Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells. In contrast, somatic cells transmit considerable epigenetic information to progenies. Paradoxically, the persistence of the somatic epigenome makes it more vulnerable to noise, since random epimutations can accumulate to massively perturb the epigenomic ground state. The rate of epigenetic perturbation depends on the genomic context, and, in particular, DNA methylation loss is coupled to late DNA replication dynamics. Epigenetic perturbation is not observed in the pluripotent state, because the rapid turnover-based equilibrium continuously reinforces the canonical state. This dynamic epigenetic equilibrium also explains how the epigenome can be reprogrammed quickly and to near perfection after induced pluripotency.

Multiple transcriptional and epigenetic changes drive differentiation of embryonic stem cells (ESCs). This study unveils an additional level of gene expression regulation involving noncanonical, cap-independent translation of a select group of mRNAs. This is driven by death-associated protein 5 (DAP5/eIF4G2/NAT1), a translation initiation factor mediating IRES-dependent translation. We found that the DAP5 knockdown from human ESCs (hESCs) resulted in persistence of pluripotent gene expression, delayed induction of differentiation-associated genes in different cell lineages, and defective embryoid body formation. The latter involved improper cellular organization, lack of cavitation, and enhanced mislocalized apoptosis. RNA sequencing of polysome-associated mRNAs identified candidates with reduced translation efficiency in DAP5-depleted hESCs. These were enriched in mitochondrial proteins involved in oxidative respiration, a pathway essential for differentiation, the significance of which was confirmed by the aberrant mitochondrial morphology and decreased oxidative respiratory activity in DAP5 knockdown cells. Further analysis identified the chromatin modifier HMGN3 as a cap-independent DAP5 translation target whose knockdown resulted in defective differentiation. Thus, DAP5-mediated translation of a specific set of proteins is critical for the transition from pluripotency to differentiation, highlighting the importance of capindependent translation in stem cell fate decisions.

NF-B induces the expression of genes involved in immune response, apoptosis, inflammation, and the cell cycle. Certain NF-B-responsive genes are activated rapidly after the cell is stimulated by cytokines and other extracellular signals. However, the mechanism by which these genes are activated is not entirely understood. Here we report that even though NF-B interacts directly with TAF II s, induction of NF-B by tumor necrosis factor alpha (TNF-␣) does not enhance TFIID recruitment and preinitiation complex formation on some NF-B-responsive promoters. These promoters are bound by the transcription apparatus prior to TNF-␣ stimulus. Using the immediate-early TNF-␣-responsive gene A20 as a prototype promoter, we found that the constitutive association of the general transcription apparatus is mediated by Sp1 and that this is crucial for rapid transcriptional induction by NF-B. In vitro transcription assays confirmed that NF-B plays a postinitiation role since it enhances the transcription reinitiation rate whereas Sp1 is required for the initiation step. Thus, the consecutive effects of Sp1 and NF-B on the transcription process underlie the mechanism of their synergy and allow rapid transcriptional induction in response to cytokines.The family of NF-B transcription factors is a central component of the cellular response to a broad range of extracellular signals, many of them are related to immunological functions and stress. NF-B controls the expression of a large number of genes including inflammatory cytokines, chemokines, immunological factors, adhesion molecules, cell cycle regulators, and pro-and antiapoptotic factors (24). A major pathway regulating NF-B activity involves its nuclear transport. In unstimulated cells, NF-B is retained in the cytoplasm in an inactive form by IB proteins. Signals that activate NF-B trigger ubiquitination and degradation of IB by the proteosome, resulting in transport of NF-B into the nucleus and transcriptional activation of responsive genes. Since IB␣ is one of the NF-B target genes, the newly synthesized IB␣ negatively regulates NF-B, thus forming an autoregulatory loop.In the nucleus, transcriptional activation by NF-B involves its association with multiple coactivators. We reported previously that the substoichiometric TFIID subunit, TAF II 105, which is enriched in B cells, interacts directly with p65/Re1A, a member of the NF-B family, and is important for activation of a subset of NF-B-dependent antiapoptotic genes in vivo (30,36,37). Likewise, other TFIID subunits such as hTAF II 250, hTAF II 80, and hTAF II 28 were reported to bind to p65/Re1A (8), although the physiological importance of these interactions was not investigated. In addition to TFIID, the coactivator protein CREB-binding protein CBP and its homolog p300 were reported to be involved in transcription activation by the p65/Re1A subunit of NF-B (6, 25). p65 was also found to interact specifically with the composite coactivator ARC/DRIP, and this complex supports NF-B-dependent transcriptional activation in v...

