Deinococcus radiodurans after exposure to radiation or prolonged desiccation. It was shown that the DdrO and IrrE proteins play a major role in regulating the expression of approximately twenty genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. At present, many questions remain, such as the number of genes regulated by DdrO. Here, we present the first ChIP-seq analysis performed at the genome level in Deinococcus species coupled with RNA-seq, which was achieved in the presence or not of DdrO. We also resequenced our laboratory stock strain of D. radiodurans R1 ATCC 13939 to obtain an accurate reference for read alignments and gene expression quantifications. We highlighted genes that are directly under the control of this transcriptional repressor and showed that the DdrO regulon in D. radiodurans includes numerous other genes than those previously described, including DNA and RNA metabolism proteins. These results thus pave the way to better understand the radioresistance pathways encoded by this bacterium and to compare the stress-induced responses mediated by this pair of proteins in diverse bacteria.Numerous genes are overexpressed in the radioresistant bacterium  Deinococcus radiodurans is one of the most resistant bacteria to genotoxic agent exposure and desiccation isolated to date [D. radiodurans is able to reconstruct an intact genome in a few hours from several hundred DNA fragments [D. radiodurans, including efficient DNA repair mechanisms [ to date ,2,3,4. Uragments . Many fachanisms ,6,7,8, achanisms ,10, and chanisms . Thus, tD. radiodurans to γ-rays, or its recovery from desiccation, results in a rapid upregulation of the expression of numerous genes [D. radiodurans encodes two LexA homologs (LexA1 and LexA2) that undergo, as in E. coli, a RecA-dependent cleavage after DNA damage, but neither LexA1 nor LexA2 are involved in the induced expression of RecA [Deinococcus, the main response pathway to genotoxic conditions is regulated by the constitutively expressed metalloprotease IrrE [The exposure of us genes ,13, evenus genes ). D. rad of RecA ,16. In Dase IrrE ,18 and tase IrrE ,20. In vase IrrE ,23,24,25ase IrrE ,25,26, aase IrrE . Recent ase IrrE  and (ii)The DdrO protein is composed of two domains: an N-terminal helix-turn-helix (HTH) XRE DNA-binding domain, itself associated with a specific structural domain at the C-terminus required for protein dimerization and for DNA binding ,29. In vddrO gene is essential for cell viability of D. radiodurans and Deinococcus deserti [D. radiodurans, an apoptotic-like response  [D. radiodurans, these two responses are mediated by common regulators, IrrE and DdrO [The  deserti ,20. InteDeinococcus species, and genes encoding IrrE/DdrO-like proteins are also present in other bacteria [Deinococcus species, suggesting the existence of a conserved radiation/desiccation response (RDR) regulon [Deinococcus species consists of at least 14–24 genes, including numerous genes involved in DNA metabolism, such as recA, ssb, gyrA, gyrB, uvrA, and uvrB, but also Deinococcus-specific genes, such as ddrA, ddrB, ddrC, ddrD, and pprA [D. radiodurans: 24 genes by Makarova et al. 2007 [ddrC [D. radiodurans DdrO was able to bind to 21 predicted RDRM motifs [The IrrE/DdrO protein pair is highly conserved in bacteria ,31. Howe regulon ,32,33. Tand pprA . Based o07 [ddrC . It has M motifs  and in vM motifs .D. radiodurans using two genome-scale approaches, i.e., ChIP-seq and RNA-seq analyses. These approaches were performed to identify additional DdrO target sites that were not predicted by previous in silico analyses. To our knowledge, we present here the first ChIP-seq analysis performed at the genome level in D. radiodurans. Alignments of DNA sequences extracted from ChIP-seq analysis were also performed to compare the consensus motif found in the predicted RDRM. As a prerequisite to a robust feature-mapping study, we resequenced our laboratory stock strain of D. radiodurans R1 ATCC 13939 to obtain an accurate reference for read alignments and gene expression quantifications.In this study, we mapped the DdrO regulon in D. radiodurans is more complex than previously thought and is composed of at least 35 genes, including genes encoding DNA and RNA metabolism proteins, such as RecG and HelD helicases, and the prokaryotic replicative DNA ligase LigA, but also new genes associated with different metabolic pathways, involved in the translation or encoding of proteins of unknown function.Our results show that the RDR regulon in E. coli strain DH5α was used as the general cloning host, and strain SCS110 was used to propagate plasmids prior to transformation of D. radiodurans [D. radiodurans strains were derivatives of the wild-type strain R1 ATCC 13939. Transformation of D. radiodurans with PCR products, genomic or plasmid DNA was performed as previously described [D. radiodurans was extracted using the NucleoSpin DNA Microbial Mini kit . D. radiodurans genomic DNA used to sequence the genome with Nanopore technologies was prepared by a lysis procedure involving a pretreatment of the cells with saturated-butanol in EDTA [Bacterial strains and plasmids are listed in iodurans . All D. escribed . Strainsescribed . The gen in EDTA . PCR ampD. radiodurans strains and derivatives were grown at 30 or 37 °C in TGY2X , or plated on TGY1X containing 1.5% agar, and E. coli strains were grown at 37 °C in Lysogeny Broth. When necessary, media were supplemented with the appropriate antibiotics used at the following final concentrations: kanamycin, 6 μg/mL; chloramphenicol, 3.5 μg/mL; hygromycin 100 μg/mL; spectinomycin, 90 μg/mL for D. radiodurans; chloramphenicol, 25 μg/mL; spectinomycin 50 μg/mL for E. coli.D. radiodurans genomic DNA from strain R1 ATCC 13939 (laboratory stock) was sequenced using Illumina NextSeq v. NS500446 , yielding 10.7 × 106 75 nt paired-end reads. This dataset was subsequently assembled with SPAdes (v3.13.1) St Petersburg University, Russia [Purified , Russia , with thD. radiodurans genomic DNA was also sequenced with Oxford Nanopore Technologies GridION (v. GXB02022–19.12.6) , yielding a total 10.87 × 109 nucleotites in 946,434 long reads (median size: 6857 nt). This reads pool was further filtered with filtlong (v. 0.2.0) (https://github.com/rrwick/Filtlong) (accessed on 10 September 2021), retaining only reads longer than 2000 nt, aligned to reference Illumina reads (see above) and totaling ~5 × 109 nt. This reads dataset was used as an input to Canu (v1.8) [In parallel, u (v1.8)  for readD. radiodurans R1 strain (GCA_000008565.1 and GCA_001638825.1). Structural RNAs were mapped on the genomic sequences with the same reference genomes using the BLASTN tool.These latter sequences were improved by mapping SPAdes-produced high quality contigs on them with BWA mem (v. 0.7.9a-r786) . SPAdes D. radiodurans R1 strain (GCA_000008565.1 and GCA_001638825.1). Structural RNAs were mapped on the genomic sequences with the same reference genomes using the BLASTN tool.CDS prediction was performed on the final assembled sequences using Prodigal (v. 2.6.3) using single mode, translation Table 11  as settiE. coli/D. radiodurans) code for a spectinomycin resistance gene, in addition to a repU gene encoding the RepU protein essential for plasmid replication in D. radiodurans. The p17235 and p17236 plasmids contain the wild-type repU gene or a TsrepU gene encoding a thermosensitive protein (RepUTs), respectively. To construct the p17238 plasmid used for conditional expression of ddrO, the native genomic ddrO gene was amplified by PCR using the NE28-NE29 primers and the PCR product was cloned into the p17236 plasmid between the BamHI/NotI sites. The same strategy was used to construct the p17237 plasmid. Following transformation of D. radiodurans strain GY14125 (non-homogenotized ΔddrOΩcat) with p17236 or p17237 plasmids, both expressing ddrO gene, the transformants were streaked several times on plates supplemented with chloramphenicol and spectinomycin, and the complete deletion from all chromosome copies of native D. radiodurans ddrO was analyzed by diagnostic PCR.The p17235 and p17236 shuttle vectors  replication, were grown at a permissive temperature (30 °C) with spectinomycin and chloramphenicol. Cells were diluted in fresh medium with antibiotics and grown at permissive temperature (30 °C) and cells in exponential growth (A650nm~0.5) were harvested by centrifugation, washed two times with TGY2X and reused to A650nm = 0.1 in fresh medium without antibiotics. Then, the temperature was shifted to 37 °C (non-permissive temperature for the thermosensitive replication plasmid). At 1, 4, 6, 8, 16, and 24 h, aliquots of 20 mL were removed for fluorescence microscopy and transcriptome analysis or Western blot analysis.ΔcDNA samples were sequenced using Illumina NextSeq v. NS500446 , yielding, on average, 22.8 × 106 50 nt. paired-end reads (±6.8 × 106 reads).For each aliquot, total RNA was isolated using the Fast RNA Pro Blue Kit  and the FastPrep-24 instrument, according to the manufacturer’s protocols. Extracted RNA was rigorously treated with TURBO DNA-free , according to the manufacturer’s instructions and the absence of DNA genomic contamination was checked by quantitative PCR (qPCR). The quality and quantity of treated RNA were analyzed using a DeNovix DS-11 spectrophotometer  and the Bioanalyzer 2100 system  with an RNA integrity number ≥6 for cDNA library preparation. The rRNA depletion and Illumina libraries were made following the Illumina protocol . The Read sequences were mapped on our reference genome sequence with BWA mem (v. 0.7.9a-r786) using default settings, and coverage values of all genomic features were computed with the bedtools “coverage” command (v2.17.0) . RNA difThe protein extractions and Western blot analyses were performed as previously described . The memD. radiodurans cells  expressing DdrO-V5 or native DdrO protein were crosslinked with 1% formaldehyde in TGY2X medium for 25 min at 30 °C with continuous shaking. Crosslinking reactions were quenched by the addition of 125 mM glycine for 15 min. Cells were harvested by centrifugation , washed twice with cold Tris Buffer Solution , and then resuspended in 3 mL of lysis buffer ). Cells were disrupted and DNA sheared using a One Shot Cell Disruptor (CellD SARL) to an average size of 100–300 bp (2 rounds of 2.4 kbar). Insoluble material was removed by centrifugation at 20,000× g for 10 min at 4 °C and the supernatant was collected in a sterile microcentrifuge tube. Then, 500 µL of supernatant fluid was added to 25 µL of pre-incubated protein G magnetic beads  with 5 µg of anti-V5 rabbit polyclonal antibody  in IP buffer . After overnight incubation at 4 °C with rotation, the immuno-precipitates were washed 5 times with washing buffers .Exponentially growing Immune complexes were eluted in 200 µL of elution buffer. The eluted samples (20 µL) were saved for control Western blots, and the remainder was incubated for 2 h at 37 °C with shaking with 100 µg/mL Proteinase K. Then, the supernatant was incubated overnight at 65 °C to reverse crosslinking with 100 µg/mL RNAse A. The DNA was purified using the PCR Clean-up kit . Three independent ChIP experiments were performed for “IP” samples.6 sequences, respectively, in addition to the “Mock” (19 × 106 sequences) and the “Input” (13 × 106 sequences) samples. The quality score was verified with FASTQC software (https://www.bioinformatics.babraham.ac.uk/projects/fastqc) (accessed on 10 September 2021) and Illumina adaptor sequences were removed with Cutadapt software (https://cutadapt.readthedocs.io/en/stable) (accessed on 10 September 2021). Sequence alignments on the genomic sequence were performed with Bowtie2 software (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) (accessed on 10 September 2021). Output SAM files were converted and indexed into BAM files, using the Samtools software (http://www.htslib.org) (accessed on 10 September 2021). They were used both for visualization with IGV, and additional conversion into BED files with Bedtools software (https://bedtools.readthedocs.io/en/latest/index.html) (accessed on 10 September 2021) providing the input file format required by bPeaks programs (Available online: https://cran.r-project.org/web/packages/bPeaks/index.html) (accessed on 10 September 2021) to perform peak calling. Searches for conserved motifs were performed by MEME and FIMO (https://meme-suite.org/meme) (accessed on 10 September 2021) with a Match p-value < 1.0 × 10−4. Prediction of E. coli-like gene promoter elements and transcription start sites in gene promoters was carried out using BPROM (http://www.fruitfly.org/seq_tools/promoter.html) (accessed on 10 September 2021) [Raw FASTQ files were obtained from sequencing three “IP” samples comprising 19, 7, and 3 × 10er 2021) . To sort650nm = 0.3), grown at 30 °C, were exposed to 1 or 5 µg/mL mitomycin C. After 3 h at 30 °C with continuous shaking, cells were harvested by centrifugation at 4 °C and the pellets washed with 1X cold saline-sodium citrate (SSC) buffer. Then, the bacteria pellets were re-suspended in 150 µL of SSC 1X with 0.4 mM protease inhibitor cocktail (Roche) and cells disrupted with a FastPrep Instrument using 0.1 g of glass beads (500 µm) and four pulses of 30 s. Cell debris were removed by centrifugation at 20,000× g for 10 min at 4 °C, and the supernatant fluid collected and placed in sterile microcentrifuge tubes. Protein concentrations were determined by Bradford assay (Bio-Rad). Proteins were subjected to electrophoresis through a 12% Glycine SDS-PAGE gel  and transferred onto a polyvinylidene difluoride (PVDF) membrane . All these experiments were performed three times as previously described [Exponentially growing bacteria  with mitomycin C , exposed (or not) to UVC at a dose rate of 3.5 J/m2/s.Bacteria were grown in TGY2X liquid medium at 30 °C to an AThe complete sequence and annotation of the genome were deposited with GenBank under accession numbers CP068791, CP068792, CP068793, and CP068794. The complete high-throughput sequence data were deposited with the Gene Expression Omnibus (GEO) data bank under accession number GSE175875 (RNA-seq and ChIP-seq).D. radiodurans strain R1 are available in databases [D. radiodurans genome of strain R1 ATCC 13939 maintained in our laboratory. We opted for Illumina NextSeq Oxford sequencing coupled with Nanopore Technologies GridION to unambiguously locate the repeated elements that may misassemble short sequences in size. Merging both sets of sequences produced an ensemble of four high quality contigs, totaling 3,578,820 nt, with a 450-fold average coverage. Among the 3230 predicted genes, 3147 encode proteins.Two genome sequences of the atabases ,49. The D. radiodurans deduced from our sequence, are closer to those published by White et al. [As shown in e et al.  than thee et al. . The lare et al.  containse et al. , with a e et al. . In addiddrO , the Mock sample (immuno-precipitated (IP) DNA of the wild-type strain), and three replicates of the DdrO-V5 IP sample were used to prepare sequencing libraries. The DNA regions over-represented in the DdrO-V5 IP sample and corresponding to potential binding sites for DdrO-V5 were identified using the bPeaks program [ program .ddrA, ddrB, ddrC, gyrA, and gyrB  within intergenic regions of genes encoding proteins , whereasand gyrB , were idand gyrB  [21,26,3mutL, and small peaks were observed in the promoter regions of recQ and sbcD that fell below the threshold used for peak detection with the bPeaks program. No peaks were detected in the region upstream of the hutU, irrI, frnE, and rsr genes. To identify candidate binding sites of the DdrO protein, the nucleotide sequences of the ChIP peaks, between 151 and 1401 in length, were compared using MEME [D. radiodurans genome. Independently, we investigated whether an RDRM was found in the promoter regions of other genes encoded by D. radiodurans. We monitored, with FIMO, their presence in a set of sequences covering the regions located between −500 and +100 nucleotides from the start of translation of all the 3147 CDS encoded by D. radiodurans. A total of 222 putative RDRM-like sequences  replication vector  and ∆ddrO/ddrO+ (prepU+) strains grown at 37 °C are denoted D37 and W37, respectively. Under our experimental conditions, the number of cells carrying the +repU vector is proportional to the increase in cell mass at 37 °C without a selective antibiotic  expressing DdrO-FLAG, PprA-HA, DdrD-HA, or RecA-HA tagged proteins. Depletion of the DdrO repressor, in cells grown at the non-permissive temperature, resulted in the complete loss of DdrO after 4 h at 37 °C  A. RNA sep-value < 0.01. Principal component analysis (PCA) confirmed that the transcriptome of the three biological replicates was clustered at each time point, showing the reproducibility of the experiments and the transcriptome patterns evolved as cells progressed through the time course of the experiment  was upregulated only after 16 h at 37 °C. The other genes, such as uvrD (dr1775) and irrI (dr0171), were not upregulated or only changed in a late stage of the experiment. In contrast, drA0151, encoding the first gene of the hut operon, was strongly downregulated in the W37 strain and in the D37 strain, but this gene was not reported as being under the control of the DdrO/IrrE proteins [Twenty genes of the RDR regulon were upregulated in the D37 strain, often from the beginning of the experimental temperature shift, with increasing fold changes as cells progressed through the time course. The proteins .D. radiodurans genes displayed a transcriptome pattern comparable to the RDR regulon genes. For this purpose, we selected genes as differentially expressed (DE) in the W37 strain in two or fewer comparisons (DE ≤ 2), and in more than three comparisons in the D37 strain (DE > 3), considering most of the profiles exhibited by the predicted RDR genes , polA, two putative helicases (DR0837 and DR1572), ligA, mutS, and recN , four genes associated with different metabolic pathways, five genes involved in translation, and seven new genes encoding proteins of unknown function  are present in the genome. According to the Chandler classification (https://www-is.biotoul.fr/index.php) (accessed on 10 September 2021) dr1296 encodes the ISDra5 and drC0017 is part of the transposable element TnDra1. Because drC0033 and dr1296 CDS exhibited 100% sequence identity, we were not able to determine, by RNA-seq, whether one of the two genes displayed an upregulation during the time course. Moreover, only a small peak was observed for drC0033 but was below the threshold established for this study.Based on these results, the DdrO regulon comprises 16 previously predicted RDR genes, mainly involved in DNA repair pathways. Moreover, three other genes involved in DNA metabolism  protein family. The deletion of dr1790 in D. radiodurans increased its membrane permeability and decreased the cell growth rate and survival upon exposure to hydrogen peroxide and radiation [In addition, 47 genes matched only two criteria. Among these, six genes were upregulated in D37 and a ChIP peak was located in each promoter region, but their sequences did not exhibit any RDRM or other conserved motif . Howeveradiation .dr0001 encoding DNA polymerase III subunit beta, were described as upregulated upon exposure to gamma rays [irrE mutant [uvrA and uvrD genes were also reported as being upregulated in the first 1.5 h when cells are exposed to large doses of gamma rays [irrE mutant. These results suggest that the expression of these genes, including uvrA and uvrD, may be augmented very early during the time course (<1 h) or are under the control of DdrO and other regulatory elements, and the simple depletion of DdrO did not modify their expression under our experimental procedures.Twenty-six other genes were only associated with a DdrO peak and also with an RDRM in their promotor region . From thmma rays , but wasE mutant . The uvrmma rays  and weresbcD, recQ, drC0012 encoding a putative transcriptional regulator, drC0033 (transposase), and dr1707 encoding DNA polymerase I).Finally, 16 genes were only found to be upregulated in D37 and not in W37, and an RDRM was detected near their respective promoter region, but no ChIP-Seq peak was reported from the ChIP-seq analysis . HoweverD. radiodurans chromatin may have locally impaired or decreased the efficiency of the ChIP experiments, and thus, these genes may also belong to the DdrO regulon. The location of the RDRM was also analyzed in the promoter region of all genes matching two or three of our criteria. The RDRM sequences were mainly located in the vicinity of the predicted position of E. coli-like −35 and −10 promoter consensus sequences (D. deserti was found both upstream (−50 bp) and downstream (+20 bp) of transcriptional start sites (TSS) potentially overlapping with the RNA polymerase binding site [A highly condensed structure of the equences . These ring site .irrE mutant after exposure to mitomycin C (MMC). For this purpose, we monitored, by Western blot analysis, the expression of C-terminal-tagged recombinant proteins. The cellular levels of five recombinant proteins  increased in wild-type cells in response to MMC, but remained constant in an ∆irrE mutant, thus corroborating our data  predicted as belonging to the RDR regulon in D. deserti [ddrR was not upregulated during our time course experiment and no peak was observed in its promoter region. The cellular levels of three recombinant proteins increased in response to MMC in wild-type cells, and also in a ∆irrE mutant , absent from the genome annotation published by Hua and Hua [dr1289, dr1572, and dr2069, encoding the RecG helicase, HelD superfamily I helicase IV, and the replicative DNA ligase LigA, respectively. In agreement with these data, transcriptome analysis of cells recovering from exposure to ionizing radiation showed no upregulation of expression of these three genes in a ∆irrE mutant compared to the wild-type strain [E. coli, RecG plays an important role in DNA repair, recombination, and replication [Bacillus subtilis or Mycobacterium smegmatis, is associated with transcriptional pathways [D. radiodurans, cells devoid of RecG exhibit a delay in growth and double strand break (DSB) repair kinetics, and a decrease in resistance to γ-irradiation and H2O2 [dr1572 mutant exhibited a greater sensitivity to H2O2, but no change in resistance to ionizing radiation and to MMC when compared to the wild-type strain [Up to 70% of the identified target genes were previously predicted to be part of the RDR regulon ,26,33. T and Hua , is idene strain . In E. clication , whereaspathways ,57. In Dand H2O2 ,59, where strain .ligA as a target gene is interesting, because DNA ligases are implicated in DNA repair and are essential in other fundamental processes within the cell [ligA promoter region should not completely repress gene expression, to thus ensure a minimum level of DNA ligase activity. However, in response to elevated amounts of DNA damage, and particularly to DSB, the basal level of LigA may not be sufficient for accurate Single Strand Annealing (SSA) [The identification of the cell . Ligase the cell . Therefong (SSA)  and Exteng (SSA) .ddrN and dr2255, encoding putative GNAT family acetyltransferases that may be involved in post-translational modification (PTM) pathways. The ∆irrE cells recovering from exposure to ionizing radiation exhibited no upregulation of ddrN expression compared to a wild-type strain [E. coli, acetylation of the chromosomal replication initiation protein DnaA leads to an arrest of DNA replication [M. tuberculosis alters the in vitro DNA-binding capacity of HU and the DNA structure, which may affect gene transcription and other protein–DNA interactions [D. radiodurans HU protein has been reported as a major actor of nucleoid compaction [D. radiodurans.We identified new DdrO target genes, e strain . PTMs ine strain . Acetylae strain . In E. clication . Moreoveractions . D. radimpaction ,66 and mdrC0017 and dr1287, encoding transposases belonging to the Tn3 family and to the ISDra5 (IS5 family), respectively, were identified as belonging to the DdrO regulon. Transposons are major actors of genome remodeling and play an important role to create diversity and to facilitate adaptation of the host to extreme environmental conditions. Insertion sequences are abundant in D. radiodurans and IS transposition is a major event in spontaneous, in addition to induced, mutagenesis [Dra2, ISDra5, ISDra3, ISDra4, and IS2621 belonging to different families  were transpositionally active in this organism under normal growth conditions and transposition was enhanced in cells recovering from DNA damage. Transposable element expression and movement are generally tightly regulated and different mechanisms control their gene expression. In E. coli, LexA protein represses expression of the Tn5 transposase gene [Surprisingly, two genes, agenesis . It has ase gene . Furtherdr2173 and the drA0165-drA0166 operon, which were strongly upregulated in response to a depletion of DdrO. These genes were not upregulated in irradiated ∆irrE cells [E. coli [D. radiodurans moeA or moeB, were not differentially expressed in D37 compared to W37. The role of these strongly upregulated unknown genes in response to DNA damage also remains to be discovered.We also identified several genes encoding proteins of unknown function, such as rE cells . Domains[E. coli  and was ddrC, ddrD, and pprA, was strongly upregulated at early times, whereas ssb, uvrD, and ddrF showed a late upregulation, suggesting that DdrO bound with more or less affinity to DNA according to the divergent RDRM sequences that may diverge. Therefore, after exposure of ionizing radiation, some genes would be upregulated earlier than others during cell recovery. It has been shown that, following extended DdrO-depletion, D. radiodurans cells were engaged in an apoptotic-like cell death (ALD) pathway leading to morphological alterations, such as larger cells, membrane blebbing, and DNA fragmentation [Caulobacter crescentus, the BapE endonuclease was reported to be involved in DNA fragmentation upon severe and extensive DNA damage [DdrO-structure and biochemicals data suggested that, in response to DNA damaging conditions, upregulation of the expression of genes of the RDR regulon is dependent on a dynamic balance between DdrO dimers bound to DNA and the IrrE-cleavable DdrO monomer forms ,28. Thusentation . In CaulA damage . BapE inA damage . InsteadD. radiodurans genome, we showed that only a small proportion of these are bound by DdrO. Based on the presence of the RDRM sequence and induction of expression in response to ionizing radiation, rsr, frnE, irrI, ddrR, and the hutU operon were previously described as part of the RDR regulon in D. radiodurans [hutU operon, rsR, frnE, and irrI is not under the control of DdrO, but also ddrR. However, we showed that the quantity of RsR, FrnE, and DdrR proteins is induced in response to exposure to MMC (ddrA, gyrA, or dr1572, were also described as being under the control of another major regulator, DdrI [D. radiodurans DNA damage response.Despite the identification of many promoter regions containing a putative RDRM sequence in the iodurans ,33. In aiodurans , we showe to MMC  in concoD. radiodurans genome encodes more than one hundred predicted transcriptional regulators, but few studies have been undertaken to identify the genes they may regulate. The RDR regulon of the radioresistant bacterium D. radiodurans, characterized here by integrative genomic analyses, paves the way for further studies to better depict the regulatory networks underlying the mechanisms that contribute to the extreme radiation tolerance of this fascinating bacterium.The


C. neoformans is the main causative agent of fungal meningitis that is responsible for about 15% of all HIV-related deaths. Although an obligate aerobic fungus, C. neoformans is well adapted to hypoxia conditions that the fungus could encounter in the host or the environment. Cryptococcus neoformans. Through genetic, proteomic, and biochemical analyses, we demonstrated that Pas2 directly interacts with another transcription factor, Rds2, in regulating cryptococcal hypoxic adaptation. The Pas2/Rds2 complex represents the key transcription regulator of metabolic flexibility. Its regulation of metabolism rewiring between respiration and fermentation is critical to our understanding of the cryptococcal response to low levels of oxygen.To aerobic organisms, low oxygen tension (hypoxia) presents a physiological challenge. To cope with such a challenge, metabolic pathways such as those used in energy production have to be adjusted. Many of such metabolic changes are orchestrated by the conserved hypoxia-inducible factors (HIFs) in higher eukaryotes. However, there are no HIF homologs in fungi or protists, and not much is known about conductors that direct hypoxic adaptation in lower eukaryotes. Here, we discovered that the transcription factor Pas2 controls the transcript levels of metabolic genes and consequently rewires metabolism for hypoxia adaptation in the human fungal pathogen  Thus, under hypoxia, HIFs increase glucose/carbon flux through glycolysis while minimizing input into the TCA cycle and oxidative phosphorylation.Oxygen, as the electron acceptor in the generation of ATP via aerobic respiration, is required for many essential biochemical reactions. Deprivation of oxygen (hypoxia) presents a physiological and pathophysiological challenge to aerobic organisms, humans and microbes alike. In mammals, cells adjust to hypoxia by rewiring the route of ATP production and redox balance, which is achieved mainly through regulation by hypoxia-inducible factors (HIFs) . Under hCandida albicans, hypoxia leads to the induction of genes in glycolysis and the repression of those in aerobic respiration  shunt, which bypasses two steps of the tricarboxylic acid (TCA) cycle  cycle . SimilarCA cycle . Althougnditions , 14, simrevisiae , Schizoses pombe , and C. albicans . Thus, lS. pombe and C. neoformans but absent in S. cerevisiae and C. albicans, while A. fumigatus contains the SREBP but not the SCAP homolog. In A. fumigatus, the SREBP SrbA regulates ergosterol biosynthesis, iron uptake, nitrate assimilation, and heme biosynthesis in response to hypoxia  binding protein (SREBP) transcription factors are known to be involved in hypoxic adaptation when the sterol level in the membrane drops in response to limited oxygen . SREBPs  hypoxia . The oth hypoxia . In C. nunctions , 14. Thetions 12–, 19.S. cerevisiae, the transcription regulators Gcr1 and Gcr2 are primarily responsible for the activation of the expression of the glycolytic genes when cells are cultured on fermentable carbon sources, while they are dispensable for growth on nonfermentable carbon sources  under normoxia and hypoxia. We first cultivated the fungus on solid yeast extract-peptone-dextrose (YPD) medium under normoxia overnight and then transferred the plates to a hypoxia chamber with the oxygen level controlled at 0.1% for 3 h. Cells that continued to be cultured under normoxia  served as controls. Cryptococcus cells were then harvested for RNA extraction and subsequent sequencing.As an obligate aerobe, P value of <0.05; log2 fold change [log2FC] greater than 1 or less than −1). In total, 356 genes were upregulated and 338 genes were downregulated in C. neoformans during growth under hypoxia relative to normoxia conditions (see CNAG_02083 (SIT2) (3.8-fold) and CNAG_06242 (CFT1) (3.6-fold) implicates an increased need for iron under hypoxia, in agreement with previously reported findings from Cryptococcus (CNAG_06063 (COX15) (2.0-fold) together with the heme binding flavohemoglobin-encoding gene CNAG_01464 (FHB1) (4-fold) were also upregulated under hypoxia  Growth of PAS2-associated strains under CoCl2 and hypoxia conditions. Download FIG S1, TIF file, 2.6 MB.PAS2 regulates cell growth under CoClnditions . (A) GroCopyright © 2021 Zhao and Lin.2021Zhao and Lin.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the SRE1 nearly abolished hypoxic growth , fermentation , gluconeogenesis (CNAG_04217), the PPP (CNAG_04025 and CNAG_06923), and the TCA cycle (CNAG_06374) were highly induced in response to hypoxia in the wild type but not the pas2Δ mutant , and a zinc finger domain . As expeΔ mutant , indicatΔ mutant , Pas2 haΔ mutant . To examant . Surprisingly, the fluorescence signal of the mCherry-tagged Pas2coreΔ protein was enriched in vacuoles instead of the nucleus  DNA binding motif present in HIFs. To test if the zinc finger binding domain is critical for the function of Pas2, we conducted site-directed mutation on the fifth conserved cysteine residue known to be critical for zinc binding activity and DNA binding activity  with mCherry-tagged Pas2 to identify its interacting partners under normoxia and hypoxia. Because Pas2 is always localized to the nucleus, we focused on the candidates that could potentially localize to the nucleus. Out of the 22 potential nuclear proteins pulled down by Pas2, 9 were normoxia specific and 10 were hypoxia specific, with 13 shared ones (CNAG_03902. Cir1 is the known regulator of iron homeostasis in C. neoformans (C. neoformans (CNAG_03902 encodes a zinc finger transcription factor that has not been studied in C. neoformans. It is homologous to Saccharomyces RDS2 (regulator of drug sensitivity), which is involved in the regulation of gluconeogenesis  . To detered ones . Functiored ones . We wereoformans , 41. CNAogenesis . However10.1128/mBio.03602-20.3FIG S32 conditions  and IDH2 (CNAG_07363) homologs, were upregulated under hypoxia in Cryptococcus  were also upregulated  yeast extract, 3% glucose, and supplements consisting of 225 mg/ml each of uracil, adenine, leucine, histidine, and lysine (600] of 3.0), and serially diluted. To analyze the growth on YES medium plus CoCl2, cell suspensions with the dilutions were spotted onto YES agar medium containing 0.6 mM CoCl2 and incubated at 30°C for 2 days. The hypoxic environment was maintained using a Biospherix C chamber with O2 levels controlled by a Pro-Ox controller and CO2 levels controlled by a Pro-CO2 controller . To test the ability of the strains to grow under hypoxia, serial dilutions of cells were spotted onto YPD agar medium and grown in the hypoxic chamber with 0.1% oxygen and 5% CO2 at 37°C for 2 days.The d lysine . Strains10.1128/mBio.03602-20.4TABLE S1Table S1, DOCX file, 0.02 MB.Key resources. Download Copyright © 2021 Zhao and Lin.2021Zhao and Lin.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the PAS2 open reading frame (ORF) in the rds2Δ and stp1Δ backgrounds, a deletion construct that contains approximately 1 kb of flanking sequences and the split dominant marker NEO and the constructs of CAS9 and guide RNAs (gRNAs) were introduced into the rds2Δ and stp1Δ recipient strains by electroporation as described previously . Nuclei were visualized by live staining with Hoechst or DAPI  stain after formaldehyde fixation as described previously .600 of 0.03. Two hundred microliters of the diluted cells was plated onto YPD plates and cultured overnight at 30°C under normoxia. Half of the plates were then transferred into the hypoxia chamber (0.1% O2 and 5% CO2 at 37°C), and half of them were transferred to the control conditions (20 to 21% O2 and 5% CO2 at 37°C). The cells were quickly sampled and snap-frozen with liquid nitrogen after 3 h. The total RNA was extracted using a PureLink RNA minikit (Life Technologies) according to the instructions of the manufacturer. Direct poly(A) RNA sequencing was performed by the Texas A&M AgriLife Genomics and Bioinformatics Service. Preliminary quality analysis of the raw FASTQ files was conducted using FastQC (For transcriptome analysis in response to hypoxia, cultures grown overnight in liquid YPD medium were diluted to an ODg FastQC , and trig FastQC  was usedg FastQC  was usedg FastQC , and theg FastQC  and GOplg FastQC .Cryptococcus strains according to a previously described method (Proteins were extracted from d method . AliquotPRJNA662775.The RNA-seq data have been deposited in the NCBI database under BioProject accession number


Helminth parasitism is a world-wide problem in livestock industries, with major impacts on health, welfare and productivity. The role of the gut microbiota in host-helminth interactions in ruminants has been extensively examined and the present study added to this body of knowledge by assessing the effects of resistance and susceptibility to helminth infection in the gastro-intestinal tract (GIT). Australian Sheep Breeding Values (ASBVs) for faecal egg count (FEC) were used to select the 10 highly helminth-susceptible (High-FEC) and 10 highly helminth-resistant (Low-FEC) sheep. FEC status was confirmed during the experiment. Using samples from the faeces and the lumen of the rumen, abomasum, duodenum, jejunum, ileum, caecum, and colon, DNA was extracted and used for 16 rRNA gene amplicon sequencing.Eubacterium, Oscillibacter, and Ruminococcus. Intersectoral-specialization zones were identified along the GIT, with the duodenum displaying major differences between the High-FEC and Low-FEC animals in values for alpha and beta diversity. After taking all samples into account and adjusting for GIT segment, the High-FEC and Low-FEC sheep differed significantly for four genera Butyrivibrio, Mycoplasma, Lachnoclostridium and Succiniclasticum. In the duodenum, the abundances of Aminipila, Lachnoclostridium and Mogibacterium differed significantly between the High-FEC and Low-FEC sheep. In the ileum, on the other hand, the genus Mycoplasma was significantly depleted in the Low-FEC group.The most frequent genera identified along the GIT were The gastro-intestinal microbial profile varies widely between helminth-resistant and helminth-susceptible sheep. Each GIT section appears to support a particular bacterial composition leading to inter-sectoral differences among the various microbial communities. The microbial populations were most rich and diverse in the duodenum of helminth-resistant sheep, comprising bacterial genera that generally ferment carbohydrates. This observation suggests that helminth-resistant sheep can reorganize the duodenal microbiome taxa which may restrict the development of parasites.The online version contains supplementary material available at 10.1186/s42523-022-00172-1. Teladorsagia circumcincta, found mainly in the abomasum, and Trichostrongylus colubriformis in the small intestine, particularly in the duodenum  can be obtained from Table S2.Additional file 5: Table S5. ANCOM test used to the identification of bacterial phyla and genera showing significant differences among segments of the gastrointestinal tract, and between samples collected from High- and Low-FEC sheep.Additional file 6: Table S6. Pairwise differences in beta diversity among GIT segments of High- and Low-FEC sheep.Additional file 7: Table S7. Results of centered log-ratio (CLR) transformation using Wilcoxon signed rank test for each bacteria taxa that differed significantly among different GIT segments from High- and Low-FEC sheep.


Tetrahymena thermophila a single orthologue of human RBBP4 and 7 proteins, RebL1, physically interacts with histone H4 and functions in multiple epigenetic regulatory pathways. Functional proteomics identified conserved functional links for Tetrahymena RebL1 protein as well as human RBBP4 and 7. We found that putative subunits of multiple chromatin-related complexes including CAF1, Hat1, Rpd3, and MuvB, co-purified with RebL1 during Tetrahymena growth and conjugation. Iterative proteomics analyses revealed that the cell cycle regulatory MuvB-complex in Tetrahymena is composed of at least five subunits including evolutionarily conserved Lin54, Lin9 and RebL1 proteins. Genome-wide analyses indicated that RebL1 and Lin54 (Anqa1) bind within genic and intergenic regions. Moreover, Anqa1 targets primarily promoter regions suggesting a role for Tetrahymena MuvB in transcription regulation. RebL1 depletion inhibited cellular growth and reduced the expression levels of Anqa1 and Lin9. Consistent with observations in glioblastoma tumors, RebL1 depletion suppressed DNA repair protein Rad51 in Tetrahymena, thus underscoring the evolutionarily conserved functions of RBBP4/7 proteins. Our results suggest the essentiality of RebL1 functions in multiple epigenetic regulatory complexes in which it impacts transcription regulation and cellular viability.Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) are two highly homologous human histone chaperones. They function in epigenetic regulation as subunits of multiple chromatin-related complexes and have been implicated in numerous cancers. Due to their overlapping functions, our understanding of RBBP4 and 7, particularly outside of Opisthokonts, has remained limited. Here, we report that in the ciliate protozoan  Saccharomyces cerevisiae an additional nuclear subunit called Hif1 (human NASP) also interacts with the HAT1-complex at roughly stoichiometric levels  and Drosophila melanogaster (p55) (Retinoblastoma-binding proteins 4 and 7 (RBBP4 and RBBP7) (Cac3 and Hat2 respectively in budding yeast), also known as Retinoblastoma Associated protein 48 (RbAp48) and 46 (RbAp46), respectively, are two highly homologous histone chaperones (∼90% sequence identity) with similar structures  that werer (p55) . RBBP4 aer (p55) ,24. For er (p55) , switch er (p55) , and poler (p55) . Furtherer (p55) ,30, and er (p55) . Within er (p55) ,32.Drosophila p55 (RBBP4) has been shown to be important for DREAM-mediated gene repression -complex . The mampression .It is increasingly recognized that epigenetic aberrations contribute to tumor initiation and development . As suchTetrahymena thermophila, a unicellular ciliate protozoan, is an excellent experimental system to study evolutionarily conserved chromatin-related processes, including gene expression and cell cycle regulation  and a somatic polyploid macronucleus (MAC), that are physically separated within the same cell , combined with previously reported RNA-seq data  of REBL1 significantly decreased cellular viability and altered the expression profile of some functionally critical genes, including RAD51. Overall, our results revealed a general role of RebL1 in multiple transcriptional regulatory pathways important for cell cycle and chromatin regulation in Tetrahymena.Here, we utilized functional proteomics and genomics approaches to characterize the single orthologue of human RBBP4 and RBBP7, RebL1, in seq data , implicaTetrahymena strains CU428 [Mpr/Mpr ] and B2086 [Mpr+/Mpr+ ] of inbreeding line B were acquired from the Tetrahymena Stock Center, Cornell University, Ithaca, NY (http://tetrahymena.vet.cornell.edu/). Cells cultured in 1× SPP were maintained axenically at 30°C as previously described  were coated with at least 5μg of the digested plasmid DNA. The DNA coated gold particles were introduced into the T. thermophila MAC using biolistic transformation with a PDS-1000/He Biolistic particle delivery system (Bio-Rad) (Epitope tagging vectors for histone escribed . Two sepBio-Rad) . The traWe cloned gateway-compatible entry clones for RBBP4 and RBBP7 into the pDEST pcDNA5/FRT/TO-eGFP vector according to the manufacturer's instructions. The cloned vectors were co-transfected into Flp-In T-REx 293 cells together with the pOG44 Flp recombinase expression plasmid. Cells were selected for FRT site-specific recombination into the genome with hygromycin  at 200 ug/mL. Expression of the gene of interest was induced by addition of doxycycline (1ug/mL) to the culture medium 24 hours before harvesting.Tetrahymena cells without tagged bait (i.e. empty cells) and HEK293 cells expressing GFP alone were used as controls in our analysis. Extensive washes between each sample (see details for each instrument type) were performed to minimize carry-over. The order of sample acquisition on the mass spectrometer was reversed for the second replicate to avoid systematic bias. On the LTQ mass spectrometer, a freshly made column was used for each sample as previously described ). For nuclease treatment, 500 units of Benzonase (Sigma E8263) was added and extracts were rotated on a Nutator for 30 min at 4°C. Whole cell extracts (WCE) were clarified by centrifugation at 16 000 × g for 30 min. The resulting soluble material was incubated with 50 μL of packed M2-agarose (Sigma) at 4°C for at least 2 hours. The M2-agarose was washed once with 10 mL IPP300 , twice with 5 mL of IP100 , and twice with 5 mL of IP100 without detergent . 500 uL of 0.5 M NH4OH was added to elute the proteins by rotating for 20 min at room temperature. Preparation of protein eluates for mass spectrometry acquisition is detailed in supplementary methods.For escribed ,70. Brie6 cells were grown independently in two batches representing two biological replicates. Cells were harvested after 24-hour induction of protein expression using doxycycline. HEK293 cell pellets were lysed in high-salt NP-40 lysis buffer  with three freeze–thaw cycles. The lysate was sonicated as described  via standard procedures. The desalted samples were analyzed with an LTQ-Orbitrap Velos mass spectrometer (ThermoFisher Scientific). Raw MS data were searched with Maxquant (v.1.6.6.0)   as descrhttps://blast.ncbi.nlm.nih.gov/Blast.cgi). SMART domain analysis (http://smart.embl-heidelberg.de/) of the predicted proteins was carried out. All MS files used in this study were deposited at MassIVE (http://massive.ucsd.edu). Additional details (including MassIVE accession numbers and FTP download links) can be found in Cytoscape  was useTetrahymena, as described previously (Tetrahymena genome (2014 annotation) using Bowtie 2 with default settings   . The occte.org/) . The clote.org/) .http://tfgd.ihb.ac.cn/)  (.ac.cn/)  and the eviously .2 during vegetative growth. The expression of the target gene was examined using Rt-qPCR analysis. To this end, we isolated total RNA from the RNAi-treated or WT Tetrahymena cells using TRIzol (Life Technologies) as per the supplier's instructions. The isolated RNA was treated with Deoxyribonuclease I . cDNA was prepared using iScript™ Reverse Transcription Supermix for RT-qPCR. qPCR was performed in technical triplicates using the cDNA prepared from three individual KD cell lines. The data were normalized to the expression levels of HTA3. Primers are indicated in RNAi vectors were constructed essentially as described . RebL1 tREBL1-RNAi treated lines in drops of media with and without 0.5 μg/mL CdCl2. After 2 days of growth, the approximate number of cells in each drop was determined and drops containing >1000 cells were considered as viable. The experiment was conducted in five independent biological replicates with 50 cells isolated in each replicate. Finally, the number of viable drops from each replicate for each condition was represented as a bean plot. We used Student's t-test to assess statistically significant difference in the averages of viable drops of examined conditions.Viability assays were carried out essentially as described . We initTetrahymena RBBP4 and RBBP7 histone chaperones by first identifying the interaction partners for the core histone H4, one of their predicted partners. The Tetrahymena genome contains two core histone H4s encoded by HHF1 and HHF2 , as well as genome-wide studies. The successful expression of the endogenously tagged proteins was confirmed by western blotting analysis in whole cell extracts (WCEs) prepared either from the HHF1- and HHF2-FZZ expressing cells or untagged wild-type Tetrahymena  for our downstream analyses.We sought to identify the and HHF2 . We generahymena . Similarrahymena , our IF rahymena . This obrahymena ,91. SincUsing our established pipeline ,70,77, wTetrahymena orthologue in greater detail.We identified TTHERM_00688660 as one of the high-confidence (FDR ≤ 0.01) H4 co-purifying proteins which shared sequence similarity with human RBBP4 and RBBP7. The co-purification with H4 of an RBBP4/7-like protein  is consistent with previous reports describing human and budding yeast orthologues to function as histone chaperones . We subsTetrahymena genome for the existence of additional RBBP4/7-like proteins with predicted WD40-repeats. Using the human RBBP4 and RBBP7 proteins to search against the Tetrahymena genome, we identified two proteins, including TTHERM_000709589 and the H4 co-purifying TTHERM_00688660 (RebL1). Both identified proteins have conserved domain architecture with an N-terminal H4-binding domain followed by six WD40 repeats , shares only weak sequence identity (∼18%) with its human and yeast counterparts; in comparison, RebL1 had an overall ∼46% sequence identity   , as asse humans) , making Tetrahymena, we carried out a phylogenetic analysis and observed that RebL1 and Wdc1 are related to the RBBP4/7-family of histone chaperones  interaction partners of RebL1 during vegetative growth  subunits of the CAF1 complex, respectively  complex . Among the remaining conjugation-specific RebL1 co-purifying partners were a Tor1-like, Asf1-interacting protein 2 (Aip2) and several unannotated proteins, including Recp1-4 (RebL1 co-purifying protein 1–4) and Ipra1 (see below), without any recognizable domains  Figure . Additios Figure . We haverahymena . We alsorahymena . The co-rahymena  reinforcs Figure .Tetrahymena. Examples of these conserved interaction partners include the Hat1-, CAF1-, and MuvB complexes , consistent with the architecture of human Lin54  cell lines and employed them for AP-MS , co-purified with both Lin9Tt-FZZ and Anqa1-FZZ as high-confidence hits   Figure , we suggTetrahymena-specific protein and lacks any recognizable domains. We named it Ipra1 (interaction partner of RebL1 and Anqa1). TTHERM_00842380 is the putative MYB-like TF (MybL1) that was also detected as a high-confidence RebL1 interaction partner during both growth and conjugation (FDR≤0.01) . TTHERM_00317260, which interacted with RebL1 during conjugation, is a ) Figure . This ob) Figure .Tetrahymena cells (5 hours post mixing the cells). Anqa1 co-purified with 20 high-confidence partners, of which 8 were common to both vegetative growth and conjugation , transcription factors TFIID (TTHERM_00160910) and TFIID subunit 5 (TTHERM_00227160), transcriptional regulatory protein Spt6 (TTHERM_01093550) and small nuclear ribonucleoprotein Sm (TTHERM_01289080). These results suggest that MuvB functions in transcription regulation during Tetrahymena development.We next identified Anqa1 PPIs in conjugating n Figure . Among sTetrahymena, we utilized our endogenously tagged RebL1 and Anqa1-FZZ cell lines and performed chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq) experiments in vegetatively growing cells . Consistent with the role of RebL1 in multiple chromatin-related processes, metagene analysis indicated that RebL1 binds throughout the gene body of its targets without any specific sequence preferences  . To inveng cells . ChIP-seferences , right.Tetrahymena genes based on their expression patterns during vegetative growth  and transcript end sites (TES) of the targeted genes Figure . Peak die growth  and corrh Figure . These rWe carried out gene ontology (GO) enrichment analysis related to molecular functions and/or biological processes. While RebL1-binding genes were not enriched in any GO terms, in keeping with its role in multiple protein complexes, Anqa1 targets were however enriched for terms associated with gene expression regulation, chromatin assembly and organization, translation regulation, ribosome biogenesis and cellular metabolic processes, suggesting a specific function in regulation of cellular processes Figure .de novo motif discovery analysis using the Anqa1 ChIP peaks. The identified Anqa1 motif appeared to share similarity with the characteristic DREAM-recruiting E2F/DP-CHR hybrid motif  being found within ±1 kb of TSS, that contain this putative DREAM-recruiting motif  and two p107/p130 (Retinoblastoma-like1/2)-like proteins  in their promoters  (P ≤ 0.05) in many tumor types (P < 0.01) compared with low expression groups  construct was introduced into vegetative Tetrahymena cells. Upon CdCl2 treatment, the RebL1 expression levels were significantly reduced in RebL1-RNAi cells in comparison with the WT and/or uninduced Tetrahymena  in wildtype Tetrahymena when treated with CdCl2   Figure , consistREBL1-RNAi dependent reduction in cellular viability could be related to indirect changes in expression levels of some functionally important genes. In human glioblastoma cells (GBMs), RBBP4 depletion results in sensitization of tumor cells to temozolomide (TMZ) in conjunction with the downregulation of DNA repair protein Rad51 , particularly in LIHC patients where increased levels of 3 out of the 5 MuvB subunits were associated with unfavourable outcomes  to be the major structurally and functionally conserved orthologue of human RBBP4 and 7 in Tetrahymena. We demonstrated that RebL1 is a component of multiple chromatin-related complexes and is important for gene expression regulation and cell growth.Effective transcriptional regulation requires the ordered assembly of multiple protein complexes on chromatin in response to specific cellular cues. In this study, we report that Drosophila salivary glands and testis have been identified  the identification of a putative E2F/DP-CHR-like DNA motif; (ii) the enrichment of Anqa1 ChIP peaks in the promoter regions of putative E2F target genes in Tetrahymena and (iii) the targeting of dpl2Δ-dependent upregulated genes by Anqa1. We propose that Tetrahymena MuvB functions in collaboration with E2F/DPs to form a DREAM-like complex that negatively regulates the expression of target genes. Although E2F DNA-binding motifs are largely conserved during evolution  of RBBP4 and RBBP7 proteins in eukaryotes.RBBP4 and RBBP7 function in multiple epigenetic complexes which might have a role in cell proliferation independent of MuvB. For example, knockdown of RBBP4 in GBM tumor cells results in suppression of the DNA-repair protein Rad51 due to disruption of the RBBP4/p300/CBP chromatin-modifying complex . Loss ofjugation ,109. It https://massive.ucsd.edu/). Additional files include the complete SAINTexpress outputs for each dataset as well as a ‘‘README’’ file that describes the details of mass spectrometry files deposition to the MassIVE repository (https://www.ncbi.nlm.nih.gov/geo/). NGS and reads files produced in this study were deposited at GEO database with unique identifier GSE156091.The mass spectrometry raw data reported in this paper are deposited at MassIVE (gkab413_Supplemental_FilesClick here for additional data file.


Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis. Here, we show that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression. During DC, however, we provide evidence that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. Our work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in  Summary: During Drosophila dorsal closure, 20E signaling acts non-canonically through an interaction between EcR and the AP-1 subunit, Jun, to control gene expression at regions containing AP-1 motifs but no EcREs. Drosophila embryo is a developmental wound-healing event in which a hole in the dorsal epidermis, occupied by a transient epithelium, the amnioserosa, is closed by migration of the epidermal flanks  of the Drosophila, though a genetic interaction has been recently uncovered during the pruning of sensory neuron dendrites  and Ultraspiracle (Usp) heterodimers to ecdysone-response elements (EcREs) to promote gene expression . Here, wendrites . Our worendrites .spook (spo) gene is the only locus encoding a member of the 20E biosynthetic pathway known to be transcribed in the amnioserosa, although other members of the pathway are expressed in the amnioserosa anlage  was largely abolished (spo or disembodied (dib), another enzyme in the 20E biosynthetic pathway, revealed abnormalities in amnioserosa morphogenesis and a failure to complete DC properly (Movies 1-3). In particular, mutants lacking 20E showed uneven contractility of the amnioserosa cells and a failure to complete amnioserosa morphogenesis, suggesting perturbation of cytoskeletal regulation. Thus, candidate genes for regulation by 20E during DC are likely regulators or components of the cytoskeleton expressed in the amnioserosa.Given that Dpp signaling to the amnioserosa is required for morphogenesis during DC, and that 20E required for DC is produced in the amnioserosa, we tested the hypothesis that Dpp regulated 20E production. An attractive mechanism for the timely production of 20E in the amnioserosa could be through the presence of all but one or two of the biosynthetic pathway members in the amnioserosa. According to this model, 20E production could be activated specifically in the amnioserosa through tissue-specific transcriptional regulation of just a couple of the pathway members. The a anlage . In tkv7bolished A,B. If 2jaguar (jar), jupiter (jup), and Z band alternatively spliced PDZ-motif protein 52 (zasp52) . jar and zasp52 have been shown to be required for scar-free DC (jup encodes for a little-studied microtubule-associated protein (zipper (zip) encodes for non-muscle myosin, which is required for cell shape change during DC, and is transcribed in a similar pattern to these three genes   , indicating regulation of zip expression by JNK signaling. We confirmed that endogenous JNK signaling was required for this process by impairing the pathway through expression of BskDN, a dominant negative form of JNK encoded by basket (bsk) .Three JNK-responsive genes were previously found to be expressed at high levels in the DME cells and amnioserosa during DC: (zasp52) . The dur-free DC , while j protein . zipper . S1M-P) . To test pathway , or a cous (hep) . Ectopicet (bsk) , which rspo or dib to determine if loss of 20E also had an effect on their transcription. jar and zasp52 expression normally disappeared from the amnioserosa by the beginning of DC in 1spo and 2dib heterozygous mutant embryos . However, expression of both genes persisted in a subset of amnioserosa cells in 1spo and 2dib homozygous mutant embryos undergoing DC . A small but statistically significant decrease in jup and zip expression within the DME cells was also observed in the mutants. Quantification of FISH signal can be found in the supplementary material . Based on these results, we conclude that 20E signaling mainly regulates the timing of the expression of at least four JNK-responsive genes in the amnioserosa during DC.We next used FISH to examine the expression patterns of the four JNK-responsive genes in embryos mutant for either  embryos A,D, whicgoing DC B,C,E,F.  mutants H,I,K,L tjar and zasp52 lose amnioserosa expression by mid-germband retraction , whereas expression of jup and zip persist longer in the tissue . We expressed BskDN in the amnioserosa to test for a requirement for JNK signaling in maintaining jup and zip transcription and found that it was not required (jup and zip transcription in both the amnioserosa and DME cells (ecdysone-inducible gene L1 (IMP-L1)  C,D (Cher(IMP-L1) , 1992. Aobserved G-J. Thesin vivo interaction using proximity ligation assay (PLA) . It is not surprising that multiple PLA signals are seen in the amnioserosa, as the amnioserosa is the site of high levels of 20E and nuclear EcR. We next assessed if the association between Ecr and Jun involved direct physical interaction using reciprocal GST pull-down assays and found that EcR could bind directly to Jun, in vitro . These assays also showed that EcR could bind to Kayak  and Jun could bind to Usp, though further work is required to confirm the in vivo relevance of these interactions. Interestingly, addition of 20E did not increase binding between Jun and EcR  , and fouay (PLA) A,B. PLA omplexes C. For thomplexes D,E. Highomplexes E′-G′. Neomplexes , showed  and EcR J,K.Fig.zip transcripts in the epidermis . 22 of these regions are in or near genes that have previously been shown to be expressed in the amnioserosa. Interestingly, EcR was picked up in the screen, thus indicating a feedback loop. In an effort to look for further evidence of joint regulation of EcR and Jun in such genes, we used chromatin immunoprecipitation sequencing (ChIP-seq) data generated by Kevin White's lab as part of the ENCODE Project Consortium  (zasp52 but no corresponding EcR binding site (We have shown that 20E is required for the expression of s of zip A (Gauhars of zip . This rensortium . These dg frames A. Thought shown) . Interesing site . Their scbt and ush, plus EcR, RhoGAP71E, and Mes2. All of these genes are expressed in the amnioserosa , and enriched in other dorsal tissues including the yolk sac and hindgut , the dorsal epidermis , and the dorsal vessel   .cbt is located in an intron of ush, but transcribed in the opposite direction. Based on prior immunostains, Cbt is expressed in yolk sac nuclei, the amnioserosa, as well as in other more ventral tissues during DC , indicating inhibition of epidermal cbt expression by 20E signaling. A previous study used reporter genes to identify a block of sequences that promoted expression in many of the tissues Cbt is found and likely constitutes the major control region for cbt , and are likely control sequences for ush , indicating promotion of ush expression by 20E signaling.uring DC . Express for cbt . This re for cbt B , suggesting that EcR operates in a positive feedback loop for 20E-mediated gene expression in the amnioserosa. RhoGAP71E expression in wild type is typically restricted to the dorsal vessel during DC, but expression was ectopically induced in the dorsal epidermis of 1spo and 2dib mutants . Interestingly, although an EcR-bound region within the RhoGAP7E locus of 20E-induced Kc167 cells was identified, the ENCODE Project Consortium data showed no binding of EcR to this region, but one instance of Usp binding .AP-1 binding motifs were found in four EcR binding regions within the 67 cells C  were on average twice the size of the average Drosophila gene (i.e. 22 kb compared to 11 kb). The large introns of the genes containing EcR-AP-1 binding regions may have provided an ideal setting for the emergence of these regulatory sequences by allowing transcription factors to experiment with their DNA binding, which could be followed by the evolution of protein–protein interactions between transcription factors fortuitously finding themselves as neighbors on DNA. This could be a mechanism for convergent evolution of steroid hormone–receptor interactions. We examined the distributions of consensus AP-1 binding motifs and ChIP-seq data for several large genes including brn-1  dissolved in ethanol, prior to fixation . The selection was inverted and the threshold was adjusted to create a black and white image, where black represented the FISH signal and white represented the background. The FISH signal was then measured as the total number of black pixels. To standardize the measurement between embryos, the number of black pixels was divided by the pixel surface area of the amnioserosa. Data were expressed as absolute values, and presented as means±s.e.m.. Student's t-tests were performed for all statistical comparisons using GraphPad. Note that the parameters used for quantification were kept constant within data sets. See Fig. S7B for an example of the quantification.Expression levels in the DME cells were quantified by measuring the intensities of the fluorescent signals derived from FISH. Heterozygous siblings of the homozygous mutant embryos served as controls for each stain, as they were treated under identical conditions within the same tube. For each embryo, the z-stacked confocal image was first converted to grayscale with Adobe Photoshop. A section of leading edge epidermis corresponding to one embryonic segment was next selected using the Rectangular Marquee tool with a fixed selection size. The fluorescence intensity of the FISH signal was then measured as mean gray value. Multiple sections of leading-edge epidermis were analyzed per embryo. Data were expressed as absolute values, and presented as means±s.e.m.. Student's 2PO4·H2O, 7 mM Na2PO4, 1.3 M NaCl, 0.1% Triton X-100, pH 7.0). Next, the embryos were incubated with 1:5 mouse anti-EcR  (PLA was performed as previously described but with modifications . Fixed ema Bank)  and 1:25ma Bank) . After tDrosophila Genomics Resource Center, IN, USA were used: EcR (RE06878), jun (LD25202), usp (LD09973), and kay (LP01201). Full-length coding regions were amplified and inserted in frame into pET-28a(+)  and/or pGEX-4T-1  to create N-terminal, His- and GST-tagged constructs, respectively. The constructs were transformed into BL21(DE3) competent cells  for expression.Preparation of tagged proteins was performed as described previously . The fol2, 150 mM NaCl, 0.1% NP-40, 10% Glycerol, 1x cOmplete protease inhibitor cocktail, pH 8.0), and the mix was incubated for 1.5 h at 4°C. In the meantime, 25 µl of Glutathione Sepharose 4B  was blocked with 1% BSA (in Buffer A) for 1 h at 4°C. The mix was then added to the blocked beads and incubated for another 1.5 h at 4°C. For testing 20E-mediated effects on binding, the appropriate amount of 20E  was also included. Following three washes with Buffer A, bound proteins were denatured and fractionated by SDS-PAGE. The presence of His-tagged, prey proteins was determined by immunoblotting with the use of the following primary antibodies: 1:150 mouse anti-EcR   to an equal volume of prey protein . The volume was then topped up to 500 µl with Buffer A   and 1:10


SAGA (Spt-Ada-Gcn5 acetyltransferase) is a highly conserved transcriptional coactivator that consists of four functionally independent modules. Its two distinct enzymatic activities, histone acetylation and deubiquitylation, establish specific epigenetic patterns on chromatin and thereby regulate gene expression. Whereas earlier studies emphasized the importance of SAGA in regulating global transcription, more recent reports have indicated that SAGA is involved in other aspects of gene expression and thus plays a more comprehensive role in regulating the overall process. Here, we discuss recent structural and functional studies of each SAGA module and compare the subunit compositions of SAGA with related complexes in yeast and metazoans. We discuss the regulatory role of the SAGA deubiquitylating module (DUBm) in mRNA surveillance and export, and in transcription initiation and elongation. The findings suggest that SAGA plays numerous roles in multiple stages of transcription. Further, we describe how SAGA is related to human disease. Overall, in this report, we illustrate the newly revealed understanding of SAGA in transcription regulation and disease implications for fine-tuning gene expression. A protein that helps add epigenetic information to genome, SAGA, controls many aspects of gene activation, potentially making it a target for cancer therapies. To fit inside the tiny cell nucleus, the genome is tightly packaged, and genes must be unpacked before they can be activated. Known to be important in genome opening, SAGA has now been shown to also play many roles in gene activation. Daeyoup Lee at the KAIST, Daejeon, South Korea, and co-workers have reviewed recent discoveries about SAGA’s structure, function, and roles in disease. They report that SAGA’s complex (19 subunits organized into four modules) allows it to play so many roles, genome opening, initiating transcription, and efficiently exporting mRNAs. Its master role means that malfunction of SAGA may be linked to many diseases. Nucleosomes bind to promoters and form a physical barrier that