Dataset DOI: 10.5061/dryad.nzs7h4545

Description of the data and file structure

This dataset was generated to test whether Polycomb (Pc) regulates sleep amount and sleep homeostasis in Drosophila and to identify the cell types and downstream genes involved. We combined cell-type–specific genetics (EB1-Gal4, R69F08-Gal4, Repo-Gal4; UAS-Pc RNAi; Kir2.1/TNT; dTrpA1; LexA/LexAop intersections) with behavioral recording under LD cycles. Sleep was measured as 24-h profiles, total sleep, and recovery after 12-h night sleep deprivation. Brain expression patterns and co-localization were assessed by immunostaining. Pc targets were mapped by TaDa (Targeted DamID) in the above populations and by RNA-seq after Pc perturbation, followed by GO/KEGG enrichment. The files provide the raw/processed data underlying the main and supplementary figures/tables.

Files and variables

Source_data_for_fig._1.xlsx

Fig. 1 Polycomb (Pc) is required for sleep and homeostasis regulation in cells expressing EB1-Gal4 and R69F08-Gal4.

To systematically elucidate the role of the epigenetic factor Pc in sleep regulation, we performed a cell-specific knockdown screen. We employed two Gal4 drivers to specifically induce the knockdown of Pc. Specifically, EB1-Gal4-induced Pc knockdown led to a reduction in both total daytime (ZT0-ZT12, Unit: minutes) and nighttime sleep (ZT12-ZT24, Unit: minutes) (Fig. 1A-B). Similarly, R69F08-Gal4-induced Pc knockdown also resulted in decreased total daytime (ZT0-ZT12) and nighttime sleep (ZT12-ZT24) (Fig. 1C-D).

To understand the role of Pc in sleep homeostasis, we employed EB1-Gal4 and R69F08-Gal4 to drive Pc RNAi expression in sleep deprivation experiments. Normally, sleep deprivation was achieved by mechanical shaking of the flies, which would lead to a recovery of sleep amount during the daytime after deprivation. Our results indicated that Pc knockdown induced by EB1-Gal4 (Fig. 1M-N) and R69F08-Gal4 (Fig. 1O-P) led to significant changes in sleep recovery after 12 hours of sleep deprivation when compared with the controls group. These findings indicate that Pc is essential for the regulation of sleep homeostasis in EB1-Gal4 and R69F08-Gal4 expressing cells.

Data information: (A-B) The sleep phenotypes of EB1-Gal4 driven Pc knockdown. (C-D) The sleep phenotypes of R69F08-Gal4 driven Pc knockdown. (M-N, O-P) For sleep deprivation experiments involving tub-Gal80^ts, conditions for the induction of Gal4 activity were performed, sleep measurement was performed at 29°C until the end of the experiment. Sleep deprivation was achieved by loading flies in DAMs monitors into the shaker fitted with a custom base. Flies were vortexed from ZT12 (Zeitgeber time 12: beginning of the dark phase) to ZT24 (Zeitgeber time 24: beginning of the light phase) at the lower intensity setting for 3 sec/min. Zeitgeber time is used to indicate the phase of the circadian rhythm in relation to a specific Zeitgeber. The 24-hour cycle is divided into 24 Zeitgeber times, where ZT0 represents the start of the light phase, and ZT12 corresponds to the start of the dark phase. The day time increase of total sleep amount (min) after sleep deprivation was calculated by subtracting the baseline daytime sleep amount of each repeat from the corresponding daytime sleep amount after the sleep deprivation. The night time decrease of total sleep amount (min) after sleep deprivation was calculated by subtracting the baseline night time sleep amount of each repeat by the corresponding night time sleep amount after the sleep deprivation. (M, O) Sleep Recovery (%) during ZT0-ZT12 was calculated by (day time increase of total sleep amount) / (night time decrease of total sleep amount). (N, P) Sleep Recovery (min) during ZT0-ZT12 was calculated by (daytime sleep amount after sleep deprivation - baseline daytime sleep amount before sleep deprivation). ZT0-ZT12 and ZT12-ZT24 (ZT denotes Zeitgeber time) indicate light and dark periods under LD (Light/Dark), respectively. Total sleep time (minutes) across circadian phases. Values indicate the total minutes of sleep recorded during the 12-hour light (ZT0-ZT12) and 12-hour dark (ZT12-ZT24) periods.

