Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium (CM). Bone morphogenetic proteins (BMPs) have previously been shown to induce hESC differentiation, in apparent contrast to mouse embryonic stem (ES) cells, in which BMP4 synergizes with leukemia inhibitory factor (LIF) to maintain self-renewal. Here we demonstrate that hESCs cultured in unconditioned medium (UM) are subjected to high levels of BMP signaling activity, which is reduced in CM. The BMP antagonist noggin synergizes with basic fibroblast growth factor (bFGF) to repress BMP signaling and sustain undifferentiated proliferation of hESCs in the absence of fibroblasts or CM. These findings suggest a basic difference in the self-renewal mechanism between mouse and human ES cells and simplify the culture of hESCs.

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally, antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails, but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here, we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally, we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example, H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique.electron transfer dissociation ͉ epigenetics ͉ posttranslational modification ͉ histone code ͉ pluripotency P luripotency-the ability to differentiate into any specialized lineage-is the hallmark of embryonic stem (ES) cells and the basis for their experimental and therapeutic potential. The precise molecular mechanisms that define pluripotency remain elusive; however, a number of recent works suggest a central role for epigenetic regulation through chromatin (1-6). Either by recruiting or shielding certain factors, modifications on histone proteins modulate a gene's local environment and thereby regulate expression (7-12). Concerted changes in histone modification states occur during differentiation (13). For example, high levels of histone H3 and H4 acetylation are characteristic of pluripotent cells in mice and the abundance of these marks decreases during differentiation (14, 15). Methylation of R2, R17, and R26 of histone H3 by CARM1 also correlates with cell fate and potency (6). Cells with higher levels of methylation, at these residues, were enriched in the embryonic part of the blastocyst. Next, specific patterns of histone H3K4me3 and H3K27me3 are observed at promoter regions of genes that are regulated during differentiation (1, 2). Finally, demethylation of H3K27me3 is required for activation of certain HOX genes essential for proper development (16,17). The demethylase responsible interacts directly with MLL 2/3 complexes, which methylate histone H3K4 (18). Taken together these experiments have shed new light on the power of epigenetic regulation within ES cells; however, the precise details and role(s) of such combinatorial PTM patterns remain largely unknown.Technological limitations in our ability to discover and quantify combinatorial histone PTMs has, and continues to, present a major obstacle. Most of our knowledge of epigenetics has been derived by antibody-based approaches. Antibodies require a priori knowledge of individual modifications, are subject to epitope occlusion, and have difficulty distingu...

The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences.

Small-RNA-mediated chromatin modifications have been widely studied in plants and S. pombe. However, direct evidence of small-RNA-guided sequence-specific chromatin alterations is scarce in animals. In C. elegans, the nuclear RNAi defective (Nrde) pathway functions to transport siRNA from the cytoplasm to the nucleus, modulate transcription elongation, induce histone H3 lysine 9 (H3K9) trimethylation, and mediate transgenerational inheritance of RNAi. Here, we show that both exogenous RNAi and NRDE-bound endogenous 22G RNAs can direct sequence-specific histone H3 lysine 27 (H3K27) trimethylation at targeted loci through the Nrde pathway. The resulting H3K27me3 status can be inherited by progeny for multiple generations. piRNAs and WAGO-1-associated siRNAs induce H3K27 methylation as well. Interestingly, CSR-1-associated endogenous siRNAs fail to trigger H3K27 methylation, whereas exogenous provision of dsRNAs can induce H3K27 methylation at the CSR-1-targeted loci via the Nrde pathway. We further observed distinct genetic requirements of H3K9 and H3K27 trimethylation. Whereas set-25 and met-2 are required for K9 methylation, mes-2 is required for K27 methylation. The depletion of mes-2 leads to a nuclear RNAi defective phenotype. These results indicate that dsRNA-triggered chromatin modification is a sequence-specific response that engages the Nrde pathway in C. elegans.