Biology subjects

Klobucar, T.

Publications and source records attributed to Klobucar, T..

3 recordsLinked to original sources

bioRxiv2021

Epiblast morphogenesis is controlled by selective mRNA decay triggered by LIN28A relocation

The embryonic progression from naive to primed pluripotency is accompanied by the rapid decay of pluripotency-associated mRNAs and a concomitant radical morphogenetic sequence of epiblast polarization, rosette formation and lumenogenesis. The mechanisms triggering and linking these events remain poorly understood. Guided by machine learning and metabolic RNA sequencing, we identified RNA binding proteins (RBPs), especially LIN28A, as primary mRNA decay factors. Using mRNA-RBP interactome capture, we revealed a dramatic increase in LIN28A mRNA binding during the naive-rosette-primed pluripotency transition, driven by its nucleolar-to-cytoplasmic translocation. Cytoplasmic LIN28A binds to 3UTRs of pluripotency-associated mRNAs to directly stimulate their decay and drive lumenogenesis. Accordingly, forced nuclear retention of LIN28A impeded lumenogenesis, impaired gastrulation, and caused an unforeseen embryonic multiplication. Selective mRNA decay, driven by nucleo-cytoplasmic RBP translocation, therefore acts as an intrinsic mechanism linking cell identity switches to the control of embryonic growth and morphogenesis.

developmental biology↗

bioRxiv2020

Sex of donor cell and reprogramming conditions predict the extent and nature of imprinting defects in mouse iPSCs

Reprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalized approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. A sensible indicator for epigenetic fidelity is genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unidentified reasons. By using a secondary reprogramming system with murine hybrid donor cells, we conducted a thorough imprinting analysis using IMPLICON in multiple female and male iPSCs generated under different culture conditions. Our results show that imprinting defects are remarkably common in mouse iPSCs causing dysregulation of the typical monoallelic expression of imprinted genes. Interestingly, the nature of imprinting defects depends on the sex of the donor cell and their respective response to culture conditions. Under serum-free conditions, male iPSCs show global hypomethylation at imprinted regions, whereas in serum conditions show focal hypermethylation at specific loci. In contrast, female iPSCs always exhibit hypomethylation defects regardless of culture conditions. These imprinting defects are more severe than the global changes in DNA methylation, highlighting the sensitivity of imprinting loci to current iPSC generation protocols. Our results reveal clear predictors underlying different types of imprinting defects in mouse iPSCs. This knowledge is essential to devise novel reprogramming strategies aiming at generating epigenetically faithful iPSCs.

molecular biology↗

bioRxiv2020

IMPLICON: an ultra-deep sequencing method to uncover DNA methylation at imprinted regions

Genomic imprinting is an epigenetic phenomenon leading to parental allele-specific expression. Dosage of imprinted genes is crucial for normal development and its dysregulation accounts for several human disorders. This unusual expression pattern is mostly dictated by differences in DNA methylation between parental alleles at specific regulatory elements known as imprinting control regions (ICRs). Although several approaches can be used for methylation inspection, we lack an easy and cost-effective method to simultaneously measure DNA methylation at multiple imprinted regions. Here, we present IMPLICON, a new high-throughput method measuring DNA methylation levels at imprinted regions with base-pair resolution and over 1000-fold coverage. We initially designed IMPLICON to look at ICRs in adult tissues of inbred mice. Then, we validated it in hybrid mice from reciprocal crosses for which we could discriminate methylation profiles in the two parental alleles. Lastly, we developed a human version of IMPLICON and detected imprinting errors in embryonic and induced pluripotent stem cells. We also provide rules and guidelines to adapt this method for investigating the DNA methylation landscape of any set of genomic regions. In summary, IMPLICON is a rapid, cost-effective and scalable method, which could become the gold standard in both imprinting research and diagnostics.

molecular biology↗