In the absence of BER, 5-caC caused a strong inhibition of the promoter activity, whereas 5-fC had almost no effect, similar to 5-methylcytosine or 5-hydroxymethylcytosine. BER of 5-caC caused a transient but significant promoter reactivation, succeeded by silencing during the following hours. Both responses strictly required thymine DNA glycosylase (TDG); however, the silencing phase additionally demanded a 5′-endonuclease (likely APE1) activity and was also induced by 5-fC or an apurinic/apyrimidinic site. We propose that 5-caC may act as a repressory mark to prevent premature activation of promoters undergoing the final stages of DNA demethylation, when the symmetric CpG methylation has already been lost. Remarkably, the downstream promoter activation or repression responses are regulated by two separate BER steps, where TDG and APE1 act as potential switches.Stepwise oxidation of the epigenetic mark 5-methylcytosine and base excision repair (BER) of the resulting 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) may provide a mechanism for reactivation of epigenetically silenced genes; however, the functions of 5-fC and 5-caC at defined gene elements are scarcely explored. We analyzed the expression of reporter constructs containing either 2′-deoxy-(5-fC/5-caC) or their BER-resistant 2′-fluorinated analogs, asymmetrically incorporated into CG-dinucleotide of the GC box  Epigenetic regulation of the genome function is crucial for concerted realization of gene expression programs during development and for maintenance of the lineage-specific gene expression patterns in adulthood. During these processes, functions of the whole genome and of the individual genes are dynamically regulated by deposition and removal or maintenance of epigenetic marks, which include specific chromatin components as well as chemically modified DNA bases ,2. 5-metRecent progress in the field of epigenetic reprogramming has revealed that 5-mC can undergo enzymatic oxidation by ten-eleven translocation (TET) family dioxygenases to 5-hydroxymethylcytosine (5-hmC) and further to 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) ,14,15. TTo directly address functional impacts of 5-hmC and its derivatives on the regulation of gene expression, these modifications can be site-specifically incorporated into a suitable reporter vector. We previously used such an approach to investigate the effects of the 5-mC oxidation products in a cyclic adenosine monophosphate response element (CRE). There, 5-mC, 5-hmC, 5-fC and 5-caC all negatively affected the promoter activity by inhibition of the cognate transcription factor CREB binding , thus cocis-regulatory element  reporter constructs containing single 5-mC, 5-hmC, 5-fC or 5-caC residues at the CpG site of an artificial promoter containing the 5′-TGGGCGGAGC-3′ GC box sequence as the only  element a. In thesequence . SynthetEGFP gene expression levels in transfected HeLa cells -fluorinated derivatives of 5-fC and 5-caC. Both modifications were incorporated into the purine-rich DNA strand as efficiently as the respective 2′-deoxy compounds (R)-fluorination entirely reversed the repressory effect of 5-fC, as the difference between the 6- and 24-h points disappeared. The expression levels were 81.2 ± 9.8% (at 6 h) and 84.6 ± 8.4% (at 24 h post-transfection), relative to the construct containing cytosine. The respective values for 2′-deoxy 5-fC were 88.0 ± 5.7% and 31.0 ± 4.1% , whereas the effect of 2′-(R)-fluorinated 5-fC was not (p = 0.62). In the case of 5-caC, the differences between the 6- and 24-h time points were, again, highly significant for deoxynucleotide (p = 1.6 × 10−4) but not for the 2′-fluorinated analog (p = 0.38). We thereby conclude that dynamic changes of the promoter activity induced by 5-fC and 5-caC are the consequence of excision of the modified bases by a DNA N-glycosylase mechanism.The observed dynamic changes of the expression of constructs carrying 5-fC and 5-caC, in contrast to 5-mC or 5-hmC, could likely be attributed to removal of these modifications from DNA. Therefore, to measure direct impacts of 5-fC and 5-caC on the GC box activity, it was necessary to eliminate their repair. Activities of DNA N-glycosylases towards their substrates, including 5-fC and 5-caC, can be efficiently inhibited by deoxyribose fluorination at the C2′ position ,31. Thisompounds a. Quanti0 ± 4.1% b. The reR)-fluorinated 5-fC and 5-caC in the GC box constructs showed stable levels of the reporter gene expression, it was now possible to quantify their direct effects on the promoter activity, based on the EGFP expression levels in transfected HeLa cells. We used 24-h values to estimate these effects, since fluorescent cell counts as well as average fluorescence intensity per cell were higher at this time point (R)-fluorinated 5-fC was minimal yet statistically significant (p = 0.035). The effect of 2′-(R)-fluorinated 5-caC was much stronger  and statistically highly significant (p = 9.3 × 10−4). Considering that GC box enhances the gene expression by a factor of 2 to 2.5 over the basal expression level GGAGCG and 5′-CATTGCGCTC[C*]GCCCACG . DNA CE-phosphoramidites Bz-dA, Bz-dC, iBu-dG, dT and Bz-mdC were obtained from Glen Research  or Link Technologies . Syntheses of the 5-hmC, 5-fC and 5-caC phosphoramidites , along wnd 5-caC , were pend 5-caC . Synthetcis-regulatory element upstream from the RNA polymerase II transcription initiation site and sustain equivalent levels of the reporter EGFP gene expression. Defined modifications targeting cytosines of the CpG dinucleotide were introduced into the purine-rich GC box strand of pGCbox-W or into the pyrimidine-rich strand of pGCbox-C, using the available sites by the Nb.BsrDI nicking endonuclease. The procedure was described in detail previously for introducing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxoG) into the same vectors [Vectors pGCbox-W and pGCbox-C, allowing substitution of the selected GC box strand with synthetic oligonucleotides, were described previously . Both ve vectors . Plasmid vectors . The eff vectors . Percent vectors . The preHeLa cells used in experiments were clones descending from HELA cervical car-cinoma cell line (German Collection of Microorganisms and Cell Cultures No. ACC 57). The derived TDG and NTH1 knockout cell lines were generated in our lab. Cells exponentially growing in 6-well plates were transfected with mixtures containing 400 ng GC box reporter vector (pGCbox-W or pGCbox-C) and 400 ng tracer pDsRed-Monomer-N1 vector , using the Effectene reagent , as described previously ,30. GC bTDG gene were generated by a CRISPR-Cas9-mediated gene-editing procedure, using the pX330-sgCas9-HF1 vector  according to the supplier’s instructions [TDG locus were designed by using CHOPCHOP online tool and subcloned into the BbsI sites [2 flasks and screened by PCR for the presence of a deletion spanning the sequence between the sgRNA-targeted sites in the exons 2 and 5 . The primers were 5′-TCCTCTGTAATCCACTCTAA (forward) and 5′-AGCTCAGCTTGAACTAGATA (reverse). Preselected clones were next screened for the presence of non-rearranged TDG alleles to eliminate the positives. The primer pair detecting the non-rearranged exon 2 was 5′-TCCTCTGTAATCCACTCTAA (forward) and 5′-ATGTCCCTACTCTGATCTTT (reverse). The remaining clones were expanded and the TDG knockout was validated by Western blotting of protein extracts with a 1:5000 dilution of the TDG rabbit polyclonal antibody #PA5-29140 (Thermo Fisher Scientific Inc.) and a 1:10,000 dilution of the IRDye® 800CW donkey anti-rabbit IgG #926-32213 . Blots were analyzed by using the Odyssey 9120 infrared imaging system (LI-COR). Stripped membranes were subsequently probed with a 1:10,000 dilution of the mouse monoclonal antibody AC88 to HSP90 # ADI-SPA-830  and a 1:10,000 dilution of the IRDye® 800CW donkey anti-mouse secondary antibody # 926-68072 (LI-COR).HeLa-derived clonal cell lines with deletion of a critical portion of the ructions , with misI sites . A pair sI sites  as a tra


O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification (PTM) linking nutrient flux through the hexosamine biosynthetic pathway (HBP) to gene transcription. Mounting experimental and clinical data implicates aberrant O-GlcNAcylation in the development and progression of cancer. Herein, we discuss how alteration of O-GlcNAc-regulated transcriptional mechanisms leads to atypical gene expression in cancer. We discuss the challenges associated with studying O-GlcNAc function and present several new approaches for studies of O-GlcNAc-regulated transcription.O-linked β-N-acetylglucosamine (O-GlcNAc) is a single sugar post-translational modification (PTM) of intracellular proteins linking nutrient flux through the Hexosamine Biosynthetic Pathway (HBP) to the control of cis-regulatory elements in the genome. Aberrant O-GlcNAcylation is associated with the development, progression, and alterations in gene expression in cancer. O-GlcNAc cycling is defined as the addition and subsequent removal of the modification by O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) provides a novel method for cells to regulate various aspects of gene expression, including RNA polymerase function, epigenetic dynamics, and transcription factor activity. We will focus on the complex relationship between phosphorylation and O-GlcNAcylation in the regulation of the RNA Polymerase II (RNAP II) pre-initiation complex and the regulation of the carboxyl-terminal domain of RNAP II via the synchronous actions of OGT, OGA, and kinases. Additionally, we discuss how O-GlcNAcylation of TATA-box binding protein (TBP) alters cellular metabolism. Next, in a non-exhaustive manner, we will discuss the current literature on how O-GlcNAcylation drives gene transcription in cancer through changes in transcription factor or chromatin remodeling complex functions. We conclude with a discussion of the challenges associated with studying O-GlcNAcylation and present several new approaches for studying O-GlcNAc regulated transcription that will advance our understanding of the role of O-GlcNAc in cancer. Cells precisely control gene expression in response to their metabolic state, the availability of building blocks and fuel, and environmental cues . EukaryoO-linked N-acetylglucosamine transferase , and removed by a single enzyme, N-acetyl-β-glucosaminidase  [O-GlcNAc cycling describes the