Qiang Tu

Global regulatory logic for specification of an embryonic cell lineage

Explanation of a process of development must ultimately be couched in the terms of the genomic regulatory code. Specification of an embryonic cell lineage is driven by a network of interactions among genes encoding transcription factors. Here, we present the gene regulatory network (GRN) that directs the specification of the skeletogenic micromere lineage of the sea urchin embryo. The GRN now includes all regulatory genes expressed in this lineage up to late blastula stage, as identified in a genomewide survey. The architecture of the GRN was established by a large-scale perturbation analysis in which the expression of each gene in the GRN was cut off by use of morpholinos, and the effects on all other genes were measured quantitatively. Several cis-regulatory analyses provided additional evidence. The explanatory power of the GRN suffices to provide a causal explanation for all observable developmental functions of the micromere lineage during the specification period. These functions are: (i) initial acquisition of identity through transcriptional interpretation of localized maternal cues; (ii) activation of specific regulatory genes by use of a double negative gate; (iii) dynamic stabilization of the regulatory state by activation of a feedback subcircuit; (iv) exclusion of alternative regulatory states; (v) presentation of a signal required by the micromeres themselves and of two different signals required for development of adjacent endomesodermal lineages; and (vi) lineage-specific activation of batteries of skeletogenic genes. The GRN precisely predicts gene expression responses and provides a coherent explanation of the biology of specification.

Gene structure in the sea urchin Strongylocentrotus purpuratus based on transcriptome analysis

A comprehensive transcriptome analysis has been performed on protein-coding RNAs of Strongylocentrotus purpuratus, including 10 different embryonic stages, six feeding larval and metamorphosed juvenile stages, and six adult tissues. In this study, we pooled the transcriptomes from all of these sources and focused on the insights they provide for gene structure in the genome of this recently sequenced model system. The genome had initially been annotated by use of computational gene model prediction algorithms. A large fraction of these predicted genes were recovered in the transcriptome when the reads were mapped to the genome and appropriately filtered and analyzed. However, in a manually curated subset, we discovered that more than half the computational gene model predictions were imperfect, containing errors such as missing exons, prediction of nonexistent exons, erroneous intron/exon boundaries, fusion of adjacent genes, and prediction of multiple genes from single genes. The transcriptome data have been used to provide a systematic upgrade of the gene model predictions throughout the genome, very greatly improving the research usability of the genomic sequence. We have constructed new public databases that incorporate information from the transcriptome analyses. The transcript-based gene model data were used to define average structural parameters for S. purpuratus protein-coding genes. In addition, we constructed a custom sea urchin gene ontology, and assigned about 7000 different annotated transcripts to 24 functional classes. Strong correlations became evident between given functional ontology classes and structural properties, including gene size, exon number, and exon and intron size.

Quantitative developmental transcriptomes of the sea urchin Strongylocentrotus purpuratus

Development depends on the precise control of gene expression in time and space. A critical step towards understanding the global gene regulatory networks underlying development is to obtain comprehensive information on gene expression. In this study, we measured expression profiles for the entire expressed gene set during sea urchin embryonic development. We confirmed the reliability of these profiles by comparison with NanoString measurements for a subset of genes and with literature values. The data show that ~16,500 genes have been activated by the end of embryogenesis, and for half of them the transcript abundance changes more than 10-fold during development. From this genome scale expression survey, we show that complex patterns of expression by many genes underlie embryonic development, particularly during the early stages before gastrulation. An intuitive web application for data query and visualization is presented to facilitate use of this large dataset.

Detecting pathogenicity islands and anomalous gene clusters by iterative discriminant analysis

We present a simple method to detect pathogenicity islands and anomalous gene clusters in bacterial genomes. The method uses iterative discriminant analysis to define genomic regions that deviate most from the rest of the genome in three compositional criteria: G+C content, dinucleotide frequency and codon usage. Using this method, we identify many virulence-related gene islands, e.g. encoding protein secretion systems, adhesins, toxins, and other anomalous gene clusters, such as prophages. The program and the whole dataset, including the catalogs of genes in the detected anomalous segments, are publicly available at http://compbio.sibsnet.org/projects/pai-ida/. This program can be used in searching for virulence-related factors in newly sequenced bacterial genomes.

