Biology subjects

Mishra, R.

Publications and source records attributed to Mishra, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

CRISPR/Cas9 and FLP-FRT mediated multi-modular engineering of the cis-regulatory landscape of the bithorax complex 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 which is collinear to their function across the AP axis. The expression pattern of Hox genes is attributed to the cis-regulatory modules (CRMs) that regulate the genes in a segment-specific manner. In the bithorax complex (BX-C), one of the two Hox complexes in Drosophila melanogaster, even the CRMs consisting of enhancers, initiators, insulators, and Polycomb/trithorax response elements are organized in order that is collinear to their function in the thoracic and abdominal region. Much of these findings are based on the analysis of hundreds of mutations in the BX-C. However, targeted genomic rearrangements comprising of duplications, inversions, etc., that can reveal the basis of collinearity and the number of regulatory modules with respect to body segments have not been reported. In the present study, we generated a series of transgenic lines with the insertion of FRT near the regulatory domain boundaries, to shuffle the CRMs associated with the posterior Hox, Abd-B, of the BX-C. Using these FRT lines, we created several alterations such as deletion, duplication, or inversion of multiple CRMs to comprehend their peculiar genomic arrangement and numbers in the BX-C.

developmental biology↗

bioRxiv2022

Symbolic quantitative cognition in wild zebrafish (Danio rerio)

Zebrafish (Danio rerio) constitute an excellent model system to investigate the neural and genetic basis of quantitative cognition We tested the ability of reward quantity discrimination of wild-type zebrafish in an associative learning task. We used an operant conditioning paradigm in which the number of horizontal lines zebrafish approached in a 2-alternative forced choice task predicted the number of food reward pellets they would receive. Zebrafish did not learn a preference for the 2-line stimulus predictive of receiving 2 food pellets. However, a subset of the fish performed significantly above chance in a discrimination task with the same apparatus, in which the 2-line stimulus was associated with the same reward but the choice of the 1-line stimulus was not rewarded. Zebrafish also showed a population level learning in a simpler spatial association task with the same apparatus. We also explored the explanatory value of alternative spatial learning hypotheses such as a Win-Stay, Lose-Shift (WSLS) strategy at the individual level for fish in navigating these spatially randomised tasks.

animal behavior and cognition↗

bioRxiv2022

Systematic dissection of phosphorylation-dependent YAP1 complex formation elucidates a key role for PTPN14 in Hippo signal integration

Cellular signaling relies on the temporal and spatial control of the formation of transient protein complexes by post-translational modifications, most notably by phosphorylation. While several computational methods have been developed to predict the functional relevance of phosphorylation sites, assessing experimentally the interdependency between protein phosphorylation and protein-protein interactions (PPIs) remains a major challenge. Here, we describe an experimental strategy to establish interdependencies between specific phosphorylation events and complex formation. This strategy is based on three main steps: (i) systematically charting the phosphorylation landscape of a target protein; (ii) assigning distinct proteoforms of the target protein to different protein complexes by electrophoretic separation of native complexes (BNPAGE) and protein/phopho correlation profiling; and (iii) genetically deleting known regulators of the target protein to identify which ones are required for given proteoforms and complexes. We applied this strategy to study phosphorylation- dependent modulation of complexes containing the transcriptional co-regulator YAP1. YAP1 is highly phosphorylated and among the most extensively connected proteins in the human interactome. It functions as the main signal integrator and effector protein of the Hippo pathway which controls organ size and tissue homeostasis. Using our workflow, we could identify several distinct YAP1 proteoforms specifically associated with physically distinct complexes and infer how their formation is affected by known Hippo pathway members. Importantly, our findings suggest that the tyrosine phosphatase PTPN14 controls the co-transcriptional activity of YAP1 by regulating its interaction with the LATS1/2 kinases. In summary, we present a powerful strategy to establish interdependencies between specific phosphorylation events and complex formation, thus contributing to the "functionalization" of phosphorylation events and by this means provide new insights into Hippo signaling.

systems biology↗

bioRxiv2022

Endogenous pancreatic microRNAs differentially target the Delta, Omicron, and Wuhan SARS-CoV-2 genomes to upregulate the Diabetes-associated genes

