The stimulatory effect of oPACAP38 was mimicked by activation of AC/cAMP and voltage-sensitive Ca2+ channel (VSCC) signaling, or induction of Ca2+ entry. In parallel, PACAP-induced PRL promoter activity was negated or inhibited by suppressing cAMP production, inhibiting PKA activity, removal of extracellular Ca2+, VSCC blockade, calmodulin (CaM) antagonism, and inactivation of CaM kinase II. Similar sensitivity to L-type VSCC, CaM and CaM kinase II inhibition were also observed by substituting cAMP analog for oPACAP38 as the stimulant for PRL promoter activity. Moreover, PACAP-induced PRL promoter activity was also blocked by inhibition of PLC signaling, attenuation of [Ca2+]i immobilization via IP3 receptors, and blockade of PI3K/P70S6K pathway. The PACAP-induced PRL promoter activation may involve transactivation of the transcription factor CREB. These results suggest that PACAP can stimulate PRL promoter activation by PAC1 mediated functional coupling of the Ca2+/CaM/CaM kinase II cascades with the AC/cAMP/PKA pathway. Apparently, other signaling pathways, including PLC/IP3 and PI3K/P70S6K cascades, may also be involved in PACAP induction of PRL gene transcription.In this study, structural analysis of grass carp prolactin (PRL) gene was performed and the signaling mechanisms for pituitary adenylate cyclase-activating peptide (PACAP) regulation of PRL promoter activity were investigated. In αT3-1 cells, PRL promoter activity could be induced by oPACAP In mammals, transcription of PRL gene is regulated by multiple factors. Dopamine is well-documented to exert inhibitory effect on PRL promoter activity via dopamine D2 receptor activation2. In contrast, thyrotropin-releasing hormone (TRH) is considered to be a PRL-releasing factor, which can stimulate PRL promoter via pit-1 transactivation3. Furthermore, sex steroids, especially estrogen, have been proposed to serve as a potent stimulator for PRL gene transcription , presumably by functional interactions of estrogen receptor (ER) and activator protein 1 (AP1) transcription factors on the respective cis-acting elements in the PRL promoter5. Unlike mammalian studies, no much information is available on PRL promoter regulation in non-mammalian vertebrates. In goldfish, dopamine inhibition of PRL promoter activity can be observed6. However, PRL transcript expression and PRL promoter activity were both suppressed by TRH treatment in the same study. This discrepancy of TRH actions between the fish model and mammals suggests that the neuroendocrine regulation of PRL gene expression might have been altered/modified during the course of vertebrate evolution.Prolactin (PRL) is a pituitary hormone with diverse function. Phylogenetic analysis of gene sequences has revealed that PRL, growth hormone (GH), and somatolactin are evolved from the same ancestral gene by gene duplication7. In mammals, the stimulatory effects of VIP on PRL secretion8 and gene expression9 have been well-documented, and multiple sites of actions, namely actions within the central nervous system 10, direct stimulation of lactotrophs at the pituitary level11, or by autocrine/paracrine mechanisms within the pituitary12 have been reported. In contrast to VIP, the biological actions of PACAP on PRL secretion have been controversial, as stimulatory13, inhibitory14, and no effect16 have been reported. In the case of stimulatory effects, e.g., in GH3 cells, PACAP can up-regulate PRL promoter activity via activation of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent signaling mechanism17. In GH4C1 cells, the stimulatory effects of PACAP and VIP on PRL gene expression are mediated through vasoactive intestinal peptide receptor 2 (VPAC1I) functionally coupled with Ras and Rap small GTPase18. VPAC receptors, formerly known as the VIP receptors, are known to bind PACAP and VIP with similar affinity, and its pharmacological properties is quite distinct from the PAC1 receptors for PACAP, which is specific for PACAP with little/very low affinity for VIP19.PACAP and vasoactive intestinal peptide (VIP) are members of the glucagon/secretin peptide family and have been reported to play roles in PRL regulation20. Furthermore, PAC1 receptors could be detected in grass carp lactotrophs. PRL secretion and PRL mRNA expression were up-regulated by PACAP but not VIP treatment via functional coupling of Ca2+/calmodulin (CaM)- and c-Jun N-terminal kinase (JNK)-dependent cascades with the cAMP/PKA pathways21. These findings suggest that PACAP but not VIP can serve as a stimulator for PRL synthesis in the carp species and this stimulatory action is mediated through activation of PAC1 but not VPAC receptors at the pituitary level. To further elucidate the mechanisms for PACAP regulation of grass carp PRL gene expression, the effect of PACAP treatment on PRL promoter activity and the post-receptor signaling cascades involved were examined in αT3-1 cells. The full gene of grass carp PRL was cloned by genome walking and the transcription initiation site was defined by primer extension. The 5′ promoter of grass carp PRL gene was then subcloned into a luciferase-expressing reporter for promoter studies in αT3-1 cells. PACAP responsive sequence of grass carp PRL promoter was mapped by 5′ deletion analysis. The functional involvement of the cAMP/PKA, Ca2+/CaM/CaM Kinase-, mitogen-activated protein kinase (MAPK)- and phosphoinositide 3-kinase (PI3K)-dependent cascades were tested with the pharmacological agents that could perturb the respective pathways. To shed light on the transcription factors mediating the genomic action of PACAP in terms of PRL gene transcription, the effect of over-expression of cAMP response element binding protein (CREB) on basal PRL promoter activity and siRNA/domain negative (DN) mutant of CREB on PACAP-induced PRL promoter activity were also investigated in αT3-1 cells.In carp model, PACAP nerve fibers can be identified in anterior pituitary overlapping with the distribution of lactotrophs22  has revealed the presence of two TATA boxes located at position −20 and −261 upstream of the transcription initiation site. Multiple cis-acting elements including the binding sites for Pit1, Sp1, AP-1, NF-kappaB, GATA1, El2, HNF-3B, TBP, Oct1, and C/EBPalpha can also be identified in the various locations spreading along the entire length of the grass carp PRL promoter . After that, intron trapping was performed using genomic DNA as the template to pull out the DNA sequence for intron I, II, III, and IV, and the associated coding exons . For primer scanning, the primer PS1 located in the 3′ downstream region of 5′ UTR was used as the anchor primer for RT-PCR with the 5′ upstream primers P(−720), P(−470), P(−210), and P(−90) located at position −720, −470, −210, and −90 of the grass carp PRL promoter, respectively Fig. . As a coary Fig. . With reTo test the functionality of the grass carp PRL promoter, a series of deletion constructs were prepared by subcloning decreasing lengths of 5′ promoter sequence of PRL gene into the luciferase-expressing reporter pGL3.Basic. These 5′ deletion constructs were transiently transfected into the rat pituitary cell line αT3-1 and the stimulatory effect on luciferase activity expression was monitored after 24-hr treatment with ovine PACAP (100 nM). In this study, the upstream promoter of the carp PRL gene, including pPRL(−1156), pPRL(−914), pPRL(−670), pPRL(−448) and pPRL(−306), acted as strong promoters in αT3-1 cells. In the absence of drug treatment, basal luciferase activity was elevated by removing the promoter sequence from −914 to −670, suggesting that inhibiting element(s) may be present within this region. The promoter activity was significantly decreased by further deletion, either between pPRL(−306) and pPRL(−166) and or between pPRL(−166) and pPRL(−124), but not shown in the region between pPRL(−124) and pPRL(−57) Fig. . These r38 (100 nM) with the peak response occurred at 24 hr after the initiation of drug treatment  with/without simultaneous treatment with the PACAP antagonist PACAP6–38 VIP.Since pPRL(−1156).Luc consistently produced high levels of both basal and PACAP-induced luciferase activity, the construct was routinely used in our transfection studies for PACAP-stimulated PRL promoter activity. In this case, PRL luciferase activity expressed in αT3-1 cells could be evaluated in a time-dependent manner by ovine PACAPent Fig. . By fixi nM Fig. . Consist23 and our recent studies in grass carp pituitary cells have shown that PACAP can trigger PRL mRNA expression via activation of pituitary PAC1 receptors21, the involvement of the cAMP-dependent cascades in PACAP induced PRL promoter activity was investigated. As a first step, αT3-1 cells were treated for 24 hrs with increasing levels of the membrane permeable cAMP analog cpt-cAMP (1–100 μM) and adenylate cyclase (AC) activator Forskolin (10–1000 nM). In these experiments, luciferase activity expression in αT3-1 cells was dose-dependently increased by cpt-cAMP  and this stimulatory effect could be totally abolished by simultaneous treatment with the AC inhibitor MDL12330A  activator Bay K8644 . In this case, PACAP  was also effective in attenuating PACAP- and Forskolin-induced luciferase activity expression in αT3-1 cells 25, the functional role of MAPK and PI3K in PACAP-induced grass carp promoter activity were also examined. In αT3-1 cells, both basal and PACAP (10 nM)-induced luciferase activity expression was not affected by simultaneous treatment with the P42/44MAPK (or Erk1/2) inhibitor U0126 2+/CaM-dependent pathways by binding to CRE sites in target gene promoters28. In grass carp pituitary cells, PACAP treatment significantly enhanced CREB phosphorylation and stimulated total CREB production , consistent with the consensus that PRL and GH are both evolved from a common ancestral gene1.The newly cloned PRL gene spans 5 kb in size and consists of 5 exons and 4 introns. All the introns identified have been confirmed to start with “GT” and terminated with “AG”, which is consistent with the ‘GT/AG” rule6. Apparently, the PRL genes of the cyprinus family, including both grass carp and goldfish, tend to have a very short 5′ UTR region. Our results, however, are at variance with that reported for human PRL gene, in which two separate transcription start sites have been identified30, with one for pituitary expression located in the proximal promoter region while the other located in the distal end of the upstream region about 5 kb away from the pituitary start site and is responsible for placental expression of PRL gene. In the proximal promoter of grass carp PRL gene, a typical TATA box with the sequence “TATAAT” was found 20 bp upstream of the transcription start site. TATA box can also be identified in the promoter region of the goldfish PRL promoter6. Similar findings have been described in the PRL gene promoter in common carp31, salmon32 and sea bream33, etc. Apparently, the presence of TATA box in the proximal region of PRL gene is a common feature for fish models.The transcription start site of grass carp PRL is located at 53 bp upstream of the transcription start site, highly comparable to the result of recent study in goldfish PRL gene21. The parallel increases in PRL protein production and PRL mRNA expression observed in our study raise the possibility that PACAP may stimulate PRL synthesis via up-regulation of PRL gene transcription. This hypothesis has been confirmed by the present study using αT3-1 cells as the host cells to examine grass carp PRL promoter activity. In our studies with αT3-1 cells, grass carp PRL promoter activity could be induced by PACAP treatment in a time- and dose-dependent manner and the PACAP responsive sequence was mapped to proximal promoter region downstream of position −306 of the grass carp PRL promoter. Within this region, consensus sequence for C/EBP alpha and AP-1 binding sites can be identified. C/EBP proteins possess domains that contain cAMP-inducible activities that are independent of direct phosphorylation by PKA35. In mammalian melanocytes, AP-1 is known to be induced by cAMP and leads to an increased tyrosinase gene expression36. The mechanisms of C/EBP alpha and AP-1 binding sites working on grass carp PRL promoter regulation and stimulatory effects induced by PACAP remain to be further clarified.Recently, we found that PACAP can stimulate PRL secretion, protein synthesis and gene expression in grass carp pituitary cells via activation of PAC1 receptor2+/CaM−, and MAPK-dependent cascades21. At the transcription level, cAMP/PKA and Ca2+/CaM/CaM-II pathways are consistently involved in the PACAP regulation on PRL promoter activity. In mammalian cell models, e.g., in human SH-SY5Y neuroblastoma cells, functional coupling of MAPK  with cAMP-dependent mechanism has been reported24. In GH3 cells, PACAP triggered ERK activation to stimulate PRL synthesis37. However, in our studies with αT3-1 cells, the inhibitors for Erk1/2, and P38MAPK were not effective in altering both basal and PACAP-induced PRL promoter activity. It is intriguing that JNK signaling was not involved in the PACAP regulation on PRL transcription, distinct from the previous findings that PACAP stimulate PRL mRNA expression through JNK activation in grass carp pituitary cells21. Apparently, the MAPK cascades are not involved in PACAP-induced grass carp PRL gene transcription. This is similar with another finding that PACAP stimulates ERK activation in αT3–1 cells, which mainly contributed to the increased DNA synthesis and cell proliferation rather than transcriptional activation of the αGSU gene38. Moreover, PACAP stimulation of grass carp PRL gene transcription also involve the activation of PI3K/p70S6K pathway, which was not exhibited in PACAP-induced PRL mRNA expression in grass carp pituitary cells21. This inconsistency may be due to the different cell types used in the individual studies. Since PKA activation is known to induce p70S6K phosphorylation, e.g., in sertoli cells after FSH stimulation39, we do not exclude the possibility that functional coupling between cAMP/PKA and PI3K/p70S6K pathway may also occur in grass carp PRL gene expression.In primary pituitary cells, PRL production induced by PACAP via functional coupling of cAMP/PKA−, Ca26. In mammalian cell models, PACAP treatment has been reported to trigger CREB phosphorylation via cAMP/PKA40, cAMP/ERK41, and/or PLC/PKC42 pathways. Over-expression of a constitutively active mutant of CREB can activate rat PRL promoter activity via both CRE and non-CRE mediated mechanisms44. Expression of a dominant-negative form of CREB (MCREB) inhibited basal and Forskolin-induced PRL promoter activity and PRL mRNA expression in rat lactotrophs45. In this study, similar to the case of the rat PRL promoter, over-expression of grass carp CREB not only elevated basal but also enhanced PACAP-induced grass carp PRL promoter activity in αT3-1 cells. The stimulatory effect of PACAP on PRL promoter activation was significantly suppressed by CREB gene silencing using siRNA as well as by over-expression of a domain negative mutant of CREB, namely the kCREB. Moreover, in grass carp pituitary cells, PACAP can stimulate CREB phosphorylation and production. These results provide evidence that CREB activation is involved in PACAP induction of PRL gene transcription in grass carp. Since CREB is the downstream phosphorylation substrate for PKA, CaMK-II as well as other signaling kinases, it is conceivable that CREB may serve as the transcription factor mediating PACAP’s action on PRL gene via the cAMP, Ca2+ and PI3K signaling cascades46. Although CRE and/or CRE-like elements (CLE) have been identified in the 5′ promoter of PRL gene in mammals 47 as well as in fish models 48, consensus sequence for CRE could not be located in the grass carp PRL promoter by TESS site search. It raises the possibility that a non-conanical CRE(s) may be present in the grass carp PRL gene and mediates CREB transactivation of PRL promoter. In mammals, CREB can form heterodimers with transcription factors of the AP-1 family49. In PC12 cells, phosphorylated CREB was found to activate c-fos transcription50. Thus alternatively, the induction effect of CREB on PRL gene transcription may be mediated by other transcription factors.CREB is a member of the leucine zipper transcription factors and well-known to be involved in the activation of cAMP-inducible genes2+]e entry triggered by the AC/cAMP/PKA pathway. The CaM/CaM K-II pathway responsible for PRL gene transcription could also be activated by PACAP stimulation of the PLC/IP3 cascades, which leads to Ca2+ release from intracellular Ca2+ stores. In this study, we have also provided evidence that the PI3K/p70S6K pathway, but not MAPK or Akt, was also involved in the post-receptor signaling of PACAP induction of PRL gene transcription. Probably, the signaling cascades triggered by PACAP stimulation could induce PRL promoter activation via transactivation of the transcription factor CREB. Although the PACAP responsive sequence in grass carp gene has been mapped to the proximal region downstream of position −306 of PRL promoter using 5′ deletion, the cis-acting elements responsible for PACAP stimulation have been yet to be identified. Further investigations are clearly warranted to delineate the functional elements in PRL promoter conferring PACAP induction of PRL gene expression in grass carp.In summary, we have cloned the full gene of grass carp PRL and characterized its genomic organization and exon/intron junctions. The 5′ promoter obtained was used to set up a luciferase reporter system in αT3-1 cells to investigate the signaling mechanisms involved in PACAP stimulation of PRL gene transcription in grass carp. In our working model Fig. , we haveCtenopharyngodon idellus) with body weight ranging from 1.5 to 2.0 kg were acquired from local markets and maintained in well-aerated 200-liter aquaria under 12 L:12D photoperiod at 18 ± 2 °C. Since the grass carp at this stage was “prepubertal” (gonadosomatic index ≤ 0.2%) and sexual dimorphism was not apparent, fish of mixed sexes were used for blood sampling and pituitary collection. During the process, the fish were anesthetized in 0.05% MS222  for blood sampling and sacrificed by spinosectomy for pituitary harvest according to the protocol (CULATR No.3890) approved for this study by the Committee for Animal Use in Teaching and Research at the University of Hong Kong (Hong Kong).One-year-old (1+) Chinese grass carps . Ovine PACAP38 was purchased from Phoenix Pharmaceuticals  and dissolved in double-distilled deionized water and stored frozen as 0.1 mM stock solution in small aliquots at −80 °C. Forskolin, H89, MDL12330A, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), A23187, Bay K8644, nifedipine, verapamil, 2-aminoethoxydiphenyl borate (2-APB), U73122, KN62, KN93, calmidazolium, TPA, GF109203, U0126, PD169316, SP600125, Ly294002, rapamycin and HIMOC were obtained from Calbiochem . The stock solutions of these pharmacological agents were prepared in a similar manner as that for ovine PACAP38 except that they were dissolved directly in dimethyl sulfoxide (DMSO). Stock solutions of test substances were diluted with prewarmed (28 °C) culture medium to appropriate concentrations 15 min prior to drug treatment. The final dilutions of DMSO were always less than 0.1% and had no effects on PRL promoter activity expressed in αT3-1 cells. Antibodies against total CREB , phosphorylated CREB , and β-actin  were used for Western blot analysis. SiRNA for CREB were purchased from Dharmacon . The vector pRC/RSV.kCREB expressing a DN mutant of CREB, namely kCREB, was obtained from the laboratory of Dr. R.H. Goodman . Other chemicals or materials used in this study were acquired from commercial sources with the highest quality available.DMEM, fetal bovine serum (FBS), antibiotic-antimycotic, OPTI-MEM, and lipofectamineTM Kit . Briefly, genomic DNA prepared was subjected to 15–18 hr digestion with EcoR V, Dra I, Pvu II, and Ssp I, respectively. After phenol:chloroform extraction, the digested DNA was ligated with GenomeWalker adaptor (Clontech) and used for nested PCR with GW1 (5′-GTAATACGACTCACTATAGGGC-3′) and GW2 (5′-ACTATAGGGCACGCGTGGT-3′) primers (for the adapter) and gene-specific primers for grass carp PRL, including GP1 (5′-GAGGGAGTGAAGTTTGTCTGA-3′) and GP2 (5′-GAGGGAGTGAAGTTTGTCTGA-3′). The PCR reactions were conducted according to the instructions of the manufacturer. After that, the PCR products were gel purified and subcloned into the pGEM®-T Easy vector for DNA sequencing. Based on the DNA sequences obtained from intron trapping and genome walking, the nucleotide sequence of the full-length gene of grass carp prolactin was compiled using MacDNasis Pro  and MacVector computer programs .Genomic DNA was isolated from the whole blood of grass carp using proteinase K digestion followed by repeated cycles of phenol:chloroform extraction. After isopropanol precipitation and desalting with 70% ethanol, genomic DNA prepared was used in intron trapping by PCR with specific primers designed based on the nucleotide sequence of grass carp PRL cDNA (GenBank No. EU074210). For the cloning of the 5′ promoter of grass carp PRL gene, genome walking was performed using a Universal GenomeWalker35S Sequi Therm EXCELTM II DNA Sequencing Kit . The samples obtained after primer extension were resolved in parallel with the sequencing ladders in 8% gel with 8 M urea by PAGE at 96 W. The gel was then vacuum-dried at 80 °C and autoradiography was performed with Biomax MR films (Kodak) at −80 °C for 2–3 days.Primer scanning and primer extension were conducted to delineate the transcription initiation site of the grass carp PRL gene. For primer scanning, total RNA was purified from the carp pituitary using TRIzol (Invitrogen) and reversely transcribed into first strand cDNA with Superscript III (Invitrogen). The RT samples were then analyzed by PCR using a 3′ anchor primer PS1 (5′-AAGTTTGTCTGAAAGTTGAGAGGGCT-3′) located in the downstream region of PRL 5′UTR and various 5′ primers located at upstream region of the PRL promoter . During the process, parallel PCR with genomic DNA as the template was used as a positive control. In the same experiment, PCR with PS1 and P0, a 5′ primer located at the 5′ end of prolactin 5′UTR, was also performed to serve as a quality control for the RT samples prepared. For primer extension, total RNA was prepared from the carp pituitary. Using a plasmid DNA containing the PRL gene fragment covering the junction between the 5′ promoter and 5′UTR as a template, sequencing ladders  were constructed using PE1 (5′-CTGCAAAGTATAGTCTAGATCCT-3′) as a primer for cycle sequencing with a 6 cells/well/0.5 ml for transfection experiments. After 15 hr incubation, transfection was performed at 37 °C for 6 hr in 50 μl OPTI-MEM with 402 ng plasmid DNA and 2.1 μl lipofectamine (Invitrogen). The plasmid DNA was composed of 200 ng pPRL.Luc, 2 ng pTK.Luc (Promega), 20 ng pEGFP-N1 (Clontech), and 50 ng Pit-I expression vector with pBssK (Invitrogen) as the carrier DNA. The renilla luciferase-expressing vector pTK.Luc was included to serve as an internal control and the GFP-expressing vector pEGFP-N1 was used to monitor the potential variations in transfection efficiency between separate experiments. For the experiments with CREB siRNA, transfection was conducted in a similar manner except that the carrier DNA was replaced by SmarkPool CREB siRNA to make up the total amount of siRNA to 40 nM/well. Parallel transfection with siRNA control alone at 40 nM/well was used as the control. After transfection, the cells were exposed to drug treatment for 24 hr and dissolved in 200 μl lysis buffer (Promega). The lysate obtained was then subjected to luciferase activity measurement using a Dual Luciferase Reporter Kit (Promega) with a LB96 Microplate Liminometer .A 1156 bp fragment of the 5′ promoter of grass carp prolactin gene was subcloned into the firefly luciferase-expressing vector pGL3.Basic (Promega) to generate pPRL(−1156).Luc. The construct was then used for 5′ deletion of PRL promoter by PCR cloning to yield pPRL.Luc reporter constructs carrying decreasing lengths of grass carp PRL promoter from position −1156 to −57. The reporter constructs with PRL promoter were used for transfection studies in αT3-1 cells. αT3-1 cells were cultured in 24-well poly-D lysine pre-coated cluster plates at a seeding density of 0.1 × 1052. Further, the C/EBPalpha from −226 to −217 and AP1 from −136 to −130 were truncated from CTTTGCATTTA to CTTTA, and from TGATTAA to TAA, respectively. The mutation and truncation constructs were then subjected to transfection and dual-luciferase assay to evaluate the effect of oPACAP38, compared with the wild-type construct [pPRL(−306).Luc] and [pPRL(−166).Luc].To test the functional roles of potential binding sites in PACAP promoter, site-directed mutagenesis to introduce loss-of-function mutations or truncations in respective sites identified in carp PRL promoter were constructed separately using pPRL(−306).Luc as the template using the KAPA HiFi™ HotStart PCR kit (Kapa Biosystems) with the site-directed primers. With the construct of [pPRL(−306).Luc] as the template, the putative C/EBPalpha from −226 to −217 and AP1 from −136 to −130 were mutated from CTTTGCATTTA to CTTTACCGTTA, and from TGATTAA to AAGGTAA, respectively to make the mutation constructs3H] inositol (DuPont/NEN) in myo-inositol free DMEM medium containing 10% fetal bovine serum. After incubation for 12 hr at 37 °C, myo-[3H] inositol labeling medium was removed, and αT3-1 cells were washed two times with prewarmed phosphate buffered saline  to remove trace of the excessive myo-[3H] inositol. The transfected cells were then incubated in DMEM containing 0.1% BSA and 10 mM LiCl (to inhibit inositol phosphate degradation) for 20 min at 37 °C before drug treatment. After that, the transfected cells were treated with ovine PACAP38, VIP, and GnRH for 45 min. Drug treatment was terminated by removing drug-containing medium followed by addition of 0.5 ml/well ice-cold perchloric acid solution  and incubated on ice for at least 5 min. The samples were then neutralized with 0.5 M KOH and incubated for 20 min at 0 °C to ensure a complete precipitation of KClO4. After a brief sedimentation, the supernatant containing total inositol phosphates was applied onto Dowex anion-exchange columns . After sample loading, these columns were washed sequentially with a “high-salt” elution buffer (0.1 M formic acid and 1 M ammonium formate) into vials containing scintillation cocktail. Levels of radioactivity in these samples were measured using a liquid scintillation counter (Beckman).Transfected αT3-1 cells were labeled with 2 μCi/well of myo-[6 cells/well in 24-well plates and incubated for 48 h with or without drug treatment53. The cells were lysed in RIPA buffer  containing a cocktail of protease and phosphatase inhibitors . Same amount of protein in each well was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted onto an Invitrogen™ PVDF membrane by low-current eletrotransfer at 50 mA for 1 h. After blocking with 5% bovine serum albumin in PBS-Tween 20 buffer (PBST) for 1 h at room temperature, the membranes were incubated with the primary antibodies against pCREB and tCREB overnight at 4 °C. After three-time washing with PBST, the membranes were re-incubated with corresponding secondary antibodies for 1 h at room temperature and introduced for signal development54.Grass carp pituitary cells were prepared by trypsin/DNase II digestion method and seeded at a density of 2.5 × 10t test or ANOVA followed by Fisher’s least significance difference (LSD) test. Differences were considered significant at p < 0.05.For transfection experiments, luciferase activity was measured in terms of “arbitrary light unit”. The data for firefly luciferase activity were transformed as a ratio of renilla luciferase activity expressed in the same sample (referred to as “FF/RL Ratio”). The normalization was conducted to control for potential variations in transfection efficiency between wells. Since no significant differences could be noted in the expression levels of renilla luciferase activity, the data were simply transformed into a percentage of the mean value in the control group (as “% Ctrl”) for statistical analysis. Data presented (Mean ± SEM) were pooled results from four to six experiments and were routinely analyzed by Student’s Supplemenary Information


