Mouse Chromosome Research Articles

Identification of a New QTL Region on Mouse Chromosome 1 Responsible for Male Hypofertility: Phenotype Characterization and Candidate Genes.

Male fertility disorders often have their origin in disturbed spermatogenesis, which can be induced by genetic factors. In this study, we used interspecific recombinant congenic mouse strains (IRCS) to identify genes responsible for male infertility. Using ultrasonography, in vivo and in vitro fertilization (IVF) and electron microscopy, the phenotyping of several IRCS carrying mouse chromosome 1 segments of Mus spretus origin revealed a decrease in the ability of sperm to fertilize. This teratozoospermia included the abnormal anchoring of the acrosome to the nucleus and a persistence of residual bodies at the level of epididymal sperm midpiece. We identified a quantitative trait locus (QTL) responsible for these phenotypes and we have proposed a short list of candidate genes specifically expressed in spermatids. The future functional validation of candidate genes should allow the identification of new genes and mechanisms involved in male infertility.

PAR-TERRA is the main contributor to telomeric repeat-containing RNA transcripts in normal and cancer mouse cells.

Telomeric repeat-containing RNA (TERRA) molecules play important roles at telomeres, from heterochromatin regulation to telomerase activity control. In human cells, TERRA is transcribed from subtelomeric promoters located on most chromosome ends and associates with telomeres. The origin of mouse TERRA molecules is, however, unclear, as transcription from the pseudoautosomal PAR locus was recently suggested to account for the vast majority of TERRA in embryonic stem cells (ESC). Here, we confirm the production of TERRA from both the chromosome 18q telomere and the PAR locus in mouse embryonic fibroblasts, ESC, and various mouse cancer and immortalized cell lines, and we identify two novel sources of TERRA on mouse chromosome 2 and X. Using various approaches, we show that PAR-TERRA molecules account for the majority of TERRA transcripts, displaying an increase of two to four orders of magnitude compared to the telomeric 18q transcript. Finally, we present a SILAC-based pull-down screen revealing a large overlap between TERRA-interacting proteins in human and mouse cells, including PRC2 complex subunits, chromatin remodeling factors, DNA replication proteins, Aurora kinases, shelterin complex subunits, Bloom helicase, Coilin, and paraspeckle proteins. Hence, despite originating from distinct genomic regions, mouse and human TERRA are likely to play similar functions in cells.

22q11.2 deletion syndrome and schizophrenia.

22q11.2 deletion is a common microdeletion that causes an array of developmental defects including 22q11.2 deletion syndrome (22q11DS) or DiGeorge syndrome and velocardiofacial syndrome. About 30% of patients with 22q11.2 deletion develop schizophrenia. Mice with deletion of the ortholog region in mouse chromosome 16qA13 exhibit schizophrenia-like abnormal behaviors. It is suggested that the genes deleted in 22q11DS are involved in the pathogenesis of schizophrenia. Among these genes, COMT, ZDHHC8, DGCR8, and PRODH have been identified as schizophrenia susceptibility genes. And DGCR2 is also found to be associated with schizophrenia. In this review, we focused on these five genes and reviewed their functions in the brain and the potential pathophysiological mechanisms in schizophrenia, which will give us a deeper understanding of the pathology of schizophrenia.

Comparative analysis of the down syndrome hippocampal non-coding RNA transcriptomes using a mouse model.

Down syndrome (DS), caused by trisomy 21, is the most common human chromosomal disorder. Hippocampal abnormalities have been believed to be responsible for the DS developmental cognitive deficits. Cumulative evidences indicated that non-coding RNAs (ncRNAs) participated in brain development and function. Currently, few was known whether dysregulated ncRNAs existed in DS whether the dysregulated ncRNAs played important pathology roles in DS. The purpose of this study was generating an overview map of the dysregulated ncRNAs in DS, including the microRNA (miRNA), long ncRNA (lncRNA) and circular RNA (circRNAs). DS mouse models are invaluable tools for further mechanism and therapy studies. The well-studied DS mouse model Dp(16)1/Yey was used in this study as it contains the trisomy of the whole human chromosome 21 syntenic region on mouse chromosomes 16. Hippocampi were isolated from pups of seven-days-old. Libraries for miRNA, lncRNA and circRNAs were constructed separately, and the next generation sequencing method was utilized. Differentially expressed (DE) miRNAs, lncRNAs and circRNAs were reported. Relative few regulating relationship were found between the DE miRNAs and DE mRNAs. LncRNAs originated from the trisomic regions expressed in clusters, but not all of them were 1.5-fold increased expressed. Dramatic DE circular RNAs were found in the DS hippocampus. The host genes of the DE circRNAs were enriched on functions which were well-known impaired in DS, e.g. long-term-potentiation, glutamatergic synapse, and GABAergic synapse. We generated the first DS developmental hippocampal ncRNA transcriptome map. This work laid foundations for further investigations on role of ncRNAs in hippocampal functions.