The SARS-CoV-2 viral genome is mutating and evolving into new variations, including the recently discovered Delta and Omicron. In this study, we used and evaluated our notion of differential targeting of SARS-CoV-2 variant genomes by microRNAs (miRNAs) of the infected human pancreas. We found that even with UTR mutations, the Delta, Omicron, and original Wuhan variations genomes would be differentially targeted by the host pancreas cells native miRNAs in the same way. The miRNAs show a difference in Minimal Free Energy (MFE) with the different viral variants genomes; however, they would still be responsible for the upregulation of the diabetes-associated genes.

genomics↗

bioRxiv2021

Deciphering the role of the Pancreatic Secretome in Covid-19 associated Multi-Organ Dysfunctions

Emerging evidence indicates an intricate relationship between the SARS-CoV-2 infection and Multi-Organ Dysfunctions (MODs). Here, we have investigated the role of the Secretome of the SARS-CoV-2 infected pancreas and mechanistically linked it with the multi-organ dysfunction using the scRNA-seq analysis. We found that acinar-specific PRSS2, REG3A, REG1A, SPINK1, and ductal-specific SPP1, MMP7 genes are upregulated in alpha, beta, delta, and mesenchyme cells. Using extensive documented experimental evidence, we validated the association of upregulated pancreatic Secretome with coagulation cascade, complement activation, renin angiotensinogen system dysregulation, endothelial cell injury and thrombosis, immune system dysregulation, and fibrosis. Our finding suggests the influence of upregulated Secretome on multi-organ systems such as Nervous, Cardiovascular, Immune, Digestive, and Urogenital systems. In addition, we report that the secretory proteins IL1B, AGT, ALB, SPP1, CRP, SERPINA1, C3, TFRC, TNFSF10, and MIF are associated with diverse diseases. Thus, suggest the role of the pancreatic Secretome in SARS-CoV-2 associated MODs.

systems biology↗

bioRxiv2021

Linking Diabetes mellitus to SARS-CoV-2 infection through differential targeting of the microRNAs in the Pancreas tissue

Coronavirus Disease 2019 (COVID-19) severity and Diabetes mellitus affect each other bidirectionally. The plus-sense single-stranded RNA (+ssRNA) genome of the SARS-CoV-2 virus can be targeted and suppressed by the host cells microRNAs (miRNAs). Using the differential gene expression analysis between the mock-infected and the SARS-CoV-2-infected pancreatic tissue, we report five Diabetes-associated genes that are upregulated due to SARS-CoV-2 infection in the hESC pancreas tissues. Ten miRNAs regulating these five genes can potentially target the SARS-CoV-2 genome. We hypothesize that the SARS-CoV-2 genome copies in the infected human pancreas cell compete with the host cells native genes in being regulated by the native miRNAs. It leads to the reduced miRNA-regulation and, thus, the upregulation of the Diabetes-associated native genes. Thus, the resultant new-onset or elevated Diabetic symptoms may worsen the condition of COVID-19 patients.

genomics↗

bioRxiv2020

Effect of earthworms in removal and fate of Antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG) during clinical laboratory wastewater treatment by vermifiltration

The wastewater treatment plants effluent has been implicated in the spread of antibiotic resistant bacteria (ARB) as these environment contains multiple selective pressures that may increase mutation rates, pathogen survivability, and induce gene transfer between bacteria. In lieu of this, the present study explored the dynamics of earthworm-microorganisms interactions on the treatment efficacy of clinical laboratory wastewater treatment by vermifiltration and the effect of earthworms in the fate of removal of pathogens and ARB. The results of the study showed that earthworms and VF associated microbial community had a significant effect on BOD and COD reduction (78-85%), pathogen removal (>99.9 %) and caused a significant shift in the prevalence pattern of ARB. Additionally, molecular profiling of ESBL (blaSHV, blaTEM and blaCTX-M), MRSA (mec-A) and Colistin (mcr-1) gene confirmed the probable mechanisms behind the resistance pattern. The microbial community diversity assists in the formation of biofilm, which helps in the removal of pathogens and results in a paradigm shift in the resistance profile of ARB and ARG, specifically most effective against drugs, targeting cell wall and protein synthesis inhibition like Ampicillin, Ticarcillin, Gentamicin and Chloramphenicol. These findings prove vermifiltration technology as a sustainable and natural treatment technology for clinical laboratory wastewater.

ecology↗

bioRxiv2020

Design, Performance Evaluation and Investigation of the Dynamic Mechanisms of Earthworm-Microorganisms interactions for wastewater treatment through Vermifiltration technology

The present study points to the relevance of the earthworms-microorganisms symbiotic and synergistic interactions that drive the wastewater treatment by identifying the most essential mechanisms underlying the removal of contaminants during vermifiltration technology. Previous studies have showed the presence of earthworms improves treatment performance of vermifilter (VF), but earthworm microbial community dynamics, their structure and functional characteristics in VF were not fully investigated. To investigate the effects of earthworms on the inherent microbial community of the VF, the present study envisages the dynamics of the complex symbiosis of earthworms & microorganisms associated to the treatment mechanisms. In this study, the design, operations and performance evaluation and influent, effluent and filter media layer were investigated for microbial diversity inside the earthworm population, along with the antimicrobial activity, enzymatic activity, and protein profiling assays. The results showed that earthworm gut microbial communities were dominated by Gammaproteobacteria, and the percentages arrived to 59-60% of the microbial species detected, while filter media layer showed presence of Firmicutes and Actinobacteria. The protein profiling of the microbiota associated with the VF showed that earthworms feeding and earthworm-microorganism interaction were responsible for enhanced treatment performance. The finding provides an insight into the complex earthworm microbial dynamics and mechanisms for wastewater treatment in VF. Furthermore, earthworm predation strongly regulated microbial biomass while improving microbial activity, and is deciphered as the possible mechanisms behind the vermifiltration technology.

ecology↗

bioRxiv2020

The formin Fmn2 is required for the development of an excitatory interneuron module in the zebrafish acoustic startle circuit

The formin family member, Fmn2, is a neuronally enriched cytoskeletal remodelling protein conserved across vertebrates. Recent studies have implicated Fmn2 in neurodevelopmental disorders, including sensory processing dysfunction and intellectual disability in humans. Cellular characterization of Fmn2 in primary neuronal cultures has identified its function in the regulation of cell-substrate adhesion and consequently growth cone translocation. However, the role of Fmn2 in the development of neural circuits in vivo, and its impact on associated behaviours have not been tested. Using automated analysis of behaviour and systematic investigation of the associated circuitry, we uncover the role of Fmn2 in zebrafish neural circuit development. As reported in other vertebrates, the zebrafish ortholog of Fmn2 is also enriched in the developing zebrafish nervous system. We find that Fmn2 is required for the development of an excitatory interneuron pathway, the spiral fiber neuron, which is an essential circuit component in the regulation of the Mauthner cell-mediated acoustic startle response. Consistent with the loss of the spiral fiber neurons tracts, high-speed video recording revealed a reduction in the short latency escape events while responsiveness to the stimuli was unaffected. Taken together, this study provides evidence for a circuit-specific requirement of Fmn2 in eliciting an essential behaviour in zebrafish. Our findings underscore the importance of Fmn2 in neural development across vertebrate lineages and highlight zebrafish models in understanding neurodevelopmental disorders. SIGNIFICANCE STATEMENTFmn2 is a neuronally enriched cytoskeletal remodelling protein linked to neurodevelopment and cognitive disorders in humans. Recent reports have characterized its function in growth cone motility and chemotaxis in cultured primary neurons. However, the role of Fmn2 in the development of neural circuits in vivo and its implications in associated behaviours remain unexplored. This study shows that Fmn2 is required for the development of neuronal processes in the acoustic startle circuit to ensure robust escape responses to aversive stimuli in zebrafish. Our study underscores the crucial role of the non-diaphanous formin, Fmn2, in establishing neuronal connectivity and related behaviour in zebrafish.

neuroscience↗

bioRxiv2020

A forward genetic approach to mapping a P-element second site mutation identifies DCP2 as a novel tumor suppressor in Drosophila melanogaster

The use of transposons to create mutants has been the cornerstone of Drosophila genetics in the past few decades. Transpositions often create second-site mutations, devoid of transposon insertion and thereby affect subsequent phenotype analyses. In a P-element mutagenesis screen, a second site mutant was discovered on chromosome 3 wherein the homozygous mutant individuals show the classic hallmarks of mutations in tumor suppressor genes including brain tumour and lethality, hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L), and thus the mutation was renamed as DCP2l(3)tb. Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5UTR coding region of DCP2, alongwith expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by Introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology this is the first instance that the mRNA decapping protein is being implicated in Drosophila tumourigenesis. Our findings therefore imply a plausible role for mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

genetics↗

bioRxiv2020

Imperative role of particulate matter in innate immunity during RNA virus infection

Sensing of pathogens by specialized receptors is the hallmark of the innate immune response. Innate immune response also mounts a defense response against various allergens and pollutants including particulate matter present in the atmosphere. Air pollution has been included as the top threat to global health declared by WHO which aims to cover more than three billion people against health emergencies from 2019-2023. Particulate matter (PM), one of the major components of air pollution, is a significant risk factor for many human diseases and its adverse effects include morbidity and premature deaths throughout the world. Several clinical and epidemiological studies have identified a key link between the PM composition and the prevalence of respiratory and inflammatory disorders. However, the underlying molecular mechanism is not well understood. Here, we investigated the influence of air pollutant, PM10 during RNA virus infections using highly pathogenic avian influenza (HPAI). We thus characterized the transcriptomic profile of lung epithelial cell line, A549 treated with PM10 prior to infection with (HPAI) H5N1 influenza virus, which is known to severely affect the lung and cause respiratory damage. We found that PM10 regulates virus infectivity and enhances overall pathogenic burden in the lung cells. Additionally, the transcriptomic profile highlights the connection of host factors related to various metabolic pathways and immune responses which were dysregulated during virus infection. Overall our findings suggest a strong link between the prevalence of respiratory illness and the air quality.

immunology↗

bioRxiv2020

Integrated role of microRNA-30e-5p through targeting negative regulators of innate immune pathways during HBV infection and SLE

Precise regulation of innate immunity is crucial for the development of appropriate host immunity against microbial infections and the maintenance of immune homeostasis. The microRNAs are small non-coding RNA, post-transcriptional regulator of multiple genes and act as a rheostat for protein expression. Here, we identified microRNA(miR)-30e-5p (miR-30e) induced by the hepatitis B virus (HBV) and other viruses that act as a master regulator for innate immune responses. Moreover, pegylated type I interferons treatment to HBV patients for viral reduction also reduces the miRNA. Additionally, we have also shown the immuno-pathological effects of miR-30e in systemic lupus erythematous (SLE) patients and SLE mouse model. Mechanistically, the miR-30e targets multiple negative regulators namely TRIM38, TANK, ATG5, ATG12, BECN1, SOCS1, SOCS3 of innate immune signaling pathways and enhances innate immune responses. Furthermore, sequestering of endogenous miR-30e in PBMCs of SLE patients and SLE mouse model respectively by the introduction of antagomir and locked nucleic acid based inhibitor significantly reduces type I interferon and pro-inflammatory cytokines. Collectively, our study demonstrates the novel role of miR-30e in innate immunity and its prognostic and therapeutic potential in infectious and autoimmune diseases.

immunology↗

bioRxiv2020

Spatial proximity of homologous centromere DNA sequences facilitated karyotype diversity and seeding of evolutionary new centromeres

Centromeres of Candida albicans form on unique and different DNA sequences but a closely related species, Candida tropicalis, possesses homogenized inverted repeat (HIR)-associated centromeres. To investigate the mechanism of centromere-type transition, we improved the fragmented genome assembly and constructed a chromosome-level genome assembly of C. tropicalis by employing PacBio sequencing, chromosome conformation capture sequencing (3C-seq), chromoblot, and genetic analysis of engineered aneuploid strains. Further, we analyzed the 3D genome organization using 3C-seq data, which revealed spatial proximity among the centromeres as well as telomeres of seven chromosomes in C. tropicalis. Intriguingly, we observed evidence of inter-centromeric translocations in the common ancestor of C. albicans and C. tropicalis. Identification of putative centromeres in closely related Candida sojae, Candida viswanathii and Candida parapsilosis indicates loss of ancestral HIR-associated centromeres and establishment of evolutionary new centromeres (ENCs) in C. albicans. We propose that spatial proximity of the homologous centromere DNA sequences facilitated karyotype rearrangements and centromere type transitions in human pathogenic yeasts of the CUG-Ser1 clade.

genetics↗

bioRxiv2019

Genetic and physical interaction of Drosophila Ino80 with Polycomb Responsive Element

The chromatin remodeling protein, dIno80 (Drosophila Ino80) regulates homeotic genes. We show that Ino80, along with Trx and ETP (Enhancer of Trithorax and Polycomb) proteins, interacts with two Polycomb/Trithorax Responsive Elements (PRE/TRE), iab-7 and bxd PRE in flies and the larval imaginal discs. In S2 cells, dIno80 localizes to the endogenous iab-7 and bxd-PREs. The localization of Ino80 and Pleiohomeotic (Pho) at the PRE is sensitive to the cellular abundance of each other; when levels of Ino80 are limiting, there is increased Pho enrichment, and Pho knock-down leads to increased enrichment of Ino80. We demonstrate that over-expression of dIno80 rescues the pupal lethality in pleiohomeotic (pho) deficient flies, which suggests that dIno80 has a role in cellular memory. The apparent competition between Pho and Ino80 for binding at the PRE indicates that Ino80 may act as a potential recruiter of the regulatory complex in addition to being a chromatin remodeler.\n\nAuthor SummaryThe null mutants of Pho and dIno80 show lethality at different stages of development in the fly, implying that they may function independent of each other. The observation that Pho-lethality can be rescued by overexpression of dIno80 with significant penetrance and that Ino80 has its own DNA binding domain, led us to predict that Ino80 may have Pho-independent functions, perhaps through non-canonical complexes. In the current study, we show that dIno80 interacts with bxd and iab-7 PRE in cooperation with Polycomb and Trithorax proteins and regulate the homeotic genes. The effect of knock-down or mutation of dIno80 results in altered phenotype in adult flies and rescue of Lac-Z expression in imaginal discs, in parallel with similar effect of Pho mutation or knock-down. We provide evidence of direct interaction of dIno80 with iab7- and bxd-PRE using chromatin immunoprecipitation. The dIno80 localization in and around the PRE sequence was enhanced in the absence of Pho, indicating competition between Pho and dIno80 for binding at the PRE.

developmental biology↗

bioRxiv2019

A comprehensive role of Toll-like receptors (TLRs) in diabetic wound healing

Diabetes is marked by delayed wound healing response and prolonged phase of inflammation. Toll-like receptors (TLRs) are the key pathogen recognition receptors known to regulate inflammation in wound healing. Therefore, we aim to investigate the role of TLRs in delayed healing of diabetic wounds. TLR (1-9) expression studies were conducted using J774 macrophage cell line. Further, primary macrophages were isolated from wound tissues of db/db mice, subjected to RT-PCR and western blot analysis. The RT-PCR expression of TLRs revealed that expression of TLR2, TLR4, TLR5, TLR6, TLR7 and TLR8 were significantly upregulated in macrophage cells, cultured in high glucose medium. Expression of TLR 1-9 were significantly upregulated in wound tissues of db/db mice. However, TLR1, TLR2 and TLR4, TLR6, TLR7 were only found upregulated in the primary macrophages. Only TLR2 and TLR4 exhibited significantly increased protein expression. Further, TLR2 and TLR4 inhibition by OxPAPC (1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine) impregnated hydrogels, resulted in a significantly increased rate of wound healing in db/db mice via decreasing the significant levels of TNF- and IL-1{beta}. These results show that increased TLR 2 and TLR 4 expression underlies delayed wound healing in diabetes and a hydrogel impregnated with OxPAPC may be a promising therapeutic strategy for the management of diabetic wounds. Our study goes closer in understanding the molecular mechanism and provides a novel treatment method in diabetic wounds.

immunology↗

bioRxiv2019

Characterisation of l(3)tb as a novel tumour suppressor allele of DCP2 in Drosophila melanogaster

Mutants provide an excellent platform for the discovery and characterization of gene functions. The present communication is a pioneering treatise on a hitherto undescribed function of the gene coding for the mRNA decapping protein 2 (DCP2) in Drosophila melanogaster. DCP2, the gene coding for the mRNA decapping enzyme, has been studied in various model organisms in the light of maintenance of transcript abundance and stability but has never been implicated in tumourigenesis. Herein, we describe the mapping and characterization of a novel tumour suppressor allele of DCP2 (CG6169), which we named as lethal(3)tumorous brain [l(3)tb]. The homozygous mutant individuals show prolonged larval life, develop larval brain tumors and are lethal in the larval/pupal stages. The tumour is characterized by the presence of increased number of superficial neuroblasts, abnormal chromosomal condensation and causes overgrowth in the wing and the eye-antennal discs of the homozygous mutant larvae, all of which are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. Our findings therefore ascribe a novel role of tumor suppression to DCP2 besides its cognate function of mRNA decapping and thereby identify it as a potential candidate for future research on tumorigenesis.

