Biology subjects

Wooten, M.

Publications and source records attributed to Wooten, M..

8 recordsLinked to original sources

bioRxiv2026

Aclarubicin disrupts RNA polymerase II progression at replication-coupled histone genes

Anthracyclines are highly effective chemotherapeutic agents that cause DNA and chromatin damage. One member of the anthracyclines, aclarubicin, has recently gained therapeutic interest due to its ability to kill cancer cells through chromatin-based mechanisms, thus avoiding the off-target effects associated with DNA damage. Despite this, the molecular mechanism of action leading to aclarubicin-induced chromatin damage remains elusive. Here we performed Cleavage Under Targets and Tagmentation (CUT&Tag) of RNA polymerase II (Pol II) and other transcriptional regulators in human cells during aclarubicin treatment. We found that aclarubicin strongly disrupts the replication-coupled histone genes, resulting in a nonproductive accumulation of Pol II-transcription machinery here beyond the levels at other genes. We attribute this sensitivity to the dense Pol II loading and rapid transcription of the histone genes, which intensify the chromatin-disrupting effects of aclarubicin at these loci. Together, our findings support the effectiveness of aclarubicin as an anticancer drug and point to the histone gene cluster as a promising target for therapeutic intervention.

molecular biology↗

bioRxiv2025

Nascent CUT&Tag captures transcription factor binding after chromatin duplication

DNA replication strips off all chromatin proteins, which must be reassembled behind the replication fork. To track chromatin reassembly on newly synthesized DNA, we developed Nascent CUT&Tag, a chromatin profiling method that directly measures transcription factor (TF) binding on nascent chromatin. We tracked the recovery of GAGA factor (GAF) and Pleiohomeotic (PHO) in Drosophila Kc167 cells. Whereas both TFs are evicted following passage of the replication fork, GAF recovers on newly synthesized DNA over a minutes-to-hours range, whereas PHO generally requires hours to fully reestablish binding. Early recovering GAF peaks are characterized by shorter GAF motifs and are associated with functions related to cell cycle progression. Conversely, late recovering peaks are characterized by longer, degenerate GAF motifs and are associated with developmental functions. GAF recovery on newly synthesized DNA requires chromatin remodeling by Brahma Associated Factor (BAF), implying that nucleosome turnover is critical to fully reestablish GAF binding.

genomics↗

bioRxiv2025

CUT&TIME captures the history of open chromatin indeveloping neurons

Chromatin structure plays a central role in defining cell identity by regulating gene expression. During development, shifts in chromatin structure facilitate changes in gene expression needed to specify distinct cell types. To understand how changes in chromatin structure influence the developmental trajectory of neural progenitor cells, we developed CUT&TIME, a technique that uses a hyperactive 6-methyl adenosine (6mA) methyltransferase pulsed in living cells to map historical chromatin accessibility genome-wide in single cells. We show that CUT&TIME produces a record of the chromatin landscape during neurogenesis in the developing retina, specifically as neural progenitors produce the major projection neuron type, retinal ganglion cells (RGCs). We further show that this method is compatible with single cell profiling technologies, which allows us to visualize and capture the diversity of chromatin states that produce RGCs. Additionally, we identify changes in promoter accessibility associated with the transition from progenitor to RGC. Together, these data demonstrate that CUT&TIME captures a historical record of chromatin structure, which can be used to identify early changes in accessibility associated with cell-fate commitment.

developmental biology↗

bioRxiv2024

Histone H4 limits transcription of the histone locus in Drosophila

In all eukaryotes DNA replication is coupled to histone synthesis to coordinate chromatin packaging of the genome. Canonical histone genes coalesce in the nucleus into the Histone Locus Body (HLB), where gene transcription and 3 mRNA processing occurs. Both histone gene transcription and mRNA stability are reduced when DNA replication is inhibited, implying that the Histone Locus Body senses the rate of DNA synthesis. In Drosophila melanogaster, the S-phase-induced histone genes are tandemly repeated in an [~]100 copy array, whereas in humans, these histone genes are scattered. In both organisms these genes coalesce into Histone Locus Bodies. We used a transgenic histone gene reporter and RNAi in Drosophila to identify canonical H4 histone as a unique repressor of histone synthesis during the G2 phase in germline cells. Using cytology and CUT&Tag chromatin profiling, we find that histone H4 uniquely occupies histone gene promoters in both Drosophila and human cells. Our results suggest that repression of histone genes by soluble histone H4 is a conserved mechanism that coordinates DNA replication with histone synthesis in proliferating cells.

genomics↗

bioRxiv2023

Aclarubicin stimulates RNA polymerase II elongation at closely spaced divergent promoters

Anthracyclines are a class of widely prescribed anti-cancer drugs that disrupt chromatin by intercalating into DNA and enhancing nucleosome turnover. To understand the molecular consequences of anthracycline-mediated chromatin disruption, we utilized CUT&Tag to profile RNA polymerase II during anthracycline treatment in Drosophila cells. We observed that treatment with the anthracycline aclarubicin leads to elevated levels of elongating RNA polymerase II and changes in chromatin accessibility. We found that promoter proximity and orientation impacts chromatin changes during aclarubicin treatment, as closely spaced divergent promoter pairs show greater chromatin changes when compared to codirectionally-oriented tandem promoters. We also found that aclarubicin treatment changes the distribution of non-canonical DNA G-quadruplex structures both at promoters and at G-rich pericentromeric repeats. Our work suggests that the anti-cancer activity of aclarubicin is driven by the effects of nucleosome disruption on RNA polymerase II, chromatin accessibility and DNA structures.

genomics↗

bioRxiv2021

Differential condensation of sister chromatids coordinates with Cdc6 to ensure distinct cell cycle progression in Drosophila male germline stem cell lineage

Stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell. During Drosophila male germline stem cell (GSC) asymmetric division, preexisting old histones H3 and H4 are enriched in the self-renewed stem daughter cell, whereas the newly synthesized H3 and H4 are enriched in the differentiating daughter cell. However, the biological consequences in the two daughter cells resulting from asymmetric histone inheritance remained to be elucidated. In this work, we track both old and new histones throughout GSC cell cycle using high spatial and temporal resolution microscopy. We find several unique features differentiating old versus new histone-enriched sister chromatids, including nucleosome density, chromosomal condensation, and H3 Ser10 phosphorylation. These distinct chromosomal features lead to their differential association with Cdc6, an essential component of the pre-replication complex, which subsequently contributes to asynchronous initiation of DNA replication in the two resulting daughter cells. Disruption of asymmetric histone inheritance abolishes both differential Cdc6 association and asynchronous S-phase entry, demonstrating that asymmetric histone acts upstream of these critical events during cell cycle progression. Furthermore, GSC defects are detected under these conditions, indicating a connection between histone inheritance, cell cycle progression and cell fate decision. Together, these studies reveal that cell cycle remodeling as a crucial biological readout of asymmetric histone inheritance, which precedes and could lead to other well-known readouts such as differential gene expression. This work also enhances our understanding of asymmetric histone inheritance and epigenetic regulation in other stem cells or asymmetrically dividing cells in multicellular organisms.

developmental biology↗

bioRxiv2020

Pruriception and neuronal coding in nociceptor subtypes in human and nonhuman primates

In human, intradermal administration of {beta}-alanine (ALA) and bovine adrenal medulla peptide 8-22 (BAM8-22) evokes the sensation of itch. Currently, it is unknown which human dorsal root ganglion (DRG) neurons express the receptors of these pruritogens, MRGPRD and MRGPRX1 respectively, and which cutaneous afferents these pruritogens activate in primate. In situ hybridization studies revealed that MRGPRD and MRGPRX1 are co-expressed in a subpopulation of TRPV1+ human DRG neurons. In electrophysiological recordings in nonhuman primates (Macaca nemestrina), subtypes of polymodal C-fiber nociceptors are preferentially activated by ALA and BAM8-22, with significant overlap. When pruritogens ALA, BAM8-22 and histamine, that activate different subclasses of C-fiber afferents, are administered in combination, human volunteers report itch and nociceptive sensations similar to those induced by a single pruritogen. Our results provide evidence for differences in pruriceptive processing between primates and rodents, and do not support the spatial contrast theory of coding of itch and pain.

neuroscience↗

bioRxiv2018

Asymmetric histone incorporation during DNA replication in Drosophila male germline stem cells

One Sentence SummaryDNA replication establishes asymmetric epigenomes SummaryOne of the most fundamental questions in developmental biology concerns how cells with identical genomes differentiate into distinct cell types. One important context for understanding cell fate specification is asymmetric cell division, where the two daughter cells establish different cell fates following a single division. Many stem cells undergo asymmetric division to produce both a self-renewing stem cell and a differentiating daughter cell1-5. Here we show that histone H4 is inherited asymmetrically in asymmetrically dividing Drosophila male germline stem cells, similar to H36. In contrast, both H2A and H2B are inherited symmetrically. By combining superresolution microscopy with the chromatin fiber method, we are able to study histone inheritance patterns on newly replicated chromatin fibers. Using this technique, we find asymmetric inheritance patterns for old and new H3, but symmetric inheritance patterns for old and new H2A on replicating sister chromatids. Furthermore, co-localization studies on isolated chromatin fibers and proximity ligation assays on intact nuclei reveal that old H3 are preferentially incorporated by the leading strand while newly synthesized H3 are enriched on the lagging strand. Finally, using a sequential nucleoside analog incorporation assay, we detect a high incidence of unidirectional DNA replication on germline-derived chromatin fibers and DNA fibers. The unidirectional fork movement coupled with the strand preference of histone incorporation could explain how old and new H3 are asymmetrically incorporated by replicating sister chromatids. In summary, our work demonstrates that the intrinsic asymmetries in DNA replication may help construct sister chromatids enriched with distinct populations of histones. Therefore, these results suggest unappreciated roles for DNA replication in asymmetrically dividing cells in multicellular organisms.

developmental biology↗