SUMMARY Epigenetic mechanisms have been proposed to play crucial roles in mammalian development, but their precise functions are only partially understood. To investigate epigenetic regulation of embryonic development, we differentiated human embryonic stem cells into mesendoderm, neural progenitor cells, trophoblast-like cells, and mesenchymal stem cells, and systematically characterized DNA methylation, chromatin modifications, and the transcriptome in each lineage. We found that promoters that are active in early developmental stages tend to be CG rich and mainly engage H3K27me3 upon silencing in non-expressing lineages. By contrast, promoters for genes expressed preferentially at later stages are often CG poor and primarily employ DNA methylation upon repression. Interestingly, the early developmental regulatory genes are often located in large genomic domains that are generally devoid of DNA methylation in most lineages, which we termed DNA methylation valleys (DMVs). Our results suggest that distinct epigenetic mechanisms regulate early and late stages of ES cell differentiation.

SUMMARY Here we show that as human embryonic stem (ES) cells exit the pluripotent state, NANOG can play a key role in determining lineage outcome. It has previously been reported that BMPs induce differentiation of human ES cells into extraembryonic lineages. Here we find that FGF2, acting through the MEK-ERK pathway, switches BMP4 induced human ES cell differentiation outcome to mesendoderm, characterized by the uniform expression of T (brachyury) and other primitive streak markers. We also find that MEK-ERK signaling prolongs NANOG expression during BMP-induced differentiation; that forced NANOG expression results in FGF independent BMP4 induction of mesendoderm; and that knockdown of NANOG greatly reduces T induction. Together, our results demonstrate that FGF2 signaling switches the outcome of BMP4 induced differentiation of human ES cells by maintaining NANOG levels through the MEKERK pathway.

Background Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells (iCMs) in situ represents a promising strategy for cardiac regeneration. A combination of three cardiac transcription factors, Gata4, Mef2c and Tbx5 (GMT), can convert fibroblasts into iCMs, albeit with low efficiency in vitro. Methods We screened 5,500 compounds in primary cardiac fibroblasts to identify the pathways that can be modulated to enhance cardiomyocyte reprogramming. Results We found that a combination of the transforming growth factor (TGF)-β inhibitor SB431542 and the WNT inhibitor XAV939 increased reprogramming efficiency eight-fold when added to GMT-overexpressing cardiac fibroblasts. The small-molecules also enhanced the speed and the quality of cell conversion, as we observed beating cells as early as 1 week after reprogramming compared to 6–8 weeks with GMT alone. In vivo, mice exposed to GMT, SB431542, and XAV939 for 2 weeks after myocardial infarction showed significantly improved reprogramming and cardiac function compared to those exposed to only GMT. Human cardiac reprogramming was similarly enhanced upon TGF-β and WNT inhibition and was achieved most efficiently with GMT plus Myocardin. Conclusions Thus, TGF-β and WNT inhibitors jointly enhance GMT-induced direct cardiac reprogramming from cardiac fibroblasts in vitro and in vivo and provide a more robust platform for cardiac regeneration.

Pluripotency, the ability of a cell to differentiate and give rise to all embryonic lineages, defines a small number of mammalian cell types such as embryonic stem (ES) cells. While it has been generally held that pluripotency is the product of a transcriptional regulatory network that activates and maintains the expression of key stem cell genes, accumulating evidence is pointing to a critical role for epigenetic processes in establishing and safeguarding the pluripotency of ES cells, as well as maintaining the identity of differentiated cell types. In order to better understand the role of epigenetic mechanisms in pluripotency, we have examined the dynamics of chromatin modifications genomewide in human ES cells (hESCs) undergoing differentiation into a mesendodermal lineage. We found that chromatin modifications at promoters remain largely invariant during differentiation, except at a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression, suggesting a hierarchy in cell fate commitment over most differentially expressed genes. We also mapped over 50 000 potential enhancers, and observed much greater dynamics in chromatin modifications, especially H3K4me1 and H3K27ac, which correlate with expression of their potential target genes. Further analysis of these enhancers revealed potentially key transcriptional regulators of pluripotency and a chromatin signature indicative of a poised state that may confer developmental competence in hESCs. Our results provide new evidence supporting the role of chromatin modifications in defining enhancers and pluripotency.

Fibroblast growth factor (FGF), transforming growth factor (TGF)/Nodal, and Insulin/insulin-like growth factor (IGF) signaling pathways are sufficient to maintain human embryonic stem cells (ESCs) and induced pluripotent stem cells in a proliferative, undifferentiated state. Here, we show that only a few FGF family members (FGF2, FGF4, FGF6, and FGF9) are able to sustain strong extracellular-signal-regulated kinase (ERK) phosphorylation and NANOG expression levels in human ESCs. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild-type protein, not receptor specificity, and that a mutated thermal-stable FGF1 sustains human ESCs and supports both differentiation and reprogramming protocols.

