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.

In situ Nuclear Matrix preparation in Drosophila melanogaster enabling genetic analysis of the nuclear architecture.

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 situ in the intact embryo or tissues of Drosophila melanogaster and its applications. We remove the chromatin to reveal underlying nuclear architectural components in organismal context. This protocol couples the power of Drosophila genetics with cell biological observation of the nuclear architecture. For complete details on the use and execution of this protocol, please refer to Pathak et al. (2022), Sureka et al. (2018), and Pathak et al. (2013).

In situ nuclear matrix preparation in Drosophila melanogaster embryos/tissues and its use in studying the components of nuclear architecture.

The 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 melanogaster embryos 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 situ in the Drosophila melanogaster and its potential applications.

Genomic organization of the autonomous regulatory domain of eyeless locus in Drosophila melanogaster.

In Drosophila, expression of eyeless (ey) gene is restricted to the developing eyes and central nervous system. However, the flanking genes, myoglianin (myo), and bent (bt) have different temporal and spatial expression patterns as compared to the ey. How distinct regulation of ey is maintained is mostly unknown. Earlier, we have identified a boundary element intervening myo and ey genes (ME boundary) that prevents the crosstalk between the cis-regulatory elements of myo and ey genes. In the present study, we further searched for the cis-elements that define the domain of ey and maintain its expression pattern. We identify another boundary element between ey and bt, the EB boundary. The EB boundary separates the regulatory landscapes of ey and bt genes. The two boundaries, ME and EB, show a long-range interaction as well as interact with the nuclear architecture. This suggests functional autonomy of the ey locus and its insulation from differentially regulated flanking regions. We also identify a new Polycomb Response Element, the ey-PRE, within the ey domain. The expression state of the ey gene, once established during early development is likely to be maintained with the help of ey-PRE. Our study proposes a general regulatory mechanism by which a gene can be maintained in a functionally independent chromatin domain in gene-rich euchromatin.

Sperm methylome alterations following yoga-based lifestyle intervention in patients of primary male infertility: A pilot study.

A majority of the cases of primary male infertility are idiopathic with the underlying molecular mechanisms contributing to the pathophysiology as yet unknown. Effects of the environment can alter the sperm epigenome thereby impacting male reproductive health. Epigenetic mechanisms are crucial to understanding health and disease, and methylome alterations are now known to have far-reaching clinical implications. Here, we report the results from our pilot study, a first of its kind analysis of the effect of the traditional practice of yoga on human sperm quality. We find marked improvement in sperm characteristics in patients of idiopathic male infertility following a supervised 21-day yoga regimen. Furthermore, next-generation sequencing-based methylome analysis reveals alterations in the sperm epigenome of these patients. We find that the practice of yoga is associated with DNA methylation changes at nearly 400 genes, 147 of which were hypermethylated while 229 were hypomethylated. These included promoters of several genes linked to maintenance of fertility and genomic integrity. This novel piece of work draws a direct link between positive lifestyle practices and male reproductive health.

Comparison of Nuclear Matrix and Mitotic Chromosome Scaffold Proteins in Drosophila S2 Cells-Transmission of Hallmarks of Nuclear Organization Through Mitosis.

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.

O-GlcNAcylation of boundary element associated factor (BEAF 32) in Drosophila melanogaster correlates with active histone marks at the promoters of its target genes.

Boundary 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.

Long Noncoding RNAs in Mammalian Development and Diseases.

Following analysis of sequenced genomes and transcriptome of many eukaryotes, it is evident that virtually all protein-coding genes have already been discovered. These advances have highlighted an intriguing paradox whereby the relative amount of protein-coding sequences remain constant but nonprotein-coding sequences increase consistently in parallel to increasing evolutionary complexity. It is established that differences between species map to nonprotein-coding regions of the genome that surprisingly is transcribed extensively. These transcripts regulate epigenetic processes and constitute an important layer of regulatory information essential for organismal development and play a causative role in diseases. The noncoding RNA-directed regulatory circuit controls complex characteristics. Sequence variations in noncoding RNAs influence evolution, quantitative traits, and disease susceptibility. This chapter presents an account on a class of such noncoding transcripts that are longer than 200 nucleotides (long noncoding RNA-lncRNA) in mammalian development and diseases.

Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole Carboxamides Enhances Silk Production.

Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5'-TTAGG-3' sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5'-TTAGG-3' sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.

Genome-wide mapping of matrix attachment regions in Drosophila melanogaster.

BACKGROUND: Eukaryotic genome acquires functionality upon proper packaging within the nucleus. This process is facilitated by the structural framework of Nuclear Matrix, a nucleo-proteinaceous meshwork. Matrix Attachment Regions (MARs) in the genome serve as anchoring sites to this framework. RESULTS: Here we report direct sequencing of the MAR preparation from Drosophila melanogaster embryos and identify >7350 MARs. This amounts to ~2.5% of the fly genome and often coincide with AT rich non-coding regions. We find significant association of MARs with the origins of replication, transcription start sites, paused RNA Polymerase II sites and exons, but not introns, of highly expressed genes. We also identified sequence motifs and repeats that constitute MARs. CONCLUSION: Our data reveal the contact points of genome to the nuclear architecture and provide a link between nuclear functions and genomic packaging.

AAGAG repeat RNA is an essential component of nuclear matrix in Drosophila.

Eukaryotic nucleus is functionally as well as spatially compartmentalized and maintains dynamic organization of sub-nuclear bodies. This organization is supported by a non-chromatin nuclear structure called the nuclear matrix. Although the precise molecular composition and ultra-structure of the nuclear matrix is not known, proteins and RNA molecules are its major components and several nuclear matrix proteins have been identified. However, the nature of its RNA component is unknown. Here we show that in Drosophila melanogaster, transcripts from AAGAG repeats of several hundred nucleotide in length are critical constituents of the nuclear matrix. While both the strands of this repeat are transcribed and are nuclear matrix associated, the polypurine strand is predominantly detected in situ. We also show that AAGAG RNA is essential for viability. Our results reveal the molecular identity of a critical RNA component of the nuclear architecture and point to one of the utilities of the repetitive part of the genome that has accumulated in higher eukaryotes.

Transposable element 'roo' attaches to nuclear matrix of the Drosophila melanogaster.

The genome of eukaryotes is organized into structural units of chromatin loops. This higher order organization is supported by a nuclear skeleton called the nuclear matrix. The genomic DNA associated with the nuclear matrix is called the matrix associated region (MAR). Only a few genome-wide screens have been attempted, although many studies have characterized locusspecific MAR DNA sequences. In this study, a MAR DNA library was prepared from the Drosophila melanogaster Meigen (Diptera: Drosophilidae) genome. One of the sequences identified as a MAR was from a long terminal repeat region of 'roo' retrotransposon (roo MAR). Sequence analysis of roo MAR showed its distribution across the D. melanogaster genome. roo MAR also showed high sequence similarity with a previously identified MAR in Drosophila, namely the 'gypsy' retrotransposon. Analysis of the genes flanking roo MAR insertions in the Drosophila genome showed that genes were co-ordinately expressed. The results from the present study in D. melanogaster suggest this sequence plays an important role in genome organization and function. The findings point to an evolutionary role of retrotransposons in shaping the genomic architecture of eukaryotes.

Complex I and energy thresholds in the brain.

Mitochondrial electron transport chain (ETC) deficiencies are thought to underlie defects in energy metabolism and have been implicated in the neurodegenerative process. In particular, reductions in complex I activities in Parkinson's disease are thought to cause bioenergetic dysfunction with subsequent degeneration of dopaminergic neurons. In terms of bioenergetics and assessing ETC-related problems in the brain, the presence of heterogeneous mitochondria has complicated matters as isolated non-synaptic mitochondria have different energy thresholds and flux control coefficients compared to isolated mitochondria of synaptic origin. The molecular mechanisms that underlie complex I deficiencies in the parkinsonian brain are unknown and are the source of intensive research. This review explores the relationship between complex I activity and energy metabolism in the brain as well as the nature of the complex I defect.

Boundary element-associated factor 32B connects chromatin domains to the nuclear matrix.

