CNTNAP2). In addition, we demonstrate that FoxP1/4 bind to the regulatory region of very low density lipoprotein receptor (VLDLR), as has been shown for FoxP2 previously. Interestingly, FoxP1/2/4 individually or in combinations regulate the promoters for SV40, zebra finch VLDLR and CNTNAP2 differentially. These data exemplify the potential for complex transcriptional regulation of FoxP1/2/4, highlighting the need for future functional studies dissecting their differential regulation in the brain.The Forkhead transcription factor FOXP2 is implicated in speech perception and production. The avian homolog, FoxP2 Fox genes perform tissue specific functions during development and mutations can cause cancer and other diseases  transcription factors comprise 19 highly evolutionary conserved, structurally related families, FoxA to FoxS. The defining feature of these genes is the Fox domain which binds to regulatory regions of target genes. Many FoxP subfamily members in vertebrates, FoxP1 to FoxP4 , encoding one of the reelin receptors, and Contactin-associated protein-like 2 (CNTNAP2) gene which codes for a neurexin called CASPR2  were bred in our colony at the Freie Universität Berlin. All procedures were performed according to the guidelines of the governmental law (TierSchG), under permits granted by the local Berlin authorities governing research involving animals. All birds were sacrificed with an Isoflurane overdose.Male zebra finches (Co-Immunoprecipitation (Co-IP) was done using Dynabeads® Protein G  following the manufacturer’s protocol with a few changes as follows. We first incubated the antibody-Dynabeads mixture for 15 min at room temperature  lysis medium for 15 min on ice. Extracts were centrifuged for 10 min at 1500 g and supernatant was stored at −80°C until used. Cell extracts and co-IP’ed proteins were separated by SDS PAGE (8%–10%), transferred to a polyvinylidene fluoride membrane , and blocked with Roti-Immunoblock for 2 h or overnight at 4°C. The membranes were then incubated with the desired antibody  25 μl of OptiMEM (GIBCO) containing 30 ng of pGL4.13 (Luciferase gene driven by the SV40 promoter which is known to be regulated by FoxP subfamily members) and 30 ng of pGL4.75  and 250 ng total vector over-expressing the different FoxPs for each well; and (b) 1 μl of Lipofectamine™ 2000 (Invitrogen) in 25 μl OptiMEM that was premixed for 5 min at RT. After combining (a) and (b) the mix was incubated for 20 min at RT, then it was added to the cells. After 4–6 h of incubation we changed the medium to 75 μl of antibiotic containing medium and incubated for further 48 h at 37°C in a CO2 incubator.For SV40 we seeded ~30000 HeLa cells in 200 μl DMEM medium (GIBCO) containing Penicillin-Streptomycin  per well of a 96-well white flat bottom plate . Transfection was performed using Lipofectamine™ 2000 (Invitrogen) following manufacturer’s protocol. Briefly, after seeding plates were incubated for 24 h at 37°C at 5% COVLDLR and CNTNAP2 we used HeK293 cells, and luciferase assays were done as described previously  following manufacturer’s protocol in an Elisa plate reader . Mean background from untransfected wells was subtracted from all other wells. We present Luciferase results as mean Relative Light Units (Luciferase RLU/Renilla RLU) calculated from the normalized values of 4–7 independent assays.The pGL4-VLDLR promoter was cloned as described .Proteins for electrophoretic mobility shift assays (EMSAs) were purified using the Protino Ni-NTA Agarose  according to manufacturer’s protocol. Protein was quantified using BCA1 (Sigma). EMSA assays were carried out as published previously, using the Oligo described for VLDLR , and therefore we used FoxP2 and FoxP4 polyclonal antibodies to pull down protein complexes, and all three antibodies for Western blot detection. As a negative control we used IgG antibodies of the same species in which the specific FoxP antibodies were raised. After IP with FoxP2 or FoxP4 antibodies, we detected co-IP’ed FoxP1 Figures . After ISince the majority of FoxP expressing cells in Area X and the surrounding striatum express FoxP1/2/4 . The SV40 promoter has a putative core consensus sequence for binding (TATTTRT) human and murine Foxp1/2/4 , in the same range as reported for mice and human FOXP1/2/4. Also, like its mammalian homologs, zebra finch FoxP4 repressed transcription stronger than FoxP1 or FoxP2 did, and FoxP2 was a weak repressor of the SV40 promoter . There were no significant differences between cells expressing FoxP1 or FoxP4 alone or in any of the combinations tested. We did observe a stronger transcriptional repression in cells expressing FoxP2 in combination with another FoxP subfamily member than when FoxP2 was expressed alone . However, we cannot rule out the formation and action of homodimers of FoxPs in those cells. Taken together, FoxP target genes in cells that express only FoxP2 may be less strongly regulated than those that are co-expressed with FoxP1 and/or FoxP4.We assessed whether the homotypic and heterotypic interaction of zebra finch FoxP proteins lead to differential regulatory activity. To do so we used the SV40 promoter driving Luciferase (VLDLR for a number of reasons: (a) it is a direct target of FoxP2 in humans, mice, and zebra finches . However, FoxP2 and FoxP4 were less effective and not significantly different from each other in their ability to activate the VLDLR promoter (p = 0.999). Combinations of FoxP1/2/4 did activate the VLDLR promoter to a similar degree . Comparing the activation of FoxP2 alone to the FoxP1/2 and FoxP1/2/4 combination (p = 0.0078 and p = 0.012 respectively) we found a significant difference on the VLDLR promoter but not with the FoxP2/4 combination (p = 0.88). There were no differences between the individual activation by FoxP4 and the combinations with FoxP1 (p = 0.10) or FoxP2 (p = 0.98), but there was a significant difference to the FoxP1/2/4 combination (p = 0.034). Taken together FoxP1/2/4 are not only repressors, but are able to bind to the VLDLR promoter and activate it differently depending on the co-expression with other FoxPs.To test whether binding of FoxP1/2/4 to the CNTNAP2 because: (a) CNTNAP2 is associated with speech disorders, as well as ASD, dyslexia and intellectual disability .To further test the regulatory ability of zebra finch FoxPs on a target gene relevant in Area X, we chose CNTNAP2 promoter contained a sequence gap located ~0.75 kb upstream of the start codon of the first exon of CNTNAP2 . FoxP1 was able to bind to the CNTNAP2 oligo and resulted in two shifted bands, consistent with the possibility that it binds in two of the three FoxP2 binding sites . Moreover, in combination, the different FoxPs also failed to regulate expression driven by the CNTNAP2 promoter, . Interestingly, combinations of FoxP4 with either FoxP1 or FoxP2 did not result in repression or activation, in contrast to FoxP1 or FoxP2 alone, suggesting that hetero-dimerization might affect the regulation of FoxPs. Taken together we show that FoxP1 and FoxP2 can bind to and regulate the CNTNAP2 promoter, with opposing activities. CNTNAP2 is an example of how FoxPs may tune the regulation of different targets genes via homo- and hetero-dimerization.To investigate whether binding of FoxP1 and FoxP2 to the r Figure . When teo Figure  FoxP4 faSV40, VLDLR and CNTNAP2 and regulate their transcriptional activity. Zebra finch FoxP1/2/4 repressed the SV40 promoter activity, as reported for mouse and human , which is in the opposite direction of the present findings with HeK293 cells. This highlights the plasticity with which FoxP proteins can regulate target genes in different cellular contexts, depending on different binding of co-factors that change the regulation of the same gene  two ways to associate or interact: the leucine zipper motif and domain swapping, e.g., the exchange of identical structural elements involving the Forkhead domain (Stroud et al., VLDLR and CNTNAP2. All zebra finch FoxP proteins studied bound to the VLDLR oligonucleotide, which was previously shown to bind to FoxP2 (Adam et al., VLDLR oligonucleotide contains a partial FOXP core sequence, ATTT (Stroud et al., ATTTAT (Wang et al., RTTTAY (Pierrou et al., CNTNAP2 oligonucleotide contains three putative FoxP2 binding sites. One TATTTAT (Enard et al., Finally, our data are the first to address neural targets regulated by FoxP1 and FoxP4. We tested the binding of two known targets of FoxP2 in zebra finches, VLDLR and CNTNAP2. Importantly, different FoxP combinations resulted in specific, differential transcriptional regulation. Together, our data demonstrate how versatile and variable FoxP regulation can be in the neural context.In summary, we show that zebra finch FoxP proteins can interact with each other in all combinations in the songbird brain. We show that all neurally expressed FoxPs have the capacity to bind to and regulate the target genes EM and CS: experiment design, analysis of data and wrote the manuscript. EM did all experiments.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We are entering an era of epigenome engineering. The precision manipulation of chromatin and epigenetic modifications provides new ways to interrogate their influence on genome and cell function and to harness these changes for applications. We review the design and state of epigenome editing tools, highlighting the unique regulatory properties afforded by these systems. Chromatin is decorated by a large array of biochemical modifications made to DNA and histone proteins . These mYet, there remains much that we do not understand about the function of the epigenome. Recently, with the advent of genomic techniques, there has been remarkable progress in our ability to map epigenomic modifications at a global scale and to correlate them with gene expression. While the roles of many chromatin modifications remain unclear, some important patterns have begun to emerge in which epigenome states have come to define key signatures of gene regulation, cell activity, and even disease states , 3. DespAt the center of these new efforts are the programmable DNA-targeting technologies behind the genome engineering revolution: zinc fingers (ZFs), transcription activator-like effectors (TALEs), and the CRISPR/Cas systems. These technologies are now being utilized for targeted epigenome editing through the recruitment of functional domains to DNA sequences of interest Fig. . ChromatTo explore and exploit the functional roles of DNA and histone modifications, new tools are being developed to selectively alter chromatin biochemistry at specific genomic loci. One striking feature of chromatin is the large biochemical diversity in the modifications and their substrates , 11. ForCells use a system of chromatin effectors and associated histone and DNA modifications to modulate and establish gene-expression states. A central goal has been to try to link these modifications to specific functional roles, such as transcriptional activation and repression , 3, 13. The advent of programmable DNA-targeting technologies, including ZFs , TALEs –18, and Sox2, in breast cancer cells through chromatin modifications [Early examples of epigenome editors include programmable DNA methyltransferases –27 and dications . The KRAications , 37. Othications  or LacI ications . Similarications .Ideally, the activity of an engineered epigenome editor is localized to a specific