The transcription factor OCT4 is fundamental to maintaining pluripotency and self-renewal. To better understand protein-level regulation of OCT4, we applied liquid chromatography-MS to identify 14 localized sites of phosphorylation, 11 of which were previously unknown. Functional analysis of two sites, T234 and S235, suggested that phosphorylation within the homeobox region of OCT4 negatively regulates its activity by interrupting sequence-specific DNA binding. Mutating T234 and S235 to mimic constitutive phosphorylation at these sites reduces transcriptional activation from an OCT4-responsive reporter and decreases reprogramming efficiency. We also cataloged 144 unique phosphopeptides on known OCT4 interacting partners, including SOX2 and SALL4, that copurified during immunoprecipitation. These proteins were enriched for phosphorylation at motifs associated with ERK signaling. Likewise, OCT4 harbored several putative ERK phosphorylation sites. Kinase assays confirmed that ERK2 phosphorylated these sites in vitro, providing a direct link between ERK signaling and the transcriptional machinery that governs pluripotency.proteomics | posttranslational regulation O CT4 is a homeobox transcription factor that was first identified for its essential role in early mammalian development (1-3). It is expressed in totipotent and pluripotent cells and down-regulated on differentiation (4-6). OCT4 is required to maintain pluripotency both in vivo and in cell culture (2,7,8), and it is indispensable for transcription factor-mediated reprogramming (9-12). Together with NANOG and SOX2, OCT4 carries out these functions by activating transcription of genes that support pluripotency and repressing genes involved in development (13)(14)(15)(16)(17).A variety of proteins, including NANOG, SOX2, and OCT4 itself, form an intricate regulatory loop that balances OCT4 expression (15,(18)(19)(20). Genomic and epigenetic studies have identified additional mechanisms that directly or indirectly influence OCT4 expression, providing a detailed view of its transcriptional regulation (21-26). OCT4 function is closely tied to its regulation, because decreasing OCT4 mRNA levels cause differentiation into trophoblast, and increasing OCT4 expression by as little as 1.5-fold causes differentiation to primitive endoderm (7). The relative stoichiometry of OCT4 and SOX2 is also important for establishing pluripotency, because the efficiency of reprogramming is dependent on the proportion of OCT4 and SOX2 transcripts (9). Thus, precise regulation of OCT4 is essential for pluripotency.Although OCT4 transcriptional regulation has been extensively studied, far less is known about its posttranslational regulation. Previous studies have speculated that phosphorylation controls OCT4 activity (27,28). For example, differences in electrophoretic mobility suggested that the homeobox domain of OCT4 is differentially phosphorylated when expressed in 293 cells compared with HeLa cells (27). Intriguingly, these different states correlated with OCT4's ability to acti...

Highlights d Integrated scRNA-, ATAC-, and ChIP-seq analyses to interrogate cardiac reprogramming d Gata4, Mef2c, and Tbx5 both facilitate and limit one another's ability to bind to DNA d Mef2c and Tbx5 bind to inaccessible chromatin and promote its remodeling d Context-specific cooperative mechanisms guide cardiac reprogramming

We have developed and implemented a sequence identification algorithm (inSeq) that processes tandem mass spectra in real-time using the mass spectrometer's (MS) onboard processors. The inSeq algorithm relies on accurate mass tandem MS data for swift spectral matching with high accuracy. The instant spectral processing technology takes ∼16 ms to execute and provides information to enable autonomous, real-time decision making by the MS system. Using inSeq and its advanced decision tree logic, we demonstrate (i) realtime prediction of peptide elution windows en masse (∼3 min width, 3,000 targets), (ii) significant improvement of quantitative precision and accuracy (~3x boost in detected protein differences), and (iii) boosted rates of posttranslation modification site localization (90% agreement in real-time vs. offline localization rate and an approximate 25% gain in localized sites). The decision tree logic enabled by inSeq promises to circumvent problems with the conventional data-dependent acquisition paradigm and provides a direct route to streamlined and expedient targeted protein analysis.T he shotgun sequencing method has rapidly evolved over the past two decades (1, 2). In this strategy eluting peptide cations have their mass-to-charge (m∕z) values measured in the MS 1 scan. Then precursor m∕z values are selected for a series of sequential tandem MS events (MS 2 ). This succession is cycled for the duration of the analysis. The process, called data-dependent acquisition (DDA), is at the very core of shotgun analysis and has not changed for over 15 y, however, MS hardware has. Major improvements in MS sensitivity, scan rate, mass accuracy, and resolution have been achieved. Orbitrap hybrid systems, for example, routinely achieve low ppm mass accuracy with MS/MS repetition rates of 5-10 Hz (3, 4). Constant operation of such systems generates hundreds of thousands of spectra in hours. These MS 2 spectra are then mapped to sequence using database search algorithms (5-7).The DDA sampling strategy offers an elegant simplicity and has proven highly useful for discovery-driven proteomics. Of recent years, however, emphasis has shifted from identification to quantification-often with certain targets in mind. In this context, faults in the DDA approach have become increasingly evident. There are two primary limitations of the DDA approach: First, is poor run-to-run reproducibility and, second, is the inability to effectively target peptides of interest (8). Hundreds of peptides often coelute so that low-level signals often are selected in one run and not the next, and selecting m∕z peaks to sequence by abundance certainly does not offer the opportunity to inform the system of preselected targets.Several DDA add-ons and alternatives have been examined. Sampling depth, for example, can be increased by preventing selection of an m∕z value identified in a prior technical replicate (PAnDA) (9). Irreproducibility can be somewhat countered by informing the DDA algorithm of the precursor m∕z values of desired targets (inclu...