Chan Zhou

Long noncoding RNAs expressed in human hepatic stellate cells form networks with extracellular matrix proteins

BackgroundHepatic fibrosis is the underlying cause of cirrhosis and liver failure in nearly every form of chronic liver disease, and hepatic stellate cells (HSCs) are the primary cell type responsible for fibrosis. Long noncoding RNAs (lncRNAs) are increasingly recognized as regulators of development and disease; however, little is known about their expression in human HSCs and their function in hepatic fibrosis.MethodsWe performed RNA sequencing and ab initio assembly of RNA transcripts to define the lncRNAs expressed in human HSC myofibroblasts. We analyzed chromatin immunoprecipitation data and expression data to identify lncRNAs that were regulated by transforming growth factor beta (TGF-β) signaling, associated with super-enhancers and restricted in expression to HSCs compared with 43 human tissues and cell types. Co-expression network analyses were performed to discover functional modules of lncRNAs, and principle component analysis and K-mean clustering were used to compare lncRNA expression in HSCs with other myofibroblast cell types.ResultsWe identified over 3600 lncRNAs that are expressed in human HSC myofibroblasts. Many are regulated by TGF-β, a major fibrotic signal, and form networks with genes encoding key components of the extracellular matrix (ECM), which is the substrate of the fibrotic scar. The lncRNAs directly regulated by TGF-β signaling are also enriched at super-enhancers. More than 400 of the lncRNAs identified in HSCs are uniquely expressed in HSCs compared with 43 other human tissues and cell types and HSC myofibroblasts demonstrate different patterns of lncRNA expression compared with myofibroblasts originating from other tissues. Co-expression analyses identified a subset of lncRNAs that are tightly linked to collagen genes and numerous proteins that regulate the ECM during formation of the fibrotic scar. Finally, we identified lncRNAs that are induced during progression of human liver disease.ConclusionslncRNAs are likely key contributors to the formation and progression of fibrosis in human liver disease.

Identification of Novel Proteins Involved in Plant Cell-Wall Synthesis Based on Protein−Protein Interaction Data

The plant cell wall is mainly composed of polysaccharides, representing the richest source of biomass for future biofuel production. Currently, the majority of the cell-wall synthesis-related (CWSR) proteins are unknown even for model plant Arabidopsis thaliana. We report a computational framework for predicting CWSR proteins based on protein-protein interaction (PPI) data and known CWSR proteins. We predict a protein to be a CWSR protein if it interacts with known CWSR proteins (seeds) with high statistical significance. Using this technique, we predicted 100 candidate CWSR proteins in Arabidopsis thaliana, 8 of which were experimentally confirmed by previous reports. Forty-two candidates have either independent supporting evidence or strong functional relevance to cell-wall synthesis and, hence, are considered as the most reliable predictions. For 33 of the predicted CWSR proteins, we have predicted their detailed functional roles in CWS, based on analyses of their domain architectures, phylogeny, and current functional annotation in conjunction with a literature search. We present the constructed PPIs covering all the known and predicted CWSR proteins at http://csbl.bmb.uga.edu/∼zhouchan/CellWallProtein/. The 42 most reliable candidates provide useful targets to experimentalists for further investigation, and the PPI data constructed in this work provides new information for cell-wall research.

pDAWG: An Integrated Database for Plant Cell Wall Genes

We have recently developed a database, pDAWG, focused on information related to plant cell walls. Currently, pDAWG contains seven complete plant genomes, 12 complete algal genomes, along with computed information for individual proteins encoded in these genomes of the following types: (a) carbohydrate active enzyme (CAZy) family information when applicable; (b) phylogenetic trees of cell wall-related CAZy family proteins; (c) protein structure models if available; (d) physical and predicted interactions among proteins; (e) subcellular localization; (f) Pfam domain information; and (g) homology-based functional prediction. A querying system with a graphical interface allows a user to quickly compose information of different sorts about individual genes/proteins and to display the composite information in an intuitive manner, facilitating comparative analyses and knowledge discovery about cell wall genes. pDAWG can be accessed at http://csbl1.bmb.uga.edu/pDAWG/.

