In embryonic stem (ES) cells, bivalent chromatin domains with overlapping repressive (H3 lysine 27 tri-methylation) and activating (H3 lysine 4 tri-methylation) histone modifications mark the promoters of more than 2,000 genes. To gain insight into the structure and function of bivalent domains, we mapped key histone modifications and subunits of Polycomb-repressive complexes 1 and 2 (PRC1 and PRC2) genomewide in human and mouse ES cells by chromatin immunoprecipitation, followed by ultra high-throughput sequencing. We find that bivalent domains can be segregated into two classes—the first occupied by both PRC2 and PRC1 (PRC1-positive) and the second specifically bound by PRC2 (PRC2-only). PRC1-positive bivalent domains appear functionally distinct as they more efficiently retain lysine 27 tri-methylation upon differentiation, show stringent conservation of chromatin state, and associate with an overwhelming number of developmental regulator gene promoters. We also used computational genomics to search for sequence determinants of Polycomb binding. This analysis revealed that the genomewide locations of PRC2 and PRC1 can be largely predicted from the locations, sizes, and underlying motif contents of CpG islands. We propose that large CpG islands depleted of activating motifs confer epigenetic memory by recruiting the full repertoire of Polycomb complexes in pluripotent cells.

In a cell-free approach to regenerative therapeutics, transient application of paracrine factors in vivo could be used to alter the behavior and fate of progenitor cells to achieve sustained clinical benefits. Here we show that intramyocardial injection of synthetic modified RNA (modRNA) encoding human vascular endothelial growth factor-A (VEGF-A) resulted in the expansion and directed differentiation of endogenous heart progenitors in a murine myocardial infarction model. VEGF-A modRNA markedly improved heart function and enhanced long-term survival of recipients. This improvement was in part due to mobilization of epicardial progenitor cells and redirection of their differentiation toward cardiovascular cell types. Direct in vivo comparison with DNA vectors, and temporal control with VEGF inhibitors, documented the markedly increased efficacy of pulse-like delivery of VEGF-A. Our results suggest that modRNA is a versatile approach for expressing paracrine factors as cell fate switches to control progenitor cell fate and thereby enhance long term organ repair.

Mammalian preimplantation embryonic development (PED) is thought to be governed by highly conserved processes. While it had been suggested that some plasticity of conserved signaling networks exists among different mammalian species, it was not known to what extent modulation of the genomes and the regulatory proteins could “rewire” the gene regulatory networks (GRN) that control PED. We therefore generated global transcriptional profiles from three mammalian species (human, mouse, and bovine) at representative stages of PED, including: zygote, two-cell, four-cell, eight-cell, 16-cell, morula and blastocyst. Coexpression network analysis suggested that 40.2% orthologous gene triplets exhibited different expression patterns among these species. Combining the expression data with genomic sequences and the ChIP-seq data of 16 transcription regulators, we observed two classes of genomic changes that contributed to interspecies expression difference, including single nucleotide mutations leading to turnover of transcription factor binding sites, and insertion of cis-regulatory modules (CRMs) by transposons. About 10% of transposons are estimated to carry CRMs, which may drive species-specific gene expression. The two classes of genomic changes act in concert to drive mouse-specific expression of MTF2, which links POU5F1/NANOG to NOTCH signaling. We reconstructed the transition of the GRN structures as a function of time during PED. A comparison of the GRN transition processes among the three species suggested that in the bovine system, POU5F1's interacting partner SOX2 may be replaced by HMGB1 (a TF sharing the same DNA binding domain with SOX2), resulting in rewiring of GRN by a trans change.

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During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG protein complex.

RAG1 and RAG2 are the two lymphoid-specific proteins required for the cleavage of DNA sequences known as the recombination signal sequences (RSSs) flanking V, D or J regions of the antigen-binding genes. Previous studies have shown that RAG1 alone is capable of binding to the RSS, whereas RAG2 only binds as a RAG1/RAG2 complex. We have expressed recombinant core RAG1 (amino acids 384-1008) in Escherichia coli and demonstrated catalytic activity when combined with RAG2. This protein was then used to determine its oligomeric forms and the dissociation constant of binding to the RSS. Electrophoretic mobility shift assays show that up to three oligomeric complexes of core RAG1 form with a single RSS. Core RAG1 was found to exist as a dimer both when free in solution and as the minimal species bound to the RSS. Competition assays show that RAG1 recognizes both the conserved nonamer and heptamer sequences of the RSS. Zinc analysis shows the core to contain two zinc ions. The purified RAG1 protein overexpressed in E.coli exhibited the expected cleavage activity when combined with RAG2 purified from transfected 293T cells. The high mobility group protein HMG2 is stably incorporated into the recombinant RAG1/RSS complex and can increase the affinity of RAG1 for the RSS in the absence of RAG2.

Bioprinting has emerged as a promising tool in tissue engineering and regenerative medicine. Various 3D printing strategies have been developed to enable bioprinting of various biopolymers and hydrogels. However, the incorporation of biological factors has not been well explored. As the importance of personalized medicine is becoming more clear, the need for the development of bioinks containing autologous/patient-specific biological factors for tissue engineering applications becomes more evident. Platelet-rich plasma (PRP) is used as a patient-specific source of autologous growth factors that can be easily incorporated to hydrogels and printed into 3D constructs. PRP contains a cocktail of growth factors enhancing angiogenesis, stem cell recruitment, and tissue regeneration. Here, the development of an alginate-based bioink that can be printed and crosslinked upon implantation through exposure to native calcium ions is reported. This platform can be used for the controlled release of PRP-associated growth factors which may ultimately enhance vascularization and stem cell migration.

During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems.