These data explain how the systemic hormone T3 acts locally during development to define future expression of hepatic genes.The type 2 deiodinase (D2) in the neonatal liver accelerates local thyroid hormone triiodothyronine (T3) production and expression of T3-responsive genes. Here we show that this surge in T3 permanently modifies hepatic gene expression. Liver-specific Dio2 inactivation (Alb-D2KO) transiently increases H3K9me3 levels during post-natal days 1–5 (P1–P5), and results in methylation of 1,508 DNA sites (H-sites) in the adult mouse liver. These sites are associated with 1,551 areas of reduced chromatin accessibility (RCA) within core promoters and 2,426 within intergenic regions, with reduction in the expression of 1,363 genes. There is strong spatial correlation between density of H-sites and RCA sites. Chromosome conformation capture (Hi-C) data reveals a set of 81 repressed genes with a promoter RCA in contact with an intergenic RCA ~300 Kbp apart, within the same topologically associating domain ( Whether thyroid hormones affect gene expression via DNA methylation is not well known. Here the authors show that type 2 deiodinase (D2) converts T4 to produce T3, which prevents DNA methylation of discrete areas in the neonatal liver. In the absence of D2, DNA methylation occurs and is associated with reduced chromatin accessibility in promoters and enhancers and affects gene expression. The genomic actions of TH signal promote localized transition of the chromatin from a tightly folded and less transcriptionally active structure known as heterochromatin to a more loosely folded and active structure known as euchromatin6. As these processes involve histone modifications, regulation of gene expression by TH is largely fluid, with the rate of transcription of TH-regulated genes fluctuating rapidly according to the levels of the biologically active T3 in the cell nucleus.Thyroid hormone (TH) regulation of gene expression involves binding to nuclear receptors (TR) that are attached or may be directed to TH responsive elements (TREs). This is followed by recruitment of transcriptional coregulators that modify histones and chromatin accessibility to transcriptional enzymes8. Methylation of H3K9, reduction of H3R17 methylation and of phosphorylation/acetylation of H3S10/K14 are all examples of specific histone modifications triggered by uTRs that inhibit transcription9.In the absence of T3, most unoccupied TRs (uTR) remain attached to TREs, where they form complexes with transcriptional co-repressors to pack the DNA. Examples of uTR-recruited co-repressors include nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors , which recruit deacetylases , methyl transferases , and kinases  to activate facultative heterochromatin formation and inhibit gene transcription10. As a result, the T3-TR not only de-represses but also trans-activates transcription of target genes1. T3-TR can also activate gene transcription via long-distance enhancer hyperacetylation, a mechanism that depends on the chromatin context11.uTRs are known to remain bound to TREs and have repressive effects on gene transcription. Nonetheless, exposure of cells to T3 directs additional TR units to TREs, and shifts TRs association from co-repressors to co-activators; these include steroid receptor coactivator (SRC) family, CBP/p300 or CARM1/SNF5, promoting transcriptional activity. In general, the chromatin modifications induced by T3-TR are the opposite of those caused by uTR, e.g., demethylation of H3K9, methylation of H3R17 and phosphorylation/acetylation of H3S10/K1413, and can affect the expression of gene clusters as opposed to a single target gene14. In general, the effects of DNA methylation on gene expression last longer when compared to histone modifications, and are involved in developmental transitions such as amphibian metamorphosis and mammalian organogenesis15, both processes typically sensitive to TH17.Less is known about TH regulation of gene expression via DNA methylation, a process that can also result from histone methylation19. Indeed, peaks of D2-T3 that enhance TH signaling are seen through this period on a tissue-specific basis3, e.g., embryonic day 17 (E17)-P1 in brown adipose tissue (BAT)20, or P15 in the cochlea21.To regulate the timing and intensity of TH signaling on an organ/tissue-specific fashion, developing cells express TH deiodinases, that can both activate (D2) or inactivate (D3) TH. In the embryo, circulating T3 is kept at relatively low levels and tissues predominantly express D3. However, D3 activity diminishes towards birth at the same time that, in some tissues, D2 activity is selectively boosted. A unique blend of D3 and D2 activities seen during this transition independently controls the levels of nuclear T3 in each tissue, hence the timing and intensity of the TH signaling. In the mouse, this process may extend well into the post-natal period given that circulating T3 levels remain low (P1≪P10) through post-natal day 10 (P10)22 that coincides with the time of C/EBPa-induced maturation of the bi-potential hepatoblasts into hepatocytes23; Dio2 expression in liver is silenced thereafter22. This spike in TH signaling is critical for post-natal liver maturation given that liver-specific Dio2 inactivation (Alb-D2KO) was associated with the formation of 1,508 CpG sites of DNA hypermethylation (H-sites) of the adult Alb-D2KO liver genome22. As a result, the adult Alb-D2KO liver response to high-fat diet (HFD) is affected22; the Alb-D2KO mice exhibit reduced susceptibility to obesity, liver steatosis, hyperlipidemia and alcoholic liver disease24.In the developing liver there is a D2-T3 peak (P1–P2) that briefly doubles T3 content26. For example, between E18.5 until adulthood, ~200,000 CpGs changed DNA methylation by >5%, and ~20,000 CpGs by >30%. These changes in DNA methylation occurred primarily in intergenic enhancer regions and coincided with the terminal differentiation of hepatoblasts into hepatocytes26.Hepatocytes normally undergo post-natal epigenetic reprogramming, including changes in DNA methylation, which are the result of terminal differentiation of hepatocyte precursorsHere, we show that during the last steps of the hepatoblast maturation, a transient surge in D2-mediated T3 signaling interferes with the epigenetic remodeling of hepatocytes, permanently modifying the transcriptome in adult mice. In the absence of this T3 surge, there is a transient  increase in H3K9me3 and permanent hypermethylation of discrete DNA areas, which are strongly associated with reduced chromatin accessibility and repression of ~1500 genes in the adult mouse liver. Gene repression involves reduction of chromatin accessibility in distant areas that operate as remote enhancers, located within the same topologically associating domain. These studies explain how D2 locally modifies T3 signaling during development to define future hepatic gene expression.25. Only 25 H-sites were among those that are normally demethylated  defective neonatal DNA demethylation and/or (ii) de novo neonatal DNA methylation, around P1-P5 but before P10, when circulating T3 reached adult levels. To resolve this, we studied the coordinates of the H-sites (Supplementary Table 22. A predominance of unoccupied thyroid hormone receptors (uTR) has been shown to foster a histone environment that has been linked to de novo DNA methylation in a number of developmental settings by recruiting co-repressors that triple-methylate H3K916. Thus, we tested if this could explain the formation of H-sites in the liver of Alb-D2KO by performing a liver ChIP-seq of H3K9me3 in P1, P5, and adult Alb-D2KO mice. Indeed, we found at least 86 H-sites in the Alb-D2KO liver transiently imbedded in H3K9me3-enriched areas that were only present in the Alb-D2KO chromatin . Here, we captured two snap shots, i.e., P1 and P5, of a process that could last until circulating T3 reached adult levels, i.e. around P1022. Thus, we next wished to define the relationship between H-sites and TRs in the liver. As we were not able to identify high-quality anti-TR antibodies that could be used to successfully prepare a ChIP-seq library in P1 and P5, we reanalyzed two sets of published liver ChIP-seq data obtained with anti-TR antibodies that are no longer available—one from adult hypothyroid mice, in which uTRs predominate over T3-TRs, and the other from T3-treated adult mice, in which T3-TRs predominate over uTRs,27. We used these datasets to assess the density of uTRs or T3-TRs in the proximity of H-site coordinates. The resulting heat-maps revealed that while only ~10% of the H-sites were flanked by uTRs in the hypothyroid liver . In contrast, the same sample of HH-sites was flanked by T3-TRs in 69 occasions  of both adult control and Alb-D2KO mouse liver nuclei   areas, and ranked them according to their ATAC-seq signal intensity and signal ratio to two typical chromatin markers H3K4me1 (enhancer) and H3K4me3 (promoter) Fig. e29,31. Ters Fig. . Of courp < 0.05; Fig. 3ap < 2.9E-14) (Supplementary Data p < 0.01) , a configuration that strongly supports a functional relationship between them. This strict criterion led us to 154 p-RCAs , selecting those that were within 5 Kbp of each other (n = 63), E2F3 (n = 32), Klf1 (n = 37) and NRF1 (n = 34) (Supplementary data 40. Using published mouse liver ChIP-seq data31 for cohesin (Rad21) and CTCF, we looked for overlaps between the 34 RCA/insulator areas and the cohesin/CTCF peaks. There were 30-CAC sites and 4 CNC-sites, highlighting the potential importance of remote regulation in our model . The correlation remained strong when only the densities of H-sites vs. p-RCA areas , setting the stage for a possible local negative effect on the promoter of the following genes: 1700047M11Rik, Gstm6, 3110082I17Rik, Mtus1, Dixdc1, Eif5, Dsp, Pde4d, Sco2, Tymp, Odf3b, Cdo1 . We confirmed this association by manually curating the TAD, p-RCA, and i-RCA data for chromosome #1, with similar results  for each chromosome. This matrix was analyzed by first mining for coordinates that were within 1 Kbp of the 154 p-RCA sites. We found 988 coordinates and 134 p-RCAs that met this strict criterion; these matrix coordinates were arbitrarily named areas-1. The remainder p-RCAs included four genes that contained an H-site near the core promoter, i.e., Sco2, Tymp, Odf3b, Gstm6. Second, we further mined the matrix and verified that these 988 areas-1 were in contact with 1060 distant areas, given that some areas-1 exhibited more than one point of contact; we arbitrarily named these distant points of contact areas-2. The average distance between areas-1 and areas-2 was ~324 Kbp (1 Kbp–2.4Mbp), well within the constraints of a TAD  (Supplementary Data We next used chromosome conformation data (Hi-C) obtained from primary liver cell culturesarea-2, the smallest distance between them averaged ~1 Mbp . These H-sites were mostly distant from core promoters and distributed similarly to the ~1550 p-RCA and ~2400 i-RCA areas; a TAD analysis revealed a highly significant association between these areas within the same TAD. These elements are likely to have disrupted the function of long-distance enhancers, which then failed to interact and activate the core promoter areas, lowering the expression of ~1300 genes involved in different aspects of liver development and function.The investigation of a dramatic phenotype seen in the Alb-D2KO mouse44. The timing and intensity of T3 signaling in developing cells is regulated via expression of deiodinases. In the embryo, circulating T3 is kept at relatively low levels and tissues predominantly express the inactivating deiodinase, i.e., D3, limiting exposure to T3. However, D3 activity diminishes towards birth at the same time that, in some tissues, D2 activity is selectively boosted. A unique blend of D3 and D2 activities seen during this transition independently controls the levels of nuclear T3 in each tissue, hence the timing and intensity of the thyroid hormone signaling.The present findings indicate that localized D2-generated T3 plays a role in the terminal maturation of the hepatocytes. This is reminiscent of the role played by D2 