Christoph Grunau

Bisulfite genomic sequencing: systematic investigation of critical experimental parameters

Bisulfite genomic sequencing is the method of choice for the generation of methylation maps with single-base resolution. The method is based on the selective deamination of cytosine to uracil by treatment with bisulfite and the sequencing of subsequently generated PCR products. In contrast to cytosine, 5-methylcytosine does not react with bisulfite and can therefore be distinguished. In order to investigate the potential for optimization of the method and to determine the critical experimental parameters, we determined the influence of incubation time and incubation temperature on the deamination efficiency and measured the degree of DNA degradation during the bisulfite treatment. We found that maximum conversion rates of cytosine occurred at 55 degrees C (4-18 h) and 95 degrees C (1 h). Under these conditions at least 84-96% of the DNA is degraded. To study the impact of primer selection, homologous DNA templates were constructed possessing cytosine-containing and cytosine-free primer binding sites, respectively. The recognition rates for cytosine (>/=97%) and 5-methylcytosine (>/=94%) were found to be identical for both templates.

Identification and resolution of artifacts in bisulfite sequencing

Bisulfite sequencing has become the most widely used application to detect 5-methylcytosine (5-MeC) in DNA, and provides a reliable way of detecting any methylated cytosine at single-molecule resolution in any sequence context. The process of bisulfite treatment exploits the different sensitivity of cytosine and 5-MeC to deamination by bisulfite under acidic conditions, in which cytosine undergoes conversion to uracil while 5-MeC remains unreactive. In this article, we address the more commonly encountered experimental artifacts associated with bisulfite sequencing, and provide methods for the detection and elimination of these artifacts. In particular, we focus on conditions that inhibit complete bisulfite-mediated conversion of cytosines in a target sequence, and demonstrate the necessity of complete protein removal from DNA samples prior to bisulfite treatment. We also include a brief summary of the experimental protocol for bisulfite treatment and tips for designing polymerase chain reaction (PCR) primers to amplify from bisulfite-treated DNA.

MethDB—a public database for DNA methylation data

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Genes Related to Ion-Transport and Energy Production Are Upregulated in Response to CO2-Driven pH Decrease in Corals: New Insights from Transcriptome Analysis

Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH≥7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO2 into HCO3 − and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation.

Histone deacetylase inhibitors induce apoptosis, histone hyperacetylation and up-regulation of gene transcription in Schistosoma mansoni

In order to explore the conservation/divergence of transcriptional regulation in the platyhelminth parasite Schistosoma mansoni, we are studying the structures and functions of transcriptional mediators and in particular histone-modifying enzymes. Reversible histone acetylation changes chromatin structure and modulates gene transcription. The removal of acetyl residues from histones and other proteins is catalyzed by histone deacetylases (HDACs) that are under increasing study as therapeutic targets, both in cancer and parasitic diseases. In order to determine the extent and importance of histone acetylation in S. mansoni, we tested the effects of three histone deacetylase inhibitors (HDACi) on both larval and adult worms in culture. Trichostatin A (TSA), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) inhibited global HDAC activity at all life-cycle stages. TSA and VPA, but not SAHA, caused mortality of schistosomula and adults, with TSA showing the most rapid effect. Moreover, TSA caused an increase in apoptosis in schistosomula shown by the TUNEL assay and an increase in caspase 3/7 activity. Both TSA and VPA were shown to cause an increase in general levels of protein acetylation in schistosomes; more particularly of histone 4 whereas histone 3 acetylation was less affected. In the case of TSA treatment this histone hyperacetylation was correlated with the increased expression of caspases 3 and 7 transcripts. Finally, quantitative chromatin immunoprecipitation showed that the proximal promoter region of the S. mansoni caspase 7 gene was hyperacetylated on histone H4 after TSA treatment.

