Defining a TFAP2C-centered transcription factor network during murine peri-implantation.

Transcription factors (TFs) play important roles in early embryonic development, but factors regulating TF action, relationships in signaling cascade, genome-wide localizations, and impacts on cell fate transitions during this process have not been clearly elucidated. In this study, we used uliCUT&RUN-seq to delineate a TFAP2C-centered regulatory network, showing that it involves promoter-enhancer interactions and regulates TEAD4 and KLF5 function to mediate cell polarization. Notably, we found that maternal retinoic acid metabolism regulates TFAP2C expression and function by inducing the active demethylation of SINEs, indicating that the RARG-TFAP2C-TEAD4/KLF5 axis connects the maternal-to-zygotic transition to polarization. Moreover, we found that both genomic imprinting and SNP-transferred genetic information can influence TF positioning to regulate parental gene expressions in a sophisticated manner. In summary, we propose a ternary model of TF regulation in murine embryonic development with TFAP2C as the core element and metabolic, epigenetic, and genetic information as nodes connecting the pathways.

The gonadal niche safeguards human fetal germline cell development following maternal SARS-CoV-2 infection.

During pregnancy, germline development is vital for maintaining the continuation of species. Recent studies have shown increased pregnancy risks in COVID-19 patients at the perinatal stage. However, the potential consequence of infection for reproductive quality in developing fetuses remains unclear. Here, we analyze the transcriptome and DNA methylome of the fetal germline following maternal severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We find that infection at early gestational age, a critical period of human primordial germ cell specification and epigenetic reprogramming, trivially affects fetal germ cell (FGC) development. Additionally, FGC-niche communications are not compromised by maternal infection. Strikingly, both general and SARS-CoV-2-specific immune pathways are greatly activated in gonadal niche cells to protect FGCs from maternal infection. Notably, there occurs an "in advance" development tendency in FGCs after maternal infection. Our study provides insights into the impacts of maternal SARS-CoV-2 infection on fetal germline development and serves as potential clinical guidance for future pandemics.

Unreprogrammed H3K9me3 prevents minor zygotic genome activation and lineage commitment in SCNT embryos.

Somatic cell nuclear transfer (SCNT) can be used to reprogram differentiated somatic cells to a totipotent state but has poor efficiency in supporting full-term development. H3K9me3 is considered to be an epigenetic barrier to zygotic genomic activation in 2-cell SCNT embryos. However, the mechanism underlying the failure of H3K9me3 reprogramming during SCNT embryo development remains elusive. Here, we perform genome-wide profiling of H3K9me3 in cumulus cell-derived SCNT embryos. We find redundant H3K9me3 marks are closely related to defective minor zygotic genome activation. Moreover, SCNT blastocysts show severely indistinct lineage-specific H3K9me3 deposition. We identify MAX and MCRS1 as potential H3K9me3-related transcription factors and are essential for early embryogenesis. Overexpression of Max and Mcrs1 significantly benefits SCNT embryo development. Notably, MCRS1 partially rescues lineage-specific H3K9me3 allocation, and further improves the efficiency of full-term development. Importantly, our data confirm the conservation of deficient H3K9me3 differentiation in Sertoli cell-derived SCNT embryos, which may be regulated by alternative mechanisms.

Transcriptome profiles and chromatin states in mouse androgenetic haploid embryonic stem cells.

Haploid embryonic stem cells (haESCs) are derived from the inner cell mass of the haploid blastocyst, containing only one set of chromosomes. Extensive and accurate chromatin remodelling occurs during haESC derivation, but the intrinsic transcriptome profiles and chromatin structure of haESCs have not been fully explored. We profiled the transcriptomes, nucleosome positioning, and key histone modifications of four mouse haESC lines, and compared these profiles with those of other closely-related stem cell lines, MII oocytes, round spermatids, sperm, and mouse embryonic fibroblasts. haESCs had transcriptome profiles closer to those of naïve pluripotent stem cells. Consistent with the one X chromosome in haESCs, Xist was repressed, indicating no X chromosome inactivation. haESCs and ESCs shared a similar global chromatin structure. However, a nucleosome depletion region was identified in 2056 promoters in ESCs, which was absent in haESCs. Furthermore, three characteristic spatial relationships were formed between transcription factor motifs and nucleosomes in both haESCs and ESCs, specifically in the linker region, on the nucleosome central surface, and nucleosome borders. Furthermore, the chromatin state of 4259 enhancers was off in haESCs but active in ESCs. Functional annotation of these enhancers revealed enrichment in regulation of the cell cycle, a predominantly reported mechanism of haESC self-diploidization. Notably, the transcriptome profiles and chromatin structure of haESCs were highly preserved during passaging but different from those of differentiated cell types.