in the post-natal development of brown adipocytes and cochlea45. At the same time, changes in the DNA methylation coincide with the hepatocyte terminal differentiation and occurs after hematopoietic stem cell migration26.In the case of hepatocytes, the short-lived, localized D2-T3 production, enriches discrete areas of the chromatin with T3-TRs, preventing accumulation of H3K9me3 and possibly the formation of the H-sites. Our H3K9me3 ChIP-seq studies in P1 and P5 Alb-D2KO livers caught the moments during which the chromatin environment could favor formation of H-sites, illustrated by the islands of H3K9me3 surrounding H-site coordinates; different timings could have been involved in the formation of the other H-sites, up until P10, when circulating T3 reached adult levels. Insulin signaling has been shown to play a similar role in the post-natal epigenetic programing of the liver, including differential DNA methylation. For example, changes in the DNA demethylation during the neonatal period were found to be essential for the ligand-activated PPARα-dependent gene regulates the hepatic fatty acid β-oxidation9 and HP1 proteins, a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure46. Indeed, the SUV39H1 was also found to cooperate with DNMTs to establish sites of de novo methylation47, which could explain the formation of the H-sites found in ALB-D2KO liver. Nonetheless, our finding that few uTRs are nearby H-sites suggests that DNA hypermethylation in these areas could be the default hepatocyte differentiation program without the modification normally caused by the D2-mediated surge in T3 and T3-TR. For example, hairless encodes a H3K9 demethylase, and is a highly T3-responsive genes48.uTRs have been shown to increase H3K9 methylation through recruitment of SUV39H1p-RCA:n-RNA-seq area along with likely long-distance i-RCA/H-site interacting chromatin areas within the same TAD. It is conceivable that H-sites initiate these processes by creating/maintaining i-RCAs and disrupting the function of the distant enhancers, subsequently depriving core promoters of transcriptional factors that activate gene transcription. Sp1–3, E2F3, Klf1, and Nrf1 were among the potential transcription factors that could be affected in the promoter p-RCAs of the downregulated hepatic genes. It is notable that TR is not one of the transcription factors identified in p-RCAs, confirming that the T3 effects observed on gene transcription are mediated in remote locations.The reduction in gene expression found in the Alb-D2KO liver is dramatic. There are probably multiple mechanisms that explain such changes, but the ones we found evidence for in the present investigation include (i) disruption of few p-RCA function by local H-sites, (ii) disruption of long-distant chromatin interactions, looping enhancers and promoters, (iii) or a combination of both. The relative proximity and similar distribution of H-sites, i-RCAs and p-RCAs within individual chromosomes is remarkable; it is an indication that indeed most of these elements exist inside the same or neighboring TAD unit, and probably exhibit a functional relationship. These concepts were illustrated in the finding of 81 genes that have a 8, which in the liver plays an essential role down-regulating T3-signaling. It is fascinating that the liver responds to a reduction in neonatal T3-signalig (due to Dio2 inactivation) and preserves T3-signaling homeostasis by inhibiting the adult expression of a TR corepressor.The analysis of the p-RCAs also revealed negative Nrf1 and Elk1 footprints in the Ncor1 promoter, one of the 146 key downregulated genes with a p-RCA in the adult Alb-D2KO liver. Ncor1 is a TR corepressorIn conclusion, the present studies revealed that during the hepatoblast-hepatocyte transition in mice, a short-lived surge in D2 expression and T3-signaling substantially modifies the hepatic transcriptome in adult animals. By acting through intergenic TRs, T3-signaling prevents discrete methylation of specific DNA sites, which would otherwise disrupt the function of areas that operate as remote enhancers. In the presence of the normal neonatal D2-T3 peak, these distant areas interact remotely with dozens of gene promoters, increasing chromatin accessibility and expression of genes involved in multiple hepatic functions. This constitutes a mechanism through which TH regulates gene expression, and explains the critical role played by deiodinases in vertebrate development.22. The mice were kept at room temperature (22 °C) under a 12-h dark/light cycle, and maintained on a chow diet .Experiments were approved by the University of Chicago Institutional Animal Care and Use Committee - protocol # 72577. C57BL/6J mice with hepatocyte-specific Dio2 inactivation (Alb-D2KO) were obtained by crossing floxed C57BL/6J D2 mice (dio2Flx) with mice expressing Cre-recombinase under the albumin promoter (Cre-ALB) 22. Only the H-sites present in both methods were considered. H-sites were annotated to gencode.vM24. using the Partek Genomic suite software 7.0.Liver hypermethylation sites (H-sites) in the Alb-D2KO mice were obtained from a previous methylome analysis that used two methods to identify H-sites: (i) Methylation-Dependent ImmunoPrecipitation followed by sequencing (MeDIP-seq) and (ii) Methylation-sensitive Restriction Enzyme digestion followed by sequencing (MRE-seq)n = 4) were studied. Each sample was made up of a pool of 3 livers, totaling 12 mice: two samples were obtained from 6 Cre control animals and 2 samples were obtained from 6 Alb-D2KO animals. Two P5 sample were studied, each made up of a pool of three livers, totaling six mice. One sample was obtained from three Cre control animals and one sample was obtained from three Alb-D2KO animals; three adult livers were processed and sequenced individually. All samples were sequenced twice in different days; replication (repeating the whole experiment) was not performed because we studied 12 P1 mice and 6 P5 mice and results in both groups were similar. So, we considered that the P5 data replicated the P1 data, in what we called neonatal data. Chromatin lysates were prepared, pre-cleared with Protein G magnetic beads, and immunoprecipitated with antibodies against the H3K9me3 . Beads were extensively washed before reverse crosslinking. Chip-enriched DNA was submitted to the Genome facility at University of Chicago for library preparation and sequence using Illumina NovaSEQ6000. FASTQ files obtained from sequencing were aligned to the Mouse mm10 genome in Partek-flow platform (Partek Inc. v.10.0.21.0509) using the BWA (v.0.7.17). The peaks were identified using MACS2 (v.2.1.1) tool, broad region and p < 0.05, compared with respective input samples and annotated using the gencode.vM24. This led us to P1: 280,580, P5: 265,920 and adult: 292,552 peaks in control livers vs. P1: 273,767, P5: 268,693 and adult: 286,124 peaks in Alb-D2KO livers. Areas of H3K9me3 enrichment unique to ALB-D2KO livers were also identified by comparing Alb-D2KO vs. controls samples, which led us to P1: 301,105, P5: 301,098, and adult: 287,032 peaks, which were filtered down to P1: 11,303, P5: 10,005 and adult: 16,730 using –log10>2.Liver ChIP-seq for H3K9me3 was performed in P1, P5 and adult control and Alb-D2KO mice, using the SimpleChIP® Plus Enzymatic Chromatin IP Kit . Liver from pups and adult control and Alb-D2KO mice were snap frozen and stored at −80 °C. Four P1 samples (28—GSM722760 (H3K4me1); GSM722761 (H3K4me3); GSM851275 (H3K27ac); GSM722762 (P300); GSM722763 (Pol2); GSM722764 (Input); chromatin-folding markers:31 Rad21 and CTCF (SRP116021) and TR27 (SRP055020). The FASTQ files were aligned to transcriptome with BWA (v.0.7.17) using the Partek flow platform (Partek Inc). The peaks were identified using MACS2 (v.2.1.1) tool (p < 0.05), compared to respective input samples and annotated using the gencode.vM24. In all instances, pre‐ and post-alignment QA/QC was done using Partek Flow (Partek Inc).The following published ChIP-seq datasets from adult mouse liver were reanalyzed and also used in the present studies: histone markers49. The final libraries were purified using Agencourt AMPure XP beads (Beckman Coulter), and quality checked using a Bioanalyzer High Sensitivity DNA Analysis kit (Agilent); concentration was measured through a qPCR-based method (KAPA Library Quantification Kit for Illumina Sequencing Platforms). Samples were pair-end sequenced with the Illumina HiSeq 4000 platform at the University of Chicago Genomics Facility. The remaining adapter sequences were removed using NGmerge (v.0.3) Sequencing reads were aligned to the Mouse mm10 genome build using BWA (v.0.7.17) and annotated using the gencode.vM24. Sequence quality was assessed using FastQC and tools from the Picard suite including CollectInsertSizeMetrics, which showed an enrichment in short fragments as expected . Run by individual sample, genrich would find only a handful of peaks, which very obviously were not due to ATAC enrichment. These regions were merged across samples into a blacklist of regions and excluded from consideration. Ultimately genrich was run using all samples grouped together while ignoring the blacklisted regions. Differential peaks were identified by overlap subtraction to find open-chromatin regions unique to each condition.Frozen livers were processed for nuclei isolation using a detergent-free kit . Nuclei integrity was verified under light microscopy Fig. . Fifty-tted Fig. . Aligned31. Signals were summarized as transcripts per kilobase millions (TPM) values in each RCA region. Those RCA regions with a TPM > 4 for either H3K4me3 or H3K4me1 were segregated into one of three categories, depending on the value of the ratio of H3K4me3 to H3K4me1. High ratios of > 1.5 were classified as a “promoter”; ratios < 0.67 were classified as “enhancer”, while intermediate values were classified as “weak promoter.” Conversely RCA regions with TPM ≤ 4 in both H3K4me3 and H3K4me1 were segregated into one of two categories depending on the relative values of ATAC-seq to H3K4me1 ratio. If the ATAC-seq signal > H3K4me1 signal, then the RCA was categorized as “insulator”. If the ATAC-seq signal < H3K4me1 signal, then the RCA was categorized as “weak insulator”.Mouse liver RCAs were classified based on the relative strengths of H3K4me1, H3K4me3, and ATAC signalsFor the motif analysis, the nucleic acid sequence of promoter ATAC-seq peak regions were extracted and masked using RepeatMasker . Motif analysis was carried out using the meme-chip command from MEME Suite (v.5.0.5) using the MOUSE/HOCOMOCOv10, MOUSE/uniprobe, MOUSE/chen2008, EUKARYOTE/jolma2013, and JASPAR/JASPAR_CORE_2016_vertebrates motif libraries. This multiple analysis tool identified known motifs, de novo motifs, characteristic distances and co-occurrences, and collapsed hits by motif similarity.RNA was isolated from mouse liver using the RNeasy Kit (Qiagen). RNA degradation was monitored using a BioAnalyzer (Agilent). Samples of total RNA with RIN > 7.5 were sent to Genome Technology Access Center at Washington University in St. Louis for library preparation and sequence. Libraries were pair-end sequenced with NovaSeqS4 (Illumina). Base-calls and demultiplexing were performed with Illumina’s bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. The FASTQ files were aligned to gencode.vM24 transcriptome with STAR (v.2.6.1d) using the Partek flow platform (Partek Inc.). All pre‐ and post-alignment QA/QC was performed in Partek Flow (Partek Inc.)  pipeline take as input FASTQ data from a Hi-C experiment and produces a filtered set of interaction pairs mapped to the reference genome (mm10). The post-pipeline analysis of the output from HICUP was done using HOMER (v.4.11)51 to create Hi-C interaction and contact matrices. Matrices were then analyzed for long-range interactions involving p-RCAs, i-RCAs as obtained from the analysis of the ATAC-seq, and H-sites.Publicly available liver Hi-C datasets (GSE65126) were analyzed using the HICUP52  was used to obtain the intersection of the H-sites and different chromatin markers on the genomic coordinates for the desired distance. The heat-maps used to analyze proximity between different chromosomes coordinates . The Integrative Genomics Viewer (IGV) 2.7.2 version was use to visualize and integrated all the genomic data analysis (http://www.broadinstitute.org/igv). The analysis of the topologically associated domains (TADs) and RCA regions (intergenic and promoter) were done using bedops (v.2.4.38)42. The results from the bedops were mined for relevant intersections using an R script.R-script using the Bioconductor IRanges packagees Figs. , were gees Figs.  were creSupplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary data 1Supplementary data 2Supplementary data 3Supplementary data 4Supplementary data 5Supplementary data 6Supplementary data 7


RPB1, coding for the RNA polymerase II large subunit. We found that this G-quadruplex-forming locus is highly evolutionarily conserved amongst plants sensu lato (Archaeplastida) that share a common ancestor more than one billion years old. Finally, we discussed a new hypothesis regarding G-quadruplexes interacting with UV light in plants to potentially form an additional layer of the regulatory network.G-quadruplexes have long been perceived as rare and physiologically unimportant nucleic acid structures. However, several studies have revealed their importance in molecular processes, suggesting their possible role in replication and gene expression regulation. Pathways involving G-quadruplexes are intensively studied, especially in the context of human diseases, while their involvement in gene expression regulation in plants remains largely unexplored. Here, we conducted a bioinformatic study and performed a complex circular dichroism measurement to identify a stable G-quadruplex in the gene  RNA polymerase II (DNA directed RNA polymerase II) is usually associated with transcription of most structural genes. Eukaryotic RNA polymerase II consists of 12 subunits encoded by different genes [Saccharomyces cerevisiae was resolved [RPB1 gene in different evolutionarily distant organisms belonging to the plant kingdom. Although regulation of gene expression is more complex, levels of active RNA polymerase II are important for the overall level of transcription. Therefore, stable G4(s) in the coding sequence of the large subunit of RNA polymerase II could significantly reduce the level of transcription. G-quadruplexes G4s) in nucleic acids are noncanonical four-stranded structures, which are different from classical double-stranded DNA (B-DNA form) described in 1953 by James Watson, Francis Crick, and Rosalind Franklin . The bass in nucla+) ions ,7,8, proa+) ions ,11, and a+) ions ,13,14,15a+) ions ,17, affea+) ions ,19,20, aa+) ions . There aa+) ions ,23. Howea+) ions  stating nt genes ,26, and resolved . The actresolved . Moreoveresolved . Recentlresolved . RNA polresolved ,32. ThusRPB1 coding sequences of various representative green plants (Viridiplantae) and their closest relative groups, Rhodophyta and Glaucophyta, which all belong to the Archaeplastida supergroup [At first, we decided to study the conservation of potential G4-forming RBP1 sequences in evolutionarily distant plants. We aligned the pergroup ; see TabRPB1 CDS sequence from Arabidopsis thaliana (NM_119746.4), the G4 locus started at nucleotide position 1257 and ended at nucleotide position 1296. For the whole genome, the identified region occupies the coordinates of chromosome 4, 16,966,308–16,966,347. The fourth guanine track is 100% conserved, while the other tracks contain a certain plasticity, which can play an important role in the resulting conformation of the formed G4  and found that the 40-bp-long potential G4 locus is the most conserved  have this locus modified by deletions and/or substitutions that disrupt G4-forming potential  of the G4Hunter prediction algorithm [Arabidopsis thaliana isoform sequencing [RPB1 gene isoform comprising exons 1–8 . It is known that G4s can induce premature transcription termination [It can be hypothesized that the coding region of the tial see . The laron rates ,46,47. Cthaliana , but no lgorithm  is used.quencing , we founmination . Howevermination .Cyanidioschyzon merolae sequence. More specifically, the RPB1 locus of this unicellular organism has low theoretical potential to form a G4 structure, and, therefore, the obtained CD signal was unexpected. This may be due to the involvement of other nucleotide residues in G4 tetrads  that comprise, for example, cytosine residue(s) [Next, we inspected the G4-forming potential of selected sequences via circular dichroism (CD) measurements a. All insidue(s) . Unfortusidue(s) ,55.RPB1 gene, we selected one representative sequence from Bathycoccus prasinos and modeled its parallel G4 structure in silico. The model is based on information obtained by CD measurement, and it mimics parameters of existing PDB structures using the 3DNus algorithm [To better visualize the structure of a parallel G4 in the lgorithm   induces DNA damage, mainly in the form of cyclobutane pyrimidine dimers . Recentlin vitro . Here, fCyanidioschyzon merolae showed a mild decrease, and Arabidopsis thaliana showed a medium decrease. In contrast, Bathycoccus prasinos or Micromonas pusilla displayed a highly pronounced decrease in molar ellipticity associated with G4 presence  from 40 model plant species that are evolutionarily distant from one another  in Arabidopsis thaliana are enclosed in  another  were cho regions . AnalyzeBathycoccus prasinos was modeled in a 3DNus environment [The potential to form G4s was predicted via the QGRS mapper  and G4scironment  using a ironment .−1 with 3 accumulations. For all CD analyses, a final concentration of 50 mM KCl was used. To denature the G4 structures, a heating rate of 10 °C·min−1 was maintained using a programmable Peltier thermostat up to 90 °C followed by cooling to 20 °C for the CD spectra measurement of renatured G4 structures. A quartz glass cell with a 10 mm path length was used for all CD measurements. The sequence of negative control (NC) was as follows: AAGGGCAAGGAGTGGAGAGTGCGCGTGAATCTCATGTGCAA (designed using the G4Killer tool) [2 UV-A and 4.1 W/m2 UV-B radiation. The control and UV-irradiated samples were compared with respect to height of the CD peak . The decrease in molar ellipticity was computed, and, for later purposes, it was expressed on the categorical scale  using the highest and lowest decreases as borders and then evenly divided into these categories. For the detailed spectrum of the UV lamp used in this study, see the All G4-forming oligonucleotides were purchased in HPLC purity from Elisabeth Pharmacon (Czech Republic) and inducted as reported earlier . CD specer tool) . To deteGel electrophoresis of the selected G4 samples was performed on a nondenaturing 15% acrylamide gel supplemented with 10 mM KCl. The gel was electrophoresed at room temperature (20 °C). After electrophoresis, the gel was stained in a bath of 0.5 µM ThT (which is a widely used fluorescent light-up probe for G4 formation ) for 15


Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1–Fhl1–Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1–Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1–Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1. Saccharomyces cerevisiae) ribosomes are produced at a rate of ∼2000 per minute and are present in ∼200 000 copies per cell. This requires not only the dedication of more than half of all RNAPII initiation events to RP or RiBi genes, but a prodigious output of ribosomal RNA (rRNA), synthesized by the combined action of RNAPI and RNAPIII. The production of RPs and rRNAs is assumed to be highly coordinated in order to ensure rapid and efficient ribosome assembly [Ribosome biogenesis is probably the most energy-intensive anabolic processes carried out in growing cells . In rapiassembly ,4. Here Work over many years has revealed a common set of TFs bound to most RPG upstream regulatory regions . The fihmo1Δ) has been reported to abolish both Fhl1 and Ifh1 binding at RPG promoters with only minor effects on transcription or cell growth [fhl1Δ cells display a severe growth defect and a profound global reduction in both rRNA and mRNA [hmo1Δ, and ifh1Δ is lethal. Furthermore, hmo1Δ cells are extremely sensitive to Ifh1 depletion compared to wild-type cells [The precise role of Hmo1 at Category I RPG promoters is still controversial. Loss of Hmo1  is thought to operate largely through mechanisms that control the association of Ifh1 with their promoters, based on the observation that stress (e.g. heat shock or inhibition of the major growth-promoting kinase TORC1 with rapamycin) causes rapid Ifh1 promoter release while leaving both Rap1 and Fhl1 binding unaffected ,12–15,29Two distinct mechanisms that prevent Ifh1 promoter binding at RPGs under stress conditions have been uncovered. The first to be identified involves a complex containing the ubiquitous casein kinase 2 (CK2), two RiBi proteins (Utp22 and Rrp7) and Ifh1, called CURI ,37. The RPL1A and RPL18B), display little or no Fhl1/Ifh1 binding [Thirteen of the 138 RPGs, which we have grouped together as Category III genes , do not  binding . Nearly  binding ; see belDespite these differences in promoter organization, particularly with respect to Category III, all RPGs display remarkably similar up-regulation in response to a glucose pulse or down-regulation in response to two different stress conditions, arrest of secretion by tunicamycin treatment or oxidative stress provoked by diamide addition . Interesin vitro [The observations described above beg the question of what activator(s) might be responsible for regulated activation of Category III RPGs. The roles of Abf1 and Fhl1 were tested by binding site mutagenesis at a limited set of promoters, where mostly modest reductions in expression were observed . Furtherin vitro  is enricin vitro . Indeed,in vitro . Thus, tNotably, the Sfp1 binding motif is also found at many RiBi gene promoters, and early studies showed that its rapid overexpression via a galactose-inducible promoter fusion leads to a spike in RiBi gene mRNA levels ,48. NeveExpression of several RiBi genes has also been shown to be partly dependent on upstream promoter binding of a GRF . Howevein vivo. As mentioned above, Sfp1 binding is detected by ChIP at Category I and II RPGs, though usually with low enrichment, but not at RiBi genes. Conversely, Sfp1 binding is readily detected by ChEC at RiBi genes, as well as an equally large group of growth-associated genes [in vitro. Furthermore, Sfp1 binding at Category I and II RPGs is strongly reduced by nuclear depletion of Ifh1. Similarly, Sfp1 binding at promoters in the G1/S regulon is abrogated by depletion of Swi4, a TF specific for these genes. Finally, the importance of the gAAAATTTTc motif for in vivo Sfp1 binding, for example at RiBi genes, has been demonstrated recently through endogenous site mutagenesis [It is worth highlighting here a striking duality in Sfp1's mode of promoter binding, as well as its detection ed genes , but noted genes , based uagenesis . Taken tagenesis .Finally, we note an additional common feature of RP and RiBi genes, namely their frequent clustering in gene pairs that display increased co-regulation under various stress conditions compared to those genes that are not present in clusters . This feRPL25 promoter does indeed abolish Fpr1 binding there. Nevertheless, there are many other promoters bound by Rap1 where Fpr1 binding is not observed. The determinants of Fpr1 binding specificity thus remain to be unraveled. Interestingly, the rapamycin binding activity of Fpr1, but not its proline isomerase activity, is required for its RPG promoter binding and transcriptional activation. However, genetic studies clearly indicated that Fpr1's effect on RPGs is independent from its interaction with TORC1 . Remark1Δ cells . Exactly1Δ cells , similar of Fpr1 .in vitro [The identification of Fpr1 as a novel TF highlights a persistent mystery regarding the binding specificity of the constellation of TFs found at RPGs. Indeed, only Rap1 binding consistently correlates with the presence of a recognizable binding motif. Fhl1, a forkhead-domain protein, recognizes a specific motif in vitro ,47,70 yein vitro . Sfp1, ain vitro ,32. Fpr1in vitro , it cannTOM1, which encodes a E3 ubiquitin ligase primarily dedicated to the degradation of unassembled RPs, profoundly impacts cellular proteostasis [Previous studies have pointed to links between ribosome biogenesis and protein homeostasis (proteostasis). For example, the primary source of ubiquitin in growing yeast, and probably most metazoan cells, comes in the form of N-terminal protein fusions to two specific ribosomal proteins ,72, thuseostasis ,74. Two eostasis ,75 have eostasis ,77. RP deostasis .http://original.disprot.org/metapredictor.php), a common feature of proteins capable of forming liquid–liquid phase separated condensates [Remarkably, then, excess unassembled RPs negatively regulate the genes that encode for them through sequestration of their key transcriptional activator, Ifh1. Significantly, Ifh1 appears to be largely composed of intrinsically disordered domains (IDRs; densates . Althougdensates . It willdensates  and acetThis transcriptional arm of RASTR appears to be a highly adaptive response since most Hsf1 target genes encode for chaperones of proteasome components and the Ifh1 activator is quite specific for RPGs, whose products are responsible for generating the stress in the first place. It is worth pointing out here that RASTR is accompanied not only by reduced solubility of RPs and Ifh1, but by condensation of a significant number of additional proteins, many of which are involved in ribosome assembly and translation ,75. A scThe notion that RASTR operates even in unstressed rapidly growing cells follows from the observation that treatment of cells with cycloheximide provokes an opposite transcriptional response: up-regulation of RPGs and down-regulation of Hsf1 target genes, suggesting that even under these conditions Ifh1 activity is mildly suppressed and Hsf1 partially actived. In fact, RASTR itself requires ongoing protein synthesis and is attenuated by cycloheximide treatment or by specific reduction of RPG transcription by nuclear depletion of Ifh1, even in cells where ribosome assembly is simultaneously perturbed . RemarkaWe recently found  that proIn summary, the two new studies outlined above ,75 illumRibosome biogenesis is a remarkably complex and energy-intensive process essential for rapid cell growth. The requirement to coordinate the production and assembly of nearly 80 ribosomal proteins with ribosomal RNA and to tune this process in response to fluctuating environmental conditions, imposes unique regulatory challenges, which are solved in yeast by the combinatorial action of multiple transcription factors deployed in more than three distinct configurations.Recent studies reveal that perturbations to ribosome biogenesis leading to an excess of unassembled ribosomal proteins induce a global yet highly focused protein homeostasis reaction, the ribosome assembly stress response. This multipronged regulatory system acts at transcriptional and post-transcriptional levels, via still poorly understood protein condensation mechanisms, to help restore cellular proteostasis.Future studies will explore the underlying molecular mechanisms that drive RASTR in yeast and provide a clearer understanding of how this response improves cell fitness under stress. We anticipate that new studies in metazoan systems will reveal the extent to which this system is evolutionarily conserved and its potential implication in development and disease, specifically in ribosomopathies but more generally in cancers, where ribosome biogenesis underlies rapid and uncontrolled proliferation.


Previous research in autism and other neurodevelopmental disorders (NDDs) has indicated an important contribution of protein-coding (coding) de novo variants (DNVs) within specific genes. The role of de novo noncoding variation has been observable as a general increase in genetic burden but has yet to be resolved to individual functional elements. In this study, we assessed whole-genome sequencing data in 2671 families with autism . We focused on DNVs in enhancers with characterized in vivo activity in the brain and identified an excess of DNVs in an enhancer named hs737.−3), and combined dataset (p = 1.1 × 10−4). Each individual with a DNV in hs737 had shared phenotypes including being male, intact cognitive function, and hypotonia or motor delay. Our in vitro assessment of the DNVs showed they all reduce enhancer activity in a neuronal cell line. By epigenomic analyses, we found that hs737 is brain-specific and targets the transcription factor gene EBF3 in human fetal brain. EBF3 is genome-wide significant for coding DNVs in NDDs  and is widely expressed in the body. Through characterization of promoters bound by EBF3 in neuronal cells, we saw enrichment for binding to NDD genes  involved in gene regulation. Individuals with coding DNVs have greater phenotypic severity  in comparison to individuals with noncoding DNVs that have autism and hypotonia.We adapted the fitDNM statistical model to work in noncoding regions and tested enhancers for excess of DNVs in families with autism. We found only one enhancer (hs737) with nominal significance in the discovery (p = 0.0172), replication (p = 2.5 × 10EBF3 that is genome-wide significant in NDDs. By assessment of noncoding variation and the genes they affect, we are beginning to understand their impact on gene regulatory networks in NDDs.In this study, we identify DNVs in the hs737 enhancer in individuals with autism. Through multiple approaches, we find hs737 targets the gene The online version contains supplementary material available at 10.1186/s40246-021-00342-3. Large-scale whole-genome sequencing (WGS) is becoming instrumental in assessing the contribution of protein-coding (coding), but more importantly noncoding variants in complex diseases . Unlike EBF3 which is enriched for coding DNVs in the hypotonia, ataxia, and delayed development syndrome (HADDS). We also identified new patients with coding variants in EBF3 and performed extensive phenotypic analysis. Combining this with phenotype data we collected for individuals with the hs737 enhancer DNVs, we found marked increases in phenotypic severity in individuals with coding than noncoding variants. This work provides critical insights into coding and noncoding DNVs at EBF3 and more generally in neurodevelopmental disorders.Autism is a complex neurodevelopmental disorder with a heritability of ~ 80% . Large c−3) and in the combined dataset (p = 1.1 × 10−4). The hs737 enhancer drives expression in the midbrain and hindbrain at embryonic day 11.5 (E11.5) ) to adulthood (40 years) and examines many regions of the brain. We wanted to determine what transcription factor may be binding to the enhancer. As we describe below, the target gene of hs737 is EBF3. We hypothesized that the transcription factor that is binding may be correlated in expression with EBF3. We find that EBF3 is expressed throughout the brain prenatally and is most highly expressed through the hindbrain in the cerebellum and cerebellar cortex  and duplications  in individuals with NDDs. None of the 19,584 control individuals contained a CNV in this enhancer  only in individuals with an NDD. To test this, we first applied two approaches to assess CNVs in hs737. We assessed the morbidity map , 36 dataWe also applied a tool to determine copy number  in 1-kbp windows across the whole genome  to our WEnhancers have well-characterized epigenetic signatures that are predictive of their activity in specific biological contexts. Thus, to examine the activity of hs737 in its native genomic context, we took advantage of available epigenomic datasets from relevant human samples. We found that hs737 has several hallmarks of neuronal enhancer activity in humans including H3K27ac enrichment in fetal brain tissue , DNaseI Hs737 was assessed for reporter activity in mouse embryos at E11.5 in the VISTA database. To further examine the activity of this element during development, we analyzed the orthologous region in the mouse genome  using epigenomic data from a mouse embryonic developmental time series recently published –45. ConsStrikingly, we found that characteristics of enhancer activity at mo-hs737 such as H3K27ac and chromatin accessibility (as measured by ATAC-seq) reach their height in brain tissue in mid to late gestation and decline at birth Fig. . This suC10orf143, CTAGE7P, EBF3, GLRX3, LINC01163, LOC728327, MGMT, and MIR4297; the only gene in this region that is constrained in the human population with a gnomAD [EBF3. To examine physical interactions between hs737/mo-hs737 and genes in this region, we analyzed two separate high-resolution Hi-C datasets examining the 3D architecture of the genome throughout mouse neuronal differentiation and human fetal corticogenesis. The mouse dataset was generated in a differentiation course from mouse embryonic stem cells (ESCs) to neural progenitor cells (NPCs) and then to cortical-like neurons (CNs) [Ebf3 promoter makes strong contacts across a large region encompassing mouse hs737 (mo-hs737) and that these interactions become stronger during differentiation to NPCs and CNs . We did not observe loops between mo-hs737 and any other genes on the chromosome.To determine the potential gene target of the hs737 enhancer, we first looked at all of the genes residing within the same topologically associating domain (TAD)  [ns (CNs)  with Hi-CNs Fig. . We usedEBF3 promoter interacts with the region of hs737 in both the GZ  and CP  samples. We note that there is a loop in CP between hs737 and a second gene GRLX3 , a gene known to be involved in multiple mitochondrial dysfunctions syndrome  and germinal zone (GZ) layers , we creaome Fig. . HoweverEBF3, we searched recent literature on coding DNVs and found EBF3 is the only gene in this TAD that has known statistical enrichment for coding DNVs in NDDs [−35, chimpanzee-human loss-of-function p = 2.26 × 10−13, denovolyzeR [−13, denovolyzeR loss-of-function p = 7.97 × 10−22) for coding DNVs using two different statistical tests. This is also the gene for the Mendelian phenotype hypotonia, ataxia, and delayed development syndrome  and hase . Of the 285 significant genes in another study [−6, OR = 1.87)  and/or transcription factor activity (DNA Binding Gene Ontology p = 6.4 × 10−11)  indicating that EBF3 may be a master-regulator of many NDD genes. Chromatin binding genes account for a sizeable fraction of DNM attributable cases of autism [EBF3 disruption could result in a milder phenotype in the spectrum, as we observe in these cases. We find further support for this by observing that EBF3 expression is highly correlated (r > |0.6|) with 116 high confidence SFARI genes (SFARI score < 3) , regulation of histone modification (FDR p = 2.12 × 10−2), chromatin remodeling (FDR p = 2.23 × 10−2), chromosome organization (FDR p = 3.00 × 10−7), chromatin organization (FDR p = 6.58 × 10−7), and positive regulation of the cell cycle process (FDR p = 3.68 × 10−2). Performing a network analysis using String-db, we find that the genes positively correlated with EBF3 have significantly more interactions than expected   and find EBF3 is part of the network with evidence from genetic interactions  in the human neuroblastoma cell line SK-N-SH . We mapper study , 26.3% o87) Fig. . Many off autism , 14, 56, 3) Fig. . Within ons Fig. .Fig. 5EIn order to understand more about the phenotypic consequences of variation in the enhancer, we reviewed de-identified phenotype information for each of the individuals with autism that had a DNV in hs737. As gene discovery in simplex autism, based on large CNVs and coding DNVs, has yielded the most findings in females and in individuals who have intellectual disability  , the firEBF3, we find that those with coding mutations in EBF3 typically have more severe phenotypic consequences  and we find significantly higher rates of autism within the hs737 DNV group when compared to the EBF3 group (p = 0.0088)  , 55 and ces Fig. . All ind88) Fig.  . The individuals with DNVs had shared phenotypes including being male, intact cognitive function, and all had hypotonia or motor delay . PrevioEBF3 as the gene targeted by hs737, in both embryonic mouse and human fetal brain, with an interaction across a distance of ~ 1.4 Mbp in the human fetal brain. EBF3 is a well-established NDD gene with genome-wide significant enrichment of coding DNVs and an established syndrome called HADDS that shares the phenotype of hypotonia with the individuals with autism in our study that had hs737 noncoding DNVs. HADDS is a severe phenotype and affects many parts of the body. This is likely because EBF3 is a transcription factor that is ubiquitously expressed in humans [EBF3 .We accessed DNV data from Wilfert et al.  through https://github.com/TNTurnerLab/fitDNM) [A list of VISTA enhancers driving the expression of their target genes in the brain was downloaded from the VISTA enhancer browser . DNVs we/fitDNM) , a methoTo test copy number variant enrichment in morbidity map , we downhttps://github.com/KiddLab/QuicK-mer2) workflow was run on WGS data to generate copy number estimates, in 1-kbp windows across the genome, in each individual. Briefly, this method utilizes a kmer-based approach to perform copy number estimation. After running QuicK-mer2, we utilized the bedtools [http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data_collections/1000G_2504_high_coverage/.The QuicK-mer2   [We downloaded ChIP sequencing data from Harms et al.  at the f Browser . We then Browser  intersec Browser  and Kapl Browser  and comprf.gov/) .https://www.encodeproject.org/files/ENCFF465SNB/@@download/ENCFF465SNB.tsv, mouse E11.5 midbrain RNAseq data was downloaded from https://www.encodeproject.org/files/ENCFF359ZOA/@@download/ENCFF359ZOA.tsv, and mouse E11.5 hindbrain data was downloaded from https://www.encodeproject.org/files/ENCFF750FTK/@@download/ENCFF750FTK.tsv. For each file, we retained all Ensembl gene identifiers and annotated them to HGNC-approved identifiers using biomart (https://m.ensembl.org/info/data/biomart/index.html). We used an expression cutoff of > 2 to call a gene as expressed and < 2 as not expressed in each region of the brain.Mouse E11.5 forebrain RNAseq data was downloaded from Neuro2a (ATCC CCL-131) were purchased from ATCC and grown under standard conditions (DMEM + 10% FBS and 1% penicillin-streptomycin).luc2 and 9 ng of Renilla luciferase vector (transfection control) were transiently transfected into the Neuro2A cell line (1 × 105 cells/well) using 3μl FuGene HD transfection reagent in 100μl of OPTI-MEM medium. Neuro2A cells were incubated for 48 h and luminescence measured using a Dual-Luciferase Reporter Assay System on a Promega GloMax luminometer. All assays were performed in triplicate for a total of six independent readings of each construct. Significance was calculated by using a two-sided t-test assuming unequal variance and used in two situations, first to compare the wild type construct to basal construct and second to compare the variant constructs to the wild type .Five hundred nanograms of firefly luciferase vector  containing the enhancer sequence cloned upstream of Transcription factor binding analysis was performed using QBiC-Pred  and selehttp://brainspan.org/static/download.html on 4 February 2021 for the developmental transcriptome which contains samples from 8 weeks after conception to 40 years. Linear regression was performed on the data set for age and brain region using R. Spearman correlations were calculated for each gene (X) and EBF3 (Y) and SFARI genes with an absolute correlation greater than 0.6 were retained for further analysis. Pathway analysis was performed using GeneMania (https://genemania.org) and String-db (https://string-db.org). GOTERM analysis was performed using http://geneontology.org where the input genes were those from cluster positively correlated with EBF3 and SFARI gene list (1/13/21 release) was used as the background.RNAseq data ) was downloaded from Phenotypes were assessed by counting the number of individuals with a given phenotype. Specifically, we assessed all probands from previous studies for a total of 13 probands in addition to 7 new probands. The 7 or more symptoms category was calculated as the total number of neurological abnormalities, other diagnoses, and the presence of craniofacial abnormalities, where 1 was added to the total number of symptoms if the individual had at least one craniofacial abnormality. A minus (−) value was taken to be the absence of the phenotype whereas NA was taken to be that the assessment was missing. Two probands were missing assessments for ataxia and were excluded from that analysis for a total of 18 individuals rather than 20. A one-sided Fisher’s exact test was used to calculate significance and odds ratio for the phenotypes and only probands who had an assessment for that phenotype were included in the calculation.Additional file 1: Table S1: Coordinates in the human genome (build 38) of VISTA enhancers driving expression in the brain.Additional file 2: Fig S1. Polygenic risk scores (PRS) for the three individuals with hs737 mutations. Fig. S2. Zoom in on the hs737 enhancer with annotations from other datasets. The enhancer is in a PsychEncode fetal enhancer, contains central nervous system DNaseI hypersensitive sites, contains conserved transcription factor binding sites, and is highly conserved across the vertebrate lineage. Also shown are the locations of the hs737 de novo mutations identified in individuals with autism. Fig S3. String-db network analysis, predicts interactions between EBF3 and SPAST, CSNK2A1, HNRNPU, CHD7, KDM6A, KDM6B.Table S2. fitDNM results for de novo mutations in VISTA enhancers driving brain expression. Table S3. Other de novo SNVs/indels seen in individuals with hs737 enhancer mutations. Table S4. Other copy number variation in individuals with hs737 de novo mutations. Table S5. Statistical significance calculations for the luciferase assays. Table S6. Expression of transcription factors potentially binding at variant locations in the hs737 enhancers.Additional file 3: Table S7:  should have the label "CNVs over hs737 in the morbidity map dataset.Additional file 4: Table S8: Protein-coding de novo mutations in EBF3 from Coe et al. 2019, Nature Genetics and Kaplanis et al. 2020, bioRxiv.Additional file 5: Table S9: should have the label "NDD genes bound by EBF3.Additional file 6: Table S10: should have the label "Phenotypes of individuals with coding and noncoding variants affecting EBF3.


During lytic infection, herpes simplex virus (HSV) 1 induces a rapid shutoff of host RNA synthesis while redirecting transcriptional machinery to viral genes. In addition to being a major human pathogen, there is burgeoning clinical interest in HSV as a vector in gene delivery and oncolytic therapies, necessitating research into transcriptional control. This review summarizes the array of impacts that HSV has on RNA Polymerase (Pol) II, which transcribes all mRNA in infected cells. We discuss alterations in Pol II holoenzymes, post-translational modifications, and how viral proteins regulate specific activities such as promoter-proximal pausing, splicing, histone repositioning, and termination with respect to host genes. Recent technological innovations that have reshaped our understanding of previous observations are summarized in detail, along with specific research directions and technical considerations for future studies. Alphaviridae, which encode their own polymerases, have evolved to globally inhibit specific events in host transcription  is like a nail.” We would like to thank Randy for his friendly personal discussions and innumerable scientific contributions and believe that ongoing research has the potential to shape that railroad spike into a tool that can be manipulated in clinical and oncolytic settings.


Barhl2 in vertebrate brain development. Specific attention is given to the transcriptional events leading to formation of the Organizer, as well as the roles and regulations of Wnt/β-catenin pathway in growth of the caudal forebrain. We present TCF/LEF activities in both embryonic and neural stem cells and discuss how alterations of this pathway could lead to tumors.Since its first discovery in the late 90s, Wnt canonical signaling has been demonstrated to affect a large variety of neural developmental processes, including, but not limited to, embryonic axis formation, neural proliferation, fate determination, and maintenance of neural stem cells. For decades, studies have focused on the mechanisms controlling the activity of β-catenin, the sole mediator of Wnt transcriptional response. More recently, the spotlight of research is directed towards the last cascade component, the T-cell factor (TCF)/Lymphoid-Enhancer binding Factor (LEF), and more specifically, the TCF/LEF-mediated switch from transcriptional activation to repression, which in both embryonic blastomeres and mouse embryonic stem cells pushes the balance from pluri/multipotency towards differentiation. It has been long known that Groucho/Transducin-Like Enhancer of split (Gro/TLE) is the main co-repressor partner of TCF/LEF. More recently, other TCF/LEF-interacting partners have been identified, including the pro-neural BarH-Like 2 (BARHL2), which belongs to the evolutionary highly conserved family of homeodomain-containing transcription factors. This review describes the activities and regulatory modes of TCF/LEF as transcriptional repressors, with a specific focus on the functions of  Understanding how the vertebrate nervous system emerges from a homogeneous layer of neuroepithelial cells, the neural plate, has long been a subject of intense fascination. Fate-mapping experiments performed at the end of the 20th century demonstrated that the primordia of the forebrain, midbrain, hindbrain, and spinal cord are all already established along the antero-posterior (AP) axis when the neural plate emerges. These studies revealed that a construction blueprint of the neural organization, and specifically that of the forebrain, is set up during gastrulation  and in flies, where a single TCF/LEF has been characterized, provided evidence that TCF/LEF act through a transcriptional switch, which either activates or represses Wnt/β-catenin target genes’ expression. This feature has been further validated in vertebrate, whose genome contains four TCF/LEF members: TCF1, TCF3, TCF4, and LEF1. In this review, the vertebrate TCF/LEF members will be referred to as TCF7 (previously TCF1), TCF7L1 (TCF3), TCF7L2 (TCF4) and LEF1 following the Human Genome Organization (HUGO) nomenclature  . Under cet genes . These dBarhl2. Specific attention is given to the transcriptional events leading to the formation of the Organizer, as well as the roles and regulations of the Wnt/β-catenin pathway in the growth of the caudal forebrain. We present core activities of TCF/LEF in Embryonic Stem Cells (ESCs) self-renewal and pluripotency, and maintenance of NSCs, as well as their identified deregulations and the emergence of cancer.In this review, we present the activities and regulatory modes of TCF as transcriptional repressors with a focus on the developmental roles of TCF/LEF proteins are the major mediators of Wnt-responsive gene transcription in the nucleus. In the absence of Wnt ligands, β-catenin is phosphorylated by the destruction complex containing Glycogen Synthase Kinase 3β (GSK3β), Casein Kinase 1 (CK1), Adenomatous Polyposis Coli (APC) tumour suppressor protein, and Axin. Phosphorylated β-catenin is targeted towards ubiquitination and further proteasome-mediated degradation. In the nucleus, inhibitory TCF/LEF members are bound on Wnt Cis-Regulary-Motifs (W-CRM), interact with co-repressors such as Gro/TLE proteins, and act as transcriptional repressors. Conversely, in Wnt-stimulated cells, the destruction complex is inhibited leading to the cytoplasmic accumulation of β-catenin and further nuclear translocation. Increase in the nuclear β-catenin levels transiently converts TCF/LEF into transcriptional activators  reviewe.http://www.stanford.edu/group/nusselab/cgi-bin/wnt/.Observations from mammalian cells , flies , and ampSaccoglossus kowalevskii , Caenorhabditis elegans (C. elegans), Drosophila melanogaster, Hydra magnipapillata, and Ciona intestinalis reveal the