Genome-wide and cell-specific epigenetic analysis challenges the role of polycomb in Drosophila spermatogenesis.

The Drosophila spermatogenesis cell differentiation pathway involves the activation of a large set of genes in primary spermatocytes. Most of these genes are activated by testis-specific TATA-binding protein associated factors (tTAFs). In the current model for the activation mechanism, Polycomb plays a key role silencing these genes in the germline precursors, and tTAF-dependent activation in primary spermatocytes involves the displacement of Polycomb from gene promoters. We investigated the genome-wide binding of Polycomb in wild type and tTAF mutant testes. According to the model we expected to see a clear enhancement in Polycomb binding at tTAF-dependent spermatogenesis genes in tTAF mutant testes. However, we find little evidence for such an enhancement in tTAF mutant testes compared to wild type. To avoid problems arising from cellular heterogeneity in whole testis analysis, we further tested the model by analysing Polycomb binding in purified germline precursors, representing cells before tTAF-dependent gene activation. Although we find Polycomb associated with its canonical targets, we find little or no evidence of Polycomb at spermatogenesis genes. The lack of Polycomb at tTAF-dependent spermatogenesis genes in precursor cells argues against a model where Polycomb displacement is the mechanism of spermatogenesis gene activation.

Research from the bedside to the lab bench & back.

Transgenic mice represent a unique opportunity in biomedical research to discover the genes underlying disease and understand how manipulating the function of single genes and proteins alters physiology in a whole animal system. These advances in biomedical research may accelerate the time between when basic discoveries are made and when the research can be 'translated', that is, when the research will positively impact the lives of patients. The purpose of this article is to present some examples of promising mouse models of human diseases.

Co-transcriptional architecture in a Y loop in Drosophila melanogaster.

The Y loops of Drosophila spermatocytes are formed by the expression of huge individual transcription units on the Y chromosome and their large size provides a unique system for the investigation of the organisation of transcription in intact nuclei. By labelling ribonucleo-protein (RNP) components, the loop chromatin and nascent transcripts in Y loop C, we reveal a highly structured organisation of RNP domains associated with nascent transcripts. We distinguish two types of RNP domain, a proximal domain that runs alongside the chromatin of loop C and a distal RNP domain that wraps around the proximal domain and the loop chromatin. The proximal domain is marked by the Pasilla protein, and separate distal subdomains are marked by the S5 antigen and Boule. We discuss the implications of this highly structured co-transcriptional architecture for the organisation of the process of transcription.

Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model.

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan. The causative nature of this mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1(+/-) knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild-type KLF1. This is the first association of a KLF1 mutation with a disease state in adult mammals and also presents the possibility of being another causative gene for HS in humans.

A comprehensive map of insulator elements for the Drosophila genome.

Insulators are DNA sequences that control the interactions among genomic regulatory elements and act as chromatin boundaries. A thorough understanding of their location and function is necessary to address the complexities of metazoan gene regulation. We studied by ChIP-chip the genome-wide binding sites of 6 insulator-associated proteins-dCTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4), and GAF-to obtain the first comprehensive map of insulator elements in Drosophila embryos. We identify over 14,000 putative insulators, including all classically defined insulators. We find two major classes of insulators defined by dCTCF/CP190/BEAF-32 and Su(Hw), respectively. Distributional analyses of insulators revealed that particular sub-classes of insulator elements are excluded between cis-regulatory elements and their target promoters; divide differentially expressed, alternative, and divergent promoters; act as chromatin boundaries; are associated with chromosomal breakpoints among species; and are embedded within active chromatin domains. Together, these results provide a map demarcating the boundaries of gene regulatory units and a framework for understanding insulator function during the development and evolution of Drosophila.

Alternative splicing modulates Ubx protein function in Drosophila melanogaster.

The Drosophila Hox gene Ultrabithorax (Ubx) produces a family of protein isoforms through alternative splicing. Isoforms differ from one another by the presence of optional segments-encoded by individual exons-that modify the distance between the homeodomain and a cofactor-interaction module termed the "YPWM" motif. To investigate the functional implications of Ubx alternative splicing, here we analyze the in vivo effects of the individual Ubx isoforms on the activation of a natural Ubx molecular target, the decapentaplegic (dpp) gene, within the embryonic mesoderm. These experiments show that the Ubx isoforms differ in their abilities to activate dpp in mesodermal tissues during embryogenesis. Furthermore, using a Ubx mutant that reduces the full Ubx protein repertoire to just one single isoform, we obtain specific anomalies affecting the patterning of anterior abdominal muscles, demonstrating that Ubx isoforms are not functionally interchangeable during embryonic mesoderm development. Finally, a series of experiments in vitro reveals that Ubx isoforms also vary in their capacity to bind DNA in presence of the cofactor Extradenticle (Exd). Altogether, our results indicate that the structural changes produced by alternative splicing have functional implications for Ubx protein function in vivo and in vitro. Since other Hox genes also produce splicing isoforms affecting similar protein domains, we suggest that alternative splicing may represent an underestimated regulatory system modulating Hox gene specificity during fly development.

Hematologic characterization and chromosomal localization of the novel dominantly inherited mouse hemolytic anemia, neonatal anemia (Nan).

One of the most commonly inherited anemias in man is Hereditary Spherocytosis (HS) with an incidence of 1 in 2000 for persons of Northern European descent. Mouse models of HS include spontaneous inherited hemolytic anemias and those generated by gene targeting. The Neonatal anemia (Nan) mouse is a novel model of HS generated by N-ethyl-N-nitrosurea mutagenesis and suffers from a severe neonatal anemia. Adult Nan mice have a lifelong hemolytic anemia with decreased red blood cell numbers, hematocrit, and hemoglobin, but elevated zinc protoporphyrin levels. Blood smears taken from Nan mice show a hypochromic anemia characterized by poikilocytosis, anisocytosis and polychromasia. The Nan phenotype can be transferred by bone marrow transplantation indicating that the defect is intrinsic to bone marrow. The hemolytic anemia in adult Nan mice can be identified by osmotic fragility testing. Examination of the erythrocyte membrane skeleton proteins (EMS) reveals a global deficiency of these proteins with protein 4.1a being completely absent. The Nan locus maps to mouse Chromosome 8 and does not co-localize with any known EMS genes. The identification of the Nan gene will likely uncover a novel protein that contributes to the stability of the EMS and may identify a new mutation for HS.

Identification of direct residue contacts in protein-protein interaction by message passing.

Understanding the molecular determinants of specificity in protein-protein interaction is an outstanding challenge of postgenome biology. The availability of large protein databases generated from sequences of hundreds of bacterial genomes enables various statistical approaches to this problem. In this context covariance-based methods have been used to identify correlation between amino acid positions in interacting proteins. However, these methods have an important shortcoming, in that they cannot distinguish between directly and indirectly correlated residues. We developed a method that combines covariance analysis with global inference analysis, adopted from use in statistical physics. Applied to a set of >2,500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for heterointeractions between sensor kinase (SK) and response regulator (RR) proteins and for homointeractions between RR proteins. The spectacular success of this approach illustrates the effectiveness of the global inference approach in identifying direct interaction based on sequence information alone. We expect this method to be applicable soon to interaction surfaces between proteins present in only 1 copy per genome as the number of sequenced genomes continues to expand. Use of this method could significantly increase the potential targets for therapeutic intervention, shed light on the mechanism of protein-protein interaction, and establish the foundation for the accurate prediction of interacting protein partners.

Insulators: linking genome architecture to gene regulation.

Insulator elements have long been associated with a proposed domain boundary function, ensuring appropriate associations between regulatory elements and transcription units through the physical organisation of the genome into looped domains. Recent experiments in Drosophila have, however, highlighted a more direct involvement of insulators in transcriptional regulation.

Co-evolving motions at protein-protein interfaces of two-component signaling systems identified by covariance analysis.

Short-lived protein interactions determine signal transduction specificity among genetically amplified, structurally identical two-component signaling systems. Interacting protein pairs evolve recognition precision by varying residues at specific positions in the interaction surface consistent with constraints of charge, size, and chemical properties. Such positions can be detected by covariance analyses of two-component protein databases. Here, covariance is shown to identify a cluster of co-evolving dynamic residues in two-component proteins. NMR dynamics and structural studies of both wild-type and mutant proteins in this cluster suggest that motions serve to precisely arrange the site of phosphoryl transfer within the complex.

Sensor complexes regulating two-component signal transduction.

Two-component signal transduction systems consisting of a sensor histidine kinase and a response regulator/transcription factor interpret a multitude of environmental and cellular signals and coordinate the expression of a wide array of genes in bacteria. Signal recognition by sensor histidine kinases is the province of a sensor complex consisting of several protein domains that together serve to augment or attenuate the activity of the histidine kinase and thereby of gene expression. Recent investigations have shown the diverse strategies bacteria use to assemble protein domains into the sensor complexes to accomplish signaling. Structural studies of such domains are leading to an understanding of the mechanisms by which sensor complexes recognize signals and regulate kinase activity.