A cascade of transcriptional repression determines sexual commitment and development in Plasmodium falciparum.

Gametocytogenesis, the process by which malaria parasites produce sexual forms that can infect mosquitoes, is essential for the transmission of malaria. A transcriptional switch of the pfap2-g gene triggers sexual commitment, but how the complex multi-step process is precisely programed remains largely unknown. Here, by systematic functional screening of a panel of ApiAP2 transcription factors, we identify six new ApiAP2 members associated with gametocytogenesis in Plasmodium falciparum. Among these, PfAP2-G5 (PF3D7_1139300) was found to be indispensable for gametocytogenesis. This factor suppresses the transcriptional activity of the pfap2-g gene via binding to both the upstream region and exonic gene body, the latter is linked to the maintenance of local heterochromatin structure, thereby preventing initiation of sexual commitment. Removal of this repressive effect through pfap2-g5 knockout disrupts the asexual replication cycle and promotes sexual commitment accompanied by upregulation of pfap2-g expression. However, the gametocytes produced fail to mature fully. Further analyses show that PfAP2-G5 is essential for gametocyte maturation, and causes the down-regulation of pfap2-g and a set of early gametocyte genes activated by PfAP2-G prior to gametocyte development. Collectively, our findings reveal a regulation cascade of gametocyte production in malaria parasites, and provide a new target for transmission blocking interventions.

The Architectural Factor HMGB1 Is Involved in Genome Organization in the Human Malaria Parasite Plasmodium falciparum.

The three-dimensional (3D) genome organization plays a critical role in the regulation of gene expression in eukaryotic organisms. In the unicellular malaria parasite Plasmodium falciparum, the high-order chromosome organization has emerged as an important epigenetic pathway mediating gene expression, particularly for virulence genes, but the related architectural factors and underlying mechanism remain elusive. Herein, we have identified the high-mobility-group protein HMGB1 as a critical architectural factor for maintenance of genome organization in P. falciparum Genome-wide occupancy analysis (chromatin immunoprecipitation sequencing [ChIP-seq]) shows that the HMGB1 protein is recruited mainly to centromeric regions likely via a DNA-binding-independent pathway. Chromosome conformation capture coupled with next-generation sequencing (Hi-C-seq) and 3D modeling analysis show that the loss of HMGB1 disrupts the integrity of centromere/telomere-based chromosome organization accompanied with diminished interaction frequency among centromere clusters. This triggers local chromatin alteration and dysregulated gene expression. Notably, the entire repertoire of the primary virulence genes (var) was completely silenced in the absence of P. falciparum HMGB1 (PfHMGB1). Furthermore, the disrupted nuclear organization was reconstituted by complementation of HMGB1, thereby rescuing the mutually exclusive expression of the var gene family. Collectively, these data demonstrate that the architectural factor HMGB1 is associated with gene expression via mediating the high-order structure of genome organization. This finding not only contributes better understanding of the epigenetic regulation of gene expression but may also provide novel targets for antimalarial strategies.IMPORTANCE Malaria remains a major public health and economic burden currently. The mutually exclusive expression of the virulence genes is associated with the pathogenesis and immune evasion of human malaria parasites in the host. The nuclear architecture provides a well-organized environment for differential gene expression in the nucleus, but the underlying mechanism remains largely unknown. In this study, we have identified the highly conserved high-mobility-group protein HMGB1 as a key architecture regulator involved in virulence gene expression by establishing high-order genome organization in the nucleus of P. falciparum Mechanistic investigation revealed that the specific interaction of HMGB1 and centromeres constructed the precisely organized nuclear architecture, which coordinated with local chromatin structure to control the singular expression of virulence genes. Hence, this protein appears to be a critical architectural regulator for the pathogenesis of malaria infection and may be a new target for the development of an intervention strategy against malaria.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

Video Snapshot: Single Image Motion Expansion via Invertible Motion Embedding.