rapid addition and removal of the sugar from intracellular proteins, which affects their interactions, stability, localization, and activity. O-GlcNAc is covalently attached to substrate proteins by a single enzyme, lcNAase) . There alcNAase) ,7,8,9. IPTMs are often regulated and/or exert their effect through an assortment of proteins known as “writers”, “readers”, and “erasers”. Simplistically, “writers” add the PTM to substate proteins, whereas “erasers” remove the modification. For example, kinases add phosphate groups to target proteins, and phosphatases remove the phosphate group from proteins. “Readers” recognize the PTM via “reader domains” and integrate the molecular and chemical information of the PTM to drive cellular processes by recruiting effector proteins and enzymes. For example, proteins with SRC homology 2 (SH2) domains recognize and bind phosphate PTMs . These “In the absence of O-GlcNAc “reader” proteins, other possibilities regarding the function of protein O-GlcNAcylation must be considered. There is considerable evidence to support the notion that O-GlcNAcylation may sterically promote or prevent protein-protein interactions. This mechanism is suggested by the wide range of biochemical changes O-GlcNAcylation induces in its protein targets, including inhibition or activation of activity, conformational changes, re-localization, and destruction ,12,13,14N-acetyl glucosamine (UDP-GlcNAc), the substrate for OGT and other glycosyltransferases, produced by the HBP [Changes in intracellular nutrient metabolite pools directly affect the level uridine-diphosphate  the HBP ,18. The  the HBP ,20,21. F the HBP ,23,24,25 the HBP . Similar the HBP . The pre the HBP . Every c the HBP ,28. Howe the HBP .At the basal level of transcription, eukaryotic gene expression is governed by three different evolutionarily conserved RNA polymerases (RNAPs). Each RNAP transcribes different types of genes in the genome. RNA polymerase I (RNAP I) transcribes ribosomal RNA genes (rRNAs). RNA polymerase II (RNAP II) transcribes a variety of DNA elements, including messenger RNAs (mRNAs), microRNAs (miRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs). RNA polymerase III (RNAP III) produces transfer RNAs (tRNAs) and 5S ribosomal RNAs (5S rRNAs) . Each RNOf all the RNAPs and their associated synthesis machinery, eukaryotic RNAP II is the most versatile, as it recognizes and catalyzes transcription from the most diverse set of gene promoters . It alsoM. musculus, Drosophila, and C. elegans, further demonstrating a role for O-GlcNAc cycling in gene transcription that seems to be evolutionally conserved in Eukarya [Using in vitro cell-free systems derived from crude HeLa cells extracts, Lewis et al. explored the function of RNAP IIγ in the transcription cycle, resulting in a revision of the early model to include O-GlcNAc cycling ,33,38,39 Eukarya .Functionally, O-GlcNAcylation of the RNAP II CTD may regulate PIC assembly, transcriptional pausing, and elongation in several possible ways. First, O-GlcNAcylation prevents phosphorylation by impeding access of kinases to the necessary serine and threonine residues of the CTD. Phosphorylation of these amino acid residues are required for RNAP II to dissociate from the PIC and critical for mRNA processing and transcription cycle progression. Second, O-GlcNAc addition and removal may promote or impede protein-protein interactions required for PIC assembly, transcriptional pausing, and elongation. Thus, this mechanism may be operative in cells to control which genes are turned off and on, or which gene splice variants are expressed under different internal cellular and external environmental conditions. Beyond RNAP II, mass spectroscopy data have identified 32 additional RNAP II transcription cycling factors that are O-GlcNAcylated . HoweverA protein factor common to all three RNAP complexes is the TBP, which is thought to serve as a PIC assembly platform in TATA-box containing promoters. TBP associates with two RNAP II transcriptional complexes, TFIID and B-TFIID ,43. EachPhosphorylation and O-GlcNAcylation have extensive crosstalk with each other ,44,45,46Cancer results from a combination of changes to the genome and epigenome . EpigeneMethylation of cytosines in DNA, particularly at promoter CpG islands, is the classical epigenetic modification that plays a critical role in transcription, affecting such downstream processes as chromosome accessibility, nucleosome positioning, and ultimately, gene expression . In humaThe polycomb group (PcG) proteins are a diverse family of epigenetic modifiers and transcriptional regulators. In mammals, PcG proteins repress Hox genes and control the expression of other genes that govern embryonic development, X chromosome inactivation, genomic imprinting, the cell cycle, and maintenance of stem cells . DiffereAmong the OGT-interacting PcG proteins, several are direct targets of OGT and thus are sensitive to O-GlcNAcylation. Pei-Wen et al. have shown that EZH2 is O-GlcNAcylated in the N-terminal region at S73, S76, S84, and S87, which protects EZH2 from ubiquitin-proteasome degradation . AdditioThe role of O-GlcNAc cycling in PcG complex formation and function has recently been evaluated in Drosophila. Using RNAi to knockdown Ogt and Oga, Akan et al. reported that PcG-mediated repression was strikingly insensitive to Oga RNAi, in contrast to Ogt RNAi, which suggested that the addition of O-GlcNAc, and not the removal, is essential for PcG complex formation and function . There aO-GlcNAc cycling exerts effects on gene transcription via protein-protein interaction with co-repressor and co-activator complexes and lineage-specific master transcription factors that function as adapter proteins to shuttle OGT and OGA to specific loci. An example of this is the Sin3A co-repressor complex, and more recently, erythroid-specific GATA-1 complexes ,78. ZhanSin3A is a core component of several transcriptional co-repressor complexes . One of The nucleosome remodeling deacetylase (NuRD) complex is a group of associated proteins with both ATP-dependent chromatin remodeling and histone deacetylase activities that relies on O-GlcNAcylation to regulate its function. NuRD was originally defined as a repressor, but more recently, it has been shown to function both as a co-activator and co-repressor . The corIn several cervical cancer cell lines, Geo et al. found that OGT knockdown and overexpression altered Snail gene expression . Snail iAlmost all RNAP II-associated transcription factors are O-GlcNAcylated, often at multiple sites . O-GlcNASpecificity protein 1 (Sp1) is a transcription factor that is frequently overexpressed in a wide variety of human cancers and contributes to malignant transformation . Sp1 is Recent studies have shown that Sox2 and Oct4, two transcription factors critical for the induction and maintenance of pluripotency, are O-GlcNAcylated. In many cancers, Sox2 and Oct4 are inappropriately activated, leading to aberrant expression of downstream target genes, which stimulates tumor growth and tumor recurrence. In mouse embryonic stem cells, Olivier-Van Stichelen et al. found that Oga gene ablation resulted in a significant elevation of Sox2 mRNA transcripts . This suSeveral studies show that increased O-GlcNAcylation is a key driver of primary and metastatic breast cancer ,105,106.The Hippo pathway is an important signaling pathway that controls organ size by regulating cell proliferation and apoptosis. Dysregulation of this pathway has been linked to tumorigenesis ,116,117.Recently, Peng et al. demonstrated that this pathway is directly regulated by O-GlcNAcylation . YAP, thThe transcription factors and co-activator described here are a small cross-section representative of transcription factors and co-activators known the be modified by OGT that function in cancer. More work is required to explore how O-GlcNAcylation of transcription factors affects the expression of genes that regulate cancer occurrence, progression, and metastasis. In the context of nutrient-regulated gene transcription, the data suggest that cells have evolved mechanisms to connect the overall metabolic state of the cell to transcription factor function. Under normal physiologic conditions, this ensures that in times of “feast and famine”, cells elicit the appropriate transcriptional response, and that uncontrolled O-GlcNAcylation of transcription factors plays a role in the onset of cancer.O-GlcNAc is a unique driver of cancer since O-GlcNAcylation affects transcription in a multitude of ways on a global level . Under nThe technical challenges for studying dynamic O-GlcNAc cycling remain a major obstacle in the field, so there is a need to develop innovative tools for O-GlcNAc studies that reduce the background noise of global O-GlcNAcylation to allow highly specific, focused experiments that yield unbiased data. Newly developed methodology and technical approaches that selectively modify O-GlcNAcylation on a single protein or hone in on the role of O-GlcNAcylation of proteins at specific DNA loci will be indispensable for elucidating the molecular mechanisms that are governed by the addition or removal of this PTM. Recently, Boulard et al. developed a new CRISPR/Cas9 approach that allows for precise targeting of OGA to any genomic sequence of interest . There iThere is a rapidly growing interest in how O-GlcNAcylation contributes to the properties of cancer cells and the progression of cancer. Dynamic O-GlcNAc cycling is commonly elevated in cancer cells. Atypical expression and activities of OGT and OGA have been reported in all human cancers studied thus far . An unde


Enhancers are widely believed to physically contact the target promoters to effect transcriptional activation. However, our understanding of the full complement of regulatory proteins and the definitive mechanics of enhancer action is incomplete. Here, we review recent findings to present some emerging concepts on enhancer action and also outline a set of outstanding questions.Differential gene expression mechanisms ensure cellular differentiation and plasticity to shape ontogenetic and phylogenetic diversity of cell types. A key regulator of differential gene expression programs are the enhancers, the gene-distal The online version contains supplementary material available at 10.1186/s13059-021-02322-1. The CRE repertoire includes the boundary elements or insulators necessary for specific spatial organization of the chromatin into topologically associated domains (TAD), the silencers, the enhancers , and the promoters. Experimental evidence strongly suggests that the distant enhancers physically contact the promoters by chromatin looping during transcription activation. However, how exactly the enhancer accomplishes the over-all act of gene activation is still unresolved—even nearly 40 years after its discovery as a 72 bp repeat element in the Simian Virus 40 genome [Metazoans—the multicellular organisms that comprise the Kingdom Animalia—began their formative journey of evolution over 635 million years ago, when multicellularity emerged from early unicellular holozoans , 2. Mult0 genome –16. In t0 genome –21.SHH gene [Enhancers are non-coding sequences in the genome that activate the expression of target genes transcribed by the RNA polymerase II (RNAPII). Enhancers can act independent of orientation, distance, and location with respect to the target gene  and can SHH gene . ReadersSHH gene . VertebrSHH gene —analogouSHH gene . EnhanceSHH gene . Thus asCapsaspora owczarzaki have identified dynamic cis-regulatory landscapes [Amphimedon queenslandica [Several lines of reasoning argue that the early holozoan relatives of the metazoans already possessed enhancer-like regulatory features. Enhancer-like elements exist in bacteria that regulate target gene expression by DNA looping , 28. Thendscapes . Among tslandica  that canslandica . Early mslandica , 40. Theslandica , suggestslandica –43. Addislandica , 45; neaslandica . Also, tslandica –51.Drosophila in 24 years [Genomic duplication events—be it tandem duplications of chromosomal regions, duplications of whole chromosomes, or whole genomes—also create enhancers. One of the copies post duplication is essentially redundant and can tolerate mutations, while the genetic integrity of the other copy is maintained. This results in complete or partial loss of the duplicated region , or gain of new functional features . If the duplicated region was a cis-regulatory module, it evolves new regulatory functions over time, resulting in the genesis of new enhancers , 53. In 24 years , while a24 years . Some en24 years –60; howe24 years . Prior t24 years . Neverth24 years .Enhancers are evolutionarily conserved in sequence and function . For exaIFNB1 gene, contain multiple interdependent domains within, such that alterations in the sequence, order, or spacing of the domains are not tolerated, and the TFs bind cooperatively as one functional unit [Two questions arise here: why do enhancers contain so many TFBSs, and why are some ultra-conserved in evolution while others are not? From the perspective of chromatin packaging, it is useful to cluster multiple TFBSs together, so that additive binding of the required TFs could progressively overcome the higher propensity of the enhancers to form nucleosomes, allowing desired gene regulation. From the perspective of cellular phenotypic diversity, a given enhancer can regulate a cognate gene in multiple cell types differentially or temporally if they express multiple different TFs that bind the enhancer. Thus, availability of TFBSs allows the different cell types to employ their specific TFs to utilize the same enhancer and yet choose the timing and magnitude of its action. The major types of enhancer architecture are the “billboard,” “TF collective,” and the “enhanceosome” models , 70. Thenal unit . These eDuring a lineage-specification event, pioneer TFs access their nucleosomal binding sites at “closed” enhancers . This piThe proteome at the enhancer can be broadly classified into five groups. These include TFs, architectural proteins like the histones, coactivators that are recruited to the enhancers via TFs, reader proteins that recognize specific modifications on the underlying histones, and finally RNAPII and other enzymes that catalyze or influence various steps to ensure stepwise transition of transcription from formation of the pre-initiation complex (PIC) to productive transcription through promoter clearance, elongation, proximal pausing, and pause release (see Table The enhancer itself undergoes transcription (discussed below) and is enriched with the RNAPII and all the general transcription factors (GTFs) , 92. NotLiving systems are surrounded by and are exposed to a wide range of environmental factors. For example, the human exposome is comprised of thousands of biotic agents and chemical compounds . One waySteroid hormone signaling presents a well-studied example of enhancer-mediated transcription regulation , 115. ThGREB1 locus [trans on a different chromosome and regulate transcription [Apart from the DNA sequence motifs and assembled proteins, non-coding RNAs comprise another integral component of enhancers . AlthougB1 locus . The magB1 locus , 147. WhB1 locus , a polyacription . Additiocription .GREB1 enhancer and promoter fragments individually do not transcribe well, but undergo robust activation when co-incubated in conditions that support maximal EPC in trans. Using this system, we observed that it is the ‘act of transcription’ at the enhancer and promoter—but not the enhancer- or promoter-derived transcripts per se—that is mutually stimulatory [To recap, not only an active enhancer itself undergoes transcription, but also the magnitude of its eRNA production mirrors the target promoter activity. Expectedly, physical contact is detected between an enhancer and promoter pair that are either dormant (not transcribing) or “active” , but not when one is dormant and the other is active . These omulatory . That isSeveral of the key features of enhancers described above are employed to identify enhancers. These approaches largely fall into five groups: (i) genome-wide maps of DNA accessibility; enrichment of epigenomic marks; occupancy of select TFs and TF clusters, coactivators and RNAPII; (ii) global transcription potential as assessed by GRO-seq, or mapping genomic TSSs by CAGE; (iii) assessment of transcription activation potential of regulatory sequences using reporter genes; (iv) targeted perturbation of the transcriptional status of the CREs by their suppression or activation; and (v) genome-wide assessment of chromatin connectomes to identify ‘in contact’ enhancer-target pairs , 154. OnCell-free methodologies  could ofHow exactly the enhancer activates target gene transcription is a persistent question in the field of eukaryotic gene regulation. To impart regulation, the enhancers must communicate with the target promoters. However, the enhancers in higher eukaryotes are physically separated along the genome from the target gene promoters. Several models have been proposed for enhancer-promoter communications. These include tracking , linking , and looping  , 161. WhAs discussed above, enhancers are dense clusters of TFBSs that assemble large protein complexes representing various structural and functional classes, and the protein-enriched enhancers greatly activate transcription from specific target promoters. Arguably, such specificity can be achieved only through direct contacts between an enhancer and promoter pair, likely through protein-protein interactions , 163. ThEmploying the 3C-based connectome approaches or fluorescence-based visualization techniques, at individual loci or at genomic scale, studies over the past two decades have revealed several important features of enhancer action as a function of EPC , delivering RNAPII, removing repressors, and facilitating pause-release  into condensates inside the cell, where not only the efficiency and fidelity of biomolecular processes are enhanced, but also the organizational and architectural specificity is attained without requiring membranous confines , 259. TwThe relative motion of the template DNA vis-à-vis the elongating RNA polymerase is an area of active debate. There are two possibilities: the RNAPII leaves the promoter after transcription initiation, pauses, and “tracks forward” along the template DNA like a locomotive as the RNA synthesis progresses. Alternatively, the RNAPII can stay tethered to nuclear structures while the template DNA “extrudes backward” during chain elongation. Nearly 40 years ago, Peter Cook and colleagues observed that the “body” of nascent RNAs are tethered to nuclear structures—presumably through the RNAPII itself . This leGREB1 locus, we observed that two gene body SRC-3-bound regions (GBS1 and GBS2) hold the enhancer and promoter in close proximity in estradiol (E2)-deprived MCF-7 cells. However, upon E2 stimulation, the GBS1 and GBS2  promptly disengage from the enhancer allowing direct EPC, and they then get back in contact with the EPC when their respective regions are undergoing transcription [NRIP1 locus as well. Recently, a strong proof for this model has come in the form of “stripes” in an exemplary nucleosome-resolution “Micro-C” connectome of the transcription-linked chromatin [Over the years, many genome-wide , 270 andcription , supporthromatin . The strTaking the above observations along with the recent transcriptional LLPS studies, we propose that the enhancer and the promoter reside within a phase-separated “transcription bubble” enriched with coactivators and the RNAPII, and that transcription elongation occurs when the downstream template DNA is dragged backward causing a progressively extruding loop Fig. a. A receGREB1 enhancer and promoter are recapitulated in vitro, inclusion of GBS1 and GBS2 fragments greatly enhance cell-free transcription of both the enhancer and promoter [An offshoot of the transcription bubble and the sunflower model described above is the tantalizing prospect of intronic regulation of enhancer action. Since the enhancer, promoter and the strategic intronic TFBS clusters  maintain near-constant contact, these nucleoprotein hubs can structurally and functionally influence each other Fig. b. Interepromoter . These rpromoter . Thus, ihow exactly enhancers activate promoters still awaits definitive answers. The mechanism of enhancer action as discussed above—that enhancer-bound TFs help recruit other TFs, GTF, coactivators, and the RNAPII holoenzyme at the promoter to stimulate transcription—is predominantly based on our knowledge of the transcription regulation in yeast, Drosophila, and mammalian model systems where the enhancer elements are in close proximity to the promoters [Despite