Phase Transition in Sequence Unique Reconstruction

In this paper, sequence unique reconstruction refers to the property that a sequence is uniquely reconstructable from all its K-tuples. We propose and study the phase transition behavior of the probability P(K) of unique reconstruction with regard to tuple size K in random sequences (iid model). Based on Monte Carlo experiments, artificial proteins generated from iid model exhibit a phase transition when P(K) abruptly jumps from a low value phase (e.g. < 0.1) to a high value phase (e.g. > 0.9). With a generalization to any alphabet, we prove that for a random sequence of length L, as L is large enough, P(K) undergoes a sharp phase transition when p ≤ 0.1015 where p = P (two random letters match). Besides, formulas are derived to estimate the transition points, which may be of practical use in sequencing DNA by hybridization. Concluded from our study, most proteins do not deviate greatly from random sequences in the sense of sequence unique reconstruction, while there are some “stubborn” proteins which only become uniquely reconstructable at a very large K and probably have biological implications.

Maintenance of macrophage transcriptional programs and intestinal homeostasis by epigenetic reader SP140

The epigenetic reader SP140, which is polymorphic in Crohn’s disease patients, regulates transcriptional programs in macrophages. PHDs crack the histone code Epigenetic readers that recognize histone modifications facilitate histone code–based transcriptional programming. Bromodomain- and plant homeodomain (PHD)–containing proteins often serve as readers of acetylation or methylation on histones, respectively. Mehta et al. have examined the function of SP140, a bromodomain- and PHD domain–containing reader in immune cells, and report that SP140 plays an essential role in repressing expression of lineage-inappropriate genes in macrophages. The authors also propose that SP140 polymorphisms associated with the development of Crohn’s disease represent functional loss of SP140. These studies advance our understanding of how epigenetic readers regulate immune responses in normal and diseased states. Epigenetic “readers” that recognize defined posttranslational modifications on histones have become desirable therapeutic targets for cancer and inflammation. SP140 is one such bromodomain- and plant homeodomain (PHD)–containing reader with immune-restricted expression, and single-nucleotide polymorphisms (SNPs) within SP140 associate with Crohn’s disease (CD). However, the function of SP140 and the consequences of disease-associated SP140 SNPs have remained unclear. We show that SP140 is critical for transcriptional programs that uphold the macrophage state. SP140 preferentially occupies promoters of silenced, lineage-inappropriate genes bearing the histone modification H3K27me3, such as the HOXA cluster in human macrophages, and ensures their repression. Depletion of SP140 in mouse or human macrophages resulted in severely compromised microbe-induced activation. We reveal that peripheral blood mononuclear cells (PBMCs) or B cells from individuals carrying CD-associated SNPs within SP140 have defective SP140 messenger RNA splicing and diminished SP140 protein levels. Moreover, CD patients carrying SP140 SNPs displayed suppressed innate immune gene signatures in a mixed population of PBMCs that stratified them from other CD patients. Hematopoietic-specific knockdown of Sp140 in mice resulted in exacerbated dextran sulfate sodium (DSS)–induced colitis, and low SP140 levels in human CD intestinal biopsies correlated with relatively lower intestinal innate cytokine levels and improved response to anti–tumor necrosis factor (TNF) therapy. Thus, the epigenetic reader SP140 is a key regulator of macrophage transcriptional programs for cellular state, and a loss of SP140 due to genetic variation contributes to a molecularly defined subset of CD characterized by ineffective innate immunity, normally critical for intestinal homeostasis.

DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC Expression to Control Definitive Endoderm Differentiation of Embryonic Stem Cells

Long noncoding RNAs (lncRNAs) exhibit diverse functions, including regulation of development. Here, we combine genome-wide mapping of SMAD3 occupancy with expression analysis to identify lncRNAs induced by activin signaling during endoderm differentiation of human embryonic stem cells (hESCs). We find that DIGIT is divergent to Goosecoid (GSC) and expressed during endoderm differentiation. Deletion of the SMAD3-occupied enhancer proximal to DIGIT inhibits DIGIT and GSC expression and definitive endoderm differentiation. Disruption of the gene encoding DIGIT and depletion of the DIGIT transcript reveal that DIGIT is required for definitive endoderm differentiation. In addition, we identify the mouse ortholog of DIGIT and show that it is expressed during development and promotes definitive endoderm differentiation of mouse ESCs. DIGIT regulates GSC in trans, and activation of endogenous GSC expression is sufficient to rescue definitive endoderm differentiation in DIGIT-deficient hESCs. Our study defines DIGIT as a conserved noncoding developmental regulator of definitive endoderm.

