Biology subjects

Dhillon, N.

Publications and source records attributed to Dhillon, N..

7 recordsLinked to original sources

bioRxiv2026

Multi-omic long-read sequencing reveals nucleosome control of transcriptional fidelity at the isoform level

Transcription and RNA processing are tightly coupled to chromatin organization, yet how nucleosome positioning is coordinated across the promoter and gene body to shape alternative isoform expression remains poorly understood. This question has been difficult to address because short-read chromatin profiling cannot simultaneously capture promoter and gene-body chromatin states on the same DNA molecule, and short-read RNA sequencing cannot resolve full-length transcripts without requiring computational assembly. To address this, we deleted two conserved chromatin remodelers, ISW1 and CHD1, in Saccharomyces cerevisiae and profiled chromatin fibers, nascent RNA, and full-length mature RNA using Oxford Nanopore long-read sequencing. We developed EpiFLAIR, a computational framework that assigns discrete long-range chromatin states across entire genes and integrates these states with isoform-level transcriptional outputs. Using EpiFLAIR, we show that single-molecule chromatin states together with sequence motifs are strong predictors of alternative TSS isoform expression. Loss of ISW1 and CHD1 produced divergent transcriptional changes by activating transcriptionally silenced promoters while repressing active ones. Single-molecule chromatin profiling suggested that this redistribution of transcriptional activity arose, in part, from an anticorrelation between gene-body and promoter chromatin accessibility along the same chromatin fiber, a finding uniquely enabled by long-read single-molecule resolution. Furthermore, disruption of regular nucleosome spacing impaired RNAPII processivity and co-transcriptional splicing, consequently altering mature RNA levels and splicing isoform composition. Together, our findings demonstrate that nucleosomes maintain transcriptional fidelity during initiation, elongation, and splicing to regulate isoform expression. More broadly, long-read single-molecule chromatin profiling represents a powerful approach for linking chromatin organization to isoform-level gene regulation.

genomics↗

bioRxiv2024

Probing chromatin accessibility with small molecule DNA intercalation and nanopore sequencing

Studies of in vivo chromatin organization have relied on the accessibility of the underlying DNA to nucleases or methyltransferases, which is limited by their requirement for purified nuclei and enzymatic treatment. Here, we introduce a nanopore-based sequencing technique called Small-Molecule Adduct sequencing (SMAdd-seq), where we profile chromatin accessibility by treating nuclei or intact cells with a small molecule, angelicin. Angelicin reacts with thymine bases in linker DNA not bound to core nucleosomes after UV light exposure, thereby labeling accessible DNA regions. By applying SMAdd-seq in Saccharomyces cerevisiae, we demonstrate that angelicin-modified DNA can be detected by its distinct nanopore current signals. To systematically identify angelicin modifications and analyze chromatin structure, we developed a neural network model, NEural network for mapping MOdifications in nanopore long-reads (NEMO). NEMO accurately called expected nucleosome occupancy patterns near transcription start sites at both bulk and single-molecule levels. We observe heterogeneity in chromatin structure and identify clusters of single-molecule reads with varying configurations at specific yeast loci. Furthermore, SMAdd-seq performs equivalently on purified yeast nuclei and intact cells, indicating the promise of this method for in vivo chromatin labeling on long single molecules to measure native chromatin dynamics and heterogeneity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/585815v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@14a0f8dorg.highwire.dtl.DTLVardef@143030eorg.highwire.dtl.DTLVardef@7d8ed4org.highwire.dtl.DTLVardef@12c3b05_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Yeast Heterochromatin Only Stably Silences Weak Regulatory Elements by Altering Burst Duration

