The general transcription factor IID (TFIID) is the first component of the preinitiation complex (PIC) to bind the core promoter of RNA polymerase II transcribed genes. Despite its critical role in protein-encoded gene expression, how TFIID engages promoter DNA remains elusive. We have previously revealed a winged-helix DNA-binding domain in the N-terminal region of the largest TFIID subunit, TAF1. Here, we report the identification of a second DNA-binding module in the C-terminal half of human TAF1, which is encoded by a previously uncharacterized conserved zinc knuckle domain. We show that the TAF1 zinc knuckle aids in the recruit of TFIID to endogenous promoters vital for cellular proliferation. Mutation of the TAF1 zinc knuckle with defects in DNA binding compromises promoter occupancy of TFIID, which leads to a decrease in transcription and cell viability. Together, our studies provide a foundation to understand how TAF1 plays a central role in TFIID promoter binding and regulation of transcription initiation. The highly-orchestrated assembly of the general transcription factors (GTFs) TFIIA, B, D, E, F, and H is responsible for properly positioning RNA polymerase II (RNAPII) at core promoters and initiating transcription. TFIID nucleates PIC formation by directly binding to promoter sequences. TFIIA further stabilizes this interaction, while TFIIB association is firmly established by TFIIF/Pol II recruitment, and TFIIE/TFIIH are essential for promoter clearance4.The formation of the preinitiation complex (PIC) is a step-wise process required for eukaryotic transcription of protein-encoded genes5. A small subset of these factors recognizes specific core promoter elements (CPEs) to orient the PIC at the transcription start site8. TFIID’s ability to selectivity recognize promoter sequences was initially thought to be largely dictated by TBP binding to the TATA box9. The primary evidence for this model centers on TBP’s ability to support a low level of transcription in the absence of TAFs, whose roles include responding to transcriptional activators11. Growing evidence supports the idea that TBP alone does not identify gene promoters, especially in higher organisms12. The majority of human protein-coding genes lack a TATA box sequence, yet TFIID is still able to engage the initiator and direct transcription from TATA-less promoters13. Therefore, other PIC components must be involved in dictating DNA specificity. A recent study demonstrated the TAF components of TFIID are vital for sequence selectivity while TBP alone showed no sequence preference14. This expands upon previous DNA footprinting analyses that illustrated an extended region of protection by TFIID compared to TBP, which was interpreted as TAFs binding near the transcriptional start site and downstream promoter sequences15.TFIID is a multi-subunit complex comprised of the TATA binding protein (TBP) and 14 TBP associated factors (TAFs) in humans2. The Initiator (Inr) is positioned at the transcription start site of over 40% of human protein-encoding genes and is enriched at TATA-less promoters17. Motif ten element (MTE), downstream promoter element (DPE), and downstream core element (DCE) are located downstream of the transcription start site and thought to contribute to promoter recognition in the absence of a TATA box19. TAF1, the largest subunit of TFIID, has been shown to associate with the DCE, a discontinuous sequence found in viral promoters as well as the human beta globin gene19. TAF1 also interacts with the Inr when in complex with TAF26. While there is a wealth of knowledge about promoter sequences, little is known about TAF/DNA binding interfaces and their mode of interaction. Characterizing these protein-DNA interactions will advance our understanding of how the entire TFIID complex mediates transcriptional initiation.The specific combination of CPEs varies from gene to gene, and their unique arrangement may help to ensure genes are expressed at specific times in response to cellular needs. Most of these elements are recognized by TAFs to enhance the interaction between TFIID and promoter DNA20. When our structure of TAF1 DUF domain bound to TAF7 was modeled into the cryo-EM structure of promoter bound TFIID, it confirmed the WH can contact downstream promoter sequences21. The cryo-EM structure further revealed a small secondary density of protein contacting the Inr, which the authors attribute to TAF1. The advancements in cryo-EM have given us a general idea of the overall shape of TFIID and insights into the topological rearrangements that ensue upon promoter binding22. Yet, further refinements are necessary to definitively resolve all the crucial interfaces between the TAF subunits of TFIID and core promoter sequences during the dynamic structural reorganization evoked by promoter engagement.Our previous work describing a winged helix (WH) located in the central DUF3591 domain provided the first look at TAF1’s promoter recognition capabilities19. The DCE is a cluster of three non-contiguous sequences. Each was shown to be important for transcription and can crosslink to TAF1; the current model suggests TAF1 directly binds the entire DCE, a region too large to be bound by the WH alone. Moreover, yeast TAF1 has been shown to contain a promoter-binding region near its C-terminus outside the WH23. This region corresponds to sequence adjacent to the double bromodomain in the human homolog, and yeast TAF1 lacking this region was unable to bind promoter DNA. Multiple DNA binding domains in TAF1 may convey plasticity to allow recognition of diverse promoter sequences and permit structural rearrangements during promoter engagement.The idea that TAF1 contains multiple DNA binding domains is supported by several pieces of evidence. In addition to structural data, TAF1 has been shown to bind two entirely distinct and separate promoter elements (i.e. Inr and DCE) implying multiple binding modulesin vitro conditions, we identified a second previously uncharacterized DNA binding module in the largest subunit of TFIID. Our study shows that TAF1 ZnK is important for cellular viability and helps to direct TFIID to endogenous core promoters through its DNA binding abilities. This work establishes a basis for dissecting the complexity of TFIID promoter recognition.Hidden between the double bromodomain and the DUF domain of TAF1, we discovered a strictly conserved CCHC zinc knuckle (ZnK) that plays a vital role in transcriptional regulation. By examining human TAF1 ZnK function under both native and ts13 mutant cell line, this domain harbors a temperature-sensitive point mutation (G716D) that causes cells to cell cycle arrest in late G1 and eventually undergo apoptosis26. We previously characterized the structural features of this domain with its binding partner TAF7 and discovered a winged-helix (WH)20. The second region of strict conservation sits between DUF3591 and the double bromodomain (2 × Bromo). Zooming in on the amino acid sequence within this region reveals an invariable Cx2Cx4Hx6C motif , using an antibody against the TAF4 subunit to immunoprecipitate the entire TFIID complex, and immunoblotting for exogenously expressed TAF1 variants using an anti-HA antibody. The presence of other TFIID subunits was examined by western blotting ; an endogenous promoter, cyclin D1 (CD1P); and a random DNA sequence (Random). We observed that TAF1 ZnA bound to all three DNA fragments  to determine if the ZnK domain binds DNA. The protein fragment used in this assay contained the annotated ZnK domain and flanking regions  so as to not exclude any potential interacting residues. Increasing concentrations of ZnA protein were incubated with three different nts Fig. . We follnts Fig. . Dissocints Fig. . InteresTo delineate the minimal ZnK DNA binding domain, TAF1 ZnA protein was incubated with IMD DNA fragment, and the mixture subsequently exposed to increasing concentrations of subtilisin protease. Digestion products were separated by SDS-PAGE, and the pattern of protein fragments compared to the apo-ZnA digestion pattern, generated in the absence of IMD incubation. Protein regions bound to DNA were protected from proteolytic cleavage and led to three stabilized species Fig. . As anti42. Strong connections are given high coupling scores and indicate the residues have a high probability of existing in the same three-dimensional space. Plotting the connections illustrates the distance between co-evolved residues and identifies modular domains within proteins. EVfold analysis revealed the ZnA construct contains two smaller subdomains: the N-terminus from aa 1234–1313, which contains the ZnK, and a C-terminal domain spanning aa 1313–1375  and ZnD (503 nM ± 67 nm) were similar to that obtained for ZnA, indicating that ZnD represents the core domain of DNA binding. As expected, mutations to the strictly conserved cysteines completely abolished binding . Firstly, several points of contact could aid in forming a secure connection between TFIID and a corresponding promoter long enough to recruit the other essential components of the transcriptional machinery. Additionally, multiple DBDs could confer TFIID the necessary flexibility to associate with a variety of core promoter sequences throughout the human genome, such that recognition of different promoters might involve different DNA binding domains. Last but not least, multiple DBDs might be important for the dynamic rearrangement of TFIID, which transitions from the conical form to the promoter bound form, as demonstrated by Cryo-EM studiesion Fig. . Either 45. The impact of the TAF1 ZnK mutations on cyclin transcription is not due to an inability to incorporate into TFIID, implying this domain is not involved in TAF interactions or the core structure of TAF1, both important for TFIID assembly. While, this study focuses on the ability of ZnK to bind nucleic acids, our findings do not exclude the possibility the ZnK may interact with other regulatory proteins. The close proximity of the ZnK to the double bromodomain suggests the ZnK could play a role in facilitating the recognition of epigenetic markings, which could influence promoter association. There is growing evidence that zinc fingers can play a dual role: supporting DNA binding and facilitating protein interactions with histones or transcriptional activators, such as those found in CBP and ZYMND1149. However, our experimental approach cannot rule out this possibility considering our transcription assays were conducted with luciferase reporter plasmids not known to incorporate histones; despite this caveat, we conclude that the impact on cyclin transcription results from a defect in active PIC formation, attributed to loss of promoter binding by the ZnK mutant containing TFIID complex. The inability to stimulate transcription from the cyclin promoters would explain why the ZnK mutant cannot rescue the ts13 growth defect in the cell complementation assay.Cyclin proteins are master regulators of the cell cycle. Their phasic expression mediates enzymatic activities required for growth and proliferation51.The mechanisms proposed in this work may also provide some insight into the pathologies of human disease states associated with TAF1 mutations by GWAS. The cBioPortal and catalog of somatic mutations in cancer (COSMIC) databases denote several mutations in the conserved CCHC residues of TAF1 including: C1285R, C1288R, H1293N, C1300W/R. While it is unknown whether these mutations are causative, they reinforce the notion that these residues are crucial for proper TAF1 function in cells. TAF1 is essential for cell viability with deletion of the gene lethal to cells. For this reason, it is doubtful that disease-associated mutations completely obliterate TAF1 function and cause global transcriptional dysfunction. More likely, TAF1 function is altered in some subtle manner that results in aberrant transcription from select genes. The role of TFIID in proliferation and cell survival makes it of particular interest to cancer biologyin vivo function of TAF1. Mutations within this region do not disrupt the stability of TAF1 or its incorporation into TFIID, but instead interfere with its ability to associate with core promoters. The loss of promoter association would result in inefficient transcription initiation. We speculate that two motifs in TAF1 provide greater flexibility within TFIID for promoter DNA sequence recognition.In summary, we uncovered a second critical DNA binding domain in TAF1, the largest subunit of the TFIID general transcription factor. The conserved zinc knuckle is essential for the full 52. Point mutations in TAF1 were introduced by site-directed mutagenesis and confirmed by DNA sequencing  supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin. For complementation assays, cells were seeded into 6-well plates, grown overnight to 70–80% confluency and transfected with 2 μg of CS2 + or TAF1 expression plasmids using polyethylenimine (PEI) transfection reagent (ratio 2.5:1 of PEI:DNA) according to previously described protocol53. Transfected cells were maintained for an additional 18–24 h at 33.5 °C, then either harvested for Western Blot analysis or shifted to nonpermissive temperature of 39.5 °C. Number of DAPI positive viable cells was determined after 36–48 h at 39.5 °C, and the percentage relative to WT-TAF1 transfected cells  was calculated.Mammalian TAF1 expression plasmids contain N-terminal HA-tagged human TAF1 coding sequence inserted downstream of the CMV promoter in CS2 + vector54. The human cyclin A2 luciferase construct has been described55. The IMD luciferase construct is previously described in Juven-Gereshon et al.56 and a gift from J. Kadonaga. HEK293 cells were grown at 37 °C in Dulbecco’s modified Eagles medium (Gibco) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin. For luciferase assays, cells were seeded into 24-well plates, grown overnight to 60%-70% confluency and cotransfected with 100 ng of CS2 + or TAF1 expression constructs and 50 ng luciferase reporter construct using FuGene transfection reagent (ratio 2.5:1 of FuGene:DNA) according to the manufacturer’s protocol (Roche). Transfected cells were maintained an additional 36 h at 37 °C, after which cells were harvested and lysed in 100 μL of 1× Passive lysis buffer (Promega). Luciferase activity was measured according to manufacturer’s protocol (Promega) on Lumat LB 9507 (Berthold Technologies) and normalized for total protein. Transcriptional activity was expressed relative to CS2+/reporter construct transfected cells (set to 1).Human cyclin D1 luciferase construct was constructed by subcloning the EcoRI to PvuII fragment of pD1-G065 (kindly provided by Yue Xiong) into the SmaI site of pGL2-basic as previously described−ΔCt.ts13 cells seeded into 35-mm dishes were grown at 33.5 °C to 60–70% confluency and transfected with 500 ng of CS2 + or HA-TAF1 expression plasmids using PEI (2.5:1 PEI:DNA ratio). Total RNA was isolated approximately 24–36 hours post-transfection and 1 μg RNA reverse transcribed in cDNA using iScript cDNA synthesis kit (BioRad). Cyclin D1 transcript levels were measuring by qPCR using SsoFast EvaGreen Supermix (BioRad) on the Applied Biosystems 7500Fast Real-Time PCR system. Relative transcript levels were determined by calculating 257. In brief, cells were lysed in 0.5 mL immunoprecipitation (IP) buffer  containing protease inhibitors and sonicated 8 times for 20 s at 40% amplitude . Nuclear extract was incubated overnight at 4 °C with either anti-HA  or mouse IgG (Abcam). Immunoprecipitated complexes were washed and eluted. Cross-links were reversed and DNA was isolated. Input DNA was purified from 100 μl extract. Fifty nanograms of input DNA and 2 μL of purified TAF1 bound DNA was amplified with SsoFast EvaGreen Supermix (Bio-Rad) using primers spanning the promoters of cyclin D1 and cyclin A2  and incubated on ice for 15 min. Cells were lysed by pushing through a 25-gauge needle 5 times. The crude nuclear pellet was isolated by centrifugation for 20 s at 12,000 g, resuspended in buffer C , and incubated for 30 min at 4 °C. After centrifugation for 5 min at 12,000 g, the supernatant/nuclear extract was incubated with anti-TAF4  or anti-HA antibody overnight at 4 °C. Ten microliters of protein A–Sepharose CL-4B  was added, and the samples were nutated for 2 h at 4 °C. Isolated proteins were washed 5 times with Buffer C and analyzed by silver stain or immunoblotting using antibodies against HA , TAF1 bromodomain , TBP , TAF4 (mAb 3A6), and TAF5 .For preparation of nuclear extracts, HEK293 cells were resuspended in buffer A  was PCR amplified from CS2 + containing full length TAF1 and cloned into pAL-GB1 using ligase independent cloning; site-directed mutagenesis was used to introduce point mutations into ZnC and ZnD constructs  was added and binding reactions were loaded onto nondenaturing 5% polyacrylamide (37.5:1 acrylamide:bis) pre-run in 0.5× TBE at 100 V for 30 min. Samples were resolved for 1.5 h at 100 V and shifted complexes detected by autoradiography. The sequences of DNAs used for EMSA are provided in Supplemental Table Purified TAF1 (ZnA-1234–1371) (7.6–61.3 pmoles) purified from 2, 1 mM DTT) by heating at 72 °C for 5 minutes and slow cooling to 25 °C. Double-stranded oligos  for 30 min then dipped into individual wells on 96-well plates (200 μL/well) containing biotinylated DNA (100 nM) in assay buffer. Unbound DNA was removed by washing and exposed streptavidin blocked with biocytin. Protein was diluted in assay buffer to 3 μM, 1 μM, 333 nm 111 nm, 37 nm, and 12 nm. Association of the protein to the probe was observed for 300 s followed by dissociation in assay buffer for 300 s. Throughout loading, association and dissociation sensors were shaking  at a constant 30 °C to promote specific interaction and to reduce non-specific binding. A reference sensor (no biotinylated DNA loaded) was included and subjected to the same procedure to control for non-specific interactions. Sensors were regenerated  between experiments and a new baseline established. Reference-subtracted data was used to calculate the equilibrium dissociation constant (Kd) by steady-state response analysis assuming 1:1 kinetics.5′-biotinylated oligonucleotides were annealed to their complement  in 1× binding buffer  for 1 h at 25 °C. Subtilisin  was added to each sample and incubated overnight on ice at 4 °C. Samples were divided in two and analyzed as follows: SDS-PAGE, coomassie stained, and scanned for analysis; SDS-PAGE, transferred to PVDF membrane, coomassie stained and stabilized fragments excised and N-terminally sequenced.TAF1 (aa 1234–1371) was exposed to limited proteolytic digestion in the presence and absence of DNA (IMD) to determine DNA-bound stabilized regions. TAF1 (625 pmol) was incubated with or without DNA (700 pmol) in 10 mM HEPES pH 7.9, 5 mM MgClAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplemental Figures and Table


The current view of neuroplasticity depicts the changes in the strength and number of synaptic connections as the main physical substrate for behavioral adaptation to new experiences in a changing environment. Although transcriptional regulation is known to play a role in these synaptic changes, the specific contribution of activity-induced changes to both the structure of the nucleus and the organization of the genome remains insufficiently characterized. Increasing evidence indicates that plasticity-related genes may work in coordination and share architectural and transcriptional machinery within discrete genomic foci. Here we review the molecular and cellular mechanisms through which neuronal nuclei structurally adapt to stimuli and discuss how the perturbation of these mechanisms can trigger behavioral malfunction. In the search for mechanisms that underlie behavioral plasticity, functional and structural changes at synapses are at the core of the theoretical framework. Processes such as long-term potentiation (LTP) or synaptogenesis are thought to be crucial for the adaptation of neuronal circuits to changing environmental conditions . Both stIn eukaryotic nuclei, DNA is wrapped around an octameric histone core comprising of two copies of each of the canonical histones H2A, H2B, H3 and H4. This basic structure, known as a nucleosome, is repeated along the double-stranded DNA, with a fifth type of histone (the linker histone H1) bridging together consecutive nucleosomes. In this fashion, long DNA strands condense with architectural proteins to form chromatin. Based on the level of compaction we can distinguish three main forms of chromatin. These forms differ biochemically with respect to the presence of specific post-translational modifications (PTMs) at the histone tails and to the binding of structural proteins. Euchromatin, a transcriptionally active form is characterized by permissive marks such as the trimethylation of histone 3 at lysine 4 (H3K4me3), and the acetylation of different lysine residues at the histone tails. In contrast, heterochromatin is a transcriptionally silent form, and is decorated by repressive epigenetic marks. It can be found in two different functional states: constitutive heterochromatin that is characterized by DNA methylation at CpGs and histone H3 trimethylation at lysine 9 (H3K9me3), and facultative heterochromatin, which, as suggested by its name, can harbor transcriptional activity and is marked by H3K27me3 .Although the folding of chromatin fibers during cell division is very similar among all cells , the spaApart from chromocenters and