C. elegans germlines that inherited only paternal chromosomes (non-Mendelian inheritance, 'red-head worms') and the germlines of their offspring vs. germlines that inherited both maternal and paternal chromosomes (Mendelian inheritance, HBR1280 control)
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ABSTRACT: RNA-seq transcriptome analysis of C. elegans germlines that inherited only paternal chromosomes (non-Mendelian inheritance, 'red-head worms') and the germlines of their offspring ('offspring of red-head worms') vs. germlines that inherited both maternal and paternal chromosomes (Mendelian inheritance, HBR1280 control). Given that epigenetic marking of sperm chromosomes is faithfully transmitted through embryo cell divisions, and that sperm epigenetic marking is important in offspring, we tested if sperm epigenetic marking alone is sufficient for proper development of the germline in offspring. We utilized a mutant that, during the first embryonic division, delivers the sperm genome to the daughter cell that generates the germline and the oocyte genome to the other daughter cell (Besseling & Bringmann, 2016). This mutant over-expresses GPR-1, a protein involved in regulation of kinetochore pulling forces. GPR-1 over-expression results in excessive pulling forces, causing the paternal and maternal pronuclei to inappropriately move to opposite poles of the 1-cell embryo instead of merging in the center of the embryo. In this mutant background, ~60% of offspring undergo atypical chromosome segregation, generating mosaic embryos whose germlines are derived entirely from sperm chromosomes (Besseling & Bringmann, 2016). To track the parental genomes, differentially tagged histone transgenes were used: a GFP-tagged histone H2B encoded in the sperm genome, and a TdTomato-tagged histone H2B encoded in the oocyte genome. The mosaic embryos whose germline inherited only sperm chromosomes (‘red-head' worms) develop into fertile adults with a normal brood size, similar to control worms, in which the germline inherited both sperm and oocyte chromosomes. RNA-seq analysis demonstrated that the germline transcriptome of ‘red-head’ worms and their offspring show few (<80 genes) and minor changes compared to control worms. These findings demonstrate that epigenetic information provided by sperm can guide proper germ cell development.
Project description:Paternal contributions to epigenetic inheritance are not well understood. We report that in C. elegans sperm, the genome is packaged in nucleosomes and carries a histone-based epigenetic memory of gene expressions during spermatogenesis. In mature sperm, genes with spermatogenesis-specific expression are marked with both active and represseive histone modifications and genes with oogenesis-enriched expression are marked with active histone modifications. We showed that genes with oogenesis-enriched expression are in fact transcribed in spermatogenic germlines. We tested if sperm chromatin marking is necessary for germ cell development in offspring that inherit both sperm and oocyte chromosomes, using male parents that either can or cannot generate H3K27me3. Males homozygous for a mutation in mes-3, which encodes a member of the worm PRC2 complex, were mated with feminized mes-3/+ heterozygous worms to produce offspring that inherited sperm chromosomes lacking H3K27me3. We call these offspring M+P- or MpPm (Maternal chromosomes are + or plus for H3K27me3, Paternal chromosomes are - or minus for H3K27me3). Most of the resulting mes-3 homozygous M+P- offspring developed into sterile adults in this sensitized genetic background. In contrast, genetically identical control offspring that received appropriate H3K27me3-marked sperm chromosomes (M+P+ or MpPp) displayed low sterility. We compared genes mis-regulated in mes-3 male germlines versus control him-8 male germlines, mature sperm from those germlines, and germlines of mes-3 mutant F1 offspring that inherited sperm chromatin lacking H3K27me3 (M+P-) versus inherited sperm chromatin with H3K27me3 (M+P+), based on RNA-seq.
Project description:C. elegans germlines that inherited only paternal chromosomes (non-Mendelian inheritance, 'red-head worms') and the germlines of their offspring vs. germlines that inherited both maternal and paternal chromosomes (Mendelian inheritance, HBR1280 control)
Project description:Worms that inherited the sperm genome lacking the repressive mark H3K27me3 (K27me3 M+P-) misexpress genes in their germlines when compared to genetically identitical worms that inherited the sperm genome with H3K27me3 (K27me3 M+P+).
Project description:The role that gamete-inherited chromatin states serve in regulating gene expression across generations is poorly understood. To interrogate how histone marks inherited on parental genomes influence gene expression in offspring, we profiled different tissue contexts in worms that inherited the sperm genome lacking the repressive mark H3K27me3. We found that worms that inherited the sperm genome lacking H3K27me3 upregulated genes in all tissues profiled, which included hermaphrodite and male germlines and mixed soma. We found that most upregulated genes were upregulated specifically from sperm alleles and in a tissue-specific manner, highlighting the importance of cellular context in determining which genes are sensitive to upregulation when H3K27me3 repression is absent. To determine whether chromatin states can impact gene expression transgenerationally, i.e. across 3 generations, we profiled gene expression and chromatin states in the germlines of worms that inherited the sperm genome lacking H3K27me3 (F1 generation) and in the germlines of their offspring (F2 generation). We found that genes upregulated in F1 germlines maintained the H3K27me3(-) state of sperm alleles and that the upregulated and H3K27me3(-) state of sperm alleles was maintained in the germlines of their offspring (F2 generation). These findings demonstrate that histone marks can serve as a transgenerational carrier.
Project description:RNA-seq transcriptome profiling of 1) C. elegans male germlines vs. oogenic germlines, and 2) male germlines, sperm, and F1 offspring germlines when offspring inherited sperm chromatin lacking H3K27me3.
Project description:PUF RNA-binding proteins control stem cells in diverse species, including mammalian, arthropod, and nematode, in addition to other biological functions. The C. elegans PUF protein FBF serves as a paradigm for metazoan PUFs. FBF is essential for the maintenance of germline stem cells but also regulates the hermpahrodite sperm/oocyte cell fate switch and is critical for the process of spermatogenesis. We have attempted to “disentangle” the different roles of FBF by comparing its targets in spermatogenic and oogenic germlines. To this end, we used FBF iCLIP to learn its binding profile in an adult hermaphrodite germline that is sexually transformed and makes only sperm due to a temperature-sensitive sex-determination mutant. As a control, we analyzed FBF iCLIP data from oogenic germlines at the same temperature. Using a modified peak calling algorithm, we identified FBF binding sites in oogenic animals at 20°C, oogenic animals at 25°C, and spermatogenic animals at 25°C. Oogenic FBF targets were similar at 20°C and 25°C. By contrast, FBF mRNA targets in spermatogenetic animals had a distinct profile, revealing sperm-specific targets that are likely critical for the FBF role in spermatogenesis. Most importantly, we found FBF bound to mRNAs regardless of germline gender. In particular, a group of 22 mRNAs clustered as bound with high frequency in a gender- and temperature-independent manner. These 22 mRNAsencode RNA-binding proteins and stem cell regulators and may be crucial for the FBF role in in stem cell maintenance.
Project description:To shed light on how proteostasis defects in the germline influence somatic tissues, we first assessed the intracellular changes induced by PGL-1 aggregation in germline cells. For this purpose, we examined the proteome of isolated germlines from C. elegans following cey-3 knockdown
Project description:According to Mendel's laws, each parent makes an equal genetic contribution to an offspring in sexually reproducing organisms. The bipolar mitotic spindle controls the equal segregation of paternal and maternal chromosomes during the first cell division. By overexpression of a single protein, GPR-1, in the maternal strain we changed the structure of the mitotic spindle from bipolar to two monopolar spindles to segregate maternal and paternal chromosomes into different cell lineages, resulting in non-mendelian segregation for entire genomes. To follow maternal and paternal segregation of the chromosomes we used red and green histone markers respectively. By mating gpr-1-overexpressing hermaphrodites with wild-type males, mendelian F1 worms that express both markers simultaneously in all tissues and non-mendelian F1 worms that express red and green markers in different tissues will be produced representing embryos with bipolar and embryos with two monopolar spindles. Thus, we show that the rules of genetic inheritance can be changed, which may inspire the formation of a new field of synthetic zoology. Transcriptional profiling was done to investigate the differences in gene expression between mendelian and non-mendelian offspring. Approximately 60 adult worms were used per sample. Four conditions were collected: hermaphrodites of the paternal strain, hermaphrodites of the maternal strain, co-segregating (mendelian) F1 after crossing of parental strains, and (non-mendelian) F1 that segregates the paternal genotype to body wall muscle, intestine + germline and the maternal genotype to the nervous system after crossing of parental strains.
Project description:With the aim to determine the extent of germline feminization in feminized male worms, we performed differential gene expression profiling of isolated gonads and whole worms between fog-1(q253) XO males and XX females.
Project description:P granules in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the mis-expression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by 1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by 2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. Four biological replicates of each condition (empty vector control, P granule RNAi, and CSR-1 RNAi germlines) were collected for total RNA.