In vivo regulatory functions have evolved from R-loops, including regulation of gene expression and telomere lengthening. However, they also exist as a form of stress, particularly when replication forks collide with the transcription machinery. New methodologies and models are being developed to delineate the biology of R-loops, including those related to cell stress-based diseases like cancer. As accumulation of R-loops is associated with disease, targeting molecular pathways that regulate their formation or removal could provide new avenues for therapeutic intervention. This review covers recent understandings of the molecular basis for R-loop formation, removal, and biological outcomes in the context of cellular stress.Exposure of genomic, single-stranded DNA (ssDNA) during transcription and replication creates opportunities for the formation of inappropriate secondary structures. Cells manage this exposure by using topoisomerases and helicases to reduce the inherent topological stress that arises from unwinding the double helix and by coating ssDNA with protective protein complexes. Interestingly, specific DNA-RNA hybrids, known as R-loops, form during transcription and exist in homeostasis throughout the genomes of prokaryotes and eukaryotes. These hybrids nucleate from guanine rich clusters in the template strand and extend across GC rich spans of transcribed genes.  A variety of topological, structural and hybridization events occur during DNA replication and gene transcription. Unwinding of the DNA double helix provides access for polymerase to a template strand, and creates torsional stress that can manifest anomalous formation of “non-traditional” moieties. One such structure, known as an R-loop , 2, occuR-loops canonically nucleate from guanine-rich clusters (G-clusters) during gene transcription , 4, and The build-up of R-loops is counteracted through a variety of mechanisms that maintain transcription bubble integrity, including nuclease activity and resolution of topological stress  speed and potentiates fork stalling, factors that contribute to R-loop nucleation. Furthermore, recruitment of the repair machinery to sites of DNA damage occurs through MOF-mediated ubiquitination , while aCollectively, the cooperative activities of transcription bubble maintenance and mRNA processing machinery resolve and prevent R-loop accumulation. Each individual component contributes to the homeostasis of R-loops, and their respective regulation determines the local prevalence of these hybrid structures throughout the genome.in vitro and in vivo. Various plasmid-based systems have been created to study the regulation of R-loop formation and removal. Known R-loop sequences are inserted into plasmids in order to examine the effect of these structures on replication or transcription in vitro.Since being first described in 1976 , severalin vitro. The R-loop structure migrates more slowly in agarose gel electrophoresis and can be visualized with ethidium bromide (EtBr) after in vitro transcription [in vitro transcription step increases sensitivity of the assay. Moreover, it eliminates signal from dsDNA that appears when using a general nucleic acid stain like EtBr. As a method to identify perturbations in the dsDNA helix, bisulfite modification can be used to detect R-loops [Classically, a series of electrophoresis techniques have been developed to identify sequences that favorably form R-loops cription , 23. Mor R-loops . Brieflyin vitro enhancement followed by sequencing (DRIVE-seq) utilizes an epitope-tagged, catalytically dead RNase H1 and affinity pulldown to recover DNA-RNA hybrids for sequencing [A powerful, genome-wide technique, known as DNA-RNA immunoprecipitation followed by sequencing (DRIP-seq), uses the S9.6 antibody to specifically pull down DNA-RNA hybrids that are subsequently identified by high throughput sequencing methods to map these structures to the genome -27. Whilquencing , 30. Depin vitro transcription. For example, the S9.6 antibody, as well as a GFP-tagged DNA-RNA hybrid binding domain of RNase H1, allow for visualization of R-loop foci with fluorescence microscopy [in situ hybridization (FISH) based techniques [Recently, IP-mass spectrometry techniques were used to identify R-loop interacting proteins in a high throughput format. The study identified the RNA helicase DHX9 and characterized its role in suppressing R-loop accumulation, reducing DNA damage, and promoting transcriptional termination . R-loopscroscopy , 32 or fchniques -35. Elecchniques .The continued development of techniques and methodologies to study R-loop dynamics and the downstream consequences of their accumulation will further detail the contribution that these structures have in gene regulation and genomic instability.in vivo role of R-loops as regulatory elements of specialized events in the eukaryotic cell. Previously, mitochondrial origins of replication contain R-loops that are used to facilitate replication of the mitochondrial DNA (mtDNA), a process likely conserved from prokaryotic ancestors [Predictably, formation of a DNA-RNA hybrid creates structural lesions in the genome that can affect replication, transcription, and recombination. Ongoing research continues to detail the ncestors . Studiesncestors , 38. In ncestors . Recentlncestors .While R-loops contribute to specialized events or structures in the human genome, they also have distinct roles in regulating transcription. In one instance, R-loops regulate epigenetic mechanisms of gene repression. When R-loops form in downstream CpG island promoter regions, they inhibit the activity of DNA methyltransferases to promote expression . In a seR-loop formation in G-Rich sequences downstream of the 3' polyadenylation (polyA) signal can promote RNA Pol II pausing . The R-lAccumulation of R-loops often results in increased cellular stress that leads to genomic instability. The current hypothesis states that stabilized, co-transcriptional R-loops disrupt RF progression during S-phase. This disruption causes stalling that can lead to fork collapse, DSBs, or incomplete replication before entry into mitosis. Recently, perturbation of the mRNA splicing, processing, and export factors have been shown to increase R-loop mediated genome instability. For example, disruption of the THO and Transcription Export (TREX) complex (discussed above) leads to a hyper-recombination phenotype in yeast and human cells , 44. IntThe deposition of R-loops at promoter and terminator regions has been shown to regulate initiation, pausing, and termination of transcription. Perturbation of these regulatory events could lead to inappropriate changes in gene expression or genetic stress. Studies indicate that mutation of the THO complex impairs elongation, possibly through the formation of R-loop intermediates that prevent RNA Pol II processivity . MoreoveDNA damage sensing and repair proteins have recently emerged as inhibitors of R-loop accumulation in eukaryotic cells  of BRCA2 in resolving R-loops has been difficult due to its contributions in DNA repair, RF stability, and other mechanisms associated with mitosis. Its inherent involvement in these processes may alone prevent R-loop formation by preventing RF collapse and recruiting the ssDNA binding protein, Rad51, to DSB sites . SeparatBoth BRCA1 (FANCS) and BRCA2 (FANCD1) are part of the Fanconi Anemia (FA) mediated DSB repair pathway. Therefore investigators elucidated that this complex may regulate genome stability through R-loop resolution. In addition to the protective functions of BRCA1 and BRCA2, the disruption of critical FA complex members FANCD2, FANCA, and FANCM lead to genomic instability and DNA damage from R-loop mediated RF collapse , 54. TheThe induction of stress due to R-loop accumulation largely results from collisions that occur between RFs and the transcriptional machinery, particularly in a head-on orientation . Genomicin vitro and associate with disease progression [R-loops can arise from a variety of cellular stresses, and lead to complications including replication defects, transcriptional irregularities, and genomic instability. The biological consequences of disrupted R-loop homeostasis has been described in multiple neurodegenerative diseases that largely associate with ataxia (Reviewed in ). GCrichgression , 59.R-loop induced disruption of gene expression occurs through distinct mechanisms in trinucleotide repeat disorders. Expansion of the repeat region in the first intron of FXN leads to R-loop formation, increased histone H3K9 dimethyl (H3K9me2) chromatin deposition, and impeded Pol II mediated transcription . ChemicaFragile X syndrome harbors a mechanism where trinucleotide expansion in the 5' UTR of the FMR1 gene leads to DNA methylation mediated silencing of this locus , 61. HowFurthermore, mutations in the machinery that maintain R-loop homeostasis also associate with neurodegenerative disease. Dysfunctional TREX or RNaseH2 are linked to Aicardi-Goutières Syndrome (AGS) while SETX mutations contribute to ataxia with oculomotor apraxia (AOA2) , 65. MutAs expected, the increased genome instability, chromosomal rearrangements, and DNA damage that occur during aberrant accumulation of R-loops is linked to oncogenesis , 67, 68 The Tudor containing protein 3 (TDRD3) plays a critical role in maintaining proper R-loop homeostasis at the c-MYC locus . TDRD3 rThis translocation commonly occurs in Burkitt's lymphoma and multiple myeloma and causes oncogenic levels of c-MYC expression. Loss-of-function in mRNP biogenesis machinery also associates with known cancers. For example, sequestration of the THO/TREX complex through Kaposi's sarcoma herpesvirus expression of ORF57 leads to increased genomic instability and subsequent malignant progression in immunodeficiency-associated models .Saccharomyces cerevisiae, the formation of R-loops at the noncoding telomere repeat-containing RNA (TERRA) locus facilitates type II survival in telomerase mutants [RAD52) dependent recombination events that result in long heterogenous tracts of telomeric repeats. Extended tracts of recombination dependent repeats appear in mammalian cells utilizing the alternative