Cloning and mapping of human PKIB and PKIG, and comparison of tissue expression patterns of three members of the protein kinase inhibitor family, including PKIA

Two novel members of the human cAMP-dependent protein kinase inhibitor (PKI) gene family, PKIB and PKIG, were cloned. The deduced proteins showed 70% and 90% identity with mouse PKIbeta and PKIgamma respectively. Both the already identified pseudosubstrate site and leucine-rich nuclear export signal motifs were defined from the 11 PKIs of different species. The PKIB and PKIG genes were mapped respectively to chromosome 6q21-22.1, using a radiation hybrid GB4 panel, and to chromosome 20q13.12-13.13, using a Stanford G3 panel. Northern-blot analysis of three PKI isoforms, including the PKIA identified previously, revealed significant differences in their expression patterns. PKIB had two transcripts of 1.9 kb and 1.4 kb. The former transcript was abundant in both placenta and brain and the latter was expressed most abundantly in placenta, highly in brain, heart, liver, pancreas, moderately in kidney, skeletal muscle and colon, and very little in the other eight tissues tested. PKIG was widely expressed as a 1.5-kb transcript with the highest level in heart, hardly detectable in thymus and peripheral blood leucocytes and was moderately expressed in the other tissues, with slightly different levels. However, PKIA was specifically expressed as two transcripts of 3.3 kb and 1.5 kb in heart and skeletal muscle. The distinct expression patterns of the three PKIs suggest that their roles in various tissues are probably different.

General approach for in vivo recovery of cell type-specific effector gene sets

Differentially expressed, cell type-specific effector gene sets hold the key to multiple important problems in biology, from theoretical aspects of developmental gene regulatory networks (GRNs) to various practical applications. Although individual cell types of interest have been recovered by various methods and analyzed, systematic recovery of multiple cell type-specific gene sets from whole developing organisms has remained problematic. Here we describe a general methodology using the sea urchin embryo, a material of choice because of the large-scale GRNs already solved for this model system. This method utilizes the regulatory states expressed by given cells of the embryo to define cell type and includes a fluorescence activated cell sorting (FACS) procedure that results in no perturbation of transcript representation. We have extensively validated the method by spatial and qualitative analyses of the transcriptome expressed in isolated embryonic skeletogenic cells and as a consequence, generated a prototypical cell type-specific transcriptome database.

Genome-wide assessment of differential effector gene use in embryogenesis.

Six different populations of cells were isolated by fluorescence-activated cell sorting from disaggregated late blastula- and gastrula-stage sea urchin embryos according to the regulatory states expressed in these cells, as reported by recombineered bacterial artificial chromosomes producing fluorochromes. Transcriptomes recovered from these embryonic cell populations revealed striking, early differential expression of large cohorts of effector genes. The six cell populations were presumptive pigment cells, presumptive neurogenic cells, presumptive skeletogenic cells, cells from the stomodeal region of the oral ectoderm, ciliated band cells and cells from the endoderm/ectoderm boundary that will give rise both to hindgut and to border ectoderm. Transcriptome analysis revealed that each of these domains specifically expressed several hundred effector genes at significant levels. Annotation indicated the qualitative individuality of the functional nature of each cell population, even though they were isolated from embryos only 1-2 days old. In no case was more than a tiny fraction of the transcripts enriched in one population also enriched in any other of the six populations studied. As was particularly clear in the cases of the presumptive pigment, neurogenic and skeletogenic cells, all three of which represent precociously differentiating cell types of this embryo, most specifically expressed genes of given cell types are not significantly expressed at all in the other cell types. Thus, at the effector gene level, a dramatic, cell type-specific pattern of differential gene regulation is established well before any significant embryonic morphogenesis has occurred. Highlighted article: Spatially distinct populations of the early sea urchin embryo display profound differences in effector gene expression before morphological differences can be distinguished.