presence, in the TCF/LEF structure, of the four major binding domains found in vertebrate  that allows TCF/LEF to bind an additional DNA motif known as the Helper site (5′-RCCGCCR-3′)  .Tcf/Lef gene is detected , the Tcf/Lef in fly (referred to as Tcf) (Most of our knowledge about TCF/LEF activity in invertebrate derives from studies performed in  as Tcf) . Consequ as Tcf) . As in v as Tcf) . Transcr as Tcf) .C. elegans, loss-of-function phenotypes indicate a dual regulatory mode for the Tcf/Lef termed POsterior Pharynx defect (POP1). An interesting mechanism has been reported for mesoderm and endoderm fate specification during embryogenesis (pop1 are detected in the MS blastomere (end1 through the recruitment of the histone deacetylase HDA-1 and UNCoordinated (UNC)-37 (the homologue of the Gro/TLE)  . In a POmal fate . Anothercription .In vertebrate, the founder members of the TCF/LEF family are TCF7  and LEF1Tcf7l2 and Lef1 transcripts are detected in the mesencephalon and the diencephalon  mutants, exhibit severe developmental alterations . The early Xenopus embryo expresses three maternally inherited tcf/lef mRNAs: tcf7, tcf7l1 and tcf7l2   in the amphibian d tcf7l2 . Tcf/Lefpression , whereasSO genes . In thishenotype . Taken ton (MBT) , and duron (MBT) .All the TCF/LEF members need to bind with nuclear co-factors to regulate target genes’ transcription. A key insight into the mechanism of Wnt transcriptional inhibition mediated by the TCF was the finding that they can directly bind to members of the Gro/TLE family of transcriptional co-repressors.Drosophila, and UNC-37 in C. elegans, both of which antagonize signaling by Wnt -rich domain (FxIxxIL), required for transcriptional repression  in vivo . The EH1 in vivo , Forkhea in vivo , Engrail in vivo  and BARH in vivo . The TF epressor . It has epressor . This inepressor .via Hairless to a complex containing Suppressor of Hairless (Su(H)) and CtBP, which represses Notch target genes, including E(spl). Upon activation of Notch signaling, the Notch intracellular domain (NICD) enters the nucleus, displaces the Gro-containing complex, recruits Mastermind (Mam) on Su(H) an interaction which further results in the transcriptional activation of E(spl). E(spl) encoded factors interact with Gro/TLE to repress proneural genes  proteins including the Hairy/Enhancer of Split (E(spl))/HES proteins, transcriptional repressors that function as downstream targets of activated Notch receptors  reviewe. In the ogenesis . The WRPogenesis . Similar complex .Xenopus embryos provide over 80% correlation between β-catenin and Gro/TLE-binding sites  activity, and/or long distance via chromatin remodeling. ChIP-seq analysis combined to RNA-seq data performed in Drosophila identified the Gro/TLE direct targets. Such analysis suggested that Gro/TLE doesn’t affect the recruitment of RNA-Pol II to the transcription start sites but further increases RNA-Pol II pausing time  promoter demonstrated that Tcf/Lef-binding sites mediate both basal repression and β-catenin-dependent activation at the W-CRM  of such transcriptomic regulations during development are still poorly understood.In conclusion, the mechanisms by which Gro/TLE mediate transcriptional repression in the presence and/or absence of TCF/LEF are still not fully understood. To add complexity, both TCF/LEF and Gro/TLE proteins are targets for developmental signals, which influence the affinity of Gro/TLE to TCF/LEF and/or W-CRM. Thereby, the developmental and cellular contexts in which Gro/TLE repression causes epigenetic regulations Xenopus, wnt1  (goosecoid (gsc) and chordin (chd), leading to the formation of the SO territory. All genes de-repressed by β-catenin in this region have been identified   , which ientified . sia andon (MBT) . In the cription . More recription , Bone Mocription . These scription .Drosophila , amphibian (Xenopus), birds (chicken), mammals , nematode (C. elegans) and S. kowalevskii among others -mutant embryos carrying a mutation in the GSK3-binding domain of Axin1, which constitutively activates Wnt signaling, show a net reduction in the telencephalic and retinal territories in favor of the diencephalic territory  is derived from the most anterior part of the neuroepithelium: the prosencephalic neural plate. Fate mapping analysis revealed that the telencephalon emerges from the most anterior part of the neural plate, whereas the diencephalon is formed within the caudal forebrain.Whereas inhibition of Wnt pathway is strictly necessary for telencephalic development  reviewe, growth nal cord . Convers neurons . Zebrafierritory . Indeed,nd Wnt2B . Wnt tarnd Wnt2B .c-Myc , develops within the diencephalic primordium -secreting Mid-Diencephalic Organizer (MDO), also known as the imordium . Within ulations . Mice laerritory . Investiyclin-D1 . Phenotypression .barhl2 transcripts are detected in the diencephalic histogenic field at late gastrula/early neurula stages in Xenopus . As the length of the S-phase in these cells remains unchanged (1.5 h), and CyclinD1 is part of the G1-S cell cycle checkpoint, Barhl2 probably acts on the length of G1 phase  inhibits its capacity to bind its target genes , phosphorylates Gro/TLE, and leads to the weakening of its repressor function, and attenuation of Gro/TLE-dependent transcriptional silencing by the E(spl) proteins, which are the effectors of the Notch cascade . VCP unfolds ubiquitinated proteins via its ATPase activity and disrupts ubiquitinated Gro3/TLE3 tetramerization, a process required for Gro/TLE to repress Wnt targets  signaling cascades . One exa cascade . Reversilization . In vivocription . Interes targets . Moreove targets . Because targets . HoweverS. kowalevskii, which is thought to be the closest species to the common ancestor at the base of the phylogenetic tree of chordates, Barhl2 ortholog gene shares close similarities in its distribution and expression patterns compared to chordates  undergo PCD during hermaphrodite embryogenesis but not in males  promoter , Wnt/β-catenin signaling appears to be actively repressed in an OCT4-dependent manner during self-renewal. In these cells, activation of Wnt signaling appears to induce loss of self-renewal, and differentiation into mesodermal lineages . Althougpromoter .Axin2, demonstrates the continued importance of persistent Wnt/β-catenin signaling for NSCs and progenitor cells emergence (NEUROD1) is a landmark of cells dropping out of self-renewal and entering neuronal commitment. Overlapping binding sites for the TCF/LEF factors and SOX2, a marker of most uncommitted cells of the CNS, are present in the promoter region of Neurod1. In this context, Wnt signaling together with removal of SOX2 triggers the expression of NEUROD1, demonstrating that the SOX2-TCF/LEF regulatory elements are critical for NEUROD1 expression, and consequently for the switch from the SOX2-mediated repression to the TCF/LEF-mediated activation, towards a neuronal fate , the most common childhood malignant brain tumor, emerges from the cerebellum and accounts for 30% of pediatric brain tumors. Integrated genomic studies allowed the identification of four types of human MB, corresponding to specific genetic alterations  is the master gene of GNPs’ development  produces the GNs that constitute the largest neuronal population in the brain. The GN population exhibits a unique developmental trait: committed GN progenitors (GNPs) are characterized by a very long period of “quiescence” occurring before birth, followed by a long proliferative phase – i.e., 2 weeks in mouse, 2 years in human - occurring after birth, before their final differentiation step  of TCF/LEF activities, including their interactions with the Barhls, are unresolved. An important point is to clarify the interactions between Barhl2, and generally the Barhls, with the different TCF/LEF members: which Barhl interacts with each of the TCF/LEF family? Are these interactions specific to the different TCF/LEF isoforms and their splice variants? What characterizes the TCF/LEF-BARHL binding interface? Is the interface evolutionarily conserved? Numerous signaling pathways interplay to orchestrate the multipotency/commitment/proliferation states of neural stem/progenitor during embryogenesis. Besides Wnt signaling, the activation and/or inhibition of TCF/LEF activity is under the influence of Notch, Shh, and MAPK/ERK pathways. The context, the repression partners, and the PTM involved in controlling these subtle levels of regulation in NSCs are still poorly understood. Exploration of via BARHL2/TCF7L1 activity. Moreover, understanding how the BARHL2/TCF7L1 modulates the open/close state of the chromatin, together with probable roles of PTM on the complex stability and its transcriptional activity, shall prove quite beneficial beyond understanding early embryogenesis. Finally, in both amphibian, and rodent, Barhl2 participates in the formation of the caudal forebrain organizer, partly through its direct activation of Shh transcription together with Otx2 (Another important unresolved question relates to how BARHL2, TCF/LEF and Gro/TLE act long distance to transcriptionally inhibit key “commitment” genes. Understanding the specificity of BARHL2 DNA-binding alone, or together with TCF/LEF, should be a first step in identifying the set of genes whose expression is silenced ith Otx2  (revieweith Otx2 . Thereby


Recent studies of transcription have revealed an advanced set of overarching principles that govern vitamin D action on a genome-wide scale. These tenets of vitamin D transcription have emerged as a result of the application of now well-established techniques of chromatin immunoprecipitation coupled to next-generation DNA sequencing that have now been linked directly to CRISPR-Cas9 genomic editing in culture cells and in mouse tissues in vivo. Accordingly, these techniques have established that the vitamin D hormone modulates sets of cell-type specific genes via an initial action that involves rapid binding of the VDR–ligand complex to multiple enhancer elements at open chromatin sites that drive the expression of individual genes. Importantly, a sequential set of downstream events follows this initial binding that results in rapid histone acetylation at these sites, the recruitment of additional histone modifiers across the gene locus, and in many cases, the appearance of H3K36me3 and RNA polymerase II across gene bodies. The measured recruitment of these factors and/or activities and their presence at specific regions in the gene locus correlate with the emerging presence of cognate transcripts, thereby highlighting sequential molecular events that occur during activation of most genes both in vitro and in vivo. These features provide a novel approach to the study of vitamin D analogs and their actions in vivo and suggest that they can be used for synthetic compound evaluation and to select for novel tissue- and gene-specific features. This may be particularly useful for ligand activation of nuclear receptors given the targeting of these factors directly to genetic sites in the nucleus. P uptake, on the other hand, is regulated in the intestine by induction of the P transporter gene SLC34A2 [NCX1, while P is regulated by SLC34A1 and SLC34A3 and indirectly via 1,25(OH)2D3’s regulatory control of hormonal PTH and FGF23 [2D3 and PTH, primarily via RANKL expression, a TNF-like factor that is produced in stroma, osteoblastic cells, and osteocytes and is essential to the formation, activation, and survival of bone-resorbing osteoclasts [FGF23, a primarily osteocyte-produced hormone [2D3 and the vitamin D hormone interchangeably) involves genetic control of Ca and P reabsorption, as described above, its actions also extend to many additional genes [2D3 is to regulate the expression of renal Cyp27b1 and Cyp24a1, two genes that, in the kidney, are responsible for the synthesis as well as turnover of 1,25(OH)2D3 [2D3 but also by FGF23, both of which suppress Cyp27b1 expression and induce Cyp24a1. These two genes are also regulated by PTH, which induces Cyp27b1 while suppressing Cyp24a1 [2D3 in the blood. Individual disruption or loss of expression of any of these hormones, either