Distribution of DAxud1 across the genome
According to results from previous reports, vertebrate DAxud1 orthologs possess transcription factor-like features, including DNA binding, acidic and transcription transactivation domains [20, 21, 28]. They have also been detected bound to specific promoters related with neural crest differentiation . Further, vertebrate and invertebrate orthologs of DAxud1 are related to the stress response and cancer through induction of apoptosis [21, 23], although, until now, this process has not been linked to its putative transcriptional function. Considering this information, we asked which groups or categories of genes are transcriptionally regulated and/or bind DAxud1 in Drosophila, using this information as a proxy to get a better perspective on the function of dAxud1. For this purpose, we performed a TaDa-Seq (DamID-seq) experiment using the method described by Southall et al. , expressing the Dam-DAxud1 fusion protein to explore the loci in which DAxud1 could reside. This was performed in imaginal wing disc tissue using the nub>Gal4 driver. Two replicates were generated per condition (third instar larvae imaginal wing disc, with Dam as a control or Dam-DAxud1). The aligned sequences were analyzed with the findpeaks script, generated by Marshall Owen . Using this method, we identified 1811 significant peaks across the Drosophila genome, representing zones where Dam-DAxud1 has a stronger, more stable positioning than the Dam signal. As shown in Fig. 1A, the Dam-DAxud1 distribution was mainly intronic, with 52.3% of peaks localizing to these gene regions. The first intron accounted for more than half of all instances of intron localization, representing 25.7% of overall genome occupancy. The other main significant locations were the intergenic and the proximal promoter regions (-1000 to +1). These regions are also the most enriched in transcription factor binding sites [31, 32]. To better understand the distribution of DAxud1 across the genome and in specific genes, we used the information from significant peaks to create a metagene profile, using gene bodies as a reference scaled to 1000 bp. The resulting metagene profile (Fig. 1B) shows a robust average signal at the promoter zone and a maximum within the 5’-half of gene bodies, confirming the previously described distribution (Fig. 1A) and suggesting a role in activating or poised gene transcription.
Several reports describe DAxud1 vertebrate orthologs as stress response genes [24, 26, 33, 34]. Therefore, we searched for significant DAxud1 binding on stress response gene loci (Additional Table S1) and found heat shock response genes as recurrent targets. Further, we generated alignment charts to visualize the distribution of peaks within this group of genes (Fig. 1C). These graphics clearly reveal that the distribution of Dam-DAxud1 specifically on hsp genes has a highly similar pattern to that observed in the metagene profile.
The intergenic, promoter, and gene-body (intron and exon) peaks were sorted and annotated for nearby genes using HOMER . Additional Table S2 shows the most relevant enrichment analysis results. Within the “biological processes” category, the two major enriched groups are related to neural development, but no groups show a significative score for stress response or tolerance. One pathway category identified involves Wnt signaling, concordant with reports that DAxud1 orthologs in mammals are linked to this signaling pathway [19, 36, 37] and apoptosis [21, 38].
Potential DAxud1 DNA binding motifs
After having found genes in which Dam-DAxud1 is enriched, we searched for recurrent binding motifs within the Dam-DAxud1 peaks. The sequence peaks had a size between 50-5000 bp and were extracted for motif find analysis from HOMER . As shown in Fig. 1D, the most recurrent DNA motif (TACATACATA), present in 1019 sequence peaks out of 1811, is novel compared with a previous report on Axud1 vertebrate orthologs , possibly due to the wide-range of sequences that the TaDa-seq experiment provides [40–42], but this information could provide insights from the chromatin context surrounding DAxud1. Using the matrix sequence, we conducted another analysis using this data and the GOMO platform, which provides information about the gene ontology (GO) of the promoter regions, using a range between -1000 and +200 bp from the TSS for each gene in the Drosophila melanogaster genome. For the TACATACATA motif, the most related GO found is “Heat shock mediated polytene chromosome puffing” (GO:0035080). This motif also found a match in the Drosophila Topoisomerase 2 gene, which encodes a protein that removes supercoils in chromatin to facilitate transcription, with an essential role in RNA polymerase pausing-release in hsps genes in the fast stress response [43, 44]. These analyses suggest that DAxud1 might play a role in the transcription of heat shock and other stress-induced genes. To confirm this last hypothesis, we performed chromatin immunoprecipitation on the hsp70 promoter with DAxud1-GFP as bait, followed by a CHIP-PCR with primers flanking the TACATACATA motif. The result confirms the presence of DAxud1 at this locus (Additional Fig. S9), reinforcing the idea that it regulates the stress response by modulating hsp gene expression and also that the regions discovered using TaDa-seq are bona fide DAxud1 binding sites representing potentially functional interactions.
DAxud1 regulates thermotolerance and lifespan in Drosophila
Our results reveal the presence of DAxud1 bound to hsp genes (Fig. 1; Additional Fig. S9), suggesting a possible role in stress tolerance, a hypothesis supported by studies on Axud1 orthologs in mammals [23, 45]. Although “stress response” is not the major GO category of DAxud1 genome occupancy (Additional Table S2), we tested whether flies with a global knockdown of this gene exhibit an altered stress response or aberrant hsps expression, given its presence in this class of genes. For this, we used flies expressing a DAxud1 RNAi construct (Vienna stock 26479, UAS-IR-DAxud1) in all tissues using the Tubulin-Gal4 driver. Flies overexpressing DAxud1 cannot be assessed in this type of experiment since ubiquitous expression of this protein is lethal . We established adult flies at 29°C for an optimal expression of the UAS RNAi construct; then, control and experimental adult flies were exposed to a heat shock of 37°C for a half-hour every day as the thermal stress condition, and their survival was measured. Knockdown of DAxud1 leads to a diminished lifespan of adult flies compared to the control genotype under the same stress condition (Fig. 2A-2B). Curiously, under control conditions (no heat shock), DAxud1 knockdown animals have a longer lifespan compared to control animals for both males and females (Figs. 2A and 2B). To confirm whether there is a fluctuation in hsp gene expression, and to relate this to the observed phenotypes, we performed qPCR analysis for different hsp genes in imaginal wing discs and salivary glands, expressing the RNAi construct or DAxud1-GFP version using the nubbin-Gal4 driver. This allowed us to study the effects of diminished DAxud1 levels in salivary glands and imaginal wing discs, tissues that have cells in an endo replication state (salivary glands) and in a mitotic state (imaginal wing discs). Figs. 2C, 2D, and 2E show the qPCR results for three hsp genes (hsp70, hsp26, hsp67). In these experiments, we can appreciate that DAxud1 knockdown increases the expression of hsp genes in control conditions, but does not potentiate the transcriptional heat shock response in either tissue. Notably, the rise of hsp mRNA expression at the control temperature can explain the longer lifespan since there is evidence that hsp overexpression generates this effect . On the other hand, DAxud1 overexpression reduces hsp basal expression at the control temperature, perhaps not inhibiting the hsp stress response but generating a milder induction (Figs. 2C-E). These results, especially those observed after DAxud1 knockdown, are in conflict with our first hypothesis in which DAxud1 could function as a transcription factor that facilitates hsp expression. Therefore, we then set out to further clarify the role of DAxud1 on the hsp70 locus.
DAxud1 exhibits a widespread presence across the genome, whereas heat shock induces its recruitment to the hsp70 locus
Further examination of the results of hsp gene expression in DAxud1 knockdown conditions (Fig. 2), suggests that DAxud1 may participate directly in hsp transcription, possibly in a repressive manner. Previous studies have reported that DAxud1 orthologs (CSRNP in mouse) have transcription factor features, including a C-terminal trans-activating domain and a DNA binding motif . However, there is no evidence indicating which type of regulatory factor it is, and its dynamics within the nuclear structure. Drosophila melanogaster’s polytene chromosomes represent a suitable model to answer these questions.
As there is no available antibody against DAxud1, we used the GFP-tagged version of DAxud1  (DAxud1-GFP) to perform immunofluorescence using an anti-GFP antibody. RNA Polymerase IIo (Hyper-phosphorylated) was used as a positive control for a chromosome attached protein and for detecting transcriptionally active loci. As seen in Fig. 3A, DAxud1-GFP on the polytene chromosome exhibits a pattern with widespread RNA Pol IIo co-distribution, which means the specific function on hsp genes may be distinct from other functions of DAxud1 in gene expression. The hsp70 locus (Fig. 3B) has been extensively analyzed with regards to its chromatin rearrangements under stress conditions. Its documented that during stress conditions such as heat shock, cytological zones known as chromatin puffs appear in hsp70 region, clearly visible with confocal microscopy, a reflection of chromatin opening for transcription and transcription factor recruitment [48, 49]. We evaluated chromatin and DAxud1-GFP dynamics on this locus (at both 17°C and 37°C, Fig. 3C) and observed that DAxud1-GFP localizes to the 87A-87B locus together with RNA Polymerase IIo after heat shock treatment. Transcription factors that relocate in this way are identified mostly as activators, for instance HSF, p-TEFb , and DSIF . From these results, we reasoned that DAxud1 is a chromatin element that relocates to the hsp70 locus during heat stress, but it does not interfere with the transcriptional induction since hsps mRNA levels rise by the same magnitude as in control, under a DAxud1 overexpression condition (Fig. 2C-E).
To confirm these results, we performed chromatin immunoprecipitation for DAxud1-GFP (Fig. 3D) to detect the presence of this factor on the hsp70 paralogs hsp70Ab and hsp70Bb/Bc, or loci 87A and 87C, respectively. According to those results, DAxud1-GFP has a clear presence on hsp70 promoters as do other heat shock transcription factors . Interestingly, DAxud1-GFP did not exhibit enrichment after oxidative stress induction by peroxide (Fig. 3D), a condition that increases hsp mRNA levels , indicating that the involvement of DAxud1 in the heat shock response is different to the mechanism evoked during oxidative stress.
