Biology subjects

Mallory, B. J.

Publications and source records attributed to Mallory, B. J..

8 recordsLinked to original sources

bioRxiv2026

Enhancer Tuning by Sequence-Specific Repressors

Enhancers integrate combinatorial inputs from sequence-specific transcription factors (TFs) and their activity must be calibrated to achieve precise spatiotemporal control of transcript dosage. Here we demonstrate that the sequence-specific repressors SNAI1 and SNAI2 (i.e. SNAIL and SLUG) quantitatively tune enhancer activity. In human neural crest cells, SNAI1/2 occupy a subset of active enhancers, where their depletion increases H3K27ac, chromatin accessibility, and enhancer regulatory potential. Changes in SNAI1/2 binding motifs contribute to enhancer divergence between human and chimpanzee, suggesting an evolutionary role for the repressor-mediated tuning. Single-molecule chromatin profiling using Deaminase-Assisted Fiber-seq (DAF-seq) reveals that individual enhancers toggle between an ensemble of open and nucleosome-dense chromatin states. While transcriptional activators increase the fraction of the open states, SNAI1/2 shift the equilibrium toward nucleosome-occupied states. This impedes binding of activator TFs, without fully repressing the enhancer. We propose that SNAI1/2 function as a molecular dimmer switch, modulating nucleosome dynamics to calibrate enhancer output.

molecular biology↗

bioRxiv2026

Single-molecule variation in telomeric sequence and structure across humans

The repetitive architectures of telomeric and subtelomeric regions have obscured studies of their genetic variation and chromatin organization across the human population. Here, we integrate near-complete diploid genome assemblies from 212 individuals with matched long-read sequencing data to construct an atlas of 316,146 telomere-spanning molecules across 12,080 chromosome-end-resolved telomere arrays. This atlas reveals that nearly every chromosome end harbors a structured and unique pattern of telomere variant repeats (TVR), or TVR code, with subtelomere-proximal TVR codes being heritable, somatically stable, and influenced by subtelomeric TAR1 regulatory elements. Despite ongoing cycles of telomere shortening and elongation in the germline, proximal TVR codes are maintained across the human population. These TVR codes expose rare telomerase-independent events that lengthen telomeres in the germline, including interchromosomal telomere exchange and recurrent internal duplications within telomere arrays. Furthermore, single-molecule chromatin fiber sequencing across 26,972 molecules spanning the telomere-subtelomere boundary confirms that TVR-rich regions adopt telomeric chromatin but introduce discrete discontinuities into otherwise compact telomeric chromatin fibers. Together, our results link chromosome-end sequence variation to telomere cap formation and telomerase-independent telomere extension mechanisms in the human germline.

genomics↗

bioRxiv2025

Principles and functional consequences of plasmid chromatinization in mammalian cells

Plasmids have fundamentally transformed how we resolve regulatory grammar across the tree of life. However, although chromatin plays an integral role in regulating the function of regulatory elements along the nuclear genome, our understanding of how, or whether, similar chromatin structures form on plasmids transfected into mammalian cells remains limited. We demonstrate that plasmid single-molecule chromatin fiber sequencing (plasmid Fiber-seq) can accurately map chromatin architectures along individual, full-length transfected plasmid molecules at near single-nucleotide resolution. Application of plasmid Fiber-seq to diverse plasmids and cell lines demonstrates that plasmids are chromatinized in a sequence-dependent organized manner and adopt a heterogeneous and incomplete chromatin architecture relative to nuclear-encoded chromatin fibers. We show that the focal occupancy of nucleosomes and transcription factors along transfected plasmids is central to their transcriptional activity within mammalian cells, and demonstrate that plasmids indeed are capable of recapitulating nuclear genome-encoded chromatin architectures, although not always. Finally, we demonstrate that combining plasmid Fiber-seq with high-throughput reporter assays can establish the molecular mechanisms underlying pathogenic non-coding variants, including disentangling the effects of transcriptional activators and repressors with near-single-nucleotide resolution. Overall, our findings reveal the principles by which plasmid-based assays can be used for accurate fine-scale mapping of chromatin-dependent regulatory grammar.

genomics↗

bioRxiv2024

Deaminase-assisted single-molecule and single-cell chromatin fiber sequencing

Gene regulation is mediated by the co-occupancy of numerous proteins along individual chromatin fibers. However, our tools for deeply profiling how proteins co-occupy individual fibers, especially at the single-cell level, remain limited. We present Deaminase-Assisted single-molecule chromatin Fiber sequencing (DAF-seq), which leverages a non-specific double-stranded DNA deaminase toxin A (SsDddA) to efficiently stencil protein occupancy along DNA molecules via selective deamination of accessible cytidines, which are preserved via C-to-T transitions upon DNA amplification. We demonstrate that DAF-seq enables [~]200,000-fold enrichment of target loci for single-molecule footprinting at near single-nucleotide resolution, enabling the precise delineation of the regulatory logic guiding neighboring proteins to cooperatively occupy chromatin fibers. Furthermore, DAF-seq enables the synchronous identification of single-molecule chromatin and genetic architectures - resolving the functional impact of rare somatic variants, as well as transitional chromatin states guiding haplotype-selective promoter actuation. Finally, we demonstrate that single-cell DAF-seq enables the accurate reconstruction of the diploid genome and epigenome from a single cell, revealing that a cells accessible regulatory landscape can diverge by as much as 63% while still retaining the cells identity. Overall, DAF-seq enables the comprehensive characterization of protein occupancy and chromatin accessibility across entire chromosomes with single-nucleotide, single-molecule, single-haplotype, and single-cell precision.

