However, unlike true tumor suppressor- or onco-genes, the SOD2 gene is not frequently lost, or rarely mutated or amplified in cancer. This allows SOD2 to be either repressed or activated contingent on context-dependent stimuli, leading to its dichotomous function in cancer. Here, we describe some of the mechanisms that underlie SOD2 regulation in tumor cells. While much is known about the transcriptional regulation of the SOD2 gene, including downregulation by epigenetics and activation by stress response transcription factors, further research is required to understand the post-translational modifications that regulate SOD2 activity in cancer cells. Moreover, future work examining the spatio-temporal nature of SOD2 regulation in the context of changing tumor microenvironments is necessary to allows us to better design oxidant- or antioxidant-based therapeutic strategies that target the adaptable antioxidant repertoire of tumor cells.While loss of antioxidant expression and the resultant oxidant-dependent damage to cellular macromolecules is key to tumorigenesis, it has become evident that effective oxidant scavenging is conversely necessary for successful metastatic spread. This dichotomous role of antioxidant enzymes in cancer highlights their context-dependent regulation during different stages of tumor development. A prominent example of an antioxidant enzyme with such a dichotomous role and regulation is the mitochondria-localized manganese superoxide dismutase SOD2 (MnSOD). SOD2 has both tumor suppressive and promoting functions, which are primarily related to its role as a mitochondrial superoxide scavenger and H However, leakage of electrons at complexes I and III can results in partial reduction of O2 to superoxide anion free radical (O2•−), a short-lived oxidant that can either directly react with biomolecules or be dismutated to H2O2 by superoxide dismutases (SOD) anthracene (DMBA) and tetradecanoylphorbol-13-acetate (TPA) activate p53 during tumor initiation and lead to SOD2 transcriptional repression, via inhibition of Sp1 binding to the proximal promoter [SOD2 gene and increased SOD2 transcription [p53 binding sites have been identified in the 5’ upstream region of the OD2 gene ,90; howepromoter ,91. As tpromoter ,87. The cription . The abocription  or wherecription . TherefoSOD2 transcription, including Nuclear Factor, Erythroid 2 Like 2 (Nrf2) and NF-κB signaling. This is linked to intra- and extra-cellular redox stress in response to changing tumor states, an example being the surge in ROS associated with matrix detachments during metastasis, as well as extracellular signals from the tumor microenvironment, including growth factors and cytokines. Prominent examples of these are discussed below. However, there are also examples where dampening of these pathways leads to SOD2 repression in cancer. Enhancer regulatory domains for a number of transcription factors are found at two main locations in the SOD2 gene, the 5′ upstream (~−1500 bp) and the intronic enhancer regions  [SOD2 transcription. SOD2 increases during tumor progression have largely been linked to stress response pathways that positively regulate  ~+1500, . In the n (AP-1) , CAMP Ren (AP-1) , p53 [90n (AP-1) , Forkhean (AP-1) , NF-κB an (AP-1) , while tn (AP-1) ,100. BinSOD2 by NF-κB was established in many studies using different experimental systems. A particular finding is that cells treated with tumor necrosis factor alpha (TNF-α) and interleukin 1 (IL-1) increase transcription of SOD2 dramatically through NF-κB binding to the intronic enhancer region (5′-GGGAATACCC-3′) located in the second intron of the SOD2 gene through interactions with CCAAT/Enhancer Binding Protein Beta (C/EBP-β) [SOD2 promoter and is associated with histone hyperacetylation [SOD2 by NF-κB, it has been demonstrated that this stress response pathway is involved in anti-apoptosis [NF-κB is a well-known stress-responsive transcription factor activated upon various stimuli including cytokines , retinoiC/EBP-β) ,100. It tylation . This hatylation . Since tpoptosis , tumor mpoptosis  and radipoptosis . TNF-α tpoptosis . Not onlpoptosis . This alpoptosis . OverallSOD2 gene has not been shown so far, algorithm-based analysis predicted SOD2 as a strong candidate of Nrf2 target genes [SOD2 could be a transcriptional target of Nrf2 [The core sequence of the antioxidant responsive element (ARE), 5′-TGACnnnGC-3′, has been found in many genes encoding detoxifying enzymes such as NAD(P)H quinone oxidoreductase (NQO1) and γ-glutamylcysteine subunits (γ-GCS) . Among met genes . Impaire of Nrf2 . The reg of Nrf2 . This hi of Nrf2 . +-dependent deacetylases, have been shown to regulate transcription of SOD2 indirectly via FoxO3a. SIRT2-mediated deacetylation of Foxo3a resulted in enhanced DNA binding and SOD2 expression under oxidative stress [SOD2 promoter, whereas expression of a SIRT3 catalytic mutant did not affect the DNA binding ability of FoxO3a [+ availability through SIRTs. FoxO3a is one of the Forkhead transcription factors in mammals and a well-known downstream target of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (PKB/Akt) signaling. Putative FoxO binding elements have been identified upstream of the SOD2 transcription start site, but the one at position −1249 is responsible for FoxO3a-dependent transcription of SOD2 in quiescent cells . In pathe stress . SIRT3 of FoxO3a . The Foxf FoxO3a . TherefoSOD2 promoter enhancer elements. For example, TPA treatment enhances CREB-1/Activating Transcription Factor 1 (ATF-1) to the CREB-like consensus binding sequence (5′-TGACGTCT-3′) at position −1258 to induce SOD2 transcription [While the above represent major stress response pathways responsible for SOD2 regulation in cancer, other transcription factors have also been implicated with elevated SOD2 expression in response to redox stress, including the aryl hydrocarbon receptor nuclear translocator (ARNT) in acute myeloid leukemia (AML) . Moreovecription .2•− has been implicated in the activation of HIF-1α [HIF-1α-dependent inhibition of SOD2 transcription has been shown in renal clear cell carcinoma .The renaf HIF-1α , decreasf HIF-1α .SOD2 gene has been shown to be Nanog and Oct4-dependent based on promoter activity assays and RNAi-mediated knockdown of the two pluripotency transcription factors [In stem cells, transcription of the  factors . Potenti factors . Inhibit factors , as prev factors . In addi factors . These sThe mRNA transcripts of SOD2 undergo various post-transcriptional modifications. One potential cause of dysregulation of SOD2 expression in cancer is mediated through miRNA. The 3′ untranslated region (UTR) of the SOD2 mRNA has the Alu-like element to which small antisense RNA binds in the cytoplasm and inhibits protein translation . High exIn contrast to the above studies, a decrease in certain miRNAs has also been linked to high SOD2 expression in tumor cells. miR-17*, generated from the 3′ arm of its miRNA precursor , downregulates the expression of antioxidant enzymes, including SOD2, glutathione peroxidase 2 (GPX2) and thioredoxin reductase 2 (TXNRD2), through binding to the putative miR-17* targeting regions of these genes . Low miRSOD2 transcription, less is known about the regulation of SOD2 activity by these modifications in cancer.Even though large-scale patient expression datasets, like The Cancer Genome Atlas (TCGA), give us important insights into the expression changes of SOD2 in different cancer types, these do not necessarily directly reflect on the activity of the enzyme. Since SOD2 can be regulated at the protein level by several post-translational modifications , there iSOD2 is a nuclear transcribed protein, which has an N-terminal mitochondrial leader sequence that targets its entry into the mitochondrial matrix. Here, it gets converted into its active form, following homotetramer assembly and Mn incorporation ,135,136.Escherichia coli (E. coli) have demonstrated that manganese SODs can mistakenly bind Fe [Each SOD2 subunit binds to a single Mn ion within its catalytic active site. In bacteria, the Mn incorporating enzyme is SOD A, which is closely related in overall primary sequence and structure to bacterial SOD B, a dismutase that utilizes Fe. Despite the striking sequence similarities, SODs are admirably metal specific. After SOD2 translocates to the mitochondrial matrix, its pre-sequence is cleaved, and subsequently, Mn incorporates into the enzyme making it fully functional ,7. Howev bind Fe ,152,153, bind Fe ,155. It  bind Fe . siRNA-m bind Fe , suggestE. coli manganese SOD A was shown to result in deleterious peroxidase activity of the enzyme [In addition to a decrease in superoxide dismutase activity, Fe incorporation into e enzyme . Interese enzyme . Howevere enzyme . Protein function is dependent on its expression level and activity. Protein expression is, however, not always an estimate of its functional activity, which profoundly depends on the post-translational modifications that also regulate protein stability. In the next section, we will describe the post-translational modifications of SOD2 that have been explored in the context of cancers, leading to an imbalance in SOD2 protein or activity and consequential changes in mitochondrial redox homeostasis. +, and it is therefore an exquisite sensor of nutrient and redox stress, which leads to decreases in NAD+/NADH ratios. Several lysine acetylation sites have been reported on SOD2 including lysine (Lys) 122, 68, 53 and 89 residues [2•−-driven activation of HIF-1α [2+) has recently been implicated in suppressing SIRT3 activity in hepatocarcinoma through decreases in the NAD+/NADH ratio [+ has the potential to regulate SOD2 activity in response to low glucose conditions and redox stress, which occur during metastatic progression, such as anchorage independence [SOD2 undergoes active acetylation and deacetylation, making it functionally inactive or active, respectively . Sirtuinresidues ,164,165.residues . Low SIRf HIF-1α ,167, leaf HIF-1α . In addif HIF-1α . Loss off HIF-1α ,169, hepf HIF-1α , glioma f HIF-1α  and B cef HIF-1α . For exaDH ratio . HoweverDH ratio . For exaDH ratio  and melaDH ratio , suggestpendence . Interespendence . Whether2+-induced de-phosphorylation [2+-dependent signaling processes may regulate SOD2 activity to modulate superoxide and hydrogen peroxide levels in the cell. In another study that investigated the effect of environmental low dose ionizing radiation, researchers showed that mitochondria-localized cyclin dependent kinase 4 (CDK4) directly phosphorylated SOD2 at Ser106, enhancing its activity and mitochondrial respiration [Listeria monocytogenes [The most common type of covalent modification is serine (Ser)/threonine/tyrosine phosphorylation, which is carried out by a diverse group of kinases within the cell and regulates the activity of most proteins. SOD2 is also a target of phosphorylation . Using prylation . This supiration . CDK1 wapiration ,176. Givpiration . Phosphoytogenes  and mitoytogenes  have beeytogenes . Taken tOxidative modifications can be associated with altering protein function, which can be reversible or irreversible. For example, reversible cysteine oxidation is a common mechanism for phosphatase inactivation. Protein hyperoxidation often leads to functional deficit of target proteins . A recenNitration is a recognized modification of SOD2  that wasE. coli, but no functional studies were performed [S-glutathionylation is the addition of glutathione at the cysteine residues of proteins and occurs during both stressed and unstressed conditions. It prevents irreversible oxidation of protein thiols and regulates diverse cellular processes . In 2006erformed . Cysteinerformed . In a moerformed . HoweverProtein turn-over is a highly regulated process carried out by the proteasomal degradation pathway where several ubiquitin ligases and deubiquitinating enzymes play a vital role. A study in coronary