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Proximity proteomics using AMPL-MS coupled to analysis for histone modification

ABSTRACT: Eukaryotic chromatin is organised in individual domains, which are defined by their location within the nuclear space and their distinct proteomic compositions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and considered to be highly dynamic. To identify proteins associated with a specific chromatin domain and to better understand how those domains are established and maintained, we established AMPL-MS (Antibody mediated proximity labelling mass spectrometry). As this method is based on antibodies and does not require the expression of fusion proteins, it constitutes a versatile and very sensitive method to characterize chromatin domains containing specific signature proteins or histone modifications. We used AMPL-MS to characterize the chromocenter as well as the chromosome territory containing the hyperactive X-chromosome in Drosophila. Our data revealed the presence of many RNA binding proteins in these domains thereby underscoring the importance of nuclear RNA in the organisation of the genome.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Axel Imhof

LAB HEAD: Prof Axel Imhof

PROVIDER: PXD044296 | Pride | 2024-05-29

REPOSITORIES: Pride

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			Action	DRS
	Rawfiles_description_Proteomexchange_Histone_modification_20180806.xlsx	Xlsx
	Ref8770_RC_01_20230523.raw	Raw
	Ref8770_RC_02_20230523.raw	Raw
	Ref8770_RC_03_20230523.raw	Raw
	Ref8770_RC_04_20230523.raw	Raw

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Project description:Background Segmental duplications (SDs) are not evenly distributed along chromosomes. The reasons for this biased susceptibility to SD insertion are poorly understood. Accumulation of SDs is associated with increased genomic instability, which can lead to structural variants and genomic disorders such as the Williams-Beuren syndrome. Despite these adverse effects, SDs have become fixed in the human genome. Focusing on chromosome 7, which is particularly rich in interstitial SDs, we have investigated the distribution of SDs in the context of evolution and the three dimensional organisation of the chromosome in order to gain insights into the mutual relationship of SDs and chromatin topology. Results Intrachromosomal SDs preferentially accumulate in those segments of chromosome 7 that are homologous to marmoset chromosome 2. Although this formerly compact segment has been re-distributed to three different sites during primate evolution, we can show by means of public data on long distance chromatin interactions that these three intervals, and consequently the paralogous SDs mapping to them, have retained their spatial proximity in the nucleus. Focusing on SD clusters implicated in the aetiology of the Williams-Beuren syndrome locus we demonstrate by cross-species comparison that these SDs have inserted at the borders of a topological domain and that they flank regions with distinct DNA conformation. Conclusions Our study suggests a link of nuclear architecture and the propagation of SDs across chromosome 7, either by promoting regional SD insertion or by contributing to the establishment of higher order chromatin organisation themselves. The latter could compensate for the high risk of structural rearrangements and thus may have contributed to their evolutionary fixation in the human genome. 2 cell lines-5 samples: 2x DNA fragmentation during apoptosis; 3x ChIP (2x H4K8ac, 1x H3) IMR90: 1x DNA fragmentation during apoptosis; 1x H4K8ac ChIP. Both hybridized on a custom designed 4x 180k oligonucleotide microarray

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Proximity proteomics using AMPL-MS coupled to analysis for histone modification

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