Biology subjects

Alexander, K. E.

Publications and source records attributed to Alexander, K. E..

3 recordsLinked to original sources

bioRxiv2026

Dfm1 promotes Ste24-dependent translocon quality control

Proteins stalled during endoplasmic reticulum translocation clog Sec61 channels and require translocon quality control (TQC) mechanisms for clearance. Genetic screens implicated the derlin Dfm1 in TQC; however, its mechanistic role remained unclear. Using engineered and disease-relevant translocon-clogging substrates in Saccharomyces cerevisiae, we show Dfm1 functions in the Ste24-dependent branch of TQC, but not Hrd1- or Ltn1-dependent pathways. Loss of DFM1 increased accumulation and toxicity of translocon-clogging proteins. Increased dosage of wild type or catalytically inactive Hrd1 or Ste24 rescued toxicity, supporting a non-enzymatic buffering function for these factors in TQC. These findings identify Dfm1 as a key TQC factor whose critical role in clearing clogged translocons can be suppressed in multiple ways. Our results suggest enhancing TQC may protect cells from proteotoxic stress caused by disease-relevant translocon-clogging substrates, with potential implications for pancreatic {beta}-cell dysfunction in diabetes. Significance StatementO_LICells rely on translocon quality control (TQC) to clear proteins that clog the Sec61 translocon, but how the derlin Dfm1 contributes to this process has remained unclear. C_LIO_LIWe demonstrate that Dfm1 functions specifically in the Ste24-dependent TQC pathway and show that increased levels of the TQC factors Hrd1 or Ste24 rescues Dfm1 deficiency even without catalytic activity, revealing an unexpected non-enzymatic buffering function. C_LIO_LIThis work defines Dfm1s mechanistic role in TQC, provides new insight into how cells preserve endoplasmic reticulum function during translocation stress, and establishes a framework for investigating conserved pathways that protect against diseases associated with translocon clogging. C_LI

cell biology↗

bioRxiv2025

Transcription can be sufficient, but is not necessary, to advance replication timing

DNA replication timing (RT) is correlated with transcription during cell fate changes but there are many exceptions and our understanding of this relationship suffers from a paucity of reductionist approaches. Here, we manipulated length and strength of transcription in hybrid-genome mouse embryonic stem cells (mESCs) at a single locus upstream of the silent, late replicating, Pleiotrophin (Ptn) gene, directly comparing RT to nascent transcription rates at engineered vs. wild-type alleles. First, we inserted four reporter genes that differ only in their promoter. Two promoters transcribed the reporter gene at high rates and advanced RT. The other two transcribed at lower rates and did not advance RT. Since these promoters may prove useful in applications where effects on RT are undesirable, we confirmed the inability of one of them to advance RT at numerous ectopic sites. We next juxtaposed these same four promoters upstream of the Ptn transcription start site where they all transcribed the 96kb Ptn gene and advanced RT to different extents correlated with transcription rates. Indeed, a doxycycline-responsive promoter, which could not advance RT when induced as a small reporter gene, elicited a rapid and reversible RT advance proportional to the rate of transcription, providing direct evidence that transcription itself can advance RT. However, deletion of the Ptn promoter and enhancer, followed by directed differentiation to neural precursors, eliminated induction of transcription throughout the entire Ptn replication domain, without preventing the switch to early replication. Our results provide a solid empirical base with which to re-evaluate many decades of literature, demonstrating that length and strength of transcription is sufficient but not necessary to advance RT. Our results also provide a robust system in which to rapidly effect an RT change, permitting mechanistic studies of the role of transcription in RT and the consequences of RT changes to epigenomic remodeling.

genomics↗

bioRxiv2023

Master transcription factor binding sites are necessary for early replication control element activity

Eukaryotic genomes replicate in a defined temporal order called the replication timing (RT) program. RT is developmentally regulated with potential to drive cell fate transitions, but mechanisms controlling RT remain elusive. We previously identified "Early Replication Control Elements" (ERCEs) necessary for early RT, domain-wide transcription, 3D chromatin architecture and compartmentalization in mouse embryonic stem cells (mESCs) but, deletions identifying ERCEs were large and encompassed many putative regulatory elements. Here, we show that ERCEs are compound elements whose RT activity can largely be accounted for by multiple sites of diverse master transcription factor binding (subERCEs), distinguished from other such sites by their long-range interactions. While deletion of subERCEs had large effects on both transcription and RT, deleting transcription start sites eliminated nearly all transcription with moderate effects on RT. Our results suggest a model in which subERCEs respond to diverse master transcription factors by functioning both as transcription enhancers and as elements that organize chromatin domains structurally and support early RT, potentially providing a feed-forward loop to drive robust epigenomic change during cell fate transitions.

molecular biology↗