The interplay between nucleosomes and transcription factors leads to programs of gene expression. Transcriptional silencing involves the generation of a chromatin state that represses transcription and is faithfully propagated through DNA replication and cell division. Using multiple reporter assays, including directly visualizing transcription in single cells, we investigated a diverse set of UAS enhancers and core promoters for their susceptibility to heterochromatic gene silencing. These results show that heterochromatin only stably silences weak and stress induced regulatory elements but is unable to stably repress housekeeping gene regulatory elements and the partial repression did not result in bistable expression states. Permutation analysis of different UAS enhancers and core promoters indicate that both elements function together to determine the susceptibility of regulatory sequences to repression. Specific histone modifiers and chromatin remodellers function in an enhancer specific manner to aid these elements to resist repression suggesting that Sir proteins likely function in part by reducing nucleosome mobility. We also show that the strong housekeeping regulatory elements can be repressed if silencer bound Sir1 is increased, suggesting that Sir1 is a limiting component in silencing. Together, our data suggest that the heterochromatic locus has been optimized to stably silence the weak mating type gene regulatory elements but not strong housekeeping gene regulatory sequences which could help explain why these genes are often found at the boundaries of silenced domains.

molecular biology↗

bioRxiv2021

A new SARS-CoV-2 lineage that shares mutations with known Variants of Concern is rejected by automated sequence repository quality control

We report a SARS-CoV-2 lineage that shares N501Y, P681H, and other mutations with known variants of concern, such as B.1.1.7. This lineage, which we refer to as B.1.x (COG-UK sometimes references similar samples as B.1.324.1), is present in at least 20 states across the USA and in at least six countries. However, a large deletion causes the sequence to be automatically rejected from repositories, suggesting that the frequency of this new lineage is underestimated using public data. Recent dynamics based on 339 samples obtained in Santa Cruz County, CA, USA suggest that B.1.x may be increasing in frequency at a rate similar to that of B.1.1.7 in Southern California. At present the functional differences between this variant B.1.x and other circulating SARS-CoV-2 variants are unknown, and further studies on secondary attack rates, viral loads, immune evasion and/or disease severity are needed to determine if it poses a public health concern. Nonetheless, given what is known from well-studied circulating variants of concern, it seems unlikely that the lineage could pose larger concerns for human health than many already globally distributed lineages. Our work highlights a need for rapid turnaround time from sequence generation to submission and improved sequence quality control that removes submission bias. We identify promising paths toward this goal.

genomics↗

bioRxiv2020

Quantitative analysis of Histone modifications in gene silencing

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. This transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown and we developed a method to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state. We show that loss of silencing required between 50% and 75% of the unacetylated histones to be replaced with acetylated histone mimics. The precise levels of unacetylated nucleosomes required varied from locus to locus and was influenced by both silencer strength and UAS enhancer/promoter strength. Simple calculations suggest that an approximately 50% reduction in the ability of acetylases to acetylate individual nucleosomes across a large domain may be sufficient to generate a transcriptionally silent region in the nucleus.

molecular biology↗

bioRxiv2019

Combinatorial analysis of Saccharomyces cerevisiae regulatory elements

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin components has led to the definition and delineation of various regulatory elements. Enhancers, promoters, 5 untranslated regions (5UTR) and transcription terminators/3 untranslated regions (3UTR) have all been defined. However, the specific contributions of each of these features as part of a regulatory unit and the functional communications between these regulatory elements remains under explored.\n\nWe built a combinatorial library of 26 different enhancers, core promoters, 5UTRs and transcription terminators/3UTRs. This library was analyzed with respect to gene expression in order to better understand the interactions between different regulatory elements. In the process we developed new methods to estimate the contribution of individual regulatory parts from just a few simple measurements. Our data show that different pairs of regulatory parts follow specific interaction rules affecting overall activity either positively or negatively. We find that while enhancers are the initiators of gene activity, core promoters modulate the levels of enhancer mediated expression. Cluster analysis based on expression show that TATA-box containing core promoters appear to increase enhancer-driven transcription to a greater extent than TATA-less promoters. Principal component analysis highlight outliers and suggest differences in mechanisms of regulation. These results provide a system to characterize regulatory elements and use these elements in the design of synthetic regulatory circuits.

synthetic biology↗

bioRxiv2018

Transfer RNA Genes Affect Chromosome Structure and Function via Local Effects

The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.

molecular biology↗