Chromatin domain boundary elements demarcate independently regulated domains of eukaryotic genomes. While a few such boundary sequences have been studied in detail, only a small number of proteins that interact with them have been identified. One such protein is the boundary element-associated factor (BEAF), which binds to the scs' boundary element of Drosophila melanogaster. It is not clear, however, how boundary elements function. In this report we show that BEAF is associated with the nuclear matrix and map the domain required for matrix association to the middle region of the protein. This region contains a predicted coiled-coil domain with several potential sites for posttranslational modification. We demonstrate that the DNA sequences that bind to BEAF in vivo are also associated with the nuclear matrix and colocalize with BEAF. These results suggest that boundary elements may function by tethering chromatin to nuclear architectural components and thereby provide a structural basis for compartmentalization of the genome into functionally independent domains.

An age-specific 35-kDa phosphoprotein binds to a repressor element in the ovalbumin gene promoter in the avian species Japanese quail.

The ovalbumin (Ov) gene is expressed in the tubular gland cells of the avian oviduct in a specific manner under the control of developmental, tissue-specific, and hormonal cues. The expression is controlled by an array of positive and negative cis-acting elements present up to 1 kb upstream of its transcription start site. Our findings presented in this communication indicate that a well-characterized repressor element may be involved in active repression of the gene during aging. At least two proteins bind to the 25-bp sequence used in the present study encompassing the COUP adjacent repressor (CAR) element. The binding of one of the trans-acting factor that interacts with the repressor element increases during aging. This is accompanied by a decrease in transcription of the gene. The binding of the factor-to-repressor element decreases when expression of the Ov gene is induced by steroid administration. The factor has an approximate molecular weight of 35 kDa and is a phosphoprotein. It loses its ability to bind to DNA upon dephosphorylation. This makes it a potential target of various kinases/phosphatases that relay the various developmental, tissue-specific, and hormonal cues. The other trans-acting factor is a single-strand specific protein that interacts with the repressor element in an age-independent manner. These two proteins acting in conjunction may be involved in the repression of the Ov gene in old female birds where the lower circulating level of steroid hormones may be acting as an age-related cue.

Subtractive differential display: a modified differential display technique for isolating differentially expressed genes.

Differential display (DD) is a novel PCR-based technique, very commonly used to study differentially expressed genes at the mRNA level. In this paper we report a modified version of this technique that we have used to study the differences between the mRNA population from brain tissue of adult and old rats. We have modified the technique to enhance reproducibility and reduce false positives and redundancy. It is fast and does not require any expensive or uncommon reagent. We choose to call it as subtractive differential display as it is a differential display performed over subtracted mRNA population. We have used this protocol successfully to clone a number of age-related differentially expressed sequences from rat brain that need to be sequenced to establish the gene identity.

DNA methylation induced changes in chromatin conformation of the promoter of the vitellogenin II gene of Japanese quail during aging.

One approach to the understanding of the molecular basis of aging in higher organisms may be to use genes whose timing and rate of expression during the life span run parallel with specific functions that can be monitored. The genes for egg proteins, such as vitellogenin (VTG), which is expressed in the liver, and ovalbumin, lysozyme etc. that are expressed in the oviduct of birds, meet these requirements. Egg laying function is dependent on the production of these proteins, which, in turn, depends on the expression of their genes. In this communication we present the age-related studies on the VTG II gene of the bird, Japanese quail. The gene is expressed only in the liver and its expression is considerably lower in old birds that do not lay eggs. Comparison of the promoter region of the gene carrying the two important cis-acting elements, estrogen responsive element (ERE) and progesterone responsive element (PRE), shows it to be 100% homologous to the corresponding region of the chicken VTG II gene. Methylation of DNA and conformation of chromatin of this region were studied, as they are known to be important for regulation of expression of genes. Our studies show that in the liver of adult female quails which lay eggs, a -CCGG- sequence located in this region is hypomethylated, and the chromatin encompassing this region of the gene is relaxed. In the old, the -CCGG- sequence is hypermethylated and the chromatin is compact. This is correlated with a decrease in the expression of the gene and decrease in egg production. Further, electrophoretic mobility shift assay (EMSA) shows that the levels/affinity of specific trans-acting factors that bind to ERE and PRE present in the region, are not different in adult and old birds. Hence the methylation status of the -CCGG- sequence that is located in-between the ERE and the PRE may be crucial for the conformation of chromatin and availability of these two important cis-acting elements for the binding of the trans-acting factors. This, in turn, may downregulate the expression of the gene in old birds.