genomics↗

bioRxiv2024

Resolving the chromatin impact of mosaic variants with targeted Fiber-seq

Accurately quantifying the functional consequences of non-coding mosaic variants requires the pairing of DNA sequence with both accessible and closed chromatin architectures along individual DNA molecules--a pairing that cannot be achieved using traditional fragmentation-based chromatin assays. We demonstrate that targeted single-molecule chromatin fiber sequencing (Fiber-seq) achieves this, permitting single-molecule, long-read genomic and epigenomic profiling across targeted >100 kilobase loci with [~]10-fold enrichment over untargeted sequencing. Targeted Fiber-seq reveals that pathogenic expansions of the DMPK CTG repeat that underlie Myotonic Dystrophy 1 are characterized by somatic instability and disruption of multiple nearby regulatory elements, both of which are repeat length-dependent. Furthermore, we reveal that therapeutic adenine base editing of the segmentally duplicated {gamma}-globin (HBG1/HBG2) promoters in primary human hematopoietic cells induced towards an erythroblast lineage increases the accessibility of the HBG1 promoter as well as neighboring regulatory elements. Overall, we find that these non-protein coding mosaic variants can have complex impacts on chromatin architectures, including extending beyond the regulatory element harboring the variant.

genomics↗

bioRxiv2024

A haplotype-resolved view of human gene regulation

Diploid human cells contain two non-identical genomes, and differences in their regulation underlie human development and disease. We present Fiber-seq Inferred Regulatory Elements (FIRE) and show that FIRE provides a more comprehensive and quantitative snapshot of the accessible chromatin landscape across the 6 Gbp diploid human genome, overcoming previously unrecognized biases in existing regulatory element catalogs. FIRE enables comprehensive detection of haplotype-selective chromatin accessibility (HSCA), exposing novel imprinted elements lacking underlying parent-of-origin CpG methylation differences, and gene regulatory modules that permit genes to escape X chromosome inactivation. We uncover that the human leukocyte antigen (HLA) locus harbors the most HSCA in immune cells, where we resolve specific transcription factor (TF) binding events disrupted by disease-associated variants. Finally, we demonstrate that the regulatory landscape of a cell is littered with autosomal somatic chromatin epimutations that are propagated by clonal expansions to create mitotically stable and non-genetically deterministic chromatin alterations.

genomics↗

bioRxiv2023

Fibertools: fast and accurate DNA-m6A calling using single-molecule long-read sequencing

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation as well as the identification of exogenously placed DNA N6-methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as co-processing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools, a state-of-the-art toolkit that features a semi-supervised convolutional neural network for fast and accurate identification of m6A-marked bases using PacBio single-molecule long-read sequencing, as well as the co-processing of long-read genetic and epigenetic data produced using either PacBio or Oxford Nanopore sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kilobase long DNA molecules with a [~]1,000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions.

bioinformatics↗

bioRxiv2023

Conservation of chromatin organization within human and primate centromeres

The focal attachment of the kinetochore to the centromere is essential for genome maintenance, yet the highly repetitive nature of satellite regional centromeres, such as those in humans, limits our understanding of their chromatin organization. We demonstrate that single-molecule chromatin fiber sequencing (Fiber-seq) can uniquely co-resolve kinetochore and surrounding chromatin architectures along point centromeres, revealing largely homogeneous single-molecule kinetochore occupancy along each chromosome. In contrast, extension of Fiber-seq to regional satellite centromeres exposed marked per-molecule heterogeneity in their chromatin organization. Regional CENP-A-marked centromere cores uniquely contain a dichotomous chromatin organization (dichromatin) composed of compacted nucleosome arrays punctuated with highly accessible chromatin patches. CENP-B occupancy phases dichromatin to the underlying alpha-satellite repeat within centromere cores, but is not necessary for dichromatin formation. Centromere core dichromatin is a conserved feature between humans despite the marked divergence of their underlying alpha-satellite organization and is similarly a conserved feature along regional centromeres that lack satellite repeats in gibbon. Overall, the chromatin organization of regional centromeres is defined by marked per-molecule heterogeneity, likely buffering kinetochore attachment against sequence and structural variability within regional centromeres. HighlightsO_LIDichotomous accessible and compacted chromatin (dichromatin) marks centromere cores C_LIO_LIHighly accessible chromatin patches punctuate sites of kinetochore attachment C_LIO_LIDichromatin can form irrespective of CENP-B occupancy C_LIO_LIConservation within centromeres is mediated at the level of chromatin, not DNA C_LI

genomics↗