Biology subjects

Vaughan, R. M.

Publications and source records attributed to Vaughan, R. M..

5 recordsLinked to original sources

bioRxiv2024

UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation

The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.

genomics↗

bioRxiv2023

Molecular dynamics simulations provide insights into ULK-101 potency and selectivity toward autophagic kinases ULK1/2

Kinase domains are highly conserved within protein kinases in both sequence and structure. Many factors, including phosphorylation, amino acid substitutions or mutations, and small molecule inhibitor binding, influence conformations of the kinase domain and enzymatic activity. The serine/threonine kinases ULK1 and ULK2 are highly conserved with N- and C-terminal domains, phosphate-binding P-loops, -C-helix, regulatory and catalytic spines, and activation loop DFG and APE motifs. Here, we performed molecular dynamics (MD) simulations to understand better the potency and selectivity of the ULK1/2 small molecule inhibitor, ULK-101. We observed stable bound states for ULK-101 to the adenosine triphosphate (ATP)-binding site of ULK2, coordinated by hydrogen bonding with the hinge backbone and the catalytic lysine sidechain. Notably, ULK-101 occupies a hydrophobic pocket associated with the N-terminus of the -C-helix. Large movements in the P-loop are also associated with ULK-101 inhibitor binding and exit from ULK2. Our data further suggests that ULK-101 could induce a folded P-loop conformation and hydrophobic pocket reflected in its nanomolar potency and kinome selectivity.

bioinformatics↗

bioRxiv2023

Analysis of histone antibody specificity directly in sequencing data using siQ-ChIP

We previously developed sans spike-in quantitative chromatin immunoprecipitation sequencing (siQ-ChIP), a technique that introduces an absolute quantitative scale to ChIP-seq data without reliance on spike-in normalization approaches. The physical model of siQ-ChIP predicted that the IP step of ChIP would produce a classical binding isotherm when antibody or epitope was titrated. Here, we define experimental conditions in which this titration is observable for antibodies that recognize modified states of histone proteins. We show that minimally sequenced points along an isotherm can reveal differential binding specificities that are associated with on- and off-target epitope interactions. This work demonstrates that the interpretation of histone post-translational modification distribution from ChIP-seq data has a dependence on antibody concentration. Collectively, these studies introduce a simplified and reproducible experimental method to generate quantitative ChIP-seq data without spike-in normalization and demonstrate that histone antibody specificity can be analyzed directly in ChIP-seq experiments.

molecular biology↗

bioRxiv2022

Theoretical and practical refinements of sans spike-in quantitative ChIP-seq with application to p300/CBP inhibition

Previously, we introduced an absolute and physical quantitative scale for chromatin immunoprecipitation followed by sequencing. The scale itself was determined directly from measurements routinely made on sequencing samples without additional reagents or spike-ins. We called this approach sans spike-in quantitative ChIP, or siQ-ChIP. In this paper we extend those results in several ways. First, we simplified the calculations defining the quantitative scale. Second, we highlight the normalization constraint implied by the quantitative scale and introduce a new scheme for generating tracks for siQ-ChIP. We next introduce some whole-genome analyses that are unique to siQ-ChIP which allow us, for example, to project the IP mass onto the genome to evaluate how much of any genomic interval was captured in the IP. We apply these analyses to p300/CBP inhibition and demonstrate that response to inhibition is a function of genomic architecture. In particular, active transcription start sites are only weakly perturbed by p300/CBP inhibition while enhancers are strongly perturbed. Similar observations have been reported in the literature, but without a quantitative scale, those observations have been misinterpreted. We discuss how the siQ-ChIP approach precludes such misinterpretations, which stem from the widespread community practice of treating unquantified and unnormalized ChIP-seq tracks as though they are quantitative.

biophysics↗

bioRxiv2019

siQ-ChIP:A reverse-engineered quantitative framework for ChIP-sequencing

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is a key technique for mapping the distribution and relative abundance of histone posttranslational modifications (PTMs) and chromatin-associated factors across genomes. There is a perceived challenge regarding the ability to quantitatively plot ChIP-seq data, and as such, approaches making use of exogenous additives, or \"spike-ins\" have recently been developed. Relying on the fact that the IP step of ChIP-seq is a competitive binding reaction, we present a quantitative framework for ChIP-seq analysis that circumvents the need to modify standard sample preparation pipelines with spike-in reagents. We also introduce a visualization technique that, when paired with our formal developments, produces a much more rich characterization of sequencing data.

genomics↗