Deciphering the regulatory principles of histone H3K36 methylation in Drosophila

Jayakrishnan, Muhunden Nallappa (2025): Deciphering the regulatory principles of histone H3K36 methylation in Drosophila. Dissertation, LMU München: Medizinische Fakultät

Abstract

Histone Post Translational Modifications (PTMs) are dynamic signatures that play a central role in genome function by influencing chromatin organization and transcriptional regulation. Their precise deposition and regulation are critical for development, and their misregulation is frequently linked to disease. While many histone modifications have been extensively studied, a major challenge remains in understanding how they interact within the chromatin landscape to influence gene activity and other biological processes. Methylation of histone H3 at lysine 36 (H3K36me1/2/3) has been implicated in transcriptional regulation and chromatin organization, with K36me3 in particular correlating with active transcription. This modification is highly conserved across species, accumulating along transcribed gene bodies. Despite this conservation, the precise relative distribution of the K36me1-3 states and their function remain unclear in certain model organisms such as Drosophila. Additionally, while the enzymes responsible for K36 methylation have been identified, their respective contributions to the establishment of K36me1, K36me2, and K36me3 remain debated. To address these gaps, ChIP-seq protocols were systematically optimized to define the genomic landscapes of K36me1/2/3 in Drosophila which revealed that each methylation state preferentially associates with distinct chromatin environments, highlighting their specialized roles. A key unresolved question is how K36 methyltransferases (HMTs) Set2, NSD and Ash1 contribute to the deposition of these modifications. Using an RNAi approach, it was observed that each HMT predominantly catalyzes methylation in specific chromatin domains with minor overlaps: Set2 deposits K36me3 at highly transcribed genes; NSD catalyzes K36me2/3 at constitutive heterochromatin in addition to weakly expressed euchromatic genes, while Ash1 deposits K36me1 at regions with enhancer signatures. Further, despite functioning largely independent of each other, evidence suggests that HMTs may indirectly influence each other’s activity and localization. K36 methylation has been implicated in both transcriptional regulation and histone modification crosstalk. To investigate these roles, RNA-seq and ChIP-seq datasets were integrated to analyze transcriptional changes and potential interactions with other chromatin marks. The results revealed a poor correlation between K36me alterations and transcriptional changes, challenging assumptions about its direct role in gene regulation. However, all three K36 HMTs were found to counteract the facultative heterochromatic mark H3K27me3 at specific loci, highlighting a broader regulatory interplay between these modifications. Finally, the functional consequences of K36 methylation often depend on reader proteins. While many of these readers have been characterized for K36me3, their affinities for K36me1/2 remain unexplored. Examining the impact of K36 HMT loss on two key readers, MSL3 and JASPer, revealed that K36me2 can partially compensate for K36me3 loss to maintain reader binding. Additionally, disruption of MSL3 binding alters H4K16 acetylation distribution and the organization of the dosage-compensated X-chromosome territory. Together, these findings provide new insights into the regulatory logic of H3K36 methylation and its functional interactions within the chromatin landscape.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Chromatin, Epigenetics, Histone modifications, H3K36, methylation, methyltransferases
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 4. August 2025
• 1. Bericht­erstatter:in: Becker, Peter