Embryonic stem (ES) cells are under precise control of both intrinsic self-renewal gene regulatory network and extrinsic growth factor-triggered signaling cascades. How external signaling pathways connect to core self-renewal transcriptional circuits is largely unknown. To probe this, we chose BMP signaling, which is previously recognized as a master control for both self-renewal and lineage commitment of murine ES cells. Here, we mapped target gene promoter occupancy of SMAD1/5 and SMAD4 on a genome-wide scale and found that they associate with a large group of developmental regulators that are enriched for H3K27 trimethylation and H3K4 trimethylation bivalent marks and are repressed in the self-renewing state, whereas they are rapidly induced upon differentiation. Smad knockdown experiments further indicate that SMAD-mediated BMP signaling is largely required for differentiation-related processes rather than directly influencing self-renewal. Among the SMAD-associated genes, we further identified Dpysl2 (previously known as Crmp2) and the H3K27 demethylase Kdm6b (previously known as Jmjd3) as BMP4-modulated early neural differentiation regulators. Combined with computational analysis, our results suggest that SMAD-mediated BMP signaling balances self-renewal versus differentiation by modulating a set of developmental regulators.

Polycomb group (PcG) proteins control organism development by regulating the expression of developmental genes. Transcriptional regulation by PcG proteins is achieved, at least partly, through the PRC2-mediated methylation on lysine 27 of histone H3 (H3K27) and PRC1-mediated ubiquitylation on lysine 119 of histone H2A (uH2A). As an integral component of PRC1, Bmi1 has been demonstrated to be critical for H2A ubiquitylation. Although recent studies have revealed the genome-wide binding patterns of some of the PRC1 and PRC2 components, as well as the H3K27me3 mark, there have been no reports describing genome-wide localization of uH2A. Using the recently developed ChIP-Seq technology, here, we report genome-wide localization of the Bmi1-dependent uH2A mark in MEF cells. Gene promoter averaging analysis indicates a peak of uH2A just inside the transcription start site (TSS) of well-annotated genes. This peak is enriched at promoters containing the H3K27me3 mark and represents the least expressed genes in WT MEF cells. In addition, peak finding reveals regions of local uH2A enrichment throughout the mouse genome, including almost 700 gene promoters. Genes with promoter peaks of uH2A exhibit lower-level expression when compared to genes that do not contain promoter peaks of uH2A. Moreover, we demonstrate that genes with uH2A peaks have increased expression upon Bmi1 knockout. Importantly, local enrichment of uH2A is not limited to regions containing the H3K27me3 mark. We describe the enrichment of H2A ubiquitylation at high-density CpG promoters and provide evidence to suggest that DNA methylation may be linked to uH2A at these regions. Thus, our work not only reveals Bmi1-dependent H2A ubiquitylation, but also suggests that uH2A targeting in differentiated cells may employ a different mechanism from that in ES cells.

Elongation of rice internodes is one of the most important agronomic traits, which determines the plant height and underlies the grain yield. It has been shown that the elongation of internodes is under genetic control and various factors are implicated in the process. Here, we report a detailed characterization of an elongated uppermost internode1 (eui1) mutant, which has been used in hybrid rice breeding. In the eui1-2 mutant, the cell lengths in the uppermost internodes are significantly longer than that of wild type and thus give rise to the elongated uppermost internode. It was found that the level of active gibberellic acids (GAs) was elevated in the mutant, whereas its growth in response to GA was similar to that of wild type, suggesting the higher level accumulation of GA in the eui1 causes the abnormal elongation of the uppermost internode. Consistently, the expression levels of several genes, which encode gibberellin biosynthesis enzymes, were altered. We cloned the EUI1 gene by map-based cloning, which encodes a putative cytochrome P450 monooxygenase, and found that EUI1 was weakly expressed in most tissues, but preferentially in young panicles. To confirm its function, transgenic experiments with different constructs of EUI1 were conducted. Overexpression of EUI1 gave rise to the GA-deficient like phenotypes, which could be partially reversed by the supplement of GA. Furthermore, apart from the alteration of expression levels of the GA biosynthesis genes, accumulation of SLR1 protein was found in the overexpression transgenic plants, indicating expression level of EUI1 is implicated in both GA-mediated SLR1 destruction and a feedback regulation in GA biosynthesis. Therefore, we proposed that EUI1 plays a negative role in GA-mediated regulation of cell elongation in the uppermost internode of rice.

The protein-protein interaction networks, or interactome networks, have been shown to have dynamic modular structures, yet the functional connections between and among the modules are less well understood. Here, using a new pipeline to integrate the interactome and the transcriptome, we identified a pair of transcriptionally anticorrelated modules, each consisting of hundreds of genes in multicellular interactome networks across different individuals and populations. The two modules are associated with cellular proliferation and differentiation, respectively. The proliferation module is conserved among eukaryotic organisms, whereas the differentiation module is specific to multicellular organisms. Upon differentiation of various tissues and cell lines from different organisms, the expression of the proliferation module is more uniformly suppressed, while the differentiation module is upregulated in a tissue- and species-specific manner. Our results indicate that even at the tissue and organism levels, proliferation and differentiation modules may correspond to two alternative states of the molecular network and may reflect a universal symbiotic relationship in a multicellular organism. Our analyses further predict that the proteins mediating the interactions between these modules may serve as modulators at the proliferation/differentiation switch.

Many fundamental questions on aging are still unanswered or are under intense debate. These questions are frequently not addressable by examining a single gene or a single pathway, but can best be addressed at the systems level. Here we examined the modular structure of the protein-protein interaction (PPI) networks during fruitfly and human brain aging. In both networks, there are two modules associated with the cellular proliferation to differentiation temporal switch that display opposite aging-related changes in expression. During fly aging, another couple of modules are associated with the oxidative-reductive metabolic temporal switch. These network modules and their relationships demonstrate (1) that aging is largely associated with a small number, instead of many network modules,(2) that some modular changes might be reversible and (3) that genes connecting different modules through PPIs are more likely to affect aging/longevity, a conclusion that is experimentally validated by Caenorhabditis elegans lifespan analysis. Network simulations further suggest that aging might preferentially attack key regulatory nodes that are important for the network stability, implicating a potential molecular basis for the stochastic nature of aging.

It has been a puzzle that genome or proteome sizes are not correlated with the complexity of the organisms. Although alternative splicing and noncoding and regulatory elements explain some of the differences, the complexity of the protein interaction network and regulatory network may provide additional explanations. Here, we collected 642 domains that mediate protein-protein interactions (PPIs) and examined the evolution of the PPI domains and its impact on organismal complexity and PPI network complexity. In agreement with previous more general studies of protein domains, a significant expansion of PPI domains per proteome was found in metazoa. We also found both the number and coverage of PPI domains per protein increased. However, a better correlation with complexity was seen with increasing PPI domain coverage per protein, so that proteins in complex organisms are more compact and specialized in PPI. Such a structural adaptation of the proteins is correlated with the number of interactions that the proteins can make in PPI networks, and seems to be a more favorable way to increase network connectivity than other structural adaptations.

Root hair formation is an important model with which to study cell patterning and differentiation in higher plants. Ethylene and auxin are critical regulators of root hair development. The role of jasmonates (JAs) was examined in Arabidopsis root hair development as well as their interactions with ethylene in this process. The results have shown that both methyl jasmonate (MeJA) and jasmonic acid (JA) have a pronounced effect on promoting root hair formation. However, the effect of MeJA and JA on root hair formation was blocked by ethylene inhibitors Ag(+) or aminoethoxyvinylglycine (AVG). The stimulatory effects of MeJA and JA were also diminished in ethylene-insensitive mutants etr1-1 and etr1-3. Furthermore, the JA biosynthesis inhibitors ibuprofen and salicylhydroxamic acid (SHAM) suppressed 1-aminocyclopropane-1-carboxylic acid (ACC)-induced root hair formation, and decreased the root hairs in seedlings of the ethylene over-producing mutant eto1-1. These results suggested that JAs promote root hair formation, through an interaction with ethylene.

A monoclonal antibody (McAb) against methyl jasmonate (MeJA) was prepared and characterized. The McAb, J2-4B, was derived from an immunogen in which the C1-COOH of jasmonic acid (JA) was conjugated to the -NH2 of keyhole limpet hemocyanin (KLH). The McAb showed a higher recognition ability to methyl esters of JA than to its free acids. The integrity of a pentenyl in JA molecule was necessary for the recognition of McAb. Hydrogenation at C-9 and C-10 (dihydrojasmonic acid, 2H-JA) or eliminating the methyl group at C-12 (JAS-25) significantly abolished the binding force of JA molecule with the McAb. Some structural or functional analogues or precursor of JA, such as cucurbic acid, theobroxide, coronatine, and linolenic acid, could not be recognized by the McAb. The McAb has been used to set up a competitive enzyme-linked immunosorbent assay (ELISA) with a linearity range from 2.06 to 500 pmol of MeJA. Using this method, the fluctuations of JA content in florets during anthesis of wheat and Italian ryegrass were analyzed. Results showed that JA level increased obviously as the florets approaching to opening, arrived at a "peak" value at full opening and decreased sharply afterwards.

PPARgamma is a dominant regulator of fat cell differentiation. However, this nuclear receptor also plays an important role in the differentiation of intestinal and other epithelial cell types. The mechanism by which PPARgamma can influence the differentiation of such diverse cell lineages is unknown. We show here that PPARgamma interacts with Hic-5, a coactivator protein expressed in gut epithelial cells. Hic-5 and PPARgamma colocalize to the villus epithelium of the small intestine, and their expression during embryonic gut development correlates with the transition from endoderm to a specialized epithelium; expression of both these factors is reduced in tumors. Forced expression of Hic-5 in colon cancer cells enhances the PPARgamma-mediated induction of several gut epithelial differentiation/maturation markers such as L-FABP, kruppel-like factor 4 (KLF4), and keratin 20. siRNA directed against Hic-5 specifically reduces PPARgamma-mediated induction of gut epithelial genes in colon cells and in an ex vivo model of embryonic gut differentiation. Finally, forced expression of Hic-5 during 3T3-L1 preadipocyte differentiation inhibits adipogenesis while inducing inappropriate expression of several mRNAs characteristic of gut epithelium in these mesenchymal cells. These results indicate that Hic5 is an important component in determining an epithelial differentiation program induced by PPARgamma.