via genetic manipulation, altered hormonal metabolism, or through disease, results in a significant impact on extracellular Ca and/or P levels in vivo, supporting their essential role in the regulation of mineral homeostasis.The fundamental actions of the steroid hormone 1,25-dihydroxyvitamin Dnd FGF23 ,8. Activeoclasts ,10,11. Reoclasts . The viteoclasts ,14,15. T hormone ,17,18. Wal genes ,20. Inde5(OH)2D3 . This re Cyp24a1 . These t2D3 regulation through genes that are essential for unique biological responses (see below). It has also been suggested that many of these cell types may also synthesize and catabolically degrade 1,25(OH)2D3 locally via Cyp27b1 and Cyp24a1, respectively [2D3. In support of this concept, the regulation of Cyp27b1 in non-renal cell types differs substantially from that in kidney. Accordingly, this gene is regulated not by PTH, FGF23, or 1,25(OH)2D3, as in kidney, but rather by a variety of inflammatory cytokine modulators [Cyp24a1, on the other hand, is not regulated in these tissues by either PTH or FGF23 as in the kidney but remains sensitive to 1,25(OH)2D3 [Cyp24a1 in these cells may be to prevent 1,25(OH)2D3 toxicity. These differences in regulation support the idea that local production of 1,25(OH)2D3 may function in these cell types in a fashion distinct from those involved in mineral metabolism. While much needs to be learned about the impact of the local production of 1,25(OH)2D3, the diverse actions of the vitamin D hormone in this broad array of tissue/cellular targets supports the concept that vitamin D may play a contributory role in biological processes that extend beyond mineral metabolism. They also imply that vitamin D deficiency could have a unique impact on these numerous biological processes. Thus, restoration of the parent vitamin or the addition of 1,25(OH)2D3 and/or a selective analogue of 1,25(OH)2D3 could be particularly useful therapeutically in diseases such as those of altered immune function or in cancer, where vitamin D may exert beneficial actions. Indeed, by promoting differentiation, 1,25(OH)2D3 not only reduces tumor cell growth but may also function to reduce inflammation through the down-regulation of inflammatory mediators [In the past decade or more, however, the actions of vitamin D have also been increasingly linked to a diverse set of important biological activities in non-renal tissues that appear to be separate from those that are involved in mineral regulation . In manydulators ,29,30. Cediators . While a2D3 might be designed to selectively target tissues involved in these secondary biology activities of vitamin D, chemists have synthesized and examined hundreds of biologically active vitamin D-like compounds for tissue-selective activity over several decades or more. The identification of compounds potentially useful therapeutically has, however, met with generally limited success. We posit that this may be due to the lack of rigorous structure/function assays in vivo that are necessary for guiding the efforts of synthetic chemists. In this article, we suggest that advances in our understanding of vitamin D action at the transcriptional level in vivo may provide not only useful structure/function relationships, but also detailed genomic analyses that could well identify the underlying mechanisms through which a novel analog might operate to manifest tissue, cell, and gene selectivity. Thus, this article represents a combination of transcriptional review, opinion piece, and proposal for incorporating new methods into the analysis of both existing as well as de novo synthesized new vitamin D chemical entities.Considering the possibility that analogues of vitamin D and/or 1,25(OH)2D3-dependent heterodimer with a nuclear accessory factor (NAF) that was required for DNA binding; subsequent experiments proved that this factor was RXR, a member of the NR family composed of three isoforms [Early studies using radioactive ligands served to both identify the existence of the VDR and to facilitate preliminary characterization and purification of the protein itself, an approach that also led similarly to the characterization of nuclear receptors (NR) for other soluble endocrine hormones . It was isoforms ,41,42,43isoforms . In totaisoforms ,45. Many2D3 and initial investigations into the utility of this hormone as a therapeutic for multiple diseases. Many of these maladies altered vitamin D metabolism and thus provoked crucial deficiencies in extracellular Ca and/or P that resulted in loss of bone mineral density and skeletal abnormalities. Classic genetic diseases included vitamin D dependent rickets type 1, later identified as arising from a mutation in the renal Cyp27b1 gene that causes crippling 1,25(OH)2D3 deficiency due to an inability to hydroxylate 25(OH)D3 at the 1α position, and vitamin D-resistant rickets due to mutations in the VDR [2D3, pioneered initially by Haussler and his colleagues [2D3 was limited in many instances, however, by its potency and narrow therapeutic window, which frequently prompted hypercalcemia through predictable actions in intestine, kidney, and bone. These clinical difficulties prompted strong efforts by chemists to synthesize analogues of vitamin D and/or 1,25(OH)2D3 that were of greater practical utility, and perhaps to identify analogues with reduced “side-effects” relative to hypercalcemia. Reviews of these efforts and the many analogues that were synthesized have been documented extensively over several decades [2D3 that were separate from those influencing mineral metabolism  became evident, chemists attempted to home in on the creation of analogues with preferential biological activities that lacked calcemic activity. These selective disease targets included those related to chronic kidney disease and its eventual consequences on the skeleton and the cardiovascular system, as well as on hyperproliferative disorders that included cancer [2D3 was predicted to ameliorate disease onset  has not been particularly effective in clinical trials [The biological actions of vitamin D to control mineral metabolism prompted the early synthesis of 1,25(OH) the VDR . Much oflleagues . The the decades . While td cancer ,51,52,53l trials ,56,57,58l trials . One mig2D3 are impacted upon the formation of active VDR complexes on DNA within the nucleus of cells. We will discuss new ways below, wherein altered or disrupted actions of the VDR at individual genes and in different tissues might be determined not only in vitro but also in vivo.How might analogues operate mechanistically? The possibilities are enormous and include such features as the individual pharmacologic, pharmacokinetic, and/or pharmacodynamic properties of the compounds themselves. Speculatively, these also include parameters impacting the distribution of the compounds based upon their interaction affinity with vitamin D binding protein (DBP) and other serum-interacting factors, requirements for activation by residual renal and non-renal enzymes, and metabolic outcomes that could lead to retention of residual vitamin D-like activity. They also include the potential for selective tissue distribution and localization, relative rates of tissue uptake, as well as unique metabolic degradation rates determined either through catabolic vitamin D enzymes or by novel degradative enzymes not known routinely to degrade vitamin D compounds. With regard to the VDR, the relative biological activity of an analogue is almost certain to be affected by its affinity for the receptor, the longevity of the ligand receptor complex at its sites of action on DNA, and/or the compound’s novel impact on VDR structure. The latter could uniquely alter receptor function at numerous downstream mechanistic levels prior to or while bound to DNA at target gene loci, thereby altering the transcriptional outcome in a potentially selective manner. While the impact of ligand-dependent pharmacodynamic issues has only briefly been considered, the impact of novel ligands on VDR structure has been an attractive hypothesis for years, and has garnered much attention, largely due to the possibility that altered VDR structure mirrors that seen for several other steroid receptors, particularly the estrogen receptor ,66,67,682D3 pretreatment, indicating that VDR represents a ligand-dependent DNA binding factor. RXR DNA binding is also modestly induced by 1,25(OH)2D3 at these VDR occupied sites as well, although RXR can be residually present even in the absence of ligand. This has suggested that prebound RXR may serve an initial role, perhaps to facilitate (affinity) and/or mark specific DNA sites for subsequent VDR binding that is dependent upon DNA half-site configuration. As RXR interacts with numerous additional NRs, it is also possible that RXR may exist at some of these sites with an alternative partner [The development of chromatin-immunoprecipitation and other genomic procedures coupled to next-generation DNA sequencing technics  and their applications have fully changed the way in which gene regulation is evaluated. This technique has widespread applications, including the capacity to identify and characterize ligand-induced transcription factor binding activity, coregulator recruitment to specific genomic sites, and to assess downstream actions such as RNA polymerase II recruitment in a generally unbiased, genome-wide fashion . These s partner . Neverth partner ,15. De n partner ,74,79. R partner ,81,82,83 partner .Since multiple enhancers may control a single gene, and the localization of transcription factors at sites on the genome does not identify the gene or genes they regulate, it is certain that enhancer location near a gene cannot be used to determine a gene target. Accordingly, the genome-wide number of VDR binding sites in a cell, for example, is not useful in determining the number or identity of genes that are regulated by the hormone. Indeed, the repeated suggestion that the VDR regulates a high percent of genes in the genome is highly misleading, since the VDR binds in a cell-specific manner to only small, although overlapping, subsets of genes in any given cell type. Furthermore, temporal delays in regulation suggest that many if not most purported target genes for vitamin D likely result from indirect secondary and tertiary activity. These evolve from vitamin D’s traditional capacity to induce autocrine and paracrine growth factors and/or endocrine hormones as well as other components that influence in turn the activity of multiple cellular signaling pathways. It would also appear that the activation of some rapidly induced genes are temporally consistent with a direct action, yet represent activities of a secondary nature as well, as appears to be the case for FGF23 . Details2D3 that result from the binding of the VDR to its site(s) of action can be assessed, and that detailed consequences arising from the activity of prospective vitamin D analogs may also be determined in an endogenous gene context. Thus, temporal relationships, tissue and cellular specificity, and gene selectivity and coregulator recruitment specificity (or lack thereof) can all be determined directly at this endogenous level. Of additional advantage is the fact that these studies can frequently benefit through an assessment of the overarching functional roles of specific enhancers deleted at the genes of interest using CRISPR/Cas9 methods in cells and in animals in vivo [2D3. These assays might now provide a rational SAR approach for driving the synthesis of new vitamin D analogues with directed tissue activities as well.The discovery of novel pathways, as well as insights into the mechanisms of existing and now well-described regulators of transcription, provide reliable avenues through which mechanistic advances can be made and new compounds with drug potential can be synthesized and assessed. Certainly, the overarching principles described in  in vivo ,91. Thus2D3. This effort focused initially upon a variety of cell types in vitro, where we identified many of the principles enumerated in We have taken a pragmatic approach to the study of gene expression and its regulation by 1,25(OH)Bone genes: We first