Advances in single-cell genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells. Current sequencing-based methods for cell lineage analysis depend on low-resolution bulk analysis or rely on extensive single-cell sequencing, which is not scalable and could be biased by functional dependencies. Here we show an integrated biochemical-computational platform for generic single-cell lineage analysis that is retrospective, cost-effective, and scalable. It consists of a biochemical-computational pipeline that inputs individual cells, produces targeted single-cell sequencing data, and uses it to generate a lineage tree of the input cells. We validated the platform by applying it to cells sampled from an ex vivo grown tree and analyzed its feasibility landscape by computer simulations. We conclude that the platform may serve as a generic tool for lineage analysis and thus pave the way toward large-scale human cell lineage discovery.

NF-B transcription factors activate genes important for immune response, inflammation, and cell survival. P-TEFb and DSIF, which are positive and negative transcription elongation factors, respectively, both regulate NF-B-induced transcription, but the mechanism underlying their recruitment to NF-B target genes is unknown. We show here that upon induction of NF-B, a subset of target genes is regulated differentially by either P-TEFb or DSIF. The regulation of these genes and their occupancy by these elongation factors are dependent on the NF-B enhancer and the core promoter type. Converting a TATA-less promoter to a TATA promoter switches the regulation of NF-B from DSIF to P-TEFb. Accumulation or displacement of DSIF and P-TEFb is dictated by the formation of distinct initiation complexes (TFIID dependent or independent) on the two types of core promoter. The underlying mechanism for the dissociation of DSIF from TATA promoters upon NF-B activation involves the phosphorylation of RNA polymerase II by P-TEFb. The results highlight a regulatory link between the initiation and the elongation phases of the transcription reaction and broaden our comprehension of the NF-B pathway.Transcription of protein-coding genes by RNA polymerase II (Pol II) is a multistep process, each step being a target for regulation and critical for the production of mature mRNA (27,29). A number of factors that control RNA Pol II elongation have been characterized in recent years. Among these are the positive elongation factor P-TEFb, which induces Pol II processivity by facilitating the transition from the early to the late elongation phase (24), and two negative elongation factors, DSIF (DRB sensitivity inducing factor) (31) and NELF (negative elongation factor) (37). In vitro, P-TEFb alleviates transcription inhibition by DSIF (25,32).NF-B is a transcription factor central to the cellular response to a broad range of extracellular signals, including inflammatory cytokines, tumor promoters, and chemotherapeutic agents. In response to these agents, NF-B induces the expression of cell cycle regulators, pro-and antiapoptotic factors, inflammatory cytokines, chemokines, adhesion molecules, and many other factors (22). In unstimulated cells, NF-B is retained in the cytoplasm by IB proteins. NF-B-activating signals trigger degradation of IB and nuclear translocation of NF-B, which result in activation of responsive genes (14). A subset of early response genes that includes IB␣ and A20 are themselves negative regulators of the NF-B pathway and so form a negative feedback loop. Transcriptional control of these genes is likely to influence the strength and the duration of the inflammatory signal.Induction of NF-B target genes is remarkably fast, and the mechanism underlying their rapid transcriptional activation was investigated previously. It was found that the promoters of NF-B-regulated genes are bound by the general transcription machinery prior to NF-B activation, and subsequent activation by NF-B increases the rate of the transcription cycl...