blocks transcription, indicating that a destabilizing interaction between DNA and histones is important for active transcription. As acetylation of lysine residues in the histone tail region neutralizes the positive charge of histones and diminishes the electrostatic interaction between histones and the negatively charged phosphodiester backbone of DNA, histone acetylation has long been considered to be generally required for active transcription3. Although Gcn5p showed HAT activity with free histones, it did not acetylate nucleosomal histones; this finding led to the discovery of functional HAT complexes containing Gcn5p4. Two HAT complexes were found in yeast: the 1.8-MDa SAGA complex and the 0.8-MDa ADA complex.SAGA (Spt-Ada-Gcn5 acetyltransferase) is a eukaryotic transcription coactivator complex that controls transcription by modifying histones. The story of SAGA started with the discovery of that the transcription coactivator Gcn5 was a nuclear histone acetyltransferase (HAT)6. STAGA and TFTC were initially regarded as distinct complexes, but more recently, they both have been increasingly recognized as corresponding to the human SAGA (hSAGA) complex. SAGA has retained its transcriptional coactivator function throughout evolution, although its specific role in transcription has been modified in some species. For example, yeast SAGA participates in the transcription of stress-inducible genes, such as heat-shock genes10, whereas hSAGA activity has been observed at ER stress-induced promoters but does not appear to be involved in the p38 MAPK pathway-mediated acetylation of histones at sodium arsenite-induced promoters11. In addition to the classical coactivator function of its HAT activity, SAGA regulates transcription via Ubp8p, which is a ubiquitin-specific protease (UBP) that catalyzes H2Bub1 deubiquitylation15. Multiple lines of evidence developed over the past decade have shown that the SAGA subcomplex critical for this DUB activity also regulates other aspects of gene expression, including the nucleocytoplasmic export of mRNAs17. Collectively, these studies suggest that the SAGA complex may comprehensively coordinate the entire gene expression process and that, conversely, malfunction of SAGA may deregulate gene expression in a manner that may be linked to various diseases.HAT complexes containing the Gcn5p homolog were also found in humans and were independently named the SPT3-TAF9-GCN5 acetyltransferase (STAGA) complex and TATA-binding protein-free TAF-containing complex (TFTC)Saccharomyces cerevisiae is designated “Gcn5p”; in Schizosaccharomyces pombe, it is “Gcn5”; and in Homo sapiens, it is “GCN5.” The nomenclature of other SAGA subunits in four representative model organisms is summarized in Table 119.Before presenting details, we declare that, in this review, the proteins discussed are designated by the standard nomenclature for the corresponding organism. For example, the Gcn5 protein in 19  was also found as a subcomplex of the SAGA complex24. The splicing module facilitates the activation and proper splicing of some SAGA-regulated transcripts, but the specific role of the SF3B subcomplex within SAGA warrants further investigation24. The compartmentalization of these functional modules allows the SAGA complex to intricately and dynamically regulate gene expression, as discussed in detail below.Yeast SAGA is composed of 19 subunits that are organized into 4 functionally distinct modules: the HAT module , the core structural module , the DUB module , and the transcription factor-binding (TF-binding) module (Tra1p)25. Biochemical and structural studies have revealed that Ada2p potentiates Gcn5p HAT activity by cooperatively binding to Gcn5p and changing its conformation to a catalytically active form. Specifically, Ada2p promotes Gcn5p binding to acetyl-CoA26. Remarkably, yeast with ADA2 deletion showed decreased telomeric silencing, indicating that Ada2p plays a specific role in maintaining genomic stability27. Deletion of GCN5 did not yield a comparable phenotype27, implying that Ada2p participates in telomeric silencing through its own targeting activity, independent of the HAT activity of Gcn5p.Gcn5p, which is one of the best-studied proteins of the GCN5-related N-acetyltransferase (GNAT) superfamily, serves as the catalytic subunit of the HAT module Fig. . Recombi28. PCAF is found in both the SAGA complex and p300/CBP, where it contributes to the activation of transcription. The most significant difference between GCN5 and PCAF is the E3 ubiquitin ligase domain of the latter, which may enable PCAF to regulate the stability of transcription factors or signaling proteins and thereby participate in diverse regulatory mechanisms30.Humans have two paralogs of the yeast Gcn5p protein, GCN5 and p300/CBP-associated factor (PCAF)S. cerevisiae31. The ADA complex consists of the SAGA HAT module and two additional subunits, Ahc1p and Ahc2p32. Although the ADA complex lacks an activator-targeting subunit, it is thought to be recruited to chromatin with relatively low specificity through the bromodomain of Gcn5p or the activator-domain-binding sites of Ada2p/Ada3p, whereby it helps maintain the global histone acetylation level. A recent study suggested that there may be an ADA-equivalent complex in Drosophila33; however, no homolog subunit of Ahc1p or Ahc2p has yet been identified in this model. Thus, it is unclear whether this proposed complex can be considered homologous to the yeast ADA complex or whether it is simply a different form of the SAGA HAT module.The HAT module is shared between the SAGA and ADA complexes in 36, although the composition of the ATAC HAT module differs slightly from that of the SAGA HAT module. Metazoans have two paralogous ADA2 proteins, ADA2a and ADA2b, which are specific to the ATAC HAT module and SAGA HAT module, respectively38. They also differ in that ATAC possesses a second HAT subcomplex, ATAC2, which is conserved in flies and mammals40. Differences in catalytic subunits and adapter protein species direct distinct HAT activities: histone H3-specific acetyltransferase activity is observed with SAGA, whereas H3/H4-specific activity is observed with ATAC42. Other subunits of the ATAC complex  have been suggested to be involved in structural or regulatory functions; a recent study partially supported this idea by showing that ATAC1 modulates the histone H3 acetyltransferase activity of the ATAC complex43.In metazoans, the HAT module is also found in the Ada2a-containing (ATAC) complex44. While SAGA prefers promoters to enhancers45, ATAC is associated with both enhancers and promoters. The following are among the yet-unanswered questions: which proteins are responsible for targeting ATAC specifically on enhancers, and which ATAC subunits interact with them?ChIP-seq data for ATAC and SAGA showed that these two HAT complexes possess overlapping and nonoverlapping binding sites46  by recruiting TBP and transmitting signals from the TF-binding module to the HAT and DUB modules. Taf6p-Taf9p, Taf10p-Spt7p, Taf12p-Ada1p, and Spt3p have histone-like fold domains (HFDs) and together form an asymmetric octamer-like fold48. A structural analysis of the SAGA core module organization showed that Taf5p is central for core module assembly and provides docking sites for histone fold pairs on its C-terminal WD40 domain49. Spt20p was found to critically stabilize the complex by providing a wedge-like structure that can be intercalated between the WD40 domain and N-terminal domain of Taf5p. Notably, a recent study on S. pombe showed that SPT20 deletion did not affect the assembly of SAGA complex subunits, except for DUBm and Tra150. These results suggest that, at least in S. pombe, Spt20 is not necessary for SAGA assembly.In yeast, Ada1p, Spt7p, and Spt20p are integral to the proper assembly of the SAGA complexHIS3 and TRP3), Spt3p and Spt8p may prevent the binding of TBP to the TATA box51. Although Spt3p is conserved in Drosophila and humans, no homolog of Spt8p has been found in metazoans. The null mutation of Spt3p results in mating-defective phenotypes, sporulation in diploids, and invasive growth in haploids; in contrast, the SPT8-null mutant shows no severe defect, suggesting that the loss of Spt8p during evolution would not have had a significant effect. In this regard, metazoan SAGA can be compared with the yeast SAGA-like (SLIK) complex, which lacks Spt8p due to truncation of the Spt8p-interacting helices in Spt7p52. Uncovering the detailed roles of the SLIK complex might improve our understanding of how SAGA has changed throughout evolution.As characterized by biochemical assays and supported by structural data, Spt3p and Spt8p are known to bind TBP, load it onto a promoter, and promote the assembly of the PIC. For genes that are inhibited by SAGA, however  images showed that the C-terminal stirrup region of TBP is bound by the Spt3p pocket, whereas the N-terminal region of TBP is in contact with Spt8p56. Similar to other PIKK family proteins, Tra1p requires chaperone and cochaperone proteins to ensure that it is properly folded and assembled on the HAT complex58. Tra1p is composed of three domains: the HEAT  domain, which harbors various binding sites for acidic activators, including Gcn4p, Gal4p, and Rap1p; the FAT  domain; and the PIKK domain60. Recent structural data suggested that Tra1p binds to the SAGA core module using a cup-shaped motif in the FAT domain50. A narrow hinge formed between Tra1 and the core module is thought to provide structural flexibility for regulatory functions60.The TF-binding module comprises a single protein, Tra1p, and forms the largest subunit (~433 kDa) of SAGA. Tra1p belongs to the phosphoinositide 3 kinase-related kinase (PIKK) family but lacks catalytic activity, making it the only pseudokinase of the PIKK family61 as part of the activator-targeting module of NuA462. The only exception to this pattern is found in S. pombe, where two paralogous genes correspond to TRA1; designated TRA1 and TRA2, these proteins are exclusively found in SAGA and NuA4, respectively63. The nonoverlapping distribution of Tra1 and Tra2 provides a way to characterize the SAGA- or NuA4-specific roles of Tra1p. In contrast to SAGA, which has other subunits that can bind TFs, none of the NuA4 subunits is known to interact with TFs. Considering that Tra1-depleted cells are viable but Tra2-depleted cells are not, the necessity of Tra1 for the viability of other organisms likely reflects the importance of the appropriate targeting of NuA4.Tra1p is also found in the NuA4 HAT complexS. cerevisiae occupy distinct facets of Tra1p, the access of activators to the Tra1p-activator-binding domains would be restricted depending on the HAT complex to which Tra1p is bound64. For instance, Tra1p and Eaf1p form the structural core of NuA465, whereas Tra1p is at periphery of the SAGA complex64, where its integrity is maintained by the core subunit. Thus, because distinct facets of Tra1p interact with HAT complexes, Tra1p might recognize different populations of TFs. A recent study on SAGA and NuA4 showed that depletion of the HAT catalytic subunits of SAGA and NuA4 led to different effects on Pol II occupancy66, implying that their targeting might be distinct. Nucleosome binding induces a structural change in the SAGA complex49 in a manner that displaces the HAT module and the DUB module from the core module, enabling them to bind to the nucleosome in the proper orientation. Considering that an activator is generally thought to be important in determining whether the coactivator binds a genomic region (and the associated nucleosomes), one possible supposition is that Tra1 may induce a conformational change in SAGA upon binding to activator. Studies have shown that nucleosomal HAT activity is more efficient when the HAT module is in a separate ADA complex than when ADA is incorporated into SAGA31, suggesting that allowing the HAT module to become more flexible upon TF binding may enable the precise regulation of the nucleosomal HAT activity of SAGA.Since SAGA and NuA4 from 67. The SAGA DUB module (DUBm) in S. cerevisiae consists of the catalytic Ubp8p (ubiquitin protease 8) subunit along with Sgf11p (SAGA-associated factor 11), Sus1p (SI gene upstream of ySa1), and Sgf73p (SAGA-associated factor 73). The deubiquitylation of H2Bub1 by SAGA was first discovered by researchers studying S. cerevisiae Ubp8p; they demonstrated that both H2B ubiquitylation and its successive deubiquitylation are essential for gene activation15. A second component of DUBm, Sgf11p, was subsequently identified and found to be required for the deubiquitylation process70. Next, Sus1p was identified as a component of both SAGA and the Sac3p-Thp1p mRNA export complex71, which was later named TREX-2. Sus1p, whose recruitment to SAGA depends on Ubp8p and Sgf11p, has also been shown to be required for the H2B-deubiquitylation activity of DUBm72. Sgf73p, which was initially identified as a novel component of SAGA through a proteomic approach73, is required for the proper