Large domains of heterochromatin direct the formation of short mitotic chromosome loops.

During mitosis chromosomes reorganise into highly compact, rod-shaped forms, thought to consist of consecutive chromatin loops around a central protein scaffold. Condensin complexes are involved in chromatin compaction, but the contribution of other chromatin proteins, DNA sequence and histone modifications is less understood. A large region of fission yeast DNA inserted into a mouse chromosome was previously observed to adopt a mitotic organisation distinct from that of surrounding mouse DNA. Here, we show that a similar distinct structure is common to a large subset of insertion events in both mouse and human cells and is coincident with the presence of high levels of heterochromatic H3 lysine nine trimethylation (H3K9me3). Hi-C and microscopy indicate that the heterochromatinised fission yeast DNA is organised into smaller chromatin loops than flanking euchromatic mouse chromatin. We conclude that heterochromatin alters chromatin loop size, thus contributing to the distinct appearance of heterochromatin on mitotic chromosomes.

Impairment of spatial memory accuracy improved by Cbr1 copy number resumption and GABAB receptor-dependent enhancement of synaptic inhibition in Down syndrome model mice

Down syndrome is a complex genetic disorder caused by the presence of three copies of the chromosome 21 in humans. The most common models, carrying extra-copies of overlapping fragments of mouse chromosome 16 that is syntenic to human chromosome 21, are Ts2Cje, Ts1Cje and Ts1Rhr mice. In electrophysiological analyses using hippocampal slices, we found that the later phase of the depolarization during tetanic stimulation, which was regulated by GABAB receptors, was significantly smaller in Ts1Cje and Ts2Cje mice than that in WT controls but not in Ts1Rhr mice. Furthermore, isolated GABAB receptor-mediated inhibitory synaptic responses were larger in Ts1Cje mice. To our knowledge, this is the first report that directly shows the enhancement of GABAB receptor-mediated synaptic currents in Ts1Cje mice. These results suggest that GABAB receptor-mediated synaptic inhibition was enhanced in Ts1Cje and Ts2Cje mice but not in Ts1Rhr mice. The Cbr1 gene, which is present in three copies in Ts1Cje and Ts2Cje but not in Ts1Rhr, encodes carbonyl reductase that may facilitate GABAB-receptor activity through a reduction of prostaglandin E2 (PGE2). Interestingly, we found that a reduction of PGE2 and an memory impairment in Ts1Cje mice were alleviated when only Cbr1 was set back to two copies (Ts1Cje;Cbr1+/+/−). However, the GABAB receptor-dependent enhancement of synaptic inhibition in Ts1Cje was unaltered in Ts1Cje;Cbr1+/+/− mice. These results indicate that Cbr1 is one of the genes responsible for DS cognitive impairments and the gene(s) other than Cbr1, which is included in Ts1Cje but not in Ts1Rhr, is responsible for the GABAB receptor-dependent over-inhibition.

Sex differences in kappa opioid receptor antinociception is influenced by the number of X chromosomes in mouse.

Kappa opioid receptor (KOR) agonists produce robust analgesia with minimal abuse liability and are considered promising pharmacological agents to manage chronic pain and itch. The KOR system is also notable for robust differences between the sexes, with females exhibiting lower analgesic response than males. Sexually dimorphic traits can be due to either the influence of gonadal hormones during development or adulthood, or due to the complement of genes expressed on the X or Y chromosome. Previous studies examining sex differences in KOR antinociception have relied on surgical or pharmacological manipulation of the gonads to determine whether sex hormones influence KOR function. While there are conflicting reports whether gonadal hormones influence KOR function, no study has examined these effects in context with sex chromosomes. Here, we use two genetic mouse models, the four core genotypes and XY*, to isolate the chromosomal and hormonal contributions to sex differences in KOR analgesia. Mice were treated with systemic KOR agonist (U50,488H) and thermal analgesia measured in the tail withdrawal assay. We found that KOR antinociception was influenced predominantly by the number of the X chromosomes. These data suggest that the dose and/or parental imprint on X gene(s) contribute significantly to the sexually dimorphism in KOR analgesia.

Type 2 diabetes susceptibility genes on mouse chromosome 11 under high sucrose environment

BackgroundBoth genetic and environmental factors contribute to type 2 diabetes development. We used consomic mice established from an animal type 2 diabetes model to identify susceptibility genes that contribute to type 2 diabetes development under specific environments. We previously established consomic strains (C3H-Chr 11NSY and C3H-Chr 14NSY) that possess diabetogenic Chr 11 or 14 of the Nagoya-Shibata-Yasuda (NSY) mouse, an animal model of spontaneous type 2 diabetes, in the genetic background of C3H mice. To search genes contribute to type 2 diabetes under specific environment, we first investigated whether sucrose administration deteriorates type 2 diabetes-related traits in the consomic strains. We dissected loci on Chr 11 by establishing congenic strains possessing different segments of NSY-derived Chr 11 under sucrose administration.ResultsIn C3H-Chr 11NSY mice, sucrose administration for 10 weeks deteriorated hyperglycemia, insulin resistance, and impaired insulin secretion, which is comparable to NSY mice with sucrose. In C3H-Chr 14NSY mice, sucrose administration induced glucose intolerance, but not insulin resistance and impaired insulin secretion. To dissect the gene(s) existing on Chr 11 for sucrose-induced type 2 diabetes, we constructed four novel congenic strains (R1, R2, R3, and R4) with different segments of NSY-derived Chr 11 in C3H mice. R2 mice showed marked glucose intolerance and impaired insulin secretion comparable to C3H-Chr 11NSY mice. R3 and R4 mice also showed impaired insulin secretion. R4 mice showed significant decreases in white adipose tissue, which is in the opposite direction from parental C3H-Chr 11NSY and NSY mice. None of the four congenic strains showed insulin resistance.ConclusionsGenes on mouse Chr 11 could explain glucose intolerance, impaired insulin secretion, insulin resistance in NSY mice under sucrose administration. Congenic mapping with high sucrose environment localized susceptibility genes for type 2 diabetes associated with impaired insulin secretion in the middle segment (26.0–63.4 Mb) of Chr 11. Gene(s) that decrease white adipose tissue were mapped to the distal segment of Chr 11. The identification of diabetogenic gene on Chr 11 in the future study will facilitate precision medicine in type 2 diabetes by controlling specific environments in targeted subjects with susceptible genotypes.

Obesity of G2e3 Knockout Mice Suggests That Obesity-Associated Variants Near Human G2E3 Decrease G2E3 Activity.

PurposeIn humans, single nucleotide polymorphisms (SNPs) near the adjacent protein kinase D1 (PRKD1) and G2/M-phase-specific E3 ubiquitin protein ligase (G2E3) genes on chromosome 14 are associated with obesity. To date, no published evidence links inactivation of either gene to changes in body fat. These two genes are also adjacent on mouse chromosome 12. Because obesity genes are highly conserved between humans and mice, we analyzed body fat in adult G2e3 and Prkd1 knockout (KO) mice to determine whether inactivating either gene leads to obesity in mice and, by inference, probably in humans.MethodsThe G2e3 and Prkd1 KO lines were generated by gene trapping and by homologous recombination methodologies, respectively. Body fat was measured by DEXA in adult mice fed chow from weaning and by QMR in a separate cohort of mice fed high-fat diet (HFD) from weaning. Glucose homeostasis was evaluated with oral glucose tolerance tests (OGTTs) performed on adult mice fed HFD from weaning.ResultsBody fat was increased in multiple cohorts of G2e3 KO mice relative to their wild-type (WT) littermates. When data from all G2e3 KO (n=32) and WT (n=31) mice were compared, KO mice showed increases of 11% in body weight (P<0.01), 65% in body fat (P<0.001), 48% in % body fat (P<0.001), and an insignificant 3% decrease in lean body mass. G2e3 KO mice were also glucose intolerant during an OGTT (P<0.05). In contrast, Prkd1 KO and WT mice had comparable body fat levels and glucose tolerance.ConclusionSignificant obesity and glucose intolerance were observed in G2e3, but not Prkd1, KO mice. The conservation of obesity genes between mice and humans strongly suggests that the obesity-associated SNPs located near the human G2E3 and PRKD1 genes are linked to variants that decrease the amount of functional human G2E3.

Temple syndrome and Kagami-Ogata syndrome: clinical presentations, genotypes, models and mechanisms.

Temple syndrome (TS) and Kagami-Ogata syndrome (KOS) are imprinting disorders caused by absence or overexpression of genes within a single imprinted cluster on human chromosome 14q32. TS most frequently arises from maternal UPD14 or epimutations/deletions on the paternal chromosome, whereas KOS most frequently arises from paternal UPD14 or epimutations/deletions on the maternal chromosome. In this review, we describe the clinical symptoms and genetic/epigenetic features of this imprinted region. The locus encompasses paternally expressed protein-coding genes (DLK1, RTL1 and DIO3) and maternally expressed lncRNAs (MEG3/GTL2, RTL1as and MEG8), as well as numerous miRNAs and snoRNAs. Control of expression is complex, with three differentially methylated regions regulating germline, placental and tissue-specific transcription. The strong conserved synteny between mouse chromosome 12aF1 and human chromosome 14q32 has enabled the use of mouse models to elucidate imprinting mechanisms and decipher the contribution of genes to the symptoms of TS and KOS. In this review, we describe relevant mouse models and highlight their value to better inform treatment options for long-term management of TS and KOS patients.

Sperm quality, aggressiveness and generation turnover may facilitate unidirectional Y chromosome introgression across the European house mouse hybrid zone.

The widespread and locally massive introgression of Y chromosomes of the eastern house mouse (Mus musculus musculus) into the range of the western subspecies (M. m. domesticus) in Central Europe calls for an explanation of its underlying mechanisms. Given the paternal inheritance pattern, obvious candidates for traits mediating the introgression are characters associated with sperm quantity and quality. We can also expect traits such as size, aggression or the length of generation cycles to facilitate the spread. We have created two consomic strains carrying the non-recombining region of the Y chromosome of the opposite subspecies, allowing us to study introgression in both directions, something impossible in nature due to the unidirectionality of introgression. We analyzed several traits potentially related to male fitness. Transmission of the domesticus Y onto the musculus background had negative effects on all studied traits. Likewise, domesticus males possessing the musculus Y had, on average, smaller body and testes and lower sperm count than the parental strain. However, the same consomic males tended to produce less- dissociated sperm heads, to win more dyadic encounters, and to have shorter generation cycles than pure domesticus males. These data suggest that the domesticus Y is disadvantageous on the musculus background, while introgression in the opposite direction can confer a recognizable, though not always significant, selective advantage. Our results are thus congruent with the unidirectional musculus → domesticus Y chromosome introgression in Central Europe. In addition to some previous studies, they show this to be a multifaceted phenomenon demanding a multidisciplinary approach.

Chromosome-scale scaffolds for the Chinese hamster reference genome assembly to facilitate the study of the CHO epigenome.

The Chinese hamster genome serves as a reference genome for the study of Chinese hamster ovary (CHO) cells, the preferred host system for biopharmaceutical production. Recent re-sequencing of the Chinese hamster genome resulted in the RefSeq PICR meta-assembly, a set of highly accurate scaffolds that filled over 95% of the gaps in previous assembly versions. However, these scaffolds did not reach chromosome-scale due to the absence of long-range scaffolding information during the meta-assembly process. Here, long-range scaffolding of the PICR Chinese hamster genome assembly was performed using high-throughput chromosome conformation capture (Hi-C). This process resulted in a new "PICRH" genome, where 97% of the genome is contained in 11 mega-scaffolds corresponding to the Chinese hamster chromosomes (2n = 22) and the total number of scaffolds is reduced by three-fold from 1,830 scaffolds in PICR to 647 in PICRH. Continuity was improved while preserving accuracy, leading to quality scores higher than recent builds of mouse chromosomes and comparable to human chromosomes. The PICRH genome assembly will be an indispensable tool for designing advanced genetic engineering strategies in CHO cells and enabling systematic examination of genomic and epigenomic instability through comparative analysis of CHO cell lines on a common set of chromosomal coordinates.

Genetic analysis of osteoblast activity identifies Zbtb40 as a regulator of osteoblast activity and bone mass.

Osteoporosis is a genetic disease characterized by progressive reductions in bone mineral density (BMD) leading to an increased risk of fracture. Over the last decade, genome-wide association studies (GWASs) have identified over 1000 associations for BMD. However, as a phenotype BMD is challenging as bone is a multicellular tissue affected by both local and systemic physiology. Here, we focused on a single component of BMD, osteoblast-mediated bone formation in mice, and identified associations influencing osteoblast activity on mouse Chromosomes (Chrs) 1, 4, and 17. The locus on Chr. 4 was in an intergenic region between Wnt4 and Zbtb40, homologous to a locus for BMD in humans. We tested both Wnt4 and Zbtb40 for a role in osteoblast activity and BMD. Knockdown of Zbtb40, but not Wnt4, in osteoblasts drastically reduced mineralization. Additionally, loss-of-function mouse models for both genes exhibited reduced BMD. Our results highlight that investigating the genetic basis of in vitro osteoblast mineralization can be used to identify genes impacting bone formation and BMD.

Genomic Structure, Evolutionary Origins, and Reproductive Function of a Large Amplified Intrinsically Disordered Protein-Coding Gene on the X Chromosome (Laidx) in Mice.

Mouse sex chromosomes are enriched for co-amplified gene families, present in tens to hundreds of copies. Co-amplification of Slx/Slxl1 on the X chromosome and Sly on the Y chromosome are involved in dose-dependent meiotic drive, however the role of other co-amplified genes remains poorly understood. Here we demonstrate that the co-amplified gene family on the X chromosome, Srsx, along with two additional partial gene annotations, is actually part of a larger transcription unit, which we name Laidx. Laidx is harbored in a 229 kb amplicon that represents the ancestral state as compared to a 525 kb Y-amplicon containing the rearranged Laidy. Laidx contains a 25,011 nucleotide open reading frame, predominantly expressed in round spermatids, predicted to encode an 871 kD protein. Laidx has orthologous copies with the rat and also the 825-MY diverged parasitic Chinese liver fluke, Clonorchis sinensis, the likely result of a horizontal gene transfer of rodent Laidx to an ancestor of the liver fluke. To assess the male reproductive functions of Laidx, we generated mice carrying a multi-megabase deletion of the Laidx-ampliconic region. Laidx-deficient male mice do not show detectable reproductive defects in fertility, fecundity, testis histology, and offspring sex ratio. We speculate that Laidx and Laidy represent a now inactive X vs. Y chromosome conflict that occurred in an ancestor of present day mice.

Subcongenic Analysis of Quantitative Trait Loci Conferring Obesity and Hypercholesterolemia on Mouse Chromosome 1

Abstract Objectives The TALLYHO (TH) mouse is a polygenic model for obesity, type 2 diabetes and hyperlipidemia. We previously established a congenic mouse strain with TH donor segment on chromosome (Chr) 1 in a C57BL/6 J (B6) background that harbors quantitative trait loci (QTL) conferring obesity and hypercholesterolemia (The B6.TH-Chr1–128Mb). The congenic mouse developed increased adiposity and hypercholesterolemia and demonstrated that distal segment of Chr 1 from TH genome is necessary to cause both phenotypes. With the goal to identify the underlying genes regulating body fat and plasma cholesterol levels on mouse Chr 1, in this study we generated and characterized subcongenic lines with smaller TH donor segments. Methods Using the original B6.TH-Chr1–128Mb congenic mouse, we fixed three overlapping subcongenic lines over the distal region of Chr 1; one containing the full distal region of TH donor genome (39Mb in size) (B6.TH-Chr1–39Mb) and two nested overlapping lines (B6.TH-Chr1–30Mb and B6.TH-Chr1–20Mb). For mapping, all three lines of subcongenic mice and control B6 mice were weaned onto either chow or high fat (HF) diets at 4 weeks of age, and maintained on the respective diets. At 14 weeks of age, body composition including fat and lean mass was measured using EchoMRI-100 whole body composition analyzer, and plasma total cholesterol levels were determined using colorimetric assays. Data were analyzed by ANOVA using GraphPad Prism 8. Results On chow, body fat mass was not significantly different among all subcongenic mice and B6 mice. On HF diets, on the other hand, all three subcongenic lines of mice had significantly larger fat mass than B6 mice without genotype differences. These results suggest that the obesity candidate region is most likely located within the unique segment retained in B6.TH-Chr1–20Mb mice, delimited from the markers D1Mit105 and D1Mit361. Plasma total cholesterol levels were also significantly higher in B6.TH-Chr1–20Mb mice than them in B6 mice both on chow and HF diets. Conclusions We have created subcongenic lines, with smaller TH donor segments from the Chr 1 congenic interval, which develop detectable phenotypes, thus narrowing target intervals for positional cloning. Funding Sources This work was supported, in part, AHA 18AIREA33960437, NIH 1 R15 DK113604–01A1.

SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat‐PCH) in mouse pre‐implantation embryos. Remarkably, pat‐PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2‐modified CBX2‐containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3‐specific chaperone DAXX to pat‐PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis‐segregation. Complementation assays show that DAXX‐mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development.

SAT-632 Involvement of NF-κB-p65 in BAG3 Regulation After Stress Stimuli

We previously identified the limb salvage QTL1 (LSQ-1) on mouse chromosome 7 as a locus that offered protection against ischemic injury following induction of hind limb ischemia (HLI) a model of experimental peripheral arterial disease (PAD) in mice. To better understand the role of the LSQ-1 locus in post ischemic adaptation we characterized several genes within this locus and identified a number of genes that were important in tissue adaptation to ischemia, including BCL2-associated athanogene 3 (BAG3). BAG3 is an anti-apoptotic protein that plays an important role in cell survival through the regulation of autophagy. BAG3 expression is induced in the gastrocnemius muscles of mice after hind limb ischemia but how ischemia regulates BAG3 expression is poorly understood. Additionally, the activation of NF-κB transcription factor is essential for cell survival after stress stimuli. We hypothesized that BAG3 upregulation following stress stimuli is regulated by NF-κB. We determined whether NF-κB is involved in BAG3-mediated survival of primary human skeletal muscle cells (HSMC) during ischemia and diabetic conditions. Within 6 hours of treatment, ischemia induced BAG3 mRNA (no ischemia vs ischemia: 1.0 ±0.09 vs 1.41±0.02; p<0.01) and protein expression (BAG3/total protein, no ischemia vs ischemia 1.0±0.01 vs 1.38±0.06; p<0.01). Knockdown of BAG3 expression by shRNA induced early cell damage in HMSC under ischemic conditions as measured by HMGB1 expression (HMGB1/total protein; control plasmid vs shRNA, 1.0±0.09 vs 1.71±0.04; p<0.01). Knockdown of p65 subunit of the NF-κB by shRNA significantly diminished BAG3 mRNA expression after ischemia (control plasmid vs shRNA: 2.11±0.18 vs 1.48±0.05; p<0.05). Moreover, treatment of HSMC with 750 uM palmitic acid (PA) and 100nM insulin for 3 days to mimic diabetic conditions significantly increased the expression of BAG3 mRNA (control vs PAL+Ins, 1.0±0.14 vs 2.27±0.08; p<0.01) and protein (BAG3/total protein, control vs PAL+Ins, 1.0±0.03 vs 1.39±0.11, p<0.01). Knocking down p65 attenuated these increase in BAG3 mRNA (PAL+Ins vs shRNA+PAL+Ins, 2.27±0.08 vs 1.56±0.02 p<0.01) and protein (BAG3/total protein; PAL+Ins vs shRNA+PAL+Ins, 1.39±0.11 vs 0.99±0.1; p<0.05) Thus, 1) BAG3 expression is important in cell survival under ischemic conditions, and 2) NF-kB plays a key role in upregulating the expression of BAG3 under diabetic and ischemic conditions.

Double Deletion of C5aR1 and C5aR2 during Streptococcal Pneumonia

Abstract The complement system is essential for the clearance of encapsulated bacteria such as Streptococcus pneumoniae (Spn), recognized as a major pathogen of pneumonia. Yet, it is not entirely clear whether C5 and its activation product C5a have beneficial or detrimental effects on the outcome of Spn infection. C5a binds to its two homologous C5a receptors (C5aR1 and C5aR2). The extent of functional overlap and role distribution between C5aR1 and C5aR2 remains enigmatic. A critical barrier to allow studies on the role distributions of C5aRs has been the absence of a mouse strain with dual deletion of both receptors - which are encoded as adjacent, linked genes on mouse chromosome 7. CRISPR/Cas9 guided gene editing was combined with zygote/pronucleus microinjections in C57BL/6J mice to generate a C5aR1/C5aR2 double-knockout strain (C5aR1/2−/−). PCR-based screening and Sanger sequencing confirmed a 12.6 kB deletion of the coding regions of C5aR1 and C5aR2 of the new mouse strain, C57BL/6J-Del( 7C5ar2-C5ar1)1Bosm. Phenotyping of C5aR1/2−/− mice was followed by functional studies including intra-tracheal injections of recombinant mouse C5a or intranasal injections of Spn TIGR4. The airway influx of Ly6G+SiglecF- neutrophils by C5a was abrogated in C5aR1/2−/− mice. Surprisingly, the dual genetic absence of C5a receptors was associated with a higher build-up of neutrophil numbers in alveolar spaces after Spn TIGR4 infection. In a transcriptome-wide screen, FACS-sorted myeloid cells from infected lungs of double and single knockout mice were subjected to RNA-sequencing. Identifying novel differentially expressed genes in the C5aR1/2−/− mice during Spn infection will help to uncover potential synergisms and redundancies of C5aR1 and C5aR2.

Regions of mouse chromosomes 2 and 11 in SJL mice contain critical alleles for resistance to Cryptococcus neoformans

Abstract Cryptococcus neoformans (Cn) remains a leading cause of deadly mycosis. While previous studies have elucidated some of the host immune processes, little is known about the fundamental host factors that determine the outcome of Cn infection. Unfortunately, all commonly used inbred mice (including the common C57BL/6) are highly susceptible to virulent Cn strains, whereas humans are resistant to Cn unless immunocompromised. Thus, we sought a murine Cn infection system that more closely models humans. Testing the susceptibility of about 20 inbred strains of mice, we found that the SJL strain is, by far, the most Cn resistant inbred mouse strain in contrast to the closely related FVB strain being completely susceptible. While the early stages of Cn infection seem similar, SJL mice eventually cleared Cn and resolved pulmonary pathology, with little CNS dissemination. In contrast, susceptible mice showed significant and increasing fungal loads in both lungs and brains with associated pathology and death. F1 mice from FVB × SJL crosses were highly resistant to Cn suggesting that the resistant phenotype is dominant. Twenty percent of N2 mice (FVB × SJL F1 mice backcrossed to FVB) survived infection suggesting the involvement of multiple loci. Gene mapping uncovered two areas of genetic linkage to Cn resistance. These areas of interest were in portions of chromosomes 2 and 11. Genomic analysis found several candidate genes which are currently under evaluation. Mapping these cryptococcal resistance gene alleles may illuminate pathways of natural resistance which could be utilized for improved therapeutic options or to identify human populations at risk for cryptococcosis, making SJL mice an important tool for understanding cryptococcosis.

Dual histone methyl reader ZCWPW1 facilitates repair of meiotic double strand breaks in male mice.

Meiotic crossovers result from homology-directed repair of DNA double-strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes tri-methylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9, as an essential meiotic recombination factor required for efficient repair of PRDM9-dependent DSBs and for pairing of homologous chromosomes in male mice. In sum, our results indicate that the evolution of a dual histone methylation writer/reader (PRDM9/ZCWPW1) system in vertebrates remodeled genetic recombination hotspot selection from an ancestral static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.