With continuous advances in DNA sequencing methods, accessibility to high-quality genomic information for all living organisms is ever-increasing. However, to interpret this information effectively and formulate hypotheses, users often require higher level programming skills. Therefore, the generation of web-based tools is becoming increasingly popular. CpG island regions in genomes are often found in gene promoters and are prone to DNA methylation, with their methylation status determining if a gene is expressed. Notably, understanding the biological impact of CpX modifications on genomic regulation is becoming increasingly important as these modifications have been associated with diseases such as cancer and neurodegeneration. However, there is currently no easy-to-use, scalable tool to detect and quantify CpX islands in full genomes. We have developed a Java-based web server for CpX island analyses that benefits from the DNA Analyzer Web server environment and overcomes several limitations. For a pilot demonstration study, we selected a well-described model organism Drosophila melanogaster. Subsequent analysis of the obtained CpX islands revealed several interesting and previously undescribed phenomena. One of them is the fact, that nearly half of long CpG islands were located on chromosome X, and that long CpA and CpT islands were significantly overrepresented at the subcentromeric regions of autosomes (chr2 and chr3) and also on chromosome Y. Wide genome overlays of predicted CpX islands revealed their co-occurrence with various (epi)genomics features comprising cytosine methylations, accessible chromatin, transposable elements, or binding of transcription factors and other proteins. CpX Hunter is freely available as a web tool at: https://bioinformatics.ibp.cz/#/analyse/cpg.

Department of Biology and Ecology Faculty of Science University of Ostrava Ostrava Czech Republic

Department of Biophysical Chemistry and Molecular Oncology Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Department of Informatics Mendel University in Brno Brno Czech Republic

Department of Informatics Mendel University in Brno Brno Czech Republic; Faculty of Mechanical Engineering Brno University of Technology Brno Czech Republic

Zobrazit více v PubMed

Deaton A.M., Bird A. CpG islands and the regulation of transcription. Genes Dev. 2011;25:1010–1022. PubMed PMC

Sae-Lee C., Barrow T.M., Colicino E., Choi S.H., Rabanal-Ruiz Y., Green D., et al. Genomic targets and selective inhibition of DNA methyltransferase isoforms. Clin. Epigenetics. 2022;14:103. PubMed PMC

Zhang X., Blumenthal R.M., Cheng X. DNA-binding proteins from MBD through ZF to BEN: recognition of cytosine methylation status by one arginine with two conformations. Nucleic Acids Res. 2024;52:11442–11454. PubMed PMC

Héberlé É., Bardet A.F. Sensitivity of transcription factors to DNA methylation. Essays Biochem. 2019;63:727–741. PubMed PMC

Wong E.M., Southey M.C., Fox S.B., Brown M.A., Dowty J.G., Jenkins M.A., et al. Constitutional methylation of the BRCA1 promoter is specifically associated with BRCA1 mutation-associated pathology in early-onset breast cancer. Cancer Prev. Res. 2011;4:23–33. PubMed PMC

Pineda M., Mur P., Iniesta M.D., Borràs E., Campos O., Vargas G., et al. MLH1 methylation screening is effective in identifying epimutation carriers. Eur. J. Hum. Genet. 2012;20:1256–1264. PubMed PMC

Edwards J.R., Yarychkivska O., Boulard M., Bestor T.H. DNA methylation and DNA methyltransferases. Epigenetics Chromatin. 2017;10:23. PubMed PMC

Du Q., Luu P.-L., Stirzaker C., Clark S.J. Methyl-CpG-binding domain proteins: readers of the epigenome. Epigenomics. 2015;7:1051–1073. PubMed

Du J., Johnson L.M., Jacobsen S.E., Patel D.J. DNA methylation pathways and their crosstalk with histone methylation. Nat. Rev. Mol. Cell Biol. 2015;16:519–532. PubMed PMC

Hughes A.L., Kelley J.R., Klose R.J. Understanding the interplay between CpG island-associated gene promoters and H3K4 methylation. Biochim. Biophys. Acta Gene Regul. Mech. 2020;1863 PubMed PMC

Bird A., Taggart M., Frommer M., Miller O.J., Macleod D. A fraction of the mouse genome that is derived from islands of nonmethylated, CpG-rich DNA. Cell. 1985;40:91–99. PubMed

