the expression of Il6, Il23a, and Cxcl1 in the nonlesional skin. Our results suggest that the absence of the NC14A domain of CoLXVII in mice causes an autoimmune response against the cutaneous basement membrane and manifests as an IL-17‒associated inflammation in the skin

Dipeptidyl peptidase 4 inhibitor‒associated bullous pemphigoid Is characterized by an altered expression of cytokines in the skin

Abstract Dipeptidyl peptidase 4 inhibitors (DPP4is), commonly used drugs for treatment of type 2 diabetes, increase the risk for bullous pemphigoid (BP). Currently, the mechanism leading to the loss of immunological tolerance of the cutaneous adhesion molecule BP180 as well as similarities and differences in disease progression between DPP4i-associated BP (DPP4i-BP) and DPP4i-independent regular BP are largely unknown. We analyzed the expression of 32 cytokines and two proteases by Luminex and ELISA assays in samples taken from lesional and nonlesional skin of patients with regular BP or DPP4i-BP and healthy controls. Cytokines mediating B-cell survival and targeting such as BAFF, CCL4, CXCL12, and IL-6 were expressed at a higher level in the lesional regular BP skin than the levels in the lesional DPP4i-BP skin. The DPP4i-BP samples had increased levels of eosinophilic cytokines CCL1, CCL17, CCL26, and IL-5, which correlated with the serum level of anti-BP180 NC16A IgG autoantibodies. The mRNA expression of BAFF, IL6, CCL1, CCL17, CCL26, and IL5 measured by qPCR correlated with the protein levels. Taken together, the cutaneous cytokine profiles were found to provide distinctive molecular fingerprints between regular BP and DPP4i-BP

Autoantibodies against the immunodominant bullous pemphigoid epitopes are rare in patients with dermatitis herpetiformis and coeliac disease

Abstract Dermatitis herpetiformis (DH) is an extraintestinal manifestation of coeliac disease (CD). Patients with DH have an elevated risk of development of another autoimmune blistering skin disease, bullous pemphigoid (BP). In this study we investigated whether patients with DH and CD (mean age for both 49 years) have circulating autoantibodies against BP180, the major BP autoantigen. ELISA tests showed that only a few DH (3/46) and CD (2/43) patients had BP180-NC16A IgG autoantibodies. Immunoblotting found that more than half of the DH samples contained IgG autoantibodies against full-length BP180. Epitope mapping with 13 fusion proteins covering the BP180 polypeptide revealed that in DH and CD patients, IgG autoantibodies did not target the NC16A or other epitopes typical of BP but recognized other intracellular and mid-extracellular regions of BP180. None of the analyzed DH and CD patients with either ELISA or immunoblotting positivity had IgG or IgA reactivity against the cutaneous basement membrane in indirect immunofluorescence analysis or skin symptoms characteristic of BP. Although only a minority of middle-aged DH patients had IgG autoantibodies against the immunodominant epitopes of BP180, our results do not exclude the possibility that intermolecular epitope spreading could explain the switch from DH to BP in elderly patients