endothelial cells isolated from type 2 diabetic mice showed augmented SOD2 ubiquitination resulting in ROS accumulation and attenuation of coronary vascular relaxation . Kim et SOD2 gene is rarely lost or mutated in cancer points to the fact that SOD2 regulation is adaptable. SOD2 regulation by stress response pathways, such as NF-κB, hence allows for transient SOD2 expression in response to changing tumor microenvironments. For example, increased oxidative stress may activate SOD2 expression specifically in response to matrix detachment [From the above studies, it is clear that aberrant regulation of SOD2 expression and activity occurs throughout cancer development and progression. We believe that the dichotomous regulation and role of SOD2 can explain its role as both a tumor suppressor in early tumorigenesis and as a tumor promoter during metastatic progression. Given that the tachment ,105, or tachment . The obstachment . Given tWhile the transcriptional regulation leading to differential SOD2 expression has been the focus of much research, it is clear that the mechanisms of SOD2 post-translational regulation are less clearly delineated in cancer. This may be of particular importance when tumor cells require rapid adaptation to changing tumor environments. For example, immediate activation of SOD2 may be required as cells detach from the primary tumor, when matrix detachment initiates rapid redox stress and nutrient deprivation. Understanding the mechanisms of tumor cell adaptability to dynamically alter their antioxidant enzyme expression and activity will allow us to better target these for therapeutic intervention. While antioxidant therapy is clearly an undesirable approach in metastatic disease, pro-oxidant intervention with the combination of targeting the mechanisms that regulate antioxidants may be a beneficial strategy for tumor types that display enhanced SOD2 activity.


Saccharomyces cerevisiae, expression of the kinetochore complex subunit Ndc80 is downregulated by a 5’ extended long undecoded NDC80 transcript isoform. Here we demonstrate a transcriptional interference mechanism that is responsible for inhibiting expression of the coding NDC80 mRNA isoform. Transcription from a distal NDC80 promoter directs Set1-dependent histone H3K4 dimethylation and Set2-dependent H3K36 trimethylation to establish a repressive chromatin state in the downstream canonical NDC80 promoter. As a consequence, NDC80 expression is repressed during meiotic prophase. The transcriptional mechanism described here is rapidly reversible, adaptable to fine-tune gene expression, and relies on Set2 and the Set3 histone deacetylase complex. Thus, expression of a 5’ extended mRNA isoform causes transcriptional interference at the downstream promoter. We demonstrate that this is an effective mechanism to promote dynamic changes in gene expression during cell differentiation.Cell differentiation programs require dynamic regulation of gene expression. During meiotic prophase in  S. cerevisiae, meiosis can be induced synchronously, allowing the study of stage-specific regulation of gene expression.Cell fate programs are driven by underlying gene regulatory networks. The budding yeast gametogenesis program, also known as sporulation, provides an ideal model for understanding principles of cell fate progression. In sporulation a diploid cell exits the mitotic program and produces four haploid gametes packaged as spores. Gametogenesis is driven by a specialized cell division called meiosis . After eThe yeast meiotic program is dynamically regulated by sequential waves of gene expression . Two masMATa or MATα) transcription of ncRNAs represses IME1 and IME4, two regulators of entry in meiosis (Noncoding RNAs (ncRNAs) and alternate mRNA isoforms are expressed throughout yeast meiosis . In addi meiosis . Through meiosis . HoweverNDC80long undecoded transcript isoform (lutiNDC80) is responsible for repressing the coding-competent NDC80 isoform (ORFNDC80) during meiotic S-phase and prophase  upstream of the NDC80 start codon. To investigate the expression pattern of the NDC80 mRNA isoforms more closely in cells undergoing meiosis, we performed northern blot analyses using a probe that recognizes both the ORF and upstream region of NDC80. Cells were first grown in rich medium overnight and then shifted to pre-sporulation medium. After a second overnight growth, cells were transferred to sporulation medium (SPO) and samples were taken at specific intervals throughout meiosis. In wild-type cells entering meiosis, both NDC80 mRNA isoforms were co-expressed . The induction of pCUP-IME1 and pCUP-IME4 ensures that cells can enter meiosis synchronously . The short NDC80 protein coding mRNA isoform is called ORFNDC80. This nomenclature is used thereafter.Our observation that the expression of the mitotic and longer  meiosis . Further isoform . Thus, tlutiNDC80 represses the downstream ORFNDC80 promoter might be related to a transcriptional interference mechanism during which intergenic transcription or transcription over promoter regions establishes a repressive chromatin state and prevents transcription factors from binding , which is homologous to human TFIIB, changed during meiosis across the NDC80 locus  when lutiNDC80 transcription occurred and cells underwent meiotic S phase, Sua7 binding to the ORFNDC80 core promoter (around −100 bp from AUG) was reduced while binding to the lutiNDC80 promoter (around −600 bp from AUG) increased   . In the and IME4 . This reORFNDC80 promoter could be due to the establishment of a repressive chromatin state. For example, transcription of an intergenic ncRNA across the SER3 promoter directs nucleosome assembly in the promoter, which is essential for SER3 repression in budding yeast  of histone H3 on micrococcal nuclease (MNase) treated chromatin extracts   and consistent with active ORFNDC80 transcription. During meiotic prophase  . In premdetails)  we deteclutiNDC80 mediated repression of ORFNDC80 also requires Set1/Set3C and Set2/Rpd3S, we measured the distribution of H3K4me2 and H3K36me3 marks at the NDC80 locus (ORFNDC80 promoter of either marks in premeiotic cells (labelled ‘2 hr’ in ORFNDC80 promoter in meiotic prophase cells (labeled ‘4 hr’ in set1∆ and set2∆). In -600-300ndc80 cells that do not express lutiNDC80, the deposition of the H3K36me3 and H3K4me2 marks in the ORFNDC80 promoter was reduced  and lysine 36 trimethylation (H3K36me3) are deposited co-transcriptionally within gene bodies by the Set1 and Set2 methyltransferases . The his80 locus . We obse reduced . Thus NDlutiNDC80 mRNA. We replaced the lutiNDC80 promoter with an inducible GAL1-10 promoter, and expressed lutiNDC80 in rich nutrient conditions in cells harboring the Gal4-ER chimeric transcription factor, which responds to β-estradiol. We observed a moderate increase of H3K4me2 in cells that harbored lutiNDC80 compared to control cells, which was independent of lutiNDC80 transcription  whose activity is induced in a concentration-dependent manner by β-estradiol (lexO) in the lutiNDC80 promoter and titrating the concentration of β-estradiol, enabled scalable transcriptional induction of lutiNDC80  and harboring three or eight lexO sites did not show a growth defect when exposed to intermediate levels of β-estradiol (15 nM), whereas control cells did  in the ORFNDC80 promoter (lutiNDC80 transcription was suppressed (ORFNDC80 promoter (pGAL-NDT80 and lutipGAL-NDC80). In addition, the growth changes as observed in the spot assays correlated well with Ndc80 protein levels , and the 5’ extended long undecoded transcript isoform (lutiNDC80), which cannot be translated into Ndc80 protein . Previous work established a role for Set2 in suppressing histone exchange and promoting nucleosome stability through chromatin remodelers  are repressed by Set2/Rpd3S whereas those with shorter overlapping transcripts (~0.9 kb), are repressed by Set1/Set3C  in the ORFNDC80 promoter is not protected by nucleosomes even during lutiNDC80 transcription  sulphate (50 µM) was added to induce IME1 and IME4 expression from the CUP1 promoter and initiate meiosis synchronously. For the synchronous experiments described in pCUP-IME1/pCUP-IME4 after two hours in SPO  yeast extract and 2.0% (w/v) peptone)+2% raffinose+2% galactose (YP-RG) and grown for another 16–18 hr. The cells were diluted to an OD600 of 0.2, grown for another 2.5–3 hr, diluted back to OD 0.2 and induced to express lutiNDC80 by the addition of 1 μM β-estradiol. 25–30 OD600 units of cells were collected for ChIP analyses at 0 hr and at 3 hr after induction.For 600 of 0.002, transferred to YP-RG and grown for another 16–18 hr. Cells were diluted to an OD600 of 0.2 in YP-RG and lutiNDC80 expression was induced by the addition of 1 μM β-estradiol. Samples were taken at 0, 3, 4.5, and 6 hr after β-estradiol addition.For the time courses in IME1 and IME4 from the CUP1 promoter. At four hours in SPO, cells were either treated with ethanol or cycloheximide (0.2 mg/ml). Samples for western blotting were taken at 15, 30, 45, 60 and 90 min after adding ethanol or cycloheximide.For 600 of 0.2. Subsequently, the cells were grown for 2.5–3 hr to reach exponential phase. The cells were diluted again to an OD600 of 0.2 and induced to express lutiNDC80 with either 10 or 20 nM of β-estradiol. Cells were collected for qPCR analysis at 0 hr and at 3 hr after induction.For 600 of 0.1, and then grown to OD600 of 0.3–0.5 at 30 °C. Three OD600 units of cells were taken as the pre-induction samples. Cells were then diluted to an OD600 of 0.1 in YPD and split into three flasks. Subsequently, β–estradiol was added to the cells with a final concentration of either 15 nM or 25 nM. Ethanol was added as to the uninduced cells. 2 hr after β–estradiol induction,~3 OD600 units of cells were taken for western analysis, and at 4 hr, another ~3 OD600 units of cells were collected. All the samples were processed according to the western blotting protocol. The OD600 of each culture was also measured when the samples were taken. Equal OD600 units of samples was loaded during the gel electrophoresis.For DAPI staining was used to monitor meiotic divisions throughout meiotic time courses. Cells were fixed in 80% (v/v) ethanol, pelleted by centrifugation and re-suspended in PBS with DAPI (1 µg/ml). Cells were sonicated for a few seconds and left in the dark at room temperature for at least 5 min. The proportion of cells containing one, two, three, or four DAPI masses was counted using a fluorescence microscope. At least two independent biological experiments were performed for each meiotic time-course experiment.2) for one hour at room temperature before flow cytometry analysis . Propidium iodide stained cells were excited with a 561 nm yellow-green laser and signals were detected using a 610/20 yellow filter. Pulse shape analysis (pulse height against pulse area) was used to exclude clumps and doublets. DNA content from single cells was estimated with a histogram of counts against pulse area. At least 50,000 cells were used for the analysis.Flow cytometry was used to monitor meiotic DNA replication as described previously . Cells wWestern blotting was used to determine Ndc80 protein levels. Protein extracts were prepared by using trichloroacetic acid (TCA) extraction protocol. In short, cells were pelleted by centrifugation  and re-suspended in 5% w/v TCA for at least 10 min. For vegetative samples, pellets were washed with TE50 buffer , then with acetone, and completely dried. For meiotic samples, the pellets were only washed with acetone and dried. Furthermore, pellets were mixed with lysis buffer ) and cells were disrupted using a mini beadbeater (BioSpec). Lysates were mixed with SDS loading buffer  and boiled for 5 min for denaturation. After polyacrylamide gel electrophoresis, proteins were transferred onto PVDF or nitrocellulose membranes using the Mini Trans-Blot Cell ). The membranes were blocked for 60 min in blocking buffer  before incubation with mouse anti-V5 ) at a 1:2000 dilution overnight at 4°C. Hxk1 loading control was detected using a rabbit anti-hexokinase antibody ) at a 1:8000 dilution overnight at 4°C. Membranes were then washed in PBST  Tween-20) and incubated with IRDye 800CW goat anti-mouse and IRDye 680RD donkey anti-rabbit secondary antibodies ) at a 1:15000 dilution. Protein levels were detected using an Odyssey Imager (LI-COR). To detect HA tagged Ime1, a mouse anti-HA antibody was used at a 1:2000 dilution ). All other steps were performed as above. At least two independent biological experiments were performed for each western blot experiment.To measure bulk histone H3 levels, membranes were blocked for 60 min in blocking buffer (5% w/v milk) before incubation with rabbit anti-H3, C terminus ) at a 1:3000 dilution overnight at 4°C. To measure bulk H3K36me3 levels, membranes were blocked for 60 min in blocking buffer  before incubation with rabbit anti-H3K36me3 ) at a 1:1000 dilution overnight at 4°C. Membranes were then washed in PBST and incubated with HRP conjugated ECL donkey anti-rabbit secondary antibodies ) at a 1:8000 dilution. After addition of ECL substrate , protein levels were detected using an ImageQuant RGB 600 machine . At least two independent biological experiments were performed for each western blot experiment.lutiNDC80 mRNA levels as described in -Nagel ). 750 ng of total RNA was reverse-transcribed using random primers and Protoscript II ), and single-stranded cDNA was quantified by real-time PCR using SYBR green mix (Life Technologies). The signals were normalized to ACT1 transcript levels. The oligonucleotide sequences used for RT-PCR experiments are displayed in To quantify lutiNDC80 signals were normalized to ACT1 transcript levels. The oligonucleotide sequences used for RT-qPCR experiments are displayed in For the RT-qPCR in NDC80 promoter and the 5’ region of the NDC80 open reading frame. Sua7-V5 binding was measured using similar procedures, except that anti-V5 agarose beads (Sigma-Aldrich) were used instead. The oligonucleotide sequences used for ChIP experiments are displayed in Chromatin immunoprecipitation (ChIP) experiments were performed as described previously. Cells were fixed in 1.0% v/v of formaldehyde for 15–20 min at room temperature and quenched with 100 mM glycine. Cells were broken using a mini beadbeater (BioSpec) and crosslinked chromatin was sheared by sonication using a Bioruptor , 7 cycles of 30 s on/off). Extracts were incubated for 2 hr or overnight at 4 ˚C with magnetic Prot A beads (Sigma) coupled with a polyclonal antibody against Histone H3 trimethyl lysine 36 , Histone H3 dimethyl lysine 4  or Histone H3 . Subsequently, reverse cross-linking was done in Tris-EDTA buffer  at 65°C overnight. After 2 hr of proteinase K treatment, samples were cleaned up and H3K36me3 enrichment was measured by real-time PCR using SYBR green mix ) and primers corresponding to the NDC80 locus, we extracted mononucleosomes using a MNase digestion protocol that was described previously followed by ChIP for histone H3  and the reaction was quenched with glycine (125 mM). Subsequently, cells were resuspended in 20 ml of buffer Z  plus β-mercaptoethanol (10 mM) and treated with 250 μg of T100 Zymolase ) for 60 min. Next, cells were resuspended in 2.5 ml NP buffer , 0.075% (w/v) Tergitol solution-type NP-40 detergent (NP-40), 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2), and extract was treated with 5, 0.625, 0.2 or 0.04 μl of MNase  for 30 min at 37°C, the reaction was quenched with EDTA (10 mM). The extract was adjusted to 0.1 M HEPES-KOH pH 7.5, 150 mM NaCl, 0.1% w/v sodium deoxycholate, and 1% w/v Triton X-100. To check for the extent of MNase digestion, 60 μl of MNase treated and untreated extracts were reverse crosslinked overnight in SDS-TE (1% (w/v) SDS, 10 mM Tris pH 8, 1 mM EDTA), treated with RNase A and purified DNA fragments were separated by gel electrophoresis. The extracts which the showed a mono-nucleosome pattern were used for ChIP with histone H3 antibodies. The ChIP was performed with 600 μl of extract as described in the chromatin immunoprecipitation section of the Materials and methods. ChIP samples were quantified by qPCR on a 7500 FAST Real-Time PCR machine ). Scanning primer pairs covering the NDC80 locus and upstream region were used for the analysis. Signals were quantified relative to untreated genomic DNA, and normalized over a primer pair directed against the PHO5 core promoter ), a DNA template and ATP (α−32P) ). The oligo-nucleotide sequences used for amplifying the NDC80, CIT1 or SCR1 templates are displayed in We adapted a northern blot protocol that was described previously . In shorORF, NDC80luti, CIT1NDC80 and SCR1 levels were estimated from northern blots using ImageJ  For ORFNDC80 levels between mutant and control strains were statistically significant.g ImageJ . The netIntensities of Ndc80 and Hxk1 bands on western blots were quantified using Image Studio Lite (LI-COR). Ndc80 levels were first normalized to Hxk1 levels and further normalized to that of the first time point on the same membrane.lutiNDC80 under control of the GAL1-10 promoter, cells were first grown on YP plus 2% glycerol (YPG) plates overnight, and then re-suspended in milliQ H2O to an OD600 of 0.2. Next, 5-fold serial dilutions were performed and diluted cells were spotted onto either YP-RG plates with no β-estradiol or YP-RG plates supplemented with1 μM β-estradiol. The cells were incubated at 30°C for 1–2 days. Note that the GAL1-10 promoter in the SK1 strain background does not directly respond to galactose. At least two independent biological experiments were performed for each spot assay experiment.For strains harboring lutiNDC80 expression is driven by LexA/lexO, cells were grown on YPD plates, re-suspended in milliQ water to an OD600 of 0.2, serially diluted as above, and then spotted onto either YPD plates with no β-estradiol or YPD plates with different concentrations of β-estradiol . The cells were incubated at 30°C for 1 day before imaging. At least two independent biological experiments were performed for each spot assay experiment.For strains harboring constructs in which  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Jessica Tyler as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Transcription of a 5' extended mRNA isoform directs dynamic chromatin changes and interference of a downstream promoter" for consideration by eLife. We realize that we are requesting a substantial amount of work to strengthen the experiments and their conclusions, but given that your manuscript is a co-submission, we considered that it was fairer to give you the opportunity to revise the paper rather than to reject it and keep the door open to a resubmission at a later date.The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. As you will see from the comments below, there was enthusiasm for this manuscript as a companion to the co-submitted manuscript from Chen et al. However, there were also several important weaknesses that need to be addressed before the paper can be published in Summary:NDC80 gene represses transcription from the NDC80ORF promoter. In the accompanying manuscript, basic features of the NDC80 regulatory system were described, including demonstration that the long mRNA, termed NDC80luti, and the ORF mRNA appear anti-correlated and that blocking transcription from the NDC80luti promoter derepresses the ORF transcript. In this manuscript, the authors find support for a transcriptional interference mechanism in which transcription from the upstream start inhibits PIC assembly at the ORF promoter and establishes a histone modification pattern at the ORF promoter (enrichment of H3K4me2 and H3K36me3) that has been previously associated with recruitment of two well characterized yeast HDACs, Set3C and Rpd3S. The authors extend their work by showing that the NDC80 regulatory system is reversible, when cells are switched from sporulation to growth media, and tunable, when the levels of the NDC80luti transcript are systematically elevated through a lexA-ER system.In this manuscript, the authors provide mechanistic insight into how an mRNA that initiates ~500bp upstream of the Demonstration of the molecular basis of the repression mechanism is a valuable complement to the accompanying paper by Chen et al. However, as the authors acknowledge, involvement of cis-repression of a promoter by an overlapping non-coding transcript via transcription-coupled histone methylation and establishment of repressive chromatin is not a novel mechanism, being established at several other genes in yeast. In addition to the examples for this cited by the authors, there is the case of the upstream ncRNA that represses SER3 by promoting nucleosome assembly over the SER3 promoter, and antisense transcripts that mediate histone methylation of the promoter for the sense transcript at FLO11. Other studies, including recent work from the Buratowski lab on a genome-wide scale, showed that noncoding transcription across promoters leads to enrichment of H3K4me2 and H3K36me3 and repression of the promoters. With that said, the study does provide insight into an interesting example of regulated transcriptional interference.ORFNDC80 promoter – to distinguish nucleosomes from non-histone chromatin protein occupancy, and to normalize changes in methylation for changes in H3 occupancy – and lack of quantification of changes in the ORFNDC80 transcript, which is required to firmly establish the role of Set2/Set3 in repressing this promoter. In addition, there are a number of instances where direct experimental evidence is insufficient to support mechanistic claims made; these instances need to be remedied by inclusion of more data or by significant rewriting of text to soften conclusions and make them more nuanced. Statistical analysis of replicate data is missing for measurements of mRNA levels by northern analysis. Loading and normalization controls are lacking in multiple figures.In addition to the need for a more thorough coverage of relevant precedents for similar examples of gene regulation (see above), there are some important control experiments lacking, notably a failure to measure by ChIP the total H3 levels in the Essential revisions:ORFNDC80 promoter region is not necessarily due to nucleosomes as it has been shown that non-histone protein binding in NDRs can also confer MNase-resistance (PMID: 28157509). For this reason, a ChIP of the MNase-resistant chromatin with histone antibodies is required to establish nucleosome versus non-histone protein occupancy.1) The increase in MNase-resistance in the 2) In lutiNDC80 TSS?3) 4) ORFNDC80 promoter in a manner dependent on the luti promoter. K4me2 levels should be included in 5) ChIP with histone antibodies is also required to normalize the H3-K36me3 and H3-K4me2 ChIP signals in ORFNCD80 transcript is elevated reduced in prophase in the double mutant relative to WT, as this seems to be limited to the 3hr timepoint, and even this increase is not evident in 6) 7) ORFNDC80 mRNA. If so, how can it be induced in the experiment of 8) The accompanying Chen et al. paper provides evidence that Ndt80 is required for induction of the short 9) 10) 11) Figure 7. The figure does not add very much to the discussion. The vertical red lines are not defined. The nucleosomes look more like Pol II molecules with modified CTDs. Please modify accordingly.ORFNDC80 transcription". Recruitment of none of these factors was tested and therefore the text needs to be modified accordingly.12) In the Discussion, the authors conclude that their data show the "co-transcriptional recruitment of Set1/Set3C and Set2/Rpd3S is essential for establishing a repressive chromatin state and inhibiting Non-essential revisions:lutiNDC80 expression influence Ndt80 occupancy in the NDC80 promoter region? Second, what is the effect of blocking