Biology subjects

Gribnau, J.

Publications and source records attributed to Gribnau, J..

28 records · Page 2Linked to original sources

bioRxiv2023

XIST dampens X chromosome activity in a SPEN-dependent manner during early human development

XIST long non-coding RNA is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female pre-implantation embryos without triggering X chromosome silencing. The long non-coding RNA XACT co-accumulates with XIST on active Xs and may antagonize XIST function. Here we used human ES cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during pre-implantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and attenuates transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional prior to XCI, confirms the existence of a transient process of X chromosome dosage compensation, and reveals that X chromosome inactivation and dampening rely on the same set of factors.

molecular biology↗

bioRxiv2023

Specific genomic targeting of the RNF12/RLIM E3 ubiquitin ligase selectively programmes developmental transcription

The E3 ubiquitin ligase RNF12/RLIM controls developmental gene expression and is mutated in the X-linked intellectual disability disorder Tonne-Kalscheuer syndrome (TOKAS). However, the mechanisms by which RNF12 E3 ubiquitin ligase activity controls specific gene expression signatures are not known. Here, we show that chromatin forms a regulatory platform for RNF12 substrate ubiquitylation and transcriptional patterning. RNF12 is recruited to specific genomic regions via a distinct consensus sequence motif, which enables targeting to key transcription factor substrate REX1. Mechanistically, RNF12 chromatin recruitment is largely REX1 independent, but is achieved via the conserved basic region (BR) adjacent to the RING domain. This region is critical for REX1 ubiquitylation on chromatin and downstream RNF12-dependent gene regulation. Furthermore, we find that RNF12 N-terminal sequences suppress chromatin recruitment and substrate ubiquitylation, uncovering a previously unappreciated autoinhibitory mechanism that governs genome targeting. Taken together, our results provide insight into mechanisms by which selective substrate targeting of an E3 ubiquitin ligase enables specific programming of gene expression.

cell biology↗

bioRxiv2022

Multi-color dSTORM microscopy in Hormad1-/- spermatocytes reveals alterations in meiotic recombination intermediates and synaptonemal complex structure

Recombinases RAD51 and its meiosis-specific paralog DMC1 accumulate on single-stranded DNA (ssDNA) of programmed DNA double strand breaks (DSBs) in meiosis. Here we used three-color dSTORM microscopy to study how the two recombinases are organized on the ssDNA at individual DSBs, using a mouse model with severe defects in meiotic DSB formation and synapsis: Hormad1-/-. In accordance with previous analyses, we most frequently observed recombinase configurations with 1 DMC1 and 1 RAD51 nanofocus (D1R1), or two DMC1 nanofoci combined with a single RAD51 nanofocus (D2R1), and established that upon synapsis, recombinase nanofoci localized closer to the synaptonemal complex (SYCP3), in both wild type and Hormad1-/- spermatocytes. In wild type, the D1R1:D2R1 frequency ratio decreased over time, but in the knockout, this ratio did not change due to increased D2R1 and decreased D1R1 foci frequency in leptotene. Also, the Hormad1-/- nanofoci were smaller. Nearest neighbor analysis of RAD51/DMC1 nanofoci showed two preferred distances at [~]300 and [~]900 nm in wild type, but only at [~]300 nm in Hormad1-/-. Combined with the lower total number of DSBs in the mutant, this suggests that a preferred distance of 900 nm represents the distance between DSB sites. We propose models with the D1R1 configuration representing a scenario with only one DSB end containing recombinases, and the other end bound by other ssDNA binding proteins, or with both ends loaded by the two recombinases, but in below-resolution proximity. Surprisingly, the data also revealed a hitherto unknown function of HORMAD1 in inhibiting the formation of coils in the synaptonemal complex. SPO11 plays a similar but weaker role in coiling and SYCP1 had the opposite effect. In conclusion, our data show that HORMAD1 affects the lifetime of recombinase-accumulation patterns at meiotic DSBs, as well as the structure of the synaptonemal complex. Author summaryMeiosis is a specialized cell division which generates haploid germ cells. In order to correctly pair homologous chromosomes in the first meiotic prophase, repair of programmed double strand breaks (DSBs) is essential. By unravelling molecular details of the protein assemblies at single DSBs, using super-resolution microscopy, we aim to understand the dynamics of repair intermediates and their functions. To this end, we investigated the localization of the two recombinases RAD51 and DMC1 in wild type and HORMAD1-deficient cells. HORMAD1 is involved in multiple aspects of homologous chromosome association: it regulates formation and repair of DSBs, and it stimulates formation of the synaptonemal complex, the macromolecular protein assembly that connects paired chromosomes. RAD51 and DMC1 enable chromosome pairing by promoting the invasions of the intact chromatids by single-stranded DNA ends that result from DSBs. We found that, in the absence of HORMAD1, RAD51 and DMC1 showed small but significant morphological and positional changes, combined with altered kinetics of specific RAD51/DMC1 configurations. We also observed changes in the structure of the synaptonemal complex in Hormad1-/ spermatocytes. This study contributes to a better understanding of the molecular details of meiotic homologous recombination and the role of HORMAD1 in meiotic prophase.

