HP1 drives de novo 3D genome reorganization in early Drosophila embryos.

Fundamental features of 3D genome organization are established de novo in the early embryo, including clustering of pericentromeric regions, the folding of chromosome arms and the segregation of chromosomes into active (A-) and inactive (B-) compartments. However, the molecular mechanisms that drive de novo organization remain unknown1,2. Here, by combining chromosome conformation capture (Hi-C), chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq), 3D DNA fluorescence in situ hybridization (3D DNA FISH) and polymer simulations, we show that heterochromatin protein 1a (HP1a) is essential for de novo 3D genome organization during Drosophila early development. The binding of HP1a at pericentromeric heterochromatin is required to establish clustering of pericentromeric regions. Moreover, HP1a binding within chromosome arms is responsible for overall chromosome folding and has an important role in the formation of B-compartment regions. However, depletion of HP1a does not affect the A-compartment, which suggests that a different molecular mechanism segregates active chromosome regions. Our work identifies HP1a as an epigenetic regulator that is involved in establishing the global structure of the genome in the early embryo.

A determining factor for insect feeding preference in the silkworm, Bombyx mori.

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference of B. mori. With the aid of a transposon-based, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) system, the GR66 locus was genetically mutated, and homozygous mutant silkworm strains with truncated gustatory receptor 66 (GR66) proteins were established. GR66 mutant larvae acquired new feeding activity, exhibiting the ability to feed on a number of plant species in addition to mulberry leaves, including fresh fruits and grain seeds that are not normally consumed by wild-type (WT) silkworms. Furthermore, a feeding choice assay revealed that the mutant larvae lost their specificity for mulberry. Overall, our findings provide the first genetic and phenotypic evidences that a single bitter GR is a major factor affecting the insect feeding preference.

Large distances separate coregulated genes in living Drosophila embryos.

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of cellular signals. Many enhancers map quite far from their target genes, on the order of tens or even hundreds of kilobases. There is extensive evidence that remote enhancers are brought into proximity with their target promoters via long-range looping interactions. However, the exact physical distances of these enhancer-promoter interactions remain uncertain. Here, we employ high-resolution imaging of living Drosophila embryos to visualize the distances separating linked genes that are coregulated by a shared enhancer. Cotransvection assays (linked genes on separate homologs) suggest a surprisingly large distance during transcriptional activity: at least 100-200 nm. Similar distances were observed when a shared enhancer was placed into close proximity with linked reporter genes in cis. These observations are consistent with the occurrence of "transcription hubs," whereby clusters (or condensates) of multiple RNA polymerase II complexes and associated cofactors are periodically recruited to active promoters. The dynamics of this process might be responsible for rapid fluctuations in the distances separating the transcription of coregulated reporter genes during transvection. We propose that enhancer-promoter communication depends on a combination of classical looping and linking models.

Sister centromere fusion during meiosis I depends on maintaining cohesins and destabilizing microtubule attachments.

Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.

Gene expression changes elicited by a parasitic B chromosome in the grasshopper Eyprepocnemis plorans are consistent with its phenotypic effects.

Parasitism evokes adaptive physiological changes in the host, many of which take place through gene expression changes. This response can be more or less local, depending on the organ or tissue affected by the parasite, or else systemic when the parasite affects the entire host body. The most extreme of the latter cases is intragenomic parasitism, where the parasite is present in all host nuclei as any other genomic element. Here, we show the molecular crosstalk between a parasitic chromosome (also named B chromosome) and the host genome, manifested through gene expression changes. The transcriptome analysis of 0B and 1B females of the grasshopper Eyprepocnemis plorans, validated by a microarray experiment performed on four B-lacking and five B-carrying females, revealed changes in gene expression for 188 unigenes being consistent in both experiments. Once discarded B-derived transcripts, there were 46 differentially expressed genes (30 up- and 16 downregulated) related with the adaptation of the host genome to the presence of the parasitic chromosome. Interestingly, the functions of these genes could explain some of the most important effects of B chromosomes, such as nucleotypic effects derived from the additional DNA they represent, chemical defense and detoxification, protein modification and response to stress, ovary function, and regulation of gene expression. Collectively, these changes uncover an intimate host-parasite interaction between A and B chromosomes during crucial steps of gene expression and protein function.