Tricyclic Antidepressants Promote Ceramide Accumulation to Regulate Collagen Production in Human Hepatic Stellate Cells

Activation of hepatic stellate cells (HSCs) in response to injury is a key step in hepatic fibrosis, and is characterized by trans-differentiation of quiescent HSCs to HSC myofibroblasts, which secrete extracellular matrix proteins responsible for the fibrotic scar. There are currently no therapies to directly inhibit hepatic fibrosis. We developed a small molecule screen to identify compounds that inactivate human HSC myofibroblasts through the quantification of lipid droplets. We screened 1600 compounds and identified 21 small molecules that induce HSC inactivation. Four hits were tricyclic antidepressants (TCAs), and they repressed expression of pro-fibrotic factors Alpha-Actin-2 (ACTA2) and Alpha-1 Type I Collagen (COL1A1) in HSCs. RNA sequencing implicated the sphingolipid pathway as a target of the TCAs. Indeed, TCA treatment of HSCs promoted accumulation of ceramide through inhibition of acid ceramidase (aCDase). Depletion of aCDase also promoted accumulation of ceramide and was associated with reduced COL1A1 expression. Treatment with B13, an inhibitor of aCDase, reproduced the antifibrotic phenotype as did the addition of exogenous ceramide. Our results show that detection of lipid droplets provides a robust readout to screen for regulators of hepatic fibrosis and have identified a novel antifibrotic role for ceramide.

Identification and characterization of m6A circular RNA epitranscriptomes

This study brings together the expanding fields of RNA modifications and circular (circ) RNAs. We find that cells express thousands of m6A methylated circRNAs, with cell-type specificity observed between human embryonic stem cells and HeLa cells. m6A-circRNAs were identified by RNA sequencing of total RNA following ribosome depletion and m6A immunoprecipitation. The presence of m6A-circRNAs is corroborated by the identification of complexes between circRNAs and YTHDF1 and YTHDF2, proteins that “read” m6A sites in mRNAs. Furthermore, m6A modifications on non-linear RNAs depend on METTL3 and METTL14, the known m6A methyltransferase “writer” complex components, suggesting that circRNAs are methylated by the same complexes responsible for m6A modification of linear RNAs. Despite sharing m6A readers and writers, m6A-circRNAs are frequently derived from exons not methylated in mRNAs. Nevertheless, m6A-mRNAs that are methylated on the same exons as those composing m6A-circRNAs exhibit less stability than other m6A-mRNA, and this circRNA-mRNA cross-talk is regulated by YTHDF2. Thus, our results expand the m6A regulatory code through identification of the first circRNA epitranscriptome.

AST: An Automated Sequence-Sampling Method for Improving the Taxonomic Diversity of Gene Phylogenetic Trees

A challenge in phylogenetic inference of gene trees is how to properly sample a large pool of homologous sequences to derive a good representative subset of sequences. Such a need arises in various applications, e.g. when (1) accuracy-oriented phylogenetic reconstruction methods may not be able to deal with a large pool of sequences due to their high demand in computing resources; (2) applications analyzing a collection of gene trees may prefer to use trees with fewer operational taxonomic units (OTUs), for instance for the detection of horizontal gene transfer events by identifying phylogenetic conflicts; and (3) the pool of available sequences is biased towards extensively studied species. In the past, the creation of subsamples often relied on manual selection. Here we present an Automated sequence-Sampling method for improving the Taxonomic diversity of gene phylogenetic trees, AST, to obtain representative sequences that maximize the taxonomic diversity of the sampled sequences. To demonstrate the effectiveness of AST, we have tested it to solve four problems, namely, inference of the evolutionary histories of the small ribosomal subunit protein S5 of E. coli, 16 S ribosomal RNAs and glycosyl-transferase gene family 8, and a study of ancient horizontal gene transfers from bacteria to plants. Our results show that the resolution of our computational results is almost as good as that of manual inference by domain experts, hence making the tool generally useful to phylogenetic studies by non-phylogeny specialists. The program is available at http://csbl.bmb.uga.edu/~zhouchan/AST.php.

Comparative analyses of distributions and functions of Z-DNA in Arabidopsis and rice.

Left-handed Z-DNA is an energetically unfavorable DNA structure that could form mostly under certain physiological conditions and was known to be involved in a number of cellular activities such as transcription regulation. We have compared the distributions and functions of Z-DNA in the genomes of Arabidopsis and rice, and observed that Z-DNA occurs in rice at least 9 times more often than in Arabidopsis; similar observations hold for other monocots and dicots. In addition, Z-DNA is significantly enriched in the coding regions of Arabidopsis, and in the high-GC-content regions of rice. Based on our analyses, we speculate that Z-DNA may play a role in regulating the expression of transcription factors, inhibitors, translation repressors, succinate dehydrogenases and glutathione-disulfide reductases in Arabidopsis, and it may affect the expression of vesicle and nucleosome genes and genes involved in alcohol transporter activity, stem cell maintenance, meristem development and reproductive structure development in rice.