Malat1 affects transcription and splicing through distinct pathways in mouse embryonic stem cells.

Malat1 is a long-noncoding RNA with critical roles in gene regulation and cancer metastasis, however its functional role in stem cells is largely unexplored. We here perform a nuclear knockdown of Malat1 in mouse embryonic stem cells, causing the de-regulation of 320 genes and aberrant splicing of 90 transcripts, some of which potentially affecting the translated protein sequence. We find evidence that Malat1 directly interacts with gene bodies and aberrantly spliced transcripts, and that it locates upstream of down-regulated genes at their putative enhancer regions, in agreement with functional genomics data. Consistent with this, we find these genes affected at both exon and intron levels, suggesting that they are transcriptionally regulated by Malat1. Besides, the down-regulated genes are regulated by specific transcription factors and bear both activating and repressive chromatin marks, suggesting that some of them might be regulated by bivalent promoters. We propose a model in which Malat1 facilitates the transcription of genes involved in chromatid dynamics and mitosis in one pathway, and affects the splicing of transcripts that are themselves involved in RNA processing in a distinct pathway. Lastly, we compare our findings with Malat1 perturbation studies performed in other cell systems and in vivo.

Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity.

MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.

Detection of transcriptome-wide microRNA-target interactions in single cells with agoTRIBE.

MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.

Historical RNA expression profiles from the extinct Tasmanian tiger.

Paleogenomics continues to yield valuable insights into the evolution, population dynamics, and ecology of our ancestors and other extinct species. However, DNA sequencing cannot reveal tissue-specific gene expression, cellular identity, or gene regulation, which are only attainable at the transcriptional level. Pioneering studies have shown that useful RNA can be extracted from ancient specimens preserved in permafrost and historical skins from extant canids, but no attempts have been made so far on extinct species. We extract, sequence, and analyze historical RNA from muscle and skin tissue of a ∼130-year-old Tasmanian tiger (Thylacinus cynocephalus) preserved in desiccation at room temperature in a museum collection. The transcriptional profiles closely resemble those of extant species, revealing specific anatomical features such as slow muscle fibers or blood infiltration. Metatranscriptomic analysis, RNA damage, tissue-specific RNA profiles, and expression hotspots genome-wide further confirm the thylacine origin of the sequences. RNA sequences are used to improve protein-coding and noncoding annotations, evidencing missing exonic loci and the location of ribosomal RNA genes while increasing the number of annotated thylacine microRNAs from 62 to 325. We discover a thylacine-specific microRNA isoform that could not have been confirmed without RNA evidence. Finally, we detect traces of RNA viruses, suggesting the possibility of profiling viral evolution. Our results represent the first successful attempt to obtain transcriptional profiles from an extinct animal species, providing thought-to-be-lost information on gene expression dynamics. These findings hold promising implications for the study of RNA molecules across the vast collections of natural history museums and from well-preserved permafrost remains.

Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut.

Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives. IMPORTANCE The malaria parasite Plasmodium falciparum causes more than half a million deaths per year. The current treatment regimen targets the symptom-causing blood stage inside the human host. However, recent incentives in the field call for novel interventions to block parasite transmission from humans to the mosquito vector. Therefore, we need to better understand the parasite biology during its development inside the mosquito, including a deeper understanding of the expression of genes controlling parasite progression during these stages. Here, we have generated single-cell transcriptome data, covering P. falciparum's development, from gamete to ookinete inside the mosquito midgut, uncovering previously untapped parasite biology, including a repertoire of novel biomarkers to be explored in future transmission-blocking efforts. We anticipate that our study provides an important resource, which can be further explored to improve our understanding of the parasite biology as well as aid in guiding future malaria intervention strategies.

