Parallel functional assessment of m6A sites in human endodermal differentiation with base editor screens.

N6-methyladenosine (m6A) plays important role in lineage specifications of embryonic stem cells. However, it is still difficult to systematically dissect the specific m6A sites that are essential for early lineage differentiation. Here, we develop an adenine base editor-based strategy to systematically identify functional m6A sites that control lineage decisions of human embryonic stem cells. We design 7999 sgRNAs targeting 6048 m6A sites to screen for m6A sites that act as either boosters or barriers to definitive endoderm specification of human embryonic stem cells. We identify 78 sgRNAs enriched in the non-definitive endoderm cells and 137 sgRNAs enriched in the definitive endoderm cells. We successfully validate two definitive endoderm promoting m6A sites on SOX2 and SDHAF1 as well as a definitive endoderm inhibiting m6A site on ADM. Our study provides a functional screening of m6A sites and paves the way for functional studies of m6A at individual m6A site level.

Negative reciprocity, not ordered assembly, underlies the interaction of Sox2 and Oct4 on DNA.

The mode of interaction of transcription factors (TFs) on eukaryotic genomes remains a matter of debate. Single-molecule data in living cells for the TFs Sox2 and Oct4 were previously interpreted as evidence of ordered assembly on DNA. However, the quantity that was calculated does not determine binding order but, rather, energy expenditure away from thermodynamic equilibrium. Here, we undertake a rigorous biophysical analysis which leads to the concept of reciprocity. The single-molecule data imply that Sox2 and Oct4 exhibit negative reciprocity, with expression of Sox2 increasing Oct4's genomic binding but expression of Oct4 decreasing Sox2's binding. Models show that negative reciprocity can arise either from energy expenditure or from a mixture of positive and negative cooperativity at distinct genomic loci. Both possibilities imply unexpected complexity in how TFs interact on DNA, for which single-molecule methods provide novel detection capabilities.

Enhanced Generation of Integration-free iPSCs from Human Adult Peripheral Blood Mononuclear Cells with an Optimal Combination of Episomal Vectors.

We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.

Expression analysis of SOX14 during retinoic acid induced neural differentiation of embryonal carcinoma cells and assessment of the effect of its ectopic expression on SOXB members in HeLa cells.

SOX14 is a member of the SOXB2 subgroup of transcription factors implicated in neural development. Although the first SOX14 gene in vertebrates was cloned and characterized more than a decade ago and its expression profile during development was revealed in various animal model systems, the role of this gene during neural development is largely unknown. In the present study we analyzed the expression of SOX14 in human NT2/D1 and mouse P19 pluripotent embryonal carcinoma cells. We demonstrated that it is expressed in both cell lines and upregulated during retinoic acid induced neural differentiation. We showed that SOX14 was expressed in both neuronal and non-neuronal differentiated derivatives, as revealed by immunocytochemistry. Since it was previously proposed that increased SOXB2 proteins level interfere with the activity of SOXB1 counteracting partners, we compared expression patterns of SOXB members during retinoic acid induction of embryonal carcinoma cells. We revealed that upregulation of SOX14 expression is accompanied by alterations in the expression patterns of SOXB1 members. In order to analyze the potential cross-talk between them, we generated SOX14 expression construct. The ectopic expression of SOX14 was demonstrated at the mRNA level in NT2/D1, P19 and HeLa cells, while an increased level of SOX14 protein was detected in HeLa cells only. By transient transfection experiments in HeLa cells we showed for the first time that ectopic expression of SOX14 repressed SOX1 expression, whereas no significant effect on SOX2, SOX3 and SOX21 was observed. Data presented here provide an insight into SOX14 expression during in vitro neural differentiation of embryonal carcinoma cells and demonstrate the effect of its ectopic expression on protein levels of SOXB members in HeLa cells. Obtained results contribute to better understanding the role of one of the most conserved SOX proteins.

Xenopus Sox3 activates sox2 and geminin and indirectly represses Xvent2 expression to induce neural progenitor formation at the expense of non-neural ectodermal derivatives.

The SRY-related, HMG box SoxB1 transcription factors are highly homologous, evolutionarily conserved proteins that are expressed in neuroepithelial cells throughout neural development. SoxB1 genes are down-regulated as cells exit the cell-cycle to differentiate and are considered functionally redundant in maintaining neural precursor populations. However, little is known about Sox3 function and its mode of action during primary neurogenesis. Using gain and loss-of-function studies, we analyzed Sox3 function in detail in Xenopus early neural development and compared it to that of Sox2. Through these studies we identified the first targets of a SoxB1 protein during primary neurogenesis. Sox3 functions as an activator to induce expression of the early neural genes, sox2 and geminin in the absence of protein synthesis and to indirectly inhibit the Bmp target Xvent2. As a result, Sox3 increases cell proliferation, delays neurogenesis and inhibits epidermal and neural crest formation to expand the neural plate. Our studies indicate that Sox3 and 2 have many similar functions in this process including the ability to activate expression of geminin in naïve ectodermal explants. However, there are some differences; Sox3 activates the expression of sox2, while Sox2 does not activate expression of sox3 and sox3 is uniquely expressed throughout the ectoderm prior to neural induction suggesting a role in neural competence. With morpholino-mediated knockdown of Sox3, we demonstrate that it is required for induction of neural tissue by BMP inhibition. Together these data indicate that Sox3 has multiple roles in early neural development including as a factor required for nogginmediated neural induction.

A transdimensional Bayesian model for pattern recognition in DNA sequences.

Identification of transcription factor binding sites (TFBSs) is essential to elucidate gene regulatory networks. This article is focused on the recognition of overpresented short patterns, called "motifs", that may correspond to regulatory binding sites in the DNA sequences upstream of genes. An integrated Bayesian model is proposed to incorporate all unknown characteristics in motif discovery, including the number of motifs, motif widths, motif compositions, the number of motif sites, and locations of motif sites. Reversible jump Markov chain Monte Carlo is used to obtain posterior inference in the transdimensional parameter space. We present a number of suggestions for graphical summarization of the posterior distribution over the complex parameter space. The basic model is extended using a third-order Markov structure for nonmotif bases and allowing positions within a motif to be switched between 2 types: "conserved" and "degenerate." We evaluate the prediction accuracy for the simulated data with 3 motifs and apply the model to upstream sequences in high signal-to-noise regions in a human ChIP-chip study. The performance of the Bayesian model is assessed using yeast data sets of various numbers of sequences and background structures, with and without true TFBSs. The performance is also compared to other computational methods, including 2 statistical approaches, AlignACE and multiple expectation maximization for motif elicitation, and 1 word numeration-based approach, yeast motif finder (YMF).