lutiNDC80 transcription on Sua7 and Ndt80 occupancy? What is the effect of blocking lutiNDC80 on nucleosome occupancy? Note: the additional ChIP experiments suggested in this point are encouraged, but are not considered essential for publication. However, if the authors choose not to strengthen this part of the study, it is essential that they substantially modulate the strength of their statements in the text to be in line with what the data actually demonstrate.1) 2) lutiNDC80 transcript. Given this redundancy, the authors should consider omitting this figure. If they prefer to retain the figure, substantial improvements are needed. Here, the corresponding Ndc80 protein data are lacking for these promoter and termination constructs; although the quality of the northern data is better here for the terminator construct. However, mRNA loading controls and quantification of the northern data from replicates is lacking and should be provided.3) luti mRNA promoter doesn't decline in prophase on induction of this transcript. This deserves some comment.4) It's surprising in luti transcript, raising questions about the location of the luti promoter. This deserves some comment.5) 6) Integrate lutiNDC80 transcription is tunable”, last paragraph: "repression" should probably be "expression".7) Subsection “Gene repression by lutiNDC80 mediated repression of ORFNDC80 is dynamic”, end of first paragraph: it would be helpful to cite explicitly the data that supports this statement.8) Subsection “NDC80 mRNAs in lutiNDC80 transcription is tunable”.9) It would be nice to see a northern analysis to examine both long and short  Essential revisions:ORF promoter region is not necessarily due to nucleosomes as it has been shown that non-histone protein binding in NDRs can also confer MNase-resistance (PMID: 28157509). For this reason, a ChIP of the MNase-resistant chromatin with histone antibodies is required to establish nucleosome versus non-histone protein occupancy.1) The increase in MNase-resistance in the NDC80NDC80 locus when lutiNDC80 is transcribed.We now performed the experiment with histone H3 ChIP on MNase treated chromatin extracts. The results are similar to our original observation. We concluded that nucleosomes bind the NDR region of the 2) In We have two potential explanations:lutiNDC80, Ume6, binds to the upstream promoter , which could exclude nucleosomes from binding to the same sequence. When lutiNDC80 is activated by Ime1-Ume6, the region remains relatively unchanged, because Ume6 binding to the promoter occurs prior to Ime1-dependent activation. Hence, the upstream promoter remains free of nucleosomes prior and during activation of lutiNDC80 transcription.1) The transcriptional repressor of lutiNDC80 and its transcription causes the +1 nucleosome to remain positioned.2) Alternatively, the Ume6 repressor keeps the +1 nucleosome in place. When Ime1 is expressed together with Ume6, it activates luti TSS?3) NDC80 promoter, which is consistent with its previous classification as a TATA-less promoter (PMID: 15006352). Sua7 binding is approximately at -100 bp from AUG, which is within the region where core promoter elements are expected in yeast (PMID: 15006352).We have attempted to look for core promoter elements, which in yeast are not so well characterized, except for the TATA element. We were not able to identify an obvious TATA element in the PAN6, which is transcribed in the opposite direction compared to NDC80. We observed that PAN6 expression increases during entry into meiosis, which may explain the reason for the broad peak of Sua7 binding.The Sua7 signal in the upstream promoter region can be explained by expression of the neighbouring gene, 4) PHO5 sequences. This control gave consistent results.We have now included examples of how we prepared the MNase treated extracts, and show the MNase digestion pattern on a gel . We haveORF promoter in a manner dependent on the luti promoter. K4me2 levels should be included in 5) ChIP with histone antibodies is also required to normalize the H3-K36me3 and H3-K4me2 ChIP signals in -600-30ndc80) in premeiotic cells and S-prophase cells (-600-500ndc80). To keep it consistent in the revised manuscript, we repeated the experiment with the -600-300ndc80mutant  We have now performed loading controls for the northern blots in orfNDC80 levels of selective time-points from three independent experiments in pCUP-IME1/IME4 system yields better synchrony. For each experiment, the wildtype and mutant samples were run on the same gel/blot/hybridization. We observed significant differences in orfNDC80expression at 3.5 and 4.5 hours between the wild-type and set2 set3 double mutant, but not the single mutants.Therefore, we only show representative experiments for SCR1 loading failed.Please note that we replaced the experiment in 7) GAL promoter using Gal4-ER fusion.The ORF mRNA. If so, how can it be induced in the experiment of 8) The accompanying Chen et al. paper provides evidence that Ndt80 is required for induction of the short NDC80orfNDC80 transcription. One is during regular mitotic cell cycle of which we do not know the corresponding transcription factors. This is also seen from the fact that orfNDC80is expressed before entry into meiosis. The second way is during meiosis by the transcription factor Ndt80. In orfNDC80 transcription is reactivated when cells are switched from meiotic entry back to mitotic growth. We propose that the transcription factors important for orfNDC80 expression in mitosis controls the re-activation during return to mitotic cell cycle. The accompanying paper by Chen et al. proposes that Ndt80 bypasses lutiNDC80 mediated repression of orfNDC80. Therefore we performed the experiment in an ndt80 mutant background, the induction of orfNDC80in an ndt80 mutant is consistent with mitotic mediated activation of orfNDC80.Our data suggest that there at least two ways to activate 9) orfNDC80 and lutiNDC80 from two independent experiments in We