ADAR1- and ADAR2-mediated regulation of maturation and targeting of miR-376b to modulate GABA neurotransmitter catabolism.

miRNAs are short noncoding RNA molecules that regulate gene expression by inhibiting translation or inducing degradation of target mRNAs. miRNAs are often expressed as polycistronic transcripts, so-called miRNA clusters, containing several miRNA precursors. The largest mammalian miRNA cluster, the miR-379-410 cluster, is expressed primarily during embryonic development and in the adult brain; however, downstream regulation of this cluster is not well understood. Here, we investigated adenosine deamination to inosine (RNA editing) in the miR-379-410 cluster by adenosine deaminase acting on RNA (ADAR) enzymes as a possible mechanism modulating the expression and activity of these miRNAs in a brain-specific manner. We show that the levels of editing in the majority of mature miRNAs are lower than the editing levels of the corresponding site in primary miRNA precursors. However, for one miRNA, miR-376b-3p, editing was significantly higher in the mature form than in the primary precursor. We found miR-376b-3p maturation is negatively regulated by ADAR2 in an editing activity-independent manner, whereas ADAR1-mediated and ADAR2-mediated editing were observed to be competitive. In addition, the edited miR-376b-3p targets a different set of mRNAs than unedited miR-376b-3p, including 4-aminobutyrate aminotransferase, encoding the enzyme responsible for the catabolism of the neurotransmitter gamma aminobutyric acid (GABA). Expression of edited miR-376b-3p led to increased intracellular GABA levels as well as increased cell surface presentation of GABA type A receptors. Our results indicate that both editing and editing-independent effects modulate the expression of miR-376b-3p, with the potential to regulate GABAergic signaling in the brain.

MapToCleave: High-throughput profiling of microRNA biogenesis in living cells.

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

Nuclear gene proximity and protein interactions shape transcript covariations in mammalian single cells.

Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.

Male swarming aggregation pheromones increase female attraction and mating success among multiple African malaria vector mosquito species.

Accumulating behavioural data indicate that aggregation pheromones may mediate the formation and maintenance of mosquito swarms. However, chemical cues possibly luring mosquitoes to swarms have not been adequately investigated, and the likely molecular incitants of these complex reproductive behaviours remain unknown. Here we show that males of the important malaria vector species Anopheles arabiensis and An. gambiae produce and release aggregation pheromones that attract individuals to the swarm and enhance mating success. We found that males of both species released significantly higher amounts of 3-hydroxy-2-butanone (acetoin), 6-methyl-5-hepten-2-one (sulcatone), octanal, nonanal and decanal during swarming in the laboratory. Feeding males with stable-isotope-labelled glucose revealed that the males produced these five compounds. A blend composed of synthetic analogues to these swarming odours proved highly attractive to virgin males and females of both species under laboratory conditions and substantially increased mating in five African malaria vectors (An. gambiae, An. coluzzii, An. arabiensis, An. merus and An. funestus) in semi-field experiments. Our results not only narrow a conspicuous gap in understanding a vital aspect of the chemical ecology of male mosquitoes but also demonstrate fundamental roles of rhythmic and metabolic genes in the physiology and behavioural regulation of these vectors. These identified aggregation pheromones have great potential for exploitation against these highly dangerous insects. Manipulating such pheromones could increase the efficacy of malaria-vector control programmes.

miRTrace reveals the organismal origins of microRNA sequencing data.

We present here miRTrace, the first algorithm to trace microRNA sequencing data back to their taxonomic origins. This is a challenge with profound implications for forensics, parasitology, food control, and research settings where cross-contamination can compromise results. miRTrace accurately (> 99%) assigns real and simulated data to 14 important animal and plant groups, sensitively detects parasitic infection in mammals, and discovers the primate origin of single cells. Applying our algorithm to over 700 public datasets, we find evidence that over 7% are cross-contaminated and present a novel solution to clean these computationally, even after sequencing has occurred. miRTrace is freely available at https://github.com/friedlanderlab/mirtrace .

Endogenous siRNAs and piRNAs derived from transposable elements and genes in the malaria vector mosquito Anopheles gambiae.

BACKGROUND: The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae. RESULTS: Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both "ping-pong" dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae. CONCLUSIONS: We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.

Transcriptome-wide analysis of microRNA expression in the malaria mosquito Anopheles gambiae.

BACKGROUND: microRNAs (miRNAs) are a highly abundant class of small noncoding regulatory RNAs that post-transcriptionally regulate gene expression in multicellular organisms. miRNAs are involved in a wide range of biological and physiological processes, including the regulation of host immune responses to microbial infections. Small-scale studies of miRNA expression in the malaria mosquito Anopheles gambiae have been reported, however no comprehensive analysis of miRNAs has been performed so far. RESULTS: Using small RNA sequencing, we characterized de novo A. gambiae miRNA repertoire expressed in adult sugar- and blood-fed females. We provided transcriptional evidences for 123 miRNAs, including 58 newly identified miRNAs. Out of the newly described miRNAs, 19 miRNAs are homologs to known miRNAs in other insect species and 17 miRNAs share sequence similarity restricted to the seed sequence. The remaining 21 novel miRNAs displayed no obvious sequence homology with known miRNAs. Detailed bioinformatics analysis of the mature miRNAs revealed a sequence variation occurring at their 5'-end and leading to functional seed shifting in more than 5% of miRNAs. We also detected significant sequence heterogeneity at the 3'-ends of the mature miRNAs, mostly due to imprecise processing and post-transcriptional modifications. Comparative analysis of arm-switching events revealed the existence of species-specific production of dominant mature miRNAs induced by blood feeding in mosquitoes. We also identified new conserved and fragmented miRNA clusters and A. gambiae-specific miRNA gene duplication. Using miRNA expression profiling, we identified the differentially expressed miRNAs at an early time point after regular blood feeding and after infection with the rodent malaria parasite Plasmodium berghei. Significant changes were detected in the expression levels of 4 miRNAs in blood-fed mosquitoes, whereas 6 miRNAs were significantly upregulated after P. berghei infection. CONCLUSIONS: In the current study, we performed the first systematic analysis of miRNAs in A. gambiae. We provided new insights on mature miRNA sequence diversity and functional shifts in the mosquito miRNA evolution. We identified a set of the differentially expressed miRNAs that respond to normal and infectious blood meals. The extended set of Anopheles miRNAs and their isoforms provides a basis for further experimental studies of miRNA expression patterns and biological functions in A. gambiae.

Drosophila mir-9a regulates wing development via fine-tuning expression of the LIM only factor, dLMO.

MicroRNAs are short non-coding endogenous RNAs that are implicated in regulating various aspects of plants and animal development, however their functions in organogenesis are largely unknown. Here we report that mir-9a belonging to the mir-9 family, regulates Drosophila wing development through a functional target site in the 3' untranslated region of the Drosophila LIM only protein, dLMO. dLMO is a transcription cofactor, that directly inhibits the activity of Apterous, the LIM-HD factor required for the proper dorsal identity of the wings. Deletions of the 3' untranslated region, including the mir-9a site, generate gain-of-function dLMO mutants (Beadex) associated with high levels of dLMO mRNA and protein. Beadex mutants lack wing margins, a phenotype also observed in null mir-9a mutants. We found that mir-9a and dLMO are co-expressed in wing discs and interact genetically for controlling wing development. Lack of mir-9a results in overexpression of dLMO, while gain-of-function mir-9a mutant suppresses dLMO expression. These data indicate that a function of mir-9a is to ensure the appropriate stoichiometry of dLMO during Drosophila wing development. The mir-9a binding site is conserved in the human counterpart LMO2, the T-cell acute leukemia oncogene, suggesting that mir-9 might apply a similar strategy to maintain LMO2 expression under a detrimental threshold.

Drosophila C-terminal binding protein, dCtBP is required for sensory organ prepattern and sharpens proneural transcriptional activity of the GATA factor Pnr.

