Biology subjects

Schooley, A.

Publications and source records attributed to Schooley, A..

5 recordsLinked to original sources

bioRxiv2025

Dynamic changes in chromosome and nuclear architecture during maturation of normal and ALS C9orf72 motor neurons

We have investigated changes in chromosome conformation, nuclear organization, and transcription during differentiation and maturation of control and mutant motor neurons harboring hexanucleotide expansions in the C9orf72 gene that cause amyotrophic lateral sclerosis (ALS). Using an in vitro reprogramming, differentiation and neural maturation protocol, we obtained highly purified populations of post-mitotic motor neurons for both normal and diseased cells. As expected, as fibroblasts are reprogrammed into iPSCs, and as iPSCs differentiate into motor neurons, chromatin accessibility, chromosome conformation, and nuclear organization change along with large-scale alterations in transcriptional profiles. We find that the transcriptome changes extensively during the first three weeks of post-mitotic neuronal maturation, with thousands of genes changing expression, but then is relatively stable for the next three weeks. In contrast, chromosome conformation and nuclear organization continue to change over the entire 6-week maturation period: chromosome territoriality increases, long-range interactions along chromosomes decrease, compartmentalization strength increases, and centromeres and telomeres increasingly cluster. In motor neurons derived from ALS patients such changes in chromosome conformation were much reduced. Chromatin accessibility changes also showed delayed maturation. The transcriptome in these cells matured relatively normally but with notable changes in expression of genes involved in lipid, sterol and mitochondrial function. We conclude that neural maturation is associated with large scale post-mitotic changes in gene expression, chromosome conformation and nuclear organization, and that these processes are defective in motor neurons derived from ALS patients carrying C9orf72 hexanucleotide repeat expansions.

neuroscience↗

bioRxiv2024

Interphase chromosome conformation is specified by distinct folding programs inherited via mitotic chromosomes or through the cytoplasm

Identity-specific interphase chromosome conformation must be re-established each time a cell divides. To understand how interphase folding is inherited, we developed an experimental approach that physically segregates mediators of G1 folding that are intrinsic to mitotic chromosomes from cytoplasmic factors. Proteins essential for nuclear transport, RanGAP1 and Nup93, were degraded in pro-metaphase arrested DLD-1 cells to prevent the establishment of nucleo-cytoplasmic transport during mitotic exit and isolate the decondensing mitotic chromatin of G1 daughter cells from the cytoplasm. Using this approach, we discover a transient folding intermediate entirely driven by chromosome-intrinsic factors. In addition to conventional compartmental segregation, this chromosome-intrinsic folding program leads to prominent genome-scale microcompartmentalization of mitotically bookmarked and cell type-specific cis-regulatory elements. This microcompartment conformation is formed during telophase and subsequently modulated by a second folding program driven by factors inherited through the cytoplasm in G1. This nuclear import-dependent folding program includes cohesin and factors involved in transcription and RNA processing. The combined and inter-dependent action of chromosome-intrinsic and cytoplasmic inherited folding programs determines the interphase chromatin conformation as cells exit mitosis.

genomics↗

bioRxiv2022

Mechanisms of insertions at a DNA double-strand break

Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay in human cells (Indel-Seq) to monitor rearrangements at the TRIM37 acceptor locus which reports indels stemming from experimentally-induced and spontaneous genome instability. Templated insertions derive from sequences genome-wide and are enriched within 100 kb of donor regions flanking a DSB. Insertions require contact between donor and acceptor loci as well as DNA-PK catalytic activity. Notably, these templated insertions originate from actively transcribed loci, underscoring transcription as a critical source of spontaneous genome instability. Transcription-coupled insertions involve a DNA/RNA hybrid intermediate and are stimulated by DNA end-processing. Using engineered Cas9 breaks, we establish that ssDNA overhangs at the acceptor site greatly stimulate insertions. Indel-Seq revels that insertions are generated via at least three distinct pathways. Our studies indicate that insertions result from movement and subsequent contact between acceptor and donor loci followed invasion or annealing, then by non-homologous end-joining at the acceptor site.

molecular biology↗

bioRxiv2021

ARP2/3- and resection-coupled genome reorganization facilitates translocations

DNA end-resection and nuclear actin-based movements orchestrate clustering of double-strand breaks (DSBs) into homology-directed repair (HDR) domains. Here, we analyze how actin nucleation by ARP2/3 affects damage-dependent and -independent 3D genome reorganization and facilitates pathologic repair. We observe that DNA damage, followed by ARP2/3-dependent establishment of repair domains enhances local chromatin insulation at a set of damage-proximal boundaries and affects compartment organization genome-wide. Nuclear actin polymerization also promotes interactions between DSBs, which in turn facilitates aberrant intra- and inter-chromosomal rearrangements. Notably, BRCA1 deficiency, which decreases end-resection, DSB mobility, and subsequent HDR, nearly abrogates recurrent translocations between AsiSI DSBs. In contrast, loss of functional BRCA1 yields unique translocations genome-wide, reflecting a critical role in preventing spontaneous genome instability and subsequent rearrangements. Our work establishes that the assembly of DSB repair domains is coordinated with multiscale alterations in genome architecture that enable HDR despite increased risk of translocations with pathologic potential.

molecular biology↗

bioRxiv2021

The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin , {beta}, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.

cell biology↗