Biology subjects

Limouse, C.

Publications and source records attributed to Limouse, C..

6 recordsLinked to original sources

bioRxiv2025

Identification of chromatin-associated RNAs at human centromeres

Centromeres are a specialized chromatin domain that are required for the assembly of the mitotic kinetochore and the accurate segregation of chromosomes. Non-coding RNAs play essential roles in regulating genome organization including at the unique chromatin environment present at human centromeres. We performed Chromatin-Associated RNA sequencing (ChAR-seq) in three different human cell lines to identify and map RNAs associated with centromeric chromatin. Centromere enriched RNAs display distinct contact behaviors across repeat arrays and generally belong to three categories: centromere encoded, nucleolar localized, and highly abundant, broad-binding RNAs. Most centromere encoded RNAs remain locally associated with their transcription locus with the exception of a subset of human satellite RNAs. This work provides a comprehensive identification of centromere bound RNAs that may regulate the organization and activity of the centromere.

genomics↗

bioRxiv2023

Genome-wide screen reveals Rab12 GTPase as a critical activator of pathogenic LRRK2 kinase

Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinsons disease. LRRK2 phosphorylates a subset of Rab GTPases, particularly Rab10 and Rab8A, and we showed previously that phosphoRabs play an important role in LRRK2 membrane recruitment and activation (Vides et al., 2022). To learn more about LRRK2 pathway regulation, we carried out an unbiased, CRISPR-based genome-wide screen to identify modifiers of cellular phosphoRab10 levels. A flow cytometry assay was developed to detect changes in phosphoRab10 levels in pools of mouse NIH-3T3 cells harboring unique CRISPR guide sequences. Multiple negative and positive regulators were identified; surprisingly, knockout of the Rab12 gene was especially effective in decreasing phosphoRab10 levels in multiple cell types and knockout mouse tissues. Rab-driven increases in phosphoRab10 were specific for Rab12, LRRK2 dependent and PPM1H phosphatase reversible; they were seen with wild type and pathogenic G2019S and R1441C LRRK2. AlphaFold modeling revealed a novel Rab12 binding site in the LRRK2 Armadillo domain and we show that residues predicted to be essential for Rab12 interaction at this site influence overall phosphoRab levels in a manner distinct from Rab29 activation of LRRK2. Our data support a model in which Rab12 binding to a new site in the LRRK2 Armadillo domain activates LRRK2 kinase for Rab phosphorylation and could serve as a new therapeutic target for a novel class of LRRK2 inhibitors that do not target the kinase domain.

biochemistry↗

bioRxiv2022

Global mapping of RNA-chromatin contacts reveals a proximity-dominated connectivity model for ncRNA-gene interactions

Non-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. Yet, the genomic targets and functions of most ncRNAs are unknown. Here we use chromatin-associated RNA sequencing (ChAR-seq) to map the global network of ncRNA interactions with chromatin in human embryonic stem cells, and the dynamic changes in interactions during differentiation into definitive endoderm. We uncover general principles governing the organization of the RNA- chromatin interactome, demonstrating that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their locus, and provide a model predicting the interactome. We uncover RNAs that interact with many loci across the genome, and unveil thousands of unannotated RNAs that dynamically interact with chromatin. By relating the dynamics of the interactome to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by a single ncRNA.

genomics↗

bioRxiv2021

From telomere to telomere: the transcriptional and epigenetic state of human repeat elements

Mobile elements and highly repetitive genomic regions are potent sources of lineage-specific genomic innovation and fingerprint individual genomes. Comprehensive analyses of large, composite or arrayed repeat elements and those found in more complex regions of the genome require a complete, linear genome assembly. Here we present the first de novo repeat discovery and annotation of a complete human reference genome, T2T-CHM13v1.0. We identified novel satellite arrays, expanded the catalog of variants and families for known repeats and mobile elements, characterized new classes of complex, composite repeats, and provided comprehensive annotations of retroelement transduction events. Utilizing PRO-seq to detect nascent transcription and nanopore sequencing to delineate CpG methylation profiles, we defined the structure of transcriptionally active retroelements in humans, including for the first time those found in centromeres. Together, these data provide expanded insight into the diversity, distribution and evolution of repetitive regions that have shaped the human genome.

genomics↗

bioRxiv2020

Identification and characterization of centromeric sequences in Xenopus laevis

Centromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

genomics↗

bioRxiv2017

Intramolecular dynamics of single molecules in free diffusion

Biomolecular systems such as multiprotein complexes or biopolymers can span several tens to several hundreds of nanometers, but the dynamics of such \"mesocale\" molecules remain challenging to probe. We have developed a single-molecule technique that uses Tracking Fluorescence Correlation Spectroscopy (tFCS) to measure the conformation and dynamics of molecular assemblies specifically at the mesoscale level (~100-1000 nm). tFCS is non-perturbative, as molecules, which are tracked in real-time, are untethered and freely diffusing. To achieve sub-diffraction spatial resolution, we use a feedback scheme which allows us to maintain the molecule at an optimal position within the laser intensity gradient. We find that tFCS is sufficiently sensitive to measure the distance fluctuations between two sites within a DNA molecule separated by distances as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted chromatin, and can assay transient protein mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the impact that tFCS can have in the study of a wide variety of biochemical processes involving mesoscale conformational dynamics.

biophysics↗