Danio rerio) remains the teleost fish of choice for biological investigations due to the vast array of molecular tools and resources available. To better understand the epigenetic regulation of autophagy, we utilized a primary myotube culture system generated from isolated myogenic precursor cells (MPCs) from zebrafish grown under starvation conditions using a media devoid of serum and amino acids. Here, we report starvation-induced regulation of several autophagy-related genes (atg) expression and profile the distribution of H3K27me3, H3K9me3, and H3K4me3 marks along lc3b, atg4b and p62/sqstm1 loci. These data support epigenetic regulation of autophagy in response to starvation that suggests a level of regulation that can be sustained for chronic conditions via chromatin modification.The zebrafish ( Summary: The role of nuclear events in the control of autophagy is underappreciated. This study demonstrates cell starvation associated gene expression changes and autophagy-specific histone modifications. In vertebrates, starvation induces muscle wasting by decreasing protein synthesis while increasing protein degradation, which is regulated via highly conserved proteolytic pathways  includinUpon starvation or other stress conditions, autophagy is induced by both a fast transcription-independent mechanism, entirely mediated by post-translational protein modifications, and a slower transcription-dependent mechanism involving a complex regulatory network . Most reHowever, it has become more than evident that the nuclear impact on autophagy is not limited to the sole action of transcription factors, but also involves epigenetic mechanisms . Histonein vitro myogenic culture system from zebrafish  in minimal media (devoid of serum and amino acids) resulted in the upregulation of several genes known to function in the autophagic pathway . Protein degradation occurs in part through the mechanisms of autophagy, a process orchestrated by autophagosome generation. As expected, the incubation of  pathway . Such an pathway . To date pathway ; howeveratg4b, p62/sqstm1 and lc3b) highly expressed in starved zebrafish myotubes  and p62/Sqstm1 locus  with reductions correlating with the increased transcription of both Atg4b and p62/sqstm1 at 24 h. Similarly, across the LC3b locus, region 2 exhibited reduced enrichment (P=0.016) of H3K9me3 and region 1 exhibited reduced enrichment of both H3K4me3 and H3K9me3 (P=0.0001) during starvation. These chromatin modification changes correspond with increased LC3b transcription upregulation at 24 h post starvation. The increased transcription of Atg4b, LC3b, and p62/Sqstm1 24 h following starvation, along with the reduction of the repressive H3K9me3 mark, suggests some degree of epigenetic regulation of these genes in starved zebrafish myotubes. To our knowledge, no data have been reported before on the enrichment in H3K9me3 at the autophagy-related loci. However, a recent study showed that H3K9 dimethylation (H3K9me2) mediated by the histone methyltransferase G9a acts as a repressor of autophagy  as well as other processes (involving DNA methylation or transcription factors) may be at play in the control of the expression of these genes. Undoubtedly, there is still a need to further understand the role histone modifications play in regulating autophagy. The zebrafish is a powerful model organism for studying numerous biological processes, and our recent characterization of an in vitro primary culture system for studying adult skeletal muscle stem cell proliferation and differentiation  were obtained from commercial suppliers and maintained in static tanks at 28°C under a 14 h light:10 h dark photoperiod on a commercial diet. Experiments were carried out in accordance with and approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Epaxial muscle from adult zebrafish was isolated and mechanically and enzymatically digested before cells were plated on poly-L-lysine HBr, laminin-coated dishes (1.5-2.0×106 cells per 35 mm dish) as previously described  and quantified by Nanodrop spectrophotometry (ThermoFisher). Purified RNAs (300 ng) were reverse transcribed using the ImProm-II™ reverse transcription system (Promega) and Oligo-dT primers (Qiagen). Thermocylcing was performed in a Bio-Rad CFX Connect Real-Time System  using Quanta PerfeCTa® Sybr® Green SuperMix as previously described  from cells using RNAzol  according to the supplier's protocol. The resultant aqueous supernatants were purified using PureLinkescribed . qPCR reescribed  were val6 per 0.65 ml of resin) and BSA (10 mg/ml), and washed . DNA was column-purified and samples were analyzed by qPCR in duplicate, as described above, with input (10%) and NTC controls. Primers were designed for autophagy-related genes  using the UCSC genome browser (http://genome.ucsc.edu) and the zebrafish ChIP-Seq data from Z-seq . Differences were considered significant at P<0.05. All statistical analyses and figure generations were completed in GraphPad Prism 7, www.graphpad.com.For chromatin immunoprecipitation (ChIP) experiments, MPCs were cultured as above until day 7 (myotubes) and then fixed following stimulation (24 h in AA+S or 24 and 48 h in MM) for 48 h in 1% methanol-free formaldehyde as previously described . For isoom Z-seq . Identif


BARX1, PAX9, LHX8, and DLX5. In addition, a novel long non-coding RNA associated with LHX8 was described. Furthermore, we identified DLX5 regulating expression of the long variant of RUNX2 transcript, which was specifically active in GFs but not in their periodontitis-affected counterparts. Knockdown of these factors in GFs resulted in altered expression of extracellular matrix (ECM) components. These results indicate activation of DLX5 and RUNX2 via its distal promoter represents a unique feature of GFs, and is important for ECM regulation. Down-regulation of these transcription factors in PAFs could be associated with their property to degrade collagen, which may impact on the process of periodontitis.Periodontitis is affecting over half of the adult population, and represents a major public health problem. Previously, we isolated a subset of gingival fibroblasts (GFs) from periodontitis patients, designated as periodontitis-associated fibroblasts (PAFs), which were highly capable of collagen degradation. To elucidate their molecular profiles, GFs isolated form healthy and periodontitis-affected gingival tissues were analyzed by CAGE-seq and integrated with the FANTOM5 atlas. GFs from healthy gingival tissues displayed distinctive patterns of CAGE profiles as compared to fibroblasts from other organ sites and characterized by specific expression of developmentally important transcription factors such as  Periodontitis is characterized by gingival inflammation accompanied by loss of supportive connective tissues for the tooth, resulting in impaired attachment of the periodontal ligament to the cementum. Periodontitis is one of the most common diseases in humans that affects over half of the adult population. Tooth loss caused by periodontitis is associated with masticatory dysfunction and poor nutritional status, and the medical cost for periodontitis and related diseases is an escalating burden to the healthcare economyBeyond the conventional view of periodontitis as an infectious disease, an increasing number of studies focus on the aberration of cellular responses in the periodontitis-affected gingival tissues. Several studies have performed comprehensive transcriptome analyses of the gingival tissues of periodontitis patients34A hallmark of periodontitis is degradation of extracellular matrices (ECM), such as collagen, between the tooth root and the alveolar bone. In various organs, the fibroblast is a central player to form the structural framework and control tissue repair by regulating ECM turnover and remodeling. Gingival fibroblasts play important roles not only in the homeostasis of gingival tissue architecture but also in the pathogenesis of periodontitis6Preventing ECM degradation in supportive connective tissues for the tooth seems a straightforward therapeutic approach for periodontitis. As a proof of concept, we have previously demonstrated that targeting PAFs by inhibition of key signaling pathways, i.e., transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), successfully protected PAF-mediated degradation of collagen in experimental models of periodontitis6To further characterize and dissect the molecular repertoire of PAFs we took advantage of the ongoing project of the Functional annotation of the mammalian genome (FANTOM) 5. FANTOM 5 is an international research consortium that has released transcriptome data for about one thousand human samples including cell lines, primary cells, and tissues, using the cap analysis of gene expression (CAGE) technology, which captures the 5′ end of capped transcripts and sequences around 27 base pairs9As part of the FANTOM5 project, we performed CAGE analyses on primary cultured human gingival fibroblasts, periodontal ligament fibroblasts, gingival epithelial cells, and epithelial cell rests of MalassezDLX5) and Runt-related transcription factor 2 (RUNX2) in its long transcript form is specific for gingival fibroblasts, and is virtually lost in PAFs. Further transcriptome analyses revealed that these identified genes are involved in the regulation of ECM. Our findings supported the hypothesis that altered molecular signals mediated by DLX5 and RUNX2 long form are associated with the aggressive phenotype of PAFs and degradation of ECM.First we investigated if gingival fibroblasts have molecular characteristics distinct from fibroblasts derived from other organs as suggested by a previous reporthttp://fantom.gsc.riken.jp/5/.This work is part of the FANTOM5 project. Data downloads, genomic tools, and co-published manuscripts are summarized online at A previous study has shown that human fibroblasts derived from different