genetics↗

bioRxiv2019

Interactome of vertebrate GAF/ThPOK reveals its diverse functions in gene regulation and DNA repair

Evolutionary conservation and lineage-specific diversification of existing proteins forms the basis for evolving complexity of protein-protein interaction networks and presumably, thereby that of organism. GAGA associated factor (GAF) belongs to BTB/POZ and zinc finger family of transcription factors and it is conserved from flies to humans. Emerging evidence shows indispensable roles of vertebrate GAF (vGAF a.k.a. ThPOK) in functionally divergent developmental processes like hematopoiesis, adipogenesis and lactation. vGAF is a sequence-specific DNA binding transcription factor with multiple context-dependent roles in gene activation/repression, enhancer-blocking and more recently, it has shown to be the part of ribonucleoprotein complexes as well. In order to understand the molecular basis of these diverse functions, we analyzed the protein-protein interactome of vGAF. This analysis shows vGAFs association with chromatin remodelers, RNA metabolic machinery, transcriptional activators/repressors, and components of DNA repair machinery, thereby provides a plausible explanation for the diverse molecular functions of vGAF. Our findings discern novel role of vGAF in several molecular processes like DNA repair and RNA metabolism. We further tested the biological significance of our protein-protein interaction data and show a novel function of vGAF in DNA repair and cell-survival after UV induced DNA damage. Consistent with these results, analysis of high-throughput RNA-seq data shows the downregulation of vGAF in samples of skin cutaneous melanoma for which the primary cause is UV induced DNA damage. These findings suggest vGAF as an early diagnostic biomarker for skin cutaneous melanoma. Taken together, our study reveals a molecular basis for the diverse functions of vGAF. We uncover its role in DNA repair and provide an explanation for key roles of such evolutionary conserved factors in processes like development and disease. O_TBL View this table: org.highwire.dtl.DTLVardef@900b68org.highwire.dtl.DTLVardef@dcdf49org.highwire.dtl.DTLVardef@c754f1org.highwire.dtl.DTLVardef@999a9dorg.highwire.dtl.DTLVardef@75c91c_HPS_FORMAT_FIGEXP M_TBL C_TBL

molecular biology↗

bioRxiv2019

Interplay of pericentromeric genome organization and chromatin landscape regulates the expression of Drosophila melanogaster heterochromatic genes

Transcription of heterochromatic genes residing within the constitutive heterochromatin is paradoxical to the tenets of the epigenetic code. Drosophila melanogaster heterochromatic genes serve as an excellent model system to understand the mechanisms of their transcriptional regulation. Recent developments in chromatin conformation techniques have revealed that genome organization regulates the transcriptional outputs. Thus, using 5C-seq in S2 cells, we present a detailed characterization of the hierarchical genome organization of Drosophila pericentromeric heterochromatin and its contribution to heterochromatic gene expression. We show that pericentromeric TAD borders are enriched in nuclear Matrix attachment regions while the intra-TAD interactions are mediated by various insulator binding proteins. Heterochromatic genes of similar expression levels cluster into Het TADs which indicates their transcriptional co-regulation. To elucidate how heterochromatic factors, influence the expression of heterochromatic genes, we performed 5C-seq in the HP1a or Su(var)3-9 depleted cells. HP1a or Su(var)3-9 RNAi results in perturbation of global pericentromeric TAD organization but the expression of the heterochromatic genes is minimally affected. Subset of active heterochromatic genes have been shown to have combination of HP1a/H3K9me3 with H3K36me3 at their exons. Interestingly, the knock-down of dMES-4 (H3K36 methyltransferase), downregulates expression of the heterochromatic genes. This indicates that the local chromatin interactions and the combination of heterochromatic factors (HP1a or H3K9me3) along with the H3K36me3 is crucial to drive the expression of heterochromatic genes. Furthermore, dADD1, present near the TSS of the active heterochromatic genes, can bind to both H3K9me3 or HP1a and facilitate the heterochromatic gene expression by regulating the H3K36me3 levels. Therefore, our findings provide mechanistic insights into the interplay of genome organization and chromatin factors at the pericentromeric heterochromatin that regulates Drosophila melanogaster heterochromatic gene expression.

genomics↗