NOD-like receptors (NLRs) localize in the cytosol to recognize intracellular pathogen products and initialize the innate immune response. However, the ligands and ligand specificity of many NLRs remain unclear. One such NLR, NLRP6, plays an important role in maintaining intestinal homeostasis and protecting against various intestinal diseases such as colitis and intestinal tumorigenesis. Here, we show that the major component of the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS), binds NLRP6 directly and induces global conformational change and dimerization. Following stimulation by ATP, the NLRP6 homodimer can further assemble into a linear molecular platform, and ASC is recruited to form higher molecular structures, indicative of a step-by-step activation mechanism. Our study sheds light on the mystery of LPS-induced inflammasome initiation, reveals the architecture and structural basis of potential pre-inflammasome, and suggests a novel molecular assembly pattern for immune receptors. Twenty-two NLRs have been identified in humans and all share typical tripartite domain architecture: the N-terminal domain recruits downstream effector molecules by homotypic protein-protein interactions; the central nucleotide-binding oligomerization  domain enables activation of the signaling complex via nucleotide-dependent oligomerization; and the C-terminal leucine-rich repeat (LRR) domain recognizes microbial ligands in a manner analogous to TLRs. Studies on NOD1, NOD2, NLRP1, NLRP3, NLRX1 and NLRC4 suggest a direct interaction between NLRs and cytoplasmic pathogen- and damage-associated molecules9, however, ligands and ligand specificity remain poorly understood for the majority of NLRs.The innate immune system is essential in the first line of defense against microbial infection, tissue injury and regulating the adaptive immune system. The initiation of innate immune responses relies on the recognition of conserved structures or products of pathogens by pattern recognition receptors (PRRs). Toll-like receptors (TLRs) and newly identified nod-like receptors (NLRs) are key PRRs in the innate immune system. TLRs recognize a wide variety of microbial products outside the cell or in intracellular endosomes and lysosomes and NLRs localize in cytosol to detect intracellular pathogen products10. Our knowledge of inflammasomes is mainly derived from studies of NLRP1, NLRP3 and NLRC4, whereby these NLRs form inflammasomes with the adaptor ASC and Caspase-1, promoting the maturation of the inflammatory cytokines IL-1b and IL-1811. NLRP6  as putatively activated oligomers  was purified from ers Fig. . The finers Fig. , and use–892 was ell Fig. . To our 24. We used analytical gel filtration to identify potential ligands of NLRP6 and characterized direct ligand binding features. A panel of various microbial components  indicates specific and high-affinity binding between LPS and NLRP6  subfamily and NLR activation may be accompanied by the exchange of ADP for ATP through the NOD domainATP Fig. . HoweverATP Fig. . FormatiATP Fig. . NLRP6 dGiven that conformational heterogeneity of NLRP6 oligomerization may play an important role in NLR activation, we examined the structure of different fractions of NLRP6 oligomers. We reconstructed a 3D EM density map of the NLRP6 tetramer with 8783 particles and the reconstructed 3D volume fit experimentally-determined class averages Fig. . The oveThe overall shape of the hexamer Fig.  and idenThe oligomerization of NLRP6 shows ATP concentration dependence Fig. . Particl30. Different chemotypes of LPS  showed similar induction of NLRP6 aggregation and subsequently ASC recruitment , consistent with our biochemical results.The DeltaVision live cell imaging system was applied to confirm the LPS binding of NLRP6 and elucidate NLRP6 oligomerization and ASC recruitment in cells Fig. . HeLa ce31, and recent studies propose that LPS can be sensed by inflammatory caspases in the cytoplasm of macrophages33. Our results provide biochemical and cellular evidences of an alternative intracellular LPS recognition mechanism through NLRP6. NLRP6 has been proposed as a multifaceted NLR, regulating both inflammasome-dependent and inflammasome-independent functions22. Recent studies found NLRP6 was involved in the regulation of autophagy and mucus granule exocytosis in goblet cells, orchestration of antimicrobial peptide secretion, epithelial regeneration, viral RNA recognition, and regulation of inflammatory signaling in myeloid cells. Currently the leading view is that each of these functions should occur in a different cell type34.LPS is a glycolipid located in the outer membrane of gram-negative bacteria. Extracellular LPS is thought to be detected exclusively by Toll-like receptor 4 (TLR4) and its co-receptors17, our results suggest that NLRP6 might act as a LPS sensor in the intestine where a high abundance of commensal bacterial exist, thus playing a crucial role in maintaining intestinal homeostasis. Cytosolic LPS may be recognized differently in macrophages33 and in intestine epithelium, consistent with different expression patterns for NLRP6. Mice lacking NLRP6 feature improved resistance to systemic infection with gram-negative Enterobacteriaceae while being more susceptible to intestinal infection with members of the same bacterial family emphasizes the concept that NLRP6 may perform highly differential functions in a cell type- and context specific manner34. Further, the oligomerization of NLRP6 triggered by LPS and ATP in a characteristic two-step mechanism appears to be a novel linear molecular platform for the recruitment of signaling partners such as ASC, and may then assemble into inflammasome or another filamentous signaling complex35.Our data confirm that LPS binds directly to the NLRP6 monomer and induces its dimerization, similar to TLR4. Considering NLRP6 is expressed highly in the intestinal epithelium and with low expression in microphages36, and also the recent filament model in which NLRP6 forms into filament with a PYD core surrounded by the NBD and the LRR domain in absence of ligand37. Indeed, due to a rigid and stable dimer formation upon high affinity ligand binding, a ring-like NLR arrangement consisting of seven re-organized monomers seems to be implausible for LPS-induced NLRP6 polymerization and activation, the process identified here appears to be a precise step-by-step mechanism , followed-up by live cell imaging. Difficulties associated with NLR expression and purification have limited the past application of this approach because it requires monomeric (ligand unbound status) full-length NLR protein to detect direct ligand binding and conformational changes and large quantities of purified NLR proteins to perform multiple experiments. However, we successfully obtained homogenous NLRP6 monomer at 2 mg/L with 99% purity. Our methods were successful in identifying NLR binders/ligands with certainty, and should be viewed as a valuable supplement to traditional ligand screening.38 and analytical gel filtration, an appropriate buffer condition  was identified out to keep NLRP6 in a monomeric state.Full-length NLRP6 fused with N-terminal His-tag followed by a maltose binding protein (MBP) tag were expressed in E. coli, and purified by Ni-NTA (QIAGEN) affinity chromatography followed by Amylose affinity chromatography (NEB). After HRV3C digestion, high purity NLRP6 protein preparations in the putative ligand binding oligomeric state were obtained. Using protein solubility test26 were selected to be incubated with NLRP6 monomer in 20:1 molar ratio, and then loading to a Superdex 200 10/300 column .To identify the potential ligand of NLRP6, a panel of microbial components including LPS(Ra)39. The image processing was using EMAN and EMAN2.Preparation of negatively stained samples and image acquisition were as described elsewhere25. Data were analyzed using Biacore 3000 and fit to a 1:1 Langmuirbinding model.In order to investigate the interaction between LPS and NLRP6, a kinetics assay was performed at 298 K using a Surface Plasmon Resonance (SPR) Biacore 3000 machine . Kinetic profiling was performed using the single cycle kinetics methodLive cell imaging was performed using a DeltaVision live cell imaging system (Applied Precision) equipped with an Olympus IX-71 inverted microscope and a 100×, 1.40 N.A. oil objective. Images were captured with 100 ms exposure times in 30 min intervals by a CoolSnap HQ2 CCD camera, and different Z sections were projected by SoftWorx suite.Supplementary Information   Supplementary Information 1 Supplementary Information2


Drosophila melanogaster, a method we call mini-INTACT.Epigenetic mechanisms play fundamental roles in brain function and behavior and stressors such as social isolation can alter animal behavior via epigenetic mechanisms. However, due to cellular heterogeneity, identifying cell-type-specific epigenetic changes in the brain is challenging. Here, we report the first use of a modified isolation of nuclei tagged in specific cell type (INTACT) method in behavioral epigenetics of Drosophila males. Social experience altered the epigenetic landscape in clusters of genes involved in transcription and neural function. Some of these alterations could be predicted by expression changes of four transcription factors and the prevalence of their binding sites in several clusters. These transcription factors were previously identified as activity-regulated genes, and their knockdown in dopaminergic neurons reduced the effects of social experience on sleep.Using ChIP-seq on mini-INTACT purified dopaminergic nuclei, we identified epigenetic signatures in socially isolated and socially enriched Drosophila as a model for cell-type-specific behavioral epigenetics and establishes that social environment shifts the epigenetic landscape in dopaminergic neurons. Four activity-related transcription factors are required in dopaminergic neurons for the effects of social environment on sleep.Our work enables the use of The online version of this article (10.1186/s12915-019-0646-4) contains supplementary material, which is available to authorized users. Drosophila melanogaster  v[1]; P{y[+t7.7] = CaryP}attP2) no insert background control vs. RNAi lines: BL29377 (Hr38); BL31900 (CrebA); BL27701 (Sr). BL36304  no insert background control vs. RNAi lines: BL42562 (CrebA); BL38276 (cbt) and BL42533 (Brms1).escribed  and wereescribed . Tissue escribed  were obtFly brains were dissected in cold 1X phosphate buffered saline (PBS) and fixed in 2% paraformaldehyde made in 1X PBS at room temperature for 1 h on a nutator, washed four times for 20 min each in PAT  at room temperature, blocked for 1 h at room temperature with blocking buffer  and incubated with primary antibodies, diluted in blocking buffer, overnight on a nutator at 4 °C. The primary antibodies used were Mouse-GFP , Rabbit-TH , and Rat-DN-cadherin . This was followed by four washes for 20 min each in PAT, and incubation overnight on a nutator at 4 °C with secondary antibodies diluted in blocking buffer. The secondary antibodies were all from Molecular Probe and used at 1:500 dilution: Alexa Fluor 488 anti-Mouse (A11029), Alexa Fluor 568 anti-Rabbit (A11036) and Alexa Fluor 633 anti-Rat (A21094). Brains were then washed four times for 20 min each in PAT at room temperature and one time for 20 min in 1× PBS and mounted with VECTASHIELD mounting medium . Samples were imaged on a Zeiss 800 confocal laser scanning microscope.TH-GAL4 which is expressed in ~ 120 neurons/brain. Drosophila males of 3X-UAS-unc84-2XGFP/TH-GAL4 genotype were either socially isolated or group housed and flash frozen during the morning activity peak. Frozen heads were collected over dry ice-cooled sieves from vortex-decapitated flies and added to 5 ml of mini-INTACT buffer consisting of 5 mM β-glycerophosphate pH 7.0, 2 mM MgCl2, 1× complete protease inhibitor cocktail (Roche 11873580001), 5 mM sodium butyrate, 0.6 mM spermidine, 0.2 mM spermine, 0.5% NP-40, and 0 .6mM β-mercaptoethanol. The suspension was passed over a modified “mini-INTACT” homogenizer, set at 1000 rpm, ten to 12 times. The Teflon homogenizer was modified such that the grooves at the bottom of the homogenizer helped push fly heads upward increasing the efficiency of homogenization and preventing sample loss  as per manufacturer’s instructions. In brief, nuclei were fixed in 1% formaldehyde for 2 min, immediately quenched with glycine and then lysed using nuclear lysis buffer with protease inhibitor cocktail at room temperature for 5 min. PBS was added to dilute the lysate-bead mix and loaded in AFA tubes (Covaris Inc. 520045) for sonication. Ultra-sonicator (Covaris Inc. E220) was used to sheer chromatin to ~ 200 bp length, and chromatin was recovered from the supernatant after magnetic separation. ChIP was performed using the following ChIP-seq grade antibodies: H3K4me3 , H3K9me3 , H3K9/K14 ac , H3K27me3 , H3K27ac , and H3K36me3 . Two biological replicates were performed for each histone mark, and input DNA was used as the control. Libraries for sequencing were prepared using MicroPlex Library Preparation kit (Diagenode C05010012) as per manufacturer’s instruction. Single-end 60 bp sequencing reads were obtained using Illumina Hi-seq 2500.TH-GAL4 with a stock carrying pJFRC105-10XUAS-IVS-nlstdTomato in VK40  and pJFRC29-10XUAS-IVS-myr::GFP-p10 in AttP40 [We isolated cell bodies of dopaminergic neurons using Fluorescence Activated Cell Sorting (FACS) during the flies’ morning activity peak. The protocol was essentially as described  with minn AttP40 . To accoDrosophila activity monitors  were kept in incubators at 25 °C with 65% relative humidity in a 12/12-h light/dark cycle. Flies were allowed one night to acclimatize to the apparatus, and activity data was collected in 1 min bins for the following 24 h as described [Flies that were previously socially isolated or group housed for 4 days were anesthetized briefly with carbon dioxide and transferred into 5 mm × 65 mm transparent plastic tubes with standard cornmeal dextrose agar media. For recording locomotor activity, escribed . One sleescribed , 109. Stescribed .Drosophila melanogaster genome [Drosophila genome release 6.02 and are included in Additional file All genomic procedures used release 6.02 of the r genome . R 3.0.3r genome . Non-parr genome  was usedr genome , follower genome . We crosr genome  and FCROr genome  packagesr genome  was usedr genome  was usedr genome  and ngs.r genome  were useK-means clustering [k-means package in R. To understand the impact of social isolation on epigenetics of genes expressed in dopaminergic neurons, a dataset of the top 40% of genes by mRNA TPM expression (5372 genes) was constructed containing normalized differences between group-housed and isolated flies for mRNA and for the six epigenetic marks. Tests using an information criterion approach (BIC) were used to determine the optimal numbers of clusters, which was k = 8 for the 5372-gene dataset. K-means clustering is a stochastic process that may yield very different results each time it is run if there is no strong pattern in the data. To determine the robustness of the gene assignments to clusters, we re-ran clustering with random seed changes to create N cluster assignments. We then compared each cluster assignment to every other (1035 = N × (N − 1)/2 comparisons for eight-cluster assignments). In each comparison, we calculated the percent overlap of a cluster in assignment i with clusters in assignment j, and reported the maximum percent overlap for that cluster. We therefore generated 8280 = 8 × 1035 comparisons. Additional file Gene Ontology (GO) analysis was done using two web tools: DAVID  and GOriustering  was doneCluster functional enrichment was determined using the DAVID 6.8 functional annotation tool  using bip-value was < 1 × 10−10. We filtered TFs by a fold change of more than 33%, which was the median fold change for the top 40% of expressed genes.We used the MEME suite of tools  to find To quantify the number of potential binding sites of each enriched motif in each gene, we used FIMO version 4  with defAdditional file 1:Design of homogenizer used in mini-INTACT. Diagram illustrating details of the homogenizer used in the mini-INTACT protocol. (PDF 637 kb)Additional file 3:Drosophila brain. The INTACT transgene (unc84-2XGFP) was driven in dopaminergic neurons (TH-GAL4) using different copy numbers of the UAS promoter and expression of GFP was compared using the same imaging settings. (A) 3X-UAS- (B) 5X-UAS- and (C) 10X-UAS-unc84-2XGFP. The 3X-UAS-unc84-2XGFP transgene most faithfully reproduced TH-GAL4 expression, while ectopic expression was observed upon further increases of the UAS copy numbers. Dopaminergic neurons were stained with anti-TH antibodies (red), INTACT transgene expression using anti-GFP antibodies (green), and N-cadherin (blue) was used as reference. See Fig. 3X-UAS-unc84-2XGFP brain imaged at higher intensity. Scale bar is 20 μm. (PDF 5243 kb)Comparison of tagged GFP expression in adult Additional file 4:3XUAS-unc84-2XGFP expression in dopaminergic neurons did not affect daytime sleep or activity over 24 h. (A) Daytime sleep measured over a 12 h period. GH males slept more than SH males during the daytime. No significant difference was observed due to tagged-GFP expression. (B) Total number of activity counts (beam breaks) over 24 h. GH are less active than SH flies as expected. No significant difference was observed due to tagged-GFP expression. N = 31–32. Unpaired t-test. (PDF 438 kb)Additional file 5:Purity assessment of dopaminergic nuclei. The table shows the number of captured green dopaminergic nuclei using bead-bound anti-GFP antibodies. Most of the contaminating red nuclei were washed away from bead-bound affinity-purified nuclei. See Fig. Additional file 6:Replicate concordance for ChIP-seq for various histone modifications. ChIP-seq replicate concordance is shown with Pearson’s correlation coefficient  calculated on Log (1 + ngs.plot) enrichment values for all six histone marks. (PDF 7315 kb)Additional file 7:Differentially marked Islands called by SICER. Table of islands called as differentially marked by SICER, with genomic location, mark levels, and mark type. (XLS 3107 kb)Additional file 8:Diffreps results. Table of regions with significant mark island differences between GH and SH males, as reported by Diffreps. (XLS 665 kb)Additional file 9:Differentially Expressed Genes. Contains TPM values for each replicate for genes called as differentially expressed by one of edgeR, CyberT, or FCros. (XLS 1458 kb)Additional file 10:Gorilla and DAVID functional analysis. The zip file contains top level html files which may be opened in a browser. These will give the Gorilla functional analysis and DAVID GO analyses referred to in the main text. (ZIP 919 kb)Additional file 11:k-means cluster overlap. The figure shows a histogram of k-means cluster overlap percentages used to calculate robustness of gene assignments to clusters. For eight clusters, the median percent overlap of a cluster in one assignment to its best match in a second assignment was 94%, and was greater than 99% 72% of the time  of genes with putative binding motifs of the TF in column 2, in each of the 8 k-means clusters (column 1). (TXT 1 kb)Additional file 13:ARG gene fold changes. Column B gives fold changes (FC) between Group and Single housed in our experiments, for 9 genes labeled ARG in Croset et al. . Additional file 15:Genes activated by cbt. Columns C-E and F-H are from Bartok et al. 2015. C-E are transcript levels in controls, F-H are transcript levels in cbt-RNAi. Columns I-O show mean normalized log2 FC between GH and SH in our experiments. Worksheet 2 gives statistics and a bar chart of the data for comparisons between repressed and activated by Cbt gene groups, for mRNA and for the 6 epigenetic marks. (XLS 145 kb)Additional file 16:Multiple regressions on TF putative binding sites. Gives details  of multiple regressions of the change in transcription for genes in the named group, based on independent variables the number of putative TF binding sites for the named TFs. (TXT 3 kb)Additional file 17:TH-GAL4 and UAS-RNAi on Δ sleep. See detailed comments at beginning of file. (TXT 6 kb)ANOVA results for RNAi knockdown effects on sleep. Results of performing Type III ANOVA to detect the interaction effect of Additional file 18:Hr38, sr, CrebA in dopaminergic neurons significantly reduced this difference. RNAi hairpins against candidate genes were present in attP2 and attP40 sites respectively, driven with TH-GAL4. Corresponding background controls were without RNAi hairpin, but with the attP2 or attP40 inserts, driven with TH-GAL4 (see the “UAS-RNAi vs. corresponding controls driven by TH-GAL4. (C) UAS-CrebA (2)-RNAi vs. control. ****, p < 0.0001, GH . (D) UAS-ARG-RNAi vs. control. **, p = 0.0059, Hr38-GH; ****, p < 0.0001, sr-GH; *, p = 0.0187, CrebA(1)-GH; *,p = 0.0378, Hr38-SH; *, p = 0.0103 . (E) UAS-cbt-RNAi vs. control, not significant; UAS-Brms1-RNAi vs. control, GH, ****, p < 0.0001; SH, p = 0.0121 . (PDF 1625 kb)Sleep over 24 h for UAS-ARG-RNAi and controls. (A) and (B) shows sleep per 30 min over 24 h associated with main Fig. see the “” sectionAdditional file 19:Brms1 by RNAi reduced social effects on daytime sleep. Brms1 is a member of Sin3A histone deacetylase complex. Knockdown of Brms1 in dopaminergic neurons was achieved by driving an RNAi transgene with TH-GAL4; controls carried empty vectors without RNAi hairpin and TH-GAL4. (A) Sleep per 30 min over 24 h for control and Brms1 knockdown in SH and GH flies. Daytime sleep is highlighted in shaded gray area for both genotypes. (B) Expressing RNAi for Brms1 in dopaminergic neurons reduced the social effect of sleep during the day (ΔSleep). Error bars are mean ± SEM. (PDF 743 kb)Knockdown of epigenetic eraser Additional file 20:Drosophila genome 6.02. Minor changes were made in Diffreps and ngs.plot local copies to add D. melanogaster genome release 6.02 to the set of genomes. (ZIP 6257 kb)Changes for Diffreps and Ngs.plot for


Genetic loci are non-randomly arranged in the nucleus of the cell. This order, which is important to overall genome expression and stability, is maintained by a growing number of factors including the nuclear envelope, various genetic elements and dedicated protein complexes. Here, we review evidence supporting roles for non-coding RNAs (ncRNAs) in the regulation of spatial genome organization and its impact on gene expression and cell survival. Specifically, we discuss how ncRNAs from single-copy and repetitive DNA loci contribute to spatial genome organization by impacting perinuclear chromosome tethering, major nuclear compartments, chromatin looping, and various chromosomal structures. Overall, our analysis of the literature highlights central functions for ncRNAs and their transcription in the modulation of spatial genome organization with connections to human health and disease. Spatial genome organization involves the 3D structure, positioning, and interactions of chromatin within the nucleus. This is a non-random process that is characterized by the regulation of various nuclear domains, topological associations, and epigenetic signatures. For example, decondensed euchromatin domains, which include active enhancer elements and are generally conducive to transcription, are found preferentially within the nuclear interior. On the other hand, heterochromatin domains are densely packed chromosome regions that are occupied by gene-silencing histone marks, which include histone H3 methylated on Lysine 27 (H3K27me3) or Lysine 9 (H3K9me2 and H3K9me3) . Such heIn fact, the nuclear genome is generally arranged within several cytologically distinct compartments. In addition to the prominent nucleolus, other nuclear compartments include the Cajal bodies, speckles, paraspeckles, and histone locus bodies. Nuclear compartments generally form via dynamic self-organization of their different constituents at sites of gene clusters . For exaDrosophila  , which cosophila , mice  which binds DNA in a sequence-specific manner . CohesinDrosophila cells, NL disruption alters LAD composition such that there is more histone H3 acetylated on Lysine 9 (H3K9Ac) and less chromatin compaction  proteins and nuclear pore complexes (NPCs) and is lined by the nuclear lamina (NL), which is a meshwork of lamin and lamin-associated