the extensive information about enhancers as sampled above, our mechanistic understanding of enhancer function remains incomplete. The key question of romoters . While tromoters . We do nAs discussed earlier, EPC is required to transmit the regulatory instruction from the enhancer onto the promoter, and yet an EPC does not guarantee transcription. Arguably, it is useful to have a preformed EPC(s) so that the gene locus is poised for transcription—waiting for a final kick start. But, what determines which set of enhancers and promoters should stay in preformed contacts, when, and for how long? Are there any universal norms that govern genome-wide EPC at non-transcribing loci, or do individual EPCs have their own molecular explanations? Are there any definitive and universal looping determinants that mediate EPC at all loci ? OSince mere EPC does not automatically ensure transcription, the preformed EPCs raise another critical question: what happens after an enhancer contacts a promoter? What triggers the transcription? Detailed genetic, biochemical, genomic, structural, and proteomic investigations into the stepwise transitions in transcription, from TBP binding and PIC assembly to promoter clearance, pausing, pause release and productive elongation, have generated a tremendous volume of information , 282–284Answering these questions would require the development of new methodologies. Arguably, isolation of genomic EPCs at sufficient temporal resolution during the transcriptional transitions and comprehensive proteome analysis thereof could provide deep insight into these questions. However, this is a formidable task because, first, we currently do not have an easy way to isolate locus-specific EPCs from the genome in sufficient quantities that would allow such detailed investigations ; prepariC. elegans, fruit fly and mammals raises the possibility that enhancer transcription is a product of transcriptional noise. However, the same observation also argues that it might be a conserved phenomenon with a purpose. This latter possibility is supported by the fact that full complements of GTFs, RNAPII, and other critical coactivators occupy active enhancers, suggesting that the resultant eRNAs might have a cellular function. The considerations described in the earlier section arguing mutual coordination between the enhancer and promoter transcription further raise the questions: why are enhancer and promoter transcription coordinated, and what is the molecular basis of that coordination? As discussed above, the “act of transcription” at an in-contact pair of enhancer and promoter is mutually stimulatory. What is the biochemical explanation of such mutualism? The subsequent questions that emerge are: why must an enhancer transcribe, and do eRNAs have any generalizable functional relevance?The fact that eRNAs are produced genome-wide in animals as diverse as The eRNAs have clearly emerged as an indisputable marker of active enhancers, and many in the field consider eRNAs as either a spontaneous byproduct of enhancer activation, or a ploy to keep the enhancer chromatin open . LEnhancers carry the regulatory instructions for a gene’s spatiotemporal expression. The enhancer-encoded instructions are communicated to the cognate gene promoters via dynamic protein-protein interactions that involve multitudes of TFs, coregulators, chromatin architectural proteins, and enzymes. Moreover, many of these proteins acquire or shed covalent modifications necessary for proper transcriptional regulation. In concert with the promoter-driven transcription, the enhancers themselves also undergo transcription, producing eRNAs of uncertain functions. These multifaceted interactions among the underlying DNA sequences at the enhancer and promoter, the assemblies of proteins, and RNA molecules likely occur in phase-separated condensates. However, global or locus-specific dynamics of these multifarious interactions leading to productive transcription are still poorly understood. The current dogma posits that the enhancer-encoded instructions are translated into a ‘TF code’ when a certain combination of TFs binds the enhancers both directly and indirectly. This combinatorial TF recruitment to the enhancers is currently thought to define the transcriptional regulation of a target gene. We opine that the combinatorial TF occupancy merely provides a platform for dynamic recruitment of the coregulators, which ultimately govern transcriptional specificity and processivity. For a more holistic and mechanistic understanding of enhancer function, it is now the time to transition from the current “TF code” of transcription to a ‘coregulator code’, where the emphasis would be to define at high temporal resolution the combinatorial coregulator repertoire that drive locus-specific transcription activation.Additional file 1. Review history.


Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in  Drosophila ovaries for the massive expression of deadhead (dhd), a gene encoding for a thioredoxin that is essential for fertility. In this paper, we have further identified two additional dhd regulators, Snr1 and Mod(mdg4) and dissected the mechanism behind hyperactivation of this gene. Using the epigenomic profiling method Cut&Run with a dedicated data analysis approach, we unexpectedly found that dhd is embedded in an unusual chromatin mini-domain featuring repressive histone modifications H3K27me3 and H3K9me3 and flanked by two regulatory elements. However, we further showed that Lid, Sin3A, Snr1 and Mod(mdg4) behave like obligatory activators of dhd independently of this mini-domain. Our study unveils how multiple broad-acting epigenomic effectors operate in non-canonical manners to ensure a critical and specialized gene activation event. These findings challenge our knowledge on these regulatory mechanisms and their roles in development and pathology.Multicellular development depends on a tight control of gene expression in each cell type. This relies on the coordinated activities of nuclear proteins that interact with DNA or its histone scaffold to promote or restrict gene transcription. For example, we previously showed that the histone modifying enzymes Lid and Sin3A/Rpd3 are required in  Gene expression is tightly controlled in eukaryotic cells by the composition, organization and dynamics of nucleosomes, consisting of an octamer of histone proteins wrapped in ~146bp of DNA. The concerted activity of protein complexes including histone chaperones, readers and writers as well as nucleosome remodelers, defines the positioning, composition and post-translational modifications of nucleosomes . The resin vivo. A privileged method is ablation or dosage manipulation of each component to measure its impact on gene expression. While these approaches can yield precious functional insight, the ubiquitous expression and wide range of activities of these factors, as well as redundancies in their interactions, make it difficult to infer their precise function. Understanding their function therefore requires identifying biologically relevant situations where disrupting these effectors impacts transcription in a critical and specific manner. We previously described one of such cases, where perturbation of the histone H3K4 demethylase Lid/KDM5 or the histone deacetylase complex Sin3A/Rpd3 in Drosophila ovaries dramatically abrogated the expression of the maternal gene deadhead (dhd), which is essential for female fertility . In. InE(z)  ovaries . Togethe domains . Also codhd mini-domain were reduced in E(z) KD ovaries and unaffected in Sin3a or mod(mdg4) KD ovaries. More surprisingly, the domain was not measurably affected in Snr1 KD ovaries, despite the fact that H3K27me3 is globally impacted by this knockdown  KDs have little if any impact on H3K27me3 at the dhd locus. Conversely, in lid KD ovaries, in which global H3K27me3 levels were unaffected, we detected an increase in H3K27me3 levels at the dhd mini-domain  or Snr1 had no effect on this enrichment  and Snr1 KD led to a strong (~63%) decrease in short fragment peak average counts genome-wide  KD did not globally affect short fragment peak counts, indicating that these factors do not play such a role  and Sin3a KDs  KD led to a similar impact on these border elements , even though it did not globally affect H3K27me3-associated elements genome-wide  is required to ensure chromatin organization of the border DNA regulatory elements at the dhd mini-domain. Remarkably, Snr1 KD led to a similar effect on border elements without affecting H3K27me3 levels at the dhd mini-domain, suggesting that Snr1 is also required for the proper organization of the dhd border elements. In striking contrast, lid KD had no detectable effect on these regulatory elements  motif, TATCGATA  (pW8-dhdWT (62E1) and combined with the dhdJ5 deficiency. In striking contrast to pW8-dhdWT, the pW8-dhdΔDRE construct was unable to rescue dhd expression, or substantially improve fertility in dhdJ5 deficient flies  . These cnt flies . The DREdhdJ5 ovaries, as well as rescue dhdJ5;;pW8-dhdWT and non-rescued dhdJ5;;pW8-dhdΔDRE ovaries. Strikingly, the 5.4kbp dhd H3K27me3 mini-domain was completely lost in dhdJ5 ovaries  KD ovaries   , insertepression  and rescpression  but was pression  and S7. dhdJ5 mutants showed that H3K9me3 was lost at the dhd-proximal half of the domain, while this mark was maintained at the dhd-distal part , driven in germ cells by a nos-Gal4 driver  and Snr1, we generated flies combining a 4 driver . We conf4 driver . Remarka RT-qPCR . Using Cons Figs  and S8B.dhd heterochromatin mini-domain does not play a repressive role in ovaries, but do not exclude that it might maintain dhd silent in other tissues. RT-qPCR analysis on dissected ovaries, testes and male and female carcasses from transgenic lines expressing dhdJ5;;pW8-dhdWT revealed dhd expression uniquely from ovaries  in organizing 3D contacts between regulatory elements and promoters  = CaryP}attP2 , P{otu-GAL4::VP16.R}1; P{GAL4-nos.NGT}40; P{GAL4::VP16-nos.UTR}MVD1 , P{GAL4::VP16-nos.UTR}MVD1 . Other stocks are: w1118, Df(1)J5/FM7c [P[Mst35Ba-EGFP] [pW8-dhdWT [Flies were raised at 25°C on standard medium. The following stocks were obtained from the Bloomington Ba-EGFP] , pW8-dhdpW8-dhdΔDRE mutant, two fragments were amplified by PCR from w1118 genomic DNA using the primers ΔDRE-1-for/ ΔDRE-1-rev and ΔDRE-2-for/ ΔDRE-2-rev (pW8-dhdWT vector  = otu-GAL4::VP16.R}1, w[*]/y[ GAL4::VP16-nos.UTR}CG6325[MVD1]/P{y[+t7.7] v[+t1.8] = TRiP. HMS00849} attP2Snr1 KD , i.eP{w[+mC] = otu-GAL4::VP16.R}1, w[*]/y[Drosophila tissues was previously described [Cut&Run in escribed . BrieflySas10 gene, which was depleted in the histone marks tested in this study. Primer sets used are provided in 0,1 ng of retrieved DNA in Cut&Run were used as template in a real time quantitative PCR assay using SYBR Premix Ex Taq II (Tli RNaseH Plus) (Takara). All qPCR reactions were performed in duplicates using Bio-Rad CFX-96 Connect system with the following conditions: 95°C for 30s followed by 40 cycles of denaturation at 95°C for 5s and annealing and extension at 59°C for 30s. As a normalization control, we processed ovary samples from each studied genotype as for Cut&Run, except the antibody and pA-MNase incubation steps were omitted and instead we incubated tissue with 10U of Micrococcal Nuclease for 30min at 37°C . Fold change in histone mark enrichment was determined relative to this whole MNase control and relative to the D. melanogaster genome using Bowtie2 (v. 2.4.2). To compare samples with identical readcount for genome coverage quantifications, we employed Downsample SAM/BAM . To obtain short fragment datasets for DNA regulatory element identification, peak calling and visualization, we selected fragments shorter than 120 bp from SAM files. These were typically a small minority of all fragments, as our Cut&Run datasets were largely dominated by nucleosome-sized fragments (150-250bp). We therefore could separately analyze modification-bearing nucleosome coverage  or putative regulatory element coverage (<120bp fragments). For genomic track visualization, we used bamCoverage from deepTools 2.0  to calculate read coverage per 25bp bin for all fragments or 10bp bin for short fragments, with paired-end extension. Peak calling was done on sorted short fragments (<120 bp) with MACS2 (v. 2.1.1.20160309) with the following parameters:–nomodel,–p-value = 0.0001,–keep-dup = all and the rest by default. To establish a high-confidence short fragment peak list we retained peaks that were present in biological replicates from the control genotype. Genome browser views screenshots were produced with the IGV software, for Cut&Run we used a 25bp bin for all fragments and a 10bp bin for short fragments (<120bp). For the midpoint-plot of fragment sizes around short fragment peaks, the length of each fragment was plotted as a function of the distance from the fragment midpoint to the summit of the peak identified by MACS2. For signal quantification at the dhd locus, normalized read counts were counted within the domain  or within border element peaks Paired-end reads were mapped to the release 6 of the Heatmaps were generated with RStudio (RStudio Team (2016). RStudio: Integrated Development for R. RStudio, Inc., Boston, MA URL) and the packages ‘gplots’ (v.3.1.1) and ‘plyr’ .pW8-dhdWT transgene sequence was done with FIMO (v. 5.3.3) [Motif scanning on the . 5.3.3)  using thS1 Figmod(mdg4) KD and Snr1 KD are efficient in the female germline. Left: RT-qPCR quantification of mod(mdg4) (top) or Snr1 (bottom) mRNA levels in Control and KD ovaries . Data from biological duplicates analyzed in technical duplicates are presented as mean ± SEM. Right: Quantification of mod(mdg4) (top) and Snr1(bottom) counts in RNA-seq data from lid, Sin3a, Snr1 and mod(mdg4) KDs downregulate dhd but do not significantly affect its neighboring genes. Quantification of counts in RNA-seq data for dhd and its neighboring genes in Control, lid, Sin3a, Snr1 and mod(mdg4) KD show that low-expressing genes in the dhd region are not or only modestly impacted by the KDs. Both duplicates are shown. C-Limited overlap in the effects of mod(mdg4) and Snr1 KDs. Hierarchical clustering of sample distance heatmap of RNA-seq samples. D-Principal component analysis for RNA-seq samples. E- lid, Sin3a, mod(mdg4) and Snr1 KD severely downregulate dhd expression. RT-qPCR quantification of dhd mRNA levels in ovaries of indicated genotypes . Two different shRNA constructs  against lid were tested. Data from biological duplicates analyzed in technical duplicates are presented as mean ± SEM.A-(TIF)Click here for additional data file.S2 Figdhd region in cultured embryonic cells and ovaries. ChIP-seq data showing the active mark H3K4me3 (yellow) [dhd region (right). Display of H3K27me3 Cut&Run , ATAC-seq  , and the(yellow) ,45 and H(yellow) ). B—Shor2 cells, ), CP190 2 cells, ), Mod Polycom2 cells, ). Cut&Ru(TIF)Click here for additional data file.S3 Figphf7 and neighboring genes. Blue arrow indicates testis-specific TSS and magenta arrow indicates ovary-specific TSS.A- Cut&Run shows the expected enrichment of H3K9me3 at pericentromeric heterochromatin. Genome browser views of H3K9me3 ChIP-seq  and Cut&(TIF)Click here for additional data file.S4 FigE(z) KD does not severely affect dhd expression. RT-qPCR quantification of dhd mRNA levels in Control and E(z) KD ovaries . Data from biological duplicates analyzed in technical duplicates are presented as mean ± SEM. B- E(z) KD and Snr1 KD affect H3K27me3 levels in nurse cells. Confocal images of representative egg chambers in Control, E(z) KD, lid KD, Sin3a KD, mod(mdg4) KD and Snr1 KD. In control ovaries, H3K27me3 staining marks somatic follicle cell nuclei, the karyosome and germline nurse cell nuclei. In E(z) KD ovaries the karyosome and nurse cells loose staining of the histone mark but follicle cells are marked normally. No notable change is observed in lid KD, Sin3a KD or mod(mdg4) KD while in Snr1 KD nurse cells staining is less intense. Scale bar 10μm. C- Cut&Run in whole ovaries captures signal from both somatic and germline cells. Genome browser views of H3K27me3 Cut&Run signal in Control and E(z) KD ovaries and H3K27me3 ChIP-seq from FACS sorted nurse cells and somatic follicle cells [E(z) KD ovaries. Lower panels show H3K27me3 domains where the signal comes almost exclusively from follicle cells and is not significantly affected in the germline E(z) KD.A-le cells . Upper p(TIF)Click here for additional data file.S5 Figdhd locus from Control and KD ovaries. Left: Dotplot showing normalized read counts of H3K27me3 Cut&Run at the dhd domain from independent biological triplicates of the indicated genotypes (duplicates for E(z) KD). B—Cut&Run qPCR yields reproducible data among replicates. Biological replicates from H3K27me3 and H3K9me3 Cut&Run-qPCR in Control and KD ovaries shown in Sin3a KD, Snr1 KD and mod(mdg4) KD affect the stability of the H3K27me3-associated regulatory elements at the dhd mini-domain. Dotplot showing normalized read counts of H3K27me3 Cut&Run <120bp fragments at dhd regulatory elements from independent biological triplicates of the indicated genotypes (duplicates for E(z) KD). 5’ and 3’ border elements are plotted separately.A—H3K27me3 Cut&Run signal at the (TIF)Click here for additional data file.S6 Figdhd domain borders align with DREF and Beaf-32 peaks.Genome browser views of ovarian H3K27me3 Cut&Run  and Dref and Beaf-32 ChIP-seq . <120 b(TIF)Click here for additional data file.S7 FigdhdJ5, dhdJ5;;pW8-dhdWT and dhdJ5;;pW8-dhdFD ovaries shown in Biological replicates of H3K27me3 and H3K9me3 Cut&Run-qPCR in Control, (TIF)Click here for additional data file.S8 Figlid KD, Sin3a KD, mod(mdg4) KD and Snr1 KD are efficient in the female germline of rescue flies. From left to right: RT-qPCR quantification of lid, Sin3a, mod(mdg4) and Snr1 mRNA levels in ovaries of the indicated genotypes . Data from biological duplicates analyzed in technical duplicates are presented as mean ± SEM. B—The dhd rescue transgene does not restore H3K27me3 in KD flies. Biological replicates of H3K27me3 Cut&Run-qPCR in the indicated genotypes shown in Sas10 gene was used as negative control and Ubx as positive control. Fold enrichment was calculated relative to Sas10. Error bars show technical variability.A—(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


The ts3agn mutation drastically increases the frequency ofectopic contacts (FEC) in specific regions of intercalary heterochromatin, suppresses learning/memory and affectslocomotion. As is shown in this study, the polytene X chromosome bands in reciprocal hybrids between ts3agn and thewild type strain Berlin are heterogeneous in modes of FEC regulation depending either on maternal or paternal geneorigin. Bioinformatic analysis reveals that FEC between X:11AB and the other X chromosome bands correlates with theoccurrence of short (~30 bp) identical DNA fragments partly homologous to Drosophila 372-bp satellite DNA repeat.Although learning acquisition in a conditioned courtship suppression paradigm is similar in hybrids, the middle-termmemory formation shows patroclinic inheritance. Seemingly, this depends on changes in miR-974 expression. Severalparameters of locomotion demonstrate heterosis. Our data indicate that the ts3agn locus is capable of trans-regulatinggene activity via POEs on the chromatin nuclear organization, thereby affecting behavior.Prognosis of neuropsychiatric disorders in progeny requires consideration of individual (1) parent-of-origineffects (POEs) relying on (2) the nerve cell nuclear 3D chromatin architecture and (3) impact of parent-specific miRNAs.Additionally, the shaping of cognitive phenotypes in parents depends on both learning acquisition and forgetting,or memory erasure. These processes are independent and controlled by different signal cascades: the first is cAMPdependent,the second relies on actin remodeling by small GTPase Rac1 – LIMK1 (LIM-kinase 1). Simple experimentalmodel systems such as Drosophila help probe the causes and consequences leading to human neurocognitive pathologies.Recently, we have developed a Drosophila model for Williams–Beuren Syndrome (WBS): a mutant  Genome plasticity is ensured by the architecture of specificnuclear loci and nuclear localization of transcriptional machinerywhere the chromatin organization is the priority-driven factor. The outcome of recent achievements in systems biologyis the notion that the plasticity of 3D chromatin architectureof nervous cell nuclei plays the leading role in cognitionand neuropsychiatric disorders . The epigeneticcomponent is still an underestimated source of psychomotordisturbances and neuronal diversity . Therefore, a new field of human biomedical researchnamed molecular cytogenetics and cytogenomics , or chromosomics  has evolved. Themain goal of chromosomics is the study of chromosomes,their 3D architecture in the interphase nucleus, the outcomesof chromosomal sub-region plasticity and gene interactions forshaping interindividual and intercellular genomic variationsin normal behavior and disease.Recently, this topical problem has turned out to be thepursuit of understanding the concomitant role of activeforgetting, since the antithesis to learning acquisition is theforgetting or memory erasure . Bothprocesses are independent and controlled by different signalcascades: learning acquisition and memory consolidationoccurs via cAMP cascade, its components being CREB andC/EBP. Active forgetting relies on actin remodeling cascaderesponsible for structural alterations of neurons and synapses:small GTPase Rac1 – LIMK1 (the key enzyme of actin remodelingLIM-kinase 1) and its phosphorylation substrateсofilin. The absence of Rac1-dependent forgetting causesthe autistic spectrum disorders. Expression changes (activeor non-active state) of LIMK1 and cofilin lead to differentneuro pathologies. The most studied example embracing allthe aforementioned facets of manifestations is Microdeletion(Deletion) Williams–Beuren Syndrome, or WBS in 7q11.23.WBS deletion leads to cardiovascular pathology, cognitivedeficit in visuospatial construction and hypersociability. This isbecause long-term synaptic plasticity and depression determinesuccessfulness of learning and memory, as well as oflocomotor behavior, and depend on epigenetic regulation of LIM-kinase 1 (LIMK1) gene, one of approximately 28 genesuncovered by WBS deletion. Epigenetic regulation of LIMK1activity involves DNA methylation, chromatin remodeling andthe noncoding RNA-mediated process .LIMK1, a member of serine/threonine (Ser/Thr) familykinases regulated by the Rho-GTPase pathway, is the keyenzyme of actin remodeling cascade. Dendritic spines areactin-rich structures, and spine dynamics is driven mainly byactin remodeling, thus sharing several molecular pathwayswith dendrite growth . Increasing sets ofevidence suggest that nuclear actin also plays a pivotal rolein transcriptional regulation and DNA repair. Interestingly,monomeric actin is a stoichiometric subunit of a variety ofchromatin remodeling complexes. Ashift between monomericand polymeric states modifies activity of histone deacetylases.agnostic locus harboringLIMK1 gene (X:11AB). This region possesses theproperties of intercalary heterochromatin. Being a hotspot ofchromosome breaks, ectopic contacts, underreplication andrecombination, the region attains strain-specific architecturemarked by single base changes and small insertion/deletions.The EMS-induced temperature-sensitive (ts) mutationts3agn carries the insertion of transposable element (TE) fromTc1/mariner superfamily ~460 bp downstream 3′UTR ofDrosophila LIMK1 gene (dlimk1), as well as A/T-rich 28 bpinsertion within intron 1 of dlimk1 capable of pairing with5′ TIR of the TE.Recently, we have developed a simple and appropriateDrosophila model for chromosomics  using the properties of the ts3agn shows a temperaturesensitivelethality at all stages of development except for theimaginal stage. At normal temperature, the adult flies showdrastic learning acquisition and memory retention defects, aswell as locomotor impairments and amyloid-like inclusions. Stressexposure (heat shock for 30 min at 37 °C) suppresses thesemanifestations . Also, ts3agnmutationleads to: (1) LIMK1 and p-cofilin increase in the adultbrain and salivary glands of 3rd instar larvae at 22–25 °C anda fall down to the level of the wild type strain Canton S at29–37 °C; (2) high level of ts-induced recombination withints3agn region; (3) 3-fold increase in frequency of non-allelicectopic contacts (FEC) within 2L arm of the chromosome 2 and in the 11В X chromosome region . Additionally, miRNAs expression including the biomarkersfor human neuropathologies is drastically reducedin ts3agn relative to the wild type strains .When maintained at 29 °C, ts3agn-specific nuclear organization is shaped in early embryogenesisalongside with formation of chromosomal heterochromatinregions. Intrinsic ts3agn FEC is maternally inherited. Therefore, ts3agn is a promisingmodel for studies on parent-of-origin effects (POEs) on progenyconsidered as significant causative factors of psychiatricdisorders . For instance, a 1.5 Mb WBSdeletion recurrently arises de novo and depends on POEs: maternalorigin leads to more severe developmental abnormalitiesand microcephaly . Moreover,when WBS deletion has a paternal origin, expression levels ofa number of genes within the WBS deletion decrease. Amongthese genes crucial for the brain development is a gene for generaltranscription factor II-I (GTF2I). It regulates transcriptionby binding to DNA and histone deacetylase (HDAC) . The main goal of the study is the analysisof POEs role in shaping quantitative traits, namely learningacquisition, memory retention, locomotion, and miRNAs expressionwhile using the advantages of the Drosophila modelfor POEs in progeny from reciprocal crosses between ts3agnand the wild type strain Berlin .To meet the requirements of chromosomics, FECs betweenthe region X:11AB and the other bands of the X chromosomemay be estimated as an indicator of chromosomal spatialorganization. This approach is justified by the existence oflate-replicating genomic territories including the underreplicatedregions of polytene chromosomes. These regions overlapwith late-replicating regions of mitotically dividing cells. Suppression of SUUR gene responsiblefor underreplication of intercalary heterochromatin and, as aconsequence, for the ectopic pairing leads to death in early embryogenesis. Additionally, to elucidatethe contribution of DNA sequence homology as componentsof epigenetic regulation in the ectopic chromatin pairing wehave developed the special software package Homology SegmentAnalysis.Drosophila stocks. The fly stocks used belong to Biocollectionof Pavlov Institute of Physiology of the Russian Academyof Sciences:• Berlin, a wild type strain;• Canton S, a wild type strain;• ts3agn, a temperature-sensitive mutation on Canton Sgenetic background within agnostic locus (X:11AB) affectingdlimk1 activity.ts3agn and Berlin were usedbecause Berlindlimk1 sequence is closer to FlyBase referencesequence . At the same time,the reciprocal hybrids ts3agn×Canton S (the genetic backgroundfor ts3agn) and Canton S×ts3agn demonstrate exactlythe same cognitive behavior as reciprocal hybrids with Berlin approving the usage of Berlin.The reciprocal hybrids between Berlin and ts3agn female and male parents and F1 female and male progeny from reciprocal crosses with theaccent on the putative hairpin formed in the X chromosomeby 28 bp A/T rich insertion within intron 1 of dlimk1 geneand 3′ end of Tc1/mariner element.Figure 1 shows the X chromosome and autosomes architecturein Flies were maintained on standard Drosophila yeast-raisinmedium at +22 ± 0.5 °С under a 12-h light/dark cycle. Forthe memory and locomotion tests, males were collected uponeclosion without narcotization and kept individually in culturevials till the behavioral experiments on the 5th dayEstimation of frequency of ectopic contacts (FECs).The aceto-orcein squash preparations were prepared fromsalivary glands of III instar D. melanogaster female larvae.20 to 30 animals were examined, therefore the number ofanalyzed chromosomes varied from 300 to 500. The examplesof ectopic contacts are presented on Fig. 2. The number ofnon-homologous contacts between the region X:11AB anddifferent bands of the X chromosome was calculated and expressedas per cent of the total number of the examined nuclei.FECs in parents and F1 reciprocal hybrids were comparedusing Student’s t-test. Identification of genes localized in theX chromosome bands forming contacts with the 11AB regionwas performed using NCBI Genome Data Viewer databasehttps://www.ncbi.nlm.nih.gov/genome/gdv/ and molecularfunction of identified genes was derived from FlyBase https://flybase.org analysis of DNA segments homology.D. melanogaster genome sequence (release 6) was taken from. Special software package HomologySegment Analysis searching the matches of short singlestrandedDNA fragments within the chromosome areasinvolved in the ectopic pairing in Drosophila has been developed.The software written in Python 3 can be freely downloadedfrom . Software version from git(commit 41719cddc6283edbd79c5bf2aee237cde48d4b7d)was used. The algorithm of the program is described in brief in. The exact run parameters for 11AB region are following: segmentanalysis.py -v -s 30 dm6.nounmapped.fa.gz :X:11982050:12772070 DmelMapTable.160615c.bedHere, dm6.nounmapped.fa.gz is Drosophila genome canonicalsequence (Ensembl release 6) and DmelMapTable.160615c.bed is chromosome bands location from Ensembldatabase converted to BED format. Both files are included insoftware repository.For other tested regions, all parameters were the sameexcept for the regions location.Preparation of miRNAs libraries and bioinformaticanalysis. The detailed description of the procedure is givenin . Extract RNA reagent was used for total RNA extraction fromadult 5 days old males. To obtain the fraction of small RNA,25 μg of total RNA were separated using 15 % polyacrylamidegel electrophoresis in the presence of Urea (8 M) followingexcision of small RNA fraction corresponding to 21–29 nts.Illumina TruSeq Small RNA prep kit  wasused for small RNA libraries preparation. Sequencing wasperformed on an Illumina HiSeq 2000 platform.The amount of mapped miRNAs reads was counted byBEDTools (v. 2.22) and mirbase annotation (r. 19) . Analysis of differentially expressed miRNAs wasperformed using edgeR (v. 3.10.2) package in R environment(v. 3.2.2) . miRNA functions werederived from miRBase . Small RNAlibraries were deposited in NCBI SRA under the numberPRJNA633483.Locomotor activity. Computer-aided automatic device forsimultaneous registration of 20 animals is described in . The experiment lasted for 1 h. Spontaneouslocomotor activity of flies was detected in a plate witheight chambers and transparent cover using high-resolutionvideo camera. Software used for locomotor activity analysisis freely available at .The following parameters of locomotion were assessed:activity index (%); run frequency (the number of run boutsin 100 seconds); running speed (mm/s). The