Methylome inheritance and enhancer dememorization construct an epigenetic gate safeguarding embryonic programs
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ABSTRACT:
PROVIDER: PRJNA734348 | ENA |
REPOSITORIES: ENA
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Project description:Unlike that of mammals, the total DNA methylome of many cold-blooded vertebrates is globally inherited from gametes to early embryos. In zebrafish, this is however accompanied by sweeping “dememorization” of enhancers prior to fertilization for sperm and just after fertilization for oocyte, as they undergo full methylation and are not demethylated again until phylotypic stage. The significance of both global methylome inheritance and enhancer dememorization in early embryos remains largely unknown. Adding to the puzzles, the zygotic mutant zebrafish of dnmt1, the major DNA methylation maintenance methyltransferase, surprisingly can develop to term. To solve the role of DNA methylation in early development, we generated zebrafish embryos derived from dnmt1-knocking down oocytes using a recently developed method OMIS (Oocyte Microinjection in situ), which successfully eliminated DNA methylation before zygotic genome activation. dnmt1-deficient embryos failed to initiate epiboly and died around gastrulation. This is in part caused by activation of immune response and p53-regulated apoptosis, likely triggered by the derepression of transposable elements. Single cell RNA-seq further revealed defective differentiation in these mutants. DNA methylation is also required for the establishment of repressive histone marks H3K27me3 and H2AK119ub. Strikingly, the loss of DNA methylation leads to extensive derepression of somatic genes and enhancers, which acquire ectopic H3K27ac, accessible chromatin, and H3K4me3. These somatic enhancers are preferentially CG-rich and are bound by CG-containing TFs. By contrast, embryonic enhancers are generally CG-poor, methylation-insensitive, and are bound by CG-less TFs. Hence, the global DNA methylome inheritance is essential for vertebrate early development, and enhancer dememorization resets an epigenetic gate that separates embryonic and somatic programs.
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Project description:Nanobody is one special type of single-domain antibody fragment with multiple advantages over traditional antibody. Our previous work established linear-double-stranded DNA (ldsDNA, or PCR amplicon) as novel biological parts for building AND gate genetic circuits in mammalian cells. During this AND-gate circuit formation process, the co-transfected up- and down-stream ldsDNAs could be linked together to form intact gene expression cassette. Here, we employed this ldsDNA-based AND-gate (LBAG) strategy to construct nanobody library in mammalian cells. The sequence complexity of complementary determining regions (CDRs) was introduced into ldsDNA by PCR amplification. After being co-transfected into mammalian cells, the up- and down- stream ldsDNAs undergo AND gate linkage and form full nanobody coding regions, containing CDR1-3. High throughput sequencing identified 22,173 unique oligonucleotide sequences in total generated by this strategy. Thus, we developed a novel method to construct nanobody library, which is a start point for building high content nanobody library in mammalian cells.
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Project description:Maternal inheritance of mitochondrial DNA (mtDNA) is highly conserved in metazoans. While many species eliminate paternal mtDNA during late sperm development to foster maternal inheritance, the regulatory mechanisms governing this process remain elusive. Through a large-scale genetic screen in Drosophila, we identified 47 mutant lines exhibiting substantial retention of mtDNA in mature sperm. We mapped one line to Poldip2, a gene predominantly expressed in the testis. Disruption of Poldip2 led to pronounced mtDNA retention in mature sperm and subsequent paternal transmission to progeny. Further investigation via imaging, biochemical analyses and ChIP assays revealed that POLDIP2 is a mitochondrial matrix protein capable of binding to mtDNA. Moreover, we uncovered that CLPX, a key component of the major mitochondrial protease, binds to POLDIP2 to co-regulate mtDNA elimination in Drosophila spermatids. This study shed light on the mechanisms underlying mtDNA removal during spermatogenesis, underscoring the pivotal role of this process in safeguarding maternal inheritance.
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Project description:Display technologies, e.g., phage, ribosome, mRNA, bacterial, and yeast-display, combine high content peptide libraries with appropriate screening strategies to identify functional peptide sequences. Construction of large peptide library and display-screen system in intact mammalian cells will facilitate the development of peptide therapeutics targeting transmembrane proteins. Our previous work established linear-double-stranded DNAs (ldsDNAs) as innovative biological parts to implement AND gate genetic circuits in mammalian cell line. In the current study, we employ ldsDNA with terminal NNK degenerate codons as AND gate input to build highly diverse peptide library in mammalian cells. Only PCR reaction and cell transfection experiments are needed to construct the library. High-throughput sequencing (HTS) results reveal that our new strategy could generate peptide library with both amino acid sequence and peptide length diversities. Our work establishes ldsDNA as biological parts for building highly diverse peptide library in mammalian cells, which shows great application potential in developing therapeutic peptides targeting transmembrane proteins.
2020-07-19 | GSE134671 | GEO