N-Ras-RGL2 axis is a distinct signaling pathway for SRF target gene expression such as Egr1 and JunB, as RGL2 Ras binding domain (RBD) significantly impaired oncogenic N-Ras-induced SRE activation. By monitoring the effect of Ras isoforms upon the change of global histone modifications in oncogenic Ras-overexpressed cells, we discovered that oncogenic N-Ras elevates H3K9ac/H3K23ac levels globally in the chromatin context. Importantly, chromatin immunoprecipitation (ChIP) assays revealed that H3K9ac is significantly enriched at the promoter and coding regions of Egr1 and JunB. Collectively, our findings define an undocumented role of N-Ras in modulating of H3 acetylation and in gene regulation.Signal transduction pathways regulate the gene expression by altering chromatin dynamics in response to mitogens. Ras proteins are key regulators linking extracellular stimuli to a diverse range of biological responses associated with gene regulation. In mammals, the three ras genes encode four Ras protein isoforms: H-Ras, K-Ras4A, K-Ras4B, and N-Ras. Although emerging evidence suggests that Ras isoforms differentially regulate gene expressions and are functionally nonredundant, the mechanisms underlying Ras specificity and Ras signaling effects on gene expression remain unclear. Here, we show that oncogenic N-Ras acts as the most potent regulator of SRF-, NF- Ras genes are found in ~25% of all human cancers, with 98% mutations at G12, G13, or G61 GTP  in assay buffer  for 10 min at 30°C. After binding to glutathione beads, the beads were washed with cold washing buffer  and were subject to liquid scintillating counting. Scatchard plot analysis was performed with linear regression using Harvard graphics.GST-fused Ras proteins .To observe DNA synthesis by oncogenic Ras isoforms, the microinjection of recombinant Ras proteins has been described previously . BrieflyCV-1 cells were grown and transiently transfected with reporter gene (100 ng) and expression vectors for oncogenic Ras isoforms along with RalGDS-RBD or RGL2-RBD for 24 hr. Cells were incubated in serum-free DMEM for 20 hr and lysed with reporter lysis buffer and assayed for luciferase activity using luciferase assay system (Promega).2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 5 mM β-glycerophosphate, 10 mM NaF, protease inhibitor, and 0.2% TritonX-100). Nuclei were isolated by centrifugation , and the resulting nuclei pellet was resuspended in buffer B . Chromatin pellet was washed three times with buffer B prior to sonication in Laemmli buffer.Chromatin was extracted as described previously . Brieflyβ-actin mRNA levels. All reactions were run in triplicate, and the data presented are the average of three independent experiments. The following primers were used to quantify target gene expression: Cyr6 (5′-CTCCCTGTTTTTGGAATGGA-3′ and 5′-TGGTCTTGCTGCATTTCTTG-3′), Egr-1 (5′-TGACCGCAGAGTCTTTTCCT-3′ and reverse 5′-AGGCACAAGGGTACAAGACAGT-3′), JunB (5′-TGGAACAGCCCTTCTACCAC-3′ and 5′-GAAGAGGCGAGCTTGAGAGA-3′), Scyl1 (5′-CTCCTCACTCACCTCCAAGC-3′and TGTCCTCTGCTGTGTCCTTG), E2F5 (5′-CTGGAGGTACCCATTCCAGA-3′ and TGTTGCTCAGGCAGATTTTG-3′), Npm1 (5′-AAAAAGCGCCAGTGAAGAAA-3′ and 5′-ACTTCCTCCACTGCCAGAGA-3′), and β-actin (5′-GTGGGGCGCCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTC-3′). For ChIP assays, cells were fixed with 1% formaldehyde and processed for immunoprecipitation using antibodies against H3 and H3K9ac as recently described [ Egr-1 (−0.4 kb) (5′-GCGACCCGGAAATGCCATAT-3′ and 5′-CCTTCTTCCCTCCTCCCAGA-3′), Egr-1 (+0.4 kb) (5′-CCCACCATGGACAACTAC CC-3′ and 5′-CCTGAGGGTTGAAGGTGCTG-3′), JunB (−0.3 kb) (5′-GCACATACTGGGACCCTCAC-3′ and 5′-TGAGTGAGGGGTTTCAGGGA-3′), JunB (+0.4 kb) (5′- AACTCCTGAAACCGAGCCTG-3′ and 5′- CGAGCCCTGACC AGAAAAGT-3′), E2F5 (+04 kb) (5′-GGGCTGCTCACTACCAAGTT-3′ and 5′-CTACACCACGCCGCTAGAC-3′), and Npm1 (+0.2 kb) (5′-TTTTGGCCCCCAAGTTACGT-3′ and 5′-TACCCCAAAGTTCAGGTGCC-3′).For qRT-PCR, total RNA was prepared using the TRIzol reagent (Invitrogen) and cDNA was prepared using M-MLV reverse transcriptase (Promega) according to the manufacturer's instructions. Real-time PCR was performed using the IQ SYBR Green Supermix and the IQ5 real-time cycler (Bio-Rad). Relative mRNA levels were normalized to escribed . The purG12V and H-RasG12V predominantly localized to the plasma membrane and cytosol whereas N-RasG12N was largely found in the perinuclear region. The differential localization of Ras isoforms suggests that these isoforms have distinct functions.In order to evaluate the differential functions of oncogenic Ras isoforms, we first investigated the subcellular localization of Ras isoforms. After cotransfection of oncogenic K-Ras or N-Ras with H-Ras into CV-1 cells, their subcellular localizations were examined by confocal microscopic analysis. As shown in G12V, K-RasG12V and N-RasG12N were increased to 63%, 48%, and 67% of injected cells, respectively , indicating no significant difference in GTP binding activities among Ras isoforms. These results suggest that the differential DNA synthesis activity of oncogenic Ras isoforms can be attributed to distinct downstream signaling pathways rather than differences in GTP binding activity.To further understand the differential roles of oncogenic Ras isoforms, we examined the effect of these isoforms on DNA synthesis using the single cell microinjection technique. We first purified constitutively active GST-fusion Ras isoforms  and thenectively . AlthougG12N > K-RasG12V > H-RasG12V). In additional experiments with other reporter constructs (AP-1 and NF-kB), we found that oncogenic N-Ras is the most potent activator  that are involved in cell cycle progression –26. In octivator . Given tG12V, N-RasG12V, or H-RasG12V and chromatin fractions were prepared from the transfected cells. As confirmed by immunoblotting, H3K9ac and H3K23ac showed an increase of 12-fold and 8-fold after the expression of oncogenic N-Ras, respectively , JunB (11-fold increase), and Scyl1 (2.5-fold increase)  and coding region (+0.4 kb). In agreement with our RT-PCR analysis, the enrichments of H3K9ac at both promoter and coding regions were significantly increased with oncogenic N-Ras transfection (3- to 4-fold increase), as compared to transfection of oncogenic K- or H-Ras transfection (less than 2-fold increase) ( Egr1 and JunB loci (data not shown). Consistent with our RT-PCR analysis, H3K9ac was minimally detected in the E2F5 gene when Ras isoforms were overexpressed  . In contncrease) . Howeverxpressed .Although Ras isoforms have a high degree of sequence homology, there is growing evidence that differences in functional properties of Ras isoforms may be implicated in cancer development , 6, 7. HOur single cell microinjection studies demonstrate that oncogenic N-Ras has a significant increase in BrdU incorporation into DNA during DNA synthesis, thereby enhancing cell proliferation. Concomitant with microinjection data, reporter assays also reveal that oncogenic N-Ras highly transactivates a set of immediate early genes implicated in cell cycle progression. In contrast, transcriptional activity driven by both H-Ras and K-Ras was less than that of N-Ras. These results suggest that oncogenic N-Ras is a more potent transcriptional activator of early cell cycle progression genes than H-Ras and K-Ras.The previous studies demonstrated that specific Ras isoforms preferentially interact with Ras effectors to trigger a variety of signaling pathways , 31. ForRecent studies have indicated that oncogenic Ras is implicated in the alteration of chromatin structures and histone modifications . SánchezSeveral questions have arisen from our study. How can oncogenic N-Ras-RGL2 axis enhance histone H3 acetylation? Which histone acetyltransferases (HATs) are linked to this pathway? Considering the previous report that oncogenic H-Ras regulates CBP and Tip60, histone acetyltransferases, affecting histone acetylations , it is pTaken together, we demonstrated that oncogenic N-Ras stimulate SRF target genes by modulating H3K9ac levels. We found that H3K9ac is highly enriched at the promoter and coding regions of target genes, thereby influencing cancer cell development. This mechanism may provide a novel therapeutic target for cancer treatment.