genomic location. One key way of controlling this is through the DNA-targeting module. Indeed, the targeting specificity of the DNA-binding module is likely to be important in defining the overall activity of an editor, specifically by directing CR activity to a specific genomic locus and thereby minimizing opportunities for off-target effects. Studies that directly compare the activity of an editor across the different classes of DNA-binding modules are lacking, but different patterns of off-target activity have been detected, for example, for KRAB fusions to ZFs and nuclease-null dCas9 –43.The genome-wide specificities of programmable DNA-binding modules, and strategies for improving them, have been the subject of considerable recent study , 44, whiThe genomic specificity of an editor can also, in some cases, be enhanced by altering the activity of the CR by changing its catalytic activity or its intrinsic interactions with binding partners, such as other regulatory proteins or DNA . For exaThe use of full-length CRs and potent transcriptional activators or repressors, such as KRAB and p65, can be effective in inducing chromatin-mediated transcriptional changes. However, these components are known to recruit multiple chromatin-modifying activities and to induce broad chromatin changes, which confound our ability to link specific modifications to specific functional roles. Addressing this issue requires epigenetic editors that have precise control over the desired chromatin-modifying activities. It also requires quantifying the biochemical specificity of an epigenetic editor, that is, quantifying the full array of modifications made to a locus that has been targeted by an editor. These modifications are inherently more challenging to quantify than genomic specificity: a comprehensive panel of DNA histone modifications must be assessed using techniques such as chromatin immunoprecipitation (ChIP) with many different antibodies.Strategies to create epigenetic editors that have improved functional or biochemical specificity have been explored. One key strategy is to truncate chromatin-modifying enzymes to their catalytic core domains. A notable recent example involved the human co-activator protein p300, which functions as a histone acetyltransferase and mediates interactions with multiple transcription factors to regulate many genes in tissues throughout the body. By fusing the catalytic core of the p300 acetyltransferase to dCas9, Hilton et al.  created A related strategy for improving the functional specificity of epigenetic editors is to remove non-catalytic domains or components from CRs in order to minimize the potential for non-specific interactions. For example, site-specifically recruiting the minimal catalytic domain of the histone methyltransferase SUV39H1 with a ZF array efficiently repressed the VEGF-A promoter, whereas full-length SUV39H1 did not cause repression . PresumaCollectively, these studies have used fusions to minimal catalytic domains to develop epigenetic editors that have improved functional specificity. Efforts to truly isolate and re-engineer the catalytic domains of CRs will be key to improving the functional specificity of epigenetic editors.In addition to improving biochemical and site specificities, several important challenges remain. Current efforts have been focused predominantly on constructing epigenome editors by fusing writer or eraser domains with DNA-targeting elements. Engineered readers remain largely underdeveloped Table . PotentiContinued work on characterizing and discovering new catalytic domains will expand the list of available parts from which to select for improved properties such as substrate specificity –71. AnotThere exist a surprisingly large number of epigenome modifications. The combinatorial interactions between these modifications and other chromatin-bound proteins increase this complexity even further. In fact, most chromatin states that are associated with regions such as active promoters and enhancers are characterized by specific combinations of chromatin modifications . Why didWith context dependency, one modification could mask, modulate, or enhance the binding interaction of a reader of a second modification. This is seen in the association of HP1 with H3K9me3, which is abolished by the dynamic and transient phosphorylation of the adjacent Ser10 residue . SimilarCombinatorial modifications could also prime a gene to follow one of multiple possible paths. Certain domains of the embryonic stem (ES) cell genome possess both activating and repressive histone modifications, known as bivalent domains; these are typically enriched at developmentally important genes , 77. It Gene expression is precisely controlled in time and space by the integration of this diverse array of PTM signals and the actions of multiple chromatin-regulating factors operating in multifactorial ways , 78. If The interactions between chromatin proteins, chromatin modifications, and the surrounding DNA sequence and chromatin state determine local transcriptional outputs. This is key for the design of functional epigenome editors because behaviors that are observed at one specific locus may not hold at another locus where the presence of existing proteins may alter the activity of a recruited epigenome editor. Therefore, one important goal for epigenome engineers is to reveal the rules of chromatin context. Accessing and deciphering these rules will require high-throughput and combinatorial techniques.There have been several in vitro methods for the rapid assessment of combinatorial and contextual properties of epigenome editors , but theIn our group, Keung et al.  fused a The simplicity of programming the CRISPR-Cas9 system to target multiple endogenous genomic loci simultaneously –84 and/oAmong the myriad modifications being written and erased on chromatin, a subset is stably inherited through mitotic or meiotic cell divisions. These epigenetically inherited modifications are important for the maintenance of gene expression patterns throughout the differentiation and developmental processes of mammals and may result in disease or cancer when mis-regulated , 91. Sevtrans- or cis-acting or a combination of both [Trans-acting mechanisms typically involve positive feedback of a transcription factor in the regulation of its own gene. This mechanism is utilized both to establish and to self-sustain a specific transcriptional state of a gene, as demonstrated in the activation and maintenance of differentiated functions of nematode sensory neurons [Cis-acting mechanisms more often involve chromatin modifications directly. DNA methylation in mammals is a prime example [A variety of different mechanisms underlie epigenetic properties but they all depend on some form of feedback. Broadly, feedback mechanisms can be  of both . Trans-a neurons , 95 and  neurons , 97. Cis example . DNA met example , 100. Wo example . Epigene example . However example , 103.Histone modifications are also involved in maintaining epigenetic regulation. For example, antagonizing groups of protein complexes, the Polycomb (PcG) and trithorax (trxG) groups, mediate the mitotic inheritance of repressive and active transcriptional states, respectively . There iIn a landmark study, Hathaway et al.  developeThe relationship between DNA methylation and H3K9 methylation, as well as other types of repressive modifications, was further investigated by Bintu et al.  in an elSchizosaccharomyces pombe [+ gene [The strong epigenetic properties of DNA methylation were confirmed in both studies. Nevertheless, studies are still attempting to confirm whether various histone modifications are truly epigenetic, that is, self-sustaining in the absence of the initial triggering signal or any necessary DNA sequence , 109. Foes pombe , 112. A  [+ gene , 112. TrArabidopsis thaliana [Many other histone modifications still remain to be tested for their epigenetic properties and many molecular details of epigenetic mechanisms remain to be discovered . These othaliana . This pathaliana .Many opportunities for exploiting the unique features of epigenetic regulation lie ahead. Researchers could work to harness any potential restricted or conditional epigenetic inheritance of histone modifications for developing “short-term” or “flexible” epigenetic memory circuitry , which cChromatin adds a unique spatial element to gene regulation at multiple scales , 116. FoDrosophila, wherein a normally active euchromatic gene is juxtaposed with heterochromatin by structural rearrangement and becomes silenced [Several molecular approaches have already been used successfully to perturb chromatin structure and these studies suggest that continued work in this area could reveal important and potentially useful regulatory principles relating to chromatin shape. For example, an ectopic repressor assay using a drug-inducible ZF-KRAB fusion protein demonstrated that KRAB-mediated repression spans tens of kilobases and is established by the long-range propagation of H3K9me3 and HP1 β . This ansilenced . Modelinsilenced , 124. ThRecent efforts have begun to directly manipulate chromatin looping and to change the three-dimensional contact profile of genes with other loci or nuclear structures Fig. . Deng anThere are other ways to induce structural perturbations in chromatin. Even a change in the direction of a small fragment (~20 bp) of DNA sequence can control transcriptional activity by reconfiguring the topology of chromatin loops . CCCTC-bStructural- or spatial-factor-dependent regulation of gene expression can also be mediated by spatially positioning genes in the nucleus. The randomly integrated reporter platform of Akhtar and colleagues , for exaIn this review, we have discussed important features that must be considered when designing functional epigenome engineering tools and current challenges that need to be addressed. The impact of recent advances in epigenome engineering has been remarkable in terms of both understanding the underlying mechanisms of epigenome regulation and designing new ways to regulate genes for future biomedical and biotechnological applications. Forward-engineering approaches allow researchers to directly interrogate the relationships between the epigenome and transcriptional function. These approaches are highly complementary to other cell biology methods and are particularly useful for systematically exploring large parameter spaces . In addiPerhaps one of the most exciting prospects in developing new epigenome editing tools is how they may alter our perspective of the function and nature of the epigenome’s complexity. Several current models depict chromatin modifications as an additional layer of regulatory nodes that act in concert with genetic networks to coordinate cellular programs . With ou


