XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

Lineage tracing and analog recording in mammalian cells by single-site DNA writing.

Studying cellular and developmental processes in complex multicellular organisms can require the non-destructive observation of thousands to billions of cells deep within an animal. DNA recorders address the staggering difficulty of this task by converting transient cellular experiences into mutations at defined genomic sites that can be sequenced later in high throughput. However, existing recorders act primarily by erasing DNA. This is problematic because, in the limit of progressive erasure, no record remains. We present a DNA recorder called CHYRON (Cell History Recording by Ordered Insertion) that acts primarily by writing new DNA through the repeated insertion of random nucleotides at a single locus in temporal order. To achieve in vivo DNA writing, CHYRON combines Cas9, a homing guide RNA and the template-independent DNA polymerase terminal deoxynucleotidyl transferase. We successfully applied CHYRON as an evolving lineage tracer and as a recorder of user-selected cellular stimuli.

Cost-efficient boundary-free surface patterning achieves high effective-throughput of time-lapse microscopy experiments.

Time-lapse microscopy plays critical roles in the studies of cellular dynamics. However, setting up a time-lapse movie experiments is not only laborious but also with low output, mainly due to the cell-losing problem (i.e., cells moving out of limited field of view), especially in a long-time recording. To overcome this issue, we have designed a cost-efficient way that enables cell patterning on the imaging surfaces without any physical boundaries. Using mouse embryonic stem cells as an example system, we have demonstrated that our boundary-free patterned surface solves the cell-losing problem without disturbing their cellular phenotype. Statistically, the presented system increases the effective-throughput of time-lapse microscopy experiments by an order of magnitude.

Dynamics and functional roles of splicing factor autoregulation.

Non-core spliceosome components are essential, conserved regulators of alternative splicing. They provide concentration-dependent control of diverse pre-mRNAs. Many splicing factors direct unproductive splicing of their own pre-mRNAs through negative autoregulation. However, the impact of such feedback loops on splicing dynamics at the single-cell level remains unclear. Here, we developed a system to quantitatively analyze negative autoregulatory splicing dynamics by splicing factor SRSF1 in response to perturbations in single HEK293 cells. We show that negative autoregulatory splicing provides critical functions for gene regulation, establishing a ceiling of SRSF1 protein concentration, reducing cell-cell heterogeneity in SRSF1 levels, and buffering variation in transcription. Most important, it adapts SRSF1 splicing activity to variations in demand from other pre-mRNA substrates. A minimal mathematical model of autoregulatory splicing explains these experimentally observed features and provides values for effective biochemical parameters. These results reveal the unique functional roles that splicing negative autoregulation plays in homeostatically regulating transcriptional programs.

Urinary metabolomics reveals the biological characteristics of early pregnancy in pigs.

BACKGROUND: Embryo implantation in sows is an important event during pregnancy. During this process, blastocysts undergo dramatic morphologic changes, and the endometrium becomes receptive. Studies have shown that developmental changes associated with the crosstalk between peri-implantation embryos and embryo-uterine are driven by various biomolecules secreted by the endometrium and embryos. In sows, changes in the uterus are also reflected in circulating body fluids and urine. Metabolomics reveals the metabolic state of cells, tissues, and organisms. In this study, we collected urine samples from large white sows during the peri-implantation period. The levels of urinary metabolites at different periods were analyzed using ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis techniques. RESULTS: A total of 32 samples were collected from 8 sows during the estrus period and at each phase of early pregnancy (9, 12, and 15 days of gestation). A total of 530 metabolites were identified with high confidence in all samples. Compared with samples collected during the estrus phase, 269 differential metabolites were found in samples obtained during early pregnancy. CONCLUSIONS: The identified metabolites included lipids and lipid-like molecules, organic acids and their derivatives, organic oxygen compounds, organoheterocyclic compounds, benzenoids, among others. Metabolites, such as choline and pregnanediol-3-glucuronide, play important roles in pregnancy in sows and other animals. These results reveal the metabolic changes in urine of sows during early pregnancy phase. The differential urinary metabolites can be used for assessing peri-implantation status in sows. Understanding these metabolic changes may promote the management of pregnant sows through various interventions such as provision of proper nutrition.