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ABSTRACTThe study of nuclear matrix (NuMat) over the last 40 years has been limited to either isolated nuclei from tissues or cells grown in culture. Here, we provide a protocol for NuMat preparation in intact Drosophila melanogasterembryos and its use in dissecting the components of nuclear architecture. The protocol does not require isolation of nuclei and therefore maintains the three-dimensional milieu of an intact embryo, which is biologically more relevant compared to cells in culture. One of the advantages of this protocol is that only a small number of embryos are required. The protocol has been extended to larval tissues like salivary glands with little modification. Taken together, it becomes possible to carry out such studies in parallel to genetic experiments using mutant/transgenic flies. This protocol, therefore, opens the powerful field of fly genetics to cell biology in the study of nuclear architecture.Summary: Nuclear Matrix is a biochemically defined entity and a basic component of the nuclear architecture. Here we present a protocol to isolate and visualize Nuclear Matrix in situin the Drosophila melanogasterand its potential applications.

ABSTRACTBoundary Element-Associated Factor 32 (BEAF 32) is a sequence specific DNA binding protein involved in functioning of chromatin domain boundaries in Drosophila. Several studies also show it to be involved in transcriptional regulation of a large number of genes, many of which are annotated to have cell cycle, development and differentiation related function. Since post-translational modifications (PTMs) of proteins add to their functional capacity, we investigated the PTMs on BEAF 32. The protein is known to be phosphorylated and O-GlcNAcylated. We mapped O-GlcNAc site at T91 of BEAF 32 and showed that it is linked to the deposition of active histone (H3K4me3) marks at transcription start site (TSS) of associated genes. Its role as a boundary associated factor, however, does not depend on this modification. Our study shows that by virtue of O-GlcNAcylation, BEAF 32 is linked to epigenetic mechanisms that activate a subset of associated genes.

Chromatin condenses several folds to form mitotic chromosomes during cell division and decondenses post-mitotically to reoccupy their nuclear territory and regain their specific transcriptional profile in a precisely lineage specific manner. This necessitates that the features of nuclear architecture and DNA topology persist through mitosis. We compared the proteome of nuclease and high salt resistant fraction of interphase nucleus known as nuclear matrix (NuMat) and an equivalent biochemical fraction in the mitotic chromosome known as mitotic chromosome scaffold (MiCS). Our study elucidates that as much as 67% of the NuMat proteins are retained in the MiCS indicating that the features of nuclear architecture in interphase nucleus are retained on the mitotic chromosomes. Proteins of the NuMat/MiCS have large dynamic range of MS signal and were detected in sub-femtomolar amounts. Chromatin/RNA binding proteins with hydrolase and helicase activity are highly enriched in NuMat as well as MiCS. Although several transcription factors involved in functioning of interphase nucleus are present exclusively in NuMat, protein components responsible for assembly of membrane-less nuclear bodies are uniquely retained in MiCS. Our study clearly indicates that the features of nuclear architecture, in the structural context of NuMat, are retained in MiCS and possibly play an important role in maintenance of cell lineage specific transcriptional status during cell division and thereby, serve as components of cellular memory.

Nuclear Matrix (NuMat) is a biochemically defined entity that provides us with a snapshot of the features of the nuclear architecture. Here, we present a protocol to isolate and visualize NuMat in situin the intact embryo or tissues of Drosophila melanogasterand its applications. We remove the chromatin to reveal underlying nuclear architectural components in organismal context. This protocol couples the power of Drosophilagenetics with cell biological observation of the nuclear architecture.