Unlike images, finding the desired video content in a large pool of videos is not easy due to the time cost of loading and watching. Most video streaming and sharing services provide the video preview function for a better browsing experience. In this paper, we aim to generate a video preview from a single image. To this end, we propose two cascaded networks, the motion embedding network and the motion expansion network. The motion embedding network aims to embed the spatio-temporal information into an embedded image, called video snapshot. On the other end, the motion expansion network is proposed to invert the video back from the input video snapshot. To hold the invertibility of motion embedding and expansion during training, we design four tailor-made losses and a motion attention module to make the network focus on the temporal information. In order to enhance the viewing experience, our expansion network involves an interpolation module to produce a longer video preview with a smooth transition. Extensive experiments demonstrate that our method can successfully embed the spatio-temporal information of a video into one "live" image, which can be converted back to a video preview. Quantitative and qualitative evaluations are conducted on a large number of videos to prove the effectiveness of our proposed method. In particular, statistics of PSNR and SSIM on a large number of videos show the proposed method is general, and it can generate a high-quality video from a single image.

Dcaf11 activates Zscan4-mediated alternative telomere lengthening in early embryos and embryonic stem cells.

Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4.

Rrp6 Regulates Heterochromatic Gene Silencing via ncRNA RUF6 Decay in Malaria Parasites.

The heterochromatin environment plays a central role in silencing genes associated with the malaria parasite's development, survival in the host, and transmission to the mosquito vector. However, the underlying mechanism regulating the dynamic chromatin structure is not understood yet. Here, we have uncovered that Plasmodium falciparum Rrp6, an orthologue of eukaryotic RNA exosome-associated RNase, controls the silencing of heterochromatic genes. PfRrp6 knockdown disrupted the singular expression of the GC-rich ncRNA RUF6 family, a known critical regulator of virulence gene expression, through the stabilization of the nascent transcripts. Mechanistic investigation showed that the accumulation of the multiple RUF6 ncRNAs triggered local chromatin remodeling in situ, which activated their adjacent var genes. Strikingly, chromatin isolation by RNA purification analysis (ChIRP-seq) revealed that a remarkable RUF6 ncRNA had interacted with distal heterochromatin regions directly and stimulated a global derepression effect on heterochromatic genes, including all variant gene families and the sexual commitment-associated regulator ap2-g gene. Collectively, Rrp6 appears to conduct the epigenetic surveillance of heterochromatic gene expression through controlling RUF6 levels, thereby securing antigenic variation and sexual commitment of malaria parasites during the infection of the host.IMPORTANCE Malaria remains a major public health and economic burden. The heterochromatin environment controls the silencing of genes associated with the fate of malaria parasites. Previous studies have demonstrated that a group of GC-rich ncRNAs (RUF6) is associated with the mutually exclusive expression of var genes, but the underlying mechanisms remain elusive. Here, through a series of genetic manipulation and genome-wide multiomics analysis, we have identified the plasmodial orthologue of RNA exosome-associated Rrp6 as an upstream regulator of RUF6 expression and revealed that the dysregulation of RUF6 upon Rrp6 knockdown triggered local chromatin alteration, thereby activating most heterochromatic genes via direct interaction of RUF6 and distal gene loci. This finding not only uncovered the in-depth mechanism of RUF6-mediated regulation of heterochromatic genes but also identified Rrp6 as a novel regulator of gene expression in human malaria parasites, which provides a new target for developing intervention strategies against malaria.

Chromatin architecture reorganization in murine somatic cell nuclear transfer embryos.