Source_data_for_fig._3.xlsx

Fig. 3 Sleep profiles after inhibiting R69F08-lexA and R69F08-Gal4 expressing cells.

To investigate the function of the EB1+/R69F08+ cells, we generated R69F08-lexA. Inhibition of R69F08-lexA expressing cells resulted in the same sleep phenotypes as inhibition of R69F08-Gal4 expressing cells (Fig. 3D-E). Moreover, sleep recovery following deprivation was abolished after inhibiting R69F08-lexA (Fig. 3G) or R69F08-Gal4 (Fig. 3I) expressing cells. In conclusion, the R69F08-lexA represents R69F08-Gal4 expressing cells in both expression pattern and function.

Data information: Sleep phenotypes (total sleep - day time during ZT0-ZT12 and total sleep - nighttime during ZT12-ZT24) caused by inhibiting the R69F08-LexA expressing cells (D). Sleep phenotypes (total sleep - daytime during ZT0-ZT12 and total sleep - nighttime during ZT12-ZT24) caused by inhibiting the R69F08-Gal4 expressing cells (E). For sleep deprivation experiments at 25°C, sleep measurement was performed at 25°C until the end of the experiment. Baseline sleep was measured for 2 days prior to sleep deprivation. Sleep deprivation was achieved by loading flies in DAMs monitors into the shaker fitted with a custom base. Flies were vortexed from ZT12 (Zeitgeber time 12: beginning of the dark phase) to ZT24 (Zeitgeber time 24: beginning of the light phase) at the lower intensity setting for 3 sec/min. Zeitgeber time is used to indicate the phase of the circadian rhythm in relation to a specific Zeitgeber. The 24-hour cycle is divided into 24 Zeitgeber times, where ZT0 represents the start of the light phase, and ZT12 corresponds to the start of the dark phase. Sleep recovery after Sleep deprivation (min) (Fig. 3G & I) was calculated by (daytime sleep amount after sleep deprivation - baseline daytime sleep amount before sleep deprivation). ZT0-ZT12 and ZT12-ZT24 (ZT denotes Zeitgeber time) indicate light and dark periods under LD (Light/Dark), respectively. Total sleep time (minutes) across circadian phases. Values indicate the total minutes of sleep recorded during the 12-hour light (ZT0-ZT12) and 12-hour dark (ZT12-ZT24) periods.

Source_data_for_fig._5.xlsx

Fig. 5 Pc is required for sleep and homeostasis regulation in Glia cells.

Knocking down of Pc using Repo-Gal4 partially recapitulated the phenotype driven by EB1-Gal4 and R69F08-Gal4. While Repo-Gal4-driven Pc knockdown partially phenocopied the sleep defects observed in EB1-Gal4-/R69F08-Gal4-marked cells (Fig. 5A-B & I), this pan-glial driver cannot isolate contributions from the specific EB1+/R69F08+ glial subpopulation. The partial overlap suggests that both neuron-glia interactions within EB1/R69F08 lineages and subtype-specific glial functions contribute to sleep regulation.

Data information: (A-B) The sleep phenotypes (Total sleep during ZT0-ZT12 and Total sleep during ZT12-ZT24) of Repo-Gal4 driven Pc knockdown. For sleep deprivation experiments involving tub-Gal80^ts, conditions for the induction of Gal4 activity were performed, sleep measurement was performed at 29°C until the end of the experiment. Sleep deprivation was achieved by loading flies in DAMs monitors into the shaker fitted with a custom base. Flies were vortexed from ZT12 (Zeitgeber time 12: beginning of the dark phase) to ZT24 (Zeitgeber time 24: beginning of the light phase) at the lower intensity setting for 3 sec/min. Zeitgeber time is used to indicate the phase of the circadian rhythm in relation to a specific Zeitgeber. The 24-hour cycle is divided into 24 Zeitgeber times, where ZT0 represents the start of the light phase, and ZT12 corresponds to the start of the dark phase. Sleep recovery (min) during ZT0-ZT12 was calculated by (daytime sleep amount after sleep deprivation - baseline daytime sleep amount before sleep deprivation) (I). ZT0-ZT12 and ZT12-ZT24 (ZT denotes Zeitgeber time) indicate light and dark periods under LD (Light/Dark), respectively. Total sleep time (minutes) across circadian phases. Values indicate the total minutes of sleep recorded during the 12-hour light (ZT0-ZT12) and 12-hour dark (ZT12-ZT24) periods.

Source_data_for_fig._6.xlsx

Fig. 6 Sleep drive is modulated through EB1-Gal4- and R69F08-Gal4-expressing cells.

In our investigation into the role of EB1-Gal4 and R69F08-Gal4 expressing cells in sleep regulation, we assessed their inhibitory phenotype using UAS-TNT and their activated phenotype using UAS-dTrpA. The inhibition of the activity of EB1-Gal4 and R69F08-Gal4 marked cells resulted in reduced overall sleep time (Fig. 6A-B). While, activation of EB1-Gal4 and R69F08-Gal4 expressing cells increased total sleep time (Fig. 6E).  

Activation of cells expressing R69F08-Gal4 alone is adequate to regulate sleep homeostasis. Consistent with the findings of Liu et al., 2016, inducing activation in R69F08-Gal4 expressing cells resulted in a sustained increase in sleep pressure. This is evidenced by the rise in daytime sleep subsequent to the activation of these cells via temperature-induced dTrpA1, even when the flies had slept well the preceding night (Fig. 6F). Conversely, activating EB1-Gal4 expressing cells had no such effect (Fig. 6F). Sleep propensity value, defined as the percentage of flies initiating sleep within five minutes after lights-off, serves as an indicator of relative sleep pressure. Results demonstrated that in contrast to the activation of EB1-Gal4 marked cells, activating R69F08-Gal4 marked cells heightened sleep pressure (Fig. 6G).

We also observed that cells expressing EB1-Gal4 and R69F08-Gal4 under sleep deprivation exhibited increased sleep duration compared to the control group (Fig. 6J). The sleep propensity value, representing the percentage of flies falling asleep within five minutes after lights-off, indicated that under sleep deprivation, activation of R69F08-Gal4 and EB1-Gal4 expressing cells results in a sustained increase in sleep pressure (Fig. 6K). Activation of R69F08-Gal4 or EB-Gal4 marked cells did not alter waking activity (Fig. 6L).

Data information: Sleep phenotypes (Total sleep during ZT0-ZT12 and Total sleep during ZT12-ZT24) caused by inhibiting the R69F08-Gal4 marked cells (A) and the EB1-Gal4 marked cells (B). (E-G) Sleep phenotypes caused by activating the R69F08-Gal4 and EB1-Gal4 marked cells. Protocol for sleep phenotypes: after being maintained at 21℃ for 1 day, flies were treated by heat pulse for 12 hr at night at 29℃ until the end of the night and then restored to 21℃ for 1 day. (E) Calculation of the sleep amount (Sleep amount during ZT12-ZT24) during dTrpA1 activation. (F) Calculation of the sleep amount (Sleep amount during ZT12-ZT24; daytime sleep at 21℃) after dTrpA1 activation. (G) Calculation of the sleep propensity in five minutes after light on. Sleep propensity value, defined as the percentage of flies initiating sleep within five minutes after lights-off, serves as an indicator of relative sleep pressure. (J-L) Sleep phenotypes caused by sleep deprivation and activating the R69F08-Gal4 or EB1-Gal4 marked cells simultaneously. Protocol for sleep deprivation: after being maintained at 21℃ for 1 day, flies were simultaneously treated by mechanical perturbation and heat pulse for 12 hr at night at 29℃ until the end of the night and then continued to 21℃ for 1 day. (J) Calculation of the sleep amount 24 hours after sleep deprivation and dTrpA1 activation. (K) Calculation of the sleep propensity in five minutes after light off. Sleep propensity value, defined as the percentage of flies initiating sleep within five minutes after lights-off, serves as an indicator of relative sleep pressure. (L) Calculation of the waking activity in 24 hours after being fully aroused. Waking activity was measured by examining activity counts in 24 hrs after being fully aroused. ZT0-ZT12 and ZT12-ZT24 (ZT denotes Zeitgeber time) indicate light and dark periods under LD (Light/Dark), respectively. Total sleep time (minutes) across circadian phases. Values indicate the total minutes of sleep recorded during the 12-hour light (ZT0-ZT12) and 12-hour dark (ZT12-ZT24) periods.

Source_data_for_fig._7.xlsx

Fig. 7 EB1-Gal4 and R69F08-Gal4 co-expressing cells played important roles in sleep homeostasis regulation.

Inhibiting EB1+/R69F08+ cells using Kir2.1, TNT or shibire resulted in reduced daytime sleep (Fig. 7A-B & D), while activating them via UAS-TrpA1 increased sleep amount (Fig. 7F). In summary, modulating the activity of cells expressing EB1-Gal4 and R69F08-Gal4, as well as their overlapping populations, elicits distinct sleep-related phenotypes. Additionally, there are no significant alterations in sleep propensity value (Fig. 7G).

To investigate the inhibition and activation effects of these cell clusters under conditions of sleep deprivation, we assessed the parameters of sleep homeostasis following interventions targeting cell inhibition and activation associated with sleep deprivation. Inhibiting EB1-Gal4 and R69F08-Gal4 expressing cells led to disrupted sleep recovery following mechanical sleep deprivation. Inhibition of EB1+/R69F08+ cells by overexpressing kir2.1 or shibire significantly reduced sleep recovery and sleep propensity (Fig. 7I-J & 7L-M). Activating EB1+/R69F08+ cells by overexpressing TrpA1 resulted in significantly increased sleep recovery and sleep propensity (Fig. 7O-P). These results suggest that activation of R69F08-Gal4 or EB1-Gal4 marked cells increases sleep pressure. Additionally, these findings imply that EB1-Gal4 and R69F08-Gal4 marked cells, along with EB1+/R69F08+ cells, are necessary for maintaining normal homeostasis.

Data information: (A-B) Sleep phenotypes caused by inhibiting the EB1-Gal4 and R69F08-lexA co-expressing cells. (D) Sleep profile for inhibition of the EB1-Gal4 and* R69F08-lexA* co-expressing cells at 29℃. Total sleep during the 24-hr inhibition. (F-G) Sleep profile during nighttime activation of EB1-Gal4 and R69F08-LexA co-expressing neurons at 29℃. (F) Calculation of the sleep amount during TrpA1 activation. (G) Calculation of the sleep propensity in five minutes after light on. Sleep propensity value, defined as the percentage of flies initiating sleep within five minutes after lights-off, serves as an indicator of relative sleep pressure. (I-J) Sleep phenotypes caused by mechanical perturbation for 12 hr at night until the end of the night. (I) Calculation of Sleep recovery  during ZT0-ZT12 after sleep deprivation. (J) Calculation of the sleep propensity in five minutes after light off. Sleep propensity value, defined as the percentage of flies initiating sleep within five minutes after lights-off, serves as an indicator of relative sleep pressure. (L-M) Protocol for sleep deprivation: after being maintained at 21℃ for 1 day, flies were simultaneously treated by mechanical perturbation and heat pulse for 12 hr at night at 29℃ until the end of the night and then continued to 29℃ for 1 day. (L) Calculation of Sleep recovery during ZT0-ZT12 after sleep deprivation. (M) Calculation of the sleep propensity in five minutes after light off. (O-P) Protocol for sleep deprivation: after being maintained at 21℃ for 1 day, flies were simultaneously treated by mechanical perturbation and heat pulse for 12 hr at night at 29℃ until the end of the night and then continued to 29℃ for 1 day. (O) Calculation of the sleep amount sleep amount during ZT0-ZT24 after dTrpA1 activation. (P) Calculation of the sleep propensity in five minutes after light off. ZT0-ZT12 and ZT12-ZT24 (ZT denotes Zeitgeber time) indicate light and dark periods under LD (Light/Dark), respectively. Total sleep time (minutes) across circadian phases. Values indicate the total minutes of sleep recorded during the 12-hour light (ZT0-ZT12) and 12-hour dark (ZT12-ZT24) periods.

Source_data_for_fig._8.xlsx

Fig. 8 EB1-Gal4 and R69F08-Gal4 expressing cells played an important role in sleep homeostasis regulation.

To investigate the inhibition effects of these cell clusters under conditions of sleep deprivation, we assessed the parameters of sleep homeostasis following interventions targeting cell inhibition associated with sleep deprivation. Inhibiting EB1-Gal4 expressing cells led to disrupted sleep recovery following mechanical sleep deprivation (Fig. 8C-D).

Data information: (C-D) Sleep recovery during the 12hr following sleep deprivation for EB1-Gal4>UAS-inactive-TNT and EB1-Gal4>UAS-strong-TNT. (C) Sleep recovery quantified as a percentage (%). Sleep recovery (%) was calculated by (day time increase of total sleep amount) / (night time decrease of total sleep amount). (D) Sleep recovery measured in absolute minutes (min). Sleep recovery (min) was calculated by (daytime sleep amount after sleep deprivation - baseline daytime sleep amount before sleep deprivation). ZT0-ZT12 (ZT denotes Zeitgeber time) indicate light period.  

Source_data_for_Fig._9.xlsx

Fig. 9 Analysis of the Pc target gene profiles identified by RNAseq and TaDa.

We identified Pc binding profiles in the EB1-Gal4 and R69F08-Gal4 expressing cells by using the Tada assay. TaDa is based on DamID, a technology designed to detect genome-wide binding sites of proteins in vivo. When coupled with the Gal4 system in Drosophila, TaDa achieves both temporal and spatial resolution. KEGG enrichment analysis revealed that this group of genes was enriched in multiple pathways (Fig. 9F).

To identify Pc-regulated targets in cells expressing EB1-Gal4 and R69F08-Gal4, we conducted RNA sequencing on head tissues with specific Pc knockdown mediated by EB1-Gal4 and R69F08-Gal4. KEGG enrichment analysis unveiled that this gene set is enriched in multiple pathways (Fig. 9G). In summary, our multi-omics approach defines Pc’s genomic targets in sleep-regulatory cells, implicating transcriptional and signaling networks in sleep homeostasis.

Data information: (F-G) The genes of Pc identified by RNAseq and TaDa were assigned to functional classes on the basis of the KEGG annotation. (F) KEGG enrichment analysis of the chimeras-defined targets of Pc identified by TaDa in the EB1-Gal4 or R69F08-Gal4 marked cells. (G) KEGG enrichment analysis of the regulated targets of Pc identified by RNAseq in the EB1-Gal4 or R69F08-Gal4 marked cells.

Source_data_for_fig._10.xlsx

Fig. 10 GO enrichment analysis of the Pc target gene profiles identified by RNAseq and TaDa.

To characterize the features of Pc's direct targets in the EB1-Gal4 and R69F08-Gal4 marked cells, we conducted combined TaDa and RNA-seq analyses.The Pc targets* identified were assigned to functional classes on the basis of the GO annotation (Fig. 10A-D). GO Enrichment analysis of the Pc targets identified by TaDa in the EB1-Gal4 and R69F08-Gal4 marked cells respectively (Fig. 10A). GO Enrichment analysis of the Pc targets identified by RNAseq in EB1-Gal4 and R69F08-Gal4 marked cells respectively (Fig. 10B). GO Enrichment analysis of functional direct targets (Pc targets identified by RNAseq and TaDa) in the R69F08-Gal4 or EB1-Gal4 marked cells (Fig. 10C). TaDa is based on DAM-ID, a technology designed to detect genome-wide binding sites of proteins in vivo. We identified Pc binding profiles using the Tada assay. GO Enrichment analysis of the gene profiles found in TaDa data, but not in RNA seq data in R69F08-Gal4 or EB1-Gal4 marked cells (Fig. 10D).