developmental biology↗

bioRxiv2021

Retrospective analysis of enhancer activity and transcriptome history

Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. Here, we utilize an inducible DCM-RNA-polymerase-subunit-b fusion protein, to label active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. We applied this DCM-time-machine (DCM-TM) technology to study intestinal homeostasis, following enterocyte differentiation back in time, revealing rapid and simultaneous activation of enhancers and nearby genes during intestinal stem cell (ISC) differentiation. We provide new insights in the absorptive-secretory lineage decision in ISC differentiation, and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes in development and differentiation.

developmental biology↗

bioRxiv2021

High throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

BackgroundDNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum-concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. ResultsThe MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell(R) RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QIAsymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (P<0.001). Vacuum-concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4, were validated by quantitative methylation specific PCR. ConclusionsThe MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

genomics↗

bioRxiv2020

SPEN is Required for Xist Upregulation during Initiation of X Chromosome Inactivation

At initiation of X chromosome inactivation (XCI), Xist is monoallelically upregulated from the future inactive X (Xi) chromosome, overcoming repression by its antisense transcript Tsix. Xist recruits various chromatin remodelers, amongst them SPEN, which are involved in silencing of X-linked genes in cis and establishment of the Xi. Here, we show that SPEN plays an important role in the initiation of XCI. Spen null female mouse embryonic stem cells (ESCs) are defective in Xist upregulation upon differentiation. We find that Xist-mediated SPEN recruitment to the Xi chromosome happens very early in XCI, and that SPEN-mediated silencing of the Tsix promoter is required for Xist upregulation. Accordingly, failed Xist upregulation in Spen-/- ESCs can be rescued by concomitant removal of Tsix. These findings indicate that SPEN is not only required for the establishment of the Xi, but is also crucial in the initiation of the XCI process.

developmental biology↗

bioRxiv2018

RLIM enhances BMP signalling mediated fetal lung development in mice

RLIM, also called RNF12, is an X-linked E3 ubiquitin ligase. It is a pleotropic protein acting as a negative regulator of LIM-homeodomain transcription factors and of SMAD7, an antagonist of the BMP and TGF{beta} pathways. Mutations in RLIM are associated with variable congenital malformations in human. The molecular mechanism causing these malformations is still poorly understood.\n\nIn this study, we used a conventional knockout (KO) mouse model to phenotypically characterize male mutants and to investigate the effect of Rlim mutation on BMP pathway signalling. Only 50% of KO male neonates survive and are significantly smaller. We show that Rlim deletion alters lung branching and maturation resulting in severe respiratory distress and neonatal death. Except for a single case of cardiac interventricular septal defect, no other malformations that mimic the human condition were observed. Western blot analysis shows that RLIM deficiency is associated with an increase of its target SMAD7 levels and a loss of SMAD1/5/9 activation. Interestingly, SMAD5 levels were diminished, suggesting another regulatory loop between RLIM and the BMP pathway.\n\nIn conclusion, RLIM is important for lung development in mice and exerts this function in part through intracellular propagation of BMP signalling.\n\nAuthor summaryRLIM is an X-linked gene encoding an enzyme that regulates a number of proteins including SMAD7, an antagonist of BMP pathway. RLIM mutations are associated with intellectual disability and several congenital malformations in boys. In mice, RLIM is thought to be nonessential for embryos development. Using a mouse model in which Rlim is deleted, we show that 50% of males carrying the deletion display respiratory distress and die short after birth. No congenital malformations mimicking the human phenotype have been observed. Histological analysis of lung samples show that Rlim is critical for late lung development and maturation. The analysis of lung samples by western blot, a technique that quantifies proteins, is consistent with a dysregulation of BMP pathway. These finding confirm the importance of RLIM for murine embryos development and provide a link between human RLIM pathology and BMP pathway.

