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Prajapati, B.

Publications and source records attributed to Prajapati, B..

10 recordsLinked to original sources

Retroviral insertions contributed to the divergence of human and chimpanzee brains

Over the past 5-7 million years, humans and chimpanzees have diverged in brain size, structural complexity, and cognitive abilities despite high conservation of protein-coding genes. Notably, the endogenization and proliferation of retroviral infections within host genomes has introduced numerous species-specific regulatory elements that have the potential to influence gene regulation. However, the role of these endogenous retroviruses in hominoid brain evolution remains unclear. A burst of lineage-specific PTERV1 retroviruses recently invaded the chimpanzee genome but are absent in humans. We conducted an epigenomic analysis of PTERV1 insertions in chimpanzee neural organoids and found that they are heavily covered by DNA methylation, representing more than 150 species-specific heterochromatin domains with the capacity to influence gene regulatory networks. We identified one such chimpanzee-specific PTERV1 insertion on chromosome 19 that blocks the expression of the long noncoding RNA LINC00662, via DNA methylation spread to the adjacent genomic region. The expression of LINC00662 was restored in chimpanzee induced pluripotent stem cells when we deleted the PTERV1 insertion using CRISPR editing. We found that LINC00662, a human-specific RNA, is highly expressed in the developing brain and plays an important role in the posttranscriptional control of neuronal maturation, axon outgrowth, and neural organoid development. In summary, our findings describe how endogenous retroviral insertions contributed to the functional divergence of the human and chimpanzee brains. This provides a new mechanism by which retroviral pandemics influenced primate brain speciation.

evolutionary biology↗

Microtubule Inhibition Triggers MYC-Mediated Immunogenic Cell Death in Breast Cancer

Oncogenic MYC promotes cancer cell proliferation, metabolism, and death, while also driving immunosuppression in the tumour microenvironment, complicating immune-based therapies. To counter MYC-driven immune evasion while leveraging MYC-dependent synthetic lethality (MYC-SL), we identified microtubule-targeting agents, including eribulin, as potent inducers of immunogenic cell death in MYChigh triple-negative breast cancer (TNBC). A screen of 528 oncology compounds using damage-associated molecular pattern (DAMP) reporters revealed that microtubule inhibitors induced key DAMPs, including HMGB1 secretion, calreticulin exposure, and double-stranded DNA release, leading to gasdermin-E associated cell death in MYChigh TNBCs. Immune cell co-culture assays showed immune activation, and patient-derived explant cultures confirmed pro-inflammatory cytokine responses. In vivo, cell-free media from eribulin-treated MYChigh murine TNBCs enhanced tumour protection in vaccination models compared to MYC-knockdown controls, linking MYC-dependent DAMP release to immunogenicity. These findings highlight a dual-function therapeutic strategy: agents that selectively induce MYC-dependent immunogenic cell death can provide both targeted cytotoxicity and local immune stimulation, thereby addressing a key limitation of conventional chemotherapeutics, offering a new approach for MYC-driven cancers.

cancer biology↗

Complex I Drives Glutamine-Dependent TCA Cycle to Support Viability of MYChigh Breast Cancer Cells

In many cancers, stably elevated MYC levels drive persistent and concerted activation of cell growth promoting anabolic programs and the cell cycle in ways that are distinct from normal cells. Therefore, synthetic-lethal strategies to target MYC reprograming of these pathways may identify new selective anticancer therapies for the treatment of MYChigh tumors. Here, we identify enhanced mitochondrial respiration as a hallmark of MYC overexpressing cancer cells. Mitochondrial respiration sustains the TCA cycle by regenerating NAD+ through complex I-mediated oxidation of NADH. Metabolic carbon tracing analysis revealed that MYC shifts TCA cycles carbon source from glucose to glutamine. Inhibition of the glutamine-fueled TCA cycle using NAD+-depleting complex I inhibitors resulted in MYC-dependent synthetic lethality in breast cancer cells. In mouse models of MYChigh tumors, persistent inhibition of tumor growth was achieved through combined inhibition of complex I and glutaminolysis. Our results suggest that the high respiration rate observed in MYChigh cells supports glutamine carbon-enriched TCA cycle, rendering MYChigh tumors selectively vulnerable to inhibitors of mitochondrial respiration and glutaminolysis.

cancer biology↗

Quiescence modulates age-related changes in the functional capacity of highly proliferative canine lung mesenchymal stromal cell populations