peripheral heterochromatin, the interphasic neuronal nucleus is structurally complex  Fig. 1b1b. BasedNuclear architecture and genome organization depend on the integrity of the nuclear envelope, a boundary that separates the cytoplasm from the nucleoplasmic reticulum. This boundary is composed of two phospholipid bilayers spanned at intervals by proteins that act as nuclear pores. The nuclear envelope is not an inert barrier, it participates in different processes including gene regulation and the transport of ions and macromolecular cargos . Its geoInternally attached to the nuclear envelope is the nuclear lamina, whose main components are the lamin proteins A/C, B1 and B2 . These pLamin-associated chromatin domains (LADs) are enriched in transcriptional and epigenetic repressors . AlthougNuclear bodies are subnuclear divisions that lack a membrane. In addition to the afore discussed chromocenters, which are the most prominent type of nuclear body in mature neurons, one can also typically find (i) a single nucleolus where rRNA transcription takes place, (ii) the Cajal bodies (CBs) that are adjacent to the nucleolus and are the site for small nuclear ribonucleic protein (snRNP) assembly, (iii) nuclear speckles that are highly enriched in splicing factors, and (iv) promyelocytic leukemia (PML) bodies that hold unknown functions  Fig. 1b1b. As diThe nucleoplasm is not an inert and homogeneous matrix filled with euchromatin fibers as once thought. Static electron microscopy images have since been challenged by the dynamic scenario revealed by molecular studies that explore short and long-range interactions between DNA sequences that are located thousands of bases apart or even in different chromosomes . The useFine submegabase 3D interactions are essential for neuronal commitment and are also likely to contribute to the regulation of gene expression during neuronal plasticity processes. We will discuss in the next section the novel techniques available and the seminal studies investigating how neuronal activation causes changes in the fine structure of the nucleoplasm.de novo long-range chromatin interactions genome-wide, and DNase I hypersensitive sites sequencing (DNase-seq), Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE)-seq and Assay for Transposase-Accessible Chromatin (ATAC)-seq allow the assessment of changes in DNA accessibility [Given their small scale, transcription-related and activity-driven dynamic changes in chromatin fibers may escape structural analyses when employing microscopy techniques, but can be tackled by molecular studies investigating long and short-range chromosomal interactions . This issibility . These nsibility , 35. Forsibility , and ultsibility .Bdnf, it has been observed that upon kainate-induced seizures there is both a weakening of its interaction with the lamina as well as the relocation of one allele from the nuclear margin to deeper areas within the nucleus [Bdnf alleles with poised RNAPII. Intriguingly, the detachment from the lamina persisted beyond the transient increase in transcription, which leaves open the possibility that this structural change could contribute to sensitization of affected neurons for ulterior reactivation [Bdnf locus has also been reported to occur in neuronal cultures after depolarization [ASCL1 (encoding for the Mash1 protein), along with other proneural genes, move from the nuclear periphery where they remain transcriptionally silent to the central nucleoplasm where they become transcribed [A key level of genome organization is the movement of genes within the interior of the nucleus. Fundamental contributions in the eighties demonstrated chromosomal movements in seizure foci of the human cortex. These movements were found to affect particularly the X chromosome although they were independent of the patient’s sex . Consist nucleus . This retivation . A similrization . These mrization . These gnscribed , 42.Enhancers are defined as regulatory sequences rich in transcription factor (TF) binding sites that regulate gene activation and are distal to the transcription start site (TSS) . They arFos in the nervous system seem to rely in the action of at least five enhancers that surround the locus and differentially respond to various stimuli  [The expression of cell type-specific and brain region-specific genes often relies on enhancer sequences that act specifically only in those cells, while being methylated and inactive elsewhere . Interesulation) .aka CCCTC-binding factor), mediator and cohesin are important components of the insulator complex that appear in distinct combinations depending on the range of interaction. CTCF and cohesin locate together in active regulatory sequences where they mediate long-range constitutive interactions. They are fundamental building blocks behind insulated chromosomal neighborhoods containing super-enhancers necessary for cell identity [Another type of regulatory sequence that relies on chromatin looping are the insulators. Insulators are described as chromatin regions that protect against the activating influence of distal enhancers associated with other genes . The proidentity . For insidentity . Whereasidentity , CTCF plidentity , 55. In identity , cohesinidentity . The invidentity , while aidentity .The term transcription factory refers to discrete foci in the eukaryotic nucleus where transcription occurs . These mde novo acetylation of SINEs located around their promoter  residues at the C-terminus domain (CTD) of RPB1, the largest subunit of the RNAPII complex . Althougter Fig. . This prter Fig. . Two houAs introduced in previous sections, the neurons in some developmental and degenerative disorders often display gross nuclear aberrations, while psychiatric disorders have been associated with more subtle changes Table . We discMED12 [FMR1 gene, which give rise to fragile X-associated tremor/ataxia syndrome (FXTAS), accumulate more heterochromatin but in smaller foci [Mutations in genes encoding proteins important for nuclear architecture  frequently result in neurodevelopmental disorders . This isMED12  that encMED12 , whereasMED12 . FurtherMED12 , while tMED12  that manMED12 , and an MED12 . Other sler foci .Another type of genetic disorders associated with abnormal nuclear architecture are laminopathies in which the nuclear lamina is prominently disrupted. This group of disorders includes Hutchinson–Gilford progeria syndrome (HGPS) that is caused by mutations in the gene encoding lamin A . IntriguDrosophila models of tauopathy and human samples from Alzheimer’s patients [Large-scale chromatin reorganization is often observed in neurons undergoing degeneration. Thus, irregularities in nuclear shape, particularly mediated by B-type lamins, have been described to precede heterochromatin relaxation, DNA damage and neurodegeneration in both patients . Furtherpatients . Other apatients .Grin2b (encoding for a subunit of the NMDA receptor) and the loss of a loop tethering the promoter to a Setdb1 target site positioned 30 kb downstream of the TSS [GRIN2B loop, is often found in schizophrenic patients and correlates with impaired working memory and schizotypic features. Notably, isogenic deletions of loop-bound sequences in mice impaired cognitive performance and decreased Grin2b expression [GAD1  and an enhancer sequence 50 Kb upstream was found reduced in the prefrontal cortex of schizophrenic patients [FKBP5, which encodes the co-chaperone FK506 binding protein 5, and enhancer sequences located in introns 2 and 7 is associated with an increased risk of developing stress-related psychiatric disorders after childhood trauma [NRG1), a putative schizophrenia susceptibility gene regulating the excitatory-inhibitory balance, are associated with an increase in NRG1 transcripts in the prefrontal cortex, suggesting that this region could function as a transcriptional enhancer. Intriguingly, the presence of these repeats correlated with an earlier age of onset of the symptoms. However, long-range interactions between the intronic sequence and the promoter remain to be experimentally proven [Aberrant chromatin loopings have been recently implicated in psychiatric disorders. For example, Akbarian and colleagues first found that overexpression of the histone methyltransferase Setdb1 caused the heterochromatinization of the promoter of  the TSS . Furtherpression . The sampatients . A simild trauma . Anothery proven . There ay proven  and impuy proven , but molGiven the difficulty of examining the specific contribution of chromatin conformation changes through gain- and loss-of-function experiments, most of the evidence discussed above is correlative. A recent study by our team investigating transgenic mice that express high levels of GFP-tagged H2B in forebrain principal neurons has provided evidence for a causal role of aberrant chromatin organization in the emergence of neuropsychiatric traits . Neuronain vivo of some enhancers carrying CTCF-binding sites relies on their relative orientation and the precise architecture of chromatin domains [Excitingly, the use of engineered transcription factors has recently demonstrated that the local manipulation of epigenetic profiles at a given gene is sufficient to control drug- and stress-evoked transcriptional and behavioral responses, thereby providing seminal evidence for a causative role for those epigenetic marks . Similar domains .As reviewed here, numerous studies have illustrated that nuclear architecture and genome topology are key for understanding neuronal function and dysfunction. Changes in subnuclear structures and chromatin loopings have been found to occur in different neuronal plasticity paradigms. Similarly, the disruption of chromatin structures is a landmark for numerous neurological disorders. Although such a disruption likely contributes to the onset of a disorder, a clear distinction between cause and consequence is still missing, except for some monogenic disorders (often associated with ID) caused by mutations in architectural proteins or regulatory sequences. Although the specific contribution of architectural proteins and the changes in 3D chromatin organization to neuroplasticity and neuropathology largely remain to be determined, new light will soon be shed now that novel techniques such as super-resolution microscopy, NGS-based techniques for the analysis of DNA conformation and CRISPR/Cas9-based epi-editing have emerged. These innovative approaches will facilitate a high resolution determination of the 3D organization of the genome, in parallel to a systems-level interrogation of the consequences of gene expression, the identification of loci associated with aberrant function, and even the manipulation of DNA conformations to promote or correct transcriptional changes.


Saccharomyces cerevisiae, nutrient depletion induces massive transcriptional reprogramming that relies upon communication between transcription factors, post-translational histone modifications, and the RNA polymerase II holoenzyme complex. Histone H3Lys4 methylation (H3Lys4 me), regulated by the Set1p-containing COMPASS methyltransferase complex and Jhd2p demethylase, is one of the most well-studied histone modifications. We previously demonstrated that the RNA polymerase II mediator components cyclin C-Cdk8p inhibit locus-specific H3Lys4 3me independently of Jhd2p. Here, we identify loci subject to cyclin C- and Jhd2p-dependent histone H3Lys4 3me inhibition using chromatin immunoprecipitation (ChIP)-seq. We further characterized the independent and combined roles of cyclin C and Jhd2p in controlling H3Lys4 3me and transcription in response to fermentable and nonfermentable carbon at multiple loci. These experiments suggest that H3Lys4 3me alone is insufficient to induce transcription. Interestingly, we identified an unexpected role for cyclin C-Cdk8p in repressing AQY1 transcription, an aquaporin whose expression is normally induced during nutrient deprivation. These experiments, combined with previous work in other labs, support a two-step model in which cyclin C-Cdk8p mediate AQY1 transcriptional repression by stimulating transcription factor proteolysis and preventing Set1p recruitment to the AQY1 locus.In the budding yeast  Saccharomyces cerevisiae is a well-defined experimental system to understand complex transcriptional responses to nutritional information. In the presence of fermentable carbons such as glucose, yeast express growth genes including ribosomal proteins, rRNA processing genes, and glycolytic enzymes, permitting increased biomass and rapid cellular divisions , is a locus-specific transcriptional regulator of stress responsive and developmental genes in yeast . Values reported are the average of three or more independent biological replicates; error bars represent the SDs.Total nucleic acids were prepared from 20 ml midlogarithmic cultures. Approximately 500 ng of total nucleic acid preparations were then treated with DNase I (New England Biolabs), followed by reverse transcription using Protoscript II reverse transcriptase (New England Biolabs) in oligo-dT primed reactions according to the manufacturer’s instructions. Subsequent qPCR reactions were prepared using the Power SYBR Master mix (Applied Biosystems) containing primers listed in Supplemental Material, Set1p occupancy was performed by incubating 500 μg of chromatin solution harboring chromosomally integrated 9-myc-Set1p or untagged wild yeast strains with 20 μl myc-conjugated agarose overnight at 4° with gentle rocking.ChIP was performed essentially as described previously  with theImmune complexes were collected using Dynabeads (ChIP-seq) or protein G agarose (ChIP-qPCR) and washed sequentially with TSE-150 and -500, LiCl/detergent, and TE. Beads were then treated with RNase A and elutions were performed by incubation in 1% SDS/TE at 65° for 15 min. Cross-links were reversed by incubating overnight at 65° followed by proteinase K (Roche) treatment according to the manufacturer’s instructions. DNA was purified using QIAquick PCR column purification  according to the manufacturer’s instructions. For qPCR, % input of each IP was calculated using a standard curve for each primer pair. ChIP-seq was performed on biological replicates; ChIP-qPCR was performed on three or more independent biological repeats with results reporting averages with error bars indicating SDs.ChIP-seq libraries were prepared on immunoprecipitated eluates by first performing end repair  and adding A bases (New England Biolabs Klenow fragment M0212) prior to adapter ligation . Adapter-ligated immunoprecipitated DNA was then purified using Agencourt AMPure XP beads (Beckman Coulter A63880). Size selection was performed by extracting bands from a 2% agarose gel  whose apparent molecular weight was ∼225 bp. Gel slices were purified using a QIAquick gel extraction kit according to the manufacturer’s instructions (QIAGEN cat. no. 28706). Materials were enriched by low cycle PCR using oligonucleotides that contain barcodes for multiplex next-generation sequencing . Library validation and quantification was performed using Agilent DNA-1000 kit on Bioanalyzer instrumentation.Next-generation sequencing was performed at the University of Pennsylvania’s functional genomics core using an Illumina HiSequation 2000 instrument to generate 50 bp single end reads.Quality control of fastq files was performed using FastQC . FastQC Strains are available upon request. Raw ChIP-seq data (.fastq) and mapped reads used for analyses reported in this manuscript are available on GEO, accession number GSE93641. cyclin C and Jhd2p inhibit histone H3Lys4 3me at the FLO11 promoter . To account for ChIP-seq biases that could result from uneven genomic coverage, we opted to use histone H3 CTD immunoprecipitations as our background control instead of “input” samples . Differential peak analyses identified 591 loci displaying a ≥twofold elevation in H3Lys4 3me levels in mutant cells relative to wild type . Conversely, we detected 465 loci with elevated H3Lys4 3me levels in wild type relative to mutant yeast; however, these peaks displayed reduced amplitudes compared to those observed in mutant yeast . The remainder of this manuscript will therefore focus on those loci displaying elevated H3Lys4 3me in cnc1Δjhd2Δ mutants.Our previous work indicated that promoter . To idenTDH1, INO1, ENO1, and AQY1 using the Integrative Genomics Viewer . Peaks were further confirmed by performing ChIP-qPCR using antibodies that recognize either histone H3Lys4 3me or total histone H3 on wild type and cnc1Δjhd2Δ mutants cultured in nonfermentable carbon. These experiments confirmed that H3Lys4 3me levels are reproducibly elevated in cnc1Δjhd2Δ mutants at the TDH1, INO1, ENO1, and AQY1 loci . Together, these data indicate that cyclin C and Jhd2p inhibit locus-specific methylation for yeast cultured in nonfermentable growth conditions.We next validated the identified H3Lys4 3me peaks by manually inspecting mapped reads from top hits including cnc1Δjhd2Δ mutants. Gene Ontology (GO) analyses of the affected loci revealed an overrepresentation of genes involved in carbon metabolism, osmotic stress, and negative regulators of pseudohyphal growth (Table S5). This is in agreement with previous reports indicating that cyclin C-Cdk8p repress the transcription of genes involved in these processes and with the constitutive pseudohyphal phenotype observed in cnc1Δjhd2Δ yeast mutants  on a genome-wide basis by evaluating all peaks and those peaks that are ≥twofold elevated in cnc1Δjhd2Δ yeast mutants . Surprisingly, we observed a broad 5′ shift in H3Lys4 3me distribution gluconeogenic genes for cnc1Δjhd2Δ yeast mutants . Together, these analyses indicate that cyclin C and Jhd2p regulate the locus-specific magnitude and distribution of H3Lys4 3me.Since genes involved in the same biological process are likely to have overlapping regulatory mechanisms, we hypothesized that any differences in H3Lys4 3me distribution may be restricted to those genes that control a singular process. To test this hypothesis, we generated H3Lys4 3me TSS curves for genes involved in one of our top hits from GO analyses, gluconeogenesis , indicating that cyclin C and Jhd2p play nonredundant roles in repressing INO1 transcription. In contrast, both cnc1Δ and cnc1Δjhd2Δ mutants cultured in nonfermentable carbon displayed similar levels of INO1 derepression , and suggest the presence of a second locus-specific signal that is required for transcriptional induction.Finally, we observed a moderate ∼twofold derepression of e carbon . Interes mutants . These rcyclin C and Jhd2p at the AQY1 locus. To do this, we performed ChIP-qPCR in wild type or yeast mutants lacking CNC1 and JHD2 in isolation or in tandem. Yeast were cultured in either fermentable or nonfermentable carbon and immunoprecipitations were directed toward histone H3 or histone H3Lys4 1me, 2me, or 3me. To allow for efficient mapping of each H3Lys4 methylation level, qPCR reactions were directed toward four regions of the AQY1 locus . Ste12p, a transcriptional activator for pseudohyphal gene transcripts, is a kinase substrate for cyclin C-Cdk8p that becomes degraded following phosphorylation (AQY1 promoter is bound directly by Ste12p as cells are grown in nutrient limited conditions that result in pseudohyphal induction (Ste12p promoter binding is a prerequisite for transcriptional induction of target genes (STE12 fail to activate AQY1 transcription during nutrient deprivation, suggesting that direct interactions between Ste12p and the AQY1 promoter are required for transcriptional induction during pseudohyphal differentiation (Why is karyotes . In suppcyclin C-Cdk8p and Jhd2p regulate AQY1 transcription. In this model, H3Lys4 methylation is inhibited by independent activities of Jhd2p, which demethylates H3Lys4, and cyclin C-Cdk8p, which prevents Set1p promoter binding, resulting in transcriptional repression (cyclin C-Cdk8p activity to allow Set1p recruitment, but H3Lys4 3me accumulation is prevented by Jhd2p (cyclin C-Cdk8p and Jhd2p activity at the AQY1 locus. This not only allows H3Lys4 3me accumulation, but also permits transcriptional activation by Ste12p, since it is no longer targeted for cyclin C-Cdk8p-mediated phosphorylation and proteolytic degradation (Our results support a stepwise model in which both cyclin C-Cdk8p-mediated H3Lys4 methylation inhibition acts via direct or indirect molecular mechanisms. Seminal work identified a role for trans-histone communication in which histone H2B ubiquitination is required for efficient H3Lys4 methylation (Paf1 complex, which is thought to serve as a molecular platform for factors that regulate histone modifications and transcription, recruits the Rad6p and Bre1p components of the H2B ubiquitin machinery. In turn, the Paf1 complex subunit Rtf1p is required for H2B ubiquitination, Set1p recruitment, and efficient H3Lys4 methylation (cyclin C-Cdk8p antagonize Rtf1p function to prevent Set1p recruitment and H3Lys4 methylation at target loci. Alternatively, cyclin C-Cdk8p may directly inhibit the COMPASS methyltransferase complex. In support of this, Brickner and colleagues recently proposed a model in which Cdk8p can directly prevent the association of the Spp1p subunit of the COMPASS complex during nutrient deprivation and transcriptional memory formation at the INO1 promoter (Cdk8p is present at this locus as Spp1p is evicted, direct kinase targets responsible for this function remain elusive. Further mechanistic studies will be required to determine which of these models can explain how cyclin C-Cdk8p control H3Lys4 3me and transcription.It remains unclear whether www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.039586/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Bidirectional transcription initiating at enhancers has been proposed to mark active enhancers and as such has been utilized to experimentally identify active enhancers Here, we show that bidirectional transcription initiation is a pervasive feature of accessible chromatin, including at enhancers, promoters, and other DNase hypersensitive regions not marked with canonical histone modification profiles. Transcription is less predictive for enhancer activity than epigenetic modifications such as H3K4me1 or the accessibility of DNA when measured both in enhancer assays and at endogenous loci. The stability of enhancer initiated transcripts does not influence measures of enhancer activity and we cannot detect evidence of purifying selection on the resulting enhancer RNAs within the human population.Our results indicate that bidirectional transcription initiation from accessible chromatin is not sufficient for, nor specific to, enhancer activity. Transcription initiating at enhancers may be a frequent by-product of promiscuous RNA polymerase initiation at accessible chromatin and is unlikely to generally play a functional role in enhancer activity.The online version of this article (doi:10.1186/s13059-017-1379-8) contains supplementary material, which is available to authorized users. Enhancers are modular, regulatory DNA elements that positively drive gene expression at a distance . They arWhile most existing enhancer discovery methods are based on a characteristic chromatin profile (high H3K4me1 and low H3K4me3) , often iThe production of RNA transcripts initiating at enhancer elements also raises the question of potential functional roles for some of these eRNAs in mediating enhancer activity. siRNA knockdowns of a number of candidate eRNAs have resulted in reduced gene expression , 17. OthIn this study, we investigate the specificity of bidirectional transcription for enhancer identification and its importance for enhancer function. We show that the initiation of both stable and unstable bidirectional transcription can frequently be detected at open chromatin regions that exhibit neither chromatin marks characteristic of enhancers nor enhancer activity. While many active enhancers do exhibit bidirectional transcription initiation, this property alone does not define enhancers. Measures of transcription initiation at candidate enhancers correlate less well with their presumptive gene targets than measures of chromatin accessibility at the same sites. Using population genetic approaches that are less perturbed by mutation rate variation than previous interspecies comparisons , we confWe systematically explored the relationship between chromatin state, transcription initiation, and DNA accessibility by focussing on four well studied cell lines , all with (i) detailed maps of chromatin modifications assimilated into chromatin state maps , (ii) CAWhile CAGE sensitively detects the initiation sites of stable transcripts, unstable transcripts can be under-detected. We identified the initiation of both stable and unstable transcripts using global run-on sequencing enriched for 5′-capped RNAs (GRO-cap) support Fig. , an analWhile much bidirectional transcription initiating at enhancers does not produce stable RNA transcripts , these rAcross these genomic contexts, the frequency of transcription initiation approximately corresponds with the level of DNA accessibility, as measured by the strength of the DHS signal . Enhancers producing stable transcripts were not significantly more active in the reporter assays than those producing only unstable transcripts (Mann-Whitney p = 0.95), suggesting that neither transcript stability nor the mature transcripts themselves are generally required for enhancer activity. That our classification of transcribed enhancers only required the support of a single CAGE or GRO-cap read, yet still showed a significantly higher activity than those enhancers with no evidence of transcription, confirmed that our choice of cutoff is biologically meaningful.To further explore the relationship between transcription initiation and enhancer activity we intersected data from 1499 high-throughput enhancer reporter assays of candidate regulatory elements in K562 cells  with meap = 0.02), demonstrating that neither bidirectional nor unidirectional initiation of transcription alone predicts enhancer activity .Sites with transcription initiation but repressive chromatin marks do not exhibit enhancer activity relative to scrambled controls  and that bidirectional transcription initiation does not seem to be predictive of enhancer activity as measured by reporter gene output .To further test our observation that bidirectional transcriptional initiation from accessible chromatin is not specifically associated with enhancer activity we carried out 47 additional reporter assays in HepG2 cells Fig. . These ecis [As enhancers are defined by their ability to positively drive gene expression in cis , we nextcis  solely ocis , regardlIn stark contrast to transcription-based correlations between regulatory elements and annotated promoters, DNase hypersensitivity measures do show clear discrimination between chromatin states in their correlation with genic transcription Fig. . DNase hWe also repeated this analysis using other approaches to identify putative regulatory element–promoter pairs, as not all enhancers target genomically adjacent promoters . When loHaving found that the stability of eRNA transcripts does not correspond to measures of enhancer activity Fig. , we tookWe have shown that low levels of transcription initiation are a common feature of accessible chromatin sites associated with enhancer activity as well as those with other functions Fig. , nor is Despite the lack of specificity for enhancers, measuring bidirectional transcription initiation is certainly not without merit, as it can be used to identify regions of open chromatin in exactly the same sample and source data in which gene expression is quantified , 12, 13.The pervasive low level initiation of transcription at highly accessible chromatin in diverse contexts suggests the resultant transcripts may be by-products rather than functional entities. There is an important distinction to be made between a molecular measure of activity where there is a detectable molecular species or event and a biological measure of function where the molecular species or event impacts an organism level phenotype. With current technologies we have the power to very sensitively detect the molecular products of the genome , buThe observation that chromatin marked enhancers work equally well as enhancers whether their associated eRNAs are relatively stable or rapidly degraded Fig.  supportsWe have shown that bidirectional transcription can be detected from all types of accessible chromatin, including those regions that have little obvious epigenetic decoration. Furthermore, this transcriptional signal alone is not sufficient to discriminate enhancers from other active regulatory regions in the genome. We propose that bidirectional transcription is predominantly a by-product of an opening of chromatin at all types of regulatory regions and, notwithstanding those published examples of functional eRNAs , the majProtein-coding, miRNA, and lincRNA annotations were extracted from the GENCODE v14 release  . We downloaded the ‘narrowPeak’ files for each cell type and considered only the intersection of both replicates in our analyses. For our cross-cell correlation analysis, the strength of DNase hypersensitivity for each region was calculated for each cell type as the summed number of reads per kilobase region per million reads mapped (RPKM) measures obtained from both replicates.DHSs for each cell type  were obthttp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeRikenCage/and all libraries from each cell type were then merged into a single BAM file.CAGE data produced by the FANTOM5 consortium  were dowGRO-cap, GRO-seq, and PRO-seq data for K562 and Gm12878 cells  were obtUnstably transcribed annotations were first identified as those with transcription initiation supported by GRO-cap evidence and where the genomic extent of overlapping DHSs do not overlap any evidence for stable transcription initiation as measured by CAGE. Subsequently, unstably transcribed regions were similarly identified separately for GRO-seq and PRO-seq evidence. Heat maps for DHS midpoints (±1 kb) were generated using ngsplot v2.61 .Productive elongation was quantified using GRO-seq data in 50-bp windows outwards from the maximally transcribed TSS (as measured by CAGE) within 250 bp of each DHS midpoint. For those DHSs overlapping annotated protein-coding and lincRNA promoters, only CAGE reads which mapped to the annotated strand were considered when measuring elongation rates. For DHSs outside annotated transcribed regions, TSSs were identified separately on each DNA strand.The expression level for regions annotated across the unified chromatin state map was quantified for each cell type as the RPKM summed across all libraries from that cell and also as the maximum RPKM from an individual TSS’s location for each region. The expression of annotated GENCODE promoters (see above) were quantified in the same way.http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlLongRnaSeq/) and individual sequencing runs were then asembled into transcripts using Cufflinks v2.2.1 [Mapped RNA-seq reads from the ENCODE project  were dows v2.2.1 , where tp value was considered. A positive correlation was recorded if the correlation coefficient was greater than 0 and p < 0.05. All other regions were considered to be nonsignificant. The uncertainty in the estimate of the percentage of positive correlations was determined by 1000 samplings of the data with replacement.We calculated the linear correlation between transcription (as scored by CAGE RPKM) or strength of accessible chromatin (as scored by the DHS RPKM) at chromatin state loci, non-chromatin state DHSs and bidirectionally transcribed-defined enhancers with the transcription (as scored by CAGE RPKM) from putative target, annotated GENCODE promoters across four cell types . These regulatory loci were paired with target promoters as (a) the nearest promoter to each locus; (b) to all promoters within 500 kb of each locus; (c) to all promoters within the same physically interacting domain at each locus; (d) to all promoters which were determined to be physically interacting with each locus. Information on the location of each physically interacting domain and pairs of physically interacting loci were obtained from the GSE63525 series at the Gene Expression Omnibus . If multp = 0.20) and therefore these two categories were merged and considered as a single null expectation for the reporter activity measured from random DNA sequences.Enhancer and repressor element activity in K562 cells was estimated as the mean expression values obtained from a parallel reporter assay . For thiAdditional validations were performed in HepG2 cells (confirmed free of mycoplasma contamination with the Lonza MycoAlert kit). HepG2 cells were sourced from the Institute of Genetics and Molecular Medicine (Edinburgh) technical services. For our HepG2 reporter assays, DHSs from each chromatin state category which were more than 1 kb beyond RepeatMasker-marked regions and GENCODE gene annotations were randomly selected. PCR primers .Derived allele frequencies were extracted from the deCODE whole-genome sequencing study of the Icelandic population . AnalogoAdditional file 1: Figure S1.Figure S2. Bidirectional transcription (as measured by CAGE) initiation around DHSs in K562 cells. Figure S3. Bidirectional transcription (as measured by CAGE) initiation around DHSs in HepG2 cells. Figure S4. Bidirectional transcription (as measured by CAGE) initiation around DHSs in Huvec cells. Figure S5. Bidirectional transcription initiation around DHSs in K562 cells. Figure S6. Bidirectional transcription (as measured by GRO-seq) initiation around DHSs in Gm12878 cells. Figure S7. Bidirectional transcription (as measured by GRO-seq) initiation around DHSs in K562 cells. Figure S8. Bidirectional transcription (as measured by PRO-seq) initiation around DHSs in K562 cells. Figure S9. Bidirectional transcription initiation (as measured by GRO-cap) across DHSs which do not overlap annotated promoters in two cell lines across various chromatin state annotations. Figure S10. Bidirectional transcription (as measured by CAGE) across DHSs which do not overlap annotated promoters in four cell lines across various chromatin state annotations. Figure S11. Productive transcription as measured by the elongation rates of transcripts initiating from various genome annotations and chromatin states. Figure S12. Correlations between chromatin state loci and bidirectionally transcribed-defined enhancers with the nearest annotated gene promoter. Figure S13. Correlations between chromatin state loci and bidirectionally transcribed-defined enhancers with annotated gene promoters within 500 kb. Figure S14. Correlations between chromatin state loci and bidirectionally transcribed-defined enhancers with annotated gene promoters within the same physical domain. Figure S15. Correlations between chromatin state loci and bidirectionally transcribed-defined enhancers with physically interacting annotated gene promoters. (PDF 1830 kb)Bidirectional transcription (as measured by CAGE) initiation around DHSs in Gm12878 cells. Additional file 2: Table S1.Counts of bidirectionally and unidirectionally transcribed DHSs defined as 250 bp around the midpoint across different chromatin state regions and gene annotations (mRNA/miRNA/lincRNA) as measured by GRO-cap, GRO-seq, and PRO-seq in K562 and, where available, Gm12878 cells. (DOC 88 kb)Additional file 3: Table S2.Counts of bidirectionally and unidirectionally transcribed DHSs defined as 250 bp around the midpoint across different chromatin state regions and gene annotations (mRNA/miRNA/lincRNA) as measured by CAGE across cell types and subcellular fractions. (DOC 343 kb)Additional file 4: Table S3.Number of chromatin state loci and bidirectionally transcribed-defined enhancers whose measure of stable transcription initiation and accessible chromatin is significantly correlated with transcription from putative annotated gene promoter targets as measured by either transcription initiation from the maximally expressed TSS (single TSS) or the sum of all annotated promoters (summed TSSs) associated with that gene. (DOC 51 kb)Additional file 5: Table S4.Counts of single-exonic and multi-exonic transcripts built by Cufflinks and filtered as described in the “Additional file 6: Table S5.Primer sequences, coordinates, and reporter construct activities measured in HepG2 cells. (DOC 294 kb)


Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy.To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells.Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway.Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.The online version of this article (10.1186/s12864-018-4533-0) contains supplementary material, which is available to authorized users. Chromatin remodeling factors and histone modifying enzymes are frequently mutated or aberrantly expressed in a wide variety of cancers, leading to altered transcription states that change cell identity, behavior, and response to therapy. In recent years, large efforts have systematically profiled epigenomes in various tissue types and diseases, including the ENCODE project –3 and thResearch (LONESTAR) consortium was created to define epigenetic factors associated with molecular changes in specific subtypes of breast cancer. We reasoned that definition of subtype specific histone modification profiles, together with definition of primary and steady state transcriptional differences, may allow identification of new targets for development of precision therapies. Therefore, we profiled distributions of 8 key histone modifications by ChIP-Seq as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes across 13 distinct cell lines that represent immortalized human mammary epithelial cells  and 5 molecular subtypes of breast cancer , growth factor expression (e.g. Her2/neu status), and specific gene expression profiles. The Lonestar Oncology Network for EpigeneticS Therapy And cer Fig. . These dDefinition of common and unique features of chromatin landscapes of all 5 breast cancer subtypesDemonstration that histone modifications associated with either enhancers or with active transcription states can discriminate between subtypesIdentification of epigenetic landscapes that define genes and pathways specifically activated or repressed in TNBCWe systematically profiled histone modification patterns and gene expression programs in a set of well characterized cell lines that represent 5 major breast cancer subtypes, including two ER positive subtypes, Luminal-A and Luminal-B, the HER2 positive subtype, and two triple-negative subtypes, TNBC-Claudin Low, and TNBC-Basal, as well as two normal immortalized breast cell lines as controls  and gene bodies (TxAct), as expected  of chromatin states that were common between cell lines within the same subtype. Enhancer states (41%), including EhAct, EhGen and EhBiv, closely followed Fig. . The dyn% of chroSince active transcription chromatin states, including both TxAct and TxFlk, represent genes with the highest expression levels , repeats/ZNF gene clusters (RpZNF) and heterochromatin (Htchr) Figs.  and 2d. Close inspection of these chromatin states signatures also identified individual genes that displayed highly specific patterns across the breast cancer subtypes. For example, both active promoter and transcription signatures Fig.  and RNA-Interestingly, although AFAP1-AS1 has not been linked to TNBC previously, it has been reported to be highly expressed and to predict poor prognosis in various types of cancers, including Barrett’s esophagus and esophageal adenocarcinoma , 10, panTo determine if AFAP1-AS1 affects the growth or aggressiveness of TNBC cells, we depleted AFAP1-AS1 expression using two siRNAs targeting different regions of AFAP1-AS1 in two TNBC cell lines, MDA-MB-231 and HCC1937 Fig. . LimitedOur analyses also identified loci that are specifically silenced in TNBC cells. One striking example is located at the bi-directional promoter of NAA60/ZNF597, which displays a complete loss of active promoter marks in both TNBC subtypes :All cell lines used for the Lonestar Consortium were purchased from ATCC  following manufacturer’s instructions. Six to eight hours after transfection, the medium was changed to normal culture medium as described above.When cells reached 60% confluence, two siRNAs targeting AFAP1-AS1  and a control siRNA were transfected separately using Lipofectamine® 2000 , following the manufacturer’s recommended procedure. 1 μg of RNA was reverse-transcribed to cDNA using a SuperScript VILO cDNA Synthesis kit  following the manufacturer’s procedure. 10 ng of cDNA was used for one qRT-PCR reaction with three technical replicates.MDA-MB-231, HCC1937 cells were transfected with control siRNA, or siRNA targeting AFAP1-AS1. After 48 h, 15,000 cells were seeded into each well of 12 well plate with three replicates for each sample and each day of counting. At indicated day, cell numbers were counted using hemocytometer.2. 10 to 15 days later, cells were washed with PBS once and fixed with Fixation buffer  for 5 min. Then cells were stained with 0.5% crystal violet for 2 h to overnight, washed, dried, and imaged.MDA-MB-231, HCC1937 cells were transfected with control siRNA, or siRNA targeting AFAP1-AS1. After 48 h, 300 cells for each sample were put into single well of a 6 well plate, with three replicates, and incubated at 37 °C with 5% COThe following antibodies were used qPCR- and sequencing-based ChIP assays in the amounts specified:Cells were grown to ~ 70–80% confluence, cross-linked with 1% formaldehyde for 10 min at 37 °C, and quenched in 125 mM glycine for 5 min at 4 °C. The cells were then collected and lysed in Farnham Lysis Buffer . The crude nuclear pellet was collected by centrifugation, resuspended in lysis buffer , and incubated on ice for 10 min. The chromatin was sheared by sonication at 4 °C using a Bioruptor 300 at the highest setting for fifteen 1-min cycles of 30 s on and 30 s off to generate chromatin fragments of ~ 200–400 bp in length. The soluble chromatin was diluted 1:10 with dilution buffer  and pre-cleared with protein A agarose beads. Five percent of the material was removed and saved as input, and the rest of the pre-cleared supernatant was incubated overnight at 4 °C with the antibody of interest and a non-specific IgG control antibody.3, 1% SDS), and the crosslinks were reversed by adding 100 mM NaCl with incubation at 65 °C overnight. The eluted material was then digested with proteinase K and RNase H to remove protein and RNA, respectively, and the enriched genomic DNA was extracted with phenol:chloroform:isoamyl alcohol followed by ethanol precipitation. The ChIPed DNA was dissolved in water and analyzed by qPCR using the enhancer- or gene-specific primers.The following day, the immune complexes were collected by adding protein A agarose beads and incubating for 2 h at 4 °C. The immunoprecipitated material was washed once with low salt wash buffer , once with high-salt wash buffer , once with LiCl wash buffer , and twice with Tris-EDTA (TE) containing 1× protease inhibitor cocktail. The immunoprecipitated material was eluted at room temperature in elution buffer  for Illumina Platforms. Ten ng of sheared DNA was used to repair the ends of the damaged fragments using a proprietary master mix. The resulted blunted fragments were 3′ A-tailed using a proprietary mixture of enzymes to allow ligation to the specific NexTflex adaptors from Bioo Scientific . Each of the steps  was followed by column clean up . After adapter ligation, DNA enrichment was performed using Kapa HiFi Hot Start Ready PCR mix, and a cocktail of primers , and purified with AmpureXP beads . The quality of the final libraries was assessed using a 2200 TapeStation (Agilent Technologies). The libraries were quantified using a Kapa Library Quantification Kit  and loaded in a flow cell for cluster generation using the Illumina cBOT (Illumina) at final concentration of 10 pM.p-value = 1e-8. To reduce the signal strength variation due to sequencing depth difference, we subsampled to a maximum of 20 million reads per replicates per sample. The reproducibility between replicates was evaluated by the