Biology subjects

Dileep, V.

Publications and source records attributed to Dileep, V..

7 recordsLinked to original sources

bioRxiv2022

Distinct effects of disease-associated TREM2 R47H/+ and T66M mutations on iPSC-derived microglia.

Genetic findings have highlighted key roles for microglia in the pathology of neurodegenerative conditions such as Alzheimers disease (AD). Distinct mutations in the microglial protein TREM2 (triggering receptor expressed on myeloid cells 2) are associated with different forms of neurodegeneration in humans; R47H/+ mutations increase AD risk, while loss-of-function mutations such as TREM2 T66M result in more severe forms of neurodegeneration. We employed gene editing and stem cell models to gain insight into the effects of these mutations on human iPSC-derived microglia. We found divergent effects of TREM2 R47H/+ and T66M mutations on gene expression, with R47H/+ cells exhibiting a pro-inflammatory gene expression signature. Both the TREM2 R47H/+ and T66M mutations caused similar impairments in microglial movement and the uptake of multiple substrates, while R47H/+ microglia were hyper-responsive to inflammatory stimuli, consistent with their gene expression signature. We developed an in vitro laser-induced injury model in neuron-microglia co-cultures, finding an impaired injury response by TREM2 R47H/+ microglia. Furthermore, in xenotransplantation experiments, mouse brains transplanted with TREM2 R47H/+ microglia exhibited reduced synaptic density. Consistently, we observed upregulation of multiple complement cascade components in TREM2 R47H/+ microglia, suggesting that inappropriate synaptic pruning may underlie the effect. Thus, these findings identify shared and distinct effects of these two TREM2 mutations on microglial gene expression and function. While the TREM2 T66M mutation impairs microglial movement and uptake processes, the TREM2 R47H/+ mutation additionally confers multiple potentially detrimental effects on human microglia, likely to underlie its association with AD.

neuroscience↗

bioRxiv2021

Neurons burdened by DNA double strand breaks incite microglia activation through antiviral-like signaling in neurodegeneration.

DNA double strand breaks (DSBs) are linked to aging, neurodegeneration, and senescence1,2. However, the role played by neurons burdened with DSBs in disease-associated neuroinflammation is not well understood. Here, we isolate neurons harboring DSBs from the CK-p25 mouse model of neurodegeneration through fluorescence-activated nuclei sorting (FANS), and characterize their transcriptomes using single-nucleus, bulk, and spatial sequencing techniques. We find that neurons harboring DSBs enter a late-stage DNA damage response marked by the activation of senescent and antiviral-like immune pathways. We identify the NFkB transcription factor as a master regulator of immune gene expression in DSB-bearing neurons, and find that the expression of cytokines like Cxcl10 and Ccl2 develop in DSB-bearing neurons before glial cell types. Alzheimers Disease pathology is significantly associated with immune activation in excitatory neurons, and direct purification of DSB-bearing neurons from Alzheimers Disease brain tissue further validates immune gene upregulation. Spatial transcriptomics reveal that regions of brain tissue dense with DSB-bearing neurons also harbor signatures of inflammatory microglia, which is ameliorated by NFkB knock down in neurons. Inhibition of NFkB or depletion of Ccl2 and Cxcl10 in DSB-bearing neurons also reduces microglial activation in organotypic brain slice culture. In conclusion, we find that in the context of age-associated neurodegenerative disease, DSBs activate immune pathways in neurons, which in turn adopt a senescence associated secretory phenotype to elicit microglia activation. These findings highlight a novel role for neurons in the mechanism of age-associated neuroinflammation. SummaryIt is unclear how age-associated DNA double strand break (DSB) accumulation in neurons influences the progression of cellular senescence and neurodegenerative disease. Here, we leverage mouse models of neurodegeneration, single-nucleus, bulk, and spatial transcriptomics from Alzheimers disease patients, mouse models, and primary neuron cultures to dissect the immune signaling pathways initiated by DSB-bearing neurons that trigger neuroinflammation.