The functional capacity of highly proliferative cell populations changes with age. Here, we report that the proliferative capacity of canine lung mesenchymal stromal cells (LMSCs) declines with increasing age of the donor. However, other functional changes such as reduced autophagy, reduced migration/wound healing, increased production of reactive oxygen species, and increased senescence are not significantly altered with increasing age. Furthermore, transcriptomic profiling suggests minimal age-related changes. These data suggest that the reduced proliferative capacity of lung LMSCs isolated from aging donors may be associated with reversible cell cycle arrest (quiescence), rather than irreversible cell cycle arrest (senescence). Similar findings have been reported in other systems, including neural and muscle stem cells that are associated with low turnover-rate tissues.

developmental biology↗

BAZ1A, an Imitation Switch (ISWI) protein, interacts and facilitates the recruitment of E2F1 to activate the E2F transcription program

ISWI (Imitation Switch) family of chromatin remodeling complexes mobilize nucleosomes to regulate DNA-template associated functions. Analysis of glioblastoma (GBM) transcriptome datasets revealed that BAZ1A (Bromodomain Adjacent to Zinc Finger Domain 1A) is a highly expressed ISWI family member. RNAi-based depletion or inhibition by a small molecule inhibited the survival/migration of glioma cells and glioma stem-like cells (GSCs), sensitized glioma cells to Temozolomide but did not affect normal astrocytes. BAZ1A-silencing arrested cells in the G1 phase and induced apoptosis. While GO analysis of BAZ1A regulated transcriptome showed enrichment of "cell cycle" related terms, GSEA showed a depletion of the "HALLMARK_E2F_TARGETS" gene set with the highest significance, indicating a nonfunctional E2F transcription program. BAZ1A silencing and overexpression experiments confirmed the requirement of BAZ1A for the E2F transcription program by identifying E2F1 as the primary target. ChIP experiments revealed that BAZ1A and E2F1 bind to the E2F1 promoter. ChIP-ReChIP demonstrated that BAZ1A-bound chromatin fragments are enriched for E2F1 protein on the E2F1 promoter at specific sites. SMARCA1/5, ATPase subunits of the ISWI family, also exhibited binding to the E2F1 promoter. BAZ1A depletion decreased the DNaseI sensitivity of E2F1 binding regions of the E2F1 promoter. Co-immunoprecipitation experiments revealed an interaction between BAZ1A and E2F1 and the presence of an E2F1-BAZ1A-SMARCA1/5 complex. Finally, BAZ1A silencing inhibited glioma tumor growth in an orthotopic xenograft mouse model. Our results demonstrate that BAZ1A containing ISWI complex recruits E2F1 to activate the E2F transcription program, thus promoting G1-S progression and highlighting BAZ1A as a potential therapeutic target.

cancer biology↗

Waves of transcription drive erythroid differentiation and launch the NRF2-activated antioxidant program

Transcriptional reprogramming drives differentiation and coordinates cellular responses. While mRNA expression in distinct cell types has been extensively analyzed, the mechanisms that control RNA synthesis upon lineage specifications remain unclear. Here, we induce erythroid differentiation in human cells, track transcription and its regulation at nucleotide-resolution, and identify molecular mechanisms that orchestrate gene and enhancer activity during erythroid specification. We uncover waves of transcription and reveal that a brief differentiation signal launches sustained and propagating changes in RNA synthesis and mRNA expression over cell divisions. NRF2, a strong trans-activator upon oxidative stress, drives erythroid differentiation without a detectable increase in reactive oxygen species. In erythroid precursors, NRF2 induces architecturally primed, differentiation-linked enhancers, and genes encoding globin and antioxidant proteins. Projecting signal-induced transcription to DNA accessibility and mRNA expression in single human bone marrow cells, reveals ordered activation of myeloid (GABPA) and erythroid (GATA1, TAL1 and HEMGN) factors in lineage-specification, followed by NRF2-triggered antioxidant response in the late erythroid cells. This study establishes molecular mechanisms that prime, execute, and temporally coordinate RNA synthesis during erythroid differentiation. Furthermore, we show that master regulators of differentiation and stress co-orchestrate erythropoiesis and produce the antioxidant machinery before erythroid cells mature to oxygen transporting enucleated erythrocytes.

genomics↗

Actin associates with actively elongating genes and binds directly to the Cdk9 subunit of P-TEFb

Nuclear actin has been demonstrated to be essential for optimal transcription, but the molecular mechanisms and direct binding partner for actin in the RNA polymerase complex have remained unknown. By using purified proteins in a variety of biochemical assays, we demonstrate a direct and specific interaction between monomeric actin and Cdk9, the kinase subunit of the positive transcription elongation factor b (P-TEFb) required for RNA polymerase II pause-release. This interaction efficiently prevents actin polymerization, is not dependent on kinase activity of Cdk9 and is not involved with releasing P-TEFb from its inhibitor 7SK snRNP complex. Supporting the specific role for actin in the elongation phase of transcription, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) reveals that actin interacts with genes only upon their active transcription elongation. This study therefore provides novel insights into the mechanisms by which actin facilitates the transcription process.

cell biology↗

DKK1, a negative regulator of Wnt signaling, is a novel inducer of neuroblastoma differentiation

BackgroundThis study aimed to analyze the role of Dickopff1 [DKK1] as a potential differentiating agent for the neuroblastoma cell line SHSY5Y and neurospheres derived from it. Materials and MethodsSHSY5Y neurospheres were formed from undifferentiated adherent cultures. The cellular properties and gene expression were used to study the effect of DKK1 on SHSY5Y-formed neurospheres. Its effect on SHSY5Y neuronal differentiation was also studied. ResultsSHSY5Y adherent undifferentiated cells were grown as neurospheres. Treatment of neurospheres resulted in their fragmentation. It also resulted in reduced mRNA expression of markers of cancer stem cells, pluripotency, and proliferation [p[≤]0.05]. DKK1 treatment also resulted in reduced mRNA expression of {beta}-catenin and TCF genes. There was significantly higher expression of neuronal differentiation genes in SHSY5Y adherent cells grown in complete DMEM media containing DKK1 as compared to cells grown without DKK1. We also found DKK1 synergized with retinoic acid-induced differentiation of neuroblastoma cells. ConclusionDKK1 was able to convincingly abrogate neurosphere formation and promote neuronal differentiation of SHSY5Y cells, including synergy with retinoic acid. This was accompanied by corresponding changes in mRNA markers of cancer stem cells, pluripotency and proliferation. These results may have implications for neuroblastoma stemness and differentiation.

neuroscience↗

Nuclear actin interactome analysis links actin to KAT14 histone acetyl transferase and mRNA splicing

In addition to its essential functions within the cytoskeleton, actin also localizes to the cell nucleus, where it is linked to many important nuclear processes from gene expression to maintenance of genomic integrity. However, the molecular mechanisms by which actin operates in the nucleus remain poorly understood. Here we have used two complementary mass spectrometry (MS) techniques, AP-MS and BioID-MS, to identify binding partners for nuclear actin. Common high-confidence interactions highlight the role of actin in chromatin remodeling complexes and identify the hATAC histone modifier as a novel actin-containing nuclear complex. Further analysis demonstrates that actin binds directly to the hATAC subunit KAT14, and modulates its histone acetyl transferase activity in vitro and in cells. BioID-MS, which can detect also transient interactions, links actin to several steps of transcription as well as to RNA processing. Alterations in nuclear actin levels disturb alternative exon skipping of the SMN2 minigene, suggesting also a functional role for actin in RNA splicing. This interactome analysis thus identifies both novel direct binding partners and functional roles for nuclear actin, as well as forms a platform for further mechanistic studies on how actin operates during essential nuclear processes.

cell biology↗

Nuclear actin is required for transcription during Drosophila oogenesis

Actin influences gene expression at multiple levels. It regulates the activity of specific transcription factors, such as myocardin-related transcription factor (MRTF), is a component of many chromatin remodelers and linked to transcription by all three RNA polymerases (Pol). However, the molecular mechanisms by which actin participates in the gene-specific vs. general transcription have remained unclear. Here we use chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) in Drosophila ovaries to demonstrate that binding of actin to the Act5C gene is not dependent on the Mrtf transcription cofactor. At the genome-wide level, actin interacts with essentially all transcribed genes and co-occupies most gene promoters together with Pol II. On highly expressed genes, actin and Pol II can be found also on the gene bodies. Manipulation of nuclear transport factors for actin leads to decreased expression of egg shell genes, demonstrating the in vivo relevance of balanced nucleo-cytoplasmic shuttling of actin for transcription.

cell biology↗