explored the genome-wide DNA binding site profile (termed a cistrome) for VDR and RXR in osteoblastic and osteocytic cell lines. These initial studies as well as others highlighted the interaction of the VDR at multiple sites near many genes involved in bone cell maturation, bone formation, biomineralization, and osteoclast-mediated bone resorption. As an example, we initially explored the effects of 1,25(OH)2D3 on the Tnfsf11 gene for RANKL in both cell types [2D3 induced the localization of both VDR and RXR to four regulatory regions that extended upstream of the gene to 76 kb. Neither VDR nor RXR bound to regions near the Tnfsf11 gene promoter. Additional factors bound to a subset of these enhancers including a master bone regulator RUNX2 and a potential pioneering factor CEBPβ [Tnfsf11 segment in mice in vivo, resulted in a blunting of both 1,25(OH)2D3 and PTH induction [Tnfsf11 promoter had no effect, confirming that this gene was not induced through promoter-proximal sequences [Tnfsf11 by these two hormones is achieved through multiple distal enhancers. Many additional genes have been explored in bone cells in this manner including the VDR itself, Mmp13, Lrp5, and Enpp1 to name a few. These and other studies therefore reveal roles for not only VDR and RXR, but other factors that are essential to 1,25(OH)2D3 action as well.ll types ,95,96. Aor CEBPβ . These fnduction . Deletioequences . Thus, t2D3 was also linked to those regulated by PTH through the frequent presence CREB at VDR/RXR binding sites [2D3 in enhancing the expression of two co-receptors essential to the Hedgehog pathway activity. This latter observation illuminated an example of the crosstalk that is provoked by 1,25(OH)2D3 and that is likely to initiate transcriptional effects secondary to those that are direct. Sites of VDR action were also characterized by the presence of active VDREs and classic epigenetic histone signatures that were indicative of dynamic regulatory chromatin architecture. At the Tnfsf11 gene, as seen in Tnfsf11 expression. In many genes, all the above events, including co-localization of coregulators, the presence of RNA polymerase II, and enrichment of H3K36me3, a histone mark present at the gene body itself and reflective of the induction of transcripts, were also seen. These features are linked directly to the transcriptional output of genes themselves. Thus, they provide direct evidence for rapid 1,25(OH)2D3 modulated transcript output that can be detected at the genomic level and correlated via RNA analysis. Finally, the impact of many of the regulatory features identified for some of the bone genes listed above were explored in the mouse in vivo through loss of expression studies, wherein regulation of the expression of the gene of interest was eliminated entirely upon deletion of the specific enhancer region. These results show that the identified enhancers and the activities they engender are linked directly to specific genetic output in vivo. This demonstration is essential, since, as indicated earlier, enhancers may be located many kilobases (kb) distal to the genes they regulate. It also provides additional opportunities to understand how non-vitamin D regulators participate with 1,25(OH)2D3 in the expression of specific bone genes. We would anticipate that vitamin D analogues would show alteration in one or more of these progressive events during activation in vivo, perhaps in a tissue-specific manner.In osteocytes, the activity of 1,25(OH)ng sites . These sFgf23 gene, despite the fact that 1,25(OH)2D3 induces Fgf23 transcripts in these cells [Fgf23 by 1,25(OH)2D3 may be indirect, perhaps through a primary DNA binding factor(s). One approach is to conduct enhancer deletion experiments in the mouse in vivo. Thus, an enhancer region that plays an extended role in 1,25(OH)2D3 induction of Fgf23, as established in vivo, could be dissected via loss of regulation studies to identify a more restricted genomic segment capable of mediating 1,25(OH)2D3 induction [2D3.Interestingly, the successful study of VDR/RXR binding across several bone cell genomes failed to identify the presence of either VDR or RXR across the extended locus that surrounds the se cells . This obnduction . This apIntestinal epithelial genes: Aside from functional studies, it has been the use of ChIP-seq analyses that has enabled a complete transition to the evaluation of gene regulation in vivo. Indeed, in addition to the advantages listed earlier, this approach provides opportunities for study at both tissue/cell- and genome-wide levels that are unparalleled. It may also facilitate a detailed study of regulation at genes of therapeutic interest using mouse disease models. 1,25(OH)2D3 action in vivo has been explored at different mechanistic levels in intestinal epithelia and in kidney cortex . In intestinal epithelial cells isolated from mice pre-injected with 1,25(OH)2D3, the VDR is bound to multiple genes across the mouse genome; its binding is enhanced by prior treatment with 1,25(OH)2D3 [S100g  and Trpv6; others are linked to intracellular Ca regulation and to the transport of elements such as manganese. The enhancers for Trpv6 were validated previously in a human intestinal cell line. VDR/RXR also induced the intestinal P transporter Slc34A2 (5(OH)2D3 ,101,102. Slc34A2 C as wellKidney Genes: Perhaps the most extensive analysis of the mechanistic actions underpinning 1,25(OH)2D3 action in vivo have been conducted recently in the kidney. Multiple genes represent targets of vitamin D action in this organ. Indeed, multiple VDR and RXR binding sites are evident at Trpv5, Trpv6, and S100g, as seen in the ChIP-seq tracks in 2D3 actions that are involved in Ca regulation in the kidney. Epigenetic chromatin activity is also seen at these sites for H3K27ac modification. VDR binding sites are also present upstream of the Slc34a1 and Slc34a3 genes that encode transporters involved in P reabsorption. Fgf23 displays actions in the kidney to regulate P transporter location, thus controlling P diuresis. These actions involve specific isoforms of the FGFRs, whose ability to bind Fgf23 is influenced by Klotho. The ability to dissect the many players involved in the regulation of genes in the kidney, bone, and intestine that are involved in the maintenance of Ca and P homeostasis is important. It suggests that they may prove useful in identifying unusual features of vitamin D analogs that have direct non-calcemic and/or P diuretic properties that may occur in a tissue-selective manner.2D3 also plays a striking role together with PTH and FGF23 in modulating the expression of Cyp27b1 and Cyp24a1 in the kidney. The Cyp27b1 gene is induced by PTH but suppressed by 1,25(OH)2D3 and FGF23, while the Cyp24a1 gene is reciprocally regulated by each of these three hormones [2D3 secretion into the blood. The basal and regulated expression of Cyp27b1 and Cyp24a1 is mediated by each of the three hormones via a complex regulatory module located within introns of two upstream genes adjacent to the Cyp27b1 locus and in an intergenic location downstream of Cyp24a1, as seen in 2D3, respectively. These same enhancers also mediate the actions of FGF23, although the factor responsible for this hormone’s activity is still unidentified. Most importantly, the entire intronic regulatory module at Cyp27b1 and some segments of the intergenic regulatory complex downstream of Cyp24a1 are unique to the kidney due to the presence of novel open chromatin structures. These open chromatin modules are absent in non-renal tissues, with the exception that several components of the Cyp24a1 module that mediates 1,25(OH)2D3 action are retained across all tissues [Cyp27b1 regulatory module eliminates basal and regulated Cyp27b1 expression selectively in the kidney, while removal of the downstream Cyp24a1 regulatory module eliminates basal, PTH, and FGF23-regulated expression selectively in the kidney. 1,25(OH)2D3 regulation of Cyp24a1 is retained in all tissues. Interestingly, treatment by PTH also leads to a striking enhancement of H3K27 acetylation (H3K27ac) at CREB-binding sites in the Cyp27b1 gene locus in the kidney but suppresses H3K27ac at CREB-binding sites in the Cyp24a1 locus 2D3, in contrast, leads to a striking suppression of H3K27ac at VDR binding sites in the Cyp27b1 gene locus in the kidney, but strongly induces H3K27ac at VDR binding sites in the Cyp24a1 locus. Thus, again, H3K27ac levels correlate uniquely with Cyp27b1 and Cyp24a1 RNA transcript regulation in the kidney. As a suppressor of Cyp27b1 and an inducer of Cyp24a1, FGF23 actions on H3K27ac mimic that of 1,25(OH)2D3 at the genes for both Cyp27b1 and Cyp24a1. Therefore, similarly to 1,25(OH)2D3, FGF23-regulated H3K27ac levels track uniquely with the downregulation of Cyp27b1 and the upregulation of Cyp24a1 transcripts as well. Consistent with the absence of these modules in Cyp27b1, no changes in H3K27ac are seen in non-renal tissues. However, 1,25(OH)2D3 does induce H3K27ac at selected sites in the Cyp24a1 gene in both renal and non-renal tissues. Finally, detection of H3K36 tri-methylation (H3K36me3) levels increase in response to PTH across the Cyp27b1 gene body but are suppressed in response to 1,25(OH)2D3 and FGF23, while detection of this same histone modification decreases in response to PTH but is increased in response to 1,25(OH)2D3 and FGF23 across the Cyp24a1 gene body. Additional factors linked to the induction or suppression of these two genes are also detected at each of the sites of regulation in both genes.As discussed in the Introduction, 1,25(OH)hormones ,90. This tissues . Accordia1 locus A,B. Thus2D3. It seems likely that a vitamin D antagonist would reveal itself rapidly under this type of molecular scrutiny as well.Exploring the effects of synthetic analogues of vitamin D using this combination of in vitro and in vivo approaches has the clear potential of revealing molecular differences relevant to unique analogue action. These differences could emerge with authentic interacting factors such as coregulators specific for individual gene targets under investigation if an analogue exerts its actions via a structural alteration that provokes a functional change in the VDR. It is now clear that many of the original hypotheses regarding the basis for coregulator recruitment at genes derived from traditional transfection methods have not been entirely correct. Since current studies are routinely conducted on a genome-wide basis, genes selected for their specific function in Ca and P homeostasis in bone could provide a focus for designing (SAR), identifying and understanding analogues that exhibit non-calcemic actions in an endogenous context relative to 1,25(OH)2D3 and their potential application to the analysis of the vitamin D mediated mechanisms in vitro and in vivo. Based on new techniques that involve ChIP-seq analysis, sites of action of the vitamin D hormone can be identified via the co-localization of the VDR and its partner RXR on endogenous DNA on a genome-wide basis in both culture cells and in tissues in vivo. Detection of gene-specific coregulators as well as the activities of these factors via epigenetic changes to histones may also be assessed. The identity of facilitator proteins that initiate the recruitment and/or distribution of RNA polymerase II across the gene body that result in nascent transcripts could also be examined. The ability to detect each of these component proteins and their activities at early time points in a largely gene-specific manner using the vitamin D hormone, as a one-time control suggests that similar studies could be used to characterize the actions of novel vitamin D analogues. This could readily be accomplished to conduct SAR based on newly synthesized compounds as well as to assess the unique mechanistic bases for unusual vitamin D analogue specificities. Indeed, this process could facilitate either the detection and/or mechanisms through which an analogue might retain a non-calcemic activity directly in vivo.The purpose of this manuscript has been to delineate new approaches to the study of mechanisms related to the transcriptional regulation of genes by 1,25(OH)