Biology subjects

Gehre, M.

Publications and source records attributed to Gehre, M..

4 recordsLinked to original sources

bioRxiv2021

A SUMO-dependent regulatory switch connects the piRNA pathway to the heterochromatin machinery in Drosophila

Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors upon binding to nascent target RNAs are poorly understood. Here, we elucidate the mechanism by which the PIWI interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. Piwi-mediated stabilization of the corepressor complex SFiNX on chromatin leads to SUMOylation of its subunit Panoramix. SUMOylation, together with an amphipathic LxxLL motif in Panoramixs intrinsically disordered repressor domain, are necessary and sufficient to recruit small ovary (Sov), a multi-zinc finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that abrogate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation, Piwi engages the heterochromatin machinery specifically at transposon loci.

molecular biology↗

bioRxiv2021

The molecular principles of Piwi-mediated co-transcriptional silencing through the dimeric SFiNX complex

Nuclear Argonaute proteins, guided to nascent target RNAs by their bound small RNAs, elicit co-transcriptional silencing through heterochromatin formation at transposon insertions and repetitive genomic loci. The molecular mechanisms involved in this process are incompletely understood. Here, we propose that the SFiNX complex, a silencing mediator downstream of nuclear Piwi-piRNA complexes in Drosophila, enables co-transcriptional silencing via the formation of molecular condensates. Condensate formation is stimulated by nucleic acid binding and requires SFiNX dimerization, mediated by the dynein light chain protein, LC8/Cutup. LC8s function within SFiNX can be bypassed with a heterologous dimerization domain, suggesting that dimerization is a constitutive feature of SFiNX. Mutations preventing LC8-mediated SFiNX dimerization result in loss of condensate formation in vitro and inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. Formation of molecular condensates might be a general mechanism that underlies effective heterochromatin establishment at small RNA target loci in a co-transcriptional manner.

molecular biology↗

bioRxiv2019

Efficient strategies to detect genome editing and integrity in CRISPR-Cas9 engineered ESCs

CRISPR-mediated genome engineering provides a powerful tool to study the function of genes and proteins. In the past decades, the advances in genome and transcriptome sequencing techniques have shed light on the genetic causes underlying many human diseases, such as neurodevelopmental disabilities or cancer. Sometimes, a single point-mutation in a protein coding gene has been identified as the primary cause of the disease. CRISPR-Cas offers the possibility to introduce or remove such a mutation of interest to understand disease mechanisms and even bears therapeutic potential. We describe the adaptation of an experimental strategy that allows the mutation of protein residues in mouse embryonic stem cells (ESCs) and propose a new screening method, Mismatch-qPCR, to reliably detect editing events in clonal cell lines as an alternative to restriction digest or Sanger sequencing. Finally, we show that RNA-Sequencing (RNA-Seq) data or low-coverage genomic sequencing data can be used to detect large chromosomal deletions and rearrangements that frequently occur at the CRISPR-targeting site.

genetics↗

bioRxiv2019

The functional landscape of the human phosphoproteome

Protein phosphorylation is a key post-translational modification regulating protein function in almost all cellular processes. While tens of thousands of phosphorylation sites have been identified in human cells to date, the extent and functional importance of the phosphoproteome remains largely unknown. Here, we have analyzed 6,801 publicly available phospho-enriched mass spectrometry proteomics experiments, creating a state-of-the-art phosphoproteome containing 119,809 human phosphosites. To prioritize functional sites, 59 features indicative of proteomic, structural, regulatory or evolutionary relevance were integrated into a single functional score using machine learning. We demonstrate how this prioritization identifies regulatory phosphosites across different molecular mechanisms and pinpoint genetic susceptibilities at a genomic scale. Several novel regulatory phosphosites were experimentally validated including a role in neuronal differentiation for phosphosites present in the SWI/SNF SMARCC2 complex member. The scored reference phosphoproteome and its annotations identify the most relevant phosphorylations for a given process or disease addressing a major bottleneck in cell signaling studies.

genomics↗