Sulfolobus solfataricus is a thermoacidophilic member of the archaea whose envelope consists of an ether-linked lipid monolayer surrounded by a protein S-layer. Protein translocation across this envelope must accommodate a steep proton gradient that is subject to temperature extremes. To better understand this process in vivo, studies were conducted on the S. solfataricus glycosyl hydrolyase family 57 α-Amylase (AmyA). Cell lines harboring site specific modifications of the amyA promoter and AmyA structural domains were created by gene replacement using markerless exchange and characterized by Western blot, enzyme assay and culture-based analysis. Fusion of amyA to the malAp promoter overcame amyAp-mediated regulatory responses to media composition including glucose and amino acid repression implicating action act at the level of transcription. Deletion of the AmyA Class II N-terminal signal peptide blocked protein secretion and intracellular protein accumulation. Deletion analysis of a conserved bipartite C-terminal motif consisting of a hydrophobic region followed by several charged residues indicated the charged residues played an essential role in membrane-association but not protein secretion. Mutants lacking the C-terminal bipartite motif exhibited reduced growth rates on starch as the sole carbon and energy source; therefore, association of AmyA with the membrane improves carbohydrate utilization. Widespread occurrence of this motif in other secreted proteins of S. solfataricus and of related Crenarchaeota suggests protein association with membranes is a general trait used by these organisms to influence external processes. Sulfolobus solfataricus is a thermoacidophilic member of the phylum Crenarchaeota. These organisms have unique envelopes including a cytoplasmic membrane comprised of a lipid monolayer surrounded by a glycosylated protein S-layer. Unlike bacteria, they do not have a cell wall made of peptidoglycan. Experimental and bioinformatic studies indicate protein translocation across archaeal cytoplasmic membranes can occur via the Secretory (Sec) pathway and the Twin-Arginine Translocation (TAT) pathway [N-terminal signal peptides for recognition and targeting to membrane-associated translocation components [N-terminal end and the signal peptide hydrophobic domain [S. solfataricus [The archaeon  pathway ,2,3. Promponents . Signal mponents . Class 1mponents . Class 2mponents . Class 3c domain ,5,6. Claataricus ,8,9.S. solfataricus, ~1.6% of the proteome (46 proteins) is predicted to consist of putative secretory proteins with N-terminal signal peptides [In eukaryotes and bacteria, Sec-mediated protein translocation can occur using co-translational or post-translational mechanisms ,10,11. Cpeptides , suggestpeptides . Althougpeptides . At the peptides ,11. DespS. solfataricus homodimeric α-amylase (AmyA) [S. solfataricus was limited by transcriptional repression of the natural promoter arising through the action of a catabolite repression system [One such model protein is the e (AmyA) . AmyA ise (AmyA) ,15,16,17e (AmyA) ,18. It be (AmyA) , includie (AmyA) ,21,22. Ge (AmyA) . Despitee (AmyA) ,24,25,26e (AmyA) . In prevn system . Data prS. solfataricus strains were grown in the basal salts medium of Allen [Archaeal strains and plasmids used in this study are listed in of Allen  as modifof Allen  at 80 °Cof Allen ,31. TrypEscherichia coli DH5α, were performed as described previously [S. solfataricus were constructed by markerless exchange [All chemicals were obtained from common chemical suppliers unless indicated otherwise. Molecular biology techniques, including DNA cloning, PCR, and plasmid transformation of eviously . Overlapeviously  and DNA eviously . Mutant exchange . StrainsmalAp::amyA (SSO1172) promoter fusion strain PBL2058 was constructed using plasmid pBN1062. A fragment containing a fusion between SSO1171 and malAp was obtained by PCR of pBN1081 using primers 1171-BamHI-F and MalAp-1172-OLE-R. Plasmid pBN1081 contained a 572 bp fragment extending 107 nt upstream of SSO1171 through 465 nt of the SSO1171 open reading frame. This region was fused to a 425 bp fragment encoding malAp that spanned regions 425 nt upstream of the malA start codon through 7 nt after the malAp transcription start site. These fused fragments were joined to the amyA open reading frame. Fragments encoding the wild type allele of amyA were obtained by PCR using primers MalAp-1172-OLE-F and 1172-BamH1-R. The SSO1171::malAp fusion to the amyA start codon was created by OLEPCR using amplicons encoding the SSO1171::malAp fusion and amyA with primers MalAp-1172-OLE-F and MalA-1172-OLE-R. The resulting SSO1171::malAp::amyA construct was amplified using primers 1171-BamHI-F and 1172-BamHI-R, inserted into the BamHI site of pPB1035 and integrated at amyA by markless exchange to create strain PBL2058. PCR and DNA sequencing confirmed the genotype of the amyA locus in strain PBL2058. PCR amplification of the amyA wild-type allele with primers SSO1171A and ApuPromR2 produced a 550 bp fragment while PCR of the recombinant allele with the same primer pair produced a 1 kb fragment.The amyA G877Stop allele and was derived by markerless exchange using plasmid pBN1063 (amyAG2692T) and strain PBL2058. The amyAG2629T fragment was amplified using primers 1172-CTER-SbfI-LF and 1172-CTER-XmaI-RR. The G2629T mutation in amyA was created by OLEPCR with primers 1172-CTER-LR and 1172-CTER-RF; both primers encode a T, instead of a G at nt 2629 of amyA, thereby removing a diagnostic NcoI site. Plasmid PB1063 was constructed by insertion of an amyAG2629T amplicon into the SbfI and XmaI sites of PB1035. PCR of amyA alleles from PBL2025 and PBL2059 using primers 1172-CTER-SbfI-LF and 1172-CTER-XmaI-RR produced a single 1.2 kb fragment. Only DNA amplified from the wild type was cut by NcoI and produced two nearly identical fragments (607 bp and 613 bp). DNA sequencing of the PCR amplicons distinguished amyA alleles.Strain PBL2059 encodes the N-terminal signal peptide deletion mutation of amyA (amyA Δ (nt 3-93) and was derived from PBL2058 using plasmid pBN1064. Primers used to amplify the amyA 5′ deletion were 1172-NTER-XmaI-LF and 1172-NTER-SphI-RR. The internal deletion of amyA was created by OLEPCR using primers 1172-NTER-LR and 1172-NTER-RF and removed a diagnostic DdeI site from within amyA. Plasmid pBN1064 was created by inserting an amyA Δ (nt 3-93) amplicon into XmaI and SphI sites of pPB1035. PCR of the wild-type amyA allele from PBL2025 with primers 1172-NTER-XmaI-LF and 1172-NTER-SphI-RR produced a fragment of 1.2 kb that was cleaved by DdeI into two fragments (647 bp and 555 bp). PCR of the amyA Δ (nt 3-93) allele from PBL2064 produced fragment of 1.1 kb that could not be digested using DdeI. DNA sequencing verified the identity of the mutant amyA allele in strain PBL2064. The amyA A2692T recombinant strain, PBL2065, was derived from strain PBL2058 using plasmid pBN1065. The amyA A2692T allele was amplified using primers 1172-CHR-SbfI-LF and 1172-CHR-XmaI-RR. The A2692T mutation was created by OLEPCR using primers 1172-CHR-LR and 1172-CHR-RF that encode a T (instead of A), 12 nt upstream of the amyA stop codon. Plasmid pBN1065 was constructed by inserting amyA A2692T amplicon into the SbfI and XmaI sites of plasmid pPB1035. PCR using primers 1172-CHR-SbfI-LF and 1172-CHR-XmaI-RR of both wild type and amyA Δ (nt 3-93) alleles produced amplicons of 1.0 kb. DNA sequencing was used to distinguish these alleles of amyA. Strain PBL2064 encodes an AmyA was purified from clarified culture supernatants as described previously . Cultureg) for 15 min to remove cells and then concentrated 20-fold by centrifugation using Centricon YM-30 filters . Cells were harvested by centrifugation (7700× g) for 15 min, resuspended in basal salts medium, and lysed by sonication as described [α-Amylase activity was determined using the dextrinization assay  with modescribed . Reactio8 cells was applied to the surface. Plates were incubated at 80N oC for 3 days and Gram iodine solution  was added to the plates to visualize zones of clearing due to starch hydrolysis.Starch plate diffusion assays used a solid medium containing basal salts and 0.6% (w/v) Gelrite (Kelco) and magnesium chloride  as described previously . Starch,g) for 15 min, and resuspended in 20 mM cacodylate buffer (pH 6.0) containing 10 mM NaCl, 0.5 mM MgSO4, and 1 mM phenylmethylsulfonyl (PMSF) prepared in isopropanol, then lysed by sonication. The S-layer and cell membrane were separated from the cytoplasmic fraction as described previously [g) for 30 min in an SW-60 rotor  to separate cytoplasmic and cell envelope fractions. The pellet containing the envelope fraction was then processed to separate membrane and S-layer components using a combination of 20 mM cacodylate buffer containing 0.5% sodium sarcosyl and 1 mM PMSF (Buffer A) and 20 mM cacodylate buffer containing 0.2% sodium dodecyl sulfate (Buffer B). Samples were resuspended in Buffer A, incubated at 37 °C for 10 min, and centrifuged (7500× g) for 15 min. The supernatant containing the solubilized membrane fraction was collected and the same procedure was repeated again using Buffer A and then twice again using Buffer B. The resulting supernatants were collected and combined. The remaining pellet containing the S-layer, was resuspended successively in 20 mM cacodylate buffer (Buffer C) and then distilled water, incubated at 37 °C for 10 min, and centrifuged (7500× g) for 15 min, then resuspended in Buffer C.Cells were harvested by centrifugation  was incubated with sonicated cell pellets for 3 h at room temperature, and blocked overnight in 2.5% (w/v) milk  in PBS buffer (10 mM NaH2PO4 and 150 mM NaCl). Nitrocellulose membranes were washed three times with PBS buffer and incubated with 1:5000 dilution of anti-amylase antibody in 0.5% (w/v) milk in PBS buffer for 2 h at room temperature.Prior to electrophoresis, samples were adjusted to 2% (w/v) SDS and 3 mM β-mercaptoethanol and boiled for 10 min. Proteins were resolved by SDS-PAGE with 10% (w/v) resolving gels and 5% (w/v) stacking gels, and PageRuler prestained molecular mass standards (Fermentas). Chemiluminescent western blot analysis was performed using the ECL system  as previously described ,32, with PBL2004 , to remo PBL2004  with modS. solfataricus P2 complete genome at NCBI (GenBank Accession number: AE006641). Sequences of putative secreted proteins in other closely related species were derived by BLASTp at NCBI using sequences from S. solfataricus strain P2.Sequences of putative secreted proteins  were obtS. solfataricus α-amylase, AmyA, had demonstrated that levels of this secreted protein were responsive to the carbon composition of the medium, a response that constituted part of a catabolite repression-like system [in vivo studies designed to study the role of AmyA motifs on protein function and secretion. To address possible transcriptional regulation, the amyA promoter was substituted using an alternative promoter. An amyA promoter fusion strain was constructed and characterized using the constitutive malAp promoter [malAp sequence including a 7 nt untranslated leader RNA were fused to the first internal methionine codon in the amyA reading frame replacing amyA sequences thought to encode the natural promoter  encoded as amyA . While ipeptides , the in itiation A. Follownditions B. Levelsnditions .To uncouple the relationship between growth and secretion, starch plate diffusion assays were conducted using a complex medium. No zone of starch degradation was detected C,D, sugg898 and Arg899  and PBL2058  were included as positive controls, while strain PBL2004 (amyA::lacS) was included as a negative control. Both strains PBL2059 and PBL2065, containing the C-terminal truncated versions of AmyA exhibited a 50 percent decreased rate of growth relative to controls (amyA+) and PBL2059 (Gly877Stop) had specific activities of 21.8 U/mg and 22.4 U/mg, respectively, thereby excluding an effect of the loss of the C-terminal motif on enzyme function. α-Amylase assays of cultures grown in a defined minimal glucose medium demonstrated there was active α-amylase activity in the culture supernatants of both strains PBL2059 and PBL2065 at levels comparable to the positive controls (amyA (panel B) but readily evident in the wild type (panel C) and cells overproducing α-Amylase as a result of promoter fusion to malAp . These data verify the localization of α-Amylase as a cell-associated surface exposed protein.Growth of the two strains was compared to controls using a defined minimal starch medium. Strains PBL2025  of s region . In unres region ,43,44. Irmicutes . The charmicutes ,47. AlteS. solfataricus, AmyA is secreted into the supernatant while a minor fraction remains membrane associated and accumulates in the cytoplasm. Ablation of the bipartite motif overcomes membrane association while secretion remains unaffected. Importantly, the loss of membrane association strongly affected the efficiency of starch utilization in a liquid medium thereby compromising growth rate. This suggests that starch hydrolysis occurring in close proximity to the cell facilitates subsequent uptake of starch hydrolytic products and in this regard resembles certain aspects of the sortase system employed by Firmicutes to maximize substrate utilization. In Bacilli, protein hydrolases can be covalently attached to the external face of the cell wall via action of the enzyme sortase [N-terminal signal peptide that targets the cytoplasmic membrane and a C-terminal cell wall sorting signal consisting of a sortase-recognition motif, LPXTG, hydrophobic domain and a positively charged C-terminus [Staphylococcus aureus demonstrated that deletion of the hydrophobic domain and positively charged tail abolished cell wall anchoring and the proteins are secreted into the culture supernatant [In wild type  sortase . These pterminus ,50. A geernatant ,50. Thisernatant . The COOernatant ,53. In aernatant . At the N-terminal deletion mutant. This result is in contrast to examples where deletion or mutation of an α-amylase did not perturb cytoplasmic accumulation [N-terminal truncation of AmyA reduces its cytoplasmic half-life by analogy to similar manipulations removing leader sequences [It had been proposed that AmyA contained a lipobox-type signal peptide, based on systematic whole-genome analysis . As showmulation ,56,57. Iequences  and Tat equences .S. solfataricus was controlled by catabolite repression but the mechanism for this effect was not determined [amyA promoter. While amino acid repression had been shown to use a transcriptional regulatory mechanism for other S. solfataricus genes [S. solfataricus strain in which AmyA production is catabolite-insensitive enables in vivo studies to be conducted independently of the carbon composition of the medium, and facilitates efforts to explore further the role of the bipartite motif as a membrane anchor. Previous studies demonstrated that repression of AmyA abundance in the extracellular environment of termined . Represstermined . In thisus genes , the mec


MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.MSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of  •We define a regulatory region governing physiological MSI2 expression in human HSPCs•USF2 and PLAG1 collaboratively control endogenous HSPC-specific MSI2 expression•MSI2 expression and stemness is maintained in culture upon USF2 and PLAG1 co-overexpression•USF2 and PLAG1 exhibit genomic co-localization and associate with autophagy genes MSI2 is an essential human hematopoietic stem and progenitor cell (HSPC) regulator, but knowledge of the mechanisms ensuring its appropriate expression in this context are lacking. Here, Hope and colleagues map the MSI2 promoter functional in hematopoietic cells and identify USF2 and PLAG1 as essential, cooperative enforcers of endogenous MSI2 expression and stemness traits in human HSPCs. Here, we report an approach to address HSC cell fate control through the systematic dissection of the MSI2 promoter functional in hematopoietic cells. Through this strategy, we have identified two TFs that function as cooperative regulators of MSI2 and that together play a key role in HSPC function.The unique ability of hematopoietic stem cells (HSCs) to self-renew while giving rise to mature cells by differentiation enables their regenerative capacity. As such, identification and characterization of key regulators of this property are of both fundamental and clinical interest. The Musashi-2 (MSI2) RNA-binding protein is now recognized as one such key HSC regulator. Its expression is highest in the primitive HSC compartment and progressively decreases upon commitment . MSI2 lon assays . In the xpressed . MSI2 haxpressed . For insxpressed . In the xpressed . Taken tcis elements governing MSI2 expression is contained within this 385 bp region we found its deletion from the full-length 3.2 kb fragment was sufficient to repress luciferase activity to the level of the promoterless reporter   Figure A. Introdreporter A.Figure MSI2-regulators. First we used in silico TF binding site prediction (MatInspector) to identify a total of 107 TF candidates representing 65 different TF families. From within this set we removed those TFs not appreciably expressed in both murine and human HSPCs, where MSI2 expression is known to be concentrated. A final refinement based on an extensive literature search to identify novel TFs, or those implicated in the regulation of stem cell function and/or leukemia, yielded a list of ten candidate regulators of primitive hematopoietic cell expression of MSI2  cells exhibited a significant 40% (1.4-fold) enhanced reporter activity compared with vector controls  contains all annotated functional domains, whereas the shorter form (PLAG1-B) is missing Zn fingers F1 and F2. A third reported slightly smaller PLAG1 translational isoform exists through translation from methionine 100 (PLAG1-S), is missing the first 17 N-terminal residues of PLAG1-B, and thus co-migrates with it on SDS-PAGE  FiguresG and S1.62 cells I. Both P62 cells J and 2K gulation L. TogethUSF2 and PLAG1 resulted in a 40% and 50% decrease in endogenous MSI2 transcription, respectively, in comparison with control cells expressing non-targeting shRNA (shLuc)  targeting  (shLuc) A–3C. In  (shLuc)  (PLAG1-B (shLuc) D. The incontrols D. These MSI2 expression. First we generated a minimal promoter reporter assay containing mutations in both the PLAG1 and USF2 consensus sites. Combinatorial binding site mutagenesis yielded a more significant reduction in minimal promoter activity than when PLAG1 or USF2 binding sites were mutated in isolation  effector CYP1B1 (MSI2 transcription at day 7 of ex vivo culture (D7) but also in the selective repression of CYP1B1 protein (and not its mRNA levels) (We have previously reported that MSI2 enforces HSPC expansion r CYP1B1 . As woul levels) E–5G. Mos levels) H–5J. Tog−16 by Fisher's exact test; de novo motif discovery on sites bound by USF2 or PLAG1. Analysis of the USF2 bound sites uncovered the expected E-box motif for USF2 as the most significantly enriched, as well as a second, less frequent G-rich motif. The motif discovery for PLAG1 revealed not only the G-rich core consensus binding motif recognized by PLAG family members  on two different clones (#4 and #5) of K562 cells co-overexpressing USF2 and FLAG-PLAG1-S . The chr members  but also members D. The mo members E provideDNMT3A, SOCS2, HOXA1, and MSI2  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of those genes that show co-occupancy, there was an abundance of genes involved in autophagy, a pathway important for stem cell control and homeostasis in the blood system  we created ranked lists with publicly available gene expression profiles assembled from sub-fractions of the human hematopoietic hierarchy ranging from primitive (Lin-CD34+) to mature (Lin+)  and thenand MSI2 A. Strikid system   was PCR amplified from BAC clone RP11-784M23 (Roswell Park Cancer Institute) and cloned upstream of firefly luciferase in pGL3-basic (Promega). The transcriptional start site is predicted to be 173 nucleotides upstream of the translational start site. 5′-Serial truncations were generated using primers listed in MSI2 promoter were retained for comparison against publicly available databases of global gene expression profiles of diverse hematopoietic subpopulations to facilitate a final selection of genes that are expressed in primitive mouse and human HSCs  online My GeneSet tool.A 3,238 bp 5′-flanking sequence upstream of the translation start site of human man HSCs . For subagenesis . PrimersRenilla luciferase) in K562 cells in triplicate. Luminescence was measured 24–36 hr after transfection using Dual-Luciferase-Assay Kit (Promega) in a FLUOstar Omega luminometer (BMG Labtech). Firefly luciferase activity was normalized to Renilla luciferase activity to adjust for transfection efficiency.Five-hundred nanograms of each clone were transiently co-transfected with 25 ng of pRL-TK  supplemented with protease inhibitor cocktail (Roche diagnostics). Lysate was spun at 14,000 rpm for 20 min on a refrigerated tabletop centrifuge. Total protein concentration was determined by Bradford assay using BSA as standard (Bio-Rad). Ten micrograms of total protein was resolved on 10% Bis-Tris PAGE. Resolved protein was transferred onto an immobilon-P polyvinylidene fluoride (PVDF) membrane (EMD Millipore) on transfer buffer  using Trans-blot Turbo apparatus (Bio-Rad). Membrane was blocked for 1 hr using LI-COR blocking buffer (LI-COR Biosciences). Primary antibodies against MSI2  β-actin , were diluted in LI-COR blocking buffer (1:1000) and incubated for 1 hr at room temperature, washed three times at 15 min intervals by using 1× TBST buffer . IRDye 680 Goat Anti-Rabbit  and IRDye 800CW Goat anti-Mouse  secondary antibodies were diluted  in LI-COR blocking buffer and incubated for 1 hr at room temperature. The membrane was washed three times at 15 min interval using 1× TBST buffer. Blots were imaged using LI-COR Odyssey imaging station (LI-COR Biosciences).PLAG1, β-ACTIN, MSI2, and CYP1B1 . Fold change in transcript level was calculated according to the 2−ΔΔCt method.Total RNA was isolated using Trizol-LS reagent (Life Technologies). cDNA synthesis and qPCR were done using the same protocol as d CYP1B1 C. PLAG1 PLAG1 (Clone Id: 30915361) and USF2 (Clone Id: 5483523) were obtained from Mammalian Gene Collections . cDNAs were PCR amplified using PrimeSTAR Max enzyme and subcloned into EcoRI/XhoI (NEB) digested MSCV-PGK/GFP vector. Primers used were: USF2-sense, CAT GCA TGG AAT TCG CCA CCA TGG ACA TGC TGG ACC, USF2-antisense, CAT GCA TGC TCG AGT CAC TGC CGG GTG CC (EcoRI and XhoI sites are underlined). To generate 3xFLAG epitope-tagged versions of the predominantly expressed variants of PLAG1-B and PLAG1-S, the respective PCR products were generated and cloned into the shuttle vector p3xFLAG-CMV10 (Sigma-Aldrich) at EcoRI/KpnI (NEB) sites. FLAG-tagged clones were PCR amplified and subcloned into MSCV-PGK/GFP vector at EcoRI/XhoI sites using MfeI/SalI (NEB) compatible end ligation. Primers used were: FLAG-PLAG1-sense, GAT CGA TCC AAT TGA TGG ACT ACA AAG ACC, FLAG-PLAG1-antisense, CAT GCA TGG TCG ACC TAC TGA AAA GCT TGA .Open reading frame cDNA clones for PLAG1 isoforms and USF2 were cloned into the bidirectional promoter pSMALB vector where the transgene is driven in one direction and mtagBFP  in the other (PLAG1-100 and USF2 were cloned consecutively into pSMALB and separated by a P2A site so that BFP positivity marks cells co-overexpressing both transgenes. shRNAs against PLAG1 and USF2 were designed using the RNAi Central tool and ligated downstream of the H1 promoter in the modified cppt-PGK-EGFP-IRES-PAC-WPRE lentiviral expression vector (he other . For co-n vector . Lentivin vector . Stable For details of ChIP and all other procedures, see All patient samples were obtained with informed consent and with the approval of the local human subject research ethics board at McMaster University.S.B. and A.K.C. contributed equally. M.S.B. designed and performed experiments, interpreted results, and wrote the manuscript. S.B. assisted with ChIP-seq experiments, performed ChIP-seq analysis, and interpreted results. A.K.C. designed and performed experiments, interpreted results, and assisted with manuscript preparation. S.R. assisted with cord blood experiments and manuscript preparation. J.S.D. designed experiments and wrote the manuscript. K.J.H. supervised the project, designed experiments, interpreted results, and wrote the manuscript.


The Arabidopsis ERFIb / RAP2.4 transcription factor family consists of eight members with highly conserved DNA binding domains. Selected members have been characterized individually, but a systematic comparison is pending. The redox-sensitive transcription factor RAP2.4a mediates chloroplast-to-nucleus redox signaling and controls induction of the three most prominent chloroplast peroxidases, namely 2-Cys peroxiredoxin A (2CPA) and thylakoid- and stromal ascorbate peroxidase (tAPx and sAPx).Arabidopsis thaliana and performed promoter binding studies.To test the specificity and redundancy of RAP2.4 transcription factors in the regulation of genes for chloroplast peroxidases, we compared the DNA-binding sites of the transcription factors in tertiary structure models, analyzed transcription factor and target gene regulation by qRT-PCR in RAP2.4, 2-Cys peroxiredoxin and ascorbate peroxidase T-DNA insertion lines and RAP2.4 overexpressing lines of All RAP2.4 proteins bound the tAPx promoter, but only the four RAP2.4 proteins with identical DNA contact sites, namely RAP2.4a, RAP2.4b, RAP2.4d and RAP2.4h, interacted stably with the redox-sensitive part of the 2CPA promoter. Gene expression analysis in RAP2.4 knockout lines revealed that RAP2.4a is the only one supporting 2CPA and chloroplast APx expression. Rap2.4h binds to the same promoter region as Rap2.4a and antagonizes 2CPA expression. Like the other six RAP2.4 proteins, Rap2.4 h promotes APx mRNA accumulation. Chloroplast ROS signals induced RAP2.4b and RAP2.4d expression, but these two transcription factor genes are (in contrast to RAP2.4a) insensitive to low 2CP availability, and their expression decreased in APx knockout lines. RAP2.4e and RAP2.4f gradually responded to chloroplast APx availability and activated specifically APx expression. These transcription factors bound, like RAP2.4c and RAP2.4g, the tAPx promoter, but hardly the 2CPA promoter.The RAP2.4 transcription factors form an environmentally and developmentally regulated transcription factor network, in which the various members affect the expression intensity of the others. Within the transcription factor family, RAP2.4a has a unique function as a general transcriptional activator of chloroplast peroxidase activity. The other RAP2.4 proteins mediate the fine-control and adjust the relative availability of 2CPA, sAPx and tAPx.The online version of this article (doi:10.1186/s12870-017-1092-5) contains supplementary material, which is available to authorized users. RELATED TO APETALA-2) transcription factors  and afterwards transferred to a growth chamber with a day / night cycle of 10 h light at 20 ± 2 °C and 14 h darkness at 18 ± 2 °C. At an age of 8 days, the seedlings were transferred to individual pots (6 cm diameter) and watered once with 0,5 g L−1 Axoris Insekten-frei . Illumination with 100–110 μmol photons*m−2*s−1 in 10 h light / 14 h dark cycles was performed with L36 W/840 Lumilux Cool White fluorescent stripes . The relative humidity was adjusted to 60% ± 5%.Seeds of .4 SALK-  and GABI.4 SALK- , of 2CP .4 SALK-  and APx .4 SALK-  and a re.4 SALK-  were strArabidopsis thaliana wild-type plants were grown aseptically on 50% MS plates supplemented with 0.5% sucrose [−2 s−1)/ 14 h dark cycles in a Percival CU41 L4 growth cabinet. Surface sterilization was performed as described in [Alternatively,  sucrose  at 10 h ribed in .flu1 mutant [Arabidopsis soil at constant light (100 μmol photons m−2 s−1). Afterwards it was transferred for 8 h into darkness and then re-illuminated with 100 μmol photons m−2 s−1 for 30 or 60 min, respectively.The 1 mutant , 41 was Arabidopsis plants were shifted to 10, 20 and 30 °C warm climate chambers for 1 week and illuminated with the same light regime as described of the standard growth conditions. To modify chlorophyll biosynthesis and photooxidative stress, one third of the 25-day-old plants grown for chlorophyll level analysis were illuminated 3 days for 4 h (starting 4 h after begin of the day period) with 750 μmol photons m−2 s−1. The plant temperature was kept at 18–20 °C with the help of a heat filtering water-bath and an optimized aeration system. The leaf temperature was controlled with an infrared thermometer during the experiment. For the prolonged dark period, the plants were covered with a light-dense shield for 23 h 1 h after the begin of the day period.For the temperature treatments, 2 week old soil grown Arabidopsis thaliana var. Col-0 wildtype plants. Per T-DNA insertion line, eight plants of the progeny were re-tested with the same PCR protocols.Genomic DNA was extracted from rosette leaves of 2–4 week-old plants, according to standard procedures and tested for the T-DNA insertions with a T-DNA border primer and primers binding approximately 500 bp up- or downstream of the proposed T-DNA insertion site  peptone, 0.1% (w/v) yeast extract, 0.5% (w/v) beef extract, 0.5% (w/v) sucrose, 0.5% (w/v) MgCl2). The strains and the agrobacteria strain GV3101 (pMP90) containing the cDNA for the p90 protein under control of the CaMV35S promoter [600 of 0.5. Before transfection, cultures of each RAP2.4 strain was mixed 60:40 with cultures of the p90 strain. After 15 min sedimentation at 3000 rpm at room temperature, the agrobacteria were resuspended in 40 mL activation buffer . After 1 h incubation at room temperature, 5% (w/v) sucrose and 0.02% (v/v) Silwet L-77  were added. Sterile plate cultures of 12 day old Arabidopsis seedlings were flooded with 40 mL of this agrobacteria suspension and six times vacuum infiltrated for 1.5 min. Afterwards, the seedlings were transferred on fresh MS plates containing 100 μg mL−1 cefotaxim and 10 μM estradiol. The plantlets were harvested after 1–2 days and immediately frozen in liquid nitrogen.Genomic DNA was isolated from plant material ground in liquid nitrogen, using the DNeasy Plant Mini Kit . The full-length cDNA sequences of the eight RAP2.4 transcription factors was amplified from the intron-free genes by PCR with gene-specific primers /FM; [−2 s−1) after 30 min dark acclimatisation, using a MINI-PAM fluorimeter .The maximum quantum efficiency of PS-II (F F0)/FM; ) was detGUS staining and GUS activity quantification were performed as described in .Arabidopsis thaliana var. Col-0 wildtype were stratified and grown on 50% MS-medium supplemented with 0.5% (w/v) sucrose, as described in [w/v) sucrose or to plates containing 50% MS-medium, 0.5% (w/v) sucrose and 100 mM NaCl. After 7 days, the root length was analyzed with EZ-Rhizo [Seeds of the KO-lines and of ribed in . Five tiEZ-Rhizo . The roo16 primer and quantitative real-time PCR was performed on a CFX96 real-time System , as described recently [ACT7  and the YLS8  transcript levels [Total RNA was extracted from rosette leaves using the Universal RNA Purification Kit (EURx). cDNA was synthesized from the mRNA, using the High Capacity Reverse Transcription Kit , and 10 μM oligo-dTrecently . Primersrecently   acetone according to [Chlorophyll levels were determined after extraction of the two largest rosette leaves in carbonate-buffered 80% (rding to .p < 0.05 or p < 0.01).Statistic test were performed with SPSS 22 and SPSS 23 . The asterisks mark significance of difference from wildtype . (PDF 87 kb)Chlorophyll levels in young leaves (< 5 mm length) in the centre of the rosettes of 4 week old RAP2.4-KO lines grown under standard conditions  and supplemented with 0, 1 and 3 mM 3-AT. (PDF 121 kb)Additional file 3:Colour map of the relative RAP2.4 transcript levels in RAP2.4 over-expressor (RAP2.4 OE) and RAP2.4 knock-out (RAP2.4 KO) lines. In each sub-figure, the darkest blue represents the strongest accumulation and the darkest red the strongest inactivation relative to the expression level in wild-type Arabidopsis. (PDF 111 kb)Additional file 4: Table S1.Primers used for T-DNA line verification (PDF 48 kb)Additional file 5: Table S2List of primers used for TOPO cloning of RAP2.4 cDNAs (PDF 47 kb)Additional file 6: Table S3.Primers used for qRT-PCR (PDF 56 kb)


Over the last 15 years, development of chromosome conformation capture (3C) and its subsequent high-throughput variants in conjunction with the fast development of sequencing technology has allowed investigators to generate large volumes of data giving insights into the spatial three-dimensional (3D) architecture of the genome. This huge data has been analyzed and validated using various statistical, mathematical, genomics, and biophysical tools in order to examine the chromosomal interaction patterns, understand the organization of the chromosome, and find out functional implications of the interactions. This review summarizes the data generated by several large-scale high-throughput chromosome conformation capture studies and the functional implications obtained from the data analyses. We also discuss emerging results on factors (both CCCTC binding factor (CTCF) related and CTCF independent) that could contribute to looping interactions. Consequently, the notion of a linear chromosomal distribution of various elements of the coding and the non-coding genome is being revisited. This has brought forth a new perspective on how genomic functions such as replication, transcription, and DNA repair would be investigated. Interestingly, the proximity of various regions of the genome, which were otherwise unintuitive, now opens new possibilities by which various aspects of gene regulation would be studied in future. Along with these, herein, we also discuss recent data suggesting the role of telomeres in looping interactions, both near and with interstitial regions of the genome. In addition, factors other than the chromatin-associated protein CCCTC binding factor (CTCF) that could mechanistically contribute to the formation of chromatin loops are also discussed.Recent advances in the field of Hi-C is a high-throughput NGS (next-generation sequencing)-based version of chromosome conformation capture assay commonly known as 3C . It provThough we obtain vast knowledge on the biology of chromosomal interactions and genome organization from Hi-C data, there are some drawbacks of the method as well. The Hi-C data, for instance, does not provide much quantitative information on the stability and strength of chromosomal interactions. Most of the reported Hi-C studies so far used asynchronized cells and did not consider the variability that the different cell cycle stages (particularly mitotic phase) would most likely introduce into the chromatin conformation. Several reviews have further described the technical aspects of these 3C-based assays and complimentary microscopy techniques .In 2009, Lieberman-Aiden et al. used karyotypically normal GM06990 human lymphoblastoid cells to generate 8.4 million reads that uniquely aligned to the human genome reference sequence in a Hi-C experiment; of these, 6.7 million reads corresponded to long-range contacts between segments of the genome > 20 Kb apart. The long-range contact reads were used to construct genome-wide contact matrices by dividing the genome into 1-Mb-sized bins. This revealed an interesting pattern comprising distinct intra- and inter-chromosomal genomic compartments with contacts that were primarily restricted within compartments . Int. Int2]. Intriguingly, probabilities of inter-chromosomal contacts show that small, gene-rich chromosomes  preferentially interact with each other. Furthermore, FISH studies also observed these chromosomes to frequently localize to the center of the nucleus , 13. On Using principal component analysis, each chromosome could be partitioned into two compartments (termed A and B) such that loci within the same compartment had correlated contact profiles. Through this correlation, even loci belonging to different chromosomes were assigned the same compartment, resulting in the whole genome being divided into two spatial compartments such that greater interactions occurred within than across compartments .It was observed that loci in compartment B had a higher tendency for close spatial localization, suggesting a relatively compact state of chromatin. On the other hand, loci within compartment A showed significant correlation with the presence of genes, higher expression, and chromatin accessibility (DNase I hypersensitivity) and were enriched for activating chromatin marks (H3K36me3). Thus, compartment A could be associated with open, accessible, and actively transcribed chromatin. Together, these suggested open and closed chromatin domains throughout the genome occupy different spatial compartments in the nucleus.At higher resolution (bin sizes less than 100 Kb), highly self-interacting regions were found to emerge, seen as triangles in the interaction matrix heat map . These rConsistent with this, fluorescent in situ hybridization (FISH) experiments revealed pairs of loci within a particular topological domain were closer in space than pairs of loci in different topological domains in spite of similar genomic distances between the loci . The genAn in situ Hi-C study using high-resolution contact matrices revealed numerous relatively small contact domains with higher intra-domain contact frequency  like topInterestingly, the in situ Hi-C data revealed six nuclear sub-compartments based on long-range interaction patterns, both intra-chromosomal and inter-chromosomal, using different approaches to clustering. On comparison with compartment A/B , two of The DI data used to identify TADs can vary substantially depending on the sliding window size selected with small window sizes giving smaller TADs and larger ones yielding larger TADs which often nest groups of smaller domains . In factChromosomal regions have been perceived as an “equilibrium globule”—a compact and densely knotted configuration originally used to describe a polymer in a poor solvent at equilibrium , 24. An Lieberman-Aiden et al. analyzed the scaling of contact probability of fractal globule and found that it is close to the contact probability observed from the Hi-C data . The preA strong enrichment of CTCF binding sites was observed at the TAD boundary regions , a prope3D genome organization [Several studies found TADs to be majorly conserved across cell types in a given organism, suggesting TADs to be stable structures of the nization , 9, 30. nization , 31–33. nization , 34, 35.These observations suggest that chromatin organization as TADs is mostly stable across cell types, within which specific structures and dynamic interactions can form to play lineage and context-specific regulatory roles contributing to molecular events associated with differentiation .Interestingly, the topological domains do not seem to be the consequence of heterochromatin formation as the detected boundaries were present in both pluripotent cells and their differentiated progeny, i.e., before and after heterochromatinization associated with cellular differentiation. Importantly, this implied that the topological domains along with boundaries delineate the endpoints of heterochromatic spreading .Furthermore, enrichment of chromatin marks associated with promoters and gene bodies and depletion of repressive chromatin marks were detected in the TAD boundaries along with enrichment of housekeeping genes, transcription start sites, and global run on sequencing signal in the boundaries. Together, these suggest a high level of transcriptional activity associated with boundary formation; however, whether boundary formation was a cause or consequence of transcriptional activity was not clear .Interestingly, Hi-C data also revealed pairs of loci that had significantly stronger interaction than any loci lying between them. These were designated as loops, and can appear within topology domains (discussed above), independently and/or across TADs. Interestingly, chromatin loops were not only conserved among human cell lines but also found to be conserved between mouse and human cells ; chromatSomewhat intriguingly, there was little or no change in promoter-enhancer looping interactions at a vast majority of TNF-alpha-responsive enhancers on TNF-alpha treatment. This suggested that in general, promoter-enhancer contacts in untreated cells, which are the existing DNA loops, did not alter upon transient activation or repression of enhancers following treatment. Interestingly, chromatin interactions involving cell type-specific enhancers are variable between cell types indicating context-specific interaction structures. This discrepancy between signal-dependent and cell type-specific enhancers correlates with H3K4me1 chromatin marks, which unlike H3K27me3 remain unchanged upon TNF-alpha treatment , 38, 39.Analysis of ENCODE ChIP-seq data revealed loci with chromatin loops were typically bound by the insulator protein CTCF 86%) and the Cohesin subunits RAD21 (86%) and SMC3 (87%) 6% and th, 40, 41.Studies by Shay and Wright’s groups have demonstrated that telomeres loop to specific loci (within 10 Mb)—a phenomenon called TPE-OLD (telomere positioning effect—over long distances). These studies showed that genes close to the telomere were silenced in young primary cells with long telomeres, but were activated when telomeres became short with cellular aging, an effect that was reversed by re-elongation of telomeres upon exogenous expression of the hTERT (human telomerase) gene. Interactions of the telomere and sub-telomere with chromosomal regions up to 10 Mb away from the telomere end were revealed using modified Hi-C and 3C experiments –47. An iThough Hi-C has given insights into the functional chromosomal organization, one can argue that this is an average, probabilistic view of chromosomal interactions with much cell-to-cell variation, such that observed domain organization and chromosomal interactions might represent just a fraction of the cells. Some recent studies attempt to address this with Hi-C at the single cell level. In the first such report, pooled data recapitulated the formation of TAD-like structures indicating these domains are robust and form the basis of chromosome conformation in each cell . HoweverThe finding that a large fraction of loop boundaries are bound by CTCF and the Cohesin subunits  led to pThe fact that CTCF motifs can act as boundary elements only when they are oriented in convergent fashion indicate dimerization of CTCF is required for stalling extruding cohesion loops. This indicates that interactions between DNA binding proteins can possibly play a role in mediating loop formation and chromatin interactions by bringing distant genomic loci together. Indeed, DNA binding proteins, YY1 and ZNF143, were found to be enriched in loop loci . Both hoApart from proteins, nucleic acids like lncRNAs and secondary DNA structures might also contribute to mediating chromosomal looping interactions. lncRNAs called as activating/enhancer RNAs were found to bind with mediator proteins (MED12 and MED1), and their knockdown led to diminished looping between the ncRNA loci and their target genes and a decrease in mediator binding to the genes , 60.cis-elements but were depleted in the vicinity of these sites. Computational analyses showed such half G quadruplexes, one from DHS promoter and one from DHS enhancer, could come together to form G quadruplexes thereby bringing together promoters and enhancers elements via looping [Another interesting context that could support loop formation in a CTCF-independent way comes from the possible involvement of DNA secondary structures such as G quadruplexes. G quadruplexes are typically formed by a stretch of DNA with 3-guanines repeated at least four times at close interval and are reported to be involved in various biological functions across life forms –64. The  looping . Several looping –70. The Deletion of a TAD boundary, on the other hand, was found to result in spreading of contacts across the deleted region and transcriptional misregulation . SeveralApart from how abrogation of TADs could lead to pathogenic phenotypes, chromatin looping interactions, even when not associated with a particular TAD, might have significant clinical implications. Recently, an interaction has been reported that might be crucial for TERT reactivation across different cancer types. The two cytosine to thymidine single-point mutations in the TERT proximal promoter (− 146 and − 124 bp from the translation start site), recurrent in several cancers, were found to create a de novo consensus binding motif for ETS transcription factor family –83. GABP3D organization of the genome impacts biological processes, it is of much interest to understand the underlying mechanistic rules governing causation of the folded architecture. Though cohesion and CTCF have been implicated in the formation of chromosomal loops, these proteins are absent in a fraction of loop loci (~ 10–14%) suggesting a role of other factors in determining how chromosomal interactions arise and how these contribute to specific cellular and context-specific functions.Many large-scale studies with vast Hi-C data have given us important insights into possible mechanisms of looping, subsequent higher order chromatin organization, and functional significance of domain formation and chromosomal interactions in different processes including transcriptional regulation. Given that these observations encourage a shift in perspective regarding how


