Proximity ligation assays of protein and RNA interactions in the male-specific lethal complex on Drosophila melanogaster polytene chromosomes

Lindehell, Henrik; Kim, Maria; Larsson, Jonas

doi:10.1007/s00412-015-0509-x

Cited by 9 publications

(13 citation statements)

References 63 publications

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“…To probe potential interactions between POF and MSL3 in D. ananassae , we used PLA essentially as described previously ( Lindehell et al 2015 ). The primary antibodies were goat anti-MSL3 and rabbit anti-POF; the corresponding PLA probes were rabbit plus and goat minus (Olink Biosciences).…”

Section: Methodsmentioning

confidence: 99%

The X-linked 1.688 Satellite in Drosophila melanogaster Promotes Specific Targeting by Painting of Fourth

et al. 2018

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Repetitive DNA, represented by transposons and satellite DNA, constitutes a large portion of eukaryotic genomes, being the major component of constitutive heterochromatin. There is a growing body of evidence that it regulates several nuclear functions including chromatin state and the proper functioning of centromeres and telomeres. The 1.688 satellite is one of the most abundant repetitive sequences in Drosophila melanogaster, with the longest array being located in the pericentromeric region of the X-chromosome. Short arrays of 1.688 repeats are widespread within the euchromatic part of the X-chromosome, and these arrays were recently suggested to assist in recognition of the X-chromosome by the dosage compensation male-specific lethal complex. We discovered that a short array of 1.688 satellite repeats is essential for recruitment of the protein POF to a previously described site on the X-chromosome (PoX2) and to various transgenic constructs. On an isolated target, i.e., an autosomic transgene consisting of a gene upstream of 1.688 satellite repeats, POF is recruited to the transgene in both males and females. The sequence of the satellite, as well as its length and position within the recruitment element, are the major determinants of targeting. Moreover, the 1.688 array promotes POF targeting to the roX1-proximal PoX1 site in trans. Finally, binding of POF to the 1.688-related satellite-enriched sequences is conserved in evolution. We hypothesize that the 1.688 satellite functioned in an ancient dosage compensation system involving POF targeting to the X-chromosome.

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Section: Methodsmentioning

confidence: 99%

The X-linked 1.688 Satellite in Drosophila melanogaster Promotes Specific Targeting by Painting of Fourth

et al. 2018

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“…for two reasons: 1) We and others have previously demonstrated a physical association between CLAMP and MLE consistent with direct binding of CLAMP to the MSL complex in males(Quinn et al 2014;Lindehell et al 2015;Albig et al 2019); 2) We hypothesized that CLAMP could regulate the distribution of MLE between the spliceosome and MSL complex to regulate MLE function in sex-specific alternative splicing in males.To determine whether CLAMP modulates MLE distribution on chromatin, we measured MLE distribution at the genomic level using Cut-and-Run(Skene et al 2018;Uyehara and McKay 2019). We performed Cut-and-Run in the presence (MTD-GAL4>UAS-GFPRNAi) and absenceRNAi) of maternal CLAMP at the pre-MZT and post-MZT embryonic stages in males.…”

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confidence: 90%

Sex-specific transcript diversity is regulated by a maternal transcription factor in earlyDrosophilaembryos

Ray

Conard

Urban

et al. 2021

Preprint

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Maternally deposited RNAs and proteins play a crucial role in initiating zygotic transcription during early embryonic development. However, the mechanisms by which maternal factors regulate zygotic transcript diversity early in development remain poorly understood. Furthermore, how early in development sex-specific transcript diversity occurs is not known genome-wide in any organism. Here, we determine that sex-specific transcript diversity occurs much earlier in development than previously thought in Drosophila, concurrent with Zygotic genome activation (ZGA). We use genetic, biochemical, and genomic approaches to demonstrate that the essential maternally-deposited pioneer factor CLAMP (Chromatin linked adapter for MSL proteins) is a key regulator of sex-specific transcript diversity in the early embryo via the following mechanisms: 1) In both sexes, CLAMP directly binds to the gene bodies of female and male sex-specifically spliced genes. 2) In females, CLAMP modulates chromatin accessibility of an alternatively-spliced exon within Sex-lethal, the master regulator of sex determination, to promote protein production. 3) In males, CLAMP regulates Maleless (MLE) distribution, a spliceosome component to prevent aberrant sex-specific splicing. Thus, we demonstrate for the first time how a maternal factor regulates early zygotic transcriptome diversity sex-specifically. We also developed a new tool to measure how splicing changes over time called time2splice.