A new role for Drosophila Aurora-A in maintaining chromosome integrity.

Aurora-A is a conserved mitotic kinase overexpressed in many types of cancer. Growing evidence shows that Aurora-A plays a crucial role in DNA damage response (DDR) although this aspect has been less characterized. We isolated a new aur-A mutation, named aur-A949, in Drosophila, and we showed that it causes chromosome aberrations (CABs). In addition, aur-A949 mutants were sensitive to X-ray treatment and showed impaired γ-H2Av foci dissolution kinetics. To identify the pathway in which Aur-A works, we conducted an epistasis analysis by evaluating CAB frequencies in double mutants carrying aur-A949 mutation combined to mutations in genes related to DNA damage response (DDR). We found that mutations in tefu (ATM) and in the histone variant H2Av were epistatic over aur-A949 indicating that Aur-A works in DDR and that it is required for γ-H2Av foci dissolution. More interestingly, we found that a mutation in lig4, a gene belonging to the non-homologous end joining (NHEJ) repair pathway, was epistatic over aur-A949. Based on studies in other systems, which show that phosphorylation is important to target Lig4 for degradation, we hypothesized that in aur-A949 mutant cells, there is a persistence of Lig4 that could be, in the end, responsible for CABs. Finally, we observed a synergistic interaction between Aur-A and the homologous recombination (HR) repair system component Rad 51 in the process that converts chromatid deletions into isochromatid deletions. Altogether, these data indicate that Aur-A depletion can elicit chromosome damage. This conclusion should be taken into consideration, since some anticancer therapies are aimed at reducing Aurora-A expression.

The Impact of Drosophila Awd/NME1/2 Levels on Notch and Wg Signaling Pathways.

Awd, the Drosophila homologue of NME1/2 metastasis suppressors, plays key roles in many signaling pathways. Mosaic analysis of the null awdJ2A4 allele showed that loss of awd gene function blocks Notch signaling and the expression of its target genes including the Wingless (Wg/Wnt1) morphogen. We also showed that RNA interference (RNAi)-mediated awd silencing (awdi) in larval wing disc leads to chromosomal instability (CIN) and to Jun amino-terminal kinases (JNK)-mediated cell death. Here we show that this cell death is independent of p53 activity. Based on our previous finding showing that forced survival of awdi-CIN cells leads to aneuploidy without the hyperproliferative effect, we investigated the Wg expression in awdi wing disc cells. Interestingly, the Wg protein is expressed in its correct dorso-ventral domain but shows an altered cellular distribution which impairs its signaling. Further, we show that RNAi-mediated knock down of awd in wing discs does not affect Notch signaling. Thus, our analysis of the hypomorphic phenotype arising from awd downregulation uncovers a dose-dependent effect of Awd in Notch and Wg signaling.

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation.

The fluorescent dye 4'-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.

How epigenome drives chromatin folding and dynamics, insights from efficient coarse-grained models of chromosomes.

The 3D organization of chromosomes is crucial for regulating gene expression and cell function. Many experimental and polymer modeling efforts are dedicated to deciphering the mechanistic principles behind chromosome folding. Chromosomes are long and densely packed-topologically constrained-polymers. The main challenges are therefore to develop adequate models and simulation methods to investigate properly the multi spatio-temporal scales of such macromolecules. Here, we proposed a generic strategy to develop efficient coarse-grained models for self-avoiding polymers on a lattice. Accounting accurately for the polymer entanglement length and the volumic density, we show that our simulation scheme not only captures the steady-state structural and dynamical properties of the system but also tracks the same dynamics at different coarse-graining. This strategy allows a strong power-law gain in numerical efficiency and offers a systematic way to define reliable coarse-grained null models for chromosomes and to go beyond the current limitations by studying long chromosomes during an extended time period with good statistics. We use our formalism to investigate in details the time evolution of the 3D organization of chromosome 3R (20 Mbp) in drosophila during one cell cycle (20 hours). We show that a combination of our coarse-graining strategy with a one-parameter block copolymer model integrating epigenomic-driven interactions quantitatively reproduce experimental data at the chromosome-scale and predict that chromatin motion is very dynamic during the cell cycle.