Bird A.P. CpG-rich islands and the function of DNA methylation. Nature. 1986;321:209–213. PubMed

Götz M., Jarriault S. Programming and reprogramming the brain: a meeting of minds in neural fate. Development. 2017;144:2714–2718. PubMed

Yates J., Boeva V. Deciphering the etiology and role in oncogenic transformation of the CpG island methylator phenotype: a pan-cancer analysis. Brief Bioinform. 2022;23 PubMed PMC

Morgan A.E., Davies T.J., Mc Auley M.T. The role of DNA methylation in ageing and cancer. Proc. Nutr. Soc. 2018;77:412–422. PubMed

Cotton A.M., Price E.M., Jones M.J., Balaton B.P., Kobor M.S., Brown C.J. Landscape of DNA methylation on the X chromosome reflects CpG density, functional chromatin state and X-chromosome inactivation. Hum. Mol. Genet. 2015;24:1528–1539. PubMed PMC

Geissler F., Nesic K., Kondrashova O., Dobrovic A., Swisher E.M., Scott C.L., et al. The role of aberrant DNA methylation in cancer initiation and clinical impacts. Ther. Adv. Med. Oncol. 2024;16 PubMed PMC

Sidler C., Kovalchuk O., Kovalchuk I. Epigenetic regulation of cellular senescence and aging. Front. Genet. 2017;8:138. PubMed PMC

Jang H.S., Shin W.J., Lee J.E., Do J.T. CpG and non-CpG methylation in epigenetic gene regulation and brain function. Genes (Basel) 2017;8:148. PubMed PMC

Gowher H., Jeltsch A. Mammalian DNA methyltransferases: new discoveries and open questions. Biochem. Soc. Trans. 2018;46:1191–1202. PubMed PMC

Ramasamy D., Deva Magendhra Rao A.K., Rajkumar T., Mani S. Non-CpG methylation—a key epigenetic modification in cancer. Brief. Funct. Genomics. 2021;20:304–311. PubMed

Fuso A., Lucarelli M. CpG and non-CpG methylation in the diet-epigenetics-neurodegeneration connection. Curr. Nutr. Rep. 2019;8:74–82. PubMed

Kim S.H., Lim S.-H., Lee A.-R., Kwon D.H., Song H.K., Lee J.-H., et al. Unveiling the pathway to Z-DNA in the protein-induced B–Z transition. Nucleic Acids Res. 2018;46:4129–4137. PubMed PMC

Santos A.S., Ramos E.S., Valente-Gaiesky V.L.S., de Melo Sene F., Manfrin M.H. Evidences of differential methylation in the genome during development in the cactophilic Drosophila species. Genesis. 2024;62 PubMed

Owen B.M., Davidovich C. DNA binding by polycomb-group proteins: searching for the link to CpG islands. Nucleic Acids Res. 2022;50:4813–4839. PubMed PMC

Weber L.M., Jia Y., Stielow B., Gisselbrecht S.S., Cao Y., Ren Y., et al. The histone acetyltransferase KAT6A is recruited to unmethylated CpG islands via a DNA binding winged helix domain. Nucleic Acids Res. 2023;51:574–594. PubMed PMC

Saravanan K.A., Kumar H., Chhotaray S., Preethi A.L., Talokar A.J., Natarajan A., et al. Drosophila melanogaster: a promising model system for epigenetic research. Biol. Rhythm Res. 2022;53:382–400.

Takai D., Jones P.A. Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc. Natl. Acad. Sci. 2002;99:3740–3745. PubMed PMC

Kuhn R.M., Haussler D., Kent W.J. The UCSC genome browser and associated tools. Brief. Bioinformatics. 2013;14:144–161. PubMed PMC

Kuhn G.C.S., Küttler H., Moreira-Filho O., Heslop-Harrison J.S. The 1.688 repetitive DNA of Drosophila: concerted evolution at different genomic scales and association with genes. Mol. Biol. Evol. 2012;29:7–11. PubMed

Bassal M.A. The interplay between dysregulated metabolism and epigenetics in cancer. Biomolecules. 2023;13:944. PubMed PMC

Öztürk-Çolak A., Marygold S.J., Antonazzo G., Attrill H., Goutte-Gattat D., Jenkins V.K., et al. FlyBase: updates to the Drosophila genes and genomes database. Genetics. 2024;227 PubMed PMC