We then asked whether imprinted or nonimprinted paternally unmethylated DMRs harbored motifs for these TFs, and found many shared motifs between the two groups. However, DMRs that are methylated in the male germ cells also share motifs with DMRs that remain unmethylated. There are recognition sequences exclusive to the unmethylated DMRs, whether imprinted or not, that correspond with cell-cycle regulators, such as p53. Thus, at least with the current available data, our results indicate a complex scenario in which TF occupancy alone is not likely to play a role in protecting unmethylated DMRs, at least during male gametogenesis. Rather, the epigenetic features of DMRs, regulatory sequences other than DMRs, and the role of DNA-binding proteins capable of endowing sequence specificity to DNA-methylating enzymes are feasible mechanisms and further investigation is needed to answer this question.Genomic imprinting is an epigenetic mechanism that affects a subset of mammalian genes, resulting in monoallelic expression depending on the parental origin of the alleles. Imprinted regions contain regulatory elements that are methylated in the gametes in a sex-specific manner . DMRs are present at nonimprinted loci as well, but whereas most regions are equalized after fertilization, methylation at imprinted regions maintains asymmetry. We tested the hypothesis that paternally unmethylated DMRs are occupied by transcription factors (TFs) present during male gametogenesis. Meta-analysis of  One of the outstanding questions in genomic imprinting is how imprints are established. Because all known imprinted loci are associated with at least one CG-rich sequence that is monoallelically methylated , DNA methylation is a reasonable surrogate for imprints . The pati.e., that in each germline, distinct elements targeted for methylation are recognized by the methylation machinery. This hypothesis is undermined by the fact that Dnmt3a and Dnmt3l, the enzymes responsible for establishing methylation imprints, do not have binding specificities  expressed in the male fetal germ cells are good candidates for blocking their binding sites from DNA methyltransferases. This hypothesis implies that DMRs contain motifs recognized by these TFs, and also that transcriptional activity could be associated with them.In the male, paternally imprinted DMRs acquire DNA methylation during a wave of genome-wide methylation that occurs between 13.5 and 15.5 d postcoitum (dpc), at the prospermatogonia stage  from both XX and XY mice. The gonads were FACS-sorted for positive EGFP expression, which indicates their germ cell origin. RNA was purified, analyzed with Affymetrix Mouse Genechip Gene 1.0 ST arrays, RMA normalized, and submitted to GEO (Accession number GSE27715). Microarrays were validated by examining expression of particular transcripts previously known to change expression levels in a sex- and/or lineage-specific manner. Multiple pairwise comparisons on the normalized array values generated gene lists that were statistically significantly (P < 0.05) enriched or depleted (>1.5-fold), relative to both other lineages and the other sex. For our experiments, gene lists were specifically taken from Dataset S2 in Original gene lists for germ cell– and sex–specifically expressed genes were obtained from ftp://ftp.informatics.jax.org/pub/reports/Affy_1.0_ST_mgi.rpt) (http://www.informatics.jax.org/batch) with Genome Location as the chosen output: chromosome, strand, start position, end position, and Gene Ontology (GO) terms . We considered promoter sequences as a 600 bp region containing 500 bp upstream of the transcription start site to 100 bp downstream of the transcription start site (TSS). For genes on the plus (+) strand, the promoter region is evaluated as (Start − 500) to (Start + 100). For genes on the minus (−) strand, the promoter region is evaluated as the reverse complement of (End − 100) to (End + 500). BED files were generated with these data and the UCSC Table Browser data retrieval tool was used to retrieve sequences in FASTA format from the UCSC Genome Browser Database (https://genome.ucsc.edu/)  IDs were retrieved from the MGI Microarray Annotation File based on probeset ID  was used (In order to discover motifs was used . As inpuhttp://meme-suite.org/) (http://meme.nbcr.net) with the JASPAR Core 2014 vertebrates motif database (205 motifs between 5 and 30 nucleotides in length). We kept only statistically significant motifs with q-values < 0.05, where q-value is defined as the minimal false discovery rate (FDR) at which a given motif is deemed significant  . As inpubased on . We convhttps://hyperbrowser.uio.no/hb/), was used to perform statistical analyses comparing the chromosomal distribution of genes in the samples (http://genome.ucsc.edu/cgi-bin/hgLiftOver) for use in Genomic HyperBrowser. To compare the chromosomal frequencies for the sex-specific genes, BED files for the groups were loaded as tracks and hypothesis testing was performed. The null hypothesis was that the expected fraction of points in track one in each chromosome is equal to the expected fraction of points in track two in each chromosome. The alternative hypothesis was that the expected fraction of points of track one in each chromosome was not equal to the expected fraction of points of track two in each chromosome. P-values were computed under the null model by preserving the total number of points in both tracks, and randomizing their positions. The test statistic used is the Z-statistic based on the observed frequencies, using pooled standard deviation. A collection of FDR-corrected P-values  per chromosome was computed. Tracks were segments treated as the middle point of every segment. In addition to statistical analyses, the NCBI Genome Decoration Page (http://www.ncbi.nlm.nih.gov/genome/tools/gdp/) was used to visualize chromosomal ideograms annotating where sex- and stage-specific genes mapped to the genome.The Genomic HyperBrowser, a web-based platform based on Galaxy ; Table S2, A and B, contain enriched motifs identified in unmasked and repeat masked promoters, respectively, of genes enriched in E12.5 XX and XY primordial germ cells; Table S2, C and D, contain enriched motifs identified in unmasked and repeat masked promoters, respectively, of genes enriched in E13.5 XX and XY primordial germ cells; Table S3 contains enriched motifs identified in randomly generated control sequences; Table S4 contains the motifs in promoters of genes active in 12.5 and 13.5 dpc primordial germ cells; Table S5 contains the full sequences of paternally unmethylated DMRs associated with analyzed imprinted genes; Table S6 contains the full sequences of paternally unmethylated DMRs associated with analyzed nonimprinted genes; and Table S7 contains the sequences of paternally methylated DMRs associated with imprinted genes analyzed in this study.Datasets used in this study are publically available and referenced within the article. Data produced by us is available as tables presented within the article and in supplemental tables. Supplemental Material, mouse primordial germ cells  with the microarray results , using the criteria for “sequence-specific DNA binding TF activity,” obtaining 543 genes . The enriched known motif search identified two false positives, motifs for Tcfap2a and Tcfap2c (Table S3). These were eliminated from the subsequent analyses.Microarray studies could fail to detect key players in transcriptional regulation expressed at low, but functionally significant levels. Thus, we took a second approach to identifying TFs that could block methylation in male germ cells . A total of 87 of these motifs are for TFs detected in male primordial germ cells and prospermatogonia by microarray, and an additional nine are present in recent RNA-seq data . in vivo deletion in mutant mouse models, associated with Nespas, Peg3, Airn, Knq1ot, and Grb10 . We identified 18 distinct motifs present in both DMRs, five of which are shared. Surprisingly, 11 of these motifs are also present either in imprinted or nonimprinted gametic DMRs that remain unmethylated paternally, and 11 of the 18 motifs are binding sites for TFs present at 12.5 dpc male germ cells or 13.5 dpc prospermatogonia  and also contains a bric-a-brac/poxvirus-zinc finger (BTB/POZ) domain, which facilitates protein-protein interactions. When Bach1 forms a heterodimer with MafK, it functions as a repressor. Both Bach1 and MafK are expressed in male primordial germ cells and spermatogonia, but the Bach1 motif is not present in nonimprinted unmethylated DMRs. Also intriguing is the motif for Zbtb33, which binds to the unmethylated consensus KAISO-binding site TCCTGCNA. Zbtb33 recruits the N-CoR repressor complex to promote histone deacetylation and the formation of repressive chromatin structures.H19 and Dlk1/Gtl2 DMRs predicted 18 TF motifs, some of which were shared with the unmethylated DMRs, and for which 11 TFs are present. The H19 DMR contains four CTCF binding sites and is methylated on the paternal allele (H19 (Loukinov 2002). This is a possible explanation, but no definitive proof for this mechanism has been put forward to date, and the microarray and RNA-seq experiments did not detect expression of CTCFL in the male primordial germ cells or prospermatogonia.The observation that weakens the hypothesis is that imprinted gametic DMRs that are paternally methylated also have motifs for TFs present in the male germ cells, but they are not protected from methylation. Analysis of the H19 and Dlk1/Gtl2 imprinted DMRs, and constitute a complex with additional repressive factors and possibly histone modifiers. Methylated DMRs not associated with imprinted genes would lack these features, rendering them susceptible to post-fertilization demethylation. There is an abundance of zinc-finger proteins and noncoding RNAs with unknown function encoded in the genome and expressed in primordial germ cells and prospermatogonia that could accommodate exclusive recognition of each DMR. These factors could also detect sequences in combination with pre-established chromatin structures unique to the imprinted DMRs, not shared with other elements that are being methylated concurrently across the genome.An alternative scenario is that protection of unmethylated DMRs by DNA-binding proteins is a default mechanism and that methylated DMRs are recognized in a sequence-specific or chromatin state–dependent manner and tagged for methylation in combination with factors that result in resistance to demethylation. For example, sequence-specific DNA-binding proteins or noncoding RNAs could guide DNA methyltransferases to the Another possibility is that unmethylated DMRs associated with imprinted genes may be engaged in stable physical contacts with other regulatory elements, or isolated in specific topological domains unavailable to methylation enzymes, thus removing them from the genome-wide reprogramming events after fertilization. Modifications to current chromosome conformation assays to analyze low cell numbers are required to further test this proposal.There are several caveats to our analysis. First, although mRNAs for specific TFs are present, it is possible that the proteins are not, due to post-transcriptional inhibition. Second, motif analysis is continuously being improved due to algorithm development and as more datasets become available, it is possible that revisiting these hypotheses in the future will yield more insight. Third, it is clear that regulatory sequences other than promoters could be involved in protection against methylation, for example, as suggested above, by direct physical contact.In conclusion, the currently available data does not provide sufficient support for the hypothesis that TFs specifically protect unmethylated DMRs during male gametogenesis without making further assumptions. Even though much progress has been made in identifying the molecular mechanisms of DNA methylation, how it is established selectively for specific CGIs is still an open question.