proteins. The nuclear lamins are important regulators of chromatin organization . Genes tmpaction . Furthermpaction . In addimpaction . For exampaction .cis) or elsewhere in the genome (trans). Here we review ncRNAs that emerge from single-copy DNA loci or repetitive DNA loci and have diverse roles in spatial genome organization, thus impacting gene expression and stability. Collectively, ncRNAs impact spatial genome organization by modulating perinuclear chromosome tethering, the formation of major nuclear compartments, chromatin looping and various chromosomal structures. These roles of ncRNAs often intersect with various other regulators of genome structure and function.In addition to nuclear compartments, TADs/LADs, the nuclear envelope and their associated protein complexes, non-coding RNAs (ncRNAs) have emerged as major regulators of spatial genome organization. ncRNAs are RNA molecules that are not translated into proteins. ncRNAs are categorized based on their size – long (>200 bp) and short (<200 bp) – and are implicated in numerous cellular processes including transcription, mRNA splicing, and protein translation . ncRNAs Kcnq1 genes. In this section we discuss roles for ncRNAs and their transcription in the control of spatial gene positioning, chromatin remodeling and nuclear compartmentalization.Single copy loci include genes required for cell function and survival and can give rise to ncRNAs that regulate higher order chromatin structure and positioning . ncRNAs Non-coding RNAs can impact the structure and function of nuclear compartments such as Cajal bodies. The latter are involved in various processes including telomerase biogenesis, 3′-end processing of histone pre-mRNAs, as well as the processing, assembly and maturation of spliceosomal small nuclear ribonucleoproteins (snRNPs) . Cajal bNEAT1 gene (nuclear enriched abundant transcript 1), which is transcribed into the long ncRNAs (lncRNAs) NEAT1_1 (Men ε) and NEAT1_2 (Men β)  , which is involved in RNA editing  . These l (Men β) . Transcrh (SFPQ) . In cont editing .NEAT1 can also regulate the subnuclear localization of growth control genes by associating with Polycomb 2 protein (Pc2), a key subunit of the chromatin-repressive PRC1 complex (TUG1 (Taurine up-regulated 1) and NEAT1, which are found in two distinct nuclear bodies. Methylated Pc2 preferentially interacts with the TUG1 ncRNA within the transcriptionally repressive Polycomb nuclear bodies, thereby silencing the Pc2-associated growth control genes. On the other hand, demethylation of Pc2 results in its preferential interaction with NEAT1, which relocates Pc2 together with its associated growth control genes to inter-chromosomal granules within which the genes can be actively transcribed. complex . MethylaNEAT1 is commonly induced upon viral infection and can regulate the transcriptional activation of various antiviral genes , which can bring an enhancer and promoter in close proximity by mediating the formation of a DNA loop, and associate with mediator complexes to drive the expression of target genes  Kim et . For exaArabidopsis thaliana, transcription of the ncRNA APOLO forms a chromatin loop encompassing the promoter of its neighboring gene, PID , limits the access of Pol II to the PID promoter, thereby regulating the transcriptional activity of this gene  in order to bring the VH, DH and JH genes in close proximity, which in turn allows the genetic rearrangements to occur J recombination and its role in B cell development.Intergenic transcription-driven chromatin looping is also implicated in lymphocyte development. In developing B cells, V(D)J recombination is required for the assembly of antigen receptors . Importato occur . Prior tentation . This inentation , which aentation . Antisengh locus . The resBCL11B (ThymoD (thymocyte differentiation factor), which mediates DNA demethylation at CTCF binding sites and subsequent activation of CTCF/cohesin-dependent chromatin looping.In developing T cells, expression of BCL11B (BAF Chromatin Remodeling Complex Subunit BCL11B) promotes expression of T-lineage-specific genes and suppresses expression of the genes associated with alternative cell fates . ActivatBCL11B  (Isoda eBCL11B . ImportaXist (X inactive specific transcript), a 17 kb lncRNA that mediates inactivation of one of the X chromosomes during early female embryonic development  is another lncRNA that is transcribed from a locus on the X chromosome . The genes are separated into distinct topological compartments based on their transcriptional profile, and during development, there exists a dynamic switch between these topological domains  is transcribed from the 5′ edge of the HOXA locus and is required for maintaining the compartmentalization of the locus. HOTTIP can associate with and target the WD repeat mixed lineage leukemia (WDR-MLL) complex across the HOXA locus to yield active histone marks. This in return maintains the active state of some of the HOXA genes. HOTTIP also physically associates with CTCF, which can bind to six conserved binding sites at HOXA and serve as an insulator  that is transcribed from the intronic region of the Kcnq1 locus of one of the parental chromosomes. Deletion of Kcnq1ot1 results in expression of the parental allele that is normally silent  as well as microsatellite repeats (telomeres). Interspersed repeats include transposable elements that are either retrotransposons or DNA transposons. Retrotransposons include LTR-retrotransposons such as HERV and non-LTR retrotransposons such as SINEs , LINEs  or SVAs. It is also important to note that some types of repeats such as human ribosomal DNA (rDNA) can be arranged in tandem repeats that are interspersed throughout the linear genome. Regardless of their relative genomic location, DNA repeats are often clustered within nuclear space. This can facilitate their transcriptional co-regulation, minimize their potential deleterious interaction with the rest of the genome and control their exposure to potentially genome-destabilizing DNA recombination and repair machineries.Repetitive DNA sequences are non-randomly arranged within the nucleus. For example, rDNA repeats are physically sequestered in the nucleolar compartment of the nucleus. This sequestration can be driven by inter- or intra-chromosomal interactions, or even direct tethering to the nuclear envelope in some organisms . Telomercis through various processes. Future studies should explore how such ncRNAs are induced under different environmental conditions.Non-coding RNAs can play a role in the spatial organization and function of rDNA through the modulation of heterochromatin formation. Transcription of rDNA into ribosomal RNA (rRNA) molecules is dependent on the varying demand for protein synthesis that cells experience in response to intracellular signals or environmental stimuli. Therefore, despite the existence of 100s of rDNA repeats in eukaryotes, only a fraction of rDNA units are transcribed, while the remainder of the repeats is epigenetically silenced. Transcriptionally active rDNA units are marked by DNA hypomethylation, H4Ac and H3K4me2, whereas inactive rDNA units are marked by promoter hypermethylation, histone H4 hypoacetylation and methylation of H3K9, H3K27, and H4K20 . DepositThe nucleolus typically exhibits a phase separation-driven tripartite organization into a fibrillar centre (FC), dense fibrillar component (DFC), and granular component (GC) . Upon exThe organization of rDNA repeat regions into epigenetically silent chromatin structures is essential to proper cellular function and alterations in this organization may be associated with human disease. For example, rDNA hypermethylation is characteristic of early Alzheimer’s disease , upregulThe telomeres at the end of linear chromosomes are often heterochromatic. In vertebrates, telomeres are composed of hexameric 5′-TTAGGG-3′ repeats that are flanked by repetitive subtelomeric regions. Telomeric and subtelomeric repeats exhibit heterochromatic marks . Loss of heterochromatin disrupts telomere length control, increases telomeric recombination and promotes premature cellular senescence . InteresConnections exist between telomere malfunction and disease. The aberrant loss of telomeric heterochromatin can trigger telomeric DNA damage responses and recombination events, which are associated with several diseases . The accIAP and LINE-1 recruit Suv4-20h2 to mediate H4K20me3 enrichment and condense chromatin at transposable elements  become enriched in the nucleolus, where they interact with the nucleolin (NCL) protein and contribute to the maintenance of nucleolar structure and function (aluRNAs, and for the related B1 transcripts in mice. Interestingly, aluRNAs can somehow target other genomic loci to the nucleolus (In addition to regulating chromatin compaction at transposable elements, transposon-associated ncRNAs can modulate the spatial organization of the nucleolus. For example, in HeLa cells, transcripts originating from intronic function  (Caudronfunction . Similarucleolus , tentaticis-acting signals that modify gene expression (LINE-1 and SVAs are causative agents in numerous other human diseases (Given the high mutagenic potential of transposable element activity, it is perhaps not surprising that these elements have been linked to disease . Transpopression . It is epression . For exapression . In addidiseases . In factdiseases . Elevatediseases . In the Centromeres are tandem repeats, which are largely assembled into heterochromatic structures and are important for kinetochore function and chromosome integrity. Centromeres are composed of centric and pericentric regions, which have different chromatin structures that are epigenetically established. CENP-A-containing centric chromatin is characterized by H3K4me3, while pericentric regions are enriched in H3K9me2, H3K9me3, H4K20, and HP1. Heterochromatin formation at centric and pericentric regions is mediated by NoRC, similar to heterochromatin formation at rDNA . In factDrosophila, centromeric SAT III ncRNAs act as a structural component of the kinetochore and are required for the recruitment of centromeric proteins (X. laevis egg extracts, loss of α-satellite ncRNAs reduces centromeric localization of the kinetochore protein Aurora-B and causes improper spindle attachment and chromosome misalignment (X. laevis suggest that centromeric ncRNAs stabilize CENP-C binding to DNA (Several classes of centromeric ncRNAs have been found to play a role in the establishment of centromeric heterochromatin and kinetochore function across a wide range of species. Importantly, centromeric heterochromatin is maintained by low levels of satellite repeat RNAs . In fissproteins . In miceproteins . Murine proteins , suggestproteins . Reducinproteins , 2012. Ilignment . Additiog to DNA . Murine g to DNA . MoreoveWhile mammalian centromeres can often co-localize with nucleoli in S phase cells, budding yeast centromeres cluster with each other at the spindle pole body, which is opposite the nucleolus . ImportaChanges to the epigenetic state of centromeres has been linked to disease . TandemlTaken together, the literature reveals numerous intersections between various types of ncRNAs and spatial genome organization in the modulation of repetitive DNA loci and their broader impact on the genome and health.In this review we have highlighted roles of ncRNAs and intergenic transcription from single copy and repetitive DNA loci in the regulation of spatial genome organization. Several ncRNAs participate in spatial genome organization through several common mechanisms of action, such as chromatin looping and heterochromatin formation, while others operate through distinct pathways such as perinuclear tethering. Deregulation of spatial genome organization is associated with developmental and age-related diseases including cancer. Although aberrant expression of ncRNAs has been implicated in disease, more direct or causal links between such ncRNAs, spatial genome organization and pathobiology should be established . Future NK and LO wrote the manuscript and prepared the figures. The manuscript was edited and updated by KM.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.


