Extensive tissue-specific expression variation and novel regulators underlying circadian behavior.

Natural genetic variation affects circadian rhythms across the evolutionary tree, but the underlying molecular mechanisms are poorly understood. We investigated population-level, molecular circadian clock variation by generating >700 tissue-specific transcriptomes of Drosophila melanogaster (w1118 ) and 141 Drosophila Genetic Reference Panel (DGRP) lines. This comprehensive circadian gene expression atlas contains >1700 cycling genes including previously unknown central circadian clock components and tissue-specific regulators. Furthermore, >30% of DGRP lines exhibited aberrant circadian gene expression, revealing abundant genetic variation-mediated, intertissue circadian expression desynchrony. Genetic analysis of one line with the strongest deviating circadian expression uncovered a novel cry mutation that, as shown by protein structural modeling and brain immunohistochemistry, disrupts the light-driven flavin adenine dinucleotide cofactor photoreduction, providing in vivo support for the importance of this conserved photoentrainment mechanism. Together, our study revealed pervasive tissue-specific circadian expression variation with genetic variants acting upon tissue-specific regulatory networks to generate local gene expression oscillations.

BRB-seq: ultra-affordable high-throughput transcriptomics enabled by bulk RNA barcoding and sequencing.

Despite its widespread use, RNA-seq is still too laborious and expensive to replace RT-qPCR as the default gene expression analysis method. We present a novel approach, BRB-seq, which uses early multiplexing to produce 3' cDNA libraries for dozens of samples, requiring just 2 hours of hands-on time. BRB-seq has a comparable performance to the standard TruSeq approach while showing greater tolerance for lower RNA quality and being up to 25 times cheaper. We anticipate that BRB-seq will transform basic laboratory practice given its capacity to generate genome-wide transcriptomic data at a similar cost as profiling four genes using RT-qPCR.

Engineered Multivalent Sensors to Detect Coexisting Histone Modifications in Living Stem Cells.

The regulation of fundamental processes such as gene expression or cell differentiation involves chromatin states, demarcated by combinatorial histone post-translational modification (PTM) patterns. The subnuclear organization and dynamics of chromatin states is not well understood, as tools for their detection and modulation in live cells are lacking. Here, we report the development of genetically encoded chromatin-sensing multivalent probes, cMAPs, selective for bivalent chromatin, a PTM pattern associated with pluripotency in embryonic stem cells (ESCs). cMAPs were engineered from a set of PTM-binding (reader) proteins and optimized using synthetic nucleosomes carrying defined PTMs. Applied in live ESCs, cMAPs formed discrete subnuclear foci, revealing the organization of bivalent chromatin into local clusters. Moreover, cMAPs enabled direct monitoring of the loss of bivalency upon treatment with small-molecule epigenetic modulators. cMAPs thus provide a versatile platform to monitor chromatin state dynamics in live cells.

Rhythmic expression of the cycle gene in a hematophagous insect vector.

BACKGROUND: A large number of organisms have internal circadian clocks that enable them to adapt to the cyclic changes of the external environment. In the model organism Drosophila melanogaster, feedback loops of transcription and translation are believed to be crucial for the maintenance of the central pacemaker. In this mechanism the cycle (or bmal1) gene, which is constitutively expressed, plays a critical role activating the expression of genes that will later inhibit their own activity, thereby closing the loop. Unlike Drosophila, the molecular clock of insect vectors is poorly understood, despite the importance of circadian behavior in the dynamic of disease transmission. RESULTS: Here we describe the sequence, genomic organization and circadian expression of cycle in the crepuscular/nocturnal hematophagous sandfly Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. Deduced amino acid sequence revealed that sandfly cycle has a C-terminal transactivation domain highly conserved among eukaryotes but absent in D. melanogaster. Moreover, an alternative form of the transcript was also identified. Interestingly, while cycle expression in Drosophila and other Diptera is constitutive, in sandflies it is rhythmic in males and female heads but constitutive in the female body. Blood-feeding, which causes down-regulation of period and timeless in this species, does not affect cycle expression. CONCLUSION: Sequence and expression analysis of cycle in L. longipalpis show interesting differences compared to Drosophila suggesting that hematophagous vector species might present interesting new models to study the molecular control of insect circadian clocks.

The biological clock of an hematophagous insect: locomotor activity rhythms, circadian expression and downregulation after a blood meal.

Despite the importance of circadian rhythms in vector-borne disease transmission, very little is known about its molecular control in hematophagous insect vectors. In Drosophila melanogaster, a negative feedback loop of gene expression has been shown to contribute to the clock mechanism. Here, we describe some features of the circadian clock of the sandfly Lutzomyia longipalpis, a vector of visceral leishmaniasis. Compared to D. melanogaster, sandfly period and timeless, two negative elements of the feedback loop, show similar peaks of mRNA abundance. On the other hand, the expression of Clock (a positive transcription factor) differs between the two species, raising the possibility that the different phases of Clock expression could be associated with the observed differences in circadian activity rhythms. In addition, we show a reduction in locomotor activity after a blood meal, which is correlated with downregulation of period and timeless expression levels. Our results suggest that the circadian pacemaker and its control over the activity rhythms in this hematophagous insect are modulated by blood intake.

Cloning and daily expression of the timeless gene in Aedes aegypti (Diptera:Culicidae).

Molecular approaches for studying biological rhythms in insects have been well investigated in the model Drosophila melanogaster, in which a number of genes have been characterized in terms of sequence, expression, protein interactions and involvement in the control of locomotor activity and eclosion rhythms. However, only scattered information is available for insect vectors of diseases. In this paper, we report the cloning and expression analysis of the clock gene timeless in the mosquito Aedes aegypti, vector of Dengue and Yellow Fever viruses. In Drosophila, timeless has a crucial role in the control of the central pacemaker and the resetting mechanism that allows the clock to synchronize with the environment light-dark cycles. Comparison of the predicted protein sequence encoded by timeless in Ae. aegypti and D. melanogaster demonstrated high similarity in some important domains, suggesting functional conservation. Analysis of the daily expression of timeless in Ae. aegypti shows a peak in mRNA abundance around the light-dark transition.

Genome-Wide Ultrabithorax Binding Analysis Reveals Highly Targeted Genomic Loci at Developmental Regulators and a Potential Connection to Polycomb-Mediated Regulation.

Hox homeodomain transcription factors are key regulators of animal development. They specify the identity of segments along the anterior-posterior body axis in metazoans by controlling the expression of diverse downstream targets, including transcription factors and signaling pathway components. The Drosophila melanogaster Hox factor Ultrabithorax (Ubx) directs the development of thoracic and abdominal segments and appendages, and loss of Ubx function can lead for example to the transformation of third thoracic segment appendages (e.g. halters) into second thoracic segment appendages (e.g. wings), resulting in a characteristic four-wing phenotype. Here we present a Drosophila melanogaster strain with a V5-epitope tagged Ubx allele, which we employed to obtain a high quality genome-wide map of Ubx binding sites using ChIP-seq. We confirm the sensitivity of the V5 ChIP-seq by recovering 7/8 of well-studied Ubx-dependent cis-regulatory regions. Moreover, we show that Ubx binding is predictive of enhancer activity as suggested by comparison with a genome-scale resource of in vivo tested enhancer candidates. We observed densely clustered Ubx binding sites at 12 extended genomic loci that included ANTP-C, BX-C, Polycomb complex genes, and other regulators and the clustered binding sites were frequently active enhancers. Furthermore, Ubx binding was detected at known Polycomb response elements (PREs) and was associated with significant enrichments of Pc and Pho ChIP signals in contrast to binding sites of other developmental TFs. Together, our results show that Ubx targets developmental regulators via strongly clustered binding sites and allow us to hypothesize that regulation by Ubx might involve Polycomb group proteins to maintain specific regulatory states in cooperative or mutually exclusive fashion, an attractive model that combines two groups of proteins with prominent gene regulatory roles during animal development.

"The Environment is Everything That Isn't Me": Molecular Mechanisms and Evolutionary Dynamics of Insect Clocks in Variable Surroundings.

Circadian rhythms are oscillations in behavior, metabolism and physiology that have a period close to 24 h. These rhythms are controlled by an internal pacemaker that evolved under strong selective pressures imposed by environmental cyclical changes, mainly of light and temperature. The molecular nature of the circadian pacemaker was extensively studied in a number of organisms under controlled laboratory conditions. But although these studies were fundamental to our understanding of the circadian clock, most of the environmental conditions used resembled rather crudely the relatively constant situation at lower latitudes. At higher latitudes light-dark and temperature cycles vary considerably across different seasons, with summers having long and hot days and winters short and cold ones. Considering these differences and other external cues, such as moonlight, recent studies in more natural and semi-natural situations revealed unexpected features at both molecular and behavioral levels, highlighting the dramatic influence of multiple environmental variables in the molecular clockwork. This emphasizes the importance of studying the circadian clock in the wild, where seasonal environmental changes fine-tune the underlying circadian mechanism, affecting population dynamics and impacting the geographical variation in clock genes. Indeed, latitudinal clines in clock gene frequencies suggest that natural selection and demography shape the circadian clock over wide geographical ranges. In this review we will discuss the recent advances in understanding the molecular underpinnings of the circadian clock, how it resonates with the surrounding variables (both in the laboratory and in semi-natural conditions) and its impact on population dynamics and evolution. In addition, we will elaborate on how next-generation sequencing technologies will complement classical reductionist approaches by identifying causal variants in natural populations that will link genetic variation to circadian phenotypes, illuminating how the circadian clock functions in the real world.

cis-regulatory requirements for tissue-specific programs of the circadian clock.

BACKGROUND: Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs CLOCK (CLK) and CYCLE (CYC) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how CLK and CYC determine tissue-specific clock programs has remained unclear. RESULTS: Here, we use a functional genomics approach to determine the cis-regulatory requirements for clock specificity. We first determine CLK and CYC genome-wide binding targets in heads and bodies by ChIP-seq and show that they have distinct DNA targets in the two tissue contexts. Computational dissection of CLK/CYC context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, we show that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by OPA and SERPENT (SRP). They act synergistically with CLK/CYC in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, we validate that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock. CONCLUSIONS: Our results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression.

Comparative genomics of gene regulation-conservation and divergence of cis-regulatory information.

We recently witnessed a tremendous increase in genomics studies on gene regulation and in entirely sequenced genomes from closely related species. This has triggered analyses that suggest a wide range of evolutionary dynamics of gene regulation, from rapid turnover of transcription-factor binding sites to conservation of enhancer function across large evolutionary distances. Many examples show that enhancers can evolve beyond recognizable sequence similarity while retaining function. However, bioinformatics approaches are increasingly able to detect conserved regulatory elements through characteristic evolutionary sequence signatures. Cis-regulatory changes are also a major source of morphological evolution, which might be facilitated by many biochemically functional elements that are selectively neutral and by the buffering function of redundant enhancers and 'shadow' enhancers.

Circadian expression of clock genes in two mosquito disease vectors: cry2 is different.

Different mosquito species show a full range of activity patterns, including diurnal, crepuscular, and nocturnal behaviors. Although activity and blood-feeding rhythms are controlled by the circadian clock, it is not yet known whether such species-specific differences in behavior are controlled directly by core clock genes or instead reflect differences in how the information of the central clock is translated into output signals. The authors have analyzed the circadian expression of clock genes in two important mosquito vectors of tropical diseases, Aedes aegypti and Culex quinquefasciatus . Although these two species show very different locomotor activity patterns and are estimated to have diverged more than 22 million years ago, they show conserved circadian expression patterns for all major cycling clock genes except mammalian-like cryptochrome2 (cry2). The results indicate that different mechanisms for cry2 regulation may exist for the two species. The authors speculate that the correlation between the differences in behavior between Ae. aegypti and Cx. quinquefasciatus and their corresponding cry2 mRNA profiles suggests a potential role for this clock gene in controlling species-specific rhythmic behavior. However, further work is needed to establish that this is the case as the different cry2 expression patterns might reflect differences between the Aedes and Culex lineages that are not directly related to changes in behavior.