genetics↗

bioRxiv2017

Allele-specific control of replication timing and genome organization during development

DNA replication occurs in a defined temporal order known as the replication-timing (RT) program. RT is regulated during development in discrete chromosomal units, coordinated with transcriptional activity and 3D genome organization. Here, we derived distinct cell types from F1 hybrid musculus X castaneus mouse crosses and exploited the high single nucleotide polymorphism (SNP) density to characterize allelic differences in RT (Repli-seq), genome organization (Hi-C and promoter-capture Hi-C), gene expression (nuclear RNA-seq) and chromatin accessibility (ATAC-seq). We also present HARP: a new computational tool for sorting SNPs in phased genomes to efficiently measure allele-specific genome-wide data. Analysis of 6 different hybrid mESC clones with different genomes (C57BL/6, 129/sv and CAST/Ei), parental configurations and gender revealed significant RT asynchrony between alleles across ~12 % of the autosomal genome linked to sub-species genomes but not to parental origin, growth conditions or gender. RT asynchrony in mESCs strongly correlated with changes in Hi-C compartments between alleles but not SNP density, gene expression, imprinting or chromatin accessibility. We then tracked mESC RT asynchronous regions during development by analyzing differentiated cell types including extraembryonic endoderm stem (XEN) cells, 4 male and female primary mouse embryonic fibroblasts (MEFs) and neural precursors (NPCs) differentiated in vitro from mESCs with opposite parental configurations. Surprisingly, we found that RT asynchrony and allelic discordance in Hi-C compartments seen in mESCs was largely lost in all differentiated cell types, coordinated with a more uniform Hi-C compartment arrangement, suggesting that genome organization of homologues converges to similar folding patterns during cell fate commitment.

cell biology↗

bioRxiv2017

Genetic mapping of species differences via "in vitro crosses" in mouse embryonic stem cells

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic dissection of trait variation. Here we describe a simple, robust and transgene-free technique to make \"in vitro crosses\" in hybrid mouse embryonic stem cells by inducing random mitotic crossovers with the drug ML216, which inhibits Bloom syndrome (BLM). Starting with an interspecific hybrid (between Mus musculus and Mus spretus) embryonic stem cell line spanning 1.5 million years of divergence, we demonstrate the feasibility of mapping enzymatic differences across species within weeks and the possibility of re-deriving whole mice. Our work shows how in vitro crosses can overcome major bottlenecks like hybrid sterility in traditional mouse breeding to address fundamental questions in evolutionary biology.\n\nImpact StatementBy mixing hybrid mouse genomes in stem cells via mitotic recombination, genetic mapping and hybrid mosaic mice can be achieved in weeks, even across species barriers.

genetics↗

bioRxiv2017

X-chromosome inactivation and its implications for human disease

In humans and other mammals, female cells carry two X-chromosomes, whereas male cells carry a single X and Y-chromosome. To achieve an equal expression level of X-linked genes in both sexes, a dosage compensation mechanism evolved, which results in transcriptional silencing of one X-chromosome in females. X chromosome inactivation (XCI) is random with respect to the parental origin of the X, occurs early during embryonic development, and is then stably maintained through a near infinite number of cell divisions. As a result of this, every female individual consists of a mosaic of two different cell populations, in which either the maternally or paternally derived X-chromosome is inactivated. As the X-chromosome harbors more than a thousand genes, of which many are implicated in human disease when mutated, this mosaicism has important disease implications. Whereas X-linked disorders are usually more severe in hemizygous males harboring a single X-chromosome, a more variable phenotype is observed in females. This variability is a direct consequence of the XCI-mosaicism, and is affected by the randomness of the XCI process. Here we review the latest insights into the regulation of this important female specific process, and discuss mechanisms that influence mosaicism in females, with a focus on the clinical consequences related to X-linked diseases in females.

genetics↗