HDAC2 in human cells. We find that while HDAC2 null cells exhibited signs of cross-regulation between HDAC1 and HDAC2, specific epigenomic phenotypes were still apparent using RNA-seq and ChIP assays. We identified specific targets of HDAC2 repression, and defined a novel class of genes that are actively expressed in a partially HDAC2-dependent manner. While HDAC2 was required for the recruitment of HDAC1 to repressed HDAC2-gene targets, HDAC2 was dispensable for HDAC1 binding to HDAC2-activated targets, supporting the notion of distinct classes of targets. Both active and repressed classes of gene targets demonstrated enhanced histone acetylation and methylation in HDAC2-null cells. Binding of the HDAC1/2-associated SIN3A corepressor was altered at most HDAC2-targets, but without a clear pattern. Overall, our study defines two classes of HDAC2 targets in human cells, with a dependence of HDAC1 on HDAC2 at one class of targets, and distinguishes unique functions for HDAC2.The transcriptional functions of the class I histone deacetylases (HDACs) HDAC1 and HDAC2 are mainly viewed as both repressive and redundant based on murine knockout studies, but they may have additional independent roles and their physiological functions in human cells are not as clearly defined. To address the individual epigenomic functions of HDAC2, here we utilized CRISPR-Cas9 to disrupt  Epigenetic regulation of gene expression is mediated by chromatin modifying complexes that orchestrate transcriptional activation or repression. For instance, the dynamic addition and removal of acetyl groups on specific lysine residues of the amino-terminal tails of histones within gene regulatory domains by specific protein complexes is a key epigenomic mechanism of transcriptional control. Net histone acetylation within chromatin regions is mediated by the interplay of histone acetyl transferases (HATs), which mediate the acetylation of lysines on histones to generally promote a transcriptionally active chromatin state, and the HDACs, which catalyze the removal of acetyl groups that is largely thought to induce a more repressive chromatin state. The Class I HDACs including HDAC1, HDAC2, HDAC3 and HDAC8, are widely expressed and are the most thoroughly studied of the HDAC families . In turnHDAC1 and HDAC2 share a high degree of amino acid identity , and form heterodimers with enhanced enzymatic activity . DependiNotably, disruption of HDAC1, HDAC2, or both can in some instances lead to decreased gene expression. For example, recent chromatin immunoprecipitation (ChIP) and transcriptomic analysis from HDAC inhibition in yeast cells or HDAC1 knockout studies in mice have shown a significant portion of downregulated genes signifying that these proteins may also work to functionally activate genes in the physiological state ,22,23. HHDAC2 to produce 293FT cells with HDAC2 loss-of-function. CRISPR-mediated production of disruptive Indels in HDAC2 successfully lowered HDAC2 protein levels to barely detectable, near background levels indicative of effectively null cells. Loss of HDAC2 does not lead to consistent compensatory increases in HDAC1 or HDAC3, but HDAC1 and HDAC2 are tightly functionally interconnected. Knockout of HDAC2 leads to both increased and decreased expression of specific genes. HDAC2 is most often required for the recruitment of HDAC1 to repressed target genes bound by HDAC2. While HDAC2 contributes to activation of some direct gene targets, its disruption leads to either no change or increased HDAC1 binding at these gene targets suggesting a compensatory function specifically at this class of targets. In both classes of targets, we find that SIN3 binding is variably affected by loss of HDAC2 at individual genes. Overall, our data support a model of two separate mechanisms of HDAC2 function as either contributing to gene repression or activation with distinct roles for HDAC1 in each case.To address HDAC2 function in human cells, here we used CRISPR-Cas9 targeting of HEK293FT cells were cultured in DMEM supplemented with 10% FBS (HyClone) and 1% L-glutamine. Transfections were conducted with Roche X-tremeGENE HP DNA transfection reagent (Roche). Media was changed 24-hours post transfection.http://crispr.mit.edu. Oligos for the guide targeting the sequence CCCATGGCGTACAGTCAAGGAGG5’ (first ATG bolded) were annealed and ligated into the Bbs1-digested pSpCas9(BB)-2A-Puro vector (Addgene) as described [HDAC2 gene targeted region using the primers: Forward CTAACCTCGAGCCCGAAACG-3’5’- and Reverse 5’-CTCGTTCTAACTGTGCCGGG-3’. The resulting PCR products were cloned into the pCR4-TOPO vector using the TOPO-TA Cloning Kit (Invitrogen) according to the manufacturer’s instructions and DNA from at least 6 colonies were sequenced per clonal line to screen for and characterize any Indels present in each. The top four possible off-target regions predicted by the website tool were also sequenced by PCR amplification of genomic DNA from each clonal line. Primer sequences for off-target region amplification are listed in Guide RNAs targeting the first exon of HDAC2 were designed using the web tool escribed . IntegraHDAC2-null lines relative to WT cells identified through EdgeR analysis of RNA-seq data were also compared to differential gene expression data produced using Cufflinks analysis of the same RNA-seq data for validation.Three plates each of wild-type (WT) cells or HDAC2-null lines #5, #14, or #15 were grown to 60 to 70% confluence and total RNA was isolated from each clonal line separately using the Nucleospin RNA Kit , with DNase treatment 5 μg RNA was submitted to the UC Davis DNA Technologies Core for library preparation utilizing the KAPA stranded mRNA-seq kit , and sequenced as single reads (SR50) on the Illumina HiSeq3000 platform. WT and each clonal line were Poly-A enriched prior to sequencing. RNA-seq reads were aligned using Tophat ,27 and qGAPDH endogenous control and analyzed via the double ΔCT method. Student’s t-test was performed to assess statistical significance. Primers are listed in RNA was reverse transcribed using the iScript cDNA Synthesis Kit (BioRad) and qPCR assays were performed in triplicate technical replicates utilizing Absolute Blue qPCR SYBR Green Master Mix (Fisher Scientific) and 5 μL cDNA on Roche Light Cycler 480 at the following conditions: 95°C for 15 seconds, 60°C for 30 seconds and 72°C for 30 seconds for 45 cycles. Expression was normalized to ChIP assays were performed as described ,34. BrieNuclear lysates were prepared according to Abcam Nuclear Fractionation protocol. Briefly, cells were scraped into ice-cold PBS and resuspended into Buffer A  and incubated for 10 minutes. Pellets were resuspended in Buffer B  for 20 minutes, centrifuged, and nuclear fraction (supernatant) was used for analysis. Protease inhibitor cocktail tablet  was added to all buffers. Histone acid extracts were prepared as described . ProteinATG of the HDAC2 gene with low probability of off-target binding. To test the predicted CRISPR-mediated knockout of HDAC2, HDAC2 protein levels were examined in the overall panel of clonal lines and each exhibited nearly undetectable HDAC2 protein levels compared to the parental WT control line  are present in these clonal lines, it is unclear whether the very faint residual protein band present in the targeted lines at approximately the same size as HDAC2 is actual residual HDAC2 protein present  or a background band. A panel of 13 HDAC2 loss-of-function clonal lines, was analyzed for HDAC1 and HDAC3 protein levels to determine if there were consistent changes in other class I HDAC family member levels with the knockout of HDAC2. We found that HDAC1 protein levels were not significantly changed across the clonal line panel  sequence designed to target the first ne panel . Howeverne panel . While tuse ESCs , the decHDAC2 gene. Sequencing indicated that all three clonal lines are hetero-allelic  carries the same 11-bp deletion 3’ of the first ATG, but in combination with both a 1-bp insertion 3’ to the first ATG and a 3-bp deletion 5’ to the first ATG in the second allele. Overall, all alleles in clones 5, 14, and 15 have at least one Indel resulting in a frameshift leading to generation of a premature stop codon that is predicted to generate null alleles. Sequencing of the top four computationally predicted off-target regions  for each clonal line determined that no Indels were present  including for the presence of Indels through PCR-sequencing of the targeted region in the -allelic . Two clo present . OverallHDAC1, HDAC2, and HDAC3 using qPCR analysis to determine the potential effects of HDAC2 disruption on expression of other class I HDAC family members. CRISPR targeting of HDAC2 resulted in a mean 50% reduction of HDAC2 RNA levels in the three clonal lines  293FT cell lines, total RNA was isolated from all lines and RNA-seq was performed in replicate samples for each line  identified 95 genes differentially regulated at an EdgeR cut-off of 0.3  the genes COL6A1, COL6A2, LMNTD2, CDKN2C, PPP1R16A, and NEFM   also correlated with increased histone acetylation in their promoter regions. ChIP assays were conducted for pan-H4-acetylation (H4ac) and H3K9 acetylation (H3K9ac). We observed elevated H3K9ac with the loss of HDAC2 at all 5 derepressed genes tested . We alsod LMNTD2 . Additio CDKN2C) . OverallHDAC2 by RNA-seq , K562  and MCF7 (GSM1010825) human cells [We used qPCR to measure mRNA levels of specific genes predicted to be downregulated by loss of  RNA-seq , observi TP53BP1 , validatits loss . We alsoan cells . StrikinHDAC2, the genes with reduced expression associated with disruption of HDAC2 did not demonstrate a loss of HDAC1 binding  tested had no consistent change in HDAC1 enrichment, indicating that these genes are not repressed in the nulls due to compensatory binding of HDAC1 in the absence of HDAC2. In contrast, an increase of HDAC1 enrichment was observed in the remaining three genes  tested. These data suggest that genes that are normally expressed in an at least partially HDAC2-dependent manner may not be bound by HDAC1-HDAC2 dimers and that at some of these targets HDAC1 may bind to partially compensate for loss of HDAC2.Notably, in contrast to the concomitant loss of HDAC1 binding that was observed for the derepressed genes (HDAC2 targets of repression) with disruption of  binding . Two of HDAC2 nulls  exhibited altered SIN3A enrichment in HDAC2-null cells, but without a clear pattern of decreased versus increased binding in the nulls .S1 FigThe DESeq2 R package was used to perform and plot PCA.(TIF)Click here for additional data file.S2 Fig(A) Quantitation of individual clones shown in (B) Nuclear lysates of a panel of 9 additional HDAC2-targeted clonal lines were analyzed for HDAC2 protein levels through Western blotting. (C) Quantitation of panel B. HDAC2 protein levels were plotted relative to WT. (D) Nuclear lysates of a panel of 13 HDAC2-targeted clonal lines, including the three characterized lines #5, #14, and #15, were analyzed for HDAC1 protein levels through Western blotting. (E) Quantitation of panel D. HDAC1 protein levels were plotted relative to WT. Mean quantitation is represented in (F) Nuclear lysates of a panel of 13 HDAC2-targeted clonal lines, including the three characterized lines #5, #14, and #15, were analyzed for HDAC3 protein levels through Western blotting. (G) Quantitation of panel F. HDAC3 protein levels were plotted relative to WT. Mean quantitation is represented in (TIF)Click here for additional data file.S3 FigHDAC2 null clones #5, #14, and #15 and PCR-amplified for the top four predicted off-target regions based on sequence complementarity to the gRNA sequence, as predicted by the website tool. The predicted off-target regions in intronic regions of BMP15(A), PARP2(B), MYL2(C), or IKBKB(D) were sequenced. All alignments were done to the hg19 reference genome. Red boxes indicate the predicted off-target sequence within the alignment.Genomic DNA was isolated from WT cells or (TIF)Click here for additional data file.S4 Fig(A) or HDAC2-activated (B) gene targets to evaluate the binding of HDAC2 or HDAC1 or enrichment of the specified histone marks at the promoter regions of the genes indicated. Enrichment was calculated by normalization to input and IgG controls and samples are plotted as mean of n = 3 with error bars representative of S.E.M.ChIP-qPCR was conducted on crosslinked chromatin of WT and clones #5, #14, and #15 with antibodies to pan-H4ac or IgG control and SYBR green qPCR analysis was conducted for HDAC2-repressed (TIF)Click here for additional data file.S5 Fig(A) with HDAC2-disruption (see (B) with HDAC2-disruption Click here for additional data file.S6 Fig(A-C) Histone acid extracts of WT or HDAC2 null clones #5, #14, and #15 were analyzed for total H4ac (A), H3K9ac (B), or H3K9me3 (C) levels through Western blotting. Image is representative of three independent Western blots. (D-F) Quantitation of the triplicate Western blot results: H4ac (D), H3K9ac (E), and H3K9me3 (F). In each case, protein levels were normalized to B-actin with LiCOR imaging software and represented relative to WT with error bars S.E.M (n = 3).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 TableDuplication rates (Dup) were determined both by Position (Pos) and Sequence (Seq).(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLS)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.


Xylanase inhibitors have been confirmed to be involved in plant defence. OsXIP is a XIP-type rice xylanase inhibitor, yet its transcriptional regulation remains unknown.OsXIP promoter exposed to rice brown planthopper Nilaparvata lugens stress, a 562 bp region (–1451 – −889) was finally identified as the key sequence for the herbivores stress response. Using yeast one-hybrid screening, coupled with chromatin immunoprecipitation analysis, a basic helix-loop-helix protein (OsbHLH59) and an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor OsERF71 directly binding to the 562 bp key sequence to activate the expression of OsXIP were identified, which is further supported by transient expression assay. Moreover, transcriptional analysis revealed that mechanical wounding and treatment with MeJA resulted in an obvious increase in transcript levels of OsbHLH59 and OsERF71 in root and shoot tissues.Herbivore infestation, wounding and methyl jasmonate (MeJA) treatment enhanced mRNA levels and protein levels of OsXIP. By analyzing different 5’ deletion mutants of OsXIP responding to stress were identified. These results reveal a coordinated regulatory mechanism of OsXIP, which may probably be involved in defence responses via a JA-mediated signaling pathway.Our data shows that two proteins as direct transcriptional activators of The online version of this article (doi:10.1186/s12896-017-0344-7) contains supplementary material, which is available to authorized users. Xylanase inhibitors (XIs) are a kind of plant-produced proteinaceous inhibitor that inhibits the activity of xylanase . RecentlTaxi-Ia expression was induced 2.5 times and the transcripts of Taxi-Ib/III and Taxi-IIb/IV rose up to 20-fold by F. graminearum infection of wheat lemma, palea and ovary , the top of which was covered with a piece of sponge. One empty cylinder was used for control plants (non-infested).OsXIP gene promoter named OP1 (−2070 bp to +52 bp) was amplified from the genomic DNA of WT with OP1-U (forward primer) and OP1-L (reverse primer). Then a series of nested 5’ deletions of OP1 fragments OP2 (−1451 bp to +52 bp), OP3 (−889 bp to +52 bp), OP4 (−569 bp to +52 bp), OP5 (−380 bp to +52 bp), OP6 (−172 bp to +52 bp), OP7 (−90 bp to +52 bp) were amplified by PCR from pMD19T-OP1 using the common reverse primer OP1-L and either the forward primers OP2-U, OP3-U, OP4-U, OP5-U, OP6-U, or OP7-U, respectively. The primers are shown in Additional file GUS (β-glucuronidase). Empty vector pBI101.3-GUS was used as a negative control (VC). All constructs were mobilized into Agrobacterium tumefaciens EHA105 and transformed into calli derived from mature seeds of rice according to a previously described protocol  that was deleted from OP3. And the 562 bp fragment contains some stress-responsive cis-acting elements, such as W-box, ARR1AT, MBS. So we speculated that the 562 bp fragment was the key sequence involved in herbivore stress response. While the 290 bp fragment (−380 bp to −90 bp) was involved in herbivore stress response, this 562 bp fragment was then used for further research in this paper.After infestation with BPH for 24 h Fig. , GUS expOsXIP promoter were obtained. The functional proteins included fructose 1, 6-bisphosphatase, HSP, photosystem, Tify domain containing protein. While only two genes LOC_Os02g02480 and LOC_Os06g09390, according to the rice genome annotation of The Institute for Genomic Research , were the candidates of regulatory proteins. Based on BLAST analysis and literatures, LOC_Os02g02480 is a basic helix-loop-helix protein (OsbHLH59) [To determine the proteins bound to the herbivores-responsive promoter region, we used the 562 bp fragment mentioned above as a bait to screen transformants from a cDNA library generated from the leaves of rice plants infested with BPH for 24 h by yeast one-hybrid (Y1H) screening system. Through initial screening, DNA sequencing and BLAST analysis, some proteins including functional proteins and regulatory proteins showing interaction with sbHLH59) ; LOC_Os0sbHLH59) .The interaction between herbivores-responsive promoter region and the corresponding complete encoding products of the two genes were re-tested by Y1H assay. The analysis showed that the two proteins interacted specially with the promoter region Fig. . MeanwhiN. benthamiana leaves alone or co-expressed with the corresponding effector vectors, respectively. It was obvious that co-expression of OsbHLH59 remarkably increased LUC expression driven by the OP2 promoter, as did expression of OsERF71 . The expression of these genes appeared to be responsive upon abiotic stress and was similar Fig. . In shooOsXIP is a wound stress-responsive gene in rice [N. lugens induction process was very similar to that caused by wounding. When the rice was infested by BPH, wounding generated first and then activated the wound-responsive gene OsXIP, thus enhancing resistance against herbivores.The phytohormone jasmonic acid (JA) play a vital role in plant defense when plants are exposed to invaders . In gene in rice . When th in rice . In our OsXIP revealed that it was a stress-induced promoter. OsXIP promoter contained stress-responsive cis-acting elements, such as ARR1AT, W-box, G-box and TGACG-motif. The W-box (TGACY), has been confirmed to be present upstream of salicylic acid or wound signal responsive genes; The G-box (CACGTG) and G-box-like  are known to the binding sites of basic helix-loop-helix (bHLH) transcription factors [OsXIP gene responded to different stress  and a repressor (r) motif between OP2 and OP3 (−1451 bp to −889 bp) and OP4 and OP5 (−569 bp to −380 bp), respectively. So a regulation model of the OsXIP gene was proposed: an enhancer site (e) may exist in −1451 bp to −889 bp, and a repressor site (r) may be located between −569 bp and −380 bp in the promoter. GUS expression in OP2 was high due to the binding of enhancer (E) to its site (e). The enhancer might preferentially bound to the promoter and facilitate the gene expression on the grounds that binding of enhancer E at e site prevents the repressor R from binding to r site by either binding with R or obstructing r site. GUS expression of OP3 and OP4 decreased because of deletion of enhancer site (e). GUS expression of OP5 and OP6 increased after deletion of the r site. Serial deletion promoter constructs between −1451 and −889 bp and between −569 and −380 bp can be constructed to determine the locations and sequences of e and r by transient expression assay and electrophoretic mobility shift assay (EMSA).OsXIP promoter were finally identified as the key sequences involved in the herbivores stress response  in the nse Fig. . By Y1H say Fig. , furtherOsXIP were found. OsbHLH59 is a member of bHLH transcription factors, which are known to bind to G-box or E-box [Wx gene [In the present study, two proteins  as transcription activators of or E-box . We analor E-box , 35; OsB[Wx gene ; OsbHLH0[Wx gene ; DPF (Os[Wx gene .Arabidopsis, the bHLH transcription factor MYC2 is a key positive transcriptional regulator of JA signaling pathway, which is inhibited by JAZ transcriptional repressors [Arabidopsis composes of the two major branches: the ERF branch and MYC branch [OsXIP. In addition, transcriptional analysis revealed mechanical wounding and MeJA induced transcriptional expression of OsbHLH59 and OsERF71 in rice (Fig. OsXIP by wounding may be regulated by the JA-mediated signaling pathway [OsXIP. This speculation is further supported by the findings that OsXIP was independent of growth and development in rice plants [In pressors , 40. Simpressors . FurtherC branch . And theC branch . Thus, te plants  and its OsXIP via directly binding to its promoter. Our discovery contributes to clarify the regulatory mechanism of OsXIP and gives us a better understanding of the function of OsXIP in plant defence.In summary, we reveal the transcriptional regulatory mechanism of OsXIP and its involvement in defense response in rice. In response to herbivore infestation, mechanical wounding or MeJA stress, OsbHLH59 and OsERF71 transcription factors promote this process by activating the expression of


BDNF remains to be elucidated. In response to different stimuli, BDNF expression can be initiated from different promoters. Several studies have suggested that the expression of BDNF is regulated by promoter methylation. An emerging theme points to the possibility that histone modifications at the BDNF promoters may link to the neurological pathology. Thus, understanding the epigenetic regulation at the BDNF promoters will shed light on future therapies for neurological disorders. The present review summarizes the current knowledge of histone modifications of the BDNF gene in neuronal diseases, as well as the developmental regulation of the BDNF gene based on data from the Encyclopedia of DNA Elements (ENCODE).Brain-derived neurotrophic factor (BDNF) is required for the development of the nervous system, proper cognitive function and memory formation. While aberrant expression of BDNF has been implicated in neurological disorders, the transcriptional regulation of  Bdnf gene  delivery in animal models of neurological and psychiatric disorders improves memory formation and the survival of neuronal cells ..Bdnf exod IV–VII D. This fccupancy . In bother I–III .Bdnf exon IX increased from the embryonic to postnatal stage [Bdnf variants are induced during development. Indeed, several studies have pointed out that expressions of different BDNF exons were detected in different brain regions during development for both rodents and humans [Bdnf transcripts. Kainic acid, a glutamate analogue, induced transcription of Bdnf exons I, IV, V VII, VIII and IXA in rat hippocampus while N-methyl-d-aspartate (NMDA) treatment identified Bdnf exons II and IV, but not I and III, as fast-reacting exons [Bdnf exon IX in the E10.5, E13.5, E14.5, E16.5 and P0 stages (Bdnf exons except VIII and IX during E10.5 to P0 and remained so until the adult stage (Bdnf promoters I–III and was replaced by H3K4me3 during development (from a break line to a continuous line). The level of H3K27me3 modification gradually diminished from E10.5 to P0 within Bdnf promoters I–VII and no enrichment was found at the adult stage (Bdnf promoter increased and serine 28 of H3K27me3 was phosphorylated by p38-mitogen-activated protein kinase (MAPK)/mitogen- and stress-activated protein kinase 1 and 2(Msk1/2), which in turn replaced EZH2 [Bdnf expression increased (RNA-seq)  compared with young mice (8 months) [Bdnf expression. For example, treatment of trichostatin A (TSA), a HDAC class I and II inhibitor, increases histone H3 and H4 acetylation  at Bdnf promoter I in Neuro-2A cells [Bdnf promoters I and IV in cortical neurons [Bdnf promoters may depend on the cell types. Another enhancer mark, H3K4me1, was reduced throughout the developmental stages  [Bdnf promoters I–III as well as V–VII contact with Bdnf promoter IV [BDNF promoters and enhancer(s) around Bdnf exons IV–VII to form a high-order chromatin structure, providing additional regulation of Bdnf. Environmental cues such as neurotransmitters trigger activity-dependent transcription in the nervous system, leading to changes of behavior. The regulation of Bdnf is also activity-dependent. Depolarization of neurons by KCl increases H3K9ac, but decreases H3K9me2 at Bdnf promoter IV [Bdnf promoter VI upon KCl stimulation [BDNF genes, it remains possible that the regulation of BDNF between human and mouse might be different.The expression of al stage . RNA-seqd humans ,16,17,18d humans . In addid humans . Furtherng exons ,35. Thes0 stages E. The hilt stage D. Howevelt stage C. The deRNA-seq) F. This r months) . The enh2A cells . The addl stages A. No enhr (CTCF) ,40. CTCFmoter IV . Thus, imoter IV . Anothermulation . AlthougBDNF by DNA methylation and non-coding RNA in neurological diseases has been reviewed in References [BDNF expression in neurological diseases is an under-investigated area of research. In the following sections, due to limited references to other neurological diseases, only histone modifications of the BDNF gene in Huntington’s disease and Alzheimer’s disease are discussed.The regulation of ferences ,45. HoweBDNF was reduced in the brain of HD patients [Bdnf in the brain of a HD animal model ameliorated HD-associated symptoms [Bdnf expression [Bdnf may be due to the alteration of the chromatin state at its promoters. Indeed, in mouse models and patients of HD, a lower level of H3K4me3 is detected at both human BDNF and rodent Bdnf promoter II compared to controls [BDNF promoter. Symmetrical arginine methylation of H2A/H4R3 (H2A/H4R3me2s) mediated by protein arginine methyltransferase 5 (PRMT5) was reduced at the BDNF promoter II in the brain of HD patients [BDNF promoter PRMT5 acts as a positive regulator. On the other hand, recruitment of the repressive chromatin remodeler may also suppress the expression of BDNF transcripts in HD. For example, RE1-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) targets the neuron-restrictive silencer element (NRSE) to repress BDNF transcript at promoter II [BDNF transcripts. REST also binds to methyl-CpG binding protein 2 (MeCP2) and the binding of MeCP2 at promoter IV reduces both human BDNF and rodent Bdnf levels in a DNA methylation–dependent manner [BDNF may underlie the reduction of BDNF compared to a normal individual.Huntington’s disease is an inherited neurological disorder that results from the degeneration of brain neurons. The expression of patients . Furthersymptoms ,48. The pression . This recontrols . Moreovecontrols . A changpatients ,52. Althpatients . Thus, imoter II . REST hamoter II ,56,57,58t manner ,60,61. TBDNF expression was observed in several independent studies [BDNF in AD patients. Infusion of amyloid fibrils, one possible pathogenic protein, into the hippocampus of rats induces HDAC2 expression, as well as HDAC2 occupancy at promoter VI of Bdnf and thus represses Bdnf expression [Bdnf promoter IV in the prefrontal cortex [BDNF can be regulated indirectly by other factors. For instance, apolipoprotein E3 (ApoE3), a transporting protein of cholesterol, promotes the nuclear export of HDAC6 and thus decreases the level of HDAC6 at BDNF promoters III and IV. Along these lines, an increased level of exon IV was also observed. Furthermore, compared to healthy controls, a higher level of HDAC6 was found in nucleus in AD patients [Alzheimer’s disease is a neurodegenerative disease that results from the abnormal aggregation of protein in neurons, causing neuronal death. In the brain of AD patients, the reduction of  studies ,63. Howepression . In an al cortex . The redpatients .Bdnf by histone modifications shows an example of how the transcription of each exon is regulated by histone modifications and the potential promoter–enhancer relay mechanism through a transcription factor or transcriptional complex. Additional data from the analysis of the higher-order chromatin structure will provide more accurate in vivo regulation of the Bdnf gene. Genome-wide analysis of high-throughput data allows the identification of new features of chromatin, which reveal the overwhelming complexity of transcriptional control. The regulation of


