| सारांश: | <p>Cytosine methylation is a pervasive DNA modification in vertebrate genomes that occurs predominantly in the context of sparsely distributed CpG dinucleotides. In contrast to the bulk genome, short genomic elements referred to as CpG islands (CGIs) feature a high GC content and clusters of non-methylated CpGs. CGIs co-localise with the majority of gene promoters where their distinct DNA sequence features enables the recruitment and assembly of transcription regulating complexes. Thus, as sites of transcription initiation, CGIs exert an important function in controlling promoter activity and gene expression. Despite their importance, it remains unclear how this is mechanistically achieved and how CGI dysfunction contributes to diseases such as cancer. This is largely due to the fact that the majority of our mechanistic insights into CGI function come from candidate-based approaches such as chromatin immunoprecipitation coupled to sequencing, which are inherently biased and limit us to investigating one protein at a time. The capacity to attribute detailed mechanistic understanding to CGI function has been hampered by our lack of a comprehensive inventory of proteins that contribute to their maintenance and function.</p>
<p>To overcome this fundamental lack of understanding, I have developed a new biochemical approach to isolate CGI chromatin from the bulk genome in order to investigate the CGI proteome in an unbiased manner using mass spectrometry. Applying next generation sequencing, I validated on the genome level that this method is suitable for specific enrichment of CGIs from mouse embryonic stem cells. Mass spectrometry data revealed many known CGI interactors, and it provided new leads for further investigation of potentially novel CGI-associated proteins. By deleting selected components of the CGI proteome, I uncovered CGI protein composition changes that pointed to new interrelations between the components of the gene regulation network at CGI promoters. Finally, using calibrated ChIP-sequencing as an orthogonal approach, I verified for selected proteins that the CGI proteome alterations were caused by changes in genomic occupancy. In conclusion, this work presents the first method to investigate the CGI proteome and its alterations comprehensively and unbiasedly in different conditions. Ultimately, this method can advance our knowledge of how CGIs contribute to gene regulation in developmental processes and pathogenesis.</p>
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