Genome wide identification of nuclear matrix associated regions and their dynamics in the developing embryos of Drosophila melanogaster

Eukaryotic genome is compartmentalized into structural and functional domains. One of the concepts of higher order organization of chromatin posits that the DNA is organized in constrained loops that behave as functional domains. A predominantly ribo-proteinaceous nucleoskeleton, termed as Nuclear matrix (NuMat) is proposed to provide the structural platform for attachment of these loops. The DNA sequence located at the base of the loops are known as the matrix attachment regions (MARs). Along with a structural role in tethering of loops, MARs are also dynamic elements, known to play a regulatory role in all nuclear processes. In the present study we have looked into the dynamics of molecular components of NuMat and MARs when the isolation process is slightly varied and during embryonic development. We have queried the effect of inclusion/removal the nuclear stabilization step during isolation of NuMat. Further we have looked into the role of RNA in nuclear architecture. Finally, we have tried to elucidate the dynamics of MARs during embryonic development of D. melanogaster. As they are functionally determined, genome-wide map of these sequences would help us to better understand the functionality of a dynamic cell.Our results from proteomic analysis of NuMat indicate that stabilization of nuclei at 37C during NuMat isolation, does not alter the quality of proteins associated, but does increase the quantity significantly. Direct sequencing of MARs shows that many new MARs are generated by stabilization. The most significant of them being the paused RNA polymerase II (Pol II) promoters. RNase A treatment leads to collapse of the NuMat with loss several proteins and also the loss of paused Pol II promoter MARs. Paused Pol II MARs are dependent on RNA transcription for NuMat association. The MARs are dynamic during development. While MARs in promoter regions and exons correlate with transcriptomic profile of the developmental stage, intronic and intergenic MARs do not relate to the expression levels of associated genes. A subset of MARs, termed as Core-MARs remain NuMat bound throughout development. The features of Core-MARs are discussed in detail.Our data reveals the role of MARs in functional compartmentalization of D. melanogaster genome and add to the current understanding of nuclear architecture and 3D organization of a functionally dynamic nucleus.