Antisense transcription can regulate sense gene expression. However, previous annotations of antisense transcription units have been based on detection of mature antisense long noncoding (aslnc)RNAs by RNA-seq and/or microarrays, only giving a partial view of the antisense transcription landscape and incomplete molecular bases for antisense-mediated regulation. Here, we used native elongating transcript sequencing to map genome-wide nascent antisense transcription in fission yeast. Strikingly, antisense transcription was detected for most protein-coding genes, correlating with low sense transcription, especially when overlapping the mRNA start site. RNA profiling revealed that the resulting aslncRNAs mainly correspond to cryptic Xrn1/Exo2-sensitive transcripts (XUTs). ChIP-seq analyses showed that antisense (as)XUT's expression is associated with specific histone modification patterns. Finally, we showed that asXUTs are controlled by the histone chaperone Spt6 and respond to meiosis induction, in both cases anti-correlating with levels of the paired-sense mRNAs, supporting physiological significance to antisense-mediated gene attenuation. Our work highlights that antisense transcription is much more extended than anticipated and might constitute an additional nonpromoter determinant of gene regulation complexity. Transcriptomics developments have led to the discovery that eukaryotic genomes are pervasively transcribed, i.e., each nucleotide (nt) can be virtually transcribed into RNA . This apcis or in trans, as shown in the budding yeast Saccharomyces cerevisiae , as they are targeted by the 5′–3′ cytoplasmic Xrn1 exoribonuclease XUTs were shown to form double-stranded (ds)RNA with their paired-sense mRNA  system and lacks the RNase III endonuclease Dicer that can process dsRNA structures into small interfering (si)RNAs (In nse mRNA . Whether(si)RNAs .Schizosaccharomyces pombe constitutes an attractive model, as it shares RNAi with higher eukaryotes . To get insights into the nature of the antisense transcripts, we define the XUT aslncRNAs landscape using RNA-seq profiling in cells devoid of the 5′–3′ cytoplasmic RNA decay pathway, and we show that they escape RNAi in fission yeast. We observe that genes with asXUTs display specific chromatin marks. Finally, subsets of asXUTs are up-regulated in cells inactivated for the conserved histone chaperone Spt6, and in diploid cells undergoing meiosis. In both conditions, asXUT accumulation correlates with paired-sense mRNA down-regulation.Here, using NET-seq, we determine the landscape of antisense transcription in Our work indicates that antisense transcription in fission yeast is much more extended than anticipated and might constitute a nonpromoter determinant for gene regulation complexity.S. pombe, we constructed NET-seq libraries from two biological replicates of WT cells, following immunoprecipitation of RNAPII . As a validation that NET-seq detected nascent transcripts, we observed signals from introns and regions beyond the polyadenylation site, which are absent from mature mRNAs . Moreover, nascent transcription signals for mRNAs and annotated ncRNAs showed high reproducibility between replicates .To determine the comprehensive landscape of antisense transcription in Supplemental Fig. S1G). The choice of these genes rather than intergenic regions was motivated by the fact that the transcriptional state of the latter remains largely unknown.To identify genes with antisense transcription, we used (i) the nascent transcription signal (RPKM) and (ii) the coverage . As a reference, we used a set of 50 untranscribed protein-coding genes, showing the lowest expression at the nascent transcription level D and a vThe nascent transcription signal for the “untranscribed” set of genes was comprised between 0.07 and 0.89 RPKM , corresponding to 1522 ncRNAs from the current version of Pombase and 487 novel noncoding transcription units (TU) recently annotated , which is consistent with previous observations based on RNA labeling  Itlabeling .S. pombe display antisense transcription, and these genes are less transcribed than those without antisense transcription. This observation is far from the original assumptions based on RNA-seq and microarray data analysis and considerably extends the antisense transcript(ion) as a general property of the gene structure.In summary, analysis of nascent transcription using NET-seq provides evidence that up to 68% of protein-coding genes in Supplemental Fig. S2A). Illustrative examples of such convergent TUs are shown in Supplemental Fig. S1B).The observation of a peak of antisense transcription in the 3′ region of genes raises the hypothesis that this could correspond to overlapping convergent TU. Among the 3455 genes with antisense nascent transcription, 2530 showed overlapping NET-seq signals with a convergent annotated TU A. This iP < 2.2 × 10−16, Wilcoxon rank-sum test), suggesting a negative correlation between the level of sense transcription and the extent of the overlap with antisense transcription.Besides, 925 genes displaying antisense transcription are not convergent to another TU. Metagene analysis revealed a different distribution of the antisense transcription signal for these 925 genes, covering most of the antisense strand A. StrikiSupplemental Fig. S2B). Globally, we also noted that levels of sense and antisense transcription are anti-correlated .To test this hypothesis, the genes with antisense transcription were separated into five subgroups, according to their coverage by the nascent antisense signal. We then compared the levels of sense transcription for these subgroups. Remarkably, sense transcription significantly decreased as the overlap by the antisense increased .Finally, overlap of the sense gene TSS appeared to be critical, as genes of which the TSS was overlapped by antisense transcription signal were significantly less transcribed than genes without antisense signal overlapping TSS . On the other hand, there were 589 genes for which the origin of the antisense signal detected at the transcriptional level could not be assigned to a convergent TU or an annotated aslncRNA.The analysis above revealed that 2530/3455 genes with antisense transcription have convergent overlapping TU. We hypothesized that at least for some of the 925 remaining genes, the antisense signal corresponds to a previously annotated aslncRNA. We retrieved 975/1286 genes with an annotated aslncRNA among the 3455 genes with antisense transcription, and 639 were common to the set of convergent genes , suggesting that the resulting antisense transcripts are unstable. In S. cerevisiae, such transcripts are predominantly targeted by the cytoplasmic 5′–3′ exoribonuclease Xrn1, the inactivation of which results in the stabilization of a class of aslncRNAs referred to as XUTs , as in S. cerevisiae (exo2Δ and a significant (P < 0.05) differential expression . Among them, 815 (49.8%) are novel transcripts; 668 overlap ≥1 nt of previously annotated lncRNAs , which was detected as a discrete transcript by northern blot , reinforcing our genomic profiling approach.To characterize the XUT aslncRNAs’ landscape in fission yeast, we performed strand-specific RNA-seq in WT and 2Δ cells A, reachirevisiae . Transcrrevisiae  identifi lncRNAs D. Figureern blot F. Other S. cerevisiae, most XUTs  are antisense to protein-coding genes in S. pombe (P = 3.60 × 10−5), cellular response to DNA damage (P = 1.84 × 10−2), and chromosome organization (P = 3.03 × 10−2), but are depleted for genes involved in translation (P = 3.46 × 10−3).As in S. pombe C. ConverSupplemental Fig. S3B). However, 309 of them might correspond to genes with antisense cryptic unstable transcripts (CUTs) targeted by the nuclear exosome  genes for which the antisense signal could not be attributed to a convergent TU or a previously annotated aslncRNA turned out to have asXUT, leaving 461 genes for which the antisense signal origin remains unknown , confirming the RNA-seq profiles . Actually, mRNAs globally accumulate in exo2Δ , or for a smaller number of cases by Rrp6 (CUTs).S. pombe raised the question of their interrelation with the RNAi machinery that could in theory generate siRNAs from mRNA/asXUT duplexes, which were shown to exist in S. cerevisiae . In addition, the reads mapped on these regions did not show the typical size (22–23 nt) expected for genuine siRNAs produced by Dicer in S. pombe .The observation that the majority of XUTs are also antisense in revisiae . Small tn asXUTs A,B. Simiexo2Δ strains are 22- to 23-nt RNAs derived from centromeric repeats .Together, these observations suggest that asXUTs do not form dsRNA or more likely that Dicer and asXUTs are in distinct subcellular compartments. Indeed, XUTs presumably localize into the cytoplasm where they are targeted by Exo2 while Dicer is restricted to the nucleus and associates to specific chromosomal regions, mainly the centromeric repeats . AccordiIn conclusion, asXUTs coexist with RNAi but are not targeted by it in fission yeast.Supplemental Fig. S5A,B). Since active transcription is often correlated with histone acetylation at the promoter of genes . This is consistent with recent observations showing that H3 acetylation is high along genes with antisense transcription in S. cerevisiae  and H3K36me3 across the gene body , which might also reflect their low transcription.Trimethylation of histone H3 lysine 4 (H3K4me3) in the 5′ region of genes and of histone H3 lysine 36 (H3K36me3) across the gene body are two other hallmarks of active transcription . In buddSupplemental Fig. 5G–J), reinforcing the idea that antisense transcription is associated to conserved specific histone modifications, and that asXUTs constitute genuine antisense TUs.Interestingly, we observed similar patterns of histone modifications across the body of the 925 nonconvergent genes for which we detected nascent antisense transcription signal using NET-seq , 746 (86%) of which are antisense  is consistent with the role of Spt6 in repressing antisense transcription .To test this, we analyzed published RNA-seq data from WT and  at 37°C . We obsentisense A. This scription . Althougspt6-1 cells could be related to any down-regulation on the paired-sense mRNA. Specifically, we observed that mRNAs with a Spt6-sensitive asXUT showed a significantly higher reduction compared to those with a Spt6-insensitive asXUT or without antisense . However, for the same reason as mentioned above, it is difficult to determine whether this effect occurs at the transcriptional or post-transcriptional level, and whether it is mediated by a change of asXUT synthesis or by the transcript itself.Next, we asked whether the overexpression of asXUTs in In summary, asXUTs’ expression is controlled by Spt6, the loss of which results in the accumulation of a large subset of asXUTs, and this correlates with a down-regulation of the paired-sense mRNA, possibly reflecting a sense/antisense attenuation mechanism.The data presented above showed that XUTs accumulate upon