Here, we summarize our recent finding that misincorporation could be a major source of transcription pausing  Elongation of transcripts by DNA-dependent RNA polymerases (RNAP) is a temporally discontinuous process: interactions with certain DNA (or RNA) sequences, DNA lesions, misincorporation events, or encounters with other DNA-bound proteins are able to trigger programmed or accidental transcriptional pausing.elemental pause state that is catalytically inactive but does not alter the translocation state of RNAP.The majority of characterized pauses are events that branch off the main elongation pathway, and are believed to start with an isomerization of the elongation complex (EC) into an Another type of RNAP pause, described by us relatively recently, is caused by slow translocation between the pre-translocated and post-translocated states following NMP incorporation.in vivo genome-wide studies recently showed, that pause sites occur with an average frequency of 1 per 100 bp in the Escherichia coli genome.−10 at the upstream edge of the RNA:DNA hybrid, and Y−1G+1, where −1 corresponds to the RNA 3′ end.Pauses that are induced by the DNA sequence are ubiquitous. Single molecule studies suggested, and A distinct type of pause is caused when the σ factor fails to disengage from the core enzyme after promoter escape, and recognizes −10-like promoter sequences in the transcribed DNA , thus causing a pause in elongation.ops pause (operon polarity suppressor) allows the EC to bind RfaH, a regulator that suppresses pausing and termination downstream the ops site.70-dependent promoter-proximal pausing of the RNAP.Specific regulatory roles have been characterized in detail for some individual pauses.E. coli, and of two other conserved non-coding RNAs, probably by preventing the formation of stable non-native structures.Pausing may influence the folding of nascent transcripts, and has been shown to be required for the correct assembly of some biologically active RNAs.lac promoter, occurring at the transition from 6- to 7-nt RNA, which is suggested to be conserved in other promoters.Pausing is the first step of both intrinsic and Rho-dependent termination,Drosophila and mammalian genes) rate limiting, and highly regulated, thus playing a critical role in regulating metazoan gene transcription.In eukaryotes, RNAPII can pause between the promoter and the first nucleosome, 30–60 nt downstream of the transcription start site.Besides the specific roles of elongation pauses in regulating the expression of specific genes, pauses are thought to be required to reduce the pace of transcription elongation, and therefore allow for the coordination of transcription with translation in bacteria.in vivo, and in vitro studies showed that the replisome can readily dislocate the RNAP and use the mRNA as a primer to continue replicating the leading strand.in vivo and in vitro.in vivo.Streptococcus pneumoniae suggested that trailing RNAPs might queue behind the stalled one, thereby forming RNAP “traffic jams,” having detrimental consequences on gene expression and forming much more potent obstacles for the replication machinery.Bacterial RNAP and the replisome move simultaneously along the same template, but the former is 12–30-fold slower than the latter.E. coli; many bacteria have only one Gre factor) can reactivate backtracked ECs by promoting a transcript cleavage reaction at the active site that generates a new 3′ end.E. coli.Different factors have been shown to play a role in removing, reactivating, or preventing backtracked ECs (and/or RNAP queues caused by them), with their effects normally becoming apparent only when one of the other pathways is disrupted.in vitro because they induce backtracking of the EC by 1 bp  has made it possible to selectively sequence transcripts that are bound to transcribing RNAP, and, therefore, to investigate genome-wide pausing E. coli and S. cerevisiae, for both wild-type and cleavage factor deficient mutants.E. coli and S. cerevisiae, respectively.−3–10−6.E. coli strains) of misincorporated ECs was reported by a different study,Recently, we analyzed transcriptional errors in earlier NET-seq data from in vivo, and, consequently, a significant source of conflicts with the replication machinery and other trailing RNAPs both in bacteria and eukaryotes , while no such bias was seen in S. cerevisiae.Interestingly, analysis of the hotspots of misincorporated ECs in −1G+1 motifs (non-template DNA sequence) increase the rate of G>A misincorporation at +1 position.−1 positions.−1 for all G>A misincorporations was, in comparison to hotspots, lower in E. coli and non-present in S. cerevisiae, suggesting that G>A misincorporation is a quite random event.The pattern of 3′ misincorporations that we observed had a strong bias toward G>A (misincorporation of A instead of G), which is consistent with previous observations.Misincorporation-induced backtracked pauses may interfere with the progression of replication forks and significantly alter the gene expression patterns, by themselves or by causing RNAP jams.


We identify more than 10,000 hubs of open chromatin interactions (HOCIs) in human cells, which are mainly active promoters and enhancers bound by many DNA-binding proteins and form interaction networks crucial for gene transcription. In addition to identifying large-scale topological structures, including topologically associated domains and A/B compartments, OCEAN-C can detect HOCI-mediated chromatin interactions that are strongly associated with gene expression, super-enhancers, and broad H3K4me3 domains.We develop a method called open chromatin enrichment and network Hi-C (OCEAN-C) for antibody-independent mapping of global open chromatin interactions. By integrating FAIRE-seq and Hi-C, OCEAN-C detects open chromatin interactions enriched by active The online version of this article (10.1186/s13059-018-1430-4) contains supplementary material, which is available to authorized users. Thess to DNA –5. Tradis to DNA , 7.Recently, several elaborate methods to identify chromatin interaction maps have been developed, including chromatin interaction analysis by paired-end tag sequencing (ChIA-PET)  and chrocis-regulatory elements, which mainly occurred among promoters and enhancers and thus regulated gene transcription. OCEAN-C is a novel tool for studying open chromatin interactions and their relationship with gene regulation.In order to overcome these limitations, we integrated the FAIRE-seq and in situ Hi-C assays and developed the open chromatin enrichment and network Hi-C (OCEAN-C) method for mapping global open chromatin interactions. By aggregating open chromatin associated with interacting partners through direct phenol-chloroform extraction, OCEAN-C enriched interactions among active We first performed in situ Hi-C and FAIRE-seq experiments using U266 multiple myeloma cells to identify genome-wide chromatin interactions and open chromatin regions , 27. As p value < 2.2e-16). Therefore, OCEAN-C peaks represent chromatin interaction hubs that form multiple interactions with a set of loci along the chromosome  and enhancers (H3K4me1 and H3K27ac).We identified 12,003 OCEAN-C peaks (median of broad size was 1.4 kb and of narrow size 232 bp) with 43.4 million valid read pairs standing for intra-chromsomal interactions in the U266 cell line. Of these, 74.3% overlapped with FAIRE-seq peaks; in contrast, only 850 peaks were determined from the same number of Hi-C reads, which barely had any intersection with OCEAN-C or FAIRE-seq peaks Fig. . The higome Fig. , demonstTo further test the reproducibility and feasibility of OCEAN-C, we examined the method in RPMI-8226 multiple myeloma cells and GM12878 lymphoblastoid cells. The three cell lines exhibited similar numbers of HOCIs and similar histone modification properties, demonstrating that HOCIs represent a common phenomenon in different cell lines  and A/B compartments. HOCIs preferentially occurred at TAD boundaries Fig. ; in contTo further explore the relationship between HOCI interactions and gene transcription, we randomly selected a chromatin region  and plotted the chromatin interactions involving HOCIs and the read depth of RNA-seq experiments in U266 cells Fig. . Genes fWe further investigated whether changes in HOCIs can explain differential gene transcription between different cell lines. We compared the gene transcription levels of two multiple myeloma cell lines (U266 and RPMI-8226) according to the three gene types defined above. Genes that have different types between the two cell lines showed significantly different gene expression, while genes that have the same types between the two cell lines showed similar transcription levels Fig. . Large dCIITA), an important gene that participates in B-cell differentiation, and examined the nearby open chromatin interactions, Hi-C heat maps, and RNA-expression levels  are associated with increased transcription elongation and enhancer activities, especially at tumor suppressor genes, and form chromatin interactions with super-enhancers , 32. In Hi-C-based methods and ChIA-PET have greatly advanced our understanding of the 3D architecture of the nucleus by uncovering TADs, compartments, and chromatin loops. Previous ChIA-PET studies illustrate that promoter–promoter interactions provide a topological basis for transcriptional regulation, and CTCF and cohesin mediate the formation of 3D genome architectures , 33. Sevcis-regulatory elements is as important as their primary sequences with regard to gene regulation. It is important to explore the interactions among cis-regulatory elements such as enhancers, promoters, and insulators to understand how they regulate gene expression. Based on OCEAN-C data, we identify HOCIs as open chromatin interaction hubs with potential regulatory functions. We demonstrate that HOCIs preferentially form open chromatin interactions, including promoter–enhancer, promoter–promoter, and enhancer–enhancer interactions, which distinguish HOCIs from other open chromatin regions. A HOCI often mediates clustered chromatin interactions and can be important for coordinated transcription of multiple genes that are nearby and faraway. In addition, OCEAN-C is feasible for investigating changes in open chromatin conformations, such as promoter–enhancer interactions, that result in differential gene expression. We demonstrate that hub genes whose promoters are HOCIs display the highest transcription activity, and changes in HOCIs between different cell lines are associated with marked changes of transcription. These findings suggest that OCEAN-C is a suitable tool for studying the activation or inactivation of developmental genes or cancer genes due to the changes in chromatin conformation.The conformation of Despite these advantages, the current version of OCEAN-C has several areas that could be improved. First, OCEAN-C is based on the Hi-C method, which only captures chromatin interactions near recognition sites of the specific restriction enzyme used. Although the four-base restriction enzymes we used have abundant cutting sites along the genome, they may miss capturing certain chromatin regions. Second, because 1–3% of the total DNA was extracted as chromatin interactions related to open chromatin, OCEAN-C needs ~ 1 million cells in order to obtain sufficient DNA for library construction, which restricts its application for clinical samples. We will continue to develop and improve OCEAN-C to overcome these limitations.We demonstrate that OCEAN-C is a powerful method for investigating open chromatin interactions and the dynamic of HOCIs in regulating gene transcription.2. The cells were cultured to 80–90% confluence and then collected and washed once with PBS. For crosslinking cells, formaldehyde was added at a final concentration of 1% at room temperature (RT) for 10 min, and then quenched with glycine (0.2 M) for 5 min. The crosslinked cells were washed once with PBS, flash-frozen by liquid nitrogen, and stored at − 80 °C for further usage.U266 cells (ATCC TIB-196), RPMI8226 cells (ATCC CCL-155), and GM12878 cells were grown in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% COThese two experiments were performed strictly in line with previously reported protocols , 27. ForU266 and RPMI8226 cells were cultured to 80–90% confluence and harvested. RNA purification and library construction were performed by Novogene  with three independent replicates for each cell line, and the differential expression analysis was performed using the TopHat-cufflinks software with the recommended parameters.6 cells) following the instructions of the in situ Hi-C method.Digestion with the MboI enzyme, filling-in with biotin-labeled dATP, and re-ligation by the T4 ligase were performed using fixed cells  and sonicated to an average DNA fragment size of 300–400 bp (Branson Sonifier 450D). The results for each 30 s of sonication were checked under a microscope until no intact cells were observed. The cells were kept on ice and foaming was avoided. The efficiency of sonication was further confirmed by agarose gels of purified DNA from a portion  of the cell lysate.The supernatants were transfered to new 1.5 ml tubes after centrifugation . To purify the open chromatin, 1 volume phenol-chloroform-isoamyl alcohol was added to each aliquot of cell lysate. After vortexing for 10 s, each aliquot was centrifuged at 13,000×g for 5 min, and the top layer was transferred to a fresh 1.5 ml tube. The phenol–chloroform–isoamyl alcohol extraction step was repeated once, after which 200 μl of chloroform–isoamyl alcohol were added to each tube to remove traces of phenol, and the aqueous layer was transferred to a new 1.5-ml tube. Next, a 1/10 volume of 3 M sodium acetate (pH 5.2), 2 volumes of 95% ethanol, and 1 μl of 20 mg/ml glycogen were added to each tube and incubated at − 80 °C for 30 min (or longer) after fully mixing the sample. Each pellet was centrifuged at 13,000×g for 15 min at 4 °C, and the DNA pellet was washed twice with 500 μl of ice-cold 70% ethanol. The DNA was dried by leaving tubes open for 5 min and re-suspended in 200 μl of 10 mM Tris-HCl (pH 7.4).DNase-free RNase A (1 μl) was added following 30 min of incubation at 37 °C, and 1 μl of proteinase K was added and incubated at 55 °C for 1 h and then at 65 °C overnight to reverse cross-linking. The DNA was collected by adding 0.9 volume of AMPure XP beads  and washed with 300 μl 10 mM Tris-HCl (pH 7.4). The concentration of DNA was measured by Qubit. The amount of purified DNA should not exceed 5% of total genomic DNA (1–3%). An optimized step can be performed to boost the yield before the biotin pull-down operation by sonicating the purified DNA with Covaris to a median fragment size of 300–500 bp.Myone Streptavidin T1 beads  were washed once with 400 μl 1 × TWB (5 mM Tris-HCl (pH 7.5), 0.05 mM EDTA, 1 M NaCl, 0.05% Tween 20), separated on a magnet, and resuspended with 300 μl 2× binding buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl). Then DNA dissolved in 300 μl 10 mM Tris-HCl (pH 7.4) was added into the bead solution and incubated at RT for 15 min with rotation. The beads were then separated on a magnet and biotinylated DNA was bound to the streptavidin beads.The library preparation processes were performed with streptavidin beads as described for the in situ Hi-C protocol. Briefly, the ends of sheared DNA were repaired and the biotin from un-ligated ends was removed, adapters were added to the A-tailed DNA fragments, and PCR was performed with eight to ten cycles using Illumina primers. Finally, DNA size selection was performed with 0.65–0.8× volume of AMPure XP beads to make sure the DNA length distributes between 300 and 500 bp. The library was quantified with Qubit and sequenced using an Illumina sequencing platform.OCEAN-C reads were mapped and filtered similarly to the situ Hi-C data. Briefly, clean reads were first trimmed to 36 bp and then mapped to genome hg19 with bowtie2, and reads with MAPQ less than 1 were discarded. If a read pair locates in the same restriction fragment (MboI), it was classified as dangling ends (inward), self-cycled (outward), or dumped pairs (same strand) and discarded. For the remaining read pairs that mapped to two different restriction fragments, if the distance between these two fragments was less than 1 kb, the read pairs were discarded due to the two ends’ close distance in sequence. The remaining read pairs were considered valid and used to call peaks and generate interaction heat maps. In the U266 cell line, the OCEAN-C peaks, which were defined as HOCIs, were determined by the ZINBA algorithm from the filtered data with the same parameters used for FAIRE-seq. In RPMI-8226 and GM12878 cell lines, HOCIs were called by ZINBA with the “pscl” method from the filtered data since the signals were too weak to be selected using the “mixture” method. HOCIs overlapping both H3K4me3 ChIP-seq peaks and gene promoters (2 kb up to genes’ transcription start sites) were defined as promoter HOCIs, and the rest which overlapping both H3k4me1 and H3K27Ac ChIP-seq peaks were defined as enhancer HOCIs. HOCI interactions, ChIA–PET interactions, gene densities, and gene transcription were examined using the WashU Epigenome Browser, and the network of HOCIs was constructed using the ggnet R package.p values in Figs. t-test with specified two-group data.The We used the rose software to identify enhancers . First, Broad H3K4me3 peaks were called from ChIP-seq data downloaded from ENCODE using MACS2 . The “gaWe use Homer  to find We use DAVID  to find Additional file 1:Figures S1–S11. (PDF 5443 kb)Additional file 2:Table S1. Summary of OCEAN-C, FAIRE-seq, Hi-C, and RNA-seq data. (PDF 19 kb)Additional file 3:Table S2. Summary of HOCI and gene expression data analysis. (PDF 22 kb)Additional file 4:Table S3. Information of public data used in this study. (PDF 20 kb)


Salvia miltiorrhiza Bunge, possesses several biological activities such as anti-inflammation, anti-cancer, neuroprotection and hypolipidemic activities. In this study, we aim to investigate the hypocholesterolemic effect of tanshinone IIA in hepatic cells. We demonstrated that tanshinone IIA significantly increased the amount of low-density lipoprotein receptor (LDLR) and LDL uptake activity in HepG2 cells at the post-transcriptional regulation. We further demonstrated that tanshinone IIA inhibited the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and mature protein, which may lead to an increase the cell-surface LDLR in hepatic cells. We further identified a regulatory DNA element involved in the tanshinone IIA-mediated PCSK9 down-regulation, which is located between the -411 and -336 positions of the PCSK9 promoter. Moreover, we found that tanshinone IIA markedly increased the nuclear forkhead box O3a (FoxO3a) level, enhanced FoxO3a/PCSK9 promoter complexes formation and decreased the PCSK9 promoter binding capacity of hepatocyte nuclear factor 1α (HNF-1α), resulting in suppression of PCSK9 gene expression. Finally, we found that the statin-induced PCSK9 overexpression was attenuated and the LDLR activity was elevated in a synergic manner by combination of tanshinone IIA treatment in HepG2 cells. Overall, our results reveal that the tanshinone IIA modulates LDLR level and activity via down-regulation of PCSK9 expression in hepatic cells. Our current findings provide a molecular basis of tanshinone IIA to develop PCSK9 inhibitors for cholesterol management.Tanshinone IIA, one of the most pharmacologically bioactive phytochemicals isolated from  Epidemiological studies have demonstrated that elevated circulating triglyceride-rich remnant lipoproteins and high concentrations of plasma low-density lipoprotein cholesterol (LDL-C) are directly correlated with the risk of atherosclerosis and cardiovascular disorders , 2. The The level of LDLR is modulated in cells at transcriptional and post-transcriptional regulation . LDLR trPCSK9 gene expression was regulated by several transcription factors such as SREBPs, hepatocyte nuclear factor 1α (HNF-1α) and forkhead box O3 (FoxO3) –15. ActiSalvia miltiorrhiza Bunge, also known as Danshen, is a traditional Chinese herbal medicine that has been used in Asian countries for the treatment of coronary artery disease, myocardial infarction, stroke, atherosclerosis and cerebrovascular disorders ; GAPDH, 5'- CATGAGAAGTATGACAACAGCCT-3' (forward) and 5'- AGTCCTTCCACGATACCAAAGT-3' (reverse)]   and Maxianalysis . The amo™ Prime Western Blotting Detection Reagent, and the signal was visualized on Amersham Hyperfilm™ ECL .Cells were seeded as described above, the medium was then changed to a DMEM/LPDS medium and the vehicle or tanshinone IIA was added for an additional 24 h. For the extraction of total cellular proteins, the cells were harvested using RIPA buffer (Thermo Fisher Scientific). For nuclear extract preparation, the cells were harvested using the NE-PER nuclear and cytoplasmic extraction reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total cellular lysates (20 μg) and nuclear extracts (20 μg) were analyzed by 10% SDS-PAGE and transferred onto a PVDF membrane . The blots were incubated with the following specific primary antibodies at 4°C for 24 h: anti-LDLR , anti-PCSK9, anti-HDAC2 , anti-N-terminus of SREBP2 , anti-HNF-1α , anti-FoxO3a  or anti-β-actin (Sigma-Aldrich). The blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at 37°C for 1 h. The proteins were detected using the Amersham ECLThe cell-surface LDLR was detected by flow cytometry analysis as previously described . Briefly®-FL-LDL (Thermo Fisher Scientific) at 37°C for 24 h. The cells were detached, washed and re-suspended in PBS and examined by flow cytometry. Data were acquired from 10,000 cells (counts). The LDL uptake data were expressed as the relative percentage of the geometric mean fluorescence intensity.The amount of LDL uptake was measured as previously described , 30. BriRenilla luciferase control vector (Promega) using the Lipofectamine® regent (Thermo Fisher Scientific) according to the manufacturer’s instructions. After transfection, the cells were treated with vehicle or tanshinone IIA for 24 h. The luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega), and the intensities were normalized to the Renilla luciferase activity.Reporter plasmids containing the PCSK9 promoter , the LDLR promoter , and the serial deletion regions of the PCSK9 promoter for the luciferase reporter assay were designated and constructed as previously described . HepG2 c5'-TCCAGCCCAGTTAGGATTTG-3' and 5'-CGGAAACCTTCTAGGGTGTG-3'[t method [The ChIP assay was performed using the ChIP Assay Kit  as previously described , 31. BriGGTGTG-3', 17. Thet method  and exprp value < 0.05 was considered