Biology subjects

Dunagin, M. C.

Publications and source records attributed to Dunagin, M. C..

9 recordsLinked to original sources

bioRxiv2025

Dynamics of Ribosomal RNA Transcription and Abundance in Normal and Leukemic Hematopoiesis

Transcription of ribosomal RNAs (rRNAs) from rDNA repeats is a critical first step of ribosome biogenesis. Often deemed a housekeeping process, its cell-type-specific regulation is largely unexplored. We profiled nascent rRNA and mature rRNAs (ribosome subunits) across mouse hematopoiesis, and observed 10-fold, cell-type-specific variation in rRNA abundance that is largely uncoupled from proliferation and translation rates. We also tested and confirmed the longstanding assumption that acute myeloid leukemia progenitors show notably higher rRNA levels than matched normal counterparts. These trends in nascent rRNA across hematopoietic contexts parallel changes in the proportion of active rDNA repeats. Absolute quantification of rRNAs unexpectedly revealed an excess of large versus small ribosomal subunits in all cells, particularly in the myeloid lineage. Finally, using an RNA Polymerase I degron system, we found that reduced rRNA transcription promotes myeloid progenitor differentiation. Collectively, our work uncovers complex dynamics and functional significance of rRNA regulation in hematopoiesis.

molecular biology↗

bioRxiv2024

AP-1 Mediates Cellular Adaptation and Memory Formation During Therapy Resistance

Cellular responses to environmental stimuli are typically thought to be governed by genetically encoded programs. We demonstrate that melanoma cells can form and maintain cellular memories during the acquisition of therapy resistance that exhibit characteristics of cellular learning and are dependent on the transcription factor AP-1. We show that cells exposed to a low dose of therapy adapt to become resistant to a high dose, demonstrating that resistance was not purely selective. The application of therapy itself results in the encoding of transient gene expression into cellular memory and that this encoding occurs for both transiently induced and probabilistically arising expression. Chromatin accessibility showed concomitant persistence. A two-color AP-1 reporter system showed that these memories are encoded in cis, constituting an example of activating cis epigenetics. Our findings establish the formation and maintenance of cellular memories as a critical aspect of gene regulation during the development of therapy resistance.

systems biology↗

bioRxiv2023

Single cell susceptibility to SARS-CoV-2 infection is driven by variable cell states

The ability of a virus to infect a cell type is at least in part determined by the presence of host factors required for the viral life cycle. However, even within cell types that express known factors needed for infection, not every cell is equally susceptible, suggesting that our knowledge of the full spectrum of factors that promote infection is incomplete. Profiling the most susceptible subsets of cells within a population may reveal additional factors that promote infection. However, because viral infection dramatically alters the state of the cell, new approaches are needed to reveal the state of these cells prior to infection with virus. Here, we used single-cell clone tracing to retrospectively identify and characterize lung epithelial cells that are highly susceptible to infection with SARS-CoV-2. The transcriptional state of these highly susceptible cells includes markers of retinoic acid signaling and epithelial differentiation. Loss of candidate factors identified by our approach revealed that many of these factors play roles in viral entry. Moreover, a subset of these factors exert control over the infectable cell state itself, regulating the expression of key factors associated with viral infection and entry. Analysis of patient samples revealed the heterogeneous expression of these factors across both cells and patients in vivo. Further, the expression of these factors is upregulated in particular inflammatory pathologies. Altogether, our results show that the variable expression of intrinsic cell states is a major determinant of whether a cell can be infected by SARS-CoV-2.

bioengineering↗

bioRxiv2023

Retrospective identification of intrinsic factors that mark pluripotency potential in rare somatic cells

Pluripotency can be induced in somatic cells by the expression of the four "Yamanaka" factors OCT4, KLF4, SOX2, and MYC. However, even in homogeneous conditions, usually only a rare subset of cells admit reprogramming, and the molecular characteristics of this subset remain unknown. Here, we apply retrospective clone tracing to identify and characterize the individual human fibroblast cells that are primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis led to increased reprogramming efficiency, identifying it as a barrier to reprogramming. Changing the frequency of reprogramming by inhibiting the activity of LSD1 led to an enlarging of the pool of cells that were primed for reprogramming. Our results show that even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.

systems biology↗

bioRxiv2022

Metastatic potential in clonal melanoma cells is driven by a rare, early-invading subpopulation

Metastasis occurs when tumor cells leave the primary tumor site and disseminate to distal organs. Even though most cells remain in the primary tumor, the circumstances by which a small fraction of them disseminate remain unclear. Here, we show that a rare, highly invasive subpopulation of melanoma cells can be detected within clonal cell lines due to non-genetic fluctuations in gene expression. The highly invasive phenotype was intrinsic to the cells, independent of their environment, and was marked by transiently high levels of SEMA3C expression, as revealed by RNA-sequencing analysis. Furthermore, the invasive subpopulation drove the bulk dissemination of tumor cells to distal locations in a mouse model of melanoma. The transcription factor NKX2.2 regulated the proportion of invasive cells in the melanoma 1205Lu cell line. Furthermore, an overall tradeoff between proliferation and invasion in single cells was observed. Our results suggest that phenotypes like metastasis may arise from intrinsic differences stemming from non-genetic fluctuations between single cells.

cancer biology↗

bioRxiv2021

CTCF blocks anti-sense transcription initiation at divergent gene promoters

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is largely unknown. Here by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters, the great majority of which bear proximal CTCF binding sites. Genome editing, chromatin conformation studies, and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its chromatin architectural function. Primary transcript RNA FISH revealed co-bursting of sense and anti-sense transcripts is disfavored, suggesting CTCF-regulated competition for transcription initiation. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.

molecular biology↗

bioRxiv2021

Systematically quantifying morphological features reveals constraints on organoid phenotypes

Organoids recapitulate complex 3D organ structures and represent a unique opportunity to probe the principles of self-organization. While we can alter an organoids morphology by manipulating the culture conditions, the morphology of an organoid often resembles that of its original organ, suggesting that organoid morphologies are governed by a set of tissue-specific constraints. Here, we establish a framework to identify constraints on an organoids morphological features by quantifying them from microscopy images of organoids exposed to a range of perturbations. We apply this framework to Madin-Darby Canine Kidney cysts and show that they obey a number of constraints taking the form of scaling relationships or caps on certain parameters. For example, we found that the number, but not size, of cells increases with increasing cyst size. We also find that these constraints vary with cyst age and can be altered by varying the culture conditions. We observed similar sets of constraints in intestinal organoids. This quantitative framework for identifying constraints on organoid morphologies may inform future efforts to engineer organoids.

systems biology↗

bioRxiv2020

Responsiveness to perturbations is a hallmark of transcription factors that maintain cell identity

Our ability to identify the particular transcription factors that maintain cell type is limited. Identification of factors by their cell type-specific expression or their participation in developmental regulation has been only modestly successful. We hypothesized that because cell type is often resilient to perturbations, the transcriptional response to perturbations would identify identity-maintaining factors. We developed Perturbation Panel Profiling (P3) as a framework for perturbing cells in dozens of conditions and measuring gene expression responsiveness transcriptome-wide. Applying P3 to human iPSC-derived cardiac myocytes showed that transcription factors known to function in cardiac differentiation and maintenance were among the most frequently up-regulated (most responsive). We reasoned that one potential function of responsive genes may be to maintain cellular identity. We identified responsive transcription factors in fibroblasts using P3 and found that suppressing their expression led to enhanced reprogramming efficiency. We propose that responsiveness to perturbations is a property of factors that help maintain cellular identity.

systems biology↗

bioRxiv2017

Intercellular mRNA Trafficking Via Membrane Nanotubes In Mammalian Cells

RNAs have been shown to undergo transfer between mammalian cells, though the mechanism behind this phenomenon and its overall importance to cell physiology is not well understood. Numerous publications have suggested that RNAs (microRNAs and incomplete mRNAs) undergo transfer via extracellular vesicles (e.g. exosomes). However, in contrast to a diffusion-based transfer mechanism, we find that full-length mRNAs undergo direct cell-cell transfer via cytoplasmic extensions, called membrane nanotubes (mNTs), which connect donor and acceptor cells. By employing a simple co-culture experimental model and using single-molecule imaging, we provide quantitative data showing that mRNAs are transferred between cells in contact. Examples of mRNAs that undergo transfer include those encoding GFP, mouse {beta}-actin, and human Cyclin D1, BRCA1, MT2A, and HER2. We show that intercellular mRNA transfer occurs in all co-culture models tested (e.g. between primary cells, immortalized cells, and in co-cultures of immortalized human and murine cells). Rapid mRNA transfer is dependent upon actin, but independent of de novo protein synthesis, and is modulated by stress conditions and gene expression levels. Hence, this work supports the hypothesis that full-length mRNAs undergo transfer between cells through a refined structural connection. Importantly, unlike the transfer of miRNA or RNA fragments, this process of communication transfers genetic information that could potentially alter the acceptor cell proteome. This phenomenon may prove important for the proper development and functioning of tissues, as well as host-parasite or symbiotic interactions.\n\nSignificanceMessenger RNA (mRNA) molecules convey genetic information within cells, beginning from genes in the nucleus to ribosomes in the cell body, where they are translated into proteins. Here, we show a novel mode of transferring genetic information from one cell to another. Contrary to previous publications suggesting that mRNAs transfer via extracellular vesicles, we provide visual and quantitative data showing that mRNAs transfer via membrane nanotubes and direct cell-to-cell contact. We predict that this process has a major role in regulating local cellular environments with respect to tissue development and maintenance, cellular responses to stress, interactions with parasites, tissue transplants, and the tumor microenvironment.\n\nAuthor contributionsG.H., A.R. and R.H.S. conceived the research and designed the experiments; C.M.E. performed and analyzed the experiments with WM983b+/-GFP, including transwell and exosomes; M.C.D. and E.E. performed and analyzed the WM983b/NIH393 co-culture experiments; G.H. performed and analyzed all other experiments; and G.H., J.E.G, A.R. and R.H.S. wrote the paper.

cell biology↗