inactivation of Exo2, and also of Spt6 for a subset of them. We asked whether XUTs could also respond to particular environmental conditions.S. pombe, we analyzed published RNA-seq data from pat1-114 diploid cells (mutated for the meiotic inhibitor Pat1), collected before and after 2, 4, 6, and 8 h of meiosis induction (P < 0.05) for 724 XUTs, in at least one time point . Among these XUTs, 561 are antisense and 441 correspond to novel transcripts, not annotated before this work.In budding and fission yeasts, subsets of lncRNAs have been shown to respond to sexual differentiation . To testnduction . We obseSupplemental Fig. S7C,D). We observed a significant difference in asXUT/mRNA correlations depending on whether the asXUT responded to meiosis induction or not , with asXUTs globally anti-correlated to sense mRNA levels when the asXUT was meiosis-responsive (median = −0.48), but not when it was unaffected (median = −0.10).Importantly, overexpression of meiosis-sensitive asXUTs correlated with paired-sense mRNAs’ down-regulation. First, we observed a global down-regulation of mRNAs for which the paired-asXUTs accumulated at least after 8 h of meiosis induction , considerably extending the current antisense catalog.Identification of aslncRNAs using RNA-seq or microarray has provided evidence for antisense transcription in many organisms, including fission yeast. However, these approaches based on mature RNA detection underestimate the accurate extent of antisense transcription. Here, using NET-seq, which currently constitutes the state of the art technique to measure nascent transcription per se, independently of post-transcriptional RNA degradation, in a strand-specific manner , we deteWhat is the effect of antisense transcription? We observed a global anti-correlation relationship with sense transcription, genes with antisense being significantly less transcribed than genes without antisense. Strikingly, levels of sense transcription decrease as the overlap by the nascent antisense transcription signal increases. This observation is consistent with previous measurements of sense mRNA synthesis using RNA labeling . In addiS. pombe, this would give at least 2824 annotated lncRNAs, with 1895 antisense, among which 1205 are XUTs. However, it is possible that the number of aslncRNAs is still underestimated since some of them might be targeted by RNA decay machineries other than Exo2, such as the nuclear exosome. Consistent with this idea, 309 of the 461 genes for which the antisense transcription signal could not be attributed to a convergent TU or an annotated aslncRNA/asXUT displayed an antisense RNA signal sensitive to Rrp6, which might correspond to CUTs.At the RNA level, the produced aslncRNAs appeared to be cryptic transcripts, mainly degraded by the Xrn1/Exo2-dependent 5′–3′ cytoplasmic RNA decay pathway. Inactivation of Exo2 resulted in the stabilization of 1638 XUTs, including 815 novel and 1205 antisense transcripts. Combined to the current annotation in cis effect  rather than from an asXUT-mediated effect in trans.XUTs are cryptic transcripts targeted by Xrn1/Exo2. One key point is to determine whether they can accumulate in biological conditions other than mutants defective for Xrn1/Exo2. If so, of particular importance would be to show any paired-sense gene regulation associated to this accumulation. In this regard, we defined a set of Spt6-sensitive asXUTs that accumulated upon inactivation of the conserved histone chaperone Spt6. More importantly, we identified a subset of asXUTs accumulated during meiosis induction. In both cases, asXUT accumulation correlated with paired-sense gene down-regulation. However, in the absence of NET-seq data that could allow precisely addressing the levels of sense and antisense transcription in these conditions, we cannot exclude that the observed down-regulation of sense mRNA levels results, at least in part, from a S. pombe. One possibility is that asXUTs and paired-sense mRNAs do not coexist within the same cells, or do not form dsRNA. In S. cerevisiae, sense/antisense pairing has been debated, as it was first reported that sense and antisense transcripts never coexist in the same cell  and YAM2397 (+ ade6-M210 ura4-D18 leu1-32 exo2Δ::kanMX4h) were purchased from Bioneer and verified by PCR on genomic DNA and northern blot. Strain YAM2402 (− exo2Δ::kanMX4h) was constructed by deleting exo2 in the YAM2400 (h−), then verified by PCR on genomic DNA. Strain YAM2492 (− rpb3-flag::natMX6h) was constructed by transformation, then verified by PCR on genomic DNA and western blot. All strains were grown at 32°C to mid-log phase in YES medium .Strains YAM2394 (rpb3-flag). Libraries were constructed as previously described (595 0.7–0.8) cells. RNAPII was immunoprecipitated using anti-FLAG M2 affinity gel (Sigma-Aldrich), then recovered upon two successive elution steps using FLAG peptide (Sigma). Ligation of DNA 3′-linker was performed from 2–3 µg of purified nascent RNA  and total RNA (input samples). Ligated RNAs were then submitted to alkaline fragmentation for 20 min at 95°C. Single-end sequencing (50 nt) of the libraries was performed on a HiSeq 2500 sequencer, giving between 20 and 50 million reads. After trimming of the 5′-adapter, reads were uniquely mapped to the reference genome (ASM294v2.30) using version 0.12.8 of Bowtie  and low total RNA levels (input samples). These genes were selected after filtering out the genes for which the signal was equal or close to zero as no (or very few) reads could be uniquely mapped, due to the high content of repetitive sequences. This was manually checked using the NET-seq signals (IP and input) and also the ChIP-seq signals (IP) for histone H3.Snapshots were produced using the VING software .A gene was considered as convergent to another TU  when (i) the distance between its annotated 3′ coordinate and the convergent TU one was <500 bp, and (ii) the nascent transcription signals overlap on ≥50 nt with a coverage ≥1 read/nt per strand, in each biological replicate.2O (Ambion) and quantified using a NanoDrop 2000c spectrophotometer. Quality and integrity of extracted RNA was checked by analysis in an RNA 6000 Pico chip in a 2100 bioanalyzer (Agilent).Total RNA was extracted from exponentially growing cells using standard hot phenol procedure, resuspended in nuclease-free H32P-labeled oligonucleotide probes  were hybridized overnight at 42°C in ULTRAhyb-Oligo hybridization buffer (Ambion).Ten micrograms of total RNA were loaded on denaturing 1.2% agarose gel and transferred to Hybond-XL nylon membrane . Supplemental Table S1.Strand-specific reverse transcription (RT) reactions were performed from at least three biological replicates, using 1 µg of total RNA and the SuperScriptII Reverse Transcriptase Kit (Invitrogen), in the presence of 6.25 µg/mL actinomycin D. For each sample, a control without RT was included. Subsequent quantitative real-time PCR was performed on technical duplicates, using a LightCycler 480 instrument (Roche). Oligonucleotides used are listed in exo2Δ) strains. Ribosomal RNAs were depleted from total RNA using the RiboMinus Eukaryote v2 Kit (Life Technologies). Depletion efficiency and quality of rRNA-depleted RNA was assessed by analysis in RNA Pico 6000 chip for 2100 Bioanalyzer (Agilent).Total RNA-seq analysis was performed from two biological replicates of the YAM2394 (WT) and YAM2397 . Paired-end sequencing (2 × 50 nt) was performed on a HiSeq 2500 sequencer. Reads were mapped using version 2.0.6 of TopHat, with a tolerance of three mismatches and a maximum size for introns of 2 kb. Tag densities were normalized on sn(o)RNA levels for all subsequent analyses.exo2Δ biological duplicates were pooled. A strand-specific signal was then computed for each nucleotide as the number of times it is covered by a read or the insert between two paired reads. After log2 transformation, the signal was smoothed using a 10–200-nt sliding window (with 10-nt increments). All genomic regions showing a smoothed log2 signal value above a threshold  were reported as segments. XUTs were defined as ≥200 nt segments that do not overlap ORF and that show a greater than twofold enrichment in exo2Δ vs WT, with a P-value (adjusted for multiple testing with the Benjamini–Hochberg procedure) <0.05 upon differential expression analysis using DESeq .Previously published poly(A) for 2 h , and fronduction  were retnduction  upon norexo2Δ) strains were constructed according to the Small RNA Sample Preparation Guide (Illumina) using 10- to 40-nt small RNAs purified from total RNA on 15% TBE–urea PAGE. Single-end sequencing (40 nt) of libraries was performed on a Genome Analyzer IIx (Illumina). After adapter sequences trimming, reads were uniquely mapped using the version 0.12.8 of Bowtie  and YAM2397  cells were fixed for 10 min at room temperature using formaldehyde , then glycine was added . Histone H3, H3K14ac and H4K5/8/12/16ac were immunoprecipitated using ab1791 (Abcam), 07-353 (Millipore), and 05-1355 (Millipore) antibodies, respectively.ChIP-seq analysis was performed from two biological replicates of YAM2394 (WT) cells. Briefly, exponentially growing (ODLibraries were prepared from 5 ng of “input” or “IP” DNA using the TruSeq ChIP Sample Preparation Kit (Illumina). Single-end sequencing (50 nt) was performed on a HiSeq 2500 sequencer. Reads were uniquely mapped using version 2.1.0 of Bowtie2 , with a t-test was used for analysis of RT-qPCR data.The Wilcoxon rank-sum test was used for the analysis of data sets showing non-normal distribution . The rrp6), GSE49573 (spt6), and PRJEB7403 . Genome browsers for visualization of processed data are publicly accessible at http://vm-gb.curie.fr/mw1.Raw sequences have been deposited to the NCBI Gene Expression Omnibus (accession number GEO: GSE72382). Accession numbers for previously published RNA-seq data are GSE64992 (Supplemental material is available for this article.