sites of the body display differential gene expression patternsUnsupervised hierarchical clustering with all DPIs revealed that primary cultures of GF and PLF established in our institute were grouped together . On the 2 fold change >2 or <−2, log2 counts per million >1, and matching single gene annotation (CAGE peaks represent transcription start sites (TSSs), and their profiling is useful to identify the locations and usage levels of TSSs. We can thereby evaluate expression levels of transcripts and promoter activities across the whole genome. To describe the CAGE profiles of GFs, CAGE tag counts of all 6 GFs were compared to those of all other 33 fibroblasts except PLFs. In total 3633 CAGE-defined promoters showed significantly higher expression, while 514 promoters displayed lower expression , Paired Box 9 (PAX9), Lim Homeobox 8 (LHX8), Distal-Less Homeobox 1 (DLX1), DLX2, DLX5, and Msh Homeobox 1 (MSX1), all of which have been shown as master regulators of mesenchymal cells during the tooth developmentNotably, highly expressed genes in GFs included BarH-Like Homeobox 1 , GSE19090 (6 GFs vs 33 others), and GSE22029  datasets, 595, 151, and 314 genes were found to be significantly enriched in GFs, respectively , DLX5, PAX9, and SIX Homeobox 1 (SIX1)  .BARX1, LHX8, and PAX9 were confirmed to be specifically expressed in GFs as compared with lung fibroblasts (LFs) and dermal fibroblasts (DF) (HOXB2 and Meis Homeobox 1 (MEIS1), both underexpressed in the FANTOM5 dataset, were not detected in GFs while expressed in LFs and DF  . In conts and DF .RUNX2, a master regulator of osteoblast differentiation and bone formationDLX5 specifically transactivates the RUNX2 distal promoter to confer cell type-specific expression of RUNX2 isoformsDLX5 is a robust GF-specific gene identified by our comparative analyses on the FANTOM5 CAGE profiles as well as 3 different microarray datasets  are transcribed from distal p1 promoter while a short form (NM_004348) uses proximal p2 promoter , and found that 34 and 16 ncRNAs showed significantly higher and lower expression in GFs, respectively 2021ectively .−82). Notably, this CAGE peak is located within 1,000 bp of the TSS of LHX8  that share the first exon and have several splicing variants. We designated these transcripts as lnc-LHX8 . In ordepression .Collectively, analyses on the CAGE profiles of GFs as compared to other fibroblasts delineated the unique transcriptional patterns of coding genes, alternative promoters, and ncRNAs.In the following analyses, we focused on gingival fibroblasts derived from patients suffering from periodontitis. Three pairs of patient-matched gingival fibroblasts were isolated from periodontitis-affected and healthy gingival tissues, and designated as periodontitis-associated fibroblasts (PAFs) and non-periodontitis-associated fibroblasts (non-PAFs). Consistent with our previous reportHierarchical clustering with all DPIs of PAFs, non-PAFs, and normal GFs revealed that both PAFs and non-PAFs tended to be grouped into the same cluster . Next weIL32), chemokine (C-X-C motif) ligand 1 (CXCL1), epiregulin (EREG), and secreted frizzled-related protein 2 (SFRP2), regulation of immune effector process such as complement component 3 (C3), and dipeptidyl peptidase-4 (DPP4), and cell adhesion such as intercellular adhesion molecule 1 (ICAM1), cadherin 18 (CDH18), and claudin 1 (CLDN1)  .CXCL1, matrix metalloproteinase-3 (MMP3), prostaglandin D2 synthase (PTGDS), SFRP2, EGF-TM7-Latrophilin-Related protein (ELTD1), IL32, and ICAM1. The heatmap of 37 genes which could be annotated both in the CAGE and GSE16134 datasets are shown in We supplemented the CAGE analysis of PAFs with publicly available microarray datasets from 241 periodontitis and 69 healthy gingival tissue samples GSE16134)43. The 4DLX5, RUNX2, PENK, and SYTL2) were listed as those highly expressed in GFs (RUNX2 short form (p2 RUNX2) was not different between PAF and non-PAFs  for RUNX2. Among them, target sequence of 236859_at is in the first exon and 5′UTR, which is specific for RUNX2 long form transcribed from p1 RUNX2. Microarray results confirmed specific knockdown of RUNX2 long form as observed in RT-qPCR experiments. Transcriptional profiling of GF4 transduced with DLX5 miR #2 , elastin (ELN), matrilin 3 (MATN3), dermatopontin (DPT), fibrillin 2 (FBN2) and vascular cell adhesion molecule 1 (VCAM1)  .DLX5 and RUNX2 distal promoters represents the unique feature of GFs, and is important for ECM regulation. Down-regulation of these transcription factors in PAFs is likely to modify the expression of ECM proteins and cell adhesion, which might be involved in the pathogenesis of periodontitis.Taken together, activation of This study evaluates the transcriptional profiles of GFs and PAFs as their pathogenic counterparts, by CAGE sequencing as part of the FANTOM5 project. We compared GFs to human fibroblasts from other tissues, which revealed the transcriptional profiles of GFs for coding genes, alternative promoters, and ncRNAs. To our knowledge, this is the first comprehensive characterization of the transcriptome characteristic for GFs.Fibroblasts are ubiquitous mesenchymal cells that play important roles in development, tissue repair, and various diseases such as fibrosing diseases and cancers. In periodontitis, fibroblasts critically contribute to its pathology by producing inflammatory cytokines and altering gingival tissue architectures12BARX1, PAX9, LHX8 and DLX5, are highly expressed in GFs. It is noteworthy that these transcription factors are also active in mesenchymal cells during tooth development. Among them, DLX5 was found to be inactivated in PAFs derived from the adult periodontitis-affected gingival tissues. This finding implies that positional memory of development is maintained in GF, and loss of GF identity is linked to pathological activation of PAFs. Transcriptional profiling of PAFs showed that genes involved in signal transduction and immune response are up-regulated , elastin, matrilin 3, dermatopontin, and fibrillin 2.Given that activation of DLX5 and RUNX2 long form, such as osteomodulin (OMD), asporin (ASPN), decorin (DCN), and osteoglycin (OGN). SLRPs are a group of proteins sharing various structural and functional similarities that have multiple roles in ECM regulationIn addition, small leucine-rich proteoglycans (SLRPs) were also found to be regulated by Asporin is found to be expressed in periodontal ligaments36DLX5-mediated alternative promoter activation of RUNX2 is crucial for ECM organization and homeostasis of the gingiva. Disruption of this signaling in PAFs may be involved in ECM degradation and impaired architecture of the gingiva of periodontitis. These findings provide novel insight into the molecular mechanisms how PAFs develop and contribute to progression of periodontitis.In conclusion, CAGE profiling characterized distinctive transcriptional features of GFs and PAFs. Loss of GF identity appeared to be linked to the PAF phenotype, and Isolation and cell culture of gingival fibroblasts from the healthy or periodontitis gingival tissues were performed as described previously711in vitro model of periodontitis according to the method described previously67Three-dimensional co-culture of gingival epithelial cells and fibroblasts were employed as an RUNX2 gene” (p1 RUNX2) since it is the first peak in terms of total read counts within the peaks associated with RUNX2. Thus, alternative promoters for the same gene were ranked by their expression levels, and termed as p1, p2, and p3. CAGE data with raw read counts were obtained from the FANTOM5 Table Extraction Tool, and were analyzed using the R Bioconductor package ‘edgeR’ for differential expression analysisThe details of CAGE library generation and clustering were described previouslyTotal RNA was extracted using the RNeasy Mini Kit . RNA-sequencing (RNA-seq) was performed at the Genome Network Analysis Support Facility (GeNAS), RIKEN CLST, Yokohama, Japan. For preparing RNA-Seq library, rRNA depletion was performed with 1 μg total RNA using TruSeq Stranded Total RNA with Ribo-Zero GoldKit . Prepared libraries were sequenced with 2 × 100 bp paired-end reads on the Illumina HiSeq 2500 sequencer (Illumina). After quality check, raw sequence reads were mapped to the hg19 genome by Tophat (version 2.0.14)GAPDH). The PCR primers are listed in Reverse transcription quantitative PCR (RT-qPCR) was performed as previously describedPublicly available microarray datasets including GSE3551DLX5 and RUNX2 long form, using BLOCK-iT™ RNAi Designer  and RUNX2 long form (miR #1), using the RNeasy Mini Kit. Microarray analysis was carried out using Affymetrix GeneChip® Human Genome U133 Plus 2.0 array, according to the manufacturer’s instructions. Expression values less than that of negative control probe were filtered out, and cut-off value of fold change compared to NC was set to 0.5 for down- and 2.0 for up-regulated genes. The dataset was deposited in the Gene Expression Omnibus database (GSE81870).Total RNA was extracted 5 d after infection of lentiviruses which carry negative control (NC) miRNA (miR) or those targeting t test for unpaired samples was used for statistical analysis. The data are expressed as means ± standard deviation (SD), and p < 0.05 was considered statistically significant.Analyses of variance or Student’s How to cite this article: Horie, M. et al. Transcriptome analysis of periodontitis-associated fibroblasts by CAGE sequencing identified DLX5 and RUNX2 long variant as novel regulators involved in periodontitis. Sci. Rep.6, 33666; doi: 10.1038/srep33666 (2016).


Chromatin 3D structure is highly dynamic and associated with many biological processes, such as cell cycle progression, cellular differentiation, cell fate reprogramming, cancer development, cellular senescence, and aging. Recently, by using chromosome conformation capture technologies, tremendous findings have been reported about the dynamics of genome architecture, their associated proteins, and the underlying mechanisms involved in regulating chromatin spatial organization and gene expression. Cellular senescence and aging, which involve multiple cellular and molecular functional declines, also undergo significant chromatin structural changes, including alternations of heterochromatin and disruption of higher-order chromatin structure. In this review, we summarize recent findings related to genome architecture, factors regulating chromatin spatial organization, and how they change during cellular senescence and aging. Aging is a complex biological process that involves multiple cellular and molecular functional declines, including cellular senescence, genomic instability, and epigenetic alterations . NumerouHigher-order chromatin organization has attracted great attention in recent years. With the advent of chromosome conformation capture (3C) methods and high-resolution nuclear microscopy, tremendous progress in our understanding of chromatin organization has been made. At the higher end of chromatin organization, interphase chromosomes form distinct territories called chromosome territories (CTs); small and active chromosomes tend to locate at the center of the nucleus, whereas inactive and heterochromatic regions are located in the nuclear periphery ,8,9. Usitrans regulatory elements, rather than as an absolute block. Nucleosomal DNA rapidly unwraps from and rewraps on the surface of the histone octamer, allowing the invasion of DNA binding proteins [From linear DNA to the 3D nucleus, chromatin organization is well characterized on both the small scale and the very large scale; however, our understanding of the intermediate levels of chromatin organization remains limited. At the finest scale, DNA is first packaged into nucleosomes, the basic subunit of chromatin. Each nucleosome includes a nucleosome core, a linker DNA, and a H1 linker histone. The structure of the eukaryotic nucleosome is generally conserved: it is composed of 147 bp of DNA coiled around a histone octamer that contains two copies of four core histone proteins, H2A, H2B, H3, and H4 ,14. Thisproteins . Histoneproteins , H3/H4 tproteins , and nucproteins . The chaproteins .At the highest scale, metaphase eukaryotic chromosomes are condensed and can be visualized under the light microscope. The morphology of metaphase chromosomes has been characterized with well-defined shapes and sizes. In contrast, interphase chromosomes are less condensed and difficult to distinguish by imaging and segmenting. Interphase chromosomes are not randomly positioned in the nuclear space but are generally segregated, with each chromosome forming distinct CTs ,22. InteThe human nucleus is functionally compartmentalized . Small cMultiple lines of evidence suggest that chromosomes are partitioned into different compartments or domains. Lieberman-Aiden et al. defined two compartments in the human genome, compartment A and compartment B, at the megabase scale using Hi-C, with the idea that domains belonging to the same compartment should have more frequent interactions . ChromatDrosophila genome at the sub-megabase scale. Boettiger et al. (2016) partitioned the Drosophila chromosomes into three distinct domain types, i.e., active, inactive, and Polycomb-repressive, according to their epigenetic states. Using super-resolution imaging, they showed that each domain type has a distinct packing density and that Polycomb-repressed domains are spatially excluded from neighboring active domains [The existence of compartment A and B have been confirmed at the single-cell level. By multiplexed FISH, these compartments are found to be spatially segregated . Similar domains .More detailed genome contact maps generated by Hi-C revealed that compartments A and B can be broken down into smaller sub-compartments  with a median size of 185 kb . Each ofThe partitioning of the genome into compartments A and B is cell type-specific. For instance, during human embryonic stem cell (ESC) differentiation, as much as 36% of the human genome undergoes compartment switching, i.e., transitions from compartment A to B, or vice versa. However, the fraction of the genome that localizes to different compartments is considerably smaller when comparing two differentiated cell types . By examChromosome compartments can be further partitioned into smaller domains, called TADs. TADs are the basic unit of chromatin 3D organization. They were first identified from mouse ES cell Hi-C data, and can be visualized in contact maps as highly self-interacting regions . Follow-Hox genes, suggesting that specific 3D interactions are limited by linear distance [With the development of super-resolution Hi-C and other 3C-based methods such as ChIA-PET, Capture Hi-C, HiChIP, and PLAC-seq, specific long-range interactions between functional loci, or chromatin looping, can be detected ,44,45,46distance . Many codistance ,49,50. Idistance .CTCF binding sites are abundant and widely distributed in bilaterian genomes, but absent in other eukaryotes . TraditiINK4a, which drives the irreversible cell cycle arrest characteristic of senescence, is encoded by the INK4/ARF locus, which contains at least three CTCF binding sites [INK4ap16 expression is negatively regulated by CTCF, which drives the formation of compact chromatin loops at its promoter [INK4ap16 silencing; however, under oncogene-induced senescence, during which CTCF is down-regulated, these loops are disrupted, resulting in high expression of INK4ap16 [IGF2) in aged and senescent cells. In both prostate cells that have undergone replicative senescence and those isolated from aged mice, decreased CTCF levels result in less binding of this protein to the IGF2 imprinted region [IGF2, and the effects are more extensive in aged individuals with prostate cancer. This suggests that CTCF-mediated imprinting pattern changes may contribute to aging-related gene expression and cancer progression [CTCF, which has been shown to be reduced during aging or senescence in various models, plays important roles in these processes. The senescence effector p16ng sites . p16INK4promoter . The higp16INK4a . CTCF ald region ,63. Redugression ,63. CTCFgression . Cells dgression .cis regulatory elements, and sister chromatid segregation during mitosis [Cohesin is a ring-shaped complex that contains four core subunits, SMC1, SMC3, RAD21, and STAG1, and several cohesin-associated proteins . It regu mitosis . Murayam mitosis . Cohesin mitosis . Cohesin mitosis , while C mitosis . Depleti mitosis . A recen mitosis .Drosophila and mammals, where the fidelity of meiotic chromosome segregation significantly declines with age. In Drosophila, a significant increase of meiotic segregation errors was observed in aged Drosophila oocytes when SMC1 is reduced [Because of the function of the cohesin complex, it is particularly essential to prevent genome instability, which is one of the hallmarks of aging . As with reduced . In mamm reduced ,76.Mediator is a large, multi-subunit complex essential for regulating transcription initiation and elongation . It is aHox gene cluster [INK4ap16 in young individuals [INK4ap16 in old tissues is partially derived from the decline of Polycomb protein BMI1, which is a potent repressor of INK4ap16 [ThymoD non-coding RNA promotes DNA demethylation at CTCF binding sites and facilitates the formation of cohesion-mediated chromatin loops between the promoter of Bcl11b and its enhancer [Polycomb proteins and non-coding RNAs are also implicated in 3D chromatin organization. In the mammalian genome, depletion of Polycomb subunits results in the disruption of chromatin loops around important developmental genes, such as  cluster ,84. Polyividuals , and thep16INK4a . In addip16INK4a . Non-codenhancer .Caenorhabditis elegans, multiple miRNAs, including lin-4, mir-71, and mir-245, promote lifespan [Numerous studies reported that noncoding RNAs are associated with lifespan. They can either be products or regulators of aging and cellular senescence. In yeast, the expression of ncRNAs from the rDNA locus, which is repressed by Sir2 under normal conditions, is significant upregulated in old individuals . In addilifespan . In mammlifespan .Cellular senescence and aging are related but distinct biological process that occur on different scales. Growing evidence shows that cellular senescence has a role in aging and is one of the major causes of age-related diseases ,93,94,95set-26 extends lifespan and moderately attenuates the decrease in histone H3 protein levels [Global loss of core histone proteins, which partially contributes to the increase of genome instability, occurs during aging and senescence  Figure A. Compeln levels . SET-26 n levels . In addin levels .Suv39h1 in progeroid cells, which was associated with a DNA repair deficiency in heterochromatin regions. They also reported that depletion of Suv39h1 in progeroid mice prolongs their lifespan and suggested that increased H3K9me3 in these mice may promote heterochromatin compaction and obstruct the access of DNA repair factors [In addition to core histone protein levels, histone modifications also play important roles in regulating aging and chromatin structures A. The ch factors . TogetheRph1 reduces the level of cryptic transcription and extends lifespan [C. elegans, lifespan can be extended by reducing H3K4me3 and increasing H3K27me3 [Drosophila, aging is associated with a loss of H3K9me3 in heterochromatin and a gain of H3K4me3 and H3K36me3 [Alterations in histone modifications are also implicated in lifespan regulation in other organisms. In budding yeast, the levels of the histone deacetylase Sir2 decrease during replicative aging, which causes an increase in H4K16 acetylation . Overexplifespan . In C. eH3K27me3 ,114,115.H3K36me3 .isw2 deletion strain, and overexpression of RAD51 extends yeast lifespan [Nucleosome structure is relatively stable, whereas the distribution of nucleosomes along chromatin is variable and can be regulated by ATP-dependent nucleosome remodeling complexes, leading to either more compact or more accessible chromatin. Deletion or inactivation of the chromatin remodeler ISW2 promotes longevity in budding yeast, in a TOR signaling-independent manner . Deletiolifespan , confirmlifespan . The chrlifespan . It inteDrosophila and Arabidopsis [Another ATP-dependent chromatin remodeling complex known to be involved in aging is the nucleosome remodeling and deacetylase (NURD) complex. The protein levels of NURD subunits RBBP4 and RBBP7 are reduced in HGPS skin fibroblasts; this is associated with lower levels of HP1, indicating global chromatin defects . Primarybidopsis . Howeverbidopsis . In summAs mentioned above, SAHFs are a special senescence-associated structure that only form in specific types of senescent cells . They arThe formation of SAHFs is one example of the disassociation between the nuclear lamina and chromatin during aging and senescence. The nuclear lamina consists two types of lamins (A and B) and associated proteins. Mutations or depletion of lamin proteins leads to severe changes, including abnormal nuclear morphology, DNA damage, and chromosomal aberrations . For exaAlthough the development of the Hi-C method greatly improved our understanding of normal genome architecture, studies that focus on chromatin higher-order structure changes during aging are limited. Overall, changes in 3D chromatin structure, such as local interaction frequencies B and chrA Hi-C experiment has also been conducted on Ras-induced senescent cells (a type of OIS), most of which contain SAHFs. No global chromosomal interaction pattern changes were observed when comparing proliferating and senescent cells. However, local interactions were reduced in constitutive heterochromatin, accompanied by the formation of specific long-range interactions with other repressed regions . The autSimilar to OIS cells, the overall genomic conformation is largely unchanged in cells that have undergone replicative senescence (RS cells); chromatin compartmentalization and TAD boundaries are generally conserved in proliferating, quiescent, and RS cells. However, a global gain of short-range interactions and a loss of long-range interactions were found in RS cells . By measThe development of Hi-C methodologies and high-resolution nuclear imaging techniques are transforming our understanding of chromatin structure and 3D genome organization. How chromatin organization influences aging and is altered by it and the related process of cellular senescence is only beginning to be explored. Decreased histone protein levels and changes in the abundance and distribution of particular histone modifications contribute to the opening of chromatin at the nucleosome level with age and senescence. This, in conjunction with the activity of chromatin remodeling enzymes, contributes to transcriptional dysregulation and, in some instances, genome instability. Consistent with the opening of chromatin, there is a loss of constitutive heterochromatin during aging and in progeroid syndromes. Heterochromatin loss is also associated with the disruption of LAD–nuclear lamina interactions, which potentially have profound effects on nuclear organization and chromatin compartmentalization. However, there have only been a few explicit studies on higher-order chromatin changes during aging and senescence; they suggest that, in most cases, such structures are overall largely preserved. TADs, the basic unit of chromatin organization, are largely intact, though there is a reduction in intra-TAD chromatin interactions. Likewise, the genome is still partitioned into compartments A and B, though compartment switching does occur both in HGPS and during cellular senescence. This compartmentalization is even maintained in cells that have undergone OIS and have formed SAHFs, in which the normally peripheral heterochromatin is localized more centrally, indicating drastic nuclear reorganization.The picture of age-associated changes in genomic organization is only beginning to emerge, and many critical questions remain unanswered. While key regulators of chromatin higher-order structure, including CTCF and cohesin, are known to decrease with age, how this affects both aging and chromatin structure is largely unknown. Chromatin looping mediated by CTCF, cohesin, and Mediator is essential for proper gene regulation, which is known to be disrupted during aging; however, age-associated changes in sub-TAD-level chromatin looping have barely begun to be explored, probably in part because of the challenges of generating enough sequencing data to obtain the needed resolution. The evidence currently suggests that, in many cases, many of the chromatin organization changes will be found at this level, though we clearly need to examine the 3D genome structure in additional aging models, particularly in primary tissue samples. Another area that deserves more attention is the role of LADs and the nuclear lamina in regulating aging, gene expression, nuclear organization, and the formation of SAHFs. Ultimately, understanding the interplay between different levels of chromatin organization, from how histone modifications contribute to chromatin looping and promote spatial segregation of heterochromatin within the nucleus to how chromatin compaction impacts short-range loop formation and gene expression, will be instrumental in unraveling the mechanisms that drive aging.


Numerous long intervening noncoding RNAs (lincRNAs) are generated from the mammalian genome by RNA polymerase II (Pol II) transcription. Although multiple functions have been ascribed to lincRNAs, their synthesis and turnover remain poorly characterized. Here, we define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells. This is based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing (mNET-seq). Notably, mNET-seq patterns specific for different Pol II CTD phosphorylation states reveal weak co-transcriptional splicing and poly(A) signal-independent Pol II termination of lincRNAs as compared to pre-mRNAs. In addition, lincRNAs are mostly restricted to chromatin, since they are rapidly degraded by the RNA exosome. We also show that a lincRNA-specific co-transcriptional RNA cleavage mechanism acts to induce premature termination. In effect, functional lincRNAs must escape from this targeted nuclear surveillance process.  •lincRNAs and pre-mRNAs are transcribed by different Pol II phospho-CTD isoforms•lincRNAs are rarely spliced and mainly non-polyadenylated•lincRNAs are stabilized in the nucleoplasm following exosome inactivation•lincRNAs are co-transcriptionally cleaved Schlackow and Nojima et al. show that mammalian pre-mRNAs and long intergenic noncoding (linc) RNAs employ radically different transcription and RNA-processing strategies. Pre-mRNAs are transcribed by defined RNA polymerase (Pol) II isoforms reflecting co-transcriptional splicing and polyadenylation. Instead, lincRNAs are mainly transcribed by deregulated Pol II and simultaneously degraded. S. cerevisiae or PROMPTs in mammals  from the human genome. These transcripts are modified by pre-mRNA processing events, such as 5′ capping, pre-mRNA splicing, 3′ end cleavage, and polyadenylation during Pol II transcription . Pre-mRN mammals . Additioed eRNAs . Finallyed eRNAs . It is tWhereas PROMPTs and enhancer RNAs (eRNAs) likely form as a consequence of Pol II accumulation at transcription initiation sites, it is more plausible that lincRNAs, with their independently defined transcription units, have specific biological significance. However, their low sequence conservation and often very low steady-state levels imply that many of these ephemeral transcripts reflect transcriptional noise . One oftDefining the TUs of lincRNAs is a challenging problem of sequence annotation. Often transcription start sites are inferred from 5′ end cap selection methods, such as CAGE  or Cap-s1S2P3T4S5P6S7) that is differentially phosphorylated during Pol II transcription  to precisely define nascent transcription across the human genome . In partcription . mNET-seWidespread lincRNAs have been defined in several comprehensive studies . AlthougPol II CTD phosphorylation states are well established to match different transcriptional stages: Ser5P (S5P) with early elongation, 5′ end capping, and active splicing and Ser2P (S2P) with later elongation and 3′ end processing . mNET-seMeta-analysis of protein coding as compared to lincRNA genes reveals significant differences in mNET-seq profiles. Both heatmaps and metagene profiles A are shoTARS and a specific lincRNA gene  is evident in mNET-seq/S5P profiles of protein-coding genes , as seenTARS D. These  regions E. We nex regions . As prev regions , Pla-B es   . The upstructure . Notablyependent G. Consisependent . Whereasfraction H. We alsepletion E. Furtheanalysis . OverallLBR  . HoweverWe predict from the widespread profiles of mNET-seq/T4P reads across lincRNA TUs that Pol II terminates sporadically across this gene class . AdditioMYC gene, S5P-specific 5′ss peaks are specifically lost with Empigen treatment, presumably because the co-immunoprecipitated spliceosome containing this splicing intermediate is now released from the Pol II complex , such as splicing intermediates or microRNA precursors . Because complex A. This w complex A. Similareatment B. Importreatment A and 5B.MALAT1 and LINC01021 in mNET-seq/S5P and S2P profiles  or the related RNase III endonuclease Dicer. Although Dicer activity is predominantly cytoplasmic, where it acts to process pre-microRNA into microRNA , nuclearCCND1 A and S6Bdetected B and S6Cdetected . HoweverMALAT1 and LINC01021  to compare protein-coding versus lincRNA TUs based on multiple parameters. Because our restricted lincRNA set displays very similar profiles to the larger antisense lncRNA set F, these sitivity A. Furthesitivity . We alsositivity B. In mansitivity .The full list of principal component (PC) values and the identified lincRNA-like protein-coding genes and protein-coding-like lincRNAs can be found in We have analyzed HeLa cell nascent transcription using mNET-seq methodology , employiWe observe less Pol II pausing over the TES region of lincRNA genes, compared to protein-coding genes B, bottomOur analysis of HeLa cell lincRNAs by subcellular RNA-seq analysis reveals a clear pathway to their rapid degradation . First, We examined the possibility that lincRNA endonucleolytic cleavage could be generally mediated by the microprocessor. Components of microprocessor, Drosha, and DGCR8 proteins cleave pre-miRNA structures co-transcriptionally . We therWe ended our bioinformatics comparison of lincRNA TUs versus protein-coding TUs by subjecting them to PCA A. RemarkDetailed protocols for mNET-seq, ChrRNA-seq, and NpRNA-seq were previously described . For mNEHg19/GRCh37 was used as a reference genome. TUs were extracted based on ENSEMBL .Heatmaps were created using the MATLAB R2015b image function. All other graphs were created using ggplot2 in R. p values are computed via a Wilcoxon test in R or a Fisher exact test in MATLAB D. PCA isM.S., with advice from T.G., performed all bioinformatics analyses. T.N. performed all molecular biology and transcriptomic experiments with help from A.D. on the microprocessor. N.J.P. and T.N. designed the project and wrote the paper with help from M.S. and M.C.-F.


Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP , which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal −1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcription. SLIM-ChIP is an attractive solution for mapping DNA binding proteins and charting the surrounding chromatin occupancy landscape at a single-cell level.  •SLIM-ChIP is a low-input, robust, high-resolution, automatable TF mapping protocol•SLIM-ChIP is applicable to a range of TFs from yeast to mammals•DNA fragments from SLIM-ChIP provide targeted footprinting at the bound site•RSC-mediated Reb1-nucleosome interactions affect promoter directionality Mapping transcription factors binding to DNA by chromatin immunoprecipitation sequencing is a key step in studying transcriptional programs. Gutin et al. introduce SLIM-ChIP, a simple, automation compatible protocol, that provides insights about the chromatin landscape at the bound sites. Using this protocol, they discover promoter architectures that enforce unidirectional transcription. Our understanding of transcription factor binding n (ChIP) . Couplinn (ChIP) . Typical9 in the original protocols) and involve several additional biochemical and/or molecular steps compared to conventional ChIP-seq. One obstacle in adapting ChIP protocols to high-throughput workflow is chromatin shearing by sonication. This obstacle can potentially be bypassed by specialized sonicators  A. We useThe SLIM-ChIP protocol starts with MNase digestion of chromatin . After sSaccharomyces cerevisiae  does not alter the signal  that are unique to ORGANIC. These peaks are different than the core peaks in three main aspects. First, more than 50% of ORGANIC-unique peaks are located within open reading frames (ORFs) compared to ∼10% of core peaks C, SecondTogether, these observations demonstrate that all three methods identify a large set of core peaks, which most likely represent sites that are highly occupied by Reb1 in a mid-log yeast population. We do observe additional peaks outside this core set. These peaks tend to have lower read coverage and thus may correspond to weaker or variable binding of Reb1 or alternatively can be due to technical biases and noise .Our current understanding is that MNase has preference to endonucleolytically cleave protein-free DNA and further digest the cleaved fragments until it encounters shielded DNA or a formaldehyde cross-linking point. Thus, we reasoned that many of our ChIP fragments should end at such sites. Indeed, when plotting the location of fragment ends flanking the Reb1 peaks, we see sharp boundaries in a 30-bp window around the motif G. These Having validated our method for Reb1, we next applied SLIM-ChIP to probe the binding pattern of Rap1 and Abf1, two additional well-studied yeast transcriptional regulators. As observed for Reb1, the binding patterns of Abf1 and Rap1 recover the expected landscape, with pronounced peaks A. Motif Our method should in principle be readily applicable to a wide range of organisms. As a concrete example, we used it to map the binding of CTCF, a key chromatin organizer, in mouse embryonic stem cells (ESCs). The resulting binding map recapitulates earlier mapping of CTCF in the same cells B. Again,The MNase digestion pattern around CTCF peaks uncovers a complex protection pattern, which is compatible with multiple zinc-finger DNA-binding domains in CTCF  Figure D. This pAn important challenge in the chromatin field is the reconstruction of the binding configurations of chromatin factors and their interactions . This problem can be tackled by methods for DNA accessibility footprinting ; howeverWe reasoned that SLIM-ChIP provides an alternative approach to focus on interactions of a target transcription factor. SLIM-ChIP captures DNA fragments enriched for the bound transcription factor. The boundaries of these fragments are largely determined by the protection pattern of the DNA as in MNase footprinting . Thus SLWe first analyzed fragment size distribution around Reb1 peaks oriented according to the TSS of the closest gene A. As expWe next examined the fragment length distribution at the level of single Reb1-binding sites B. ClearlBinding sites at clusters 3 and 4 are characterized by bimodal distribution of fragment lengths in which the long fragments likely represent an interaction of Reb1 with a proximal nucleosome. This is more apparent in cluster 4, which is heavily skewed toward long fragments (nucleosome interaction) and insensitive to MNase levels B. To furReb1 promotes NDR formation partially by recruiting the essential RSC chromatin remodeling complex, which can evict or slide nucleosomes away from Reb1-binding sites . We usedReb1 is known to activate transcription , likely NET-seq patterns also reveal pronounced short aborted transcripts immediately adjacent to Reb1 at Cluster 4 sites B, arrow.locked) sites is due to physical constraints, such as DNA-binding proteins, that limit the ability of the nucleosome to move away from the Reb1 site  shows that SLIM-ChIP is in excellent agreement with previous results and yet it is much simpler to perform.The combination of MNase digestion and recovery of variable fragment lengths allows us to gain important insights about binding events. Effectively, by combining MNase footprinting  with SLIAltogether, SLIM-ChIP is an attractive solution for mapping DNA-binding events that allows us to examine and interrogate the DNA occupancy landscape at a single-molecule level in the context of the binding event. This has the potential to elucidate mechanisms of transcriptional regulation that are not observed in the absence of informative fragment length data.See All FLAG-tagged transcription factors strains were done in BY4741 background by inserting a 5xGly-FLAG His3MX6 cassette immediately before the target gene stop codon. In auxin depletion strains, an auxin-inducible degradation domain was genomically inserted immediately before the target gene stop codon in 2721 cells . In all Mouse R1 ESCs (cell line) were grown on gelatin and maintained in ESC medium .Cells were fixed with 1% formaldehyde for 15 min. Yeast cells were treated with zymolyase to generate spheroplasts prior to lysis and MNase treatment. Lysed spheroplasts or mouse ESCs (mESCs) were treated with different amounts of MNase to digest chromatin into nucleosomes.MNase-digested chromatin was allowed to bind to antibodies for 2–4 hr. Paramagnetic protein G dynabeads were added for an additional hour, and the beads were extensively washed. Bound chromatin was ligated to barcoded DNA adapters, and chromatin was reverse cross-linked and pooled. Barcoded chromatin was amplified with Illumina next-generation-sequencing compatible primers, and DNA libraries were paired-end sequenced by Illumina NextSeq 500. For MNase nucleosome mapping, MNase-digested chromatin was reverse cross-linked and isolated by 2× SPRI beads, and MNase sequencing libraries were prepared as described previously .Paired-end reads were mapped to the yeast (sacCer3) and mouse (mm9) genomes using bowtie2 default parameters, except for setting of maximal fragment size of 2,000 bp. Peak calling was performed using MACS algorithm (version 2.1.1) . Motif dThe location of the motif within the peak region was determined. The motif-containing regions were then oriented according to the motif directionality and aligned around its center. Peaks without a motif were discarded from this analysis.The closest TSS to each peak summit was found. The genomic regions were then oriented according to the gene directionality and aligned around the TSS. Peaks that were >256 bp away from the nearest TSS were discarded from this analysis.Finally, the sum of read coverage in the aligned regions was plotted.The peaks used for the comparison were reconstructed from the short reads (<80 bp) of the low-MNase sample B. The ChEach peak was represented by a “fragment length vector” containing the fraction of reads mapped to the peak region in every length bin . These vectors were clustered using K-means algorithm (K = 4), generating 4 distinct clusters C. Peaks Raw NET-seq data was downloaded from the original manuscript . For NETNucleosome coverage plots were generated by taking the center of all mapped fragments and extending them by 25 bp in both directions. Peaks that reside in telomeres were discarded from this analysis.


In addition, midregion PTHrP appears to be imported in the nucleoplasm of cultured MDA-MB231 cells and in vitro, it can bind chromatin of metaphase spread preparations and also an isolated 20-mer oligonucleotide, thereby appearing endowed with a putative transcription factor–like DNA-binding ability. The object of this review is to discuss collectively and critically both precedent and more updated data obtained in the lab, the latter arising from assays on DNA status, and gene and protein expression patterns of treated cells, aiming to check whether the cytotoxicity of the peptide may result from a reprogramming of gene expression towards apoptotic death or, instead, it is to be ascribed to an unprogrammed perturbation of cell functions.PTHrP is a polyhormone undergoing proteolytic processing into smaller bioactive forms, comprising an N-terminal peptide, which is the mediator of the “classical” PTH-like effect, as well as midregion and C-terminal peptides. The midregion PTHrP domain (38-94)-amide was found to restrain growth and invasion