Biology subjects

Reinholdt, L.

Publications and source records attributed to Reinholdt, L..

7 recordsLinked to original sources

bioRxiv2025

The mouse pangenome reveals the structural complexity of the murine protein coding landscape

We present the first mouse pangenome consisting of 17 high-quality inbred mouse strain genomes with complete annotation. This collection includes 12 widely used classical laboratory strains and 5 wild-derived strains. We have fully resolved previously incomplete genomic regions, including the major histocompatibility complex (MHC), the defensin cluster, T-cell receptor, and Ly49 complexes. Hundreds of non-reference genes identified in previous publications not found in GRCm39, like Defa1, Raet1a, and Klra20 (Ly49T), were localised in the new reference genomes. We conducted the first genome-wide scan of variable number tandem repeats (VNTRs) within the coding regions of mice, identifying over 400 genes with VNTR polymorphisms up to more than 600 repeat copies and repeat units reaching 990 nucleotides. Our strain-specific annotations enhance RNA-Seq analyses, as demonstrated in PWK/PhJ, where we observed a 5.1% improvement in read mapping and expression level differences in 2.1% of coding genes compared to using GRCm39.

genomics↗

bioRxiv2024

The structural diversity of telomeres and centromeres across mouse subspecies revealed by complete assemblies

It is over twenty years since the publication of the C57BL/6J mouse reference genome, which has been a key catalyst for understanding mammalian disease biology. However, the mouse reference genome still lacks telomeres and centromeres, contains 281 chromosomal sequence gaps, and only partially represents many biomedically relevant loci. We present the first T2T mouse genomes for two key inbred strains, C57BL/6J and CAST/EiJ. These T2T genomes reveal significant variability in telomere and centromere sizes and structural organisation. We add an additional 213 Mbp of novel sequence to the reference genome containing 517 protein-coding genes. We examined two important but incomplete loci in the mouse genome - the pseudoautosomal region (PAR) on the sex chromosomes and KRAB zinc finger proteins (KZFPs) loci. We identified distant locations of the PAR boundary, different copy number and sizes of segmental duplications, and a multitude of amino acid substitution mutations in PAR genes.

genomics↗

bioRxiv2023

The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3 end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3 processing are deployed across human tissues, with nearly half of multitranscript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

genomics↗

bioRxiv2021

An allelic series of spontaneous mutations in Rorb cause a gait phenotype, retinal abnormalities, and transcriptomic changes relevant to human neurodevelopmental conditions

Rorb encodes the Retinoic Acid Receptor-related orphan receptor beta. Mutations in either of the two transcripts of Rorb cause defects in multiple systems, including abnormal photoreceptor abundance and morphology in the retina and a characteristic "high-stepper" or "duck-like" gait arising from dysfunction of interneurons in the spinal cord. Rorb is also important for cortical development and cell fate specification in mice. Rorb variants segregate with epilepsy and comorbidities such as intellectual disability in numerous clinical cases. Here we describe five mouse strains with spontaneous mutations in Rorb identified by their gait phenotype. These mutations affect different domains and isoforms of Rorb, which correspond to the spectrum of anatomical and physiological phenotypes exhibited by these mice. Gene set analysis in Rorb mutants implicates pathways associated with development and nervous system function, and differential gene expression analysis indicates changes in numerous genes related to epilepsy, bipolar disorder, and autism spectrum disorder (ASD). Many of these genes and their protein products are known to interact during synapse formation and neuronal activity. These findings further illuminate the role of Rorb in nervous system development, provide further evidence for an association between Rorb and several neurological conditions, and describe an allelic series of Rorb mutant mice that will be useful for dissecting thalamocortical afferent (TCA) development, neural cell fate determination, and as animal models exhibiting transcriptomic shifts in neurological conditions such as epilepsy, bipolar disorder, and ASD. {blacksquare}Five mutant mice with a characteristic high-stepper gait phenotype have mutations in Rorb {blacksquare}These allelic series mutations manifest in a spectrum of anatomical and physiological abnormalities {blacksquare}Gene expression data suggest involvement of pathways related to neurodevelopmental disorders

genetics↗

bioRxiv2021

Deficiency of SYCP3-related XLR3 Disrupts the Initiation of Meiotic Sex Chromosome Inactivation in Mouse Spermatogenesis

In mammals, the X and Y chromosomes share only small regions of homology called pseudo-autosomal regions (PAR) where pairing and recombination in spermatocytes can occur. Consequently, the sex chromosomes remain largely unsynapsed during meiosis I and are sequestered in a nuclear compartment known as the XY body where they are transcriptionally silenced in a process called meiotic sex chromosome inactivation (MSCI). MSCI mirrors meiotic silencing of unpaired chromatin (MSUC), the sequestration and transcriptional repression of unpaired DNA observed widely in eukaryotes. MSCI is initiated by the assembly of the axial elements of the synaptonemal complex (SC) comprising the structural proteins SYCP2 and SYCP3 followed by the ordered recruitment of DNA Damage Response (DDR) factors to effect gene silencing. However, the precise mechanism of how unsynapsed chromatin is detected in meiocytes is poorly understood. The sex chromosomes in eutherian mammals harbor multiple clusters of SYCP3-like amplicons comprising the Xlr gene family, only a handful of which have been functionally studied. We used a shRNA-transgenic mouse model to create a deficiency in the testis-expressed multicopy Xlr3 genes to investigate their role in spermatogenesis. Here we show that knockdown of Xlr3 in mice leads to spermatogenic defects and a skewed sex ratio that can be traced to MSCI breakdown. Spermatocytes deficient in XLR3 form the XY body and the SC axial elements therein, but are compromised in their ability to recruit DDR components to the XY body. Author SummaryA key event in the production of sperm is the pairing and synapsis of homologous chromosome pairs to facilitate genetic recombination during the first meiotic division. Chromosomal abnormalities that undermine pairing and synapsis in spermatocytes trigger a checkpoint that leads to removal of the abnormal cells via programmed cell death. In mammals, the sex chromosomes, X and Y, lack homology through most of their length and remain largely unpaired. To avoid triggering the checkpoint the X and Y are sequestered in a specialized nuclear compartment called the XY body. DNA damage response (DDR) proteins are recruited to the XY body in a highly ordered progression leading to repression of all gene transcription from the sex chromosomes. We show that knocking down expression of the X-linked SYCR3-like gene, Xlr3, disrupts sex chromosome gene silencing by interfering with recruitment of DDR factors, leading to compromised sperm production.

genetics↗

bioRxiv2019

The genome of C57BL/6J \"Eve\", the mother of the laboratory mouse genome reference strain

Isogenic laboratory mouse strains are used to enhance reproducibility as individuals within a strain are essentially genetically identical. For the most widely used isogenic strain, C57BL/6, there is also a wealth of genetic, phenotypic, and genomic data, including one of the highest quality reference genomes (GRCm38.p6). However, laboratory mouse strains are living reagents and hence genetic drift occurs and is an unavoidable source of accumulating genetic variability that can have an impact on reproducibility over time. Nearly 20 years after the first release of the mouse reference genome, individuals from the strain it represents (C57BL/6J) are at least 26 inbreeding generations removed from the individuals used to generate the mouse reference genome. Moreover, C57BL/6J is now maintained through the periodic reintroduction of mice from cryopreserved embryo stocks that are derived from a single breeder pair, aptly named C57BL/6J Adam and Eve. To more accurately represent the genome of todays C57BL/6J mice, we have generated a de novo assembly of the C57BL/6J Eve genome (B6Eve) using high coverage, long-read sequencing, optical mapping, and short-read data. Using these data, we addressed recurring variants observed in previous mouse studies. We have also identified structural variations that impact coding sequences, closed gaps in the mouse reference assembly, some of which are in genes, and we have identified previously unannotated coding sequences through long read sequencing of cDNAs. This B6Eve assembly explains discrepant observations that have been associated with GRCm38-based analyses, and has provided data towards a reference genome that is more representative of the C57BL/6J mice that are in use today.

genomics↗

bioRxiv2018

Multiple laboratory mouse reference genomes define strain specific haplotypes and novel functional loci

The most commonly employed mammalian model organism is the laboratory mouse. A wide variety of genetically diverse inbred mouse strains, representing distinct physiological states, disease susceptibilities, and biological mechanisms have been developed over the last century. We report full length draft de novo genome assemblies for 16 of the most widely used inbred strains and reveal for the first time extensive strain-specific haplotype variation. We identify and characterise 2,567 regions on the current Genome Reference Consortium mouse reference genome exhibiting the greatest sequence diversity between strains. These regions are enriched for genes involved in defence and immunity, and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. Several immune related loci, some in previously identified QTLs for disease response have novel haplotypes not present in the reference that may explain the phenotype. We used these genomes to improve the mouse reference genome resulting in the completion of 10 new gene structures, and 62 new coding loci were added to the reference genome annotation. Notably this high quality collection of genomes revealed a previously unannotated gene (Efcab3-like) encoding 5,874 amino acids, one of the largest known in the rodent lineage. Interestingly, Efcab3-like-/- mice exhibit severe size anomalies in four regions of the brain suggesting a mechanism of Efcab3-like regulating brain development.

genomics↗