Determinants of Chromosome Architecture: Insulator Pairing in cis and in trans.

The chromosomes of multicellular animals are organized into a series of topologically independent looped domains. This domain organization is critical for the proper utilization and propagation of the genetic information encoded by the chromosome. A special set of architectural elements, called boundaries or insulators, are responsible both for subdividing the chromatin into discrete domains and for determining the topological organization of these domains. Central to the architectural functions of insulators are homologous and heterologous insulator:insulator pairing interactions. The former (pairing between copies of the same insulator) dictates the process of homolog alignment and pairing in trans, while the latter (pairing between different insulators) defines the topology of looped domains in cis. To elucidate the principles governing these architectural functions, we use two insulators, Homie and Nhomie, that flank the Drosophila even skipped locus. We show that homologous insulator interactions in trans, between Homie on one homolog and Homie on the other, or between Nhomie on one homolog and Nhomie on the other, mediate transvection. Critically, these homologous insulator:insulator interactions are orientation-dependent. Consistent with a role in the alignment and pairing of homologs, self-pairing in trans is head-to-head. Head-to-head self-interactions in cis have been reported for other fly insulators, suggesting that this is a general principle of self-pairing. Homie and Nhomie not only pair with themselves, but with each other. Heterologous Homie-Nhomie interactions occur in cis, and we show that they serve to delimit a looped chromosomal domain that contains the even skipped transcription unit and its associated enhancers. The topology of this loop is defined by the heterologous pairing properties of Homie and Nhomie. Instead of being head-to-head, which would generate a circular loop, Homie-Nhomie pairing is head-to-tail. Head-to-tail pairing in cis generates a stem-loop, a configuration much like that observed in classical lampbrush chromosomes. These pairing principles provide a mechanistic underpinning for the observed topologies within and between chromosomes.

Comparative Proteomic Analysis of Posterior Silk Glands of Wild and Domesticated Silkworms Reveals Functional Evolution during Domestication.

The wild silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild silkworms on the third and fifth days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that, with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild silkworms were enriched in the ribosome pathway, which is closely related to cell size and translation capacity. Together, these results suggest that functional evolution of the PSG during domestication was driven by reinforcing the advantageous pathways to increase the synthesis efficiency of silk proteins in each cell and thereby improve silk yield.

New chromosomal evidence for the origin of Triatoma infestans populations from Brazil.

In this study, the karyometry of different Triatoma infestans populations from different states of Brazil was analyzed and compared with those of a population from Cochabamba. No significant differences were found between the population from Cochabamba and those from Brazil. These results are consistent with the origin of the T. infestans populations of Brazil by a founder effect from Cochabamba. Moreover, these findings also confirm that a founder effect occurred during the dispersal of T. infestans populations in different Brazilian states.

Evidence for a gene involved in multiple and diverse rearrangements in the Drosophila genus.

In Drosophila, chromosomes have been extensively reorganized during evolution, with most rearrangements affecting the gene order in chromosomal elements but not their gene content. The level of reorganization and the evidence for breakpoint reuse vary both between and within elements. The subito gene stands out as a gene involved in multiple rearrangements both because of its active single-gene transposition and because it is the nearest gene to diverse rearrangements breakpoints. Indeed, subito has undergone three single-gene transpositions and it is the nearest gene to the breakpoints of other single-gene transpositions and of two chromosomal inversions. Given that subito is involved in meiosis and therefore active in the female germ line, the high number of nearby fixed breakages might be related among others to the presumed high accessibility of the subito region to the machinery associated with double-strand breaks repair. A second important contributor would be the reduced and simple regulatory region of subito, which would imply that a fraction of the rearrangements originating from subito nearby breakages would have not affected either its pattern or timing of expression and would have, thus, not resulted in reduced fitness.

Heterochromatin characterization and ribosomal gene location in two monotypic genera of bloodsucker bugs (Cimicidae, Heteroptera) with holokinetic chromosomes and achiasmatic male meiosis.

