Biology subjects

Fogarty, E. A.

Publications and source records attributed to Fogarty, E. A..

8 recordsLinked to original sources

bioRxiv2026

Mi-2 and E93 coordinate robust cell cycle exit with terminal differentiation through enhancer decommissioning

Many postmitotic tissues coordinate robust cell cycle exit with the progression of terminal differentiation. While the signals involved in initiating cell cycle exit during terminal differentiation for some tissues have been described, less is known about the mechanisms that maintain a stable, non-cycling state. We previously found that a subset of rate-limiting cell cycle genes are subject to developmentally controlled changes in chromatin accessibility after cell cycle exit, suggesting chromatin remodelers may play a key role in maintaining a robust postmitotic state during terminal differentiation. Here we show that the general chromatin remodeler Mi-2 is required to ensure a stable postmitotic state in Drosophila eyes and wings. Mi-2-mediated remodeling coordinates cell state transitions during development, as compromising Mi-2 function slows the chromatin accessibility and gene expression dynamics that are critical for tissue maturation. Mi-2 and a temporally expressed transcription factor involved in terminal differentiation, E93, each decommission chromatin at developmentally dynamic regions near a limited set of shared target genes, including the early differentiation factor Broad and the rate-limiting mitotic regulator string (cdc25c). Our evidence supports a model where Mi-2 and E93 act in vivo to decommission a newly identified distal string enhancer that is active during cell cycle exit, to coordinate the transition to a robust postmitotic state with the progression of terminal differentiation. Temporally regulated enhancer decommissioning at the string locus, depending on both an instructive temporal factor and an enzymatic remodeler, provides a molecular explanation for the long-term, nearly irreversible postmitotic state observed in many tissues as terminal differentiation progresses.

developmental biology↗

bioRxiv2024

Deciphering gene regulatory programs underlying functionally divergent naive T cell subsets

Naive CD8+ T cells are a heterogeneous population, with different subsets possessing distinct functions and kinetics upon activation. However, the gene regulatory circuits differentiating these naive subsets are not well studied. In this work, we analyzed a large collection of public and newly generated RNA-seq and ATAC-seq profiles of different subsets of naive CD8+ T cells, revealing significant differences in the gene regulatory landscapes between subsets. We leveraged these data by employing a network inference algorithm, Inferelator, to identify the transcriptional regulatory circuits active in each subset. The predicted transcriptional network of the naive CD8+ T cell pool was validated by multiple orthogonal approaches, including CUT&Tag and Micro-C. Interestingly, our network analysis revealed a novel role for Eomes in promoting effector cell differentiation in specific cell subsets. Moreover, we uncovered multiple novel regulators across a variety of subsets and discovered several modules of genes that were co-regulated by shared sets of transcription factors in distinct subsets. Collectively, our data defines the gene regulatory programs differentiating naive CD8+ T cells and facilitates the identification of novel transcription factors that may alter the propensity of naive CD8+ T cells to become effector or memory cells after infection.

systems biology↗

bioRxiv2024

Transcriptional repression and enhancer decommissioning silence cell cycle genes in postmitotic tissues.

The mechanisms that maintain a non-cycling status in postmitotic tissues are not well understood. Many cell cycle genes have promoters and enhancers that remain accessible even when cells are terminally differentiated and in a non-cycling state, suggesting their repression must be maintained long term. In contrast, enhancer decommissioning has been observed for rate-limiting cell cycle genes in the Drosophila wing, a tissue where the cells die soon after eclosion, but it has been unclear if this also occurs in other contexts of terminal differentiation. In this study, we show that enhancer decommissioning also occurs at specific, rate-limiting cell cycle genes in the long-lived tissues of the Drosophila eye and brain, and we propose this loss of chromatin accessibility may help maintain a robust postmitotic state. We examined the decommissioned enhancers at specific rate-limiting cell cycle genes and show that they encode dynamic temporal and spatial expression patterns that include shared, as well as tissue-specific elements, resulting in broad gene expression with developmentally controlled temporal regulation. We extend our analysis to cell cycle gene expression and chromatin accessibility in the mammalian retina using a published dataset, and find that the principles of cell cycle gene regulation identified in terminally differentiating Drosophila tissues are conserved in the differentiating mammalian retina. We propose a robust, non-cycling status is maintained in long-lived postmitotic tissues through a combination of stable repression at most cell cycle gens, alongside enhancer decommissioning at specific rate-limiting cell cycle genes. HighlightsIn long-lived postmitotic Drosophila tissues, most cell cycle genes retain accessible chromatin despite persistent transcriptional downregulation. Cell cycle genes with accessible enhancers remain activatable during terminal differentiation, suggesting their repression must be continuously maintained in the postmitotic state. Long-lived postmitotic tissues decommission enhancers at specific, rate-limiting cell cycle genes in a developmentally regulated manner. Genome-wide enhancer identification performed in cell culture misses many developmentally dynamic enhancers in vivo. Decommissioned enhancers at cell cycle genes include shared and tissue-specific elements that in combination, result in broad gene expression with temporal regulation. The principles of cell cycle gene regulation identified in Drosophila are conserved in the mammalian retina.

developmental biology↗

bioRxiv2024

A novel regulatory pathway recognizes and degrades transcripts with long 3' untranslated regions

Quantitative control of gene expression is fundamental to cellular function, and post-transcriptional regulation is a consequential, additional layer of control over protein output. Within mammalian mRNAs, the 3' untranslated region (3'UTR) acts as a hub of regulatory control, typically mediated by regulatory sequence elements within the 3'UTR. We have found that expression from transcripts with long 3'UTRs is strongly repressed compared to those with short 3'UTRs, and this phenomenon appears to be independent of sequence elements within the 3'UTR. The repression increases with 3'UTR length and is substantial; reporters with 2,000 nucleotide long 3'UTRs are repressed >10-fold compared to 400 nucleotide 3'UTRs. Conversely, increasing the length of the coding region has no effect on expression, demonstrating that it is 3'UTR length, not transcript length, that elicits repression. Reporters with different length 3'UTRs show no difference in transcription rates nor translation efficiency but have clear differences in mRNA half-lives and nucleocytoplasmic distribution, indicating repression of long-3'UTR transcripts is mediated by accelerated RNA decay. However, this transcript degradation does not involve the nonsense-mediated decay (NMD) pathway nor other canonical RNA decay pathways. In order to identify trans factors involved in 3'UTR-length-mediated decay, we quantified the differences in proteins bound to reporters with long and short 3'UTRs, and identified over 100 differentially associated factors. Knockdowns of two differentially associating proteins, the mRNA nuclear export factors DDX39B and ZC3H11A, indicate they play a role in repression of mRNAs with long 3'UTRs. This study establishes a novel length-dependent regulatory feature of 3'UTRs, which potentially regulates many genes.

genetics↗

bioRxiv2023

A TOPBP1 Allele Causing Male Infertility Uncouples XY Silencing Dynamics From Sex Body Formation

Meiotic sex chromosome inactivation (MSCI) is a critical feature of meiotic prophase I progression in males. While the ATR kinase and its activator TOPBP1 are key drivers of MSCI within the specialized sex body (SB) domain of the nucleus, how they promote silencing remains unclear given their multifaceted meiotic functions that also include DNA repair, chromosome synapsis and SB formation. Here we report a novel mutant mouse harboring mutations in the TOPBP1-BRCT5 domain. Topbp1B5/B5 males are infertile, with impaired MSCI despite displaying grossly normal events of early prophase I, including synapsis and SB formation. Specific ATR-dependent events are disrupted including phosphorylation and localization of the RNA:DNA helicase Senataxin. Topbp1B5/B5 spermatocytes initiate, but cannot maintain ongoing, MSCI. These findings reveal a non-canonical role for the ATR-TOPBP1 signaling axis in MSCI dynamics at advanced stages in pachynema and establish the first mouse mutant that separates ATR signaling and MSCI from SB formation.

cell biology↗

bioRxiv2022

Single-cell transcriptomics of the immune system in ME/CFS at baseline and following symptom provocation

ME/CFS is a serious and poorly understood disease. To understand immune dysregulation in ME/CFS, we used single-cell RNA-seq (scRNA-seq) to examine immune cells in cohorts of patients and controls. Post-exertional malaise (PEM), an exacerbation of symptoms following strenuous exercise, is a characteristic symptom of ME/CFS. Thus, to detect changes coincident with PEM, we also performed scRNA-seq on the same cohorts following exercise. At baseline, ME/CFS patients displayed dysregulation of classical monocytes suggestive of inappropriate differentiation and migration to tissue. We were able to identify both diseased and more normal monocytes within patients, and the fraction of diseased cells correlated with metrics of disease severity. Comparing the transcriptome at baseline and post-exercise challenge, we discovered patterns indicative of improper platelet activation in patients, with minimal changes elsewhere in the immune system. Taken together, these data identify immunological defects present at baseline in patients and an additional layer of dysregulation following exercise. HighlightsME/CFS is a debilitating disease with unknown causes. Here, we provide, for the first time, an extensive single cell resolution dataset detailing the gene expression programs of circulating immune cells of ME/CFS cases at baseline and after symptom provocation. We were able to detect robust dysregulation in certain immune cells from patients, with dysregulation of classical monocytes manifesting the strongest signal. Indeed, the fraction of aberrant monocytes in ME/CFS patients correlated with the degree of disease severity. Surprisingly, platelet transcriptomes were also altered in ME/CFS, and they were the only component of the immune system that showed large-scale changes following symptom provocation.

immunology↗

bioRxiv2022

A tissue dissociation method optimized for ATAC-seq and CUT&RUN in Drosophila pupal tissues.

Chromatin accessibility, histone modifications and transcription factor binding are highly dynamic during Drosophila metamorphosis and drive global changes in gene expression as larval tissues differentiate into adult structures. Unfortunately, the presence of pupa cuticle on many Drosophila tissues during metamorphosis prevents enzyme access to cells and has limited the use of enzymatic in situ methods for assessing chromatin accessibility and histone modifications. Here, we present a dissociation method for cuticle-bound pupal tissues that is optimized for use with ATAC-Seq and CUT&RUN to interrogate chromatin accessibility and histone modifications. We show this method provides comparable chromatin accessibility data to the non-enzymatic approach FAIRE-seq, with only a fraction of the amount of input tissue required. This approach is also compatible with CUT&RUN, which allows genome-wide mapping of histone modifications with less than 1/10th of the tissue input required for more conventional approaches such as Chromatin Immunoprecipitation Sequencing (ChIP-seq). Our protocol makes it possible to use newer, more sensitive enzymatic in situ approaches to interrogate gene regulatory networks during Drosophila metamorphosis.

developmental biology↗

bioRxiv2020

Robust partitioning of microRNA targets from downstream regulatory changes

The biological impact of microRNAs (miRNAs) is determined by their targets, and robustly identifying direct miRNA targets remains challenging. Existing methods suffer from high falsepositive rates and are unable to effectively differentiate direct miRNA targets from downstream regulatory changes. Here, we present an experimental and computational framework to deconvolute post-transcriptional and transcriptional changes using a combination of RNA-seq and PRO-seq. This novel approach allows us to systematically profile the regulatory impact of a miRNA. We refer to this approach as CARP: Combined Analysis of RNA-seq and PRO-seq. We apply CARP to multiple miRNAs and show that it robustly distinguishes direct targets from downstream changes, while greatly reducing false positives. We validate our approach using Argonaute eCLIP-seq and ribosome profiling, demonstrating that CARP defines a comprehensive repertoire of targets. Using this approach, we identify miRNA-specific activity of target sites within the open reading frame. Additionally, we show that CARP facilitates the dissection of complex changes in gene regulatory networks triggered by miRNAs and identification of transcription factors that mediate downstream regulatory changes. Given the robustness of the approach, CARP would be particularly suitable for dissecting miRNA regulatory networks in vivo.

genomics↗