Pearson correlation of peak intensities  transcription start sites, (B) gene bodies, (C) enhancer peaks. (D) Genomic distribution of histone modification ChIP-seq tags. (E) Hierarchical clustering of histone modification ChIP-seq peak signals. (PDF 936 kb)Additional file 2:Table S1. ChIP-seq sample alignment summary. (XLSX 66 kb)Additional file 3:Table S2. ChIP-seq sample replicates correlation. (XLSX 61 kb)Additional file 4:Figure S2. (A) Clustering of chromatin states model learned on individual cells showing same enrichment pattern that can recover the chromatin state jointly learned using all 13 cells. (B) RNA-seq expression levels for genes associated with different chromatin states. (C) Cumulative fractions of chromatin state counts versus number of samples. Larger area under curve indicates more variability across breast cancer cells. (PDF 1549 kb)Additional file 5:Figure S3. ChromHMM model of 15 chromtain states defined by all 8 histone modifications. (PDF 358 kb)Additional file 6:Figure S4. Unsupervised clustering of histone modification occupancy in highly variable regions  and enriched genomic regions ., showing subtype specificity and reproducibility between replicates. (PDF 2936 kb)Additional file 7:Table S5. List of genes with subtype specific active enhancer states. (XLS 212 kb)Additional file 8:Figure S5. Spearman correlation of H3K36me3 occupancy and gene expression levels in all samples. (PDF 489 kb)Additional file 9:Table S4. List of genes with subtype specific active transcription states. (XLS 44 kb)Additional file 10:Table S7. Subtype gene signatures of active transcription states and active transcription flanking states. (XLSX 23 kb)Additional file 11:Table S8. Significant pathways of subtype gene signatures of active transcription states and active transcription flanking states. (XLSX 18 kb)Additional file 12:Table S9 Significant upstream regulators of subtype gene signatures of active transcription states and active transcription flanking states. (XLSX 16 kb)Additional file 13:Figure S6. Subtype expression patterns of TCGA breast cancer samples. (PDF 359 kb)Additional file 14:Table S6. List of genes with subtype specific repressive polycomb domain states. (XLS 10 kb)Additional file 15:Figure S7. Chromatin state landscape of depleted H3K27me3 signals at NLRP gene cluster in normal-like celllines. (PDF 595 kb)Additional file 16:Figure S8. Chromatin state landscapes of depleted H3K4me3 signals in the bi-directional promoter of NAA60/ZNF597 in TNBC subtype celllines. (PDF 328 kb)Additional file 17:Table S10. RNA-seq gene expression read count. (XLSX 8751 kb)Additional file 18:Table S3. Emission probability and transition probability of Chromatin States defined by ChromHMM. (XLSX 46 kb)


Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4  confirmed that the mutation alters the binding of regulatory complex, leading to a significant decrease of both GDNF and GDNF-AS mRNA expression levels. This work in canine spontaneous forms of human neuropathies allowed the identification of a novel gene GDNF and its regulation mechanism, not yet described in human HSAN, opening the field of clinical trials to benefit both canine and human medicine.In this study, we present a canine neuropathy characterized by insensitivity to pain in the feet, sometimes combined with self-mutilation described in four sporting breeds. This particular phenotype has the clinical hallmarks of human Hereditary Sensory Autonomic Neuropathies (HSAN). As we hypothesized that a monogenic recessive disorder was shared between these breeds, we performed a Genome Wide Association Study (GWAS) to search for the genetic causes and found one homozygous chromosomal region in affected dogs. High-throughput sequencing of this region allowed the identification of a point mutation upstream to the  SPTCL1 or SPTCL2 , vesicular transport  or neurotrophic factors and their tyrosine kinase receptors  [Inherited peripheral neuropathies are neurodegenerative diseases of the peripheral nervous system (PNS), including the Charcot-Marie-Tooth diseases (CMT) and Hereditary Sensory and Autonomic Neuropathies (HSANs) . They ar, NGF-B) –8. AlthoInterestingly, while neurological diseases such as HSANs are rare in humans, some dogs breeds spontaneously develop such diseases with high frequencies . Indeed,GDNF gene , a neurotrophic factor involved in neuronal development and adult neuronal survival [GDNF-AS) that is transcribed in the opposite direction of GDNF. Comparative genomic analysis with the human orthologous genomic region led us to hypothesize that the mutation disrupts a regulatory region, such as an enhancer, controlling GDNF expression. We then performed quantitative PCR analyses and showed lower expression levels of both GDNF and GDNF-AS RNAs in affected dogs compared to unaffected dogs. By gel shift assays, we further showed that the mutation modified the affinity of a regulatory complex. Such findings highlight the GDNF/GDNF-AS partnership as a subtle regulatory mechanism and relevant candidates to search for human HSAN mutations and potentially to develop targeted therapies.We thus investigated these affected breeds as a potential opportunity to identify new genes for unexplained cases of human HSANs. We first started the genetic study with the French Spaniel breed and identified one locus strongly associated with the disease, on canine chromosome 4, and refined the locus by intersecting the haplotypes of the other affected breeds. Using this strategy, we highlighted a common homozygous haplotype of 1.8 Mb  shared by all affected dogs in the four sporting breeds. Next, we performed targeted DNA sequencing of the locus in four affected and four unaffected dogs. We found 478 variants which were individually removed by Allele-Specific PCR except one located upstream of the survival –25. Moreet al, no signs of autonomic disturbances were detected by neurological exams [We first focused the genetic analysis on the French Spaniel breed. The collection of 173 blood samples with pedigree information allowed us to design a large pedigree and to confirm the monogenic recessive inheritance of the disease in this breed as previously described . Affecteal exams  and moto−16; 100,000 = 10−5) , which appeared as a new and good candidate for HSAN  using a linear mixed-model method accounting for population stratification and relatedness, with 49 French spaniels (21 affected showing insensitivity to pain in their feet and 28 unaffected dogs) . Followi = 10−5) . Since a = 10−5) . This infor HSAN . Indeed,survival –25 and wGDNF gene . To validate this variant, we sequenced over 900 unaffected dogs from 130 different breeds and observed that the mutated allele was never detected in these dogs, thus excluding a polymorphism. We also checked the allelic distribution of this variant in a panel of 250 sporting dogs of known clinical status and we observed a perfect correlation between the clinical status of dogs and their genetic status, confirming the strong association of the variant with the disease in the analyzed sporting breeds. Interestingly, heterozygous dogs did not present any clinical signs and only one dog was homozygous mutated with only insensitivity to pain . Even if it was lying in a not yet described genomic region, we considered this variant as potentially causal and further named it “AMS variant” for Acral Mutilation Syndrome variant.To identify the disease-associated variants (InDel and SNP), we chose to enlarge the homozygous candidate region to 3 Mb (70.3 Mb to 73.3 Mb on CanFam3 assembly) and performed a targeted sequencing . Four afGDNF locus, we mapped the canine 3 Mb locus targeted for sequencing  onto the human genome (GRCh38) using the LiftOver tools available on the UCSC genome browser [GDNF-AS) is annotated upstream of GDNF [To unravel the genomic context surrounding the  browser ,27 and t browser  Fig 2)..GDNF loc of GDNF , transcrGDNF as in human .To refine the annotation of the canine locus, we used 33 RNA sequencing data produced from 25 different tissues of seven breeds . The seqin human . Next, tin human . Remarkain human . The lin RNA-seq  and is mGDNF-AS and the four closest protein-coding genes i.e. GDNF, WDR70, NUP155 and C5orf42  but not with the other protein-coding genes . These results, together with the close proximity of GDNF and GDNF-AS transcription start sites (TSSs), reinforced the relationship between both genes and pointed toward the presence of a bi-directional promoter [It has been shown that lincRNAs transcribed from a bi-directional promoter could regulate the level of expression of the corresponding neighbor mRNAs –39. We t C5orf42 . We founpromoter ,40,41.GDNF/GDNF-AS  and showed that it matches the human genome  with 84.2% of nucleic acid identity . Indeed, DRG are interesting, as it was previously recognized as harboring the predominant lesions in these breeds [GDNF and GDNF-AS together with the neighboring genes WDR70, NUP155, and SLC1A3. QRT-PCR revealed that the GDNF mRNA level was significantly decreased (60%) in affected dogs . We also showed that the expression levels of the other genes of the locus were not altered (GDNF-AS expression level (80%) in this tissue in agreement with the hypothesis that GDNF-AS and GDNF share a bi-directional promoter highlighted by correlated expression shown by RNA-seq and genomic analyses . We first used increasing amounts of nuclear extract from HeLa cells with duplex of wild-type or mutated sequences. We showed a high-affinity specific interaction of a Nuclear Extract (NE) complex with the double-stranded oligonucleotide containing the wild-type sequence but a low-affinity with the sequence containing the AMS variant . In addiGDNF, we hypothesized a regulatory effect of this variant on GDNF. We then showed a strong correlation between the presence of the mutation and the decreased expression levels of both GDNF and its divergent long non-coding RNA GDNF-AS. Finally, we demonstrated the impact of the mutation on the binding of a complex confirming the hypothesis of a regulatory element. With this work, we provide relevant arguments to explore GDNF/GDNF-AS partnership in human patients with unexplained HSAN.This study presents the identification of the mutation involved in a severe form of hereditary sensory autosomal neuropathy (HSAN) shared by four sporting breeds having a common history. The identification of a 1.8 Mb homozygous region on chromosome 4 by GWAS, with only one variant perfectly segregating with the disease, illustrates the advantages of the canine model in genetics. Since this variant is located 90 kb upstream of the neurotrophic factor We started this project focusing our study on dogs with self-mutilations using a precise clinical questionnaire and we quickly detected that all dogs with self-mutilations also presented insensitivity to pain, not always reported. This important clinical sign led us to improve the genetic analysis. Indeed, we found additional affected dogs without self-mutilations but related to cases with self-mutilations. These dogs showed the “affected” homozygous haplotype, while owners did not detect the insensitivity to pain. This observation reflects the difficulty