The oocyte cytoplasm can reprogram the somatic cell nucleus into a totipotent state, but with low efficiency. The spatiotemporal chromatin organization of somatic cell nuclear transfer (SCNT) embryos remains elusive. Here, we examine higher order chromatin structures of mouse SCNT embryos using a low-input Hi-C method. We find that donor cell chromatin transforms to the metaphase state rapidly after SCNT along with the dissolution of typical 3D chromatin structure. Intriguingly, the genome undergoes a mitotic metaphase-like to meiosis metaphase II-like transition following activation. Subsequently, weak chromatin compartments and topologically associating domains (TADs) emerge following metaphase exit. TADs are further removed until the 2-cell stage before being progressively reestablished. Obvious defects including stronger TAD boundaries, aberrant super-enhancer and promoter interactions are found in SCNT embryos. These defects are partially caused by inherited H3K9me3, and can be rescued by Kdm4d overexpression. These observations provide insight into chromatin architecture reorganization during SCNT embryo development.

Increase in DNA Damage by MYCN Knockdown Through Regulating Nucleosome Organization and Chromatin State in Neuroblastoma.

As a transcription factor, MYCN regulates myriad target genes including the histone chaperone FACT. Moreover, FACT and MYCN expression form a forward feedback loop in neuroblastoma. It is unclear whether MYCN is involved in chromatin remodeling in neuroblastoma through regulation of its target genes. We showed here that MYCN knockdown resulted in loss of the nucleosome-free regions through nucleosome assembly in the promoters of genes functionally enriched for DNA repair. The active mark H3K9ac was removed or replaced by the repressive mark H3K27me3 in the promoters of double-strand break repair-related genes upon MYCN knockdown. Such chromatin state alterations occurred only in MYCN-bound promoters. Consistently, MYCN knockdown resulted in a marked increase in DNA damage in the treatment with hydroxyurea. In contrast, nucleosome reorganization and histone modification changes in the enhancers largely included target genes with tumorigenesis-related functions such as cell proliferation, cell migration, and cell-cell adhesion. The chromatin state significantly changed in both MYCN-bound and MYCN-unbound enhancers upon MYCN knockdown. Furthermore, MYCN knockdown independently regulated chromatin remodeling in the promoters and the enhancers. These findings reveal the novel epigenetic regulatory role of MYCN in chromatin remodeling and provide an alternative potential epigenetic strategy for MYCN-driven neuroblastoma treatment.

Accurate annotation of accessible chromatin in mouse and human primordial germ cells.

Extensive and accurate chromatin remodeling is essential during primordial germ cell (PGC) development for the perpetuation of genetic information across generations. Here, we report that distal cis-regulatory elements (CREs) marked by DNase I-hypersensitive sites (DHSs) show temporally restricted activities during mouse and human PGC development. Using DHS maps as proxy, we accurately locate the genome-wide binding sites of pluripotency transcription factors in mouse PGCs. Unexpectedly, we found that mouse female meiotic recombination hotspots can be captured by DHSs, and for the first time, we identified 12,211 recombination hotspots in mouse female PGCs. In contrast to that of meiotic female PGCs, the chromatin of mitotic-arrested male PGCs is permissive through nuclear transcription factor Y (NFY) binding in the distal regulatory regions. Furthermore, we examined the evolutionary pressure on PGC CREs, and comparative genomic analysis revealed that mouse and human PGC CREs are evolutionarily conserved and show strong conservation across the vertebrate tree outside the mammals. Therefore, our results reveal unique, temporally accessible chromatin configurations during mouse and human PGC development.

Sin3a-Tet1 interaction activates gene transcription and is required for embryonic stem cell pluripotency.

Sin3a is a core component of histone-deacetylation-activity-associated transcriptional repressor complex, playing important roles in early embryo development. Here, we reported that down-regulation of Sin3a led to the loss of embryonic stem cell (ESC) self-renewal and skewed differentiation into mesendoderm lineage. We found that Sin3a functioned as a transcriptional coactivator of the critical Nodal antagonist Lefty1 through interacting with Tet1 to de-methylate the Lefty1 promoter. Further studies showed that two amino acid residues (Phe147, Phe182) in the PAH1 domain of Sin3a are essential for Sin3a-Tet1 interaction and its activity in regulating pluripotency. Furthermore, genome-wide analyses of Sin3a, Tet1 and Pol II ChIP-seq and of 5mC MeDIP-seq revealed that Sin3a acted with Tet1 to facilitate the transcription of a set of their co-target genes. These results link Sin3a to epigenetic DNA modifications in transcriptional activation and have implications for understanding mechanisms underlying versatile functions of Sin3a in mouse ESCs.