Controlled Chaos of Polymorphic Mucins in a Metazoan Parasite (Schistosoma mansoni) Interacting with Its Invertebrate Host (Biomphalaria glabrata)

Invertebrates were long thought to possess only a simple, effective and hence non-adaptive defence system against microbial and parasitic attacks. However, recent studies have shown that invertebrate immunity also relies on immune receptors that diversify (e.g. in echinoderms, insects and mollusks (Biomphalaria glabrata)). Apparently, individual or population-based polymorphism-generating mechanisms exists that permit the survival of invertebrate species exposed to parasites. Consequently, the generally accepted arms race hypothesis predicts that molecular diversity and polymorphism also exist in parasites of invertebrates. We investigated the diversity and polymorphism of parasite molecules (Schistosoma mansoni Polymorphic Mucins, SmPoMucs) that are key factors for the compatibility of schistosomes interacting with their host, the mollusc Biomphalaria glabrata. We have elucidated the complex cascade of mechanisms acting both at the genomic level and during expression that confer polymorphism to SmPoMuc. We show that SmPoMuc is coded by a multi-gene family whose members frequently recombine. We show that these genes are transcribed in an individual-specific manner, and that for each gene, multiple splice variants exist. Finally, we reveal the impact of this polymorphism on the SmPoMuc glycosylation status. Our data support the view that S. mansoni has evolved a complex hierarchical system that efficiently generates a high degree of polymorphism—a “controlled chaos”—based on a relatively low number of genes. This contrasts with protozoan parasites that generate antigenic variation from large sets of genes such as Trypanosoma cruzi, Trypanosoma brucei and Plasmodium falciparum. Our data support the view that the interaction between parasites and their invertebrate hosts are far more complex than previously thought. While most studies in this matter have focused on invertebrate host diversification, we clearly show that diversifying mechanisms also exist on the parasite side of the interaction. Our findings shed new light on how and why invertebrate immunity develops.

Applying ecological and evolutionary theory to cancer: a long and winding road

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.

Frequent DNA hypomethylation of human juxtacentromeric BAGE loci in cancer

The BAGE (B melanoma antigens) sequence family contains 15 nearly identical sequences that are in the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. BAGE loci are expressed in male germ tissue and in a high percentage of cancers and cancer cell lines. We analyzed the DNA methylation state of the sequences in or near the promoters of the BAGE loci by a quantitative bisulfite and PCR‐based assay (multiplex COBRA) using MboI and HphI in 18 somatic tissue samples, 4 testis and 4 sperm samples, and 48 tumors and tumor cell lines. In 94% of the control somatic tissue samples, DNA was highly methylated in the analyzed regions. In contrast, 98% of tumor DNA samples displayed hypomethylation. Also, DNA from testes and sperm was hypomethylated in at least one of the BAGE loci. BAGE transcripts were observed in only 47% of the analyzed tumor samples. Consequently, we propose BAGE hypomethylation as a new, highly informative epigenetic biomarker for the diagnosis of cancer, whose hypomethylation in cancer may be causally related to that of juxtacentromeric satellite DNA.

An improved version of the DNA methylation database (MethDB)

Cytosine methylation is a characteristic property of prokaryotic and eukaryotic genomes. In the latter, it is indispensable for a healthy development of the organism and uncontrolled changes in the distribution of 5-methylcytosine (5mC) have been linked to severe disorders, in particular cancer. The growing scientific interest in DNA methylation has led to a considerable amount of data about this epigenetic phenomenon. In order to make these data readily available, we have established a dedicated database. The DNA Methylation database (MethDB) is currently the only public database for DNA methylation (http://www.methdb.net). This constantly growing database has become a key resource in the field of DNA methylation research. The database contains currently methylation patterns, profiles and total methylation content data for 46 species, 160 tissues and 72 phenotypes coming from a total of 6667 experiments (as of September 4, 2002). About 14% of the data have not been published elsewhere. These data can be conveniently searched and represented in different ways. Recently, we have included an on-line submission tool that permits the scientific public to directly enter new data into MethDB.

A family of variable immunoglobulin and lectin domain containing molecules in the snail Biomphalaria glabrata

Technical limitations have hindered comprehensive studies of highly variable immune response molecules that are thought to have evolved due to pathogen-mediated selection such as fibrinogen-related proteins (FREPs) from Biomphalaria glabrata. FREPs combine upstream immunoglobulin superfamily (IgSF) domains with a C-terminal fibrinogen-related domain (FreD) and participate in reactions against trematode parasites. From RNAseq data we assembled a de novo reference transcriptome of B. glabrata to investigate the diversity of FREP transcripts. This study increased over two fold the number of bonafide FREP subfamilies and revealed important sequence diversity within FREP12 subfamily. We also report the discovery of related molecules that feature one or two IgSF domains associated with different C-terminal lectin domains, named C-type lectin-related proteins (CREPs) and Galectin-related protein (GREP). Together, the highly similar FREPs, CREPs and GREP were designated VIgL (Variable Immunoglobulin and Lectin domain containing molecules).