lengthening of telomere (ALT) pathway during bypass of senescence. Therefore, accumulation of R-loops at TERRA may also play a role in promoting ALT activation in some types of cancer.In  mutants . The surin vivo, we may discover new disease associations of R-loops.Together, these models provide a wide variety of mechanisms where stress associates with increased R-loop formation and can lead to progressive neurodegenerative disease or cancer. The diversity of these mechanisms lends credence to the importance of proper R-loop homeostasis. Redundancy in the machinery that resolves R-loops suggests these structures are dynamic and widespread (studies indicate that R-loops occupy up to 5% of the genome ). As we Emerging evidence demonstrates the importance of proper R-loop homeostasis. These structures provide critical regulatory functions for gene expression and, in some cases, gene rearrangements. However, their formation and resolution must be tightly regulated to prevent topological events that disrupt replication through impaired RF progression. Such impairment provides an avenue for genome instability and contributes to the development of progressive diseases like cancer. Moreover, prolonged R-loop occupancy exposes ssDNA to genotoxic stress, which produces deleterious effects on gene expression.Future examination of R-loops in cancer models should determine whether the seemingly independent functions of BRCA1 and BRCA2 are coordinated to prevent RF collapse through FANC-complex-mediated mechanisms. Specifically, research should elucidate whether BRCA1 recruits SETX to unwind R-loops while BRCA2 acts to traverse the lesion in a manner to complete replication and allow DNA repair to occur at a later phase. Moreover, delineating the coordination between RNase H, helicases, and topoisomerases to prevent R-loop accumulation could provide insight into why specific cell types are susceptible to diseases associated with improper R-loop homeostasis.Finally, the link between the epigenetics and sites of R-loop formation has been established, but further examination of the machinery that recruits chromatin-modifying enzymes is warranted. Discovery of mechanisms that link R-loop homeostasis to chromatin modification would lend further clues into the development of specific cancers or neurodegenerative diseases. Specifically, the field would benefit from identification of R-loop “readers” that allow cells to detect these structures and modify chromatin to repress gene expression or promote transcriptional termination.R-loops provide an interesting new source of replicative and transcriptional stress that clearly have disease-causing consequences. As new methodologies and models are developed to study their regulation and homeostasis, researches will continue to integrate this under-studied structure into cell biology and pathogenesis.


MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We also provide evidence that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses RDT1 transcription until it is removed from the promoter in response to a dsDNA break at the MAT locus induced by HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating-type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating-type switching, thus providing a novel model for dissecting condensin function in vivo.The NAD +-dependent protein deacetylase and defining member of the sirtuin protein family. It was identified 40 years ago in the budding yeast, Saccharomyces cerevisiae, as a factor that silences the cryptic mating-type loci, HML and HMR. These heterochromatic cassettes are utilized as templates for mating-type switching, whereby a programmed DNA double-strand break at the MATa or MATα locus is repaired by gene conversion to the opposite mating-type. This directional switching is called donor preference, which in MATa cells, is driven by a cis-acting DNA element called the recombination enhancer (RE). It was believed the only role for Sir2 in mating-type switching was silencing HML and HMR. However, we now show that Sir2 also regulates expression of a small gene (RDT1) in the RE that is activated during mating-type switching. The promoter of this gene is also bound by the condensin complex, and deleting this region of the RE drastically changes chromosome III structure and alters donor preference. The RE therefore appears to function as a complex locus control region (LCR) that links transcriptional control to chromatin architecture, thus providing a new model to investigate the underlying mechanistic principles of programmed chromosome architectural dynamics.Sir2 is a highly conserved NAD For exiewed in ).HML and HMR play a critical role in mating-type switching. Haploid cells of the same mating-type cannot mate to form diploids, the preferred cell type in the wild. Therefore, in order to facilitate mating and diploid formation, haploid mother cells switch their mating-type by expressing HO endonuclease, which introduces a programmed DNA double-strand break (DSB) at the MAT locus [HML or HMR as a donor template for gene conversion [HO is deleted from most standard lab strains in order to maintain them as haploids, so expression of HO from an inducible promoter such as PGAL1 is commonly used to switch mating-types during strain construction [AT locus . The brenversion , 7. Thistruction .MATα cells preferentially switch to MATa using HMR as the donor. However, while both silent mating loci can be utilized as a donor template, usage of HML by MATa cells requires a 2.5 kb intergenic region located ~17 kb from HML called the recombination enhancer (RE) [α2 binding site (DPS1) and multiple Fkh1 binding sites [MATa cells, requiring Mcm1 and Fkh1 activity at their respective binding sites [MATα cells due to expression of transcription factor α2 from MATα [α2) to repress MATa-specific genes [HML in close proximity with MATa by interacting with threonine-phosphorylated H2A (γ-H2AX) and Mph1 DNA helicase at the HO-induced DNA DSB [There is a “donor preference” directionality to mating-type switching such that ~90% of the time, the HO-induced DSB is repaired to the opposite mating-type [cer (RE) . Donor png sites . The RE ng sites –12. The rom MATα , which f DNA DSB , 15.HML and HMR has two known functions related to mating-type switching. First, HML and HMR must be silenced in haploids to prevent formation of the a1/α2 heterodimer, which would otherwise inactivate haploid-specific genes such as HO [HML and HMR blocks cleavage by HO, thus restricting its activity to the fully accessible MAT locus [RDT1. Here, Sir2 was found to repress the MATa-specific transcription of RDT1, which is also translated into a small 28 amino acid peptide. RDT1 expression is also dramatically upregulated during mating-type switching when Sir2 is lost from the RDT1 promoter and instead associates with the HO-induced DNA DSB at MATa. Furthermore, eliminating Sir2/condensin recruitment to the RDT1 promoter disrupts chromosome III architecture such that donor preference is partially impaired. The RDT1 promoter region therefore functions like a classic locus control region (LCR) in MATa yeast cells, regulating localized transcription as well as long-range chromosome interactions.Sir2-dependent silencing of ch as HO . Second,AT locus , 18. Hersir2Δ strains in which the condensin subunit Smc4 was C-terminally tagged (13xMyc)  that specifies donor preference of mating-type switching in MATa cells [MATa-specific  known to have an HML silencing defect [HML was also deleted  . To avoi for GFP . The speantibody , with ensilencer . Sir2-desilencer . The ~2.Ta cells , 13. QuaxMyc . Tospecific , which wruitment . Similarg defect  reduced  deleted . Sir2 anKAR4 (YCL055W)-proximal 700 bp domain defined by an Mcm1/α2 binding site (DPS1) [α2 binding site (DPS2) and a small gene of unknown function called RDT1 [RDT1 coincided with the smallest of several putative non-coding RNAs (ncRNA) previously reported as being transcribed from the RE, but not annotated in SGD (MATa) and BY4742 (MATα) revealed that RDT1 expression was indeed MATa specific  , 11, 13.led RDT1  . Accordpromoter , consistpromoter . Further did not . While Hne tails . Since Rase HDA1 , 26, we e mutant . RDT1 ex deleted . An evenparalogs . On the n mutant . Taken tRDT1 promoter by precisely deleting a 100bp DNA sequence underlying the shared enrichment region (coordinates 30701–30800), while not disturbing the adjacent Mcm1/α2 site  upstream of the Sir2/condensin ChIP-seq peaks an ideal candidate, because it had not previously been ascribed a function other than redundancy with DPS1 for donor preference [MCM1 is lethal, so alternatively, we precisely deleted the 2nd Mcm1/α2 binding site  and the RDT1 promoter (defined as the Sir2/condensin peaks) was significantly reduced in the binding site mutant. These results suggest that Mcm1 nucleates a complex that recruits the SIR and condensin complexes to the RDT1 promoter in MATa cells, and also provides a possible mechanism of blocking the recruitment in MATα cells due to the interaction of Mcm1 with α2.Because Sir2 and condensin were not present at the eference . DeletinRibosome Detected Transcript-1 (RDT1) was originally annotated as a newly evolved gene whose transcript was associated with ribosomes and predicted to have a small open reading frame of 28 amino acids [RDT1 and the putative non-coding R2 transcript were the same   of HMRα-B. Therefore, although RDT1 gene expression strongly correlates with switching, a specific function for its gene product remains elusive. Therefore, we shifted our attention to a possible function for the RDT1 promoter.Since o method , and thee by PCR . No signaber lab , whereby(HMRα-B) . FollowiRDT1 transcriptional regulation during mating-type switching was reminiscent of classic locus control regions (LCR) that modulate long-range chromatin interactions [RDT1 promoter region functions as an LCR to modulate long-range chromatin interactions of chromosome III. To test this hypothesis, we performed Hi-C analysis with WT, sir2Δ and the 100bpΔ strains. Genomic contact differences between the mutants and WT were quantified using the HOMER Hi-C software suite [HML (bin 2) and HMR (bin 29) loci. Interestingly, HML (bin 2) also appeared to sample the entire right arm of chromosome III, with the interaction frequency increasing as a gradient from CEN3 to a maximal observed interaction at HMR, thus also encompassing the MATa locus at bin 20. This distinct interaction pattern was completely disrupted in the sir2Δ mutant, whereas some telomere-subtelomere contacts were retained in the 100bpΔ mutant  and HMR (bin 29) again formed the dominant interaction pair off the diagonal in WT, which was lost in the sir2Δ or 100bpΔ mutants (HMR (bin 29) and MATa (bin 20) appeared in both mutants , though still significantly different from WT . In thi late G1 . Therefowitching  suggestsing RNAs , 27. DisRDT1 in mating-type regulation or other cellular processes, the promoter region of this gene clearly controls the structure of chromosome III. Three-dimensional chromatin structure has long been proposed to influence donor preference [RDT1, changes chromosome III conformation without a dramatic change in donor preference [RDT1 promoter bound by the SIR and condensin complexes. Importantly, deleting this small region not only altered chromosome III structure, but also had a significant effect on donor preference, though not as strong as the sir2Δ mutation.While we do not yet know the molecular function of eference , 41. Howeference , 13, 42.RDT1 expression with loss of Sir2/condensin binding at its promoter during mating-type switching, together with the loss of HML-HMR interaction in the 100bpΔ mutant, makes this site intriguingly similar to classic locus control regions (LCRs) in metazoans, which are cis-acting domains that contain a mixture of enhancers, insulators, chromatin opening elements, and tissue-specificity elements [st Mcm1/α2 binding site (DPS1) appears to be important for this activity in MATa cells [MATa cells by silencing HMLALPHA2 expression, which prevents transcriptional repression by an Mcm1/α2 heterodimer, and by protecting the HML template from HO cleavage. Similarly, the loss of Sir2 also represses RDT1 expression and condensin recruitment in the right half of the RE due to HMLALPHA2 expression. It remains possible that SIR-dependent heterochromatin formation also directly contributes to the HML-HMR interaction through clustering. More clearly, however, Sir2 directly represses RDT1 through localized histone deacetylation. How the loss of RDT1 regulation and condensin recruitment changes chromosome III structure in the sir2Δ mutant remains unknown, but we propose that the HMR-MATa interaction is a default state, while the HML-HMR association has to be actively maintained by condensin and likely additional factors co-localized to this element, as well as SIR-dependent heterochromatin.The coordination of elements . The minTa cells . We findHML locus, such that deleting the left half of the RE specifically stabilizes HML silencing in MATa cells [RDT1 promoter to encroach and somehow enhance the heterochromatic structure at HML. Under this scenario, the left half of the RE could be insulating HML from the chromosomal organizing activity that occurs at the RDT1 promoter.Interestingly, there also appears to be a functional relationship between the RE and silencing at the Ta cells . The mecRDT1 promoter is a major condensin binding site identified by ChIP-seq  toward HMR, suggesting that HML “samples” the right arm of chromosome III. Once brought in contact, HML and HMR would then remain associated due to their heterochromatic states and shared retention at the nuclear envelope [HMR association with MATa, but since the 100bpΔ mutation has no effect on the timing of switching, we do not believe that condensin at the RDT1 promoter functions directly in the homologous recombination process. Rather, general Brn1 subunit depletion could slow the switching process by affecting chromosome III flexibility or conformational dynamics.The ChIP-seq , and givle assay . The mecwith HMR . HOMER aenvelope . FormatiRDT1 promoter exclusively in MATa cells, though it is not clear if they bind at the same time, or are differentially bound throughout the cell cycle. Since DPS2 was required for Sir2 and condensin recruitment, and derepression of HMLALPHA2 from HML also eliminated binding, we hypothesized and then demonstrated , and yeast condensin does not appear to have a consensus DNA binding sequence [RDT1 promoter indicates an A/T rich region with consensus binding sites for the transcription factors Fkh1/2 and Ash1, each of which regulates mating-type switching [CLB2 promoter during stress [RDT1 repression in daughter cells. Mcm1 activity in MATa cells could also indirectly establish a chromatin environment that is competent for Sir2/condensin recruitment, rather than direct recruitment through protein-protein interactions. In MATa cells, Mcm1 appears to prevent the strong nucleosome positioning across the RE that occurs in MATα cells [RDT1 promoter LCR therefore provides an outstanding opportunity for dissecting mechanisms of condensin loading and function.Condensin, and Sir2 each strongly associated with the nstrated  that McmTα cells . This rasequence . Closer witching , 48, 49.g stress . Ash1 isg stress , raisingTα cells , and indTα cells . FurtherTα cells , though SIR2, or HST1 open reading frames (ORFs) were deleted with kanMX4 using one-step PCR-mediated gene replacement. HML was deleted and replaced with LEU2. Precise deletions of the 100bp condensin/Sir2 binding site within the RDT1 promoter (chrIII coordinates 30701–30800), DPS2 (chrIII coordinates 30557–30626), or the RDT1 ORF (chrIII coordinates 30910–30996) were generated using the delitto perfetto method [SIR2, BRN1, or SMC4 genes were C-terminally tagged with the 13xMyc epitope (13-EQKLISEEDL). Deletion and tagged gene combinations were generated through genetic crosses and tetrad dissection, including Brn1 tagged with a V5-AID tag (template plasmids kindly provided by Vincent Guacci). All genetic manipulations were confirmed by PCR, and expression of tagged proteins confirmed by western blotting. The pGAL-HO-URA3 expression plasmid was kindly provided by Jessica Tyler [Yeast strains were grown at 30°C in YPD or synthetic complete (SC) medium where indicated. The o method . EndogenSir2 ChIP-seq was previously described . For othsir2Δ backgrounds were subtracted from the WT SMC4-13xMyc and sir2Δ SMC4-13xMyc peaks, respectively, using BEDTools “intersect” with the–v option. The resulting normalized peaks were annotated using BEDTools “closest” with the -t all option specified, and in combination with a yeast gene list produced from USCS genome tables. The annotated peaks were then analyzed for GO terms using YeastMine (yeastmine.yeastgenome.org).Biological duplicate fastq files were concatenated together and reads mapped to the sacCer3 genome using Bowtie with the following options:—best,—stratum,—nomaqround, and—m10 . The res2O and stored at -80°C. Thawed cells were resuspended in 5 ml of 1X NEB2 restriction enzyme buffer (New England Biolabs) and poured into a pre-chilled mortar containing liquid N2. Nitrogen grinding was performed twice as previously described [600 of 12 in 1x NEB2 buffer. 500 μl of cell lysate was used for each Hi-C library as follows. Lysates were solubilized by the addition of 50 μl 1% SDS and incubation at 65°C for 10 min. 55 μl of 10% TritonX-100 was added to quench the SDS, followed by 10 μl of 10X NEB2 buffer and 15 μl of HindIII  to digest at 37°C for 2 hr. An additional 10 μl of HindIII was added for digestion overnight. The remainder of the protocol was based on previously published work with minor exceptions [2O and treated with RNAse A for 45 min. Following treatment with T4 DNA polymerase to remove biotinylated DNA ends that were unligated, the samples were concentrated with a Clean and Concentrator spin column  and sheared to approximately 300bp with a Diagenode Bioruptor. Biotinylated fragments were captured with 30 μl pre-washed Streptavidin Dynabeads (Invitrogen), then used for library preparation. Hi-C sequencing libraries were prepared with reagents from an Illumina Nextera Mate Pair Kit (FC-132-1001) using the standard Illumina protocol of End Repair, A-tailing, Adapter Ligation, and 12 cycles of PCR. PCR products were size selected and purified with AMPure XP beads before sequencing with an Illumina Miseq  or Hiseq . Raw and mapped reads are deposited at GEO (GSE92717).Log-phase cultures were cross-linked with 3% formaldehyde for 20 min and quenched with a 2x volume of 2.5M Glycine. Cell pellets were washed with dHescribed , and theceptions . In shorhttp://mirnylab.bitbucket.org/hiclib/index.html. Briefly, reads were mapped using the iterative mapping program, which uses Bowtie2 to map reads and iteratively trims unmapped reads to increase the total number of mapped reads. Mapped reads were then parsed into an hdf5 python data dictionary for storage and further analysis. Mapped reads of the same strains were concatenated using the hiclib library’s “Merge" function. Both individual and concatenated mapped reads have been deposited in GEO. Mapped reads were then run through the fragment filtering program using the default parameters as follows: filterRsiteStart(offset = 5), filterDuplicates, filterLarge, filterExtreme . Raw heat maps were further filtered to remove diagonal reads and iteratively corrected using the 03 heat map processing program. Finally, the iteratively corrected heatmaps were normalized for read count differences by dividing the sum of each row by the sum of the max row for a given plot, which drives all values towards 1 to make individual heatmaps comparable.Iteratively corrected heatmaps were produced using python scripts from the Mirny lab hiclib library, Observed/Expected heatmaps were created using HOMER Hi-C analysis software on the BAM file outputs from the iterativemapping program of the hiclib library python package . Tag dirMATα) and BY4741 (MATa) backgrounds, respectively. Reads were then mapped to the sacCer3 genome using Bowtie2 with no further processing of the resulting BAM files visualized in this paper.RNA-Seq data was acquired from GEO accessions GSE73274  and GSE5PDC1 intergenic region that is not recognized by HindIII, in addition to PCR of the un-crosslinked control for all tested looping interaction primer pairs.Chromosome Conformation Capture (3C) was performed in a similar manner to Hi-C with a few exceptions due to assay-specific quantification via quantitative real-time PCR rather than sequencing. Most notably, digested DNA ends were not filled in with dCTP-biotin before ligation and an un-crosslinked control library was created for each 3C library. Furthermore, all PCR reactions were normalized for starting DNA concentration using a ACT1 mRNA, with this ratio then normalized to 1.0 for a specific strain or condition indicated for each experiment.Total RNA (1 μg) was used for cDNA synthesis with oligo(dT) and Superscript II reverse transcriptase as previously described . ExpressProteins were blotted using standard TCA extraction followed by SDS-PAGE as previously described . Myc-tagURA3, pre-cultured in SC-ura + raffinose (2%) medium overnight, re-inoculated into the same medium (OD600 = 0.05) and then grown into log phase. Galactose (2%) was added to induce HO expression for 45 min. Glucose (2%) was then added and aliquots of the cultures were harvested at indicated time points. Genomic DNA was isolated and 10 ng used for PCR amplification. MATα was detected using primers JS301 and JS302. The SCR1 gene on chromosome V was used as a loading control (primers JS2665 and JS2666). PCR products were separated on a 1% agarose gel stained with ethidium bromide and then quantified using ImageJ. Donor preference with strains containing HMRα-B was performed as previously described [MATa was amplified with primers Yalpha105F and MATdist-4R from genomic DNA 90 after switching was completed (90 min), and then digested with BamHI. Ethidium stained bands were quantified using ImageJ. For the conditional V5-AID degron strains, degradation of V5-AID-fused Brn1 protein was induced by addition of 0.5 mM indole-3-acetic acid .For tracking the efficiency of switching, strains were transformed with pGAL-HO-escribed . BrieflyMATa, HML, and HMR, the WT and sir2Δ strains containing the pGAL-HO-URA3 plasmid were induced with galactose for 0 to 2 hrs following growth in raffinose. Genomic DNA was then isolated and PCR across the HO-cleavage site performed with primers specific to each locus, and SCR1 used as a loading control. MATa was detected with JS301 and JS854, HML with JS3101 and JS3103, and HMR with JS3097 and JS3100. PCR was performed in the linear range and bands on ethidium stained agarose gels quantified with ImageJ. Real-time qPCR of HML and HMR with the same genomic DNA was performed with primers JS3101-JS3103, and JS3097-JS3098, respectively.For assaying HO cleavage across S1 Fig(A) IGV screenshot of compiled raw RNA-seq read data from BY4741 (MATa) and BY4742 (MATα) strains. The top two blue peaks represent Smc4-myc and Sir2-myc ChIP-seq reads. (B) Quantitative ChIP assay showing additional SIR complex subunit enrichment at the RDT1 promoter. Signals are relative to input. (C) RT-qPCR showing effects of deleting SIR2 and/or HST1 on RDT1 expression (relative to ACT1) when HML is present or deleted . The WT RDT1/ACT1 ratio is normalized to 1.(TIF)Click here for additional data file.S2 Fig(A) Western blot showing steady state Sir2 protein levels in WT (ML1), sir2Δ (ML25), and 100bpΔ (ML275) strains. (B) Western blot with anti-Myc detection of Brn1-13xMyc or Sir2 in WT (ML149), sir2Δ (ML161), and 100bpΔ (ML322) strains. (C) Western blot with anti-Myc detection of Smc4-13xMyc or Sir2 in WT (ML152), sir2Δ (ML160), and 100bpΔ version.(TIF)Click here for additional data file.S3 Fig(A) Schematic diagram depicting the location of the DPS2 sequence deletion relative to other elements with the RE, with the deleted chromosome III coordinates indicated in red. (B) Quantitative ChIP of native Sir2 in WT and dps2Δ strains. (C) Quantitative ChIP of Brn1-Myc in WT and dps2Δ strains. @RDT1 promoter indicates enrichment at the Sir2/condensin peak. ChIP signals are plotted relative to the input signal. (**p<0.005).(TIF)Click here for additional data file.S4 Fig(A) Representative time course of switching from MATa to MATα in WT (ML440) and rdt1Δ (ML443) strains. PCR products are specific to MATα and an SCR1 loading control. (B) Quantitation of the average MATα/SCR1 PCR signal ratio from 3 biological replicates. (C) Schematic of chromosome III in the donor preference reporter strain harboring an artificial HMRα-B cassette as the donor for switching, which introduces a unique BamHI site to the MAT locus. (D) Representative ethidium bromide stained agarose gel of BamHI-digested MATα PCR products after mating-type switching in WT (XW652) and rdt1Δ (MD30) strains. The bottom two bands represent HMR-derived switching. (E) Quantitation of average MATα-B utilization from 3 biological replicates.(TIF)Click here for additional data file.S5 Fig(A) Quantitative mating assay for WT (ML1) and 100bpΔ (ML275) strains. (B) Quantitative ChIP assay showing Sir2 enrichment at HML-I in WT (ML1) and 100bpΔ (ML275) strains. (**p<0.005).(TIF)Click here for additional data file.S6 Fig(A) PCR amplification of MATa, HML, HMR, and SCR1 loci from MATa WT (ML440) and sir2Δ (MD29) strains containing the pGAL-HO-URA3 expression vector. Times after HO induction with galactose are indicated. A representative experiment is depicted. (B) Quantitation of mean MATa PCR signal, relative to the SCR1 control, from 3 independent biological replicates. Error bars indicate standard deviation. (C) Quantitation of mean HML PCR signal, as done for panel B. (D) Quantitation of mean HMR PCR signal, as done for panel B. (E and F) Real-time qPCR signal, relative to SCR1 control, for HML and HMR, respectively. The PCR signal at time 0 is normalized to 1.0 in each panel.(TIF)Click here for additional data file.S7 Fig(A) Western blot time course of auxin induced degradation of Brn1-V5. Time indicates minutes after addition of auxin. (B) ChIP assay showing Brn1-V5 enrichment at the RDT1 promoter with and without auxin addition. The Brn1-V5 signal is relative to input and the ratio from an untagged control strain is normalized to 1. (C) RT-qPCR of RDT1 expression following 30 or 60 minutes of Brn1 depletion by auxin. (D) RT-qPCR of HMLALPHA2 expression following 30 or 60 min of Brn1 depletion by auxin. In panels C and D, the signals are relative to ACT1 control and normalized to 1.0 without auxin.(TIF)Click here for additional data file.S1 TableThis Excel spreadsheet lists the systematic ORF names of all genes that were closest to Sir2-dependent condensin peaks, as chosen using MACS.(XLSX)Click here for additional data file.S2 TableSaccharomyces cerevisiae strains used in this study, along with their genotypes and source.List of all (DOCX)Click here for additional data file.S3 TableList of oligodeoxynucleotides used in this study.(DOCX)Click here for additional data file.


The ability of a cell to dynamically switch its chromatin between different functional states constitutes a key mechanism regulating gene expression. Histone mark “readers” display distinct binding specificity to different histone modifications and play critical roles in regulating chromatin states. Here, we show a plant-specific histone reader SHORT LIFE (SHL) capable of recognizing both H3K27me3 and H3K4me3 via its bromo-adjacent homology (BAH) and plant homeodomain (PHD) domains, respectively. Detailed biochemical and structural studies suggest a binding mechanism that is mutually exclusive for either H3K4me3 or H3K27me3. Furthermore, we show a genome-wide co-localization of SHL with H3K27me3 and H3K4me3, and that BAH-H3K27me3 and PHD-H3K4me3 interactions are important for SHL-mediated floral repression. Together, our study establishes BAH-PHD cassette as a dual histone methyl-lysine binding module that is distinct from others in recognizing both active and repressive histone marks. Histone mark reader proteins bind to particular histone modifications and regulate chromatin state. Here, Qian et al. show that the SHORT LIFE reader has a unique ability to recognize both activating and repressive histone marks and that these interactions enable SHORT LIFE to repress flowering in plants. Aberrant modification patterns are associated with a wide variety of developmental defects and diseases13. One major type of epigenetic modification is DNA methylation, the addition of a methyl group to DNA cytosine bases. Post-translational modification (PTM) to histone proteins is another epigenetic mark and forms complex interconnections with DNA methylation17. Epigenetic modifications are added, removed, and recognized by specific proteins and/or protein domains named “writer”, “eraser”, and “reader”, respectively, in which the “reader” modules can serve as the molecular interpreters to translate the complex PTM codes to certain functional outputs and to transmit these signals to downstream effectors20.Epigenetic modification is an important gene regulatory mechanism conserved in plants, animals, and fungi and impacts many biological processes including genome defense, imprinting, proper development and physiology, and environmental responses21. Plant homeodomain (PHD) fingers are diverse chromatin-binding modules for various histone marks, including methylated, unmethylated, and acetylated lysine residues with different sequence contexts22. Chromodomain family proteins display strong binding preference for methylated histone lysine residues23. Bromo-adjacent homology (BAH) is an evolutionarily conserved binding module known to recognize distinct histone modifications24. Interestingly, many reader proteins contain multiple histone recognition domains that often exist in tandem and function in multivalent chromatin binding to elicit high specificity and avidity to the appropriate epigenetic landscapes26.Emerging evidence suggests that chromatin reader domains exhibit distinct binding specificity to different histone PTMs and this specific PTM recognition contributes to either repressive or active chromatin states to modulate gene expression. For example, bromodomain functions mainly as acetyl-lysine recognition motifs28. Similarly, Arabidopsis CHROMOMETHYLASE3 (CMT3) contains a BAH domain and a chromodomain, both interacting with the repressive mark H3K9me229. Interestingly, the PHD-bromo cassette of TRIM33 can recognize both the silencing mark H3K9me3 and the active mark H3K18ac30. In plants, the histone mark readers are generally similar to their animal counterparts but with some plant-specific features. Plant histone reader proteins often have unique reader domain combinations and different reader-mark pairs32.Currently, the majority of multivalent reader proteins engages similar PTM states: either active or repressive histone marks. For example, human BPTF recognizes the active histone marks H3K4me3 and H4K16ac by its PHD and bromo domains, respectively37. Despite the similar domain structures, the two paralogs SHL and EBS adopt different mechanisms regulating flowering by repressing distinct floral genes33. Genetically, while EBS regulates the expression of a floral integrator FLOWERING LOCUS T (FT), SHL has been shown to repress another floral integrator SUPPRESSION OF OVEREXPRESSION OF CO1 (SOC1) to inhibit flowering35. Despite the repressive role of SHL, the molecular mechanism underlying SHL floral repression remains largely unexplored. Here, our biochemical and X-ray structural studies show that SHL is capable of recognizing an active histone mark H3K4me3 and a repressive mark H3K27me3 independently via its PHD and BAH domain, respectively. Chromatin immunoprecipitation followed by sequencing shows that a subset of SHL-bound genes was co-marked with H3K4me3 and H3K27me3. Mutations abolishing BAH-H3K27me3 and PHD-H3K4me3 binding cause a failure of SHL in complementing the early shl flowering phenotype, suggesting that SHL binding to H3K27me3 and H3K4me3 is functional in vivo. Collectively, we establish a single protein-mediated dual histone mark recognition and provide insights into establishing and maintaining proper chromatin landscapes.In Arabidopsis, the BAH and PHD dual domain-containing proteins SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS) function in chromatin-mediated floral repression and seed dormancy33. PHD of Arabidopsis SHL (AtSHL) was previously shown to bind to H3K4me2/3 in vitro by pull down assays33. Prior studies showed that BAH domains are also histone mark readers24. To explore the function of BAH and further identify the histone binding mode of SHL, we purified the full-length recombinant AtSHL protein and screened it on a histone peptide microarray38. We found that AtSHL bound H3K4me3  of 15.1 µM between AtSHL and H3K27me3 peptides  assay and found a dissociation constant , which has similar PHD and BAH domains , similar to other canonical methyl-lysine readers39. In addition, Tyr41, Trp63, and Met31 form a shallow hydrophobic pocket to accommodate the methyl group of H3A25 K27me3 complex at 3.2 Å resolution Fig. . The pep3cKS consensus motif, H3P30 at the n + 3 position of H3K27me3 (but not H3G12 at the n + 3 position of H3K9me3) can be specifically recognized by PtSHL, suggesting a sequence-specific recognition mechanism. Mutations of His88 (H88A) or Asp90 (D90A) involved in H3P30 recognition as well as mutations of the aromatic residues responsible for H3K27me3 recognition , abolished the binding of PtSHL with H3K27me3 K27me3 peptide pre-saturated PtSHL and H3(1–15)K4me3 peptide yielded a binding affinity of 27.6 μM Fig. , similarN-value of 1.17 K4me3K27me3 peptide yielded a binding affinity of 16.9 μM and a binding .17 Fig. , indicat.17 Fig. , suggestshl when compared to the wild-type  distributed in 2707 genes  only partially rescued the early shl-2 flowering phenotype  and PHD (F141A/Y148A) that abolished SHL binding to H3K27me3 and H3K4me3, respectively Figs.  and 3d. l-2 Fig. . We founype Fig. . To ruleype Fig. . These r40 mutant as well as an H3K4me3 methyltransferase ARABIDOPSIS TRITHORAX1 (ATX1) mutant42. We found that SHL chromatin association at several target genes was significantly decreased along with reduced H3K27me3 levels in the clf28 mutant ant Fig. , suggestant Fig. , despiteant Fig. . The par45. Our biochemical and structural analyses provide the molecular basis of H3K27me3 recognition by BAH and H3K4me3 by PHD finger. This represents an example of a dual recognition mode for two antagonistic histone methylation marks mediated by one single protein. Previously, only multi-protein complexes were known to display H3K4me3-H3K27me3 crosstalk46. Thus, the BAH-PHD dual binding cassette may represent a regulatory circuit to mediate the chromatin switching between an active and repressive state.An epigenetic paradox is the ability to maintain a stable epigenetic state for many cellular generations, yet also to be flexibly switched to an alternative chromatin state at any stage. Specific recognition of histone marks by “reader” proteins constitutes an important mechanism in establishing and maintaining proper epigenetic landscapes. In this study, we identified SHL as a dual histone methyl-lysine reader that is distinct from other reader proteins in recognizing both H3K4me3 and H3K27me3, two well-known histone marks with opposing roles in balancing chromatin and transcriptional states24, with the histone marks recognized by BAH domains representing a diversified set. Maize ZMET2, mouse ORC1, and Arabidopsis ORC1b utilize their BAH domains to recognize H3K9me2, H4K20me2, and unmodified H3, respectively  to repress flowering33. We find that H3K27me3 and CLF are also important for SHL-mediated floral repression. SHL was unable to bind SOC1 chromatin in the absence of CLF and H3K27me3  have chromatin states carrying both H3K4me3 and H3K27me3 Fig.  and theime3 Fig. . We furtme3 Fig. , consist33, several chromatin proteins contain both BAH and PHD domains including a mammalian histone methyltransferase ASH1L implicated in leukemia53 and yeast transcription factor SNT2 involved in fungi respiration and oxidative stress responses54. It is possible that the BAH-PHD cassette of these proteins or other dual domain-containing proteins might similarly recognize both active and repressive marks to balance proper chromatin landscapes to fine-tune the expression of crucial development factors.The unique BAH-PHD dual histone binding module is functionally conserved as demonstrated by similar binding affinity and specificity of Arabidopsis and Populus SHL with H3K27me3 and H3K4me3. Although SHL homology has yet-to-be identified in animalsshl-2 (GT442) allele is in the Ler background and was a gift from Dr. Manuel Piñeiro . The clf-28 (SALK_139371) and atx1-2 (SALK_149002), in the Col-0 background, were gifts from Dr. Richard Amasino (UW-Madison). Seeds were sown in soil and kept at 4 °C for two days before transferring to long-day (16 hrs light/ 8 hrs dark) conditions at 22 °C.The ® Site-Directed Mutagenesis kit  and were transformed into the shl-2 background via agrobacterium-mediated floral dip method. T1 transgenic plants of Basta resistance were used for leaf counting at the long-day condition. The pSHL::SHL-3xFLAG construct was transformed into atx1-2 mutants and T1 plants containing the transgenes were used for ChIP-qPCR experiments. The pSHL::SHL-3xFLAG was crossed into a clf28 mutant and basta resistant F2 plants with clf28 null mutant background were used for ChIP-qPCR experiments (Supplementary Table Genomic DNA of SHL with 1kb promoter was amplified and cloned into pENTR/D-TOPO (Invitrogen), recombined into the pEarleyGate302 binary vectors to create an epitope-tagged 3xFLAG fusion (pSHL::SHL-3xFLAG), and transformed into Columbia-0 background. Western blotting and ChIP-seq experiments were conducted using tissues from the homozygous T3 transgenic plants. The wild type AtSHL, BAH (W63L/Y65A), and PHD mutants (F141A/Y148A) were generated in the pSHL::SHL-3×FLAG backbone using a Q5Escherichia coli strain BL21(DE3) RIL (Stratagene). The protein expression was induced by adding IPTG to a final concentration of 0.2 mM upon the OD600 of cell culture reaching to 0.7, followed by overnight induction at 20 °C. The recombinant protein was purified using a HisTrap column . The hexahistidine-sumo tag was cleaved by ulp1 protease and removed by a second step HisTrap column. The AtSHL protein was further purified using a Superdex G200 column . The PtSHL was cloned, expressed, and purified using the same strategy as AtSHL. All mutant versions of SHL were generated using a PCR based method and expressed and purified using the same protocol as wild type SHL protein. The peptides were purchased from the GL Biochem Company (Shanghai).The full-length AtSHL was cloned into a pET-Sumo vector to fuse a hexahistidine-sumo tag to the target protein. The plasmids were transformed into 55. Specifically, the peptide array slide was blocked with blocking solution  at 4 °C overnight. After washing three times with 1 ml TTBS buffer , the slide was incubated with 500 nM GST-tagged recombinant SHL in 1 ml binding buffer  at RT for 2 h. The slide was then washed with TTBS three times and incubated with an anti-GST antibody  in TBS with 1% BSA for 1 h at RT. After washing with TTBS three times, the slide was incubated with an anti-rabbit IRDye 650 secondary antibody  in 1 ml TBS buffer with 1% BSA for 1 h. The slide was washed with TTBS three times, dried by centrifugation, and imaged at dual wavelengths of 532 and 635 nm on Axon GenePix 4000B (Molecular Devices). The laser power was set to 100%, with automatic gain adjustment (0.05% saturation tolerance) for dual photomultipliers. Features in each block were defined by manual adjustment of 13 × 13 grid  to cover every spot. Signal intensities were quantified by GenePix Pro 6.1 software (Molecular Devices). For each spot, the mean intensities for 635 nm wavelength were used for subsequent analysis. For each peptide species, an average was calculated from three replicate spots. The signal at 532 nm wavelengths was used as a control to identify the misprinting events. Detailed information for peptides on the array can be found in Supplementary Data Peptide microarray assay was performed as following a previous studyThe 0.5 nM biotinylated histone peptides were first incubated with 62.5 μl magnetic streptavidin bead slurry (NEB) pre-washed with 1 ml peptide binding buffer  for 1 h at 4 °C. The peptide-bound beads were washed twice with 1 ml of peptide binding buffer and then incubated with 2 μg GST-tagged SHL proteins in the peptide binding buffer for 3 h at 4 °C. After washing three times with binding buffer, protein-bead complex was boiled in 1XSDS loading buffer , subjected to SDS-PAGE gel, and detected with an anti-GST antibody . The raw images of the western blots can be found in Supplementary Fig. 56. A summary of the statistics of the data collection for the two complexes is listed in Table The PtSHL protein was incubated with the histone peptides with molar ratio of 1:4 at 4 °C for 30 min. The PtSHL in complex with an H3(1-15)K4me3 peptide was crystallized in a condition of 0.2 M potassium acetate and 20% PEG3350. The PtSHL in complex with an H3(20–36)K27me3 peptide was crystallized in a condition of 0.2 M lithium sulfate, 2.0 M ammonium sulfate, and 0.1 M CAPS, pH 10.5. Both crystals were cryo-protected into the reservoir solution supplemented with 15% glycerol and flash cooled into liquid nitrogen. The diffraction data were collected at the beamline BL19U1 of the National Center for Protein Sciences Shanghai (NCPSS) at the Shanghai Synchrotron Radiation Facility (SSRF) and processed using the program HKL2000/3000 package57, with the structures of PHD finger of BPTF (PDB code: 2F6N) and BAH domain of PBAF (PDB code: 1W4S) as search models58. The manual model building and structure refinement were carried out using the programs Coot and Phenix59, respectively. Throughout the refinement, the geometry of structure models was monitored using the program Procheck60. The structure PtSHL-H3K27me3 was determined using the same protocol as the PtSHL1-H3K4me3 complex. The statistics of the structural refinement are summarized in Table 62.The structure of PtSHL1 in complex with H3K4me3 was solved using the molecular replacement method as implemented in the program PhenixThe interaction between SHL and histone peptides was monitored on a Microcal PEAQ-ITC instrument  at 20 °C. An additional artificial tyrosine residue was added to the C-terminus of the peptide in the ITC experiment for accurately measuring the concentration of peptide by OD280. The protein was dialyzed against a buffer of 100 mM NaCl and 20 mM Tris-HCl, pH 8.0 for 3 h and the lyophilized peptides were dissolved into the same buffer. The titration data were fit using the program Origin 7.0. The titration between AtSHL and H3(1-15)K4me3 peptide yielded serious precipitation, although we tried different pH, salt concentrations, and temperatures.FLAG epitope-tagged proteins were detected with horseradish peroxidase (HRP) conjugated anti-FLAG antibody . The following histone antibodies were used: H3 , H3K4me3 , and H3K27me3 . All western blots were developed using ECL Plus Western Blotting Detection System  and chemiluminescent imaging using an Imagequant LAS 4000 . The raw images of the western blots can be found in Supplementary Fig. 63. Two grams above ground tissues of three-week old plants were ground into powder in liquid nitrogen and cross-linked in nuclei isolation buffer I ) with 1% formaldehyde for 20 min at room temperature. Crosslinking was stopped with 2 M glycine and the homogenate was filtered through miracloth . Samples were pelleted and re-suspended with 0.3 ml of Nuclear Lysis Buffer  and 0.7 ml of ChIP Dilution Buffer  before shearing the chromatin by sonication.AtSHL ChIP was performed as following the instruction from a previously published study3) and reverse cross-linked at 65 °C overnight. After proteinase K and RNase treatment, DNA was purified by the standard phenol–chloroform method and was used for quantitative PCR  was performed as described above. For re-ChIP, the DNA-protein complex was eluted twice with 250 μl of 150 ng/μl 3×FLAG peptide  15 min at RT. The eluted SHL–DNA complex was equally divided into two tubes and incubated with 5 μl of either anti-H3K4me3  or anti-H3K27me3  antibody for 1 h at 4 °C. The samples were then incubated with pre-washed 40 μl magnetic protein A beads  overnight at 4 °C. The beads were washed with low salt buffer , high salt buffer , LiCl buffer , and TE Buffer . The DNA-protein complex was then eluted with Elution Buffer  and custom PERL scripts were used for further analysis. ChIP-seq peaks of H3K4me3 and H3K27me3 were obtained from66. In SHL genome-wide distribution pattern, log2 value of normalized ChIP read density relative to wild-type Col reads was calculated and binned in 100 kb intervals. For plots of H3K4me3 and H3K27me3 level over genes, each gene was divided into 20 intervals  separately for gene body, 2 kb upstream of the transcription start site, and 2 kb downstream of the transcription termination site. All statistical analysis and figures were generated using R (3.2.3). The total reads obtained are listed in Supplementary Table ChIP-seq libraries were constructed using an Ovation Ultralow DR Multiplex System  and sequenced using a HiSeq2000 (Illumina) in the UW-Madison Biotechnology Center. Sequencing reads were aligned to the Arabidopsis TAIR10 genome using Bowtie2 (v2.1.0) with default parameters5ZNP for PtSHL-H3K4me3 complex and 5ZNR for PtSHL-H3K27me3 complex. ChIP-seq data were deposited into GEO with the accession number GSE111780.Coordinates and structure factors have been deposited in the RCSB Protein Data Bank with the accession codes: Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Here, we demonstrate that, based on expression of Flt3, CD172a, CD13, and CD4, a precise identification of bovine blood conventional DC type 1 and 2 , plasmacytoid DC (pDC), and monocytes is possible with cDC1 being Flt3+CD172adimCD13+CD4−, cDC2 being Flt3+CD172a+CD13−CD4−, pDC being Flt3+CD172adimCD13−CD4+, and monocytes being Flt3−CD172ahighCD13−CD4−. The phenotype of these subsets was characterized in further detail, and a subset-specific differential expression of CD2, CD5, CD11b, CD11c, CD14, CD16, CD26, CD62L, CD71, CD163, and CD205 was found. Subset identity was confirmed by transcriptomic analysis and subset-specific transcription of conserved key genes. We also sorted monocyte subsets based on their differential expression of CD14 and CD16. Classical monocytes (CD14+CD16−) clustered clearly apart from the two CD16+ monocyte subsets probably representing intermediate and non-classical monocytes described in human. The transcriptomic data also revealed differential gene transcription for molecules involved in antigen presentation, pathogen sensing, and migration, and therefore gives insights into functional differences between bovine DC and monocyte subsets. The identification of cell-type- and subset-specific gene transcription will assist in the quest for “marker molecules” that—when targeted by flow cytometry—will greatly facilitate research on bovine DC and monocytes. Overall, species comparisons will elucidate basic principles of DC and monocyte biology and will help to translate experimental findings from one species to another.A clear-cut delineation of bovine  Bothbona fide DC can be classified into three subsets, each of them depending on different transcription factors for their development  has been described containing a distinctive transcriptome  and in their phenotypic plasticity depending on tissue localization and inflammatory state  of cattle have been classified into two subsets . As an anticoagulant, citrate-based Alsever's solution  was used. The blood sampling was performed in compliance with the Swiss animal protection law and approved by the animal welfare committee of the Canton of Bern, Switzerland, license number BE102/15. For PBMC isolation, blood was first centrifuged at 1,000 × g for 20 min. Then the buffy coat was collected and diluted with PBS to a ratio of 1 to 1 before being layered onto lymphocyte separation medium . After centrifugation , PBMC were collected and washed twice with cold PBS containing 1 mM EDTA . A final washing step was done at 250 × g (8 min) to remove platelets.Blood of cows  was collected at the Clinic for Ruminants  or at the Institute of Virology and Immunology  by puncturing the jugular vein. For sorting experiments and transcriptomic analyses, six different cows were used . Primary antibodies and secondary reagents are listed in Table 6 cells were recorded in the “large-cell” gate.Six-color phenotyping of DC subsets was performed in 96-well U-bottom microtiter plates with 2 × 10+CD4+, putative cDC1 as Flt3+CD4−CD13+CD172alow, and putative cDC2′ as Flt3+CD4−CD13−CD172a+ using a FACS Aria (BD Biosciences).For sorting of putative DC subsets, freshly isolated bovine PBMC were enriched for Flt3 expression by magnetic-activated cell sorting  using His-tagged bovine recombinant Flt3L followed by anti-His-PE, and anti-PE magnetic beads (both Miltenyi Biotec). Enriched DC (~60-fold) were stained with anti-CD4, anti-CD13, and anti-CD172a and corresponding secondary antibodies anti-mouse IgG2a-PECy7, anti-mouse IgG1-Alexa488, and anti-mouse IgG2b-Alexa647 , CD172a+CD14+CD16+ (intM), CD172a+CD14−CD16high (ncM) and CD172a+CD14−CD16−Flt3+ (cDC2′′) populations were sorted using a FACS Aria (BD Biosciences). All sorted subsets had a purity of at least 97%.In order to sort for monocyte subsets together with cDC2, a two-step staining was performed with 3 x 10http://www.ebi.ac.uk/ena) under the accession number PRJEB28324.FACS-sorted cell subsets were frozen to minus 80°C in TRIzol (ThermoFisher) for later RNA extraction. Total RNA was extracted using the Nucleospin RNA kit (Macherey Nagel), as recently described for porcine DC subsets . Quality+ cDC1, blood CD1c+ cDC2, blood pDC, blood CD14+ monocytes) and mouse . All gen = 1,000 genes each from the top and bottom of the lists. The relative similarity among the cell types was generally consistent for other values of n . To assess the statistical significance of the similarity scores, the observed values were compared with a null distribution obtained by reshuffling the genes. Because invariant genes do not influence the similarity score, the middle 60% of genes were excluded from the permutations.Each cell-type specific signature from cattle was compared with all human, mouse and pig signatures using the R package OrderedList v.1.44.0 . This towww.inkscape.org).Figures were prepared using FlowJo version 10 , GraphPad Prism version 7.03 for Windows , R version 3.4.2, and Inkscape  were sorted together with cDC2 , intermediate (intM), and non-classical (ncM) monocytes throughout figures and text. The cDC2 subset sorted together with the monocyte subsets was labeled cDC2′′ to keep it apart from the cDC2 subset sorted together with pDC and cDC1, which was labeled cDC2′. The gating strategies for both sortings are shown in Figure ITGAM (CD11b). Transcription of CD163 was limited to CD14+CD16− cM. The chemokine receptor CX3CR1 was found to be mainly transcribed by CD16+ monocytes, and to a lesser extent by cDC2  clustered together, but distinctively apart from CD14+CD16− cM. To our surprise, the transcriptomes of cDC2′ and cDC2′′ show consistent differences  showed a high transcription rate in cM whereas RIG-I and MDA5 for detection of viral RNA showed a high level of transcription in CD16+ monocytes (intM and ncM), though inter-individual variation was found to be high.Transcriptomic data revealed pronounced differences between subsets in regard to gene transcription related to pathogen recognition, migration, antigen presentation and antimicrobial activity Figures , 8. We fCCR1 and CCR2 transcripts. Non-classical monocytes lacked the transcription of CCR2 and transcribed very low levels of CCR1, but together with intM they transcribed the highest levels of CX3CR1 , showed the highest transcription level in pDC and classical monocytes, and was only weakly transcribed in cDC1. Intermediate monocytes and ncM showed high transcription rates of ITGA4, ITGB1, PECAM1, ITGAL, and ITGB2  and 24 genes downregulated . For cDC2 (cDC2′ and cDC2′′), only transcripts for five genes were found to be exclusively upregulated and only ECE1 was found to be at least 5-fold weaker transcribed compared to all other subsets. In cM, we found specific transcription of genes involved in phagocytosis  and antimicrobial activity . In total, 46 genes were up- and 6 genes downregulated in cM. Intermediate monocytes and ncM were treated as one subset, as—with the criteria mentioned above—no genes were found to be specifically transcribed when they were treated as separate subsets. Together, intM and ncM showed specific transcription of genes related to angiogenesis  and transcripts involved in the classical pathway of complement activation , with 60 genes specifically up- and 7 genes specifically downregulated . Many of the pDC-enriched genes are associated with B-cell development and function. Among the genes that were specifically downregulated in pDC were the inflammasome-associated genes ADORA3, TSPAN5, SEMA4B and six different solute carrier genes. Transcription of genes coding for C-type lectins was found to be specific not only for cDC1 (CLEC9A) and cDC2 (CLEC10A), but also for cM . Transcripts of SEMA7A were specifically enriched in intM and ncM, whereas SEMA4A transcription was found to be specifically downregulated in these two monocyte subsets.Molecule classes repetitively found to be differentially transcribed across subsets and reported to be relevant for immune responses are illustrated as heat maps in Figure The subset-specific transcription patterns of these genes allow insights into functional specialization of subsets, which, when conserved across species, may be of particular relevance for subset-specific contributions to immune responses.+CD4+CD13− and Flt3+CD4−CD13+, respectively. The identification of cDC2 remains challenging in all species due to their phenotypic similarity to monocytes, and the paucity of cDC2-specific key genes. We therefore directly compared the transcriptome of monocyte subsets and putative cDC2. Accordingly, we employed two different gating strategies for cDC2. One based on the expression of Flt3 and the exclusion of pDC and cDC1, that is Flt3+CD4−CD13−, and the other based on the expression of Flt3 and CD172a, and the exclusion of monocytes, that is Flt3+CD172a+CD14−CD16−. Both putative cDC2 populations contained high levels of FCER1A transcripts and transcribed IRF4 in the almost complete absence of IRF8 transcripts, supporting their correct identification .Bovine DC subsets have been addressed in numerous studies, looking at blood –37, 39, i et al.  followed− subset . These s− subset , all bloGibson et al.  could deAXL in bovine cDC2, a gene associated with a DC precursor subset recently detected in human blood by single-cell sequencing of lineage−HLA-DR+ cells  we observed on bovine pDC in comparison to the other DC subsets has also been described for murine pDC in tissues and might be related to high iron demand of pDC upon rapid and large-scale production of type I interferons  has been shown to function as a co-inhibitory receptor favoring tolerogenic responses  and also expresses high levels of CD62L, allowing this subset to access lymph nodes directly from the blood via HEV, as reported for murine cM under inflammatory and steady-state conditions (+CD16− cM (+ monocytes expressing CD14 (intermediate) and monocytes almost lacking CD14 expression . Analogous to the human subsets, we have also sorted intM and ncM, however they were found to be very similar in their transcriptomic profile, as determined by PCA, and may therefore not represent functionally distinct subsets in cattle. Our data demonstrate that CD16+ monocytes (intM and ncM) in cattle most probably resemble Ly6C/Gr1− monocytes in mice, which were suggested to preferentially give rise to alternatively activated macrophages that contribute to tissue regeneration  family, which seems to be—at least in part—conserved across species. tivation , 85, 86.tivation  and, as tivation . Althougtivation , the rolPurinergic receptors can evoke pro- and anti-inflammatory immune responses when binding extracellular nucleotides released by cellular stress or apoptosis . SignalihighCD82low, whereas late activated BMDC showed the inverse phenotype, with a migratory, cytoskeletal and antigen presentation machinery optimized for activating naïve T cells. Steady-state bovine classical DC completely lack transcription of CD82, but clearly contain mRNA of CD37 , suggesting that the findings in mice might be transferable to cattle. For the tetraspanins CD9, CD37, CD63, CD81, CD82, and CD151 it is suggested that they play an important role in regulating the function of DC surface receptors (CD9, a tetraspanin that is otherwise broadly expressed in leukocytes and was shown to function in the stabilization of the immunological synapse (In mice, the tetraspanins CD82 and CD37 have been described to be involved in migration and antigen presentation of DC . Non-acteceptors . In agre synapse .SEMA4B, it may be speculated that they assist SEMA4B-producing T cells in regulating basophil responses. SEMA4A transcripts were enriched in both bovine pDC and cDC1. SEMA4A has been shown to induce activation and differentiation of T cells into antigen-specific subsets (+ monocytes, has been described as a potent autocrine activator of human monocytes (+ monocytes.Semaphorins play important roles in guiding immune responses . We foun subsets , and to  subsets . Also SE subsets  and to p subsets . Finallyonocytes , howeverIn summary, a clear-cut identification of bovine DC and their delineation from monocytes, as presented here, provides the basis for future studies on their functions and their involvement in disease pathogenesis, which will enable a compilation of strategies to improve vaccines and immunotherapeutics. This work also represents the basis for future work addressing transcriptomic signatures and functions during infection and inflammation, which includes different lymphoid and non-lymphoid tissues. In addition, comparative studies on DC function in mammals provide a deeper understanding of DC biology in general, as they reveal conserved and species-specific characteristics of DC subsets.The animal experiments were performed according to the local law and were approved by the Ethical Committee for Animal Experiments of the Canton of Bern.ST performed laboratory work, analyzed the data, and wrote the manuscript. AB and GB performed laboratory work and analyzed data. IK and RB performed bioinformatic analyses. AS designed and supervised the overall project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ehrlichia chaffeensis is an obligate intracellular tick-borne bacterium that causes human monocytic ehrlichiosis. Studying Ehrlichia gene regulation is challenge, as this and related rickettsiales lack natural plasmids and mutagenesis experiments are of a limited scope. E. chaffeensis contains only two sigma factors, σ32 and σ70. We previously developed Escherichia coli surrogate system to study transcriptional regulation from RNA polymerase (RNAP) containing Ehrlichia σ32 or σ70. We reported that RNAP binding motifs of E. chaffeensis genes recognized by σ32 or σ70 share extensive homology and that transcription may be initiated by either one of the sigma factors, although transcriptional efficiencies differ. In the current study, we investigated mapping the E. chaffeensis dnaK gene promoter using the pathogen σ32 expressed in E. coli lacking its native σ32. The E. coli surrogate system and our previously described in vitro transcription system aided in defining the unique −10 motif and spacer sequence of the dnaK promoter. We also mapped σ32 amino acids/domains engaged in its promoter regulation in E. chaffeensis. The data reported in this study demonstrate that the −10 and −35 motifs and spacer sequence located between the two motifs of dnaK promoter are critical for the RNAP function. Further, we mapped the importance of all six nucleotide positions of the −10 motif and identified critical determinants within it. In addition, we reported that the lack of C-rich sequence upstream to the −10 motif is unique in driving the pathogen-specific transcription by its σ32 from dnaK gene promoter. This is the first study in defining an E. chaffeensis σ32-dependent promoter and it offers insights about how this and other related rickettsial pathogens regulate stress response genes. Ehrlichia chaffeensis, a Gram-negative, obligate intracellular tick-borne rickettsial pathogen, causes human monocytic ehrlichiosis (HME)  . HME as is (HME) . Furtherled deer . The patled deer . HME patled deer . In addint years . Though ffeensis . and simnaplasma , the gennaplasma . Severalronments . HoweverEscherichia coli possesses seven σ factors, while 109 σ factors are identified in Sorangium cellulosum  (the primary housekeeping σ70 gene) and rpoH (ECH_0655)  (Regulation of gene expression in bacteria is primarily controlled at the transcription. An RNA polymerase (RNAP) core enzyme with a sigma (σ) factor offers a simple and valid mechanism for bacteria to rapidly accommodate to diverse environmental changes by suitably modifying the transcriptional profiles . Typicalllulosum . Obligatllulosum . For exa32 gene)   encoding for DnaK protein is transcribed primarily by σ32  provides abundant knowledge regarding gene regulation from its σ32- and σ70-bound RNAP, while such knowledge for both σ32-and σ70 bound RNAP in other Gram-negative bacteria, particularly for α-proteobacteria, including for pathogenic organisms is very limited. Importantly, it is unclear how intracellular pathogens, such as E. chaffeensis, regulate gene expression to overcome the host stress and adapt to host environmental changes within its arthropod (tick) and vertebrate hosts. To extend knowledge on how E. chaffeensis regulates its gene expression, we continue investigations in defining the functions of its RNAP holoenzyme comprising σ32 or σ70. Transcription derived by an RNAP typically implicates in recognizing and binding to DNA sequence motifs of a promoter; −10 and −35 regions, and the spacer sequences located between the two motifs of a gene promoter  is inactivated , BL21(DE3) , and CAG57101  and the modified plasmid was referred as the pSAKT. The pSAKT-Ech_rpoH (previously known as pSAKT32- Ech_rpoH) contained E. chaffeensis rpoH . The names of the modified pSAKT-Ech_rpoH are provided in CAG57101 . SeveralCAG57101 , pQF50K-CAG57101  and pMT5CAG57101 . Geneticpromoter . The E. sis rpoH . E. chaflacZ) with a pMB1 origin of replication and with a kanamycin resistance gene, was reported earlier . The names of all engineered plasmids were listed in For  earlier . ConstruE. coli strain CAG57101 transformed with the recombinant plasmids were grown as in early reports . E. coli cultures of CAG57101 in LB medium were grown overnight with appropriate antibiotic supplements which were diluted 1:100 into a fresh medium containing appropriate antibiotics and the growth was continued for 2 h. Subsequently, cultures were then induced with 1 mM IPTG for 3 h before harvesting, when OD at 600 nm reached between 0.6 and 0.8. Lysates were prepared and used to measure β-galactosidase enzyme activity using a β-Gal Assay Kit . All experiments were performed three independent times with independently grown cultures; specific activity of β-galactosidase was calculated as outlined in the kit protocol.The  reports . Briefly32  mixed with 10-fold molar excess of purified recombinant E. chaffeensis σ32 and kept in ice for 30 min prior to using for the reactions. The transcription reactions were performed at 37°C for 20 min, and the reactions were terminated by adding 7 μl of stop solution . Six microliters each of the samples were resolved on a 6% polyacrylamide sequencing gel with 7 M urea, then gels were transferred to a Whatman paper, dried and 162 nucleotide transcripts were visualized by exposing an X-ray film to the gels. The transcripts were quantified using ImageJ software1.In vitro transcription reactions were performed in 10 μl reaction mixture containing 0.1 picomoles each of the supercoiled plasmid DNA as the template and using RNAP holoenzyme containing recombinant E. chaffeensis σ32 . The holMultiple DNA alignments were done using the programs Clustal X version 2.0 with default parameters .t-test, and a P-value < 0.05 was considered significant with a single asterisk.Statistical analyses were performed using Student’s 70-bound RNAP holoenzyme transcription from σ70-dependent promoters in E. chaffeensis  compared to the dnaK wildtype (WT). The reduction of the β-galactosidase activity for the −10 motif deletion was similar to the negative control where the promoter segment was absent (NP).Our prior studies demonstrated that −35 motif, but not −10 motif, is required for the σffeensis . We alsoloenzyme . To testnsis σ32 . The −10E. chaffeensis promoters recognized by its σ32 (TTGTAT) is similar to the consensus −35 motif of σ32-dependent promoters in E. coli (TTGAAA) and similarly it shares extensive homology to −35 motif for the genes recognized by σ70 (TTGNTT) (E. chaffeensis promoters recognized by its σ32 (TATATN) is also similar to its consensus −10 motif recognized by its σ70 (TATTNT), however, it differs significantly from the consensus −10 motif of E. coli σ32-dependent promoters (CCCCATWT) , whereas A2G mutation caused decline of two thirds of activity similar to T1A substitution. In the third and fourth positions, changes to any other nucleotide had the greatest impact in promoter activity decline (78–98% reduction). Mutations in the fifth position to T5G or T5C had a greater impact (a near 90% decline), while reduction was less apparent for the T5A that is similar to the mutations in the first position T. In the sixth position, only C6G mutation resulted in significant decline in the promoter activity to 35%.The consensus sequence of −35 motif for (TTGNTT) . The conCCCATWT) . While dffeensis , such deactivity . These nund RNAP , the −10activity . We haveE. coli surrogate system, we performed in vitro transcription assays using several randomly selected promoter mutation constructs; the assays were performed using RNAP holoenzyme reconstituted with the recombinant E. chaffeensis σ32. We selected five dnaK −10 motif mutants for this experiment and compared the results with the wildtype promoter. Both wildtype and mutated versions of dnaK promoter segments were cloned into the G-less cassette and used as templates in the in vitro transcription assays , or with a high GC content spacer while keeping the spacer length constant (GC), or by increasing the spacer sequence lengths from 17 bp to 18, 19, or 20 bps or by decreasing it to 16, 15, or 14 bp and finally by deleting the spacer sequence completely. These different spacer mutant constructs were depicted in E. coli surrogate system (CAG57101)  . The CP AG57101) .E. coli revealed that the −10 motif of σ32-dependent promoters is recognized by few amino acids within the regions 2.3, 2.4, and 3.0 of σ32 protein  at position 112 in E. coli σ32 is also implicated for its contribution to −13C recognition and interaction with its σ32-dependent promoters  at position 130 in region 3.0 of E. coli σ32 is similarly implicated for the recognition of the C-rich region for the σ32-promoters; groE and grpE , while F110 substitution to A or E  in E. chaffeensis σ32 did not significantly alter the promoter function  at position 104 within the 2.3 region of E. coli σ32 is identified as critical for its structural integrity and activity of σ32  {tyrosine (Y)} is present at the position homologous to F104. Similarly, A111 of E. coli σ32 is implicated for its binding to core RNAP (E. chaffeensis σ32 activity (88% reduction). Likewise, substitution of A109 to glutamine (Q) in E. chaffeensis σ32 that is homologous to A111of E. coli σ32 resulted in a significant decline of activity (about 31% of WT level). Previous studies in E. coli demonstrate that substitution of F136A within the region 3.0 reduces the interaction between core RNAP and σ32 thus leading to 80% decline in promoter activity for its groE gene . Together, E. chaffeensis σ32 substitution mutation experiments allowed the identification of critical functional domains engaged in σ32-bound RNAP interactions with the dnaK promoter, including in confirming that the C-rich region is not critical for its function.Prior studies in  protein . To detecoli σ32 . Tryptoped by it . In E. cromoters . Phenylaand grpE . Glutamifunction . Similary of σ32 . In E. core RNAP . Y102 toroE gene . A similE. coli and other γ- proteobacterial gene promoters, −10 motif of E. chaffeensis σ32-dependent dnaK promoter (TATATN) is distinct in lacking a C-rich sequence upstream to −10 motif  were replaced with either two Cs or four Cs (E. chaffeensis −10 motif to be more similar to the E. coli consensus −10 motif (CCCCATWT) (E. coli surrogate system expressing either E. chaffeensis σ32 (Ech- σ32) or E. coli σ32 (Eco-σ32). Compared to the wildtype dnaK promoter, both the 2C and 4C mutants caused drastic decline in the β-galactosidase expression by as much as 15-fold for E. chaffeensis σ32  . The modnsis σ32 . On the E. coli. Obligate intracellular bacteria typically have reduced genomes and consequently their genomes also have limited numbers of sigma factors. For example, E. chaffeensis genome has only two sigma factor genes encoding for a constitutive and an alternative sigma factor, σ70 and σ32, respectively    is also similar to E. coli consensus −35 motif (E. chaffeensis σ32-dependent promoters (TATATN), however, differs substantially from the consensus E. coli−10 motif (CCCCATNT)   are simictively) . We reporomoters . The pre35 motif . The −10CCCATNT) . The conbacteria , while −gulation . Such seromoters . Thus, wactivity . In partactivity . Consistr region .E. chaffeensis dnaK−10 and −35 motifs and the spacer sequence upstream to it by performing detailed mutational analysis. Furthermore, this study confirmed that the C-rich region–specific interactions between a gene promoter and σ32 protein domains, known to be critical for E. coli, are absent in E. chaffeensis, which makes the pathogen gene regulation distinct, as judged from characterizing the dnaK gene promoter.In summary, we mapped The datasets generated for this study are available on request to the corresponding author.RG conceived and directed the research design. HL contributed to the research plan and performed the experiments. HL and RG evaluated the data and prepared the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.