Biology subjects

Buchert, E. M.

Publications and source records attributed to Buchert, E. M..

3 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

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↗

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↗