Assignment of a member of the ribosomal protein S6 kinase family, RPS6KA5, to human chromosome 14q31-->q32.1 by radiation hybrid mapping.

The mitogen-activated protein kinase (MAPK) family members can be classified according to their characteristics of either being strongly activated by growth factors such as polypeptide growth factor and tumour-promoting phorbol esters, or being strongly activated by stress signals and proinflammatory cytokines (Cohen, 1997). The latter group therefore is often referred to as stress-activated protein kinases (SAPKs). MAPKAP-K1 isoforms were the substrates for MAPK1/ERK1 and MAPK2/ERK2 (Sturgill et al., 1988; Zhao et al., 1995) while MAPKAP-K2 and MAPKAP-K3 were substrates for SAPK2/ p38 (Freshney et al., 1994; Rouse et al., 1994; Clifton et al., 1996; Mclaughlin et al. 1996). Recently, a 3,831-bp cDNA encoding a putative ribosomal protein S6 kinase has been cloned from a human brain cDNA library in our laboratory and was deposited into GenBank with accession no. AF090421. The deduced protein (hereafter designated as RPS6KA5, i.e. ribosomal protein S6 kinase, 90 kDa, polypeptide 5, by the HUGO Nomenclature Committee) contains two Ser/Thr catalytic domains located in its N-terminal and C-terminal respectively and shows 63.8% homology with mouse protein mMSK2 (GenBank accession no. AF074714). Northern blot revealed that RPS6KA5 had a 4.4-kb transcript, which was widely expressed in various tissues. This protein, however, was recently reported by another group with the name MSK1 (Deak et al., 1998). It was found that MSK1 could be directly activated by both MAPK/ERKs and SAPK2/p38, and it was also suggested that MSK1, rather than MAPKAP-K1 or MAPKAP-K2/K3, mediated activation of the transcription factors CREB and ATF by either growth factors or stress signals (Deak et al., 1998). Up to now, no study reveals the chromosome location of MSK1/RPS6KA5. Here, we report the assignment of MSK1/ RPS6KA5 by radiation hybrid mapping to human chromosome 14q31→q32.1.

Assignment of human GADD45G to chromosome 9q22.1-->q22.3 by radiation hybrid mapping.

The growth arrest and DNA damage inducible (GADD) genes represent a family of genes that were identified on the basis of rapid induction by treatment with DNA-damaging agents or by certain growth arrest conditions (Fornace et al., 1988). GADD45, in particular, is a group of genes that are induced by a certain subset of environmental stresses, such as methyl methanesulfonate (MMS), ultraviolet, and ionizing radiation (Fornace et al., 1992). It has been reported that GADD45 played a role in negative growth control, including cell cycle arrest, DNA repair, and/or apoptosis (Liebermann et al., 1998). Recently, two cDNA sequences, which are 1378 bp and 1060 bp, respectively were isolated in our laboratory (GenBank Accession No. AF087853 and AF087883). The cDNA nucleotide sequences predict two proteins of 160 amino acids and 159 amino acids, which were recently reported as GADD45ß and GADD45Á (Takekawa et al., 1998). Northern blot analysis of mRNA from human multiple tissues (MTN I and II, Clontech) detects predominant mRNA species about 1.4 kb for GADD45ß and 1.35 kb for GADD45Á. The GADD45ß is expressed in most tissues, with the exception of thymus, small intestine, and brain, whereas the expression of GADD45Á was most abundant in the heart, placenta, skeletal muscle, prostate, testis, and ovary. Recent evidence suggested these GADD45like proteins were able to activate MTK1 (a human kinase MAPKKK) kinase activity, both in vivo and in vitro, via binding to an N-terminal domain of MTK1, which is upstream of both the p38 and JNK (c-Jun N-terminal kinase) MAPK pathway involved in apoptosis (Chen et al., 1996). This is consistent