Hongshan Guo

Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells

Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing (RNA-Seq) analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

The DNA methylation landscape of human early embryos

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing

DNA methylation is crucial for a wide variety of biological processes, yet no technique suitable for the methylome analysis of DNA methylation at single-cell resolution is available. Here, we describe a methylome analysis technique that enables single-cell and single-base resolution DNA methylation analysis based on reduced representation bisulfite sequencing (scRRBS). The technique is highly sensitive and can detect the methylation status of up to 1.5 million CpG sites within the genome of an individual mouse embryonic stem cell (mESC). Moreover, we show that the technique can detect the methylation status of individual CpG sites in a haploid sperm cell in a digitized manner as either unmethylated or fully methylated. Furthermore, we show that the demethylation dynamics of maternal and paternal genomes after fertilization can be traced within the individual pronuclei of mouse zygotes. The demethylation process of the genic regions is faster than that of the intergenic regions in both male and female pronuclei. Our method paves the way for the exploration of the dynamic methylome landscapes of individual cells at single-base resolution during physiological processes such as embryonic development, or during pathological processes such as tumorigenesis.

The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells

Germ cells are vital for transmitting genetic information from one generation to the next and for maintaining the continuation of species. Here, we analyze the transcriptome of human primordial germ cells (PGCs) from the migrating stage to the gonadal stage at single-cell and single-base resolutions. Human PGCs show unique transcription patterns involving the simultaneous expression of both pluripotency genes and germline-specific genes, with a subset of them displaying developmental-stage-specific features. Furthermore, we analyze the DNA methylome of human PGCs and find global demethylation of their genomes. Approximately 10 to 11 weeks after gestation, the PGCs are nearly devoid of any DNA methylation, with only 7.8% and 6.0% of the median methylation levels in male and female PGCs, respectively. Our work paves the way toward deciphering the complex epigenetic reprogramming of the germline with the aim of restoring totipotency in fertilized oocytes.

Whole-genome analysis of 5-hydroxymethylcytosine and 5-methylcytosine at base resolution in the human brain

Background5-methylcytosine (mC) can be oxidized by the tet methylcytosine dioxygenase (Tet) family of enzymes to 5-hydroxymethylcytosine (hmC), which is an intermediate of mC demethylation and may also be a stable epigenetic modification that influences chromatin structure. hmC is particularly abundant in mammalian brains but its function is currently unknown. A high-resolution hydroxymethylome map is required to fully understand the function of hmC in the human brain.ResultsWe present genome-wide and single-base resolution maps of hmC and mC in the human brain by combined application of Tet-assisted bisulfite sequencing and bisulfite sequencing. We demonstrate that hmCs increase markedly from the fetal to the adult stage, and in the adult brain, 13% of all CpGs are highly hydroxymethylated with strong enrichment at genic regions and distal regulatory elements. Notably, hmC peaks are identified at the 5′splicing sites at the exon-intron boundary, suggesting a mechanistic link between hmC and splicing. We report a surprising transcription-correlated hmC bias toward the sense strand and an mC bias toward the antisense strand of gene bodies. Furthermore, hmC is negatively correlated with H3K27me3-marked and H3K9me3-marked repressive genomic regions, and is more enriched at poised enhancers than active enhancers.ConclusionsWe provide single-base resolution hmC and mC maps in the human brain and our data imply novel roles of hmC in regulating splicing and gene expression. Hydroxymethylation is the main modification status for a large portion of CpGs situated at poised enhancers and actively transcribed regions, suggesting its roles in epigenetic tuning at these regions.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc−/−) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos

Circular RNAs (circRNAs) are a new class of non-polyadenylated non-coding RNAs that may play important roles in many biological processes. Here we develop a single-cell universal poly(A)-independent RNA sequencing (SUPeR-seq) method to sequence both polyadenylated and non-polyadenylated RNAs from individual cells. This method exhibits robust sensitivity, precision and accuracy. We discover 2891 circRNAs and 913 novel linear transcripts in mouse preimplantation embryos and further analyze the abundance of circRNAs along development, the function of enriched genes, and sequence features of circRNAs. Our work is key to deciphering regulation mechanisms of circRNAs during mammalian early embryonic development.

Profiling DNA methylome landscapes of mammalian cells with single-cell reduced-representation bisulfite sequencing

The heterogeneity of DNA methylation within a population of cells necessitates DNA methylome profiling at single-cell resolution. Recently, we developed a single-cell reduced-representation bisulfite sequencing (scRRBS) technique in which we modified the original RRBS method by integrating all the experimental steps before PCR amplification into a single-tube reaction. These modifications enable scRRBS to provide digitized methylation information on ∼1 million CpG sites within an individual diploid mouse or human cell at single-base resolution. Compared with the single-cell bisulfite sequencing (scBS) technique, scRRBS covers fewer CpG sites, but it provides better coverage for CpG islands (CGIs), which are likely to be the most informative elements for DNA methylation. The entire procedure takes ∼3 weeks, and it requires strong molecular biology skills.

DNA methylation and chromatin accessibility profiling of mouse and human fetal germ cells.

Chromatin remodeling is important for the epigenetic reprogramming of human primordial germ cells. However, the comprehensive chromatin state has not yet been analyzed for human fetal germ cells (FGCs). Here we use nucleosome occupancy and methylation sequencing method to analyze both the genome-wide chromatin accessibility and DNA methylome at a series of crucial time points during fetal germ cell development in both human and mouse. We find 116 887 and 137 557 nucleosome-depleted regions (NDRs) in human and mouse FGCs, covering a large set of germline-specific and highly dynamic regulatory genomic elements, such as enhancers. Moreover, we find that the distal NDRs are enriched specifically for binding motifs of the pluripotency and germ cell master regulators such as NANOG, SOX17, AP2γ and OCT4 in human FGCs, indicating the existence of a delicate regulatory balance between pluripotency-related genes and germ cell-specific genes in human FGCs, and the functional significance of these genes for germ cell development in vivo. Our work offers a comprehensive and high-resolution roadmap for dissecting chromatin state transition dynamics during the epigenomic reprogramming of human and mouse FGCs.

Single-cell DNA methylome sequencing of human preimplantation embryos

DNA methylation is a crucial layer of epigenetic regulation during mammalian embryonic development1–3. Although the DNA methylome of early human embryos has been analyzed4–6, some of the key features have not been addressed thus far. Here we performed single-cell DNA methylome sequencing for human preimplantation embryos and found that tens of thousands of genomic loci exhibited de novo DNA methylation. This finding indicates that genome-wide DNA methylation reprogramming during preimplantation development is a dynamic balance between strong global demethylation and drastic focused remethylation. Furthermore, demethylation of the paternal genome is much faster and thorough than that of the maternal genome. From the two-cell to the postimplantation stage, methylation of the paternal genome is consistently lower than that of the maternal genome. We also show that the genetic lineage of early blastomeres can be traced by DNA methylation analysis. Our work paves the way for deciphering the secrets of DNA methylation reprogramming in early human embryos.An analysis of single-cell DNA methylome sequencing data from human preimplantation embryos finds evidence for de novo methylation. Methylation reprogramming at this stage is a balance between global demethylation, which is faster in the paternal genome, and focused remethylation.