The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning.

Insulator sequences guide the function of distantly located enhancer elements to the appropriate target genes by blocking inappropriate interactions. In Drosophila, five different insulator binding proteins have been identified, Zw5, BEAF-32, GAGA factor, Su(Hw) and dCTCF. Only dCTCF has a known conserved counterpart in vertebrates. Here we find that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlapped with dCTCF binding sites and interacts with dCTCF. Binding of dCTCF to targets requires CP190 in many cases, whereas others are independent of CP190. Analysis of the bithorax complex revealed that six of the borders between the parasegment specific regulatory domains are bound by dCTCF and by CP190 in vivo. dCTCF null mutations affect expression of Abdominal-B, cause pharate lethality and a homeotic phenotype. A short pulse of dCTCF expression during larval development rescues the dCTCF loss of function phenotype. Overall, we demonstrate the importance of dCTCF in fly development and in the regulation of abdominal segmentation.

Features of protein-protein interactions in two-component signaling deduced from genomic libraries.

As more and more sequence data become available, new approaches for extracting information from these data become feasible. This chapter reports on one such method that has been applied to elucidate protein-protein interactions in bacterial two-component signaling pathways. The method identifies residues involved in the interaction through an analysis of over 2500 functionally coupled proteins and a precise determination of the substitutional constraints placed on one protein by its signaling mate. Once identified, a simple log-likelihood scoring procedure is applied to these residues to build a predictive tool for assigning signaling mates. The ability to apply this method is based on a proliferation of related domains within multiple organisms. Paralogous evolution through gene duplication and divergence of two-component systems has commonly resulted in tens of closely related interacting pairs within one organism with a roughly one-to-one correspondence between signal and response. This provides us with roughly an order of magnitude more protein pairs than there are unique, fully sequenced bacterial species. Consequently, this chapter serves as both a detailed exposition of the method that has provided more depth to our knowledge of bacterial signaling and a look ahead to what would be possible on a more widespread scale, that is, to protein-protein interactions that have only one example per genome, as the number of genomes increases by a factor of 10.

Whole genome microarray analysis of gene expression in an imprinting center deletion mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is caused by loss of paternally expressed genes in the 15q11-q13 region. To further characterize alterations in gene expression in this classical obesity syndrome we used whole genome microarrays to study a PWS mouse model resulting from a paternally derived imprinting center (IC) deletion (PWS IC deletion). These mice die generally within 2-3 days of life (reflective of failure to thrive in infants with PWS) and therefore, the analysis was performed on RNA extracted from the whole brain of PWS IC deletion mice and normal littermates at less than 24 hr after birth. Of more than 45,000 probes examined, 26,471 (59%) were detected for further analysis, and 69 had a significant change in expression of at least 1.5-fold and a false discovery rate (FDR) of 5%. Eight of the genes with differential expression were imprinted and from the PWS critical region (PWSCR). The three genes with the highest expression in the PWS IC mice were pro-opiomelanocortin (Pomc) and two transcripts of unknown function. Pomc knockout mice have been shown to develop obesity. Therefore, elevated Pomc RNA in PWS IC deletion neonatal mice may be an important genetic factor in the survival of these mice as it may affect eating behavior. Interestingly, Mc5r, a melanocortin receptor known to directly respond to Pomc expression changes, was upregulated as well. Mc5r is known to be involved with thermoregulation which is reportedly abnormal in PWS infants. These observations support a role for Pomc and the network of genes involved in regulating energy homeostasis in the early clinical findings of failure to thrive observed in PWS. Other notable patterns include three previously unstudied transcripts that are expressed only from the paternal allele under regulatory control of the IC and include AK013560, BB3144814, and BB182944 (whose genes are located in the mouse PWSCR on chromosome 7B). As expected, all the known paternally expressed genes from the PWSCR had detection signals below the threshold in the PWS IC deletion mice but were clearly detectable in control littermates. Several of the genes in this study were further examined by quantitative reverse transcription-PCR (RT-PCR) to confirm their expression status. Further analysis of gene expression in these mice may lead to novel pathways affected in PWS. These results, along with other recent reports, suggest that the cumulative effect of modest changes in expression of many genes, especially genes involved in energy metabolism, contribute to the failure to thrive of infants with PWS.

Improvement in survival and muscle function in an mdx/utrn(-/-) double mutant mouse using a human retinal dystrophin transgene.