The peripheral nervous system is required for animals to detect and to relay environmental stimuli to central nervous system for the information processing. In Drosophila, the precise spatial and temporal expression of two proneural genes achaete (ac) and scute (sc), is necessary for development of the sensory organs. Here we present an evidence that the transcription co-repressor, dCtBP acts as a negative regulator of sensory organ prepattern. Loss of dCtBP function mutant exhibits ectopic sensory organs, while overexpression of dCtBP results in a dramatic loss of sensory organs. These phenotypes are correlated with mis-emerging of sensory organ precursors and perturbated expression of proneural transcription activator Ac. Mammalian CtBP-1 was identified via interaction with the consensus motif PXDLSX(K/R) of adenovirus E1A oncoprotein. We demonstrated that dCtBP binds directly to PLDLS motif of Drosophila Friend of GATA-1 protein, U-shaped and sharpens the adult sensory organ development. Moreover, we found that dCtBP mediates multivalent interaction with the GATA transcriptional activator Pannier and acts as a direct co-repressor of the Pannier-mediated activation of proneural genes. We demonstrated that Pannier genetically interacts with dCtBP-interacting protein HDAC1, suggesting that the dCtBP-dependent regulation of Pannier activity could utilize a repressive mechanism involving alteration of local chromatine structure.

Drosophila dLMO-PA isoform acts as an early activator of achaete/scute proneural expression.

The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). Here we investigate, through new genetic tools, the function of dLMO, the Drosophila LIM only transcription factor that was already known to control wing development. We show that dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA(-) deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB(-) deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. We found that dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression.

Long-distance interactions between regulatory elements are suppressed at the end of a terminally deficient chromosome in Drosophila melanogaster.

In Drosophila melanogaster, broken chromosome ends behave as real telomeres and are believed to be covered with telomere-specific chromatin. It has been shown previously that the telomeric chromatin represses normal activity of enhancers that regulate yellow expression in wings and body cuticle. In this paper, we have found that a modified yellow promoter is fully active in the wing and body cuticle when it is located at the chromosome end, which is evidence that the telomeric chromatin does not repress transcription. Substitution of the yellow core promoter region, including TATA and Inr, with the promoter regions of the eve, hsp70 (TATA-containing), and white (TATA-less) promoters does not affect the ability of the promoter to be cis- or trans-activated by the yellow enhancers if the heterologous promoter is located at a distance of about 6 kb from the chromosome end. The best characterized Drosophila insulator found in the gypsy retrotransposon can specifically repress the yellow promoter at a distance when one component of the insulator complex, Mod(mdg4)-67.2 protein, is inactive. We have also found that, in the mod(mdg4) mutant background, the gypsy insulator can repress the heterologous promoters, indicating that the core promoter elements are not critical for specificity of repression. However, long-distance functional enhancer-promoter and gypsy-promoter interactions were suppressed when the distance between the yellow promoter and the end of the deficient chromosome was less than 6 kb. These results suggest that Drosophila telomeric chromatin does not generally repress transcription but is somehow involved in suppression of some long-distance interactions between regulatory elements.

Transposition of Regulatory Elements by P-Element-Mediated Rearrangements in Drosophila melanogaster.

Previously we described highly unstable mutations in the yellow locus, induced by the chimeric element and consisting of sequences from a distally located 1A unique genomic region, flanked by identical copies of an internally deleted 1.2-kb P element. Here we show that a sequence, which is part of the yellow 1A region, can be transmitted to the AS-C by successive inversion and reinversion generated by yellow- and AS-C-located P elements. The chimeric element contains a regulatory element from the 1A region that specifically blocks yellow wing and body enhancers and simultaneously stimulates yellow expression in bristles. These results suggest that P-element-generated chimeric elements may play a certain role in rapid changes of regulatory regions of genes during evolution.

The Drosophila LIM-homeo domain protein Islet antagonizes pro-neural cell specification in the peripheral nervous system.

The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix-loop-helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in clusters of cells (pro-neural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorso-central enhancer (DC) of ac/sc. We identify the LIM-homeo domain (LIM-HD) transcription factor Islet (Isl) by genetic screening and investigate its role in the thoracic pre-patterning. We show that isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) pro-neural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases pro-neural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC pro-neural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction. We propose that sensory organ pre-patterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying pre-patterns within the different pro-neural clusters.

An endogenous Su(Hw) insulator separates the yellow gene from the Achaete-scute gene complex in Drosophila.

The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Here we show that two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute.

Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa.

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. We show here that this communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes and we show that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. Our study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication.