Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene (), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact expression in fly heads; the biggest upstream deletion reduces peak RNA levels and RNA cycling amplitude to about 15% of normal, and there are similar effects on protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels.

Comparison of TRIBE and STAMP for identifying targets of RNA binding proteins in human and cells.

RNA binding proteins (RBPs) perform a myriad of functions and are implicated in numerous neurological diseases. To identify the targets of RBPs in small numbers of cells, we developed TRIBE, in which the catalytic domain of the RNA editing enzyme ADAR (ADARcd) is fused to an RBP. When the RBP binds to an mRNA, ADAR catalyzes A to G modifications in the target mRNA that can be easily identified in standard RNA sequencing.

Neural connectivity molecules best identify the heterogeneous clock and dopaminergic cell types in the adult brain.

Our recent single-cell sequencing of most adult circadian neurons indicated notable and unexpected heterogeneity. To address whether other populations are similar, we sequenced a large subset of adult brain dopaminergic neurons. Their gene expression heterogeneity is similar to that of clock neurons, i.

Comment on "Circadian rhythms in the absence of the clock gene ".

Ray (Reports, 14 February 2020, p. 800) recently claimed temperature-compensated, free-running mRNA oscillations in liver slices and skin fibroblasts. We reanalyzed these data and found far fewer reproducible mRNA oscillations in this genotype.

A transcriptomic taxonomy of circadian neurons around the clock.

Many different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of circadian neurons, we optimized a single-cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression.

Allatostatin-C/AstC-R2 Is a Novel Pathway to Modulate the Circadian Activity Pattern in Drosophila.

Seven neuropeptides are expressed within the Drosophila brain circadian network. Our previous mRNA profiling suggested that Allatostatin-C (AstC) is an eighth neuropeptide and specifically expressed in dorsal clock neurons (DN1s). Our results here show that AstC is, indeed, expressed in DN1s, where it oscillates.

Striking circadian neuron diversity and cycling of alternative splicing.

Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, we used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries.

A Screening of UNF Targets Identifies , a Novel Regulator of Circadian Rhythms.

Behavioral circadian rhythms are controlled by multioscillator networks comprising functionally different subgroups of clock neurons. Studies have demonstrated that molecular clocks in the fruit fly are regulated differently in clock neuron subclasses to support their specific functions (Lee et al., 2016; Top et al.

RNA-seq analysis of Drosophila clock and non-clock neurons reveals neuron-specific cycling and novel candidate neuropeptides.

Locomotor activity rhythms are controlled by a network of ~150 circadian neurons within the adult Drosophila brain. They are subdivided based on their anatomical locations and properties. We profiled transcripts "around the clock" from three key groups of circadian neurons with different functions.

Circadian neuron feedback controls the Drosophila sleep--activity profile.

Little is known about the ability of Drosophila circadian neurons to promote sleep. Here we show, using optogenetic manipulation and video recording, that a subset of dorsal clock neurons (DN1s) are potent sleep-promoting cells that release glutamate to directly inhibit key pacemaker neurons. The pacemakers promote morning arousal by activating these DN1s, implying that a late-day feedback circuit drives midday siesta and night-time sleep.

Genome-wide features of neuroendocrine regulation in Drosophila by the basic helix-loop-helix transcription factor DIMMED.

Neuroendocrine (NE) cells use large dense core vesicles (LDCVs) to traffic, process, store and secrete neuropeptide hormones through the regulated secretory pathway. The dimmed (DIMM) basic helix-loop-helix transcription factor of Drosophila controls the level of regulated secretory activity in NE cells. To pursue its mechanisms, we have performed two independent genome-wide analyses of DIMM's activities: (i) in vivo chromatin immunoprecipitation (ChIP) to define genomic sites of DIMM occupancy and (ii) deep sequencing of purified DIMM neurons to characterize their transcriptional profile.

Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction.

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. Here we use chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, we identified wingless (wg), a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), as a potential Repo target gene.

The transcription factor Mef2 links the Drosophila core clock to Fas2, neuronal morphology, and circadian behavior.

The transcription factor Mef2 regulates activity-dependent neuronal plasticity and morphology in mammals, and clock neurons are reported to experience activity-dependent circadian remodeling in Drosophila. We show here that Mef2 is required for this daily fasciculation-defasciculation cycle. Moreover, the master circadian transcription complex CLK/CYC directly regulates Mef2 transcription.

CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in Drosophila.

A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription factors CLOCK (CLK) and CYCLE (CYC) activate the transcription of direct target genes like period (per) and timeless (tim). They encode the proteins PER and TIM, respectively, which repress CLK/CYC activity.

Nascent-Seq reveals novel features of mouse circadian transcriptional regulation.

A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA).

Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila.

To determine the prevalence of cotranscriptional splicing in Drosophila, we sequenced nascent RNA transcripts from Drosophila S2 cells as well as from Drosophila heads. Eighty-seven percent of the introns assayed manifest >50% cotranscriptional splicing. The remaining 13% are cotranscriptionally spliced poorly or slowly, with ∼3% being almost completely retained in nascent pre-mRNA.

Drosophila CLOCK target gene characterization: implications for circadian tissue-specific gene expression.

CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night.

Dynamic PER repression mechanisms in the Drosophila circadian clock: from on-DNA to off-DNA.

Transcriptional feedback loops are central to the generation and maintenance of circadian rhythms. In animal systems as well as Neurospora, transcriptional repression is believed to occur by catalytic post-translational events. We report here in the Drosophila model two different mechanisms by which the circadian repressor PERIOD (PER) inhibits CLOCK/CYCLE (CLK/CYC)-mediated transcription.

Genome-wide identification of targets of the drosha-pasha/DGCR8 complex.

Drosha is a type III RNase, which plays a critical role in miRNA biogenesis. Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely recognize and cleave miRNA precursor RNAs or pri-miRNA hairpins cotranscriptionally. To identify RNAs processed by Drosha, we used tiling microarrays to examine transcripts after depletion of drosha mRNA with dsRNA in Drosophila Schneider S2 cells.

The nuclear exosome and adenylation regulate posttranscriptional tethering of yeast GAL genes to the nuclear periphery.

GAL genes and other activated yeast genes remain tethered to the nuclear periphery even after transcriptional shutoff. To identify factors that affect this tethering, we designed a plasmid-based visual screen. Although many factors affected GAL tethering during transcription, fewer specifically affected posttranscriptional tethering.

Protein characterization of Saccharomyces cerevisiae RNA polymerase II after in vivo cross-linking.

To characterize proteins associated with active transcription complexes, we purified RNA polymerase II (pol II) from Saccharomyces cerevisiae after fixing live cells with formaldehyde. The approach mimics ChIP and requires solubilizing cross-linked complexes with sonication. Pol II was affinity-purified, and associated proteins were identified by MS.

Sus1, Sac3, and Thp1 mediate post-transcriptional tethering of active genes to the nuclear rim as well as to non-nascent mRNP.

Errors in the mRNP biogenesis pathway can lead to retention of mRNA in discrete, transcription-site-proximal foci. This RNA remains tethered adjacent to the transcription site long after transcriptional shutoff. Here we identify Sus1, Thp1, and Sac3 as factors required for the persistent tethering of such foci (dots) to their cognate genes.

3'-end formation signals modulate the association of genes with the nuclear periphery as well as mRNP dot formation.

Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site.

Biochemical analysis of TREX complex recruitment to intronless and intron-containing yeast genes.

The TREX complex is involved in both transcription elongation and mRNA export and is recruited to nascent transcription complexes. We have examined Yra1p, Sub2p and Hpr1p recruitment to nine genes of varying lengths and transcription frequencies. All three proteins increase from the 5' to the 3' ends of the four intronless genes examined.

An alpha-tubulin mutant demonstrates distinguishable functions among the spindle assembly checkpoint genes in Saccharomyces cerevisiae.

Cells expressing a mutant allele of alpha-tubulin, tub1-729, are cold sensitive and arrest as large-budded cells with microtubule defects. The cold sensitivity of tub1-729 is suppressed by extra copies of a subset of the mitotic checkpoint genes BUB1, BUB3, and MPS1, but not MAD1, MAD2, and MAD3. This suppression by checkpoint genes does not depend upon their role in the MAD2-dependent spindle assembly checkpoint.