Biology subjects

Lardo, S. M.

Publications and source records attributed to Lardo, S. M..

3 recordsLinked to original sources

bioRxiv2026

FACT depletion results in a temporal cascade of chromatin disruption preceding transcriptional collapse in stem cells

The histone chaperone FACT is essential for chromatin organization and stem cell identity, yet the sequence of events through which FACT maintains chromatin integrity remains unclear. We generate a high-resolution temporal map of events leading to chromatin destabilization and cell identity loss in murine embryonic stem cells following dTAG-mediated depletion of FACT. Loss of FACT initiates a rapid cascade, with nucleosome phasing disruption and reduced H3K4me3 beginning within 10 minutes in a transcription-dependent manner. This architectural destabilization enables pluripotency transcription factors to ectopically occupy gene bodies. FACT loss results in RNA Polymerase II accumulation within 30 minutes and altered H3K36me3 deposition preceding a global transcriptional collapse at 2 hours of FACT depletion. FACT restoration fails to rescue these defects, demonstrating they are largely irreversible. Our findings define a temporal hierarchy in which FACT loss drives progressive deterioration of chromatin architecture, leading to transcriptional collapse in stem cells.

genomics↗

bioRxiv2024

A direct interaction between the Chd1 CHCT domain and Rtf1 controls Chd1 distribution and nucleosome positioning on active genes

The nucleosome remodeler Chd1 is required for the re-establishment of nucleosome positioning in the wake of transcription elongation by RNA Polymerase II. Previously, we found that Chd1 occupancy on gene bodies depends on the Rtf1 subunit of the Paf1 complex in yeast. Here, we identify an N-terminal region of Rtf1 and the CHCT domain of Chd1 as sufficient for their interaction and demonstrate that this interaction is direct. Mutations that disrupt the Rtf1-Chd1 interaction result in an accumulation of Chd1 at the 5 ends of Chd1-occupied genes, increased cryptic transcription, altered nucleosome positioning, and concordant shifts in histone modification profiles. We show that a homologous region within mouse RTF1 interacts with the CHCT domains of mouse CHD1 and CHD2. This work supports a conserved mechanism for coupling Chd1 family proteins to the transcription elongation complex and identifies a cellular function for a domain within Chd1 about which little is known.

molecular biology↗

bioRxiv2021

FACT maintains pluripotency factor expression through gene-distal regulation in embryonic stem cells

The FACT complex is a conserved histone chaperone with essential roles in transcription and histone deposition. FACT is essential in pluripotent and cancer cells, but otherwise dispensable for most mammalian cell types. FACT deletion or inhibition can block induction of pluripotent stem cells, yet the mechanism through which FACT regulates cell fate decisions remains unclear. To determine this mechanism, we used inducible depletion of FACT subunit SPT16 in murine embryonic stem cells paired with genomic factor localization, nascent transcription, and chromatin accessibility analyses. Over a timecourse of SPT16 depletion, nucleosomes invade loci bound by master pluripotency factors and gene-distal DNaseI hypersensitive sites. Simultaneously, transcription of Pou5f1 (OCT4), Sox2, Nanog, and enhancer RNAs produced at the genes associated enhancers are downregulated, suggesting that FACT regulates expression of the pluripotency factors themselves. We find that FACT maintains cellular pluripotency through a precise nucleosome-based regulatory mechanism for appropriate expression of both coding and non-coding transcripts associated with pluripotency.

molecular biology↗