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“…These components have been recovered in other large-scale pulldowns and represent peripheral binding partners that may participate in dosage compensation without necessarily binding directly to the bait. Indeed proximity ligation experiments indicate that JIL-1 contacts both MSL1 and MSL2 but not MSL3 (13).…”

Section: Resultsmentioning

confidence: 99%

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

Zee

Alekseyenko

McElroy

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Posttranslational modifications (PTMs) are key contributors to chromatin function. The ability to comprehensively link specific histone PTMs with specific chromatin factors would be an important advance in understanding the functions and genomic targeting mechanisms of those factors. We recently introduced a cross-linked affinity technique, BioTAP-XL, to identify chromatin-bound protein interactions that can be difficult to capture with native affinity techniques. However, BioTAP-XL was not strictly compatible with similarly comprehensive analyses of associated histone PTMs. Here we advance BioTAP-XL by demonstrating the ability to quantify histone PTMs linked to specific chromatin factors in parallel with the ability to identify nonhistone binding partners. Furthermore we demonstrate that the initially published quantity of starting material can be scaled down orders of magnitude without loss in proteomic sensitivity. We also integrate hydrophilic interaction chromatography to mitigate detergent carryover and improve liquid chromatography-mass spectrometric performance. In summary, we greatly extend the practicality of BioTAP-XL to enable comprehensive identification of protein complexes and their local chromatin environment.chromatin complexes | affinity pulldown | liquid chromatography-mass spectrometry | BioTAP-XL | histone modifications C hromatin encompasses the subset of proteins and RNAs in complex with DNA to form the chromosomes within eukaryotic nuclei. As the primary protein constituents of chromatin, histones organize genomic DNA into nucleosomes and display an extensive array of posttranslational modifications (PTMs) (1). It is increasingly evident that specific histone PTMs are selectively recognized by specific nonhistone proteins that are themselves regulators of many nuclear events such as epigenetic silencing (2, 3). Consequently, defects in these chromatin components often manifest in a number of human diseases (4).As specific interactions between modified histones and nonhistone proteins help distinguish complexes participating in distinct processes, the identification of protein and modification-dependent interactions provides key insights into chromatin biology. Various chromatography and pulldown methods have been developed to identify binding partners. In a typical native pulldown experiment, nuclei from hypotonically lysed cells are digested with micrococcal nuclease (MNase). Extracted chromatin is subjected to immunoprecipitation to isolate the desired complexes. If using a tag such as FLAG, complexes are then competitively eluted and identified. Several drawbacks to native pulldowns as described here are that MNase leaves behind a substantial insoluble nuclear pellet and degrades associating RNAs, that salt extraction of the digested nucleosomes risks dissociation of the tagged bait from its interacting partners, and that most tags do not have sufficient affinity for their respective antibody to permit highly stringent washes. The result is often an incomplete picture of the chromatin...

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Proximity ligation assays of protein and RNA interactions in the male-specific lethal complex on Drosophila melanogaster polytene chromosomes

Cited by 9 publications

References 63 publications

The X-linked 1.688 Satellite in Drosophila melanogaster Promotes Specific Targeting by Painting of Fourth

The X-linked 1.688 Satellite in Drosophila melanogaster Promotes Specific Targeting by Painting of Fourth

Sex-specific transcript diversity is regulated by a maternal transcription factor in earlyDrosophilaembryos

Streamlined discovery of cross-linked chromatin complexes and associated histone modifications by mass spectrometry

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