The genetic code-the binding specificity of all transfer-RNAs-defines how protein primary structure is determined by DNA sequence. DNA also dictates when and where proteins are expressed, and this information is encoded in a pattern of specific sequence motifs that are recognized by transcription factors. However, the DNA-binding specificity is only known for a small fraction of the~1400 human transcription factors (TFs). We describe here a high-throughput method for analyzing transcription factor binding specificity that is based on systematic evolution of ligands by exponential enrichment (SELEX) and massively parallel sequencing. The method is optimized for analysis of large numbers of TFs in parallel through the use of affinity-tagged proteins, barcoded selection oligonucleotides, and multiplexed sequencing. Data are analyzed by a new bioinformatic platform that uses the hundreds of thousands of sequencing reads obtained to control the quality of the experiments and to generate binding motifs for the TFs. The described technology allows higher throughput and identification of much longer binding profiles than current microarray-based methods. In addition, as our method is based on proteins expressed in mammalian cells, it can also be used to characterize DNA-binding preferences of full-length proteins or proteins requiring post-translational modifications. We validate the method by determining binding specificities of 14 different classes of TFs and by confirming the specificities for NFATC1 and RFX3 using ChIP-seq. Our results reveal unexpected dimeric modes of binding for several factors that were thought to preferentially bind DNA as monomers.

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IMPORTANCEThe long-term health outcomes and symptom burden of COVID-19 remain largely unclear.

Domesticated ungulate pluripotent embryonic stem (ES) cell lines would be useful for generating precise gene-modified animals. To date, many efforts have been made to establish domesticated ungulate pluripotent ES cells from early embryos without success. Here, we report the generation of porcine-induced pluripotent stem (iPS) cells using drug-inducible expression of defined factors. We showed that porcine iPS cells expressed alkaline phosphatase, SSEA3, SSEA4, Tra-1-60, Tra-1-81, Oct3/4, Nanog, Sox2, Rex1 and CDH1. Pig iPS cells expressed high levels of telomerase activity and showed normal karyotypes. These cells could differentiate into cell types of all three germ layers in vitro and in teratomas. Our study reveals properties of porcine pluripotent stem cells that may facilitate the eventual establishment of porcine ES cells. Moreover, the porcine iPS cells produced may be directly useful for the generation of precise gene-modified pigs.

Eukaryotic chromosomes are folded into higher-order conformations to coordinate genome functions. In addition to long-range chromatin loops, recent chromosome conformation capture (3C)-based studies have indicated higher levels of chromatin structures including compartments and topologically associating domains (TADs), which may serve as units of genome organization and functions. However, the molecular machinery underlying these hierarchically three-dimensional (3D) chromatin architectures remains poorly understood. Via high-throughput assays, including in situ Hi-C, DamID, ChIP-seq, and RNA-seq, we investigated roles of the Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU), a nuclear matrix (NM)-associated protein, in 3D genome organization. Upon the depletion of HNRNPU in mouse hepatocytes, the coverage of lamina-associated domains (LADs) in the genome increases from 53.1% to 68.6%, and a global condensation of chromatin was observed. Furthermore, disruption of HNRNPU leads to compartment switching on 7.5% of the genome, decreases TAD boundary strengths at borders between A (active) and B (inactive) compartments, and reduces chromatin loop intensities. Long-range chromatin interactions between and within compartments or TADs are also significantly remodeled upon HNRNPU depletion. Intriguingly, HNRNPU mainly associates with active chromatin, and 80% of HNRNPU peaks coincide with the binding of CTCF or RAD21. Collectively, we demonstrated that HNRNPU functions as a major factor maintaining 3D chromatin architecture, suggesting important roles of NM-associated proteins in genome organization.

Cell fate commitment of pre-implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. However, the intercellular signals that control fate determination of these cells remain obscure. Human embryonic stem cells (hESCs) provide a unique model for studying human early embryonic development. We have previously shown that Activin/Nodal signaling contributes to maintaining pluripotency of hESCs, which are derivatives of the inner cell mass. Here we further demonstrate that the inhibition of Activin/Nodal signaling results in the loss of hESC pluripotency and trophoblast differentiation, similar to BMP4-induced trophoblast differentiation from hESCs. We also show that the trophoblast induction effect of BMP4 correlates with and depends on the inhibition of Activin/Nodal signaling. However, the activation of BMP signaling is still required for trophoblast differentiation when Activin/Nodal signaling is inhibited. These data reveal that the early lineage segregation of hESCs is determined by the combinatorial signals of Activin/Nodal and BMP.

In order to address the biological function of GlcNAc N-deacetylase/N-sulfotransferase-1 (NDST-1), we disrupted the NDST-1 gene by homologous recombination in mouse embryonic stem cells. The NDST-1 null mice developed respiratory distress and atelectasis that subsequently caused neonatal death. Morphological examination revealed type II pneumocyte immaturity, which was characterized by an increased glycogen content and a reduced number of lamellar bodies and microvilli. Biochemical analysis further indicated that both total phospholipids and disaturated phosphatidylcholine were reduced in the mutant lung. Our data revealed that NDST-1 was essential for the maturation of type II pneumocytes and its inactivation led to a neonatal respiratory distress syndrome.z 2000 Federation of European Biochemical Societies.

Human embryonic stem (ES) cells possess the potential to differentiate into all the cell types of the human body and provide potential applications in regenerative medicine [1]. However, the concerns of immune rejection hamper transplantation therapies using human ES cells. To avoid the complications of immune rejection, diverse methods, such as somatic nuclear transfer (also called therapeutic cloning) and fusion of somatic cells with human ES cells [2], have been attempted to produce patient-specific pluripotent stem cells. Most of these approaches have resulted in little success. The generation of human iPS cells (induced pluripotent stem cells) from somatic cells with defined transcription factors makes it possible to produce patientspecific ES-like stem cells for therapeutic purposes [3,4]. Two sets of four-factors, OCT4, SOX2, C-MYC, KLF4 reported by Yamanaka's laboratory and OCT4, SOX2, NANOG, LIN28 reported by Thomson's laboratory, have been shown to reprogram human somatic cells to pluripotency with similar efficiency (10-20 iPS cell colonies from 0.1 million initial fibroblasts) [3,4]. We speculated that C-MYC and KLF4 might synergize with Thomson's 4 factors (OCT4, SOX2, NANOG, LIN28) to reprogram the human somatic cells. Our present study shows that a combination of 6 transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, significantly increases the efficiency of generating iPS cells from human somatic cells.The human genes encoding the transcription factors, OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4, were cloned into a lentiviral vector to produce lentivirus.Half million human newborn foreskin fibroblasts were transduced with the lentivirus carrying GFP (serving as negative control), or a cocktail of lentivirus carrying 4 factors (OCT4, NANOG, SOX2 and LIN28) or 6 factors (OCT4, NANOG, SOX2, LIN28, C-MYC and KLF4). Twenty-four hours later, the cells were dissociated with trypsin, transferred to five flasks (0.1 million initial cells per flask) coated with murine embryonic fibroblast (MEF) feeder, and cultured in human embryonic stem cell media. Colonies with a human ES cell-like morphology (iPS cell colonies) first became visible 12 days after transduction with 4 factors, whereas iPS cell colonies became visible 7 days after transduction with 6 factors. In order to quantify the efficiency of the reprogramming, the cells in one flask for each combination of factors were fixed on day 17 to analyze the alkaline phosphatase expression. A total of 16±3 colonies from 0.1 million initial fibroblast cells transduced with 4 factors were alkaline phosphatase-positive, 166±6 colonies from 0.1 million initial fibroblast cells transduced with 6 factors were alkaline phosphatase-positive, and no colonies were observed in GFP-lentivirus transduced controls ( Figure 1A). The efficiency of 6 factors is 10.4-fold more than that of 4 factors. The iPS colonies generated by transduction of 4 factors were picked on day 26. As reported by Yu et al. [4], these cells expressed alkaline phosphatase and undifferentia...