Biology subjects

Reed, K. S. M.

Publications and source records attributed to Reed, K. S. M..

6 recordsLinked to original sources

bioRxiv2026

Re-establishment of TAD boundary organization during DNA replication

A ubiquitous feature of higher order genome organization is the presence of topologically associating domains (TADs). The chromatin architectural proteins cohesin and CTCF are known critical organizers of TADs, but the mechanisms of TAD establishment and maintenance, including their accurate duplication during genome replication, are not well characterized. To address this gap, we used high-throughput imaging-based CRISPR/Cas9 knock-out screening to discover chromatin factors involved in maintenance and establishment of TADs. Among the cellular factors that affect TAD organization, we found enrichment for cell cycle proteins, especially components of the DNA replication machinery. Accordingly, we demonstrate that TADs undergo temporary unfolding during S-phase DNA replication and that interference with progression through replication impedes restoration of normal TAD structure. Mechanistically, inhibition of the RPA complex prevents cohesin and CTCF binding, delays post-replication TAD re-folding and affects TAD folding in non-cycling cells. These results provide novel insights into how TAD structures are re-established during genome duplication.

cell biology↗

bioRxiv2026

Acute degron-mediated RUNX1 loss reprograms enhancer activity to epigenetically drive epithelial destabilization and initiate cancer hallmarks

The RUNX1 transcription factor mediates cell-type specific gene expression. RUNX1 suppression and perturbations are recurrently associated with breast tumor initiation and progression. However, the mechanisms governing the dual roles of RUNX1 in sustaining the mammary epithelial phenotype while epigenetically suppressing initiation of cancer-compromised gene expression are poorly understood. To address this, we used the power of degron-mediated acute, selective, and complete RUNX1 ablation in human mammary epithelial cells. RUNX1 mediates promoter and distal enhancer-driven expression of a gene cohort. Dynamic epigenomic responsiveness upon RUNX1 ablation reveals a rapid and selective decrease in chromatin accessibility and H3K27ac at RUNX1-bound enhancers, but not promoters. While differentially initiated and expressed genes contacted by RUNX1-bound enhancers are enriched in pathways involved in epithelial maintenance and stemness, genes with RUNX1-promoter occupancy support DNA damage responsiveness. Modified cell morphology, metabolic control, increased breast cancer stemness, plasticity, anchorage-independent survival, chemoresistance, and perturbed DNA damage reactivity are observed upon RUNX1 ablation. Together, these findings define RUNX1 as an epigenetic tumor suppressor that maintains epithelial cell state by preserving enhancer activity and preventing gene expression associated with hallmarks of cancer.

cancer biology↗

bioRxiv2025

TAD boundary architecture and gene activity are uncoupled

Topologically associating domains (TADs) are prominent features of genome organization. A proposed function of TADs is to contribute to gene regulation by promoting chromatin interactions within a TAD and by suppressing interactions between TADs. Here, we directly probe the structure-function relationship of TADs by simultaneously assessing the behavior of TAD boundaries and gene activity at the single-cell and -allele level using high-throughput imaging. We find that while TAD boundaries pair more frequently than non-boundary regions, these interactions are infrequent and are uncorrelated with transcriptional activity of genes within the TAD. Similarly, acute global transcriptional inhibition or gene-specific activation does not alter TAD boundary proximity. Furthermore, while loss of the cohesin component RAD21 alters gene activity, disruption of TAD boundaries by depletion of the architectural chromatin protein CTCF is insufficient to alter expression of genes within the TAD. These results suggest that TAD boundary architecture and gene activity are largely uncoupled.

cell biology↗

bioRxiv2025

Genome reorganization and its functional impact during breast cancer progression

Cancer progression involves extensive alterations in epigenetic and gene expression programs, but the accompanying changes in higher-order genome organization remain less well understood. Using high-resolution Micro-C mapping in the MCF10 cell model of breast cancer, we profiled chromatin compartments, topologically associated domains, and chromatin loops. We find large-scale compartmental shifts occur predominantly in early stages of cancer development, with more fine-scale structural changes in TADs and looping accumulating during the later transition to metastasis. Relating these chromatin features to gene expression and enhancer-associated histone marks revealed that many differentially expressed genes are physically connected to distal regulatory elements. While enhancer-promoter contact frequency and distal enhancer activity correlated with gene expression, strong changes in chromatin looping were relatively infrequent during progression, suggesting that alterations in chromatin contacts are not globally necessary but may facilitate gene regulation at a subset of genes. These results elucidate the connection between gene regulation and genome remodeling in a cell-based cancer progression model.

genomics↗

bioRxiv2022

Temporal analysis suggests a reciprocal relationship between 3D chromatin structure and transcription

To infer potential causal relationships between 3D chromatin structure, enhancers, and gene transcription, we mapped each feature in a genome-wide fashion across eight narrowly-spaced timepoints of macrophage activation. Enhancers and genes connected by loops exhibited stronger correlations between histone H3K27 acetylation and expression than can be explained by genomic distance or physical proximity alone. Changes in acetylation at looped distal enhancers preceded changes in gene expression. Changes in gene expression exhibit a directional bias at differential loop anchors; gained loops are associated with increased expression of genes oriented away from the center of the loop, while lost loops were often accompanied by high levels of transcription with the loop boundaries themselves. Taken together, these results are consistent with a reciprocal relationship in which loops can facilitate increased transcription by connecting promoters to distal enhancers while high levels of transcription can impede loop formation. HIGHLIGHTSO_LILPS + IFN{gamma} triggers genome-wide changes in chromatin looping, enhancer acetylation, and gene expression C_LIO_LILooped enhancer-promoter pairs exhibit ordered and correlated changes in acetylation and expression C_LIO_LIChanges in gene expression exhibit a directional bias at differential loop anchors C_LIO_LILost loops are associated with high levels of transcription within loop boundaries C_LI

genomics↗

bioRxiv2021

Synthetic amyloid beta does not induce a robust transcriptional response in innate immune cell culture systems

Alzheimers disease (AD) is a progressive neurodegenerative disease that impacts nearly 400 million people worldwide. The accumulation of amyloid beta (A{beta}) in the brain has historically been associated with AD, and recent evidence suggests that neuroinflammation plays a central role in its origin and progression. These observations have given rise to the theory that A{beta} is the primary trigger of AD, and induces proinflammatory activation of immune brain cells (i.e. microglia), which culminates in neuronal damage and cognitive decline. In order to test this hypothesis, many in vitro systems have been established to study A{beta}-mediated activation of innate immune cells. Nevertheless, the transcriptional resemblance of these models to the microglia in the AD brain has never been comprehensively studied on a genome-wide scale. To address this, we used bulk RNA-seq to assess the transcriptional differences between in vitro cell types used to model neuroinflammation in AD, including several established, primary and iPSC-derived immune cell lines (macrophages, microglia and astrocytes) and their similarities to primary cells in the AD brain. We then analyzed the transcriptional response of these innate immune cells to synthetic A{beta}. We found that human induced pluripotent stem cell (hIPSC)-derived microglia (IMGL) are the in vitro cell model that best resembles primary microglia. Surprisingly, synthetic A{beta} does not trigger a robust transcriptional response in any of the cellular models analyzed, despite testing a wide variety of A{beta} formulations, concentrations, and treatment conditions. Finally, we found that bacterial LPS and INF{gamma} activate microglia and induce transcriptional changes similar to those observed in disease associated microglia present in the AD brain, suggesting the potential suitability of this model to study AD-related neuroinflammation.

neuroscience↗