to diagnose insensitivity to pain in dogs, which contributed to the spreading of this severe disorder in the four related breeds ,19,21. TGDNF gene appeared as an excellent candidate. Indeed, GDNF  promotes the axon development and the survival of sensory neurons at different stages of the development in mice and chicken model [in vitro that after injury, nearly 100% of the sensory neurons are rescued by the production or injection of GDNF [GDNF leading to the decrease of the number of sensory neurons and then their death, as previously observed in affected dogs [With a high coverage targeted sequencing of the locus (365x), we identified one variant perfectly associated with the phenotype, and absent in more than 900 unaffected dogs. This variant located upstream of the en model –25. More of GDNF ,46,52,53ted dogs –20. ThisGDNF-AS, described in human [GDNF gene. While the GDNF mRNA sequence is well conserved between human, mouse and dog, the canine GDNF-AS transcript sequences diverge significantly from the human sequences at the nucleotide level  and no GDNF-AS are yet annotated in mouse. However, the whole locus is well conserved at the syntonic and nucleotide levels between human and dog. Regarding shared lncRNAs functions between species in spite of low sequence conservation, several well-described lncRNAs  exhibit conserved biological functions even though nucleotide conservation is only limited to small patches and their exon/intron structure is not maintained [The genomic analyses of the human and dog orthologous regions revealed the presence of long non-coding RNA transcripts in both species. One of them, in human , containintained .GDNF-AS, which is the only exon shared by all of the 12 isoforms annotated by our RNA-seq data. Strikingly, another neurotrophic factor, BDNF (Brain Derived Neurotrophic Factor precursor), playing similar functions as GDNF, also presents a similar genomic feature than GDNF, with the presence of a lincRNA transcribed in the opposite direction. The parallel between BDNF and GDNF is interesting because they both stimulate the growth and differentiation of new neurons and support the survival of existing neurons in central and peripheral nervous systems [NTRK2 gene, which has a paralog NTRK1, already annotated as mutated in HSAN type IV [BDNF-AS increases BDNF levels in vivo [GDNF-AS increases the expression level of GDNF in human HEK293T cells [GDNF and GDNF-AS partnership could fine-tune the expression of GDNF and that the AMS variant disturbs this relationship at a specific developmental stage [An interesting point is the fact that the AMS variant is located in the last exon of  systems ,52,55. M type IV ,57. In a in vivo ,58. Moda3T cells . We coulal stage .GDNF [GDNF-AS, both GDNF and GDNF-AS transcripts are weakly expressed in affected dogs as compared to unaffected dogs  neurons were described as potential target of GDNF ,62. In aGDNF . Thus, tted dogs . This imGDNF regulation is modulated by a long distance interaction between the region containing the AMS variant and the promoter. As the luciferase reporter assay did not validate this hypothesis, we checked if the AMS variant could inhibit the enhancer activity by preventing the binding of a transcription complex. The EMSA experiment clearly showed that a complex binding the wild-type sequence has less affinity for the mutated sequence. The fact that the mutation of a single nucleotide, in a 20-nucleotides sequence, inhibits the fixation of regulatory elements comforts the causal effect of the variant. It will be interesting to further investigate the nature of the complex as we noticed that the AMS variant introduces a change of one nucleotide in the conserved binding motif recognized by NEUROD1  mice [GDNF acts in a dose-dependent manner as suggested by GDNF-/- mice model [While the complete knock-out er birth ,64, the spection  and no mut) mice ,64. Thesce model .GDNF/GDNF-AS partnership represents a very precise regulation mechanism for the mRNA expression of GDNF. Indeed, since we showed that the mutation (in the last exon of GDNF-AS) dramatically diminishes both the expression of GDNF/GDNF-AS, by probably disrupting a regulatory element, the AMS variant most probably leads to a decrease of the GDNF expression, preventing a correct growth and maintenance of the small fibers from the knees down to the paws. To date, no mutation in the GDNF gene or in its regulatory regions has been described in human HSAN patients. This work illustrates the power of the canine model in genetics and shows the potential implication of GDNF in such neurological diseases, revealing the importance to explore this gene and its regulatory elements as serious candidates in human HSAN patients.In conclusion, considering the parallel with BDNF function, our RNA expression analyses, and dog/human comparative genomic analyses, we hypothesize that the http://dog-genetics.genouest.org). The work with dog samples was approved by the CNRS ethical board, France (35-238-13) for UMR6290.All dogs were client-owned and no harmful procedures were performed, so there was no animal experimentation according to the European legal definition . Blood and biopsies were obtained as part of routine clinical procedures for diagnostic purposes, approved by the CNRS ethical board (France), and were sent by veterinarians and French Veterinary Schools. The biological samples were obtained from the ‘Cani-DNA_CRB’, which is part of the CRB-Anim infrastructure, ANR-11-INBS-0003  according to the manufacturer’s instructions. Epidemiological and clinical data were collected using a dedicated questionnaire for each affected dog. Dogs over three years old without any symptoms were considered unaffected. Complementary neurological diagnoses  were done by neurologists, in the Veterinary Schools of Lyon (France) and St-Hyacinthe (Canada).Using the Illumina Canine HD 173k (BeadChip), genotyping was performed at the Centre National de Génotypage  in the frame of European LUPA project in 62 dogs: 49 French spaniels (21 affected and 28 unaffected dogs). This design contained 2 subpopulations: one from France and one from Canada, each including 3 small families (parents + 2 sibling including one affected and one unaffected), all other dogs were unrelated to one another at the grandparent level. To reduce the 3 Mb locus, additional dogs were genotyped including 5 German Short-haired Pointers , 4 unrelated English Pointers (1 affected and 3 unaffected dogs) and 4 unrelated English Springer Spaniels . Using the PLINK software (v1.06–1.07)  for SNP Genomic library sample preparation was performed using the Illumina paired-end library sample preparation kit . Sample preparation was carried out by Integragen  according to the manufacturer’s instructions (Agilent), using 4μg of genomic DNA. We used two unrelated affected dogs in French Spaniel (FS), one affected German Shorthaired Pointer (GP) and one affected English Springer Spaniel . For conwww.bioinformatics.nl/primer3plus/) [GDNF gene and the variant on 16 affected and 16 unaffected French Spaniel using the Type-it Microsatellite PCR kit (Qiagen) on C1000 and S1000 thermocyclers (Bio-Rad). PCR products were cleaned using the Illustra Exostar-1 Step reagent (Dutscher) and sequenced by Sanger method using the BigDye Terminator v3.1 cycle sequencing kit (Thermo Fisher Scientific). Electrophoresis of the products was realized on a 370 ABI sequencer. Sequences were analyzed using the PhredPhrap and Consed software’s [All primers used were designed using Primer3plus (r3plus/) . We re-ftware’s –76. The GDNF-AS long non-coding RNA (lincRNA), we analyzed 33 RNA sequencing data produced from 25 different tissues of 7 breeds (i.e. not annotated in Ensembl) was measured using three complementary programs CPC, CPAT, PLEK [https://www.r-project.org), we computed all pairwise Pearson correlations for the 5 representative genes in the AMS locus i.e. GDNF-AS, GDNF, WDR70, NUP155, and C5orf42 , WRD70 (TFS Ref: Cf02651565_m1), NUP155 (TFS Ref: Cf02644933_m1) and SLC1A3 (TFS Ref: Cf02702629_m1) and we also specifically designed other probes for GDNF-AS (PPIB (Peptidylprolyl Isomerase B) was used as the reference gene (Thermo Fisher Scientific Reference: Cf02629556_m1). Each sample was measured in triplicate and each qRT-PCR was carried out three times by different experimenters. Relative amounts of the transcript were determined using the ΔΔCt method [https://www.r-project.org), we determined the significance of the variation of expression level for all genes.RNA was extracted from tissues, using the NucleoSpin RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. We used two affected French Spaniel (brother and sister) and two unrelated dogs for controls, all already genotyped on the Illumina Canine HD 173k. Reverse transcription was performed on 1 μg of total RNA using the High-capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The total Dorsal Root Ganglia cDNA of the canine er pairs . qRT-PCR GDNF-AS . Canine t method . Expresset al [Clones were created to be centered on the AMS variant and correspond to a fragment of 2359 bp containing two SINE, two LINE, one LTR, and eight SNVs. PCR was performed from the genomic DNA of two dogs: one affected dog carrying the mutation and rare alleles for 8 SNVs, and one unaffected dog with the same haplotype as the dog genome reference. The PCR fragments were cloned into the pTAL Luciferase plasmid  by DNA ligation or by homologous recombination using the Gibson assembly MasterMix . All primer sequences used to prepare these constructs are given in et al .HeLa nuclear extract were purchased from the Computer Cell Culture Center S.A. (Belgium) and prepared according to Dignam et al . SY5Y ce32P-g ATP. Labeled oligonucleotides were purified on a Sephadex G-25 column. Unlabeled complementary oligonucleotides were then annealed to form a WT-duplex or Mut-duplex in hybridization buffer  by incubation for 1 min at 95°C and gradually cool down to room temperature. Complete annealing of the radioactive probe was checked by electrophoresis on 20% native acrylamide gel. For complex formation, 40fmoles (15000cpm) of duplex was incubated with various amount of nuclear extracts in a total volume of 10μl containing 25 mM HEPES (pH 7.5), 150 mM KCl, 5 mM dithiothreitol (DTT), 10% glycerol, 1 μg of poly (dI-dC). Incubation was performed for 30 min on ice prior to loading. For competition experiments, the mixture was preincubated for 30 min at 4°C with 2 pmoles of unradiolabeled competitor duplex, before addition of the radiolabeled duplex. Complexes were then fractionated onto a 5% polyacrylamide (38:2) gel with 2.5% (v/v) glycerol, 0.5 mM EDTA and 22.25 mM Tris-borate (pH 8.3) by electrophoresis at 4°C in 0.5 mM EDTA, 0.25%glycerol and 22.25 mM Tris-borate (pH 8.3). Free and bound duplexes were visualized with a phosphoimager using the ImageJ software.Probe labeling and duplex formation: 10 pmoles of primers wild type sequence (WT: 5’- TGTTGTCTTTGCTGCTGTCATGATGG-3’) and mutated sequence (Mut: 5’- TGTTGTCTTTGCTACTGTCATGATGG-3’) were 5'-end labeled using T4-PNK (Promega M4101) and 20μCi of S1 File(TXT)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.