Members of the family Cimicidae (Heteroptera: Cimicomorpha) are temporary bloodsuckers on birds and bats as primary hosts and humans as secondary hosts. Acanthocrios furnarii (2n=12=10+XY, male) and Psitticimex uritui (2n=31=28+X1X2Y, male) are two monotypic genera of the subfamily Haematosiphoninae, which have achiasmatic male meiosis of collochore type. Here, we examined chromatin organization and constitution of cimicid holokinetic chromosomes by determining the amount, composition and distribution of constitutive heterochromatin, and number and location of nucleolus organizer regions (NORs) in both species. Results showed that these two bloodsucker bugs possess high heterochromatin content and have an achiasmatic male meiosis, in which three regions can be differentiated in each autosomal bivalent: (i) terminal heterochromatic regions in repulsion; (ii) a central region, where the homologous chromosomes are located parallel but without contact between them; and (iii) small areas within the central region, where collochores are detected. Acanthocrios furnarii presented a single NOR on an autosomal pair, whereas P. uritui presented two NORs, one on an autosomal pair and the other on a sex chromosome. All NORs were found to be associated with CMA3 bright bands, indicating that the whole rDNA repeating unit is rich in G+C base pairs. Based on the variations in the diploid autosomal number, the presence of simple and multiple sex chromosome systems, and the number and location of 18S rDNA loci in the two Cimicidae species studied, we might infer that rDNA clusters and genome are highly dynamic among the representatives of this family.

Interactions among Polycomb domains are guided by chromosome architecture.

Polycomb group (PcG) proteins bind and regulate hundreds of genes. Previous evidence has suggested that long-range chromatin interactions may contribute to the regulation of PcG target genes. Here, we adapted the Chromosome Conformation Capture on Chip (4C) assay to systematically map chromosomal interactions in Drosophila melanogaster larval brain tissue. Our results demonstrate that PcG target genes interact extensively with each other in nuclear space. These interactions are highly specific for PcG target genes, because non-target genes with either low or high expression show distinct interactions. Notably, interactions are mostly limited to genes on the same chromosome arm, and we demonstrate that a topological rather than a sequence-based mechanism is responsible for this constraint. Our results demonstrate that many interactions among PcG target genes exist and that these interactions are guided by overall chromosome architecture.

Proteome analysis of silkworm, Bombyx mori, larval gonads: characterization of proteins involved in sexual dimorphism and gametogenesis.

Sexual dimorphism is initialed by the components of the sex determination pathway and is most evident in gonads and germ cells. Although striking dimorphic expressions have been detected at the transcriptional level between the silkworm larval testis and the ovary, the sex-dimorphic expressions at the protein level have not yet been well characterized. The proteome of silkworm larval gonads was investigated using a shotgun-based identification. A total of 286 and 205 nonredundant proteins were identified from the silkworm testis and ovary, respectively, with a false discovery rate (FDR) lower than 1%. Only 40 and 16 proteins were previously identified, and 246 and 189 proteins were newly identified in the silkworm testis and the ovary, respectively. The gametogenesis mechanism of silkworm was demonstrated using the protein expression profile and bioinformatics analysis. Cellular retinoic acid binding protein (CRABP) showed to be highly abundant in testis, while tubulins were abundant in ovary. Several homologies of Drosophila essential proteins for gametogenesis were identified in silkworm, such as male meiotic arrest gene product ALY and VISMAY in testis, and maternal mRNA localization protein exuperantia and SQUID in ovary. The gene ontology (GO) annotation and pathway analysis provide system-level insights into the sexual dimorphism and gametogenesis.

On the fractal geometry of DNA by the binary image analysis.

