Biology subjects

Chandrashekar, H.

Publications and source records attributed to Chandrashekar, H..

5 recordsLinked to original sources

bioRxiv2024

FISHnet: Detecting chromatin domains in single-cell sequential Oligopaints imaging data

Sequential Oligopaints DNA FISH is an imaging technique that measures higher-order genome folding at single-allele resolution via multiplexed, probe-based tracing. Currently there is a paucity of algorithms to identify 3D genome features in sequential Oligopaints data. Here, we present FISHnet, a graph theory method based on optimization of network modularity to detect chromatin domains and boundaries in pairwise distance matrices. FISHnet uncovers cell type-specific domain-like folding patterns on single alleles, thus enabling future studies aiming to elucidate the role for single-cell folding variation on genome function.

molecular biology↗

bioRxiv2024

The chromatin conformation landscape of Alzheimer's disease

We have been investigating epigenetic alterations in the brain during human aging and Alzheimers disease (AD), and have evidence for histone acetylation both protecting the aging epigenome and driving AD. Here we extend our studies to chromatin architecture via looping studies, and with binding studies of key proteins required for looping: CTCF and RAD21. We detected changes in CTCF and RAD21 levels and localization, finding major changes in CTCF in AD compared to fewer changes in healthy aging. In our study of 3D genome conformation changes, we identified stable topological associating domains (TADs) in Old and AD; in contrast, in AD, there is loss of interaction at genomic sites/loops within TADs, likely reflecting the loss of CTCF. We identified genes and potential transcription factor binding at the loops that are lost in AD. in addition, we found enrichment of CTCF peak losses for AD eQTLs, suggesting that architectural dysfunction has a role in Alzheimers. Functional experiments lowering the homologues of several key genes in a Drosophila model of A{beta}42 toxicity exacerbate neurodegeneration. Taken together, these data indicate both functional protections and losses occur in the Alzheimers brain genome compared to normal aging.

neuroscience↗

bioRxiv2024

A multi-looping chromatin signature predicts dysregulated gene expression in neurons with familial Alzheimer's disease mutations

Mammalian genomes fold into tens of thousands of long-range loops, but their functional role and physiologic relevance remain poorly understood. Here, using human post-mitotic neurons with rare familial Alzheimers disease (FAD) mutations, we identify hundreds of reproducibly dysregulated genes and thousands of miswired loops prior to amyloid accumulation and tau phosphorylation. Single loops do not predict expression changes; however, the severity and direction of change in mRNA levels and single-cell burst frequency strongly correlate with the number of FAD-gained or -lost promoter-enhancer loops. Classic architectural proteins CTCF and cohesin do not change occupancy in FAD-mutant neurons. Instead, we unexpectedly find TAATTA motifs amenable to binding by DLX homeodomain transcription factors and changing noncoding RNAPolII signal at FAD-dynamic promoter-enhancer loops. DLX1/5/6 mRNA levels are strongly upregulated in FAD-mutant neurons coincident with a shift in excitatory-to-inhibitory gene expression and miswiring of multi-loops connecting enhancers to neural subtype genes. DLX1 overexpression is sufficient for loop miswiring in wildtype neurons, including lost and gained loops at enhancers with tandem TAATTA arrays and singular TAATTA motifs, respectively. Our data uncover a genome structure-function relationship between multi-loop miswiring and dysregulated excitatory and inhibitory transcriptional programs during lineage commitment of human neurons homozygously-engineered with rare FAD mutations.

molecular biology↗

bioRxiv2023

Cell type-specific loops linked to RNA polymerase II elongation in human neural differentiation

DNA is folded into higher-order structures that shape and are shaped by genome function. The role for long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNAPolII) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitors (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNAPolII initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops, promoter-promoter loops, or unlooped. Genes transitioning from repression to RNAPolII initiation exhibit slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNAPolII-mediated elongation during neural cell fate transitions. HighlightsO_LIThousands of loops are decommissioned and gained upon human iPSC differentiation to NPCs/neurons C_LIO_LIGenes transitioning from initiated-to-elongated exhibit robust mRNA upregulation when connected in cell type-specific promoter-enhancer loops C_LIO_LIGenes transitioning from repressed-to-initiated exhibit slight increases in mRNA levels independent of loop status C_LIO_LIUpon short-term RNAPolII degradation, loops formed by elongated genes are more severely disrupted than those anchoring initiated genes C_LI

genomics↗

bioRxiv2021

A new contact killing toxin permeabilizes cells and belongs to a large protein family

Vibrio cholerae is an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts, V. cholerae uses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequenced V. cholerae isolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate both V. cholerae and Escherichia coli cells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VI Permeabilizing Effector Vibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm of E. coli permeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine that tpiV, the gene located immediately downstream of tpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins. IMPORTANCEBacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness and pathogenicity of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.

microbiology↗