TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency.

Transition from primed to naive pluripotency is associated with dynamic changes in transposable element (TE) expression and demethylation of imprinting control regions (ICRs). In mouse, ICR methylation and TE expression are each regulated by TRIM28; however, the role of TRIM28 in humans is less clear. Here, we show that a null mutation in TRIM28 causes significant alterations in TE expression in both the naive and primed states of human pluripotency, and phenotypically this has limited effects on self-renewal, instead causing a loss of germline competency. Furthermore, we discovered that TRIM28 regulates paternal ICR methylation and chromatin accessibility in the primed state, with no effects on maternal ICRs. Taken together, our study shows that abnormal TE expression is tolerated by self-renewing human pluripotent cells, whereas germline competency is not

A protein assembly mediates Xist localization and gene silencing

Nuclear compartments have diverse roles in regulating gene expression, yet the molecular forces and components that drive compartment formation remain largely unclear. The long non-coding RNA Xist establishes an intra-chromosomal compartment by localizing at a high concentration in a territory spatially close to its transcription locus and binding diverse proteins to achieve X-chromosome inactivation (XCI). The XCI process therefore serves as a paradigm for understanding how RNA-mediated recruitment of various proteins induces a functional compartment. The properties of the inactive X (Xi)-compartment are known to change over time, because after initial Xist spreading and transcriptional shutoff a state is reached in which gene silencing remains stable even if Xist is turned off. Here we show that the Xist RNA-binding proteins PTBP1, MATR3, TDP-43 and CELF1 assemble on the multivalent E-repeat element of Xist and, via self-aggregation and heterotypic protein–protein interactions, form a condensate in the Xi. This condensate is required for gene silencing and for the anchoring of Xist to the Xi territory, and can be sustained in the absence of Xist. Notably, these E-repeat-binding proteins become essential coincident with transition to the Xist-independent XCI phase, indicating that the condensate seeded by the E-repeat underlies the developmental switch from Xist-dependence to Xist-independence. Taken together, our data show that Xist forms the Xi compartment by seeding a heteromeric condensate that consists of ubiquitous RNA-binding proteins, revealing an unanticipated mechanism for heritable gene silencing

X Chromosome Dosage Compensation During Human Early Embryonic Development

Mammalian embryonic development is one of the most complex biological processes that involves multiple epigenetic events. One of the major epigenetic processes is X chromosome dosage compensation in female mammalian cells harboring two Xchromosomes. The balance of X-linked gene levels between male and female cells can be achieved by X chromosome inactivation (XCI) or X chromosome dampening (XCD). Most of our knowledge about this complex process comes from mouse studies. Through decades of research, it has been established that the long-noncoding RNA Xist orchestrates XCI in mice. However, recent publications revealed intriguing epigenetic differences of the X chromosome and Xist function between mouse and human. In human, a different form of dosage compensation acts in preimplantation embryos and results in a dampening, but not silencing, of genes on both X chromosomes (XCD). Additionally, the localization of XIST lncRNA to the dampened X chromosome, indicates that XIST RNA can be expressed without inducing silencing, which has never been observed in the mouse. Similar to the human pre-implantation embryo, XIST is also expressed in female human primordial germ cells (hPGCs) after the inactive X chromosome is reactivated. In our work, we also demonstrate that similar to preimplantation human blastocysts X chromosome dampening is taking place in hPGCs in vivo. Additionally, using single cell RNA sequencing we demonstrate that expression of XIST in female hPGCs correlates with downregulation of X linked genes. Lastly, we demonstrate that primate specific lncRNA XACT, that has been described in the literature as pluripotency specific lncRNA, is also explicitly expressed from both active X chromosomes in hPGCs.To address the question whether XIST is mediating X chromosome dampening we have performed functional experiments in na ve human embryonic stem cell (ESC) lines that capture the preimplantation state of the X with XIST RNA expression from the active X chromosome and X chromosome dampening (XCD). We show that XIST deletion results in upregulation of dampened X linked genes. Additionally, we demonstrate for the first time that XIST spreads to specific autosomal regions in naive pluripotent stem cells and regulate their levels. However, it still remains unclear whether XIST has a role in regulating autosomal genes during early development in vivo. Taken together, our studies reveal insights into how epigenetic mechanisms differ between mouse and human, increase our knowledge of X chromosome regulation, and create strong bases to understand transmission of X-linked diseases through the generations

FGFR3 is expressed by human primordial germ cells and is repressed after meiotic initiation to form primordial oocytes.

Human germ cell development is a highly regulated process beginning soon after embryo implantation with the specification of primordial germ cells (PGCs) and ending in adulthood with the differentiation of gametes. Here, we show that fibroblast growth factor receptor 3 (FGFR3) is expressed by human PGCs during the first and second trimester, becoming repressed as PGCs differentiate into primordial oocytes. Using fluorescence-activated cell sorting (FACS) with antibodies that recognize FGFR3 followed by single-cell RNA sequencing, we show that isolating FGFR3-positive cells enriches for human PGCs. Taken together, FGFR3 could be used in future studies as a strategy to identify maturing PGCs in vitro

Quantitative 3D structured illumination microscopy of nuclear structures

3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week

Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). Human pre-implantation embryos are an exception, in which dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities to naive pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X chromosomes are active and express the long non-coding RNAs X active coating transcript (XACT) and X inactive specific transcript (XIST)-the master regulator of XCI-which are silenced after entry into meiosis. We find that XACT is a hPGC marker, describe XCD associated with XIST expression in hPGCs and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Furthermore, we found a unique mechanism of X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline

TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency.

Transition from primed to naive pluripotency is associated with dynamic changes in transposable element (TE) expression and demethylation of imprinting control regions (ICRs). In mouse, ICR methylation and TE expression are each regulated by TRIM28; however, the role of TRIM28 in humans is less clear. Here, we show that a null mutation in TRIM28 causes significant alterations in TE expression in both the naive and primed states of human pluripotency, and phenotypically this has limited effects on self-renewal, instead causing a loss of germline competency. Furthermore, we discovered that TRIM28 regulates paternal ICR methylation and chromatin accessibility in the primed state, with no effects on maternal ICRs. Taken together, our study shows that abnormal TE expression is tolerated by self-renewing human pluripotent cells, whereas germline competency is not

TRIM28-Regulated Transposon Repression Is Required for Human Germline Competency and Not Primed or Naive Human Pluripotency

Summary: Transition from primed to naive pluripotency is associated with dynamic changes in transposable element (TE) expression and demethylation of imprinting control regions (ICRs). In mouse, ICR methylation and TE expression are each regulated by TRIM28; however, the role of TRIM28 in humans is less clear. Here, we show that a null mutation in TRIM28 causes significant alterations in TE expression in both the naive and primed states of human pluripotency, and phenotypically this has limited effects on self-renewal, instead causing a loss of germline competency. Furthermore, we discovered that TRIM28 regulates paternal ICR methylation and chromatin accessibility in the primed state, with no effects on maternal ICRs. Taken together, our study shows that abnormal TE expression is tolerated by self-renewing human pluripotent cells, whereas germline competency is not