Together, the data obtained here, have described the genome-wide distribution of DAxud1, its influence on hsps transcription, and its effects on lifespan. However, how does DAxud1 exert its function and what is its relevance for cell physiology? In the hsp70 gene, there is a special chromatin configuration, known as the pausing complex, in which the activated RNA Polymerase II pauses the transcription process at positions +30 to +50 bp of the gene, and remains stalled as a “poised polymerase” . This poised polymerase forms a complex with two main components, NELF and DSIF, whose role is to maintain the polymerase in the paused state. Transcription resumes when the p-TEFb complex phosphorylates NELF and DSIF, releasing the active polymerase for resumption of elongation . hsp70 genes have been used as transcriptional pausing models because the resumption of transcription occurs quickly when heat shock or stress signals reach the cell . Considering the data on the position and behavior of DAxud1 on the hsp70 gene after heat shock and that elements of the pausing complex on hsp70 exhibit a similar pattern of reorganization on chromatin during heat shock [4, 18], we conjectured that DAxud1 might interact with the pausing complex.
This hypothesis is also supported by RNA-seq evidence (Additional Figs. S3 and S4), in which DAxud1 overexpression increases hsp70 RNA synthesis only at the 5’ end of the gene, with an incomplete synthesis of hsp70 mRNAs, specifically on hsp70B paralogs, which display high reads only between +1 and +100 region. This effect was not detected in qPCRs performed in Figs. 2C-E because the primers were designed to detect the 3’ side. In addition, this role in pausing is also supported by the evidence of double-hybrid experiments with Drosophila proteins, which demonstrated a physical interaction between DAxud1 and Spt5 , a member of the DSIF complex, with a pivotal role in the RNA Polymerase transcriptional pausing and elongation complex . Notably, the DAxud1 overexpression wing phenotype is reversed in a heterozygous spt5 mutant background (Additional Fig. S6). Another component of the pausing machinery is the NELF complex (NELF-A, NELF-B, NELF-C/D, NELF-E), a stabilizer factor of the stalled polymerase in basal conditions. These components dissociate from the polymerase when transcription is resumed after heat shock , and the loss of function of some of its components generates higher levels of hsp70 , which is similar to our observations in the case of DAxud1 knockdown (Fig. 2C). Considering this information, we tested whether DAxud1 influences RNA Polymerase II location within the hsp70 gene body . Chromatin immunoprecipitation (CHIP) for RNA Pol II was performed under different DAxud1 levels of expression (Fig. 4A). In the RNA Pol II CHIP, we find that, in the case of DAxud1 knockdown, its levels decrease uniformly in the hsp70 gene locus, and there is no change in the 5’/3’ rate of RNA Pol II compared to the control and heat shock conditions (Fig. 4B). On the other hand, DAxud1 overexpression generates an increase of this ratio, more so than in the control condition, due to an increase of RNA Pol II on 5’ end of the hsp70 gene body (Fig 4A).
To find out whether DAxud1 interacts with pausing factors, we performed a co-immunoprecipitation assay in imaginal wing disc expressing NELF-B-HA and DAxud1-GFP. In this assay (Fig. 4C), a fraction of NELF-B-HA was co-immunoprecipitated with DAxud1-GFP, indicating a physical interaction between these two proteins, suggesting they could act in the same process in hsp70 expression.
The information provided by the physical and genetic interaction of DAxud1 with NELF-B and Spt5, respectively, strongly suggests that it could be influencing RNA Polymerase II dynamics on the hsp70 gene. To confirm this, we performed a CHIP for RNA Polymerase on the 5’ and 3’ regions of the hsp70 gene, under DAxud1 overexpression and knockdown conditions. The results, presented in Fig. 4, reveal that DAxud1 overexpression generates an apparent pausing effect on RNA Polymerase II distribution (Fig. 4), coincident with extensive upregulation of the 5’ RNA levels of hsp70 (Additional Figs. S3 and S4).
Promoter-proximal pausing in hsp genes allows a rapid expression after heat shock induction due to pausing elements as DSIF/NELF complexes. Also, this rapid expression has a shut-off mechanism to restore hsp70 mRNA levels after the heat shock condition is reverted (heat shock recovery), and there is evidence that supports the role of NELF complex in the shut-off mechanism . We tested the physical interaction between NELF-B and DAxud1 (Fig. 4C) as well as its genetic interaction (Additional Fig. S7), allowing us to further clarify the role of DAxud1 in hsp70 shut-down levels during heat shock recovery. To evaluate the effect of DAxud1 in this process, we performed qPCR for hsp70 mRNA 40’ after a 20’ heat shock (37°), as shown in Fig. 4D, in DAxud1 knockdown salivary glands. It is possible to observe that hsp70 induction has an apparently slower turn-over than in control animals (Fig. 4D), suggesting that DAxud1 plays a role in shutting-down hsp70 locus induction, and confirming DAxud1 has a synergistic function with NELF-B in the transcriptional pausing of hsp70 as well as the complex stabilization for further heat shock stimulus.