assembly of DUBm onto the SAGA complex and the recruitment of TREX-2 to SAGA. A physical interaction between SAGA and TREX-2 is essential for targeting the transcriptional machinery to the periphery of the nuclear pore complex (NPC) in a phenomenon known as “gene gating”75.Histone H2B ubiquitylation has been shown to be essential for many chromatin-based actions, but its role in transcription regulation is undoubtedly the most thoroughly studied. Works from many laboratories have revealed the existence of cotranscriptional H2B ubiquitylation–deubiquitylation cycles and their roles in directly stimulating transcription elongation69, and its zinc-finger (ZnF)-UBP domain cannot bind to free ubiquitin68, suggesting that Ubp8p activity may be subject to allosteric regulation promoted by a nonsubstrate partner (likely another DUBm subunit). The minimal DUBm structure that confers full deubiquitylation of the nucleosome consists of Ubp8p, Sgf11p, Sus1p, and an N-terminal fragment of Sgf73p (amino acids 1–104)74. Several studies have shed light on the structural basis for the deubiquitylation activity of DUBm78. Sgf11p was found to activate Ubp8p through its C-terminal ZnF domain, which directly interacts with the C-terminal catalytic domain of Ubp8p. An interaction between Sus1p and the helix of Sgf11p and between Sus1p and the ZnF domain of Ubp8p yields an entirely closed conformation of DUBm that seems to stabilize its activity. Sgf73p facilitates the coupling of the “catalytic lobe” and the “assembly lobe” of DUBm, which act together as a unified module through its C-terminal fragment, which is required to connect DUBm with the remainder of SAGA79. Overall, the four subunits seem to have a highly intertwined arrangement. Recent structural studies elucidated the chromatin-binding interface of DUBm80, and these findings suggested that there are interactions between the ZnF domain of Sgf11p and the conserved acidic patch in histone H2A/H2B81 and between the catalytic domain of Ubp8p and histone H2B. However, the crystal structure assessed in the abovementioned reports did not include the SCA7 domain within Sgf73p, which contains the second ZnF domain and was previously reported as a nucleosome-binding domain82. Notably, a recent in vitro study suggested that a fine-tuned regulatory mechanism for generating sophisticated ubiquitylation patterns might involve the competition of DUBm with Bre1p for binding the acidic patch in the nuclear core particle83.Studies have shown that Ubp8p alone cannot carry out the deubiquitylation reactionDrosophila melanogaster86 and H. sapiens89. In contrast, the ATXN7 (the human ortholog of yeast Sgf73p) gene has undergone diversification during evolution and is found as two paralogs, ATXN7L1 and ATXN7L2, in Mus musculus and H. sapiens90. Both paralogs have been described as being part of the SAGA complex, as assessed through proteomic analysis91. Another subunit of DUBm, ATXN7L3 (the human ortholog of yeast Sgf11p), also has a paralog called ATXN7L3B. However, ATXN7L3B localizes to the cytoplasm and does not associate with either SAGA or TREX-2, although it interacts with ENY2 (the human ortholog of yeast Sus1p)92.DUBm is clearly conserved in S. cerevisiae, the 19S regulatory particle (19S RP) of the proteasome facilitates the separation of functional DUBm from SAGA through a physical interaction93. Consistent with this finding, studies from different laboratories have shown that DUBm can function as an independent module. For example, it reportedly remained stable following the loss of dAtxn7  in D. melanogaster86 and upon knockdown of SUPT20H  in H. sapiens11. In striking contrast to the effect seen in yeast, a few studies showed that H2B ubiquitylation decreased following the loss of dAtxn7 and ATXN7L3 in metazoans90. Thus, DUBm may maintain its integrity without SAGA or even lose its initial role as a ubiquitin protease and thus, likely has an alternative, its SAGA-independent function.DUBm has been shown to dissociate from SAGA under certain conditions. In 96. H2B ubiquitylation may also directly regulate transcription elongation by changing the chromatin structure. Although the role of H2B ubiquitylation in nucleosome stabilization seems to suggest the opposite effect98, several biochemical studies have shown that H2B ubiquitylation disrupts chromatin compaction and promotes an open chromatin structure101. H2B deubiquitylation has also been shown to have a profound effect on subsequent H3 methylation and productive transcription103. Notably, Ubp8p and Rad6p/Bre1p may interact with elongating RNA polymerase II, travel along with it into the coding regions104, and act predominantly on gene bodies45. These findings collectively suggest that repeated and transient cycles of H2B ubiquitylation and deubiquitylation occur in the wake of RNA polymerase II during transcription elongation and function as a key checkpoint for optimal gene activation.Histone H2B ubiquitylation facilitates transcription elongation by cooperating with FACT to support efficient nucleosome reassemblyS. cerevisiae. Deubiquitylation of this ubiquitylated H2B is mediated by DUBm; this modification is necessary for the subsequent recruitment of Ctk1p104, which phosphorylates Ser2 in the RNA polymerase II CTD  complex is recruited to RNA polymerase II, with which it interacts through pSer5 in the RNA polymerase II CTD . This PAF recruitment facilitates Rad6p/Bre1p-mediated H2B ubiquitylation in CTD Fig. . pSer2 c71 this functional and physical interaction between the two complexes enables SAGA to function in maintaining genome stability and coordinating mRNA export107  displayed defects in axial skeletal development123. SAGA is also important for maintaining the stemness of stem cells. The well-known target of GCN5 acetyltransferase is c-Myc, which is a pluripotency factor known to be important in the early reprogramming phase of pluripotency induction124. Thus, the activation of c-Myc by GCN5 overexpression and enhanced SAGA recruitment facilitates reprogramming. Similarly, a GCN5 overexpression gain-of-function mutation has been identified as a source of c-Myc-driven cancers in various tissues130. SAGA activates the transcription of c-Myc and is recruited to c-Myc-targeted genes through interactions between c-Myc and SAGA subunits 133. Since many c-Myc-dependent genes are related to cell proliferation and cell growth134, this interaction may further accelerate malignancies.Owing to the importance of SAGA in proper gene expression, malfunction of SAGA subunits is likely to cause developmental defects or diseases136. More recent studies have shown that USP22 is not only a marker of aggressive tumors but also a tumor inducing factor. USP22 plays an essential role in regulating gene activation and cell growth and in maintaining genome integrity. Usp22 itself regulates transcription through its deubiquitylation activity and is also involved in regulating several TFs, including the androgen receptor137, the oncogene c-MYC88, and the tumor suppressor p53138. USP22 was also shown to act as a key factor in cell cycle progression through the G1 phase by controlling CCND1 ubiquitylation in non-small-cell lung cancer139. Further, USP22 has been linked to the maintenance of genome integrity by modulating the stability of TRF1, which regulates the length of a telomere and whose misregulation causes chromosomal abnormalities and cell death140. Interestingly, the depletion of GCN5 does not alter TRF1 mRNA expression, and the expression of catalytically inactive GCN5 does not affect TRF1 protein expression, but the loss of GCN5 alters the TRF1 protein level because it impairs the associations of USP22 and ATXN7L3 with SAGA. Overall, the evidence indicates that overexpression of USP22 in cancer leads to the abnormal activation of several pathways involved in cell survival and tumorigenesis. Thus, the development of new cancer therapies targeting USP22 may yield promising results.Another remarkable subunit of SAGA that appears to be highly engaged in cancer is USP22, which is the catalytic subunit of DUBm. USP22 was initially identified in microarray screens as a member of an 11-gene “death-from cancer” signature that can be used to predict tumor recurrence, metastasis, highly aggressive tumors, and poor prognosis for people with one of several types of cancers141. Several studies have indicated that polyQ-expanded ATXN7 (polyQ-ATXN7) is incorporated into SAGA143 and affects its function. Nuclear inclusions formed by misfolded polyQ-ATXN7 have been found in the cell nuclei of SCA7 patients; these nuclear inclusions also contain other proteins, including the SAGA components GCN5144, USP22145, and ATXN7L3146. PolyQ-ATXN7 has been suggested to further compromise the integrity of SAGA, as it was found to alter the stability of subunits such as GCN5 and STAF36142. Although another study failed to observe a change in the level of GCN5 in the polyQ-ATXN7-containing SAGA, the levels of other key SAGA subunits  were reduced147. PolyQ-ATXN7 has also been shown to decrease the HAT activity of SAGA in some studies147, while others have found that polyQ-ATXN7 increases H2B ubiquitylation without decreasing H3 acetylation146. Further research is needed to resolve this discrepancy. Although GCN5 depletion may also contribute to the severity of SCA7 phenotypes, it is not sufficient to drive SCA7 in a mouse model148. As the loss of GCN5 affects the stability of DUBm140, this observation may suggest that the HAT-independent activity of SAGA plays a regulatory role in SCA7.In addition to its contribution to cancer development, the DUBm subunit ATXN7 is closely related to neurodegenerative disease. An expansion of a highly conserved polyQ motif in the amino-terminal region of ATXN7 causes spinocerebellar ataxia type 7 (SCA7)SAGA was initially found to be important for both transcriptional activation and elongation, and more recent studies have shown that the complex is also important for mRNA export, especially through the DUBm. SAGA has also been reported to contribute to the biogenesis, quality control, and export of mRNPs. Collectively, these findings show that the SAGA complex intricately coordinates gene expression from the activation step through elongation and export. It seems likely that the structural organization of its functional modules allows SAGA to execute comprehensive and dynamic regulation of gene expression.149, but recent reports have suggested that the SAGA complex plays a more general role in gene expression150. Nonetheless, the highly regulated genes, whose expression has been suggested to be more dependent on SAGA than TFIID, tend to have TSS-upstream nucleosomes with higher occupancy and less well-defined positioning compared to those of housekeeping genes. For the proper expression of these genes, chromatin-level regulation is required to resolve the competition between the nucleosome and the TF. Indeed, several studies have demonstrated interactions between SAGA and chromatin remodelers. For example, the SAGA-acetylated nucleosome is displaced by SWI/SNF, and Chd1p151 is thought to physically associate with SAGA152. These studies seem to suggest that SAGA and these chromatin remodelers cooperate to regulate transcription. Additional studies are needed to examine the possible functional links between SAGA and other chromatin remodelers critical for regulating the chromatin structure of the promoter. Understanding the selectivity of specific remodelers and the detailed mechanisms underlying their coordinated action will help researchers elucidate how SAGA functions to fine-tune gene expression.It was long believed that SAGA and TFIID selectively regulate the expression of distinct gene groupsBased on the current evidence, it is tempting to speculate that mammalian SAGA, ADA, and ATAC function to acetylate histone H3 (and possibly H4) at specific genomic locations through the distinct recruitment mechanisms and/or subunit contexts of each complex. Genome-wide studies to investigate the global localization of each complex in a mammalian system will provide the fundamental information needed to verify this hypothesis. Furthermore, comparative genomics of the localization of yeast SAGA and mammalian HAT complexes may provide insight into how the roles of SAGA have been modified throughout evolution.To improve our understanding of the functions of SAGA, researchers should seek to resolve the complete structure of SAGA. The structures of some modules have been published, but the literature lacks their functional configurations when bound to chromatin as part of the whole complex and in different cellular contexts. In addition, the process and mechanisms through which SAGA is assembled from individual subunits to an intact 19-protein complex require much more research. Information on whether each subunit undergoes a conformational change upon binding to one another, upon binding of a TF, and/or during the assembly of the PIC will provide invaluable insights into the overall function of SAGA.