Dmitri Papatsenko

Computational Models for Neurogenic Gene Expression in the Drosophila Embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. Here, we apply similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. We propose that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo.

How the Dorsal gradient works: Insights from postgenome technologies

Gradients of extracellular signaling molecules and transcription factors are used in a variety of developmental processes, including the patterning of the Drosophila embryo, the establishment of diverse neuronal cell types in the vertebrate neural tube, and the anterior–posterior patterning of vertebrate limbs. Here, we discuss how a gradient of the maternal transcription factor Dorsal produces complex patterns of gene expression across the dorsal–ventral (DV) axis of the early Drosophila embryo. The identification of 60–70 Dorsal target genes, along with the characterization of ≈35 associated regulatory DNAs, suggests that there are at least six different regulatory codes driving diverse DV expression profiles.

Short fuzzy tandem repeats in genomic sequences, identification, and possible role in regulation of gene expression

MOTIVATION Genomic sequences are highly redundant and contain many types of repetitive DNA. Fuzzy tandem repeats (FTRs) are of particular interest. They are found in regulatory regions of eukaryotic genes and are reported to interact with transcription factors. However, accurate assessment of FTR occurrences in different genome segments requires specific algorithm for efficient FTR identification and classification. RESULTS We have obtained formulas for P-values of FTR occurrence and developed an FTR identification algorithm implemented in TandemSWAN software. Using TandemSWAN we compared the structure and the occurrence of FTRs with short period length (up to 24 bp) in coding and non-coding regions including UTRs, heterochromatic, intergenic and enhancer sequences of Drosophila melanogaster and Drosophila pseudoobscura. Tandems with period three and its multiples were found in coding segments, whereas FTRs with periods multiple of six are overrepresented in all non-coding segment. Periods equal to 5-7 and 11-14 were characteristic of the enhancer regions and other non-coding regions close to genes. AVAILABILITY TandemSWAN web page, stand-alone version and documentation can be found at http://bioinform.genetika.ru/projects/swan/www/ SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Modeling Familial Cancer with Induced Pluripotent Stem Cells

In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.

Organization of developmental enhancers in the Drosophila embryo

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. Here, we investigate the regulatory ‘grammar’ of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid–Bicoid, Hunchback–Hunchback, Bicoid–Dorsal, Bicoid–Caudal and Dorsal–Twist. Direct evidence is presented for the importance of Bicoid–Dorsal linkage in the integration of the anterior–posterior and dorsal–ventral patterning systems. Hunchback–Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. We also present evidence that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. We conclude that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo.

Divisional History and Hematopoietic Stem Cell Function during Homeostasis

Summary We investigated the homeostatic behavior of hematopoietic stem and progenitor cells (HSPCs) temporally defined according to their divisional histories using an HSPC-specific GFP label-retaining system. We show that homeostatic hematopoietic stem cells (HSCs) lose repopulating potential after limited cell divisions. Once HSCs exit dormancy and accrue divisions, they also progressively lose the ability to return to G0 and functional activities associated with quiescent HSCs. In addition, dormant HSPCs phenotypically defined as multipotent progenitor cells display robust stem cell activity upon transplantation, suggesting that temporal quiescence is a greater indicator of function than cell-surface phenotype. Our studies suggest that once homeostatic HSCs leave dormancy, they are slated for extinction. They self-renew phenotypically, but they lose self-renewal activity. As such, they question self-renewal as a characteristic of homeostatic, nonperturbed HSCs in contrast to self-renewal demonstrated under stress conditions.

Dual regulation by the Hunchback gradient in the Drosophila embryo

The regulation of segmentation gene expression is investigated by computational modeling using quantitative expression data. Previous tissue culture assays and transgene analyses raised the possibility that Hunchback (Hb) might function as both an activator and repressor of transcription. At low concentrations, Hb activates gene expression, whereas at high concentrations it mediates repression. Under the same experimental conditions, transcription factors encoded by other gap genes appear to function as dedicated repressors. Models based on dual regulation suggest that the Hb gradient can be sufficient for establishing the initial Kruppel (Kr) expression pattern in central regions of the precellular embryo. The subsequent refinement of the Kr pattern depends on the combination of Hb and the Giant (Gt) repressor. The dual-regulation models developed for Kr also explain some of the properties of the even-skipped (eve) stripe 3+7 enhancer. Computational simulations suggest that repression results from the dimerization of Hb monomers on the DNA template.

A rationale for the enhanceosome and other evolutionarily constrained enhancers.

Summary Metazoan enhancers direct localized stripes, bands and cell-specific patterns of gene expression during development (for example [1]). A typical enhancer is a 500 base pair DNA segment that contains clustered binding sites for two or more sequence-specific transcription factors. Roughly half of all enhancers are located somewhere in the 5′ flanking region of the associated transcription unit, while the other half are distributed among introns, 3′ flanking regions or even protein-coding sequences (for example [2]). Bioinformatics studies suggest that enhancers usually contain a flexible arrangement of binding sites (for example [3]). Here, we present a model to explain why a special subset of enhancers contains a fixed organization.

ClusterDraw web server

ClusterDraw is a program aimed to identification of binding sites and binding-site clusters. Major difference of the ClusterDraw from existing tools is its ability to scan a wide range of parameter values and weigh statistical significance of all possible clusters, smaller than a selected size. The program produces graphs along with decorated FASTA files. ClusterDraw web server is available at the following URL: http://flydev.berkeley.edu/cgi-bin/cld/submit.cgi

Enhancer responses to similarly distributed antagonistic gradients in development.

Formation of spatial gene expression patterns in development depends on transcriptional responses mediated by gene control regions, enhancers. Here, we explore possible responses of enhancers to overlapping gradients of antagonistic transcriptional regulators in the Drosophila embryo. Using quantitative models based on enhancer structure, we demonstrate how a pair of antagonistic transcription factor gradients with similar or even identical spatial distributions can lead to the formation of distinct gene expression domains along the embryo axes. The described mechanisms are sufficient to explain the formation of the anterior and the posterior knirps expression, the posterior hunchback expression domain, and the lateral stripes of rhomboid expression and of other ventral neurogenic ectodermal genes. The considered principles of interaction between antagonistic gradients at the enhancer level can also be applied to diverse developmental processes, such as domain specification in imaginal discs, or even eyespot pattern formation in the butterfly wing.