Rhodobacter sphaeroides has two sets of flagellar genes, fla1 and fla2, that are responsible for the synthesis of two different flagellar structures. The expression of the fla2 genes is under control of CtrA. In several α-proteobacteria CtrA is also required for the expression of the flagellar genes, but the architecture of CtrA-dependent promoters has only been studied in detail in Caulobacter crescentus. In many cases the expression of fla genes originates from divergent promoters located a few base pairs apart, suggesting a particular arrangement of the cis-acting sites.R. sphaeroides fla2 genes and analyzed in detail two regions containing the divergent promoters flgB2p-fliI2p, and fliL2p-fliF2p. Binding sites for CtrA of these promoters were identified in silico and tested by site directed mutagenesis. We conclude that each one of these promoter regions has a particular arrangement, either a single CtrA binding site for activation of fliL2p and fliF2p, or two independent sites for activation of flgB2p and fliI2p. ChIP experiments confirmed that CtrA binds to the control region containing the flgB2 and fliI2 promoters, supporting the notion that CtrA directly controls the expression of the fla2 genes. The flgB and fliI genes are syntenic and show a short intercistronic region in closely related bacterial species. We analyzed these regions and found that the arrangement of the CtrA binding sites varies considerably.Here we characterized several control regions of the fla2 divergent promoters showing that CtrA promotes transcriptional activation using more than a single architecture.The results in this work reveal the arrangement of the The online version of this article (10.1186/s12866-018-1264-y) contains supplementary material, which is available to authorized users. Escherichia coli and Salmonella enterica, the basal body includes the MS-, P- and L-rings. Flagellar biogenesis requires the expression of more than 40 genes, and the assembly of this structure proceeds outwardly from proximal to distal end [E. coli and S. enterica the transcriptional activator FlhD/FlhC is required to express the early flagellar genes (Class II) which encode the proteins required to form the basal body and the hook. The genes encoding the sigma factor FliA and its specific anti-sigma factor, FlgM also belong to this class. Once the basal body and the hook are assembled, FlgM is exported out of the cell and FliA associates with the core RNA polymerase to recognize class III promoters [Vibrio, Pseudomonas), the flagellar gene expression hierarchy shows a different architecture and special features. In several species the sigma factor RpoN together with an activator protein promote the expression of the early flagellar genes, and the late genes are dependent on FliA. In these systems the expression of late flagellar genes also requires the export of FlgM [Sinorhizobium meliloti two members of the LuxR family, VisN and VisR [rem, which encodes an OmpR-like transcriptional activator that promotes the expression of class II flagellar genes [flaA, flaB, and flaC as well as several genes encoding chemotactic receptors are directly activated by the conserved transcriptional regulator CtrA [S. meliloti is still unknown. In Caulobacter crescentus, the flagellar expression hierarchy has been thoroughly studied. In this case, CtrA activates the expression of the class II genes that encode the proteins of the MS ring and the flagellar export apparatus as well as the regulatory proteins RpoN and FlbD. Upon completion of the export apparatus and the MS-ring, FlbD is phosphorylated by an unknown kinase and together with the sigma factor RpoN promote transcription of class III/IV genes, whose products form the axial rod, the L- and P-rings, the hook and the flagellar filament [Many bacteria use rotating flagella for locomotion, the motor is embedded in the membrane and couples the influx of specific ions to the generation of rotational force. The bacterial flagellum can be divided in a basal body that includes the motor, a filament that acts as a propeller, and a universal joint also known as the hook which links the basal body and the filament. The basal body is the most complex structure that includes an axial rod and three or more rings, depending on the bacterial species. In stal end , 2. In mstal end –5. The f of FlgM . Only a and VisR , bring aar genes . The exptor CtrA . The mecfilament , 10.Rhodobacter sphaeroides is an α-proteobacterium with two flagellar systems of different phylogenetic origin [fla1 genes are expressed and direct the assembly of a single subpolar flagellum [fla1 genes of R. sphaeroides were acquired by horizontal transfer. In contrast, the gene products of the vertically inherited fla2 genes enable, under particular conditions, the synthesis of several polar flagella [fla1 genes follows a hierarchical expression pattern in which the early genes are expressed under the control of RpoN1 and the activator proteins FleQ and FleT whereas the late genes are dependent on FliA [fla2 genes is dependent on the two-component system (TCS) formed by the hybrid histidine kinase, CckA, the histidine phosphotransferase, ChpT, and the response regulator CtrA [−/Fla2+ phenotype [c origin . Under tlagellum . It has flagella , 13. The on FliA . In conttor CtrA . The sighenotype , 15, 16.The CckA/ChpT/CtrA TCS, is broadly distributed among α-proteobacteria. In some bacterial species this system is essential given that CtrA regulates the expression of genes that control the cell cycle; in contrast in many other bacteria, CtrA is not essential. In general, genes controlled by CtrA are enriched in certain functional categories, i.e. cell motility, signal transduction, and cell wall/membrane envelope biogenesis .C. crescentus the architecture of the CtrA promoters has been recently analyzed at genomic level by means of a global 5’ RACE protocol combined with a search for CtrA binding sites using a weight position matrix generated from ChIP experiments. From these studies it was observed that there are two classes of CtrA binding motifs, a full site represented by the sequence TTAA (N7) TTAA, and a half site (TTAA) [In e (TTAA) . Previoue (TTAA) . In addie (TTAA) . The rele (TTAA) .ctrA from Rickettsia prowazekii is partially functional in C. crescentus, and that ctrA from C. crescentus is functional in Sinorhizobium meliloti [For other bacteria, it was suggested that CtrA could have similar recognition sites based on the fact that related motifs have been identified upstream of genes controlled by CtrA , 21–24. meliloti , 26.In many species, the flagellar genes presumably controlled by CtrA are expressed as divergent transcriptional units, and in many cases, the control region encompasses less than 100 bp –29.fla2 promoters flgB2p-fliI2p and fliF2p-fliL2p. The architecture of these regulatory regions was characterized using 5’-RACE, site directed mutagenesis and chromatin immunoprecipitation (ChIP) assay. From these analyses we conclude that CtrA directly activates the fla2 promoters using more than a single architecture.In this work we analyzed two divergent promoter regions carrying the All plasmids and bacterial strains used in this work are listed in Table R. sphaeroides was grown in Sistrom’s minimal medium [Escherichia coli was grown in LB medium [R. sphaeroides, kanamycin (25 μg/ml), tetracycline (1 μg/ml), spectinomycin (50 μg/ml). For E. coli, kanamycin (50 μg/ml), tetracycline (12 μg/ml) ampicillin (100 μg/ml), spectinomycin (50 μg/ml), chloramphenicol (20 μg/ml), gentamycin (30 μg/ml).l medium . When inB medium  at 37 °CRoutine genetic manipulations were performed as described elsewhere . RestricflgB2 and fliI2 as well as the 5′ region of each gene was replaced with the omega-SpcR cartridge. For this a chromosomal region of 2250 bp was amplified using the oligonucleotides DinterB2For and DinterI2Rev. The PCR product encompassing from the end of flgC2 to the end of fliI2, was cloned in pCR2.1-TOPO, digested with StuI and religated. The StuI digestion allowed the removal of two internal fragments, one of 392 and the other of 126 bp. The resulting linearized plasmid was self-joined to bring together the C-terminal half of flgB2 with the C-terminal half of fliI2. This plasmid was purified, digested with StuI and ligated with the omega-SpcR cartridge obtained from the pBOR plasmid. The complete fragment was then subcloned into pJQ200mp18 [E. coli S17–1 and subsequently transferred to R. sphaeroides by conjugation [R. sphaeroides, the double-recombination event was selected on LB agar plates in the presence of spectinomycin and 5% sucrose. The mutant was verified by PCR.The intercistronic region between Q200mp18 . This pljugation . Since pfla2 promoters for the genes flgB2, fliI2, fliF2, fliL2 and flgE2 was amplified by PCR using the oligonucleotides indicated in Table uidA gene that encodes for the β-glucuronidase. In pRK415/uidA, the uidA-aadA cassette was cloned in the BamHI site, the orientation of the fragment was selected in such a manner that the uidA gene can be transcribed from a promoter cloned as an EcoRI-SstI fragment.The regulatory region of the Cell-free extracts from exponential phase cultures grown photoheterotrophically were tested for β-glucuronidase activity following the previously reported protocol , 35. As flgB2p, fliI2p, fliL2p, and fliI2p were used as templates. The presence of the mutation was verified by sequencing.Mutagenesis was performed essentially following the method of Kunkel  with an flgB2, fliI2, fliF2, fliL2, and flgE2, the primers shown in Table Bacterial cells from cultures grown to mid-log phase in 0.2% casamino acids, were collected at 4 °C and immediately used to extract the complete pool of RNA using the RiboPure-Bacteria kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Residual DNA in the samples was removed using DNaseI (Roche). To determine the 5′ end of the mRNA transcripts of ctrA was amplified using the oligonucleotides ctrABADFwSac and ctrABADRvEco. The amplification product was cloned into pBAD/HisA (Invitrogen). This construction was introduced