Data information: (A-D) GO enrichment is measured by p.adjust and GeneRatio in this pathway. p.adjust value is the p value corrected by multiple hypothesis test, the range of q value is [0,1], the closer it is to zero, the more significant the enrichment is. GeneRatio refers to the ratio of the number of binding genes or differentially expressed genes in different pathways to all annotated total genes.

Source_data_for_Fig._11.xlsx

Fig. 11 Functional classifications of Pc target genes enriched in the EB1-Gal4 and R69F08-Gal4 marked cells.

The targets of Pc identified by RNAseq and TaDa are shown by Venn diagrams (Fig. 11A). The distribution of common targets of Pc found in both RNAseq and TaDa (TaDa is based on DAM-ID, a technology designed to detect genome-wide binding sites of proteins in vivo. We identified Pc binding profiles using the Tada assay) are shown by Volcano plot (Fig. 11B-C). Panel B and Panel C indicated the distribution of Pc targets in R69F08-Gal4 marked cells (Fig. 11B) and EB1-Gal4 marked cells (Fig. 11C). KEGG Enrichment analysis of functional direct targets (Pc targets  identified by both RNAseq and TaDa) in the R69F08-Gal4 or EB1-Gal4 marked cells (Fig. 11D). KEGG Enrichment analysis of the gene profiles found in TaDa data, but not in RNA seq data in the R69F08-Gal4 or EB1-Gal4 marked cells (Fig. 11E).

Data information: Log2FC represents the logarithm of the difference multiple of the expression level of a gene in two samples. FDR (False Discovery Rate) is a statistically adjusted value following multiple testing corrections. It indicates the expected proportion of false positives (false discoveries) among all genes identified as differentially expressed. A smaller FDR value suggests higher reliability of the result. The use of -log10(FDR) is intended to represent small, significant FDR values, thereby more intuitively showing the significance level of gene differential expression: the higher the Log2FC value, the more significant the gene's differential expression. This study used significance threshold is FDR < 0.05 (B-C). KEGG enrichment is measured by p.adjust and GeneRatio in this pathway. p.adjust value is the p value corrected by multiple hypothesis test, the range of q value is [0,1], the closer it is to zero, the more significant the enrichment is. GeneRatio refers to the ratio of the number of binding genes or differentially expressed genes in different pathways to all annotated total genes (D-E).

Source_data_for_fig._12.xlsx

Fig. 12 Enrichment analysis of Pc target genes in glial and neurons.

Enrichment analysis of Pc targets expressed in either glia or neurons revealed their distribution among different pathways (Fig. 12C-E). These findings position Pc as a pan-cellular epigenetic regulator, with targets spanning neural and glial functions in sleep circuitry. KEGG Enrichment analysis of Pc targets exhibiting expression in glia cells (Pc targets identified by both RNAseq or TaDa) (Fig. 12C). KEGG Enrichment analysis of Pc targets exhibiting expression in neurons (Fig. 12D). KEGG Enrichment analysis of Pc targets exhibiting expression in both glia and neurons (Fig. 12E).

Data information: (C-E) KEGG enrichment is measured by p.adjust and GeneRatio in this pathway. p.adjust value is the p value corrected by multiple hypothesis test, the range of q value is [0,1], the closer it is to zero, the more significant the enrichment is. GeneRatio refers to the ratio of the number of binding genes or differentially expressed genes in different pathways to all annotated total genes.

Source_data_for_fig._13.xlsx

Fig. 13 Heightened sensitivity of Pc target gene expression to sleep deprivation upon Pc knockdown.

Quantitative RT-PCR analysis of Pc targets in control and treatment groups. Replicate # indicates biological replicates, with each replicate comprising > 25 individual fly heads. Sleep dep indicates Sleep deprivation (Fig. 13A’-H’).

Source_data_for_fig._14.xlsx

Fig. 14 Pc target genes that are involved in neurotransmission are required for sleep homeostasis regulation in EB1-Gal4 and R69F08-Gal4 expressing cells.

Sleep Recovery at ZT0-ZT12 (%) phenotypes after sleep deprivation when the genes involved in neurotransmission are knocked down using EB1-Gal4 and R69F08-Gal4 respectively (Fig. 14A). Sleep recovery at ZT0-ZT12 (min) phenotypes after sleep deprivation when the genes involved in neurotransmission are knocked down using EB1-Gal4 and R69F08-Gal4 respectively (Fig. 14B). Sleep amount (ZT0-ZT24) when the genes involved in neurotransmission are knocked down using EB1-Gal4 and R69F08-Gal4 respectively (Fig. 14C). ZT0-ZT12 and ZT12-ZT24 (ZT denotes Zeitgeber time) indicate light and dark periods under LD (Light/Dark), respectively.

Data information: For sleep deprivation experiments at 25°C, sleep measurement was performed at 25°C until the end of the experiment. Baseline sleep was measured for 2 days prior to sleep deprivation. Sleep deprivation was achieved by loading flies in DAMs monitors into the shaker fitted with a custom base. Flies were vortexed from ZT12 (Zeitgeber time 12: beginning of the dark phase) to ZT24 (Zeitgeber time 24: beginning of the light phase) at the lower intensity setting for 3 sec/min. Zeitgeber time is used to indicate the phase of the circadian rhythm in relation to a specific Zeitgeber. The 24-hour cycle is divided into 24 Zeitgeber times, where ZT0 represents the start of the light phase, and ZT12 corresponds to the start of the dark phase. The day time increase of total sleep amount (min) after sleep deprivation was calculated by subtracting the baseline daytime sleep amount of each repeat from the corresponding daytime sleep amount after the sleep deprivation. The night time decrease of total sleep amount (min) after sleep deprivation was calculated by subtracting the baseline night time sleep amount of each repeat by the corresponding night time sleep amount after the sleep deprivation. Sleep recovery (%) was calculated by (day time increase of total sleep amount) / (night time decrease of total sleep amount) (A). Sleep recovery (min) was calculated by (daytime sleep amount after sleep deprivation - baseline daytime sleep amount before sleep deprivation) (B). 

Source_data_for_fig._15.xlsx

Fig. 15 Gene intersection size of the Pc targets identified by TaDa and ChIP.

Gene intersection size of Pc targets identified by RNAseq and TaDa (TaDa is based on DAM-ID, a technology designed to detect genome-wide binding sites of proteins in vivo. We identified Pc binding profiles using the Tada assay) in EB1-Gal4 and R69F08-Gal4 marked cells comparing with ChIPseq data, which was reported in the Schwartz et al., 2006. ChIP (Chromatin Immunoprecipitation) is an experimental method used to study protein-DNA interactions. 

Source_data_for_fig._S1.xlsx

Fig. S1 Effect of Pc knockdown on the survival of EB1-Gal4 and R69F08-Gal4 expressing cells.

EB1-Gal4 expressing cells after induce the Pc RNAi in the same condition with control group in 3-5-day-old flies. Cells expressing RFP denote red fluorescent protein. The calculation of EB1-Gal4 expressing cells number (The number of EB neurons) (Fig. S1G). R69F08-Gal4 expressing cells after induce the Pc RNAi in the same condition with control group by imaging. Cells expressing RFP denote red fluorescent protein. The calculation of R69F08-Gal4 expressing cells number (The number of EB neurons) (Fig. S1K).

Figures are stored in Zenodo at https://doi.org/10.5281/zenodo.17038126

Code/software

No original code was produced; the software used is described in the Methods section above.

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Data was derived from the following sources:

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