Duchenne muscular dystrophy is a progressive muscle disease characterized by increasing muscle weakness and death by the third decade. mdx mice exhibit the underlying muscle disease but appear physically normal with ordinary lifespans, possibly due to compensatory expression of utrophin. In contrast, double mutant mice (mdx/utrn(-/-)), deficient for both dystrophin and utrophin die by approximately 3 months and suffer from severe muscle weakness, growth retardation, and severe spinal curvature. The capacity of human retinal dystrophin (Dp260) to compensate for the missing 427 kDa muscle dystrophin was tested in mdx/utrn(-/-) mice. Functional outcomes were assessed by histology, EMG, MRI, mobility, weight and longevity. MCK-driven transgenic expression of Dp260 in mdx/utrn(-/-) mice converts their disease course from a severe, lethal muscular dystrophy to a viable, mild myopathic phenotype. This finding is relevant to the design of exon-skipping therapeutic strategies since Dp260 lacks dystrophin exons 1-29.

Iron metabolism mutant hbd mice have a deletion in Sec15l1, which has homology to a yeast gene for vesicle docking.

Defects in iron absorption and utilization lead to iron deficiency and anemia. While iron transport by transferrin receptor-mediated endocytosis is well understood, it is not completely clear how iron is transported from the endosome to the mitochondria where heme is synthesized. We undertook a positional cloning project to identify the causative mutation for the hemoglobin-deficit (hbd) mouse mutant, which suffers from a microcytic, hypochromic anemia apparently due to defective iron transport in the endocytosis cycle. As shown by previous studies, reticulocyte iron accumulation in homozygous hbd/hbd mice is deficient despite normal binding of transferrin to its receptor and normal transferrin uptake in the cell. We have identified a strong candidate gene for hbd, Sec15l1, a homologue to yeast SEC15, which encodes a key protein in vesicle docking. The hbd mice have an exon deletion in Sec15l1, which is the first known mutation of a SEC gene homologue in mammals.

Genomic analysis of heat-shock factor targets in Drosophila.

We have used a chromatin immunoprecipitation-microarray (ChIP-array) approach to investigate the in vivo targets of heat-shock factor (Hsf) in Drosophila embryos. We show that this method identifies Hsf target sites with high fidelity and resolution. Using cDNA arrays in a genomic search for Hsf targets, we identified 141 genes with highly significant ChIP enrichment. This study firmly establishes the potential of ChIP-array for whole-genome transcription factor target mapping in vivo using intact whole organisms.

Positional cloning of the Ttc7 gene required for normal iron homeostasis and mutated in hea and fsn anemia mice.

Genes playing essential roles in iron homeostasis have yet to be identified. We report the discovery of a strong candidate gene affecting iron homeostasis in two allelic anemia mouse mutants: hea (hereditary erythroblastic anemia) and fsn (flaky skin). To clone this novel gene positionally, we established a large backcross, which generated a critical region of seven genes from which only one gene exhibited a mutation in hea mice. This was a deletion in Ttc7 (tetratricopeptide repeat domain 7) extending from exon 1 to exon 14. Correspondingly, the allelic variant fsn mice showed a mutation of an ETn retrotransposon integration into intron 14 of the Ttc7 gene, which results in an abnormal Ttc7 RNA transcript. TTC7 is a member of the TPR repeat protein family known to interact with other proteins, to facilitate transport, and to act as chaperone or scaffolding proteins. We speculate that TTC7 plays an important role in iron transport.

Chromosomal localization, hematologic characterization, and iron metabolism of the hereditary erythroblastic anemia (hea) mutant mouse.

Understanding iron metabolism has been enhanced by identification of genes for iron deficiency mouse mutants. We characterized the genetics and iron metabolism of the severe anemia mutant hea (hereditary erythroblastic anemia), which is lethal at 5 to 7 days. The hea mutation results in reduced red blood cell number, hematocrit, and hemoglobin. The hea mice also have elevated Zn protoporphyrin and serum iron. Blood smears from hea mice are abnormal with elevated numbers of smudge cells. Aspects of the hea anemia can be transferred by hematopoietic stem cell transplantation. Neonatal hea mice show a similar hematologic phenotype to the flaky skin (fsn) mutant. We mapped the hea gene near the fsn locus on mouse chromosome 17 and show that the mutants are allelic. Both tissue iron overloading and elevated serum iron are also found in hea and fsn neonates. There is a shift from iron overloading to iron deficiency as fsn mice age. The fsn anemia is cured by an iron-supplemented diet, suggesting an iron utilization defect. When this diet is removed there is reversion to anemia with concomitant loss of overloaded iron stores. We speculate that the hea/fsn gene is required for iron uptake into erythropoietic cells and for kidney iron reabsorption.