into Rosetta and a culture of this strain grown at mid-log phase was induced with 0.2% L-arabinose for 4 h at 37 °C. Cells were collected by centrifugation and resuspended in 1/100 of the original volume in phosphate buffered saline (PBS) 0.058 M Na2HPO4, 0.017 M NaH2PO4, 0.068 M NaCl, pH 7.4. The cell suspension was sonicated in an ice bath for five bursts of 10 s. Cell debris were removed by centrifugation and the supernatant was mixed with Ni-NTA-agarose beads (Quiagen) and incubated for 1 h on ice in the presence of 20 mM imidazole. The beads were loaded into a polypropylene column (1 ml of capacity) and washed with PBS/30 mM imidazole/200 mM NaCl. The protein was eluted using PBS containing 250 mM imidazole.Six three weeks old BALB/C female mice, were immunized intraperitoneally with 20 μg of His6X-CtrA protein (for each mice) in incomplete Freund’s adjuvant, and 3 weeks later reimmunized. Two weeks later the mice were bled and antisera were obtained by low-speed centrifugation. The specificity of the anti-CtrA antibodies was tested by immunoblotting following standard protocols ; in thisR. sphaeroides, grown in Sistrom’s medium with 0.2% casamino acids, were transferred to a 125 ml Erlenmeyer flask when the OD600 reached 0.5. Immediately, 0.41 ml of 37% of freshly prepared formaldehyde was added and incubated for 10 min at 30 °C with slow shaking. After this time, the flasks were incubated on ice for 30 min. The cells were washed three times with PBS buffer and then resuspended in 250 μl of PBS with protease inhibitors . The sample was incubated at room temperature with lysozyme (80 μg/ml) for 15 min; after this time, 2.5 μl of Triton X-100 and 5 μl of EDTA 50 mM were added, and carefully mixed. After 10 min, the sample was sonicated 10 times for 8 s (30% duty cycle) on ice. Cell debris were removed by centrifugation at 10,000 x g for 5 min. The supernatant was transferred to a clean tube containing 100 μl of a mixture of Protein A Sepharose/DNA/BSA (100 μg of sonified herring sperm DNA/100 μg of BSA). The sample was incubated at 4 °C on a rocking platform shaker, for 2 h, and centrifuged for 3 min at 550 x g. The supernatant was transferred to a clean tube, and an aliquot of 20 μl was withdrawn and labeled as input. The remaining sample was mixed with 4 μl of anti-CtrA antibody and incubated overnight at 4 °C on a rocking platform shaker. The complexes were captured by adding 100 μl of Protein A-Sepharose/DNA/BSA and continuing incubation for 2 h. The immunoprecipitated material was washed twice in low, and high salt buffers , twice in LiCl buffer  and twice in TE buffer . The sample was resuspended in 500 μl of elution buffer  and incubated overnight at 65 °C to reverse the cross-linking. Input samples were also incubated overnight at 65 °C in elution buffer. The samples were treated for 30 min with 50 μg of RNase for 30 min at 37 °C, and subsequently with 120 μg of proteinase K for 30 min. DNA was purified using the QIAquick PCR purification kit. The procedure was carried out three times for AR1 and EA1 strains carrying the plasmid pRK_flgB2p::uidA-aadA; and twice for AR1 carrying pRK_flgB2p − 10::uidA-aadA or pRK_flgB2p − 35::uidA-aadA.ChIP was carried out according to the protocol previously reported  with minflgB2-fliI2 region in the ChIP samples, we carried out a PCR reaction and evaluated the amount of product at different cycles of the reaction. The DNA products were analyzed by acrylamide gel electrophoresis and quantified using Image J software [To assess the presence of the software . The datfla2 genes was analyzed using a position weight matrix (PWM) built from the regulatory region of 54 genes identified to be controlled by CtrA in C. crescentus [http://embnet.ccg.unam.mx/rsat). Sequence alignments were carried out using MUSCLE [The DNA sequence containing the escentus  with theg MUSCLE .R. sphaeroides WS8N genome consists of two chromosomes and two plasmids. Most of the fla2 genes are clustered in a region of approximately 32.3 Kb localized in chromosome I . Eleven hits mapped within coding regions, suggesting a high rate of false negatives.The fliL2-fliF2, and flgB2-fliI2 in a plasmid that carries the reporter gene uidA (pRK415/uidA), in order to create a transcriptional fusion between these promoters and the uidA gene, which encodes for the enzyme β-glucuronidase. To test the activity of the promoters fliL2p, fliF2p, flgB2p and fliI2p, these regions were cloned in both orientations regarding uidA. These constructions were introduced to the AM1 mutant strain (for simplicity this strain will be named as wild type from now on) and its ΔctrA derivative (EA1 strain). The amount of β-glucuronidase produced by each strain was determined.To evaluate the functional relevance of some of the identified sites, we cloned the intercistronic regions located between ctrA) strain  strain, but not in the EA1  and EA1 strains. A high level of β-glucuronidase was detected in AM1 but not in EA1 (data not shown).To validate our plasmid system, we cloned the fliL2p, fliF2p, flgB2p, fliI2p and flgE2p, we proceeded to determine the transcriptional start site (TSS) by 5’-RACE. The TSS was established by sequencing five independent clones from each sample. For these promoters, the initiation nucleotide was always a purine . For fliI2p, the algorithm predicted one CtrA-binding site (score 7.1), which starts upstream of the sequence shown in Fig. As can be observed in Fig. flgB2p, fliI2p, fliF2p and fliL2p such as the putative CtrA binding site or the putative − 10 promoter region, we proceeded to mutagenize the nucleotides shown in Fig. To gain insight on the role of the sequences identified from the alignments of flgB2p introducing additional changes along the regulatory region . These results lead us to the conclusion that the functional site for CtrA binding must be the sequences highlighted in yellow in Fig. For ter Fig. , indicatflgB2 and fliI2 are transcribed from divergent promoters as well as fliF2 and fliL2; nonetheless, from the above results we realized that the architecture of these promoter regions is different. Figure flgF2p and fliL2p, whereas flgB2p and fliI2p are activated from independent CtrA binding sites.As mentioned before, fla2 promoters by gel electrophoresis mobility shift assay (EMSA) and DNAse I footprinting were unsuccessful; therefore, we decided to undertake an in vivo approach using chromatin immunoprecipitation (ChIP) in order to reveal the binding of CtrA to the fla2 promoters. For these experiments we used the plasmid pRK415/uidA carrying flgB2p in the AR1 strain, in which the chromosomal region corresponding to flgB2p and fliI2p was deleted.Attempts to detect binding of His6X-CtrA to different flgB2p immunoprecipited by α-CtrA antibodies was detected by semi-quantitative PCR. A strong increase of the PCR product was observed in the sample obtained from AR1 as compared with that obtained from EA1 (ΔctrA), indicating that CtrA binds to this region , there are two independent CtrA-binding sites in this region. This is supported by the fact that site-directed mutagenesis of the CtrA binding site of flgB2p  do not conform to the consensus that has been previously reported for other bacteria, given that for flgB2p and fliI2p, the left and right elements of the CtrA binding site are located only 6 bp apart. As explained in the results section, for flgB2p, the other possible sites for CtrA binding were unlikely to be functional, given that one overlapped with the TSS, and the other showed a very low score. For fliI2p the best CtrA binding site that was identified bioinformatically was not supported by the experiments reported in this work. Therefore, we presume that for these promoters CtrA is able to activate transcription from atypical binding sites. A possible scenario could be that a very short intercistronic region with divergent promoters could be particularly affected by negative supercoiling (undertwisting) of the DNA. This conformation would be due to the activity of the divergent transcription from these promoters, enabling a better recognition of the CtrA binding site regardless of the fact that the left and right elements are positioned only 6 bp apart. A similar spacing between the left and right TTAA elements of the CtrA binding site has been described for Magnetospirillum magneticum [ctrA binding sites of the ctrA P1 and P2 promoters of C. crescentus. In this case the P1 promoter that is negatively controlled by CtrA, could have the ungapped variant for CtrA binding; however, for the CtrA-activated P2 promoter, the contribution of the left and right elements spaced by 6 bp was verified by an electrophoretic mobility shift assays [It should be stressed that the proposed CtrA-binding sites for these promoters  are encoded by genes that are dependent on RpoN and the activator protein FlbD [R. sphaeroides none of its four different RpoN proteins [fla2 genes (unpublished results). Moreover, the gene encoding FlbD is absent in the species of Rhodobacteraceae shown in Fig. R. sphaeroides and other species of this genus, the early constituents of the flagellar structure including the periplasmic components are encoded by genes that could be directly activated by CtrA.In his site . This diein FlbD . In R. sproteins  is involfla2 genes of R. sphaeroides. The identification of the transcriptional start site for several fla2 promoters allowed us to identify several conserved features such as a conserved A at the − 11 position and a purine at the initiation start site. The CtrA binding sites were tested by site directed mutagenesis and it was found that CtrA activates the expression of the divergent promoters fliL2p-fliFp2 using a single binding site, whereas for the divergent promoters flgB2p-fliI2p CtrA activates transcription from two independent sites. In addition, we found that the CtrA binding site could also be functional when the left and right elements of the full motif are 6 bp apart.In this work we show that CtrA directly activates the expression of the Additional file 1:fla2 cluster of Rhodobacter sphaeroides using the PWM reported for Caulobacter crescentus. Hits found by RSAT in the fla2 cluster of R. sphaeroides. (XLSX 40 kb)CtrA-binding sites predicted in the


In vitro, exposure to the BMP2 and BMP4 ligands is sufficient to increase neurogenesis from AH-NSPCs in a WNT dependent manner while decreasing oligodendrogenesis. Moreover, BMP2/4 and WNT3A, a key regulator of adult hippocampal neurogenesis, cooperate to further enhance neuronal production. Our data point to a mechanistic convergence of the BMP and WNT pathways at the level of the T-cell factor/lymphoid enhancer factor gene Lef1. Altogether, we provide evidence that BMP signalling is an important regulator for the neuronal fate specification of AH-NSPCs cultures and we show that it significantly cooperates with the previously described master regulator of adult hippocampal neurogenesis, the WNT signalling pathway.Neuronal production from neural stem cells persists during adulthood in the subgranular zone of the hippocampal dentate gyrus. Extracellular signals provided by the hippocampal microenvironment regulate the neuronal fate commitment of the stem cell progeny. To date, the identity of those signals and their crosstalk has been only partially resolved. Here we show that adult rat hippocampal neural stem and progenitor cells (AH-NSPCs) express receptors for bone morphogenetic proteins (BMPs) and that the BMP/P-Smad pathway is active in AH-NSPCs undergoing differentiation towards the neuronal lineage.  The activation of quiescent neural stem cells to give rise to progenitor cells, their expansion, neuronal cell fate determination, migration and full differentiation are the main sequential phases required for proper adult neurogenesis. Local niche signals from the neural stem cell microenvironment dynamically regulate all these phases [reviewed in3]. Among these signals, short-range niche morphogens, including ligands from the WNT and Bone morphogenetic protein (BMP)/Growth and Differentiation Factor (GDF) families, are thought to play a prominent modulatory role.Neurogenesis in mammals persists throughout adult life in specific brain locations or “niches” due to the existence of reservoirs of largely quiescent neural stem cells (NSCs) that can generate new neurons through a series of intermediate progenitors. The two main niches in rodents are located in the subgranular zone (SGZ) of the hippocampal dentate gyrus and in the ventricular-subventricular zone or subependymal zone (SEZ) of the lateral ventricles  1:500 , Polyclonal Goat Anti-SOX2 1:300 , Rabbit monoclonal anti-Ki67 1:50 , Monoclonal Anti-MAP2 (2a + 2b) clone AP-20 1:300 , Monoclonal anti-ID1 (B-8) 1:100 , Cy3 Goat anti-Mouse 1:2000 , Cy3 Donkey anti-Rabbit 1:2000 , Alexa 488 Donkey anti-Rabbit 1:500 , Biotinylated Donkey Horse anti-Goat 1:200  and Cy5 Streptavidin 1:400 . The RIP monoclonal antibody developed by S. Hockfield was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa. DNA was stained with 4,6-diamidino-2-phenylindole (DAPI) 10 µg/ml (Sigma). Samples were processed with Dabco/Mowiol 1/20 (Sigma/Merck), and analysed with an ImageA1 Axio microscope (Carl Zeiss).−ΔΔCt method24. Primer sequences are available upon request.RNA was extracted from cells or 2-month old wild type (CD-1) mice using Illustra RNAspin Mini Kit  and measured with a Microplate Reader (Infinite M200 TECAN). cDNA was obtained by reverse-transcription (RT) employing PrimeScrip RT Reagent kit (Takara). Gene expression was determined by quantitative polymerase chain reaction (qPCR) in a LightCycler480 (Roche) using SYBR PremixEX Taq (2×) (Takara) and the corresponding forward and reverse primer for each gene, following the manufacturer protocol. Data were analysed according to the 2Cell extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane following the manufacturer’s protocol (Bio-Rad). Membranes were incubated with 5% nonfat milk or 3% BSA (Sigma) in TBST for 60 min. Primary and secondary antibodies used for the western-blot assays are as follows: Phospho-Smad1/Smad5/Smad8 antibody , Smad1/5/8 (N-18)-R antibody , Neuronal Class III β-Tubulin antibody , Glial Fibrillary Acidic Protein antibody , Goat Doublecortin (C-18) antibody , Phospho-LRP6 (Ser1490) antibody , β-actin antibody , Donkey anti-Rabbit ECL 1:50000 , Sheep anti-Mouse ECL 1:50000 , Donkey anti-Goat HRP 1:10000 .http://www.ebi.ac.uk/Tools/msa/clustalw2) using default settings and the alignment viewer AliView. The accession numbers of the BMP protein sequences analysed in mouse were as follows: BMP2 (NP_031579.2), BMP4 (AAH13459.1), BMP5 (NP_031581.2), BMP6 (NP_031582.1), BMP7 (NP_031583.2), BMP8A (NP_031584.1) and BMP8B (NP_031585.2). The 5 kb sequence upstream of the initial ATG start codon from the Rattus norvegicus Lef1 gene was retrieved (giІ389673387:821409826409), and promoter and transcriptional factor binding sites analysis were carried out using Genomatix Promoter Inspector and MatInspector tool (Genomatix Software Suite v3.0).Phylogenetic Tree distances between BMPs were calculated using CLUSTAL-W2 (2 (Biotools), 240 µM dNTP Mix (Invitrogen) and Lef1 promoter primers (sequences available upon request).AH-NSPC were treated with BMP4 (25 ng/ml) for 6 h. Proteins and DNA were crosslinked for 10 mins with formaldehyde 37% (Panreac) to a final concentration of 1%. Extracted material was sonicated using a UP50H Ultrasonic Processor (Hielscher), sonication conditions were as follow: 30% amplitude, 10 sec x20 pulses. Proteins were pulled down using 60 µl Protein A agarose/Salmon Sperm DNA (Millipore) and 10 µg of Smad4 Antibody (H-552) . ChIP Dilution Buffer, Low Salt Immune Complex Wash Buffer, Hight Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer, TE Buffer and SDS Lysis Buffer were prepared as described in Chromatin Immunoprecipitation (ChIP) Assay Kit (Upstate). Immunoprecipitated material was amplified by PCR using DNA polymerase (Biotools), 10× Standard Reaction Buffer with 2 mM MgClt test. All the values correspond to average ± sem, and those with a P value < 0.05 were considered significant . Arcsen transformation was carried out for the statistical analysis of percentages.The statistical significance of the difference between means for the kinetics experiments was assessed by one-way ANOVA, using the Tukey test as post-hoc comparison. To determine the significance of the BMP2/4 and WNT3A synergic effect we used two-way ANOVA analysis of the percentage of neurons. The significance between means of the remaining experiments was calculated using paired 2-tailed Student t (cycle threshold) values, we found that most BMP ligands and receptors are expressed at the mRNA level in the adult DG  using a combination of Retinoic Acid (RA) and Forskolin (FSK), a well-established treatment that facilitates the acquisition of the neuronal fate . After RNA extraction and cDNA synthesis, the gene expression pattern of the BMP type 1 receptors  and the BMP type 2 receptors  was determined by quantitative RT-PCR and referred to the housekeeping genes Sdha  and decreased expression of the immature intermediate filament gene Nestin (Nes) were used to confirm the differentiation of the cultures . Our data show that BMP2 and BMP4 induce a marked dose-dependent increase in the percentage of TuJ1+ neurons, while BMP7 has a very mild effect  compared to the control condition . Out of the SOX2+ cells, only 16.4 ± 3.7% were cycling (Ki67+) in the presence of BMP4, suggesting that most of the AH-NSPCs that did not engage in the differentiation programme stayed quiescent19.Since AH-NSPCs express different receptor combinations throughout differentiation, we next analysed whether BMP ligands with different affinities for those receptors influence neurogenesis. To this end, we cultured AH‐NSPCs in the absence of mitogenic stimulation but in the presence of an increasing concentration of several prototypic BMP ligands: BMP2 and BMP4 recombinant proteins, belonging to the Dpp subgroup that preferentially bind to BMPR1A; and BMP7 recombinant protein, belonging to the 60 A family that is best bound by BMPR1B and ACVR1ect Fig. . BMP2/4 2/4 Fig. . In addi2/4 Fig. , suggestBmpr1a is markedly expressed in undifferentiated AH‐NSPCs and decreases throughout the neuronal differentiation process19 . At BMP2 and WNT3A concentrations of 25 ng/ml, a marked raise in TuJ1+ cells was elicited with a 19-fold increase in the number of neurons when both ligands were present, relative to the 5- and 3.5-fold increases elicited by BMP2 or WNT3A treatments alone, respectively  concomitant with increasing concentrations of the canonical WNT signalling inhibitor XAV93927 to prevent endogenous canonical WNT signalling -binding sites in their promotersein Fig. .Figure 7Lef1 gene promoter region to check for the presence of conserved BMP regulatory elements. We searched the rat genome databases, retrieved the 5 kb sequence upstream of the initial Lef1 ATG start codon located in Chr2 and performed a theoretical analysis of the sequence using promoter inspector tools. We found a validated TATA-less promoter sequence spanning from −501 to +100 relative to the translational start site ATG codon at +1  and a second theoretical promoter region  within the first intron of the Lef1 gene  lacking a consensus TATA box and a second intronic promoter (P2) located downstream of exon 130. In human, the P1 promoter drives the expression of a full-length LEF1 polypeptide; the P2 promoter instead produces a truncated protein that lacks the beta-catenin binding domain and supresses WNT signalling30. From these two promoter sequences, we selected the upstream −501/+100 P1 promoter for further analysis and scanned the sequence for transcription factor binding sites (TFBS). Out of the 154 total TFBS sites mapped to the proximal upstream promoter region, two corresponded to canonical SMAD protein binding sites. The SMAD binding sites were located at position −302 (“5′-GTCT-3′ SBE” or SMAD4 binding element) and −261 (“GCCG-like motif” or GC-rich SMAD1/5 binding element) from the start codon, and were recognized as putative binding sites for P-SMAD/SMAD4 dimers  assay with anti-SMAD4 antibodies, followed by PCR analysis with primer pairs amplifying the region containing the putative SMAD binding sites  was overexpressed in AH-NSPCs. The dnBMPR1A, with a non-functional intracellular domain but a functional extracellular domain that traps the endogenously produced BMP ligands, increased astroglial differentiation of the cultures and this was likely mediated by the concomitant increase in the expression of BMPR1B in the cultures36.As for the contribution of the specific subtypes of BMP receptors, the gene expression patterns obtained suggest that BMPR1A receptor has an early role in the differentiation process, while BMPR1B and ACVR1 may possibly have a late role. The early role of BMPR1A is consistent with the results obtained in the differentiation assays employing the preferred BMPR1A ligands (BMP2/4) and with the retroviral experiments in which we overexpressed a constitutive active form of BMPR1A. Moreover, we found that the differentiation levels attained under an initial and transient exposure to BMP2/4 were similar to those obtained with a continuous exposure to the ligands, further indicating that the signalling inducing the neuronal fate occurs in the first stages of the differentiation process and thus is likely transduced by the most highly expressed type 1 receptor in those initial stages, BMPR1A. Our results partly complement a previous study10. We found that the BMP pro-neurogenic effect requires basal levels of endogenous WNT signalling and that the BMP and WNT pathway show a synergistic interaction. When combining the BMP and WNT ligands at non-saturating concentrations, the effect of the two taken together was greater than the sum of their separate effects at the same doses. In the light of these data, it seemed likely that the two signalling pathways would share components of the same mechanistic route.To gain insight into potential mechanisms underlying the BMP pro-neurogenic action uncovered in our assay, we examined its dependency on WNT signalling, a master regulator of adult hippocampal neurogenesis38. The TCF/LEF family of transcription factors mediate the downstream effects of canonical Wnt/β-catenin cascade and has four members in mammals, LEF1 being the most well characterized39. Given LEF1 is expressed in adult hippocampal neural stem cells and is required for neurogenesis40, and since the expression of Lef1 can be activated by BMP4 in BMP-mediated inductive tissue interactions28, we focused on the Lef1 gene as a possible convergence point between the BMP and the WNT routes. We identified functional BMP regulatory elements on the Lef1 promoter, we found that Lef1 expression was increased shortly after BMP exposure and we demonstrated that overexpressing Lef1 is sufficient to enhance neurogenesis, uncovering a novel mechanistic link between the BMP/WNT signalling pathways operating in adult hippocampal neural stem and progenitor cells. A similar mechanism could be active as well during the development of the hippocampal dentate gyrus, since it has been recently reported that Lef1 expression at embryonic stages is regulated by the BMP type 1 receptor ACVR141.The BMP and WNT pathways have been shown to interact with each other at multiple levels, for instance, via the binding of both SMAD4 and TCF/LEF transcription factors to the promoter and enhancer regions of certain target genes20 but possibly also the early stages of the differentiation cascade. At least in vitro, the pro-neurogenic canonical WNT signalling interplays with, and is subjected to regulation by, BMP signalling. This may be achieved by increasing the expression of the transcription factor LEF1. Although future in vivo studies are needed to further evaluate the effect of BMP signalling in fate specification, our in vitro model already shows that the role of BMPs/BMPRs during adult neurogenesis is much more complex than was previously thought.In summary, signalling through the BMP type 1 receptor BMPR1A plays a relevant role in the modulation of adult hippocampal neurogenesis, regulating not only NSC quiescenceSupplementary information


As the cost of high-throughput sequencing technologies decreases, genome-wide chromatin accessibility profiling methods such as the assay of transposase-accessible chromatin using sequencing (ATAC-seq) are employed widely, with data accumulating at an unprecedented rate. However, accurate inference of protein occupancy requires higher-resolution footprinting analysis where major hurdles exist, including the sequence bias of nucleases and the short-lived chromatin binding of many transcription factors (TFs) with consequent lack of footprints.Here we introduce an assay termed cross-link (XL)-DNase-seq, designed to capture chromatin interactions of dynamic TFs. Mild cross-linking improved the detection of DNase-based footprints of dynamic TFs but interfered with ATAC-based footprinting of the same TFs.XL-DNase-seq may help extract novel gene regulatory circuits involving previously undetectable TFs. The DNase-seq and ATAC-seq data generated in our systematic comparison of various cross-linking conditions also represent an unprecedented-scale resource derived from activated mouse macrophage-like cells which share many features of inflammatory macrophages.The online version of this article (10.1186/s13072-019-0277-6) contains supplementary material, which is available to authorized users. Understanding of tissue-specific transcriptional regulome requires the knowledge about DNA sequence-specific chromatin interactions of transcription factors which are active in the given cell context. ChIP-seq has been widely adopted and currently the most common method of profiling the regulatory landscape of a transcription factor . RelatedGenomic TF footprinting, an alternate method for identifying occupancy of a large number of TFs in one DNase-seq or ATAC-seq sample, had the potential to circumvent many of these issues with ChIP-based assays , 11, 23.However, serious limitations have dampened early enthusiasms for using the chromatin accessibility analysis methods to identify TF occupancy in an unbiased high-throughput manner. First, the enzymes used to probe chromatin (DNase I and Tn5) in these assays were found to have non-negligible DNA sequence preferences for their reaction, which complicates the assumption that these nucleases non-specifically sample accessible nucleotides , 17, 34.Another problem with genomic TF footprinting was the lack of sufficient protection of DNA from TFs whose chromatin binding is short-lived (referred to as “dynamic TFs” in this study) , 33, 34.We sought to systematically assess the effects of various cross-linking procedures on the genomic footprints of dynamic TFs in the same chromatin material. For a fixed source of chromatin, we chose a cell state in which numerous TFs are directly interacting with chromatin in a cascade of gene regulatory actions. Since the chromatin sample is prepared from a cell population containing snapshots of these dynamic interactions, we reasoned that this would be a rich platform to assess changes in footprint depths of many TFs simultaneously. To this end, immortalized mouse macrophage-like RAW264.7 cells were used, where many dynamic TFs, including NF-κB and AP-1, are activated in response to bacterial products such as lipopolysaccharide (LPS). This cell context allows a large number of TFs occupying the chromatin, thereby providing an ideal platform for assessing TF footprint characteristics. We chose this cell system also because of the rich information about TF regulatory networks that the new data will help uncover in a physiologically important innate immune cell type. RAW264.7 cells have chromatin profiles which are similar to those of primary macrophages (data not shown) , which aWith the same chromatin material from LPS-stimulated RAW264.7, we varied the duration and the concentration of the cross-linking agent formaldehyde to determine the cross-linking parameters which may affect footprinting characteristics of dynamic TFs Fig. . Based oWe have performed the modified DNase-seq, termed cross-link (XL)-DNase-seq and generated a panel of sequencing libraries. The enrichment, complexity, and quality of each library were confirmed, and all the libraries were subject to ultra-deep paired-end read sequencing  curves may also be useful especially for unbalanced binary outcome data. The PR curves generally preserved the top-performing samples (0.1% 30 s XL-DNase-seq among DNase samples and native among ATAC-seq samples), with the intermediate rankings varying somewhat over different recall ranges . This and other pitfalls known to affect footprinting analysis  suggest Previous studies have pinpointed nuclear receptors, such as GR and ER, as a special class of TFs because their chromatin binding produces “anti-footprints”, i.e., a slight enhancement of (instead of protection from) cleavage at the binding motif elements , 9, 34. We have focused on interrogating cross-linking duration to identify an optimal cross-linking condition, because a previous study of cross-linking kinetics found that TF occupancy, observed by ChIP, largely depends on the cross-linking duration but is rather insensitive to the formaldehyde concentration . Hence, We have determined the effects of various cross-linking protocols on TF binding predictions based on footprints in XL-ATAC-seq or XL-DNase-seq data, in quantitative and objective terms. We demonstrate that XL-DNase-seq improves footprintability of certain TFs, while the same analysis revealed that XL-ATAC-seq fails to enhance footprinting, compared to the native protocol. These findings will critically inform investigators as they utilize the methodologies. For example, while ATAC-seq is widely popular due to its simplicity and small-scale cell yields required for the assay, XL-DNase-seq may offer unique advantages if the study calls for higher-resolution occupancy information of TFs. In addition, our dataset represents an unprecedented-scale resource for the epigenetics and immunology communities.Additional file 1. Supplementary table and methods.Additional file 2. Supplementary figures.