neuroscience↗

bioRxiv2021

High throughput genome-wide single cell protein:DNA binding site mapping by targeted insertion of promoters (TIP-seq)

Assessing cell to cell, and importantly, chromosome to chromosome, heterogeneity in cellular phenotypes is a central goal of modern cell biology. However, chromatin profiling in single cells has been extremely challenging, and single chromosome profiling has not been achieved. In cases where single cell methods have shown promise, success has been mainly limited to histone proteins and/or require highly specialized equipment or cell type specific protocols and are relatively low throughput. Here, we have combined the advantages of tagmentation, linear amplification and combinatorial indexing to produce a high throughput single cell DNA binding site mapping method that does not require specialized equipment and is capable of multiplexing several samples/ target proteins in one experiment. Targeted Insertion of Promoters (TIP-seq) uses Tn5 fused to protein A (as with CUT&Tag) to insert a T7 RNA polymerase promoter into sites adjacent to an antibody bound to a chromatin protein of interest, followed by linear amplification of flanking DNA with T7 polymerase, cDNA preparation and PCR indexing. Tip-seq provides [~]10-fold higher unique reads and thus higher coverage per single cell compared to state-of-the-art methods. We apply TIP-seq to map histone modifications, RNA PolII and CTCF binding sites in single human and mouse cells. TIP-seq will also be adaptable for other platforms, such as 10X genomics and ICELL8. In summary, TIP-seq provides a high-throughput, low-cost method for single cell protein mapping, that yields substantially higher coverage per cell and signal to noise than existing methods.

genomics↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2018

Identification of cis elements for spatio-temporal control of DNA replication

The temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis-elements regulating spatio-temporal control of replication have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide delay in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These \"early replication control elements\" (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis-regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.\n\nHighlightsO_LIcis-elements (ERCEs) regulate large scale chromosome structure and function\nC_LIO_LIMultiple ERCEs cooperatively control domain-wide replication\nC_LIO_LIERCEs harbor prominent active chromatin features and form CTCF-independent loops\nC_LIO_LIERCEs enable genetic dissection of large-scale chromosome structure-function.\nC_LI

molecular biology↗

bioRxiv2017

Single-cell replication profiling reveals stochastic regulation of the mammalian replication-timing program.

In mammalian cells, distinct replication domains (RDs), corresponding to structural units of chromosomes called topologically-associating domains (TADs), replicate at different times during S-phase1-4. Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments5,6. Although replication origins are selected stochastically, such that each cell is using a different cohort of origins to replicate their genomes7-12, replication-timing is regulated independently and upstream of origin selection13 and evidence suggests that replication timing is conserved in consecutive cell cycles14. Hence, quantifying the extent of cell-to-cell variation in replication timing is central to studies of chromosome structure and function. Here we devise a strategy to measure variation in single-cell replication timing using DNA copy number. We find that borders between replicated and un-replicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus. Nonetheless, measurable variation was evident. Surprisingly, we detected a similar degree of variation in replication timing from cell-to-cell, between homologues within cells, and between all domains genome-wide regardless of their replication timing. These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.

genomics↗

bioRxiv2017

An Integrative Framework For Detecting Structural Variations In Cancer Genomes

Structural variants can contribute to oncogenesis through a variety of mechanisms, yet, despite their importance, the identification of structural variants in cancer genomes remains challenging. Here, we present an integrative framework for comprehensively identifying structural variation in cancer genomes. For the first time, we apply next-generation optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole genome sequencing to systematically detect SVs in a variety of cancer cells.\n\nUsing this approach, we identify and characterize structural variants in up to 29 commonly used normal and cancer cell lines. We find that each method has unique strengths in identifying different classes of structural variants and at different scales, suggesting that integrative approaches are likely the only way to comprehensively identify structural variants in the genome. Studying the impact of the structural variants in cancer cell lines, we identify widespread structural variation events affecting the functions of non-coding sequences in the genome, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel 3D chromatin structural domains.\n\nThese results underscore the importance of comprehensive structural variant identification and indicate that non-coding structural variation may be an underappreciated mutational process in cancer genomes.

genomics↗