Biology subjects

Flemington, E.

Publications and source records attributed to Flemington, E..

5 recordsLinked to original sources

bioRxiv2026

A BRRF1-CCR4-NOT axis underlies conserved transcriptome-wide loss of splicing fidelity during gammaherpesvirus reactivation

Gammaherpesvirus reactivation drives a collapse of host mRNA splicing fidelity that extends across the transcriptome, with exon skipping affecting up to [~]57% of expressed genes, exceeding the effects of depletion of any of 186 splicing factors. Combining five Epstein-Barr virus (EBV) and Kaposis sarcoma-associated herpesvirus (KSHV) reactivation systems across B cell and epithelial models with deep poly(A)+ RNA sequencing of purified lytic cells, we find that most induced isoforms are predicted to undergo nonsense-mediated decay or to lose conserved protein domains, broadly compromising cell cycle, innate immune and RNA-processing pathways. The phenotype arises independently of viral DNA replication, indicating early host remodeling. A screen of EBV early genes identifies BRRF1 as a key driver: through a CIY(Y/E) motif conserved in KSHV ORF49, BRRF1 engages the nuclear CCR4-NOT complex through its CNOT9 and CNOT1 subunits, hijacking this canonically cytoplasmic deadenylation hub for nuclear disruption of host splicing.

pathology↗

bioRxiv2026

KSHV miR-K12-9 Induces Transformation of Immortalized and Primary Endothelial Cells

Like most herpesviruses, KSHV encodes multiple microRNAs (miRNAs). Collectively, they comprise an important mechanism through which the virus maintains latency and persists in cells. At the same time, individual miRNAs can also play distinct, nonredundant roles. Past experiments with single miRNA knockout viruses showed that miR-K12-9, in particular, filled a unique niche. Endothelial cells latently infected with the miR-K12-9 knockout grew to be many times larger than WT-infected cells and proliferated at a significantly slower rate. Their ability to migrate was slowed as well. RNA-seq identified nearly 8,500 differentially expressed genes between miR-K12-9 knockout- and WT-infected cells. To further study miR-K12-9, we generated Telomerase-Immortalized Microvascular Endothelial (TIME) cells expressing either miR-K12-9 or a control miRNA from a lentivirus. Unexpectedly, after approximately one month in culture, unmistakable morphological changes began to occur in two of the three miR-K12-9-expressing cell lines. These smaller, more rounded cells proliferated rapidly and swiftly took over the two cultures. Given this result, we proceeded to characterize all the lentivirus-transduced cell lines in various assays focused on oncogenesis. When looking at colony formation in soft agar, only those two miR-K12-9-expressing cell lines produced colonies, indicating a loss of contact inhibition. NOD/SCID mice injected with the two cell miR-K12-9-expressing cell lines developed tumors while those receiving other cell lines did not. To confirm reproducibility of these results, we transduced both TIME and primary endothelial cells (HUVECs) with the miR-K12-9 and control lentiviruses. Once again, approximately half of the cell lines expressing miR-K12-9 showed hallmark phenotypes of transformation. We are currently characterizing the miR-K12-9 targetome in the transduced cell lines and mouse tumors using bulk and single-cell RNA-seq. This should yield insights into the underlying mechanism and required cofactors of miR-K12-9-induced transformation. To our knowledge, this is the first description of transformation of endothelial cells by a viral miRNA.

cancer biology↗

bioRxiv2025

Clustering of Tandem Inverse Duplications in Centromeric and Subtelomeric Chromosomal Regions of Aspergillus flavus

The formation of inverted repeats frequently initiates rearrangements associated with gene amplification. Among these rearrangements are inverted triplications (TRP/INVs) that are associated with multiple disease states in humans. We have used the filamentous fungus Aspergillus flavus as a model system to analyze spontaneous DNA rearrangements using high-depth third-generation sequencing of DNA isolated from vegetatively cultured strains. Analysis of sequence data identified a class of infrequent and transient rearrangements having structures typical of TRP/INVs. These TRP/INVs form both unprocessed and processed species and have a variably sized deletion at the junction between direct and inverted sequences. We found that TRP/INVs are enriched in heterochromatic centromeric and subtelomeric A+T-rich regions, suggesting that this process is a source of genetic instability in these domains. Consistent with this finding, A+T-rich regions contain elevated levels of direct, inverted, and perfect palindromic repeats. Inverted junctions contain palindromic sequences that are typically associated with TRP/INVs. A closer examination of the class of genomic palindromes found at inverted junctions revealed a broad distribution, with the majority lying in AT-rich centromeric sequences. The distribution of TRP/INVs in centromeric and subtelomeric domains mirrors the frequency of palindromes, indicating that palindrome abundance is likely to be a driver of TRP/INVs. A further examination of predicted pairing patterns in palindrome-like structures suggests that these structures may function as sites of DNA resolution, strand transfer, and replicative stalling. These results are consistent with a replication-based model in which palindromes, under conditions of replication stress, facilitate the formation of TRP/INV structures. These studies also represent, to our knowledge, the first characterization of spontaneous TRP/INV formation, likely to underlie the complex rearrangements associated with higher eukaryotic genomic instability and disease states. AUTHOR SUMMARYInverted triplications (TRP/INVs), coupled with gene amplification, are sources of genetic instability. However, little is known about how these species arise spontaneously prior to selective pressures. We have established a fungal model system that uses long-read third-generation sequencing to identify low-abundance rearrangements in the absence of an intentional selection. We identified and characterized multiple classes of TRP/INVs flanked by palindromic sequences. These rearrangements were concentrated in the AT-rich centromeric and subtelomeric regions, suggesting that these sites are regulated recombinational hotspots. Palindromes that are also associated with TRP/INVs are also most prevalent in A+T-rich centromeric regions, suggesting that the abundance of palindromes, both as a source of DNA damage and fork stalling, is a driver of TRP/INV formation. These studies provide the first evidence (to our knowledge) of the spontaneous formation of TRP/INVs during DNA replication, which is likely to precede more complex sources of rearrangements associated with oncogenesis and other disease states in higher eukaryotes.

genetics↗

bioRxiv2022

Reversal of splicing infidelity is a pre-activation step in B cell differentiation

B cell activation and differentiation is central to the adaptive immune response. Changes in exon usage can have major impacts on cellular signaling and differentiation but have not been systematically explored in differentiating B cells. We analyzed exon usage and intron retention in RNA-Seq data from subsets of human B cells at various stages of differentiation, and in an in vitro laboratory model of B cell activation and differentiation (Epstein Barr virus infection). Blood naive B cells were found to have an unusual splicing profile, with unannotated splicing events in over 30% of expressed genes. Splicing changed substantially upon naive B cell entry into secondary lymphoid tissue and before activation, involving significant increases in exon commitment and reductions in intron retention. These changes preferentially involved short introns with weak splice sites and were likely mediated by an overall increase in splicing efficiency induced by the lymphoid environment. The majority of transcripts affected by splicing changes showed restoration of encoded conserved protein domains and/or reduced targeting to the nonsense-mediated decay pathway. Affected genes were enriched in functionally important immune cell activation pathways such as antigen-mediated signaling, cell cycle control and mRNA processing and splicing. Functional observations from donor B cell subsets in progressive states of differentiation and from timecourse experiments using the in vitro model suggest that these widespread changes in mRNA splicing play a role in preparing naive B cells for the decisive step of antigen-mediated activation and differentiation.

immunology↗

bioRxiv2019

Defining the dynamic chromatin landscape of nephron progenitors

Six2+ cap mesenchyme cells, also called nephrons progenitor cells (NPC), are precursors of all epithelial cell types of the nephron, the filtering unit of the kidney. Current evidence indicates that perinatal \"old\" NPC have a greater tendency to exit the progenitor niche and differentiate into nascent nephrons than their embryonic \"young\" counterpart. Understanding the underpinnings of NPC aging may offer insights to rejuvenate old NPC and expand the progenitor pool. Here, we compared the chromatin landscape of young and old NPC and found common features reflecting their shared lineage but also intrinsic differences in chromatin accessibility and enhancer landscape supporting the view that old NPC are epigenetically poised for differentiation. Annotation of open chromatin regions and active enhancers uncovered the transcription factor Bach2 as a potential link between the pro-renewal MAPK/AP1 and pro-differentiation Six2/b-catenin pathways that might be of critical importance in regulation of NPC fate. Our data provide the first glimpse of the dynamic chromatin landscape of NPC and serve as a platform for future studies of the impact of genetic or environmental perturbations on the epigenome of NPC.\n\nSummary statementHilliard et al. investigated the chromatin landscape of native Six2+ nephron progenitors across their lifespan. They identified age-dependent changes in accessible chromatin and regulatory regions supporting the view that old nephron progenitors are epigenetically poised for differentiation.

developmental biology↗