Wutz A., Gribnau J. X inactivation Xplained. Curr. Opin. Genet. Dev. 2007;17:387–393. PubMed

Sharp A.J., Stathaki E., Migliavacca E., Brahmachary M., Montgomery S.B., Dupre Y., et al. DNA methylation profiles of human active and inactive X chromosomes. Genome Res. 2011;21:1592–1600. PubMed PMC

Conrad T., Akhtar A. Dosage compensation in Drosophila melanogaster: epigenetic fine-tuning of chromosome-wide transcription. Nat. Rev. Genet. 2012;13:123–134. PubMed

Sokolov V., Kyrchanova O., Klimenko N., Fedotova A., Ibragimov A., Maksimenko O., et al. New Drosophila promoter-associated architectural protein Mzfp1 interacts with CP190 and is required for housekeeping gene expression and insulator activity. Nucleic Acids Res. 2024;52:6886–6905. PubMed PMC

Kaushal A., Mohana G., Dorier J., Özdemir I., Omer A., Cousin P., et al. CTCF loss has limited effects on global genome architecture in Drosophila despite critical regulatory functions. Nat. Commun. 2021;12:1011. PubMed PMC

Cavalheiro G.R., Girardot C., Viales R.R., Pollex T., Cao T.B.N., Lacour P., et al. CTCF, BEAF-32, and CP190 are not required for the establishment of TADs in early Drosophila embryos but have locus-specific roles. Sci. Adv. 2023;9 PubMed PMC

Duan J., Rieder L., Colonnetta M.M., Huang A., Mckenney M., Watters S., et al. CLAMP and Zelda function together to promote Drosophila zygotic genome activation. eLife. 2021;10 PubMed PMC

Yao B., Li Y., Wang Z., Chen L., Poidevin M., Zhang C., et al. Active N6-methyladenine demethylation by DMAD regulates gene expression by coordinating with polycomb protein in neurons. Mol. Cell. 2018;71:848–857.e6. PubMed PMC

McKowen J.K., Avva S.V.S.P., Maharjan M., Duarte F.M., Tome J.M., Judd J., et al. The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex. G3 (Bethesda) 2022;12 PubMed PMC

Lloyd J.P.B., Lister R. Epigenome plasticity in plants. Nat. Rev. Genet. 2022;23:55–68. PubMed

Lemmens B., van Schendel R., Tijsterman M. Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers. Nat. Commun. 2015;6:8909. PubMed PMC

Belotserkovskii B.P., Neil A.J., Saleh S.S., Shin J.H.S., Mirkin S.M., Hanawalt P.C. Transcription blockage by homopurine DNA sequences: role of sequence composition and single-strand breaks. Nucleic Acids Res. 2013;41:1817–1828. PubMed PMC

Mérel V., Boulesteix M., Fablet M., Vieira C. Transposable elements in Drosophila. Mobile DNA. 2020;11:23. PubMed PMC

Haney R.A., Feder M.E. Contrasting patterns of transposable element insertions in Drosophila heat-shock promoters. PLoS One. 2009;4 PubMed PMC

Brázda V., Kolomazník J., Lýsek J., Bartas M., Fojta M., Šťastný J., et al. G4Hunter web application: a web server for G-quadruplex prediction. Bioinformatics. 2019;35:3493–3495. PubMed PMC

Brázda V., Kolomazník J., Lỳsek J., Hároníková L., Coufal J., Št’astnỳ J. Palindrome analyser–A new web-based server for predicting and evaluating inverted repeats in nucleotide sequences. Biochem. Biophysical Res. Commun. 2016;478:1739–1745. PubMed

Tang D., Chen M., Huang X., Zhang G., Zeng L., Zhang G., et al. SRplot: a free online platform for data visualization and graphing. PLoS One. 2023;18 PubMed PMC

Zou Z., Ohta T., Oki S. ChIP-Atlas 3.0: a data-mining suite to explore chromosome architecture together with large-scale regulome data. Nucleic Acids Res. 2024;52:W45–W53. PubMed PMC

Szklarczyk D., Kirsch R., Koutrouli M., Nastou K., Mehryary F., Hachilif R., et al. The STRING database in 2023: protein–protein association networks and functional enrichment analyses for any sequenced genome of interest. Nucleic Acids Res. 2023;51:D638–D646. PubMed PMC