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Proximity ligation assays of protein and RNA interactions in the male-specific lethal complex on Drosophila melanogaster polytene chromosomes
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Jan Larsson)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Jan Larsson)
Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). (Jan Larsson)
2015 (Engelska)Ingår i: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 124, nr 3, s. 385-395Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

In Drosophila, the male-specific lethal (MSL) complex specifically targets the male X chromosome and participates in a twofold increase in expression output leading to functional dosage compensation. The complex includes five proteins and two non-coding RNAs (ncRNAs). A number of additional associated factors have also been identified. However, the components' roles and interactions have not been fully elucidated. The in situ proximity ligation assay (PLA) provides a sensitive means to determine whether proteins and other factors have bound to chromosomes in close proximity to each other, and thus may interact. Thus, we modified, tested, and applied the assay to probe interactions of MSL complex components on polytene chromosomes. We show that in situ PLA can detect and map both protein-protein and protein-ncRNA interactions on polytene chromosomes at high resolution. We further show that all five protein components of the MSL complex are in close proximity to each other, and the ncRNAs roX1 and roX2 bind the complex in close proximity to MLE. Our results also indicate that JIL1, a histone H3 Ser10 kinase enriched on the male X chromosome, interacts with MSL1 and MSL2, but not MSL3 of the MSL complex. In addition, we corroborate proposed interactions of the MSL complex with both CLAMP and TopoII.

Ort, förlag, år, upplaga, sidor
2015. Vol. 124, nr 3, s. 385-395
Nyckelord [en]
dosage compensation, protein interaction, polytene chromosomes, MSL-complex
Nationell ämneskategori
Genetik
Forskningsämne
genetik
Identifikatorer
URN: urn:nbn:se:umu:diva-100658DOI: 10.1007/s00412-015-0509-xISI: 000360288200009PubMedID: 25694028Scopus ID: 2-s2.0-84940451246OAI: oai:DiVA.org:umu-100658DiVA, id: diva2:793226
Forskningsfinansiär
Vetenskapsrådet, 621-2012-2165Cancerfonden, 2011/382Tillgänglig från: 2015-03-06 Skapad: 2015-03-05 Senast uppdaterad: 2023-03-24Bibliografiskt granskad
Ingår i avhandling
1. Jack of all trades, master of none: the multifaceted nature of H3K36 methylation
Öppna denna publikation i ny flik eller fönster >>Jack of all trades, master of none: the multifaceted nature of H3K36 methylation
2023 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Alternativ titel[sv]
Molekylär mångsysslare : komplexiteten kring H3K36 metylering
Abstract [en]

Post-translational modifications of histones enable differential transcriptional control of the genome between cell types and developmental stages, and in response to environmental factors. The methylation of Histone 3 Lysine 36 (H3K36) is one the most complex and well-studied histone modifications and is known to be involved in a wide range of molecular processes. Commonly associated with active genes and transcriptional elongation, H3K36 methylation also plays a key role in DNA repair, repression of cryptic transcription, and guiding additional post-translational modifications to histones, genomic DNA, and RNA. In Drosophila melanogaster, trimethylated H3K36 has also been linked to dosage compensation of the single male X chromosome as a binding substrate for the Male-Specific Lethal (MSL) complex. However, this model has been challenged by structural and biochemical studies demonstrating higher MSL complex affinity for other methylated lysines. There is an additional system of chromosome-specific gene regulation in D. melanogaster where transcription from the small heterochromatic fourth chromosome is increased by Painting of fourth (POF), a protein specifically binding nascent RNA on the fourth chromosome. The fourth chromosome is thought to have been an ancestral X chromosome that reverted into an autosome. POF mediating high transcription levels from an autosome is believed to be a remnant of an ancient sex-chromosome dosage compensation mechanism. 

Proximity ligation assays revealed no interaction between MSL complex components and methylated H3K36. This finding was corroborated by RNA sequencing of H3K36 methylation impaired mutants: the transcriptional output of the male X chromosome was unaffected in mutants where Lysine 36 on Histone 3 was replaced by an Arginine, abolishing methylation of this site. However, we found that knocking out Set2, which encodes the methyltransferase responsible for H3K36 trimethylation, significantly reduced X-linked transcription relative to autosomal transcription. This strongly suggests the existence of previously unrecognized alternate Set2 substrates. Interestingly, we also found that Ash1- and NSD-mediated methylation of H3K36 was required to maintain high expression from chromosome four. 

Recent studies have also implicated H3K36 methylation in the silencing of transposon activity in somatic cells. By analyzing the transcription of transposable elements and Piwi-interacting RNAs (piRNAs), we identified dimethylation of H3K36 by Set2 as the main methylation mark involved in this process and showed that dual-stranded piRNA clusters are preferentially activated upon disturbing the methylation machinery. These findings extends the long list of processes dependent on functional H3K36 methylation.

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2023. s. 47
Nyckelord
H3K36, histone methylation, dosage compensation, chromosome-specific gene regulation, transposable elements, PIWI/piRNA biosynthesis, Set2, Ash1, NSD, Histone 3.3, post-translational modifications, histone modifications, epigenetics, proximity ligation assay, Drosophila
Nationell ämneskategori
Biokemi och molekylärbiologi Genetik Bioinformatik (beräkningsbiologi)
Forskningsämne
genetik; molekylärbiologi; biofarmaci
Identifikatorer
urn:nbn:se:umu:diva-201602 (URN)978-91-7855-952-7 (ISBN)978-91-7855-953-4 (ISBN)
Disputation
2023-01-27, Astrid Fagraeus-salen (A103), byggnad 6A, Umeå, 09:00 (Engelska)
Opponent
Handledare
Tillgänglig från: 2023-01-05 Skapad: 2022-12-19 Senast uppdaterad: 2023-01-02Bibliografiskt granskad

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