Nuclear architecture and the structural basis of mitotic memory.

The nucleus is a complex organelle that hosts the genome and is essential for vital processes like DNA replication, DNA repair, transcription, and splicing. The genome is non-randomly organized in the three-dimensional space of the nucleus. This functional sub-compartmentalization was thought to be organized on the framework of nuclear matrix (NuMat), a non-chromatin scaffold that functions as a substratum for various molecular processes of the nucleus. More recently, nuclear bodies or membrane-less subcompartments of the nucleus are thought to arise due to phase separation of chromatin, RNA, and proteins. The nuclear architecture is an amalgamation of the relative organization of chromatin, epigenetic landscape, the nuclear bodies, and the nucleoskeleton in the three-dimensional space of the nucleus. During mitosis, the nucleus undergoes drastic changes in morphology to the degree that it ceases to exist as such; various nuclear components, including the envelope that defines the nucleus, disintegrate, and the chromatin acquires mitosis-specific epigenetic marks and condenses to form chromosome. Upon mitotic exit, chromosomes are decondensed, re-establish hierarchical genome organization, and regain epigenetic and transcriptional status similar to that of the mother cell. How this mitotic memory is inherited during cell division remains a puzzle. NuMat components that are a part of the mitotic chromosome in the form of mitotic chromosome scaffold (MiCS) could potentially be the seeds that guide the relative re-establishment of the epigenome, chromosome territories, and the nuclear bodies. Here, we synthesize the advances towards understanding cellular memory of nuclear architecture across mitosis and propose a hypothesis that a subset of NuMat proteome essential for nucleation of various nuclear bodies are retained in MiCS to serve as seeds of mitotic memory, thus ensuring the daughter cells re-establish the complex status of nuclear architecture similar to that of the mother cells, thereby maintaining the pre-mitotic transcriptional status.

CRISPR/Cas9 and FLP-FRT mediated regulatory dissection of the BX-C of Drosophila melanogaster.

The homeotic genes or Hox define the anterior-posterior (AP) body axis formation in bilaterians and are often present on the chromosome in an order collinear to their function across the AP axis. However, there are many cases wherein the Hox are not collinear, but their expression pattern is conserved across the AP axis. The expression pattern of Hox is attributed to the cis-regulatory modules (CRMs) consisting of enhancers, initiators, or repressor elements that regulate the genes in a segment-specific manner. In the Drosophila melanogaster Hox complex, the bithorax complex (BX-C) and even the CRMs are organized in an order that is collinear to their function in the thoracic and abdominal segments. In the present study, the regulatorily inert regions were targeted using CRISPR/Cas9 to generate a series of transgenic lines with the insertion of FRT sequences. These FRT lines are repurposed to shuffle the CRMs associated with Abd-B to generate modular deletion, duplication, or inversion of multiple CRMs. The rearrangements yielded entirely novel phenotypes in the fly suggesting the requirement of such complex manipulations to address the significance of higher order arrangement of the CRMs. The functional map and the transgenic flies generated in this study are important resources to decipher the collective ability of multiple regulatory elements in the eukaryotic genome to function as complex modules.

Homeotic Genes: Clustering, Modularity, and Diversity.

Hox genes code for transcription factors and are evolutionarily conserved. They regulate a plethora of downstream targets to define the anterior-posterior (AP) body axis of a developing bilaterian embryo. Early work suggested a possible role of clustering and ordering of Hox to regulate their expression in a spatially restricted manner along the AP axis. However, the recent availability of many genome assemblies for different organisms uncovered several examples that defy this constraint. With recent advancements in genomics, the current review discusses the arrangement of Hox in various organisms. Further, we revisit their discovery and regulation in Drosophila melanogaster. We also review their regulation in different arthropods and vertebrates, with a significant focus on Hox expression in the crustacean Parahyale hawaiensis. It is noteworthy that subtle changes in the levels of Hox gene expression can contribute to the development of novel features in an organism. We, therefore, delve into the distinct regulation of these genes during primary axis formation, segment identity, and extra-embryonic roles such as in the formation of hair follicles or misregulation leading to cancer. Toward the end of each section, we emphasize the possibilities of several experiments involving various organisms, owing to the advancements in the field of genomics and CRISPR-based genome engineering. Overall, we present a holistic view of the functioning of Hox in the animal world.

Drosophila Choline transporter non-canonically regulates pupal eclosion and NMJ integrity through a neuronal subset of mushroom body.

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/ß core (α/ßc) and ϒ neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/ßc and ϒ neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.