Expression of homothorax and extradenticle mRNA in the legs of the crustacean Parhyale hawaiensis: evidence for a reversal of gene expression regulation in the pancrustacean lineage

In Drosophila leg development, the extradenticle (exd) gene is expressed ubiquitously and its co-factor homothorax (hth) is restricted to the proximal leg portion. This condition is conserved in other insect species but is reversed in chelicerates and myriapods. As the region of co-expression does not differ in the two groups and transcripts from both are necessary for function, this difference in expression is likely to be functionally neutral. Here, we report the expression patterns of exd and hth in a crustacean, the amphipod shrimp Parhyale hawaiensis. The patterns in P. hawaiensis are similar to the insect patterns, supporting the close relationship between crustaceans and insects in the taxon Tetraconata. However, mRNA expression of exd in P. hawaiensis is weak in the distal leg parts, thus being intermediate between the complete lack of distal exd expression in chelicerates and myriapods and the strong distal exd expression in insects. Our data suggest that the reversal of the gene expression regulation of hth and exd occurred in the pancrustacean lineage

Progressive Polycomb Assembly on H3K27me3 Compartments Generates Polycomb Bodies with Developmentally Regulated Motion

Polycomb group (PcG) proteins are conserved chromatin factors that maintain silencing of key developmental genes outside of their expression domains. Recent genome-wide analyses showed a Polycomb (PC) distribution with binding to discrete PcG response elements (PREs). Within the cell nucleus, PcG proteins localize in structures called PC bodies that contain PcG-silenced genes, and it has been recently shown that PREs form local and long-range spatial networks. Here, we studied the nuclear distribution of two PcG proteins, PC and Polyhomeotic (PH). Thanks to a combination of immunostaining, immuno-FISH, and live imaging of GFP fusion proteins, we could analyze the formation and the mobility of PC bodies during fly embryogenesis as well as compare their behavior to that of the condensed fraction of euchromatin. Immuno-FISH experiments show that PC bodies mainly correspond to 3D structural counterparts of the linear genomic domains identified in genome-wide studies. During early embryogenesis, PC and PH progressively accumulate within PC bodies, which form nuclear structures localized on distinct euchromatin domains containing histone H3 tri-methylated on K27. Time-lapse analysis indicates that two types of motion influence the displacement of PC bodies and chromatin domains containing H2Av-GFP. First, chromatin domains and PC bodies coordinately undergo long-range motions that may correspond to the movement of whole chromosome territories. Second, each PC body and chromatin domain has its own fast and highly constrained motion. In this motion regime, PC bodies move within volumes slightly larger than those of condensed chromatin domains. Moreover, both types of domains move within volumes much smaller than chromosome territories, strongly restricting their possibility of interaction with other nuclear structures. The fast motion of PC bodies and chromatin domains observed during early embryogenesis strongly decreases in late developmental stages, indicating a possible contribution of chromatin dynamics in the maintenance of stable gene silencing

Expression of zebrafish pax6b in pancreas is regulated by two enhancers containing highly conserved cis-elements bound by PDX1, PBX and PREP factors

BACKGROUND: PAX6 is a transcription factor playing a crucial role in the development of the eye and in the differentiation of the pancreatic endocrine cells as well as of enteroendocrine cells. Studies on the mouse Pax6 gene have shown that sequences upstream from the P0 promoter are required for expression in the lens and the pancreas; but there remain discrepancies regarding the precise location of the pancreatic regulatory elements. RESULTS: Due to genome duplication in the evolution of ray-finned fishes, zebrafish has two pax6 genes, pax6a and pax6b. While both zebrafish pax6 genes are expressed in the developing eye and nervous system, only pax6b is expressed in the endocrine cells of the pancreas. To investigate the cause of this differential expression, we used a combination of in silico, in vivo and in vitro approaches. We show that the pax6b P0 promoter targets expression to endocrine pancreatic cells and also to enteroendocrine cells, retinal neurons and the telencephalon of transgenic zebrafish. Deletion analyses indicate that strong pancreatic expression of the pax6b gene relies on the combined action of two conserved regulatory enhancers, called regions A and C. By means of gel shift assays, we detected binding of the homeoproteins PDX1, PBX and PREP to several cis-elements of these regions. In constrast, regions A and C of the zebrafish pax6a gene are not active in the pancreas, this difference being attributable to sequence divergences within two cis-elements binding the pancreatic homeoprotein PDX1. CONCLUSION: Our data indicate a conserved role of enhancers A and C in the pancreatic expression of pax6b and emphasize the importance of the homeoproteins PBX and PREP cooperating with PDX1, in activating pax6b expression in endocrine pancreatic cells. This study also provides a striking example of how adaptative evolution of gene regulatory sequences upon gene duplication progressively leads to subfunctionalization of the paralogous gene pair

Genome-Wide Tissue-Specific Occupancy of the Hox Protein Ultrabithorax and Hox Cofactor Homothorax in Drosophila

The Hox genes are responsible for generating morphological diversity along the anterior-posterior axis during animal development. The Drosophila Hox gene Ultrabithorax (Ubx), for example, is required for specifying the identity of the third thoracic (T3) segment of the adult, which includes the dorsal haltere, an appendage required for flight, and the ventral T3 leg. Ubx mutants show homeotic transformations of the T3 leg towards the identity of the T2 leg and the haltere towards the wing. All Hox genes, including Ubx, encode homeodomain containing transcription factors, raising the question of what target genes Ubx regulates to generate these adult structures. To address this question, we carried out whole genome ChIP-chip studies to identify all of the Ubx bound regions in the haltere and T3 leg imaginal discs, which are the precursors to these adult structures. In addition, we used ChIP-chip to identify the sites bound by the Hox cofactor, Homothorax (Hth). In contrast to previous ChIP-chip studies carried out in Drosophila embryos, these binding studies reveal that there is a remarkable amount of tissue- and transcription factor-specific binding. Analyses of the putative target genes bound and regulated by these factors suggest that Ubx regulates many downstream transcription factors and developmental pathways in the haltere and T3 leg. Finally, we discovered additional DNA sequence motifs that in some cases are specific for individual data sets, arguing that Ubx and/or Hth work together with many regionally expressed transcription factors to execute their functions. Together, these data provide the first whole-genome analysis of the binding sites and target genes regulated by Ubx to specify the morphologies of the adult T3 segment of the fly

Insights into Hox Protein Function from a Large Scale Combinatorial Analysis of Protein Domains

Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA), we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences

Segment-Specific Neuronal Subtype Specification by the Integration of Anteroposterior and Temporal Cues

To address the question of how neuronal diversity is achieved throughout the CNS, this study provides evidence of modulation of neural progenitor cell “output” along the body axis by integration of local anteroposterior and temporal cues

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region.

The poliovirus mutant 5NC-11 has a 4-base insertion at position 70 within the 5' untranslated region and is deficient in RNA synthesis. Revertants from 5NC-11 were isolated, showing a partial recovery of wild-type levels of RNA synthesis. The 5' noncoding region of those revertants contained the mutation intact; mix-and-match experiments with the cDNA from these revertants revealed that a restricted region within the 3C gene was the site of the suppressing mutations in the revertants. The suppressors were point mutations, confirmed by introducing them into the 3C gene by site-directed mutagenesis. Although complementation studies indicated that the suppressors were cis active, we believe that protein changes rather than RNA sequence alterations are responsible for the suppression because RNA changes that did not alter protein sequence had no effect, whereas various protein alterations were suppressive. The results therefore imply that protein 3C interacts with the 5' end of the RNA and may play a role in RNA replication

Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.

The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA