(Abstracted from Nat Genet 2017;49:1502–1510) After fertilization, gametes undergo dramatic epigenomic reprogramming to initiate embryonic development. Retrotransposons are repetitive elements that form almost half of the mammalian genome; some are transcribed after fertilization.
Abstract The genome is packaged and organized in an ordered, non-random manner and specific chromatin segments contact nuclear substructures to mediate this organization. While transfer RNA genes (tDNAs) are essential for the generation of tRNAs, these loci are also binding sites for transcription factors and architectural proteins and are thought to play an important role in the organization of the genome. In this study, we investigate the role of tDNAs in genomic organization and chromosome function by editing a chromosome so that it lacks any tDNAs. Surprisingly our analyses of this tDNA-less chromosome show that loss of tDNAs does not grossly affect chromosome folding or chromosome tethering. However, loss of tDNAs affects local nucleosome positioning and the binding of SMC proteins at these loci. The absence of tDNAs also leads to changes in centromere clustering and a reduction in the frequency of long range HML-HMR heterochromatin clustering. We propose that the tDNAs primarily affect local chromatin structure that result in effects on long-range chromosome architecture.
During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice.
Accurate chromosome segregation is dependent on the centromere-specific histone H3 isoform known generally as CenH3, or as Cse4 in budding yeast. Cytological experiments have shown that Cse4 appears at extracentromeric loci in yeast cells deficient for both the CAF-1 and HIR histone H3/H4 deposition complexes, consistent with increased nondisjunction in these double mutant cells. Here, we examined molecular aspects of this Cse4 mislocalization. Genome-scale chromatin immunoprecipitation analyses demonstrated broader distribution of Cse4 outside of centromeres in cac1Δ hir1Δ double mutant cells that lack both CAF-1 and HIR complexes than in either single mutant. However, cytological localization showed that the essential inner kinetochore component Mif2 (CENP-C) was not recruited to extracentromeric Cse4 in cac1Δ hir1Δ double mutant cells. We also observed that rpb1-1 mutants displayed a modestly increased Cse4 half-life at nonpermissive temperatures, suggesting that turnover of Cse4 is partially dependent on Pol II transcription. We used genome-scale assays to demonstrate that the CAF-1 and HIR complexes independently stimulate replication-independent histone H3 turnover rates. We discuss ways in which altered histone exchange kinetics may affect eviction of Cse4 from noncentromeric loci.
Abstract Disclosure: R. Cannarella: None. O.J. Rando: None. R.A. Condorelli: None. C. Sandrine: None. S. Romano: None. A. Guglielmino: None. Q. Yin: None. H. Gustafsson: None. F. Mancuso: None. C. Bellucci: None. G. Luca: None. S. La Vignera: None. A.E. Calogero: None. Background: Sperm RNAs can regulate early embryo development. A potential key regulator of growth is insulin-like growth factor 2 (IGF2), a highly conserved paternally-imprinted gene involved in cell growth and proliferation that is expressed in human spermatozoa. Animal research supports the positive impact of IGF2-supplemented cultures on embryo quality. However, the role of sperm-carried IGF2 in the early development of the human embryo is unknown. Objective: To study whether sperm-carried IGF2 mRNA influences the morphokinetics of the human embryo. Design: Prospective, uncontrolled, observational pilot study. Setting: University-affiliated fertility center. Subjects: We enrolled 16 patients undergoing assisted reproductive techniques (ART) for idiopathic or male factor infertility and 10 healthy fertile controls with proven paternity. Couples with female factor infertility due to reduced oocyte reserve were excluded, while those with tubal factor or anovulation were included. Main Outcome Measure(s): After clinical use, an aliquot of the sperm samples employed for ART was used to evaluate IGF2 mRNA levels. Embryo kinetics were recorded and correlated with the amount of IGF2 mRNA, correcting for female age, body mass index (BMI), anti-Müllerian hormone (AMH) levels, and oocyte quality. To provide mechanistic insights into the human data, a transcriptome analysis was performed of parthenotes obtained from twelve-week-old superovulated mice injected with Igf2 mRNA plus Gfp mRNA (experimental group, n=20) or Gfp mRNAs (control group, n=20). Pathway enrichment analysis was then performed using g:Profiler g:GOSt and EnrichR. Results: Sperm IGF2 mRNA negatively correlated with time to 2-cell stage (t2), t3, t4, t5, and time to expanded blastocyst (tEB), independently of maternal age, BMI, AMH, and oocyte quality. IGF2 mRNA index >4.9 predicted the ability of the embryo to reach the blastocyst stage at day 5, with a sensitivity of 100% and specificity of 71.6% (AUC 0.845; p<0.001). The distribution of sperm IGF2 levels differs significantly between morphologically good or poor embryos, with the lowest levels more frequently associated with morphologically poor embryos (61.9%) than with good ones (38.1%) (p=0.0091). Furthermore, sperm-derived IGF2 levels were significantly lower in subfertile men compared to fertile controls with proven paternity. Transcriptome analysis of Igf2-injected embryos demonstrated differential expression of 101 genes activating pathways regulating early embryo development. Conclusion: Sperm-carried IGF2 mRNA can support early embryo development, independently of female factors. Larger studies are needed to confirm this finding and to investigate its role as a clinical marker to predict the chances of obtaining blastocysts that can be implanted in infertile couples undergoing ART. Presentation: 6/1/2024