statistically significant.All experiments were replicated at least three times, and the values are expressed as the mean ± SD. The results were analyzed using one-way ANOVA with Dunnet’s post hoc test, and a p<0.01) -containing medium and treated with vehicle (0.1% DMSO) or tanshinone IIA (1–20 μM) for 24 h. The cell viability was determined by MTT assay. As shown in p<0.01) . Moreovep<0.01), respectively , respectively. We further examined the effect of tanshinone IIA on the transcriptional activity of PCSK9 promoter by transfecting cells with a PCSK9 promoter reporter plasmid (PCSK9-p(-1459 /-4)) and a Renilla luciferase control plasmid, as described in Materials and Methods section. The result showed that the PCSK9 promoter activity was significantly reduced in the 5 and 10 μM tanshinone IIA-treated cells compared to the vehicle-treated cells (p<0.01)  . Furtherp<0.01). Additionally, we found that the PCSK9 promoter activity was strongly attenuated in those cells transfected with the reporter plasmid PCSK9-p(-335/-4). These data indicate that the DNA region located between the -411 and -336 positions on PCSK9 proximal promoter is a critical regulatory element for tanshinone IIA-mediated PCSK9 down-regulation. It has been reported that this region of the PCSK9 promoter contains an HNF-1α binding site, a SREBP2 binding element , as well as a FoxO3a binding site [To investigate the molecular mechanism underlying the tanshinone IIA-mediated PCSK9 down-regulation, the regulatory DNA elements of the PCSK9 promoter that are responsive to the tanshinone IIA-mediated PCSK9 reduction were further identified. HepG2 cells were transfected with reporter plasmids containing serial deletions of the PCSK9 promoter  Fig 5A)Fig 5A) fing site , 15, 33 in vivo interaction of FoxO3a with the PCSK9 promoter in HepG2 cells was examined using a ChIP assay, as described in Materials and Methods section. As shown in To investigate whether these transcription factors are involved in the tanshinone IIA-mediated PCSK9 down-regulation, the effect of tanshinone IIA on the levels of nuclear HNF-1α, SREBP2 and FoxO3a proteins were evaluated in HepG2 cells. As shown in Statin treatment may increase the PCSK9 gene expression and attenuate the therapeutic ability for lowering the plasma LDL, thus, we further investigated whether a combination of tanshinone IIA and statins alter the PCSK9 expression and LDL uptake in HepG2 cells. The cytotoxic effect of a tanshinone IIA and statins combination was evaluated in HepG2 cells. As shown in In the present study, we demonstrate that tanshinone IIA markedly down-regulates PCSK9 gene expression, which is associated with increases in the amount of cell-surface LDLR and its LDL-uptake activity in HepG2 cells. We show that tanshinone IIA dramatically elevates the nuclear abundance of the FoxO3a and its interaction with PCSK9 promoter, which may result in the inhibition of HNF-1α/PCSK9 promoter complex formation and the down-regulation of PCSK9 gene expression . MoreoveThe hepatic LDLR expression is involved in the regulation of plasma cholesterol homeostasis. It is known that patients with familial hypercholesterolemia, commonly mutations in the LDLR, experience high levels of LDL-C and premature atherosclerosis . ElevateIn addition to transcriptional control, the LDLR is also modulated by PCSK9 or the inducible degrader of LDLR (IDOL) at post-transcriptional regulation , 39. The+-dependent deacetylase sirtuin 6 (Sirt6) for the suppression of SREBP2 expression and modulation of cholesterol biosynthesis in a mouse model [In the present study, we demonstrated that the PCSK9 mRNA is down-regulated by tanshinone IIA through the inhibition of promoter activity. The tanshinone IIA-responsive element was characterized and defined to the proximal promoter region (-411 to -336). Within this region of the PCSK9 promoter, there are three potential transcriptional factor binding sites, including the HNF-1α binding element , the stese model . In addise model . In thisse model . Nho et se model . WhetherStatins and ezetimibe are the most common and effective drugs for lowering plasma cholesterol levels and improving outcomes from coronary artery diseases –54. StatIn conclusion, we report that tanshinone IIA exerts the promising effect of PCSK9 down-regulation. Our data elucidates that tanshinone IIA mediates the suppression of PCSK9 in a FoxO3a-related manner, leading to an increase in LDL uptake in hepatic cells. Our findings provide a molecular basis of tanshinone IIA for the development of PCSK9 inhibitors for cholesterol homeostasis maintenance.


Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis.Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA  Hottip. Moreover, using multiple approaches, including lncRNA depletion, genetic manipulation of Hottip locus, transcriptional activation and premature termination of lncRNA transcript along with RNA localization, we demonstrate that Hottip lncRNA regulates expression of neighbouring Hoxa genes.Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including regulation of gene expression. However, the mechanism through which they regulate gene expression is not clearly understood. Here we show that a transcriptional co activator—Psip1 specifically regulates the expression of the lncRNA  In cpression 35]. Nas. NasHottpression . Togethepression , these rPsip–/–MEFs with the p52 isoform of Psip1 led to an increase in expression of posterior Hoxa genes , concomitant with the increase in expression of these 3' Hoxa genes upon p52 or Hottip depletion  in limb mesenchymal cells, leaving the Hottip promoter intact [Hottip. qRT-PCR analysis of ChIRPed RNA showed specific enrichment for Hottip RNA , demonstrating that misregulation of 3' Hoxa genes in the absence of Hottip  using 11ttip RNA . qPCR anip ChIRP . Hottip tip Figs  and 3B iHottipΔ cells is due to loss of cis -regulatory elements located within the deleted region, rather than loss of the Hottip RNA per se. Hottip is known to function at the site of its synthesis (in cis) and it fails to activate target genes when expressed ectopically from a retroviral construct [Hottip in ES cells where Hottip and Hox gene clusters are repressed by polycomb complexes, to study the effect of lncRNA activation in cis or trans. We have previously shown that targeted recruitment of an ectopic activator (Vp16) to silent loci in murine ES cells (mESCs) can overcome this repression[HOTTIP which is transcribed bi-directionally from the HOXA13 CpG island promoter [Hottip or Hoxa13 –47 specir Hoxa13 . CRISPR a13, a11 and a10), but not anterior Hoxa and Hoxd genes upon dcas9-VP160 mediated Hottip activation in mESCs, relative to transfection with dCas9 recruitment alone (no VP160)  . In contnaltered . Further network  suggestiWT cells , pointinHottip lncRNA molecule from the act of lncRNA transcription at the Hottip locus, for up-regulation of 5’ Hoxa genes, we used CRISPR-Cas9-mediated homologous recombination to insert a 49 bp synthetic polyadenylation (polyA) cassette[Hottip transcription start site (TSS) in 14fp cells  clones demonstrated that spliced Hottip RNA was strongly reduced in all three polyA knockin lines  compared to WT  cells[HOTTIP/HOXA13 are regulated by PSIP1, and the role of PSIP1 and HOTTIP in oncogenesis.By inserting a polyadenylation cassette at the 5’ end of d Hoxa13 –51. ReceCC) cells, furtherHOTTIP[in cis does not require the lncRNA molecule itself. Instead their effect is mediated by enhancer-like activity of underlying DNA elements in the lncRNA locus, the act of transcription and/or splicing of lncRNAs[Noncoding transcription at enhancer elements has been associated with enhancer activity,54. HoweHOTTIP,58. Howef lncRNAs–62.The recent controversies in the lncRNA field demand thorough investigations to distinguish the role of lncRNA molecules from enhancer- like function of the DNA elements which encode them, and from the process of transcription and splicing of these loci. Our studies presented here show how these facets of lncRNA regulation and function can be dissected at one well-studied lncRNA locus. With the ever increasing number of lncRNAs annotated in genomes using high-throughput sequencing technologies, the list of these transcripts with unknown mechanisms of upstream transcriptional regulation and downstream functional mechanism is growing and there will be the need to develop more high-throughput methods for the rigorous testing of lncRNA function and mechanism of action.Cervical dislocation was used as a euthanasia method and all mouse experiments were performed under the Animals (Scientific Procedures) Act 1986' and were approved by the University of Edinburgh ethical committee (TR-38-16) and performed under UK Home Office license number PPL 60/4418.Psip1–/–and its corresponding WT MEFs[Psip–/–MEFs is previously described[g WT MEFs,63 were g WT MEFs and wereg WT MEFs. Psip1/pdescribed.Hottip  targeting Psip1/p52, Psip1/p75 and Hottip  were traGapdh. Details of the oligos are given in the Reverse transcription followed by quantitative PCR (RT-qPCR) was performed as described previously. BrieflymHottip in 10.5 dpc mouse embryos were performed as previously described[Hottip cDNA including T7 (sense) and T3 (Antisense) promoter sequences are given in RNA in situ hybridization for described. DetailsHox array and data was normalized as described previously[ChIP was performed as described previously, using areviously.Hottip RNA were designed using the web tool from Stellaris FISH Probe Designer (https://www.biosearchtech.com/support/education/stellaris-rna-fish) Biosearch Technologies, CA, USA). Eleven biotinylated oligos were synthesized by Sigma-Aldrich . Paired guide RNAs (gRNAs)   were desend site . gRNAs wHottip and Hoxa13 , gRNA (25ng/ul) and repair template DNA (75ng/ul)  at the 75ng/ul) . The emb75ng/ul) . Total RS1 FigHottip in 10.5d embryo (right). Image from Psip1 RNA in situ hybridization for 11.5d embryos from Embrys resource (left) http://www.emouseatlas.org/emagewebapp/pages/emage_general_query_result.jsf. (B) Similar to Gapdh, of Hoxa genes, along with Psip1/p52, Psip1/p75, and Hottip RNA, in limb cells transduced with independent shRNAs (sh2’s in Hottip gene (yellow), Sanger sequencing data confirms 49 bp polyA signal sequence insertion (highlighted in yellow) and flanking Hottip sequence. (E) Similar to Gapdh, fold change in expression of Hottip, Hoxa13, a11, a10, a9 and Hoxc13 in two wild type (WT1 and WT2) and one polyA knock-in 12.5 dpc whole embryo at Hottip locus.(A) Whole mount RNA in situ hybridization of sh2’s in  targetin(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 TableNucleotide sequence of single stranded oligonucleotide used as homology directed repair templated used for insertion of a synthetic polyA signal sequence to hottip locus. 75 base nucleotide homology arms are shaded in grey, 49base polyA signal is shaded in yellow. GuideRNA binding site is in red, PAM site is in blue.(DOCX)Click here for additional data file.