LIPA alterations, are based on the evaluation of LAL activity. In dry blood spots (DBS) submitted for LAL‐D diagnostics (the screening cohort) over a two‐year period or obtained from a cohort of retrospective LAL‐D patients, we measured: (1) LAL activity using a two‐reaction assay with 4‐methylumbelliferone palmitate  and Lalistat‐2, a specific LAL inactivator; (2) total lipase (TL) activity by a 1‐hour kinetic 4‐MU‐Palm cleavage reaction . The TL activity was expressed as the area under the kinetic curve after 1 hour (TL‐AUC1h) of the reaction and presented as the median (min‐max). LAL activity was reduced in 30/537 individuals from the screening cohort, among which LIPA sequencing revealed six patients and one carrier. Overall, 16 (89%) individuals among six novel and 12 retrospective LAL‐D patients carried at least one c.894G>A mutation (six were homozygous). The TL‐AUC1h in nonLAL‐D specimens with normal LAL activity (n = 90) was unambiguously higher (9471 [4015‐23 585] RFU*h/punch) compared to LAL‐D patients, including six new and nine retrospective patients (1810 [357‐2608] RFU*h/punch). Importantly, in 13/15 examined nonLAL‐D specimens with reduced LAL activity the TL‐AUC1h was above a threshold of 2652 RFU*h/punch. Applying this threshold, the TL‐AUC1h index discriminated all LAL‐D patients (100% sensitivity) and 103/105 nonLAL‐D specimens (98% specificity). Given that there is no need for Lalistat‐2 and two parallel enzymatic reactions in conjunction with high sensitivity and specificity, the kinetic assay seems to be practical for LAL‐D screening.Laboratory diagnostics of lysosomal acid lipase deficiency (LAL‐D), a rare disorder associated with Lysosomal acid lipase deficiency responsible for Wolman disease and cholesterol ester storage disease could be reliably detected using a kinetic assay of total lipase activity with a fluorogenic substrate. LAL is an acid hydrolase of cholesteryl esters; thus, body lipid metabolism disturbances are characteristic of this disease.Lysosomal acid lipase deficiency (LAL‐D) is an autosomal recessive disorder that relates to a mutation of the LIPA sequencing.LAL‐D diagnostics are based on detecting reduced enzymatic activity toward a lipase substrate supported by With the advent of enzyme replacement therapy by a recombinant human LAL, sebelipase alfa, LAL‐D has become a potentially treatable disease. A number of clinical trials have demonstrated amelioration of clinical and laboratory manifestations of LAL‐D with increased life expectancy.LIPA alterations found in 18 Russian LAL‐D patients.Herein, we report the two‐year experience of performing the Lalistat‐2‐dependent LAL activity assay for LAL‐D diagnostics in a cohort of individuals screened clinically for LAL‐D signs and describe a kinetic, Lalistat‐2‐independent modification of the conventional LAL activity assay and evaluate its suitability for LAL‐D diagnostics. Additionally, we provide the description of 2All described laboratory procedures were performed at the Laboratory Department, National Medical Research Center for Children's Health, Moscow.2.1For the implementation of the Lalistat‐2‐dependent LAL activity assay,2.2This cohort included DBS obtained from individuals, who were suspected of having LAL‐D after clinical screening , and submitted to our laboratory for the measurement of LAL activity between June 2016 and July 2018.2.3This cohort included LAL‐D affected individuals, who were either diagnosed at or attending our institutions, including the National Medical Research Center for Children's Health (Moscow), the Federal Research Center for Nutrition and Biotechnology (Moscow), and Filatov's Children Hospital No.5 (St. Petersburg), starting in 2012. DBS for the present analyses were obtained during a LAL‐D diagnostic workflow or routine patient checkup.2.4For all analyses presented in this study, DBS were prepared by spotting EDTA venous blood on filter paper (Whatman 903). Fresh DBS were analyzed within three days upon preparation and were archived at −25°C in individual plastic bags with a desiccant pouch for further storage.2.5This assay was carried out essentially as described by Hamilton et al2.61h) of the 4‐MU‐Palm cleavage reaction. Total lipase AUC1h (TL‐AUC1h) was calculated with Magellan software  and expressed as relative fluorescence units (RFU)*1 hour per punch. All TL‐AUC1h values were corrected for the time point of 0 minute by subtracting the RFU value of the 1st measurement from the other measurements in the same specimen. All kinetic assays were performed in duplicate.For the kinetic assay, 40 μL of DBS water extracts and 150 μL of the reaction buffer described above were mixed in a 96‐well, flat bottom black plate. The plate was immediately placed into the Infinite 200 M plate reader, which was prewarmed to 37°C, and the kinetic measurement was started. Changes in fluorescence generated by the release of 4‐MU upon the lipase‐mediated 4‐MU‐Palm cleavage were recorded at 5‐minutes intervals. Initially, to keep the assay time in accordance with the Hamilton method,2.7LIPA were sequenced on an ABI 3500 DNA Sequencer (Applied Biosystems). Nucleotide sequences were analyzed using the Sequencing Analysis software version 5.4 (Applied Biosystems). Alamut 3.2 software  was used for the alignment analysis and mutation identification.Genomic DNA was extracted from DBS using the QuickGene DNA Tissue Kit S . All exonic regions along with the flanking intronic regions of 2.8U test; correlation between LAL activity and age was assessed by means of the Spearman's r coefficient. The tests were considered statistically significant at P < .05.Statistical analysis was performed using IBM SPSS Statistics for Windows, version 21.0 . Median values were compared using the Mann‐Whitney 33.1In 2016, we implemented the LAL assay described by Hamilton et al3.2LIPA sequencing was performed in all 30 specimens and revealed six individuals with pathogenic mutations affecting both LIPA alleles and one individual carrying a pathogenic mutation in one LIPA allele with an intact second allele. In the remaining 23 individuals with reduced LAL activity, no significant LIPA sequence alterations were detected. Thus, six new genetically confirmed LAL‐D cases were detected in the examined cohort. Detailed characteristics of the affected individuals are provided below.Using the implemented Lalistat‐2‐dependent LAL activity assay, we analyzed 537 dB specimens submitted from 57 cities from all over Russia between June 2016 and July 2018. The specimens were obtained from individuals of variable age, ranging from ≤1 months to 66 years; 239 (44.5%) specimens were from children ≤ 5 years of age. In 30 (5.6%) of these 537 dB, LAL activity was below the defined lower reference boundary of 0.07 nmol 4‐MU/punch/1 hour. r = −.371, P < .001) and was significantly increased in children ≤ 5 years of age . This finding suggested that, in addition to LAL, the activity of other enzyme(s) responsible for 4‐MU‐Palm cleavage could be suppressed in LAL‐D patients, thus reducing the total lipase activity.While implementing the Lalistat‐2‐dependent LAL assay in our laboratory, we found that the total lipase activity  in healthy individuals and LAL‐D patients did not intersect . The addition of Lalistat‐2 inhibited 62% of the total lipase activity in specimens from healthy individuals, leaving 38% attributed to nonLAL lipases. In LAL‐D specimens lacking LAL, Lalistat‐2 suppressed <1% of the total lipase activity, implying that the cleavage of 4‐MU‐Palm in LAL‐D specimens was mediated almost exclusively by nonLAL lipases, upon which Lalistat‐2 had no effect. Interestingly, absolute values of nonLAL lipase activity in healthy individuals were nearly 20% higher than those in LAL‐D patients  was measured in 5‐minutes intervals for 1 hour without the addition of Lalistat‐2. The rate of 4‐MU release was clearly higher in nonLAL‐D specimens than that in LAL‐D specimens ; the values of TL‐AUC1h did not intersect between the two groups. A lower limit boundary for TL‐AUC1h, 2652 RFU*h/punch, was defined as 28% of the median TL‐AUC1h found in nonLAL‐D specimens with LAL > 0.07 nmol 4‐MU/punch/1 hour, analogous to the lower limit for LAL corresponding to 28% of the median LAL activity in healthy individuals (see above). The examined 15 LAL‐D patients had TL‐AUC1h values below this threshold  90 dB randomly selected out of 507 nonLAL‐D specimens with LAL activity > 0.07 nmol 4‐MU/punch/1 hour, 2) all available nonLAL‐D specimens with LAL activity < 0.07 nmol 4‐MU/punch/1 hour 15/23), and (3) DBS from 15 LAL‐D patients. As shown in Table  all avai, and (3)i) from repeated analyses of DBS with normal and low TL‐AUC1h over a period of three days. Importantly, the kinetic assay exhibited low TL‐AUC1h CVi values both in normal and LAL‐D specimens . In comparison, the Lalistat‐2‐dependent LAL assay in our hands was less precise, demonstrating CVi values for normal and patient LAL activities of 18% and 109%, respectively.To evaluate the precision of the kinetic assay, we calculated the interday variation  and a recent review4The objectives of this study were 2‐fold. First, we analyzed the results of LAL activity measurements in a large cohort of individuals with suspected LAL‐D and demonstrated the sufficient accuracy of the Lalistat‐2‐independent kinetic modification of the total lipase activity assay in detecting LAL‐D affected individuals. Second, we provided molecular genetic data of 18 LAL‐D patients, including six individuals diagnosed during this study. To the best of our knowledge, this is the first extensive report describing LAL‐D patients from Russia.1h, calculated in a single enzymatic kinetic reaction, without adding Lalistat‐2. Given that there is no need for Lalistat‐2, the existence of two parallel enzymatic reactions and a reduced assay time in conjunction with high sensitivity, specificity, and reproducibility, the kinetic assay seems to be practical for LAL‐D screening.In general, the Lalistat‐2‐dependent LAL assay demonstrated good analytical performance, exhibiting high sensitivity and specificity. However, this assay incorporates two enzymatic reactions, which unavoidably increase total assay error, as has been discussed elsewhere.LIPA alterations. A reduced enzymatic activity in DBS is usually ascribed to poor specimen quality.In 4.3% of the examined DBS, we observed a low LAL activity without detectable LIPA have been identified to this point.LIPA alteration. Almost 90% of the patients carried the c.894G>A mutation, which is the most frequently reported LIPA allele in LAL‐D affected individuals.Reportedly, 98 disease‐causing variants in In conclusion, the described Lalistat‐2‐independent assay seems to represent a practical and accurate approach for investigating LAL activity within the LAL‐D diagnostic workflow.N.M. designed the study, supervised the laboratory work, analyzed the data and wrote the manuscript. E.B., T.S., N.V., A.S., O.G., and K.S. contributed to the conception and design of the study. E.B. performed enzymatic assays. A.P. and K.S. carried out genetic examination. T.S., N.V., A.S., and O.G. conducted clinical diagnostics and patient characterization. All authors contributed to data analysis, data interpretation, and reviewed drafts of the manuscript and approved the final draft for submission.Nikolay Mayanskiy, Alexander Pushkov, Tatiana Strokova, Nikolay Vlasov, Andrej Surkov, Olga Gundobina have received a speaker honorarium from Alexion Pharma LLC, Russia. Ekaterina Brzhozovskaya and Kirill Savostianov declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from all participants or their legally authorized representatives for being included in the study.