The multifractal analysis of binary images of DNA is studied in order to define a methodological approach to the classification of DNA sequences. This method is based on the computation of some multifractality parameters on a suitable binary image of DNA, which takes into account the nucleotide distribution. The binary image of DNA is obtained by a dot-plot (recurrence plot) of the indicator matrix. The fractal geometry of these images is characterized by fractal dimension (FD), lacunarity, and succolarity. These parameters are compared with some other coefficients such as complexity and Shannon information entropy. It will be shown that the complexity parameters are more or less equivalent to FD, while the parameters of multifractality have different values in the sense that sequences with higher FD might have lower lacunarity and/or succolarity. In particular, the genome of Drosophila melanogaster has been considered by focusing on the chromosome 3r, which shows the highest fractality with a corresponding higher level of complexity. We will single out some results on the nucleotide distribution in 3r with respect to complexity and fractality. In particular, we will show that sequences with higher FD also have a higher frequency distribution of guanine, while low FD is characterized by the higher presence of adenine.

Genome-wide profiling of forum domains in Drosophila melanogaster.

Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Most forum domains are 50-200 kb in length. We mapped forum domain termini using FISH on polytene chromosomes and we performed genome-wide mapping using a Drosophila melanogaster genomic tiling microarray consisting of overlapping 3 kb fragments. We found that forum termini very often correspond to regions of intercalary heterochromatin and regions of late replication in polytene chromosomes. We found that forum domains contain clusters of several or many genes. The largest forum domains correspond to the main clusters of homeotic genes inside BX-C and ANTP-C, cluster of histone genes and clusters of piRNAs. PRE/TRE and transcription factor binding sites often reside inside domains and do not overlap with forum domain termini. We also found that about 20% of forum domain termini correspond to small chromosomal regions where Ago1, Ago2, small RNAs and repressive chromatin structures are detected. Our results indicate that forum domains correspond to big multi-gene chromosomal units, some of which could be coordinately expressed. The data on the global mapping of forum domains revealed a strong correlation between fragmentation sites in chromosomes, particular sets of mobile elements and regions of intercalary heterochromatin.

A BEAF dependent chromatin domain boundary separates myoglianin and eyeless genes of Drosophila melanogaster.

Precise transcriptional control is dependent on specific interactions of a number of regulatory elements such as promoters, enhancers and silencers. Several studies indicate that the genome in higher eukaryotes is divided into chromatin domains with functional autonomy. Chromatin domain boundaries are a class of regulatory elements that restrict enhancers to interact with appropriate promoters and prevent misregulation of genes. While several boundary elements have been identified, a rational approach to search for such elements is lacking. With a view to identifying new chromatin domain boundary elements we analyzed genomic regions between closely spaced but differentially expressed genes of Drosophila melanogaster. We have identified a new boundary element between myoglianin and eyeless, ME boundary, that separates these two differentially expressed genes. ME boundary maps to a DNaseI hypersensitive site and acts as an enhancer blocker both in embryonic and adult stages in transgenic context. We also report that BEAF and GAF are the two major proteins responsible for the ME boundary function. Our studies demonstrate a rational approach to search for potential boundaries in genomic regions that are well annotated.

TCAGG, an alternative telomeric sequence in insects.

The TTAGG repeat, the only determined telomerase-dependent sequence in the Insecta, is generally reputed to be the canonical telomeric motif within the class. By studying the distribution of telomeric DNAs in 30 coleopteran beetles using Southern hybridization, BAL 31 DNA end-degradation assay and fluorescence in situ hybridization, we showed that arrays built of a TCAGG repeat substitute for (TTAGG)n sequences in all tested species within the superfamily Tenebrionoidea. We also provided the experimental evidence that (TCAGG)n repeats represent the terminal sequences on all chromosomes of the model species Tribolium castaneum. (TCAGG)n repeats are therefore promoted as the first sequence-motif alternative to TTAGG-type chromosome ends in insects. Detection of species negative for both TTAGG and TCAGG reveals that, although widespread, these motifs are not ubiquitous telomeric sequences within the order Coleoptera. In addition, Timarcha balearica proved to be a species that harbors (TTAGG)n repeats, but not at telomeric positions, thus further increasing the complexity of telomeric DNAs. Our experiments discarded CTAGG, CTGGG, TTGGG, and TTAGGG variants as potential replacements in TTAGG/TCAGG-negative species, indicating that chromosome termini of these beetles comprise other form(s) of telomeric sequences and telomere maintenance mechanisms.