Biology subjects

Kiefer, L.

Publications and source records attributed to Kiefer, L..

4 recordsLinked to original sources

bioRxiv2026

Engineered Allosteric Control Enhances Specificity and Potency of CRISPR-CasX-based Epigenetic Repressors

Epigenetic silencers are targeted therapeutics capable of persistently silencing genes without altering DNA sequence. However, many current silencers rely on direct fusion of a constitutively active DNA methyltransferase catalytic domain, that may cause off-target DNA methylation and cellular toxicity. In contrast, endogenous DNA methyltransferases contain a regulatory domain (ADD) that requires recognition of a permissive histone state before DNA methylation can occur. We reinstate this sequential proofreading logic in a compact CasX-based epigenetic repressor, generating Epigenetic Long-term CasX Repressors (ELXRs) that read local chromatin state before writing DNA methylation. In ELXRs, a repressor domain converts active chromatin into a state that unlocks the allosteric DNA methyltransferase. This creates a sequential, multi-gated process that preferentially restricts DNA methylation to the target site. Surprisingly, implementing this proofreading mechanism improved not only specificity, but also activity. ELXRs decreased off-target methylation up to 10-fold and rescued DNMT3A-dependent growth defects, with transcriptome profiling showing up to 150-fold fewer dysregulated genes, while also increasing on-target repression up to 4-fold across multiple loci. In mouse models, lipid nanoparticle delivery produced potent, durable PCSK9 silencing with precise promoter methylation. Together, these results demonstrate that coupling molecular recognition to effector activity through sequential proofreading can simultaneously improve specificity and potency.

molecular biology↗

bioRxiv2023

Improving the study of RNA dynamics through advances in RNA-seq with metabolic labeling and nucleotide-recoding chemistry

RNA metabolic labeling using 4-thiouridine (s4U) captures the dynamics of RNA synthesis and decay. The power of this approach is dependent on appropriate quantification of labeled and unlabeled sequencing reads, which can be compromised by the apparent loss of s4U-labeled reads in a process we refer to as dropout. Here we show that s4U-containing transcripts can be selectively lost when RNA samples are handled under sub-optimal conditions, but that this loss can be minimized using an optimized protocol. We demonstrate a second cause of dropout in nucleotide recoding and RNA sequencing (NR-seq) experiments that is computational and downstream of library preparation. NR-seq experiments involve chemically converting s4U from a uridine analog to a cytidine analog and using the apparent T-to-C mutations to identify the populations of newly synthesized RNA. We show that high levels of T-to-C mutations can prevent read alignment with some computational pipelines, but that this bias can be overcome using improved alignment pipelines. Importantly, kinetic parameter estimates are affected by dropout independent of the NR chemistry employed, and all chemistries are practically indistinguishable in bulk, short-read RNA-seq experiments. Dropout is an avoidable problem that can be identified by including unlabeled controls, and mitigated through improved sample handing and read alignment that together improve the robustness and reproducibility of NR-seq experiments.

biochemistry↗

bioRxiv2022

Cohesin erases genomic-proximity biases to drive stochastic Protocadherin expression for proper neural wiring

Clustered Protocadherin (Pcdh) proteins act as cell-surface recognition barcodes for neural circuit formation. Neurites expressing the same barcode repel each other, but this mechanism is deployed in two different ways. For instance, convergence of olfactory sensory neuron (OSN) projections requires stochastic expression of distinct Pcdh isoforms in individual cells, while tiling of neural arbors of serotonergic neurons (5-HTs) requires expression of the same isoform, Pcdhc2. Despite their essential role, however, the molecular mechanisms of cell-type specific Pcdh barcoding remain a mystery. Here, we uncover a new role of cohesin: that of regulating distance-independent enhancer-promoter interactions to enable random Pcdh isoform choice via DNA loop extrusion in OSNs. Remarkably, this step mediates DNA demethylation of Pcdh promoters and their CTCF binding sites, thus directing CTCF to the chosen promoter. In contrast, the uniform pattern of Pcdh expression in 5-HTs is achieved through conventional cohesin-independent, distance-dependent enhancer/promoter interactions, that favor choice of the nearest isoform. Thus, cell-type specific cohesin deployment converts a distance-dependent and deterministic regulatory logic into a distance-independent and stochastic one. We propose that this mechanism provides an elegant strategy to achieve distinct patterns of Pcdh expression that generate wiring instructions to meet the connectivity requirements of different neural classes.

genomics↗

bioRxiv2021

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3 splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

cancer biology↗