full record wasdivided into 1 s quanta, and the mean speed of fly movementin each quantum was calculated. If the result was less thanthe threshold value (5 mm/s), the fly was considered to beresting during this time quantum; otherwise, it was considered moving. Neighboring quanta with similar movement patternwere merged in intervals of moving and resting. Activity indexis determined as a time spent in movement. Running speedis an average fly speed, determined using only intervals ofmovement. The Kruskal–Wallis analysis of variance with themultiple comparison of mean ranks was used to compare allthe experimental groups.Learning acquisition and middle-term memory formationin Drosophila males. Detailed description of learning/memory assessments in conditioned courtship suppressionparadigm (CCSP) and specially designed software for observationand statistical analysis (randomization test) of learningindices based on courtship indices is given in . CCSP employs the natural stimuli of Drosophilacourtship. Both virgin and fertilized females emit an aphrodisiacpheromone, attracting a naïve male without courtshipexperience. However, a fertilized female rejects a male atthe courtship stage of attempted copulation via emitting anaversive pheromone. Repetitive rejections during 30 mintraining provoke a kind of learned helplessness when a malestops courting another female. This courtship suppressionmight last for one hour when test female is virgin and foreight hours when fertilized. Males with defective memoryformation continue to court after such a training as vigorously,as naïve males.The courtship index was calculated for each male. The learning index (LI)was computed according to the formula:Analysis of spatial nuclear organizationdelimited to FECs formedby the X chromosome region 11ABBerlin, ts3agn and their hybrids are presented in Table 1. Thecolumns 1–4 show the pattern of FECs between the 11AB regionand other X chromosome regions pertinent to listedstrains. The absence of significant differences of FECs betweencolumns, i.e. 1 vs 2 and 3 vs 4 indicates matroclinicinheritance, 2 vs 3 indicates hybrid-specific frequencies, 2 vs 4and 1 vs 3 pinpoints the patroclinic inheritance. The polytenechromosome bands in hybrids demonstrate differences inFECs, a part of them showing either matroclinic propertiesor properties of the father strain. In certain bands, FECs aresimilar in reciprocal hybrids or depend on the direction of a cross, but significantly differfrom that of parents.Spatial nuclear organization was analyzed using microscopicimages of polytene chromosomes in larvae salivaryglands. The results of comparative analysis of FECs betweenthe 11AB region and the other X chromosome regions in Berlin, ts3agn and their hybrids are presented in Table 1. Thecolumns 1–4 show the pattern of FECs between the 11AB regionand other X chromosome regions pertinent to listedstrains. The absence of significant differences of FECs betweencolumns, i.e. 1 vs 2 and 3 vs 4 indicates matroclinicinheritance, 2 vs 3 indicates hybrid-specific frequencies, 2 vs 4and 1 vs 3 pinpoints the patroclinic inheritance. The polytenechromosome bands in hybrids demonstrate differences inFECs, a part of them showing either matroclinic propertiesor properties of the father strain. In certain bands, FECs aresimilar in reciprocal hybrids or depend on the direction of a cross, but significantly differfrom that of parents.NCBI Genome Data Viewer software helped to reveal thegenes located in the X chromosome bands forming the ectopiccontacts. Based on the assumption that shared location ofgenes determines their functional features ,it was worth elucidating what biological processes are underthe influence of epigenetic factors related to allelic parentof-origin. Therefore, the grouping of genes implied theirinvolvement in control of a certain biologic process .ts3agn×Berlin relative to the reciprocalhybrid are genes for motor proteins  and sensoryreceptors . Genes involved in neurodevelopment,oxidative-reduction process and proliferation do not present inBerlin×ts3agn progeny. Heterosis, when the number of genesinvolved in hybrid-specific contacts is more than 2-fold higherthan in both parents, is manifested for oxidative-reductionprocess (3), signal transduction (4), proliferation (9) and noncodingRNAs (13).Figure 3, a presents provisional definition of the biologicalprocesses controlled by genes within bands forming contactswith 11AB region either in matroclinic or in reciprocal hybridspecificmanner. Among the functional groups of genes havingincreased number in ts3agn×Berlin relative to thereciprocal hybrid), reactive oxygen species metabolic process, and metabolism . Sensory perception andsignal transduction groups do not present in Berlin×ts3agn.The biological processes and genes involved in ectopiccontacts with 11AB in a mode of father strain are shown inFig. 3, b. Among them are the genes responsible for chromatinremodeling ; metabolism ; cell proliferation ); chromatine remodeling . Reparation/recombination group does not present in Berlin×ts3agn.Data on biological processes and the number of genes involvedin contacts with 11AB region exclusively in hybridsor with FECs prevailing those of parents (FEC heterosis), areshown on Fig. 3, c. The ratio of gene numbers for Profiles of the localized fragment frequencies (LFFs)Using our specially designed software Homology SegmentAnalysis, we analyzed correlation between ectopic pairingand distribution of small identical fragments within contactingregions.The region X:11AB (~790 kb) involved in DrosophilaX – X:11AB ectopic pairing was selected as the source ofsmall 30 nt fragments, and the profile of X:11AB localizedfragment frequencies (LFFs) was constructed for the X chromosome. The region X:14B–15B was selected asa control region having almost equal length (~790 kb).ts3agn×Berlin FEC (X:11AB). Probably,20 nt fragments are too short to display DNA homologyspecifically associated with ectopic pairing. The increasein fragments length from 20 to 50 nt leads to decrease inLFF (X:14B–15B) – FEC (X – X:11AB) unspecific correlation and simultaneous increase in LFF – FEC (X:11AB) specificcorrelation. However, there is a lack of LFF (50 nt) – ECFcorrelation for ts3agn. Hence, 30 nt fragment length seems tobe optimal in search for the identical fragments within thecandidate chromosomal regions involved in ectopic pairing.Both LFF and the FEC (X – X:11AB) distributions significantlydiffer from normal. Therefore, we calculated Spearman’srank-order correlation coefficients (rS) for LFF andthe strain-specific FEC (X – X:11AB) (Table 2). A positivecorrelation between LFF and FEC is observed within the region of interest X:11AB. The influence of the fragment lengthon rS value has been tested. For 30 nt fragments, rS is highlysignificant for all Drosophila strains ( p < 0.001). As to LFF(X:14B–15B), there is no significant correlation with FEC(X – X:11AB) ( p ≥ 0.01) pointing to specificity of analysisof DNA homology within the region of ectopic pairing. For 20 nt fragments, there is a false-positive correlation betweenLFF (X:14B–15B) and Berlin FEC and thelocalization frequencies for the several specific X:11AB 30 ntfragments having the highest number of occurrences (NO)in the X chromosome (Table 3). The maximal correlation isevident in the part of 372 bp middle repetitive DNA sequence). The majorityof fragments with significant positive rS values appear to bethe parts of a ~50 bp repeat (marked with #) having significantself-complementarity. This sequence also shows an almostcomplete identity to another part of 372 bp repeat. Such repeatswith slight sequence variations occur in both DNA strands ofthe X chromosome. rS is much lower for (-at-)15 repeat and isnearly absent for (-gt-)15 (-tc-)15 and (-ca-)15 repeats, althoughtheir NO can be higher. Noteworthy, all rS values for 372 bprepeat fragments are lower compared to rS for the whole setof the localized fragments (see Table 2), hence all of themseemingly impact ectopic pairing.To find out what DNA sequences impact the ectopic pairingthe most, we calculated rS between miRNAs expression profileBerlin, ts3agn and their reciprocalhybrids demonstrates significant differences betweenmales of parent strains and hybrids in content of 44 miRNAs.For the reciprocal hybrids, the heat maps of miRNAs expressionare presented in Fig. 5. Among 44 miRNAs 10 miRNAsbelong to the same cluster of testis-specific miRNAs . However, only miR-980 and miR-974are involved in memory processes. Expression of miR-980suppresses Drosophila memory ,while lowered expression of miR-974 impairs memory formationAnalysis of miRNAs expression in The heat map represents the RPM-normalized and log2-transformed counts of miRNAs reads with z-scale normalizationof the rows. Thirty percent of low-expressed miRNAswere removed from further analysis. Only the miRNAs withaltered content in a hybrid compared to at least one parentare shown. analysis of parent strainsand their reciprocal hybridsLocomotor behavior. Figure 6 presents the parameters oflocomotion. As to activity indices, they are significantly differentin ts3agn and Berlin, both hybrids differ from ts3agn andBerlin×ts3agn differs from Berlin. Running speed and runfrequency in ts3agn and Berlin are similar. Both hybrids differfrom ts3agn and Berlin×ts3agn from Berlin. However, thehybrid running speed exceeds that of parents, demonstratingheterosis. Comparatively to parents, run frequency in hybridsis intermediate. At the same time, any alterations in locomotorparameters in hybrids are similar and unidirectional.Learning acquisition and memory formation.In all strains,CIs of males decrease after training with fertilized femalescompared to naïve flies . Learning/memory scoresin reciprocal hybrids show that memory formation (3 hoursafter training), but not learning acquisition (0 hours aftertraining), demonstrates patroclinic inheritance .Berlin strain in crossests3agn×Berlin, learning and 3-hour memory do not differfrom LIs of Berlin, but do differ from LIs of ts3agn. Patroclinicinheritance is evident in the reciprocal hybrid Berlin×ts3agn.In this case, 3-hour LIs in Berlin×ts3agn and ts3agn are similar.The patroclinic inheritance cannot be associated with theY chromosome, as it is likely to be similar in Berlin, ts3agnand Canton S . Also, it cannot be attributed to the X chromosomes,since they are different in Berlin and ts3agn×Berlin.Seemingly, this may be caused by some paternal epigeneticfactors, such as miRNAs in cytoplasm of male sperm.When male parent originates from Discussionts3agn gene and epigenetic factors (POEs) in spatial nucleararchitecture, learning/memory formation and spontaneouslocomotor activityStudies of genome-wide associations between DNA polymorphismsand phenotypic traits have revealed genetic variantspredisposing to different mental diseases. Findings pinpointingthe role of POEs in genetic risk for neuropathology opennew possibilities for therapy and preventive medicine . In this study, we estimated FECs in reciprocalhybrids considering an impact both of genetic variants ofdlimk1 gene.Our assumption was that FEСs partly reflect the restrictedhomology of short DNA sequences in different, seemingly“non-homologous” regions.To exploit the advantages given by the model, we delimitedthe analysis of spatial nuclear organization to FECs formedby the X chromosome region 11AB harboring As shown in this study for the short identic fragments withinthe contacting regions, FECs correlate with LFFs. Althoughthe correlation is rather moderate (~0.35), it is highly specificfor 30 nt fragments. Many factors affect ectopic pairing,such as DNA homology, the distance between the interactingregions and epigenetic factors causing the interstrainFECs differences . Our computationalalgorithm concerns only fragments aligned with the X chromosomewithout gaps. This reveals the partial homology ofinteracting bands. Thus, the interacting chromosomal areasmay be significantly larger than 30 or 50 nts. Although differentmechanisms are involved in ectopic pairing, includingPOEs, our data indicate the significant role of DNA sequenceitself. However, as FEC–LFF correlation is mainly observedfor specific DNA fragments, their pairing mediated by someproteins or non-coding RNAs cannot be ruled out.Most of the found 30–50 nt fragments are similar to theD. melanogaster dispersed 372 bp A/T-rich noncoding repeat. This moderately repeated sequence islocated in the euchromatin of the X chromosome between theregions 4 and 14A in ~300–400 copies per haploid genome.The 372 bp repeat is a part of 1.688 g/cm3 class of satelliteDNA (1.688X repeats) . siRNAfrom the 1.688X repeats is involved in dosage compensationin recognition of the X and autosomal chromatin, therebydelimiting activities of male-specific lethal (MSL) complexto sex chromosomes through up-regulation of the X chromosome.ts3agn, FECs in thesebands significantly decrease. In this case maternal control ofspatial localization and therefore, of gene expression is geneticallydetermined. These are genes controlling membranereceptor regulation (PPYR1) and signal transduction (gce),chromatin remodeling , axon guidance andchemosensory jumping behavior (acj6 ).The data obtained consider the common mechanisms ofectopic contacts formation and dosage compensations. Seemingly,POEs might influence the spatial chromatin organization,thereby affecting behavioral performances.This indicates that each Drosophila strain possesses itsown pattern of ectopic contacts with the region 11AB. Thepolytene chromosome bands are heterogeneous in their modesof regulation of ectopic pairing. Apart of them is regulated bygenes of either maternal, or paternal origin. A separate classis comprised of regions manifesting only hybrid properties.Similar РОЕs were observed for the pattern of methylation andnucleosome distribution within the imprinted loci in humansand plants .In both reciprocal crosses, bands 7A, 9A and 13B displaythe maternal properties. When mother is This indicates that each Drosophila strain possesses itsown pattern of ectopic contacts with the region 11AB. Thepolytene chromosome bands are heterogeneous in their modesof regulation of ectopic pairing. Apart of them is regulated bygenes of either maternal, or paternal origin. A separate classis comprised of regions manifesting only hybrid properties.Similar РОЕs were observed for the pattern of methylation andnucleosome distribution within the imprinted loci in humansand plants .ts3agn, FECs in thesebands significantly decrease. In this case maternal control ofspatial localization and therefore, of gene expression is geneticallydetermined. These are genes controlling membranereceptor regulation (PPYR1) and signal transduction (gce),chromatin remodeling , axon guidance andchemosensory jumping behavior (acj6 ).In both reciprocal crosses, bands 7A, 9A and 13B displaythe maternal properties. When mother is ts3agn×Berlin, the number of genes with knownfunctions contacting with 11AB with maternal-specific frequencyis 2-fold higher than in reciprocal cross. Possibly, thisis due to the ts3agn-specific miRNAs pattern of expression.The role of miRNAs in maternal inheritance and expressionin embryogenesis is sparsely studied. As we have shownearlier, the expression level of miR-9, miR-34 and miR-124differs in ts3agn from that in Berlin, Canton S and Oregon-R. miR-9 and miR-124 are alsoexpressed in early development (0–12 hrs) , miR-34 is detected in embryos till zygotic reduction. As known, the switch from maternal tozygotic development program occurs between the second andthe third hours of embryonic stage, hence miRNA found inearly development have maternal origin . ThesemiRNAs targets are Swi/Snf-like complex, neural-progenitorspecificnpBAF, repressor-element-1-silencing transcriptionfactor (REST belonging to 1 class of histone deacetylases(HDAC1/2) and silent information regulator 1 (SIRT1) –3 class of NAD+-dependent histone deacetylases involved inheterochromatin formation, Bourassa, Ratan, 2014). They areinvolved in neurogenesis, dendrite morphogenesis and axonguidance which depend on global chromatin remodeling.In the cross ts3agn×Berlinectopic contacts between 11AB and regions containing genesinvolved in chromosome remodeling are formed with frequencycharacteristic for the maternal genome. The products ofthese genes are: Tip60 – histone acetyltransferase, HDAC6 –histone deacetylase; mxc – regulator of histone synthesis ofPolycomb group; Top1 – DNA topoisomerase. However, onlytwo regions containing genes HDAC6 and Top1 are presentin cross Berlin×ts3agn.Therefore, it is not surprising that in cross ts3agn-like matroclinicmode of inheritance is pertinent to genes responsible for actinand microtubules-binding proteins with motor function andneurodevelopment. Noteworthy, the state of actin remodelingdetermining neurologic manifestations is a diagnostic featureof ts3agn .Noteworthy, new knowledge about topologically associatingdomains (TADs) indicates that polytene, diploid, andembryonic TADs condensation along the chromosome axisis just the same everywhere . Moreover,comparison of TADs with 3D chromatin organization revealedby the Hi-C method confirms that the interphase nucleusspatial organization into TADs is directly represented by bandingpattern of polytene chromosomes .Therefore, this allows to bridge the ratio of genes forming ectopic contacts in a mode of either maternal, or paternal strainin reciprocal crosses and their physiologic manifestations.The later might result from alterations in the 11AB regionarchitecture. As shown in Fig. 3, a, the The regions with FECs similar in reciprocal hybrids, butdiffering from parents, i. e. manifesting hybrid properties,contain a large set of genes responsible for motor functions.ts3agn:meiosis, reparation, recombination; transcription factors; metabolism;proliferation; actin-binding proteins, microtubuleassociatedproteins.Figure 3, c shows genes and biological processes for chromosomalregions forming ectopic contacts with X:11AB onlyin hybrids or mainly in hybrids compared to parents. Theseprocesses are pertinent to main manifestations of ts3agn the chromosomalbands 8D, 12E, and 19D demonstrate FECs significantly exceedingthese of parents. These bands contain genes involvedin taste and odor perception and neurodevelopment, in particularof the mushroom bodies of the brain. The other examplesof father strain manifestations might result from activities oftrans-acting factors, such as miRNAs .Interestingly, in the cross Berlin×ts3agn and in progenyof Berlin×ts3agn (impaired 3-hour memory) and is similar towild type in the ts3agn×Berlin cross . Likely,miR-974 might act as trans-acting factor presumed to regulategenes in patroclinic mode. The prevailing role of the paternalgenome in memory formation is evident in Canton S and ts3agnreciprocal hybrids .miR-974 is involved in memory processes: its loweredexpression impairs memory formation .Decrease in its content in olfactory neurons and the mushroombody V2 neurons promotes 3-hour memory. Noteworthy, thecontent of miR-974 is decreased both in agnostic locusmight belong to the class of quantitative trait loci (QTL) .Taken together, our data indicate that the agnostic LIMK1 gene is a good candidate for linkingthe neuronal activity  and genetic apparatus . Additionally, quite recent andunexpected findings  reveal a new targetof intellectual disabilities: learning acquisition and memory erasure (forgetting) are governed by different signal cascades,correspondently cAMP-dependent and actin remodeling cascadesmall GTPase Rac1 – LIMK1 (the key enzyme of actinremodeling LIM-kinase 1) and its phosphorylation substrateсofilin. The absence of Rac1-dependent forgetting causesthe autistic spectrum disorders. Expression changes (activeor non-active state) of LIMK1 and cofilin lead to differentneurological disorders. Therefore, in the tradition of Russiangenetic school , the agnostic genemight be a functional link between genetic and cytogeneticprocesses within the nervous system and serve as a model forelucidating both the maternal and paternal modes of transgenerationalinheritance.One of the requirements of predictive and personalizedmedicine is consideration of POEs for prognosis of clinicalphenotype of many multifactorial neuropsychiatric disorders.These different and individual manifestations of cognitiveabilities and motor functions in patients with the same disease,i. e. behavioral plasticity, results from genome plasticity provokedby 3D chromatin architecture of the nerve cells nuclei.The evolutionary gene conservation approves the usage ofsimple low cost, fast and efficient models as Drosophila toprobe the causes, consequences and mechanisms of pathologyleading to human disease . 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