Kick-starting the zygotic genome: licensors, specifiers, and beyond.

Zygotic genome activation (ZGA), the first transcription event following fertilization, kickstarts the embryonic program that takes over the control of early development from the maternal products. How ZGA occurs, especially in mammals, is poorly understood due to the limited amount of research materials. With the rapid development of single-cell and low-input technologies, remarkable progress made in the past decade has unveiled dramatic transitions of the epigenomes, transcriptomes, proteomes, and metabolomes associated with ZGA. Moreover, functional investigations are yielding insights into the key regulators of ZGA, among which two major classes of players are emerging: licensors and specifiers. Licensors would control the permission of transcription and its timing during ZGA. Accumulating evidence suggests that such licensors of ZGA include regulators of the transcription apparatus and nuclear gatekeepers. Specifiers would instruct the activation of specific genes during ZGA. These specifiers include key transcription factors present at this stage, often facilitated by epigenetic regulators. Based on data primarily from mammals but also results from other species, we discuss in this review how recent research sheds light on the molecular regulation of ZGA and its executors, including the licensors and specifiers.

Stepwise de novo establishment of inactive X chromosome architecture in early development.

X chromosome inactivation triggers a dramatic reprogramming of transcription and chromosome architecture. However, how the chromatin organization of inactive X chromosome is established de novo in vivo remains elusive. Here, we identified an Xist-separated megadomain structure (X-megadomains) on the inactive X chromosome in mouse extraembryonic lineages and extraembryonic endoderm (XEN) cell lines, and transiently in the embryonic lineages, before Dxz4-delineated megadomain formation at later stages in a strain-specific manner. X-megadomain boundary coincides with strong enhancer activities and cohesin binding in an Xist regulatory region required for proper Xist activation in early embryos. Xist regulatory region disruption or cohesin degradation impaired X-megadomains in extraembryonic endoderm cells and caused ectopic activation of regulatory elements and genes near Xist, indicating that cohesin loading at regulatory elements promotes X-megadomains and confines local gene activities. These data reveal stepwise X chromosome folding and transcriptional regulation to achieve both essential gene activation and global silencing during the early stages of X chromosome inactivation.

Stable maternal proteins underlie distinct transcriptome, translatome, and proteome reprogramming during mouse oocyte-to-embryo transition.

BACKGROUND: The oocyte-to-embryo transition (OET) converts terminally differentiated gametes into a totipotent embryo and is critically controlled by maternal mRNAs and proteins, while the genome is silent until zygotic genome activation. How the transcriptome, translatome, and proteome are coordinated during this critical developmental window remains poorly understood. RESULTS: Utilizing a highly sensitive and quantitative mass spectrometry approach, we obtain high-quality proteome data spanning seven mouse stages, from full-grown oocyte (FGO) to blastocyst, using 100 oocytes/embryos at each stage. Integrative analyses reveal distinct proteome reprogramming compared to that of the transcriptome or translatome. FGO to 8-cell proteomes are dominated by FGO-stockpiled proteins, while the transcriptome and translatome are more dynamic. FGO-originated proteins frequently persist to blastocyst while corresponding transcripts are already downregulated or decayed. Improved concordance between protein and translation or transcription is observed for genes starting translation upon meiotic resumption, as well as those transcribed and translated only in embryos. Concordance between protein and transcription/translation is also observed for proteins with short half-lives. We built a kinetic model that predicts protein dynamics by incorporating both initial protein abundance in FGOs and translation kinetics across developmental stages. CONCLUSIONS: Through integrative analyses of datasets generated by ultrasensitive methods, our study reveals that the proteome shows distinct dynamics compared to the translatome and transcriptome during mouse OET. We propose that the remarkably stable oocyte-originated proteome may help save resources to accommodate the demanding needs of growing embryos. This study will advance our understanding of mammalian OET and the fundamental principles governing gene expression.

Translatome and transcriptome co-profiling reveals a role of TPRXs in human zygotic genome activation.

Translational regulation plays a critical role during the oocyte-to-embryo transition (OET) and zygotic genome activation (ZGA). Here, we integrated ultra-low-input ribosome profiling (Ribo-lite) with messenger RNA sequencing to co-profile the translatome and transcriptome in human oocytes and early embryos. Comparison with mouse counterparts identified widespread differentially translated gene functioning in epigenetic reprogramming, transposon defense, and small RNA biogenesis, in part driven by species-specific regulatory elements in 3' untranslated regions. Moreover, PRD-like homeobox transcription factors, including TPRXL, TPRX1, and TPRX2, are highly translated around ZGA. TPRX1/2/L knockdown leads to defective ZGA and preimplantation development. Ectopically expressed TPRXs bind and activate key ZGA genes in human embryonic stem cells. These data reveal the conservation and divergence of translation landscapes during OET and identify critical regulators of human ZGA.

Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development.

In mammals, translational control plays critical roles during oocyte-to-embryo transition (OET) when transcription ceases. However, the underlying regulatory mechanisms remain challenging to study. Here, using low-input Ribo-seq (Ribo-lite), we investigated translational landscapes during OET using 30-150 mouse oocytes or embryos per stage. Ribo-lite can also accommodate single oocytes. Combining PAIso-seq to interrogate poly(A) tail lengths, we found a global switch of translatome that closely parallels changes of poly(A) tails upon meiotic resumption. Translation activation correlates with polyadenylation and is supported by polyadenylation signal proximal cytoplasmic polyadenylation elements (papCPEs) in 3' untranslated regions. By contrast, translation repression parallels global de-adenylation. The latter includes transcripts containing no CPEs or non-papCPEs, which encode many transcription regulators that are preferentially re-activated before zygotic genome activation. CCR4-NOT, the major de-adenylation complex, and its key adaptor protein BTG4 regulate translation downregulation often independent of RNA decay. BTG4 is not essential for global de-adenylation but is required for selective gene de-adenylation and production of very short-tailed transcripts. In sum, our data reveal intimate interplays among translation, RNA stability and poly(A) tail length regulation underlying mammalian OET.

Kinetics and mechanisms of mitotic inheritance of DNA methylation and their roles in aging-associated methylome deterioration.

Mitotic inheritance of the DNA methylome is a challenging task for the maintenance of cell identity. Whether DNA methylation pattern in different genomic contexts can all be faithfully maintained is an open question. A replication-coupled DNA methylation maintenance model was proposed decades ago, but some observations suggest that a replication-uncoupled maintenance mechanism exists. However, the capacity and the underlying molecular events of replication-uncoupled maintenance are unclear. By measuring maintenance kinetics at the single-molecule level and assessing mutant cells with perturbation of various mechanisms, we found that the kinetics of replication-coupled maintenance are governed by the UHRF1-Ligase 1 and PCNA-DNMT1 interactions, whereas nucleosome occupancy and the interaction between UHRF1 and methylated H3K9 specifically regulate replication-uncoupled maintenance. Surprisingly, replication-uncoupled maintenance is sufficiently robust to largely restore the methylome when replication-coupled maintenance is severely impaired. However, solo-WCGW sites and other CpG sites displaying aging- and cancer-associated hypomethylation exhibit low maintenance efficiency, suggesting that although quite robust, mitotic inheritance of methylation is imperfect and that this imperfection may contribute to selective hypomethylation during aging and tumorigenesis.

A genetically engineered Escherichia coli that senses and degrades tetracycline antibiotic residue.

Due to the abuse of antibiotics, antibiotic residues can be detected in both natural environment and various industrial products, posing threat to the environment and human health. Here we describe the design and implementation of an engineered Escherichia coli capable of degrading tetracycline (Tc)-one of the commonly used antibiotics once on humans and now on poultry, cattle and fisheries. A Tc-degrading enzyme, TetX, from the obligate anaerobe Bacteroides fragilis was cloned and recombinantly expressed in E. coli and fully characterized, including its K m and k cat value. We quantitatively evaluated its activity both in vitro and in vivo by UV-Vis spectrometer and LC-MS. Moreover, we used a tetracycline inducible amplification circuit including T7 RNA polymerase and its specific promoter PT7 to enhance the expression level of TetX, and studied the dose-response of TetX under different inducer concentrations. Since the deployment of genetically modified organisms (GMOs) outside laboratory brings about safety concerns, it is necessary to explore the possibility of integrating a kill-switch. Toxin-Antitoxin (TA) systems were used to construct a mutually dependent host-plasmid platform and biocontainment systems in various academic and industrious situations. We selected nine TA systems from various bacteria strains and measured the toxicity of toxins (T) and the detoxifying activity of cognate antitoxins (A) to validate their potential to be used to build a kill-switch. These results prove the possibility of using engineered microorganisms to tackle antibiotic residues in environment efficiently and safely.