have now included a loading control and normalized the ChIP signals to histone H3. We have quantified the levels of both 10) We now included Ndc80 protein levels for 11) Figure 7. The figure does not add very much to the discussion. The vertical red lines are not defined. The nucleosomes look more like Pol II molecules with modified CTDs. Please modify accordingly.We have now changed the figure, accordingly.ORF transcription". Recruitment of none of these factors was tested and therefore the text needs to be modified accordingly.12) In the Discussion, the authors conclude that their data show the "co-transcriptional recruitment of Set1/Set3C and Set2/Rpd3S is essential for establishing a repressive chromatin state and inhibiting NDC80We have removed the terms Set1/Set3C and Set2/Rpd3S from most parts of the manuscript, and mention Set2 and Set3 only. We discuss Set1/Set3C and Set2/Rpd3S only in the Discussion and Introduction.Non-essential revisions:luti expression influence Ndt80 occupancy in the NDC80 promoter region? Second, what is the effect of blocking NDC80luti transcription on Sua7 and Ndt80 occupancy? What is the effect of blocking NDC80luti on nucleosome occupancy? Note: the additional ChIP experiments suggested in this point are encouraged, but are not considered essential for publication. However, if the authors choose not to strengthen this part of the study, it is essential that they substantially modulate the strength of their statements in the text to be in line with what the data actually demonstrate.1) orfNDC80 promoter. We tested sequence specific transcription factors that have been implicated in regulating NDC80 expression by genome-wide studies: Hcm1, Swi4, and Fkh1. In addition, we have tried to ChIP for basal transcription factors such as: Taf1 and TBP. Unfortunately, none the factors gave a reproducible ChIP signal. One explanation is that orfNDC80 expression quite low during mitosis (only a few mRNA copies per cell). This makes it is challenging to obtain robust ChIP signals. We have modulated our statements and only talk about TFIIB (as this is only factor that worked) and not preinitiation complex.We agree that ChIP for more factors is desirable. We have tried to ChIP for several factors at the 2) We agree that our model predicts an increase in histone acetylation. However, given that we had put a lot of effort and time in completing the other essential and non-essential comments, we decided to test this further in a follow up study. We have modulated our conclusions in the Results section and refer the Set2 and/or Set3. We only refer to the Set1/Set3C and Set2/Rpd3S pathways in the Discussion.luti transcript. Given this redundancy, the authors should consider omitting this figure. If they prefer to retain the figure, substantial improvements are needed. Here, the corresponding Ndc80 protein data are lacking for these promoter and termination constructs; although the quality of the northern data is better here for the terminator construct. However, mRNA loading controls and quantification of the northern data from replicates is lacking and should be provided.3) We have removed 4) It's surprising in lutiNDC80 and transcription causes the +1 nucleosome to remain positioned.We agree that it is somewhat surprising that there was no shift in nucleosome occupancy around that +1 positon. Given that this a PCR based assay, we cannot exclude that there are subtle changes that we missed. Given that Ume6 binds the region prior to its activation, perhaps the Ume6 repressor keeps the +1 nucleosome in place. When Ime1 is expressed together with Ume6, it activates 5) PAN6 transcribed in the divergent direction is directly adjacent lutiNDC80. Its expression increases upon meiotic entry, which may explain the shift in peak. See also point 3 of Essential revisions.6) Integrate Has been added, accordingly.luti transcription is tunable”, last paragraph: "repression" should probably be "expression".7) Subsection “Gene repression by NDC80Has been changed, accordingly.luti mediated repression of NDC80ORF is dynamic”, end of first paragraph: it would be helpful to cite explicitly the data that supports this statement.8) Subsection “NDC80We now changed the section:ORFNDC80 promoter is not protected by nucleosomes even during lutiNDC80 transcription  in the cription , which mlutiNDC80 mediated repression can be rapidly reversed  It would be nice to see a northern analysis to examine both long and short NDC80 mRNAs in orfNDC80 mRNA band will most likely increase when lutiNDC80 mRNA is highly expressed, owing to the close proximity of the transcripts. Therefore we have measured Ndc80 protein levels in cells harbouring no or 8 copies of lexO sites in the lutiNDC80 promoter instead. We used different concentrations of β-estradiol to measure the effect on Ndc80 protein levels. The differences in growth inhibition observed in the spot assay were also reflected at the Ndc80 protein level (see We agree that this is would further support our data. However, the experiment is not trivial. The northern blot can be tricky to quantify because the background signal around the relatively weak evel see .


Histone proteins and transcription factors (TFs) play critical roles in gene transcription and development of multicellular organisms. Although antibody mediated protein isolation couple with mass spectrometry approach has been a standard method to identify TF interacting partners and characterize their functional molecular complexes, it becomes urge to develop a robust method to functional characterize how these transcription factors act during biological process in the post-human genome project era. Here, Dr. Zhao and his colleagues in the National Heart, Lung, and Blood Institute of NIH develop a sensitive and robust strategy to globally identify and characterize in vivo protein–protein interactions termed bait protein–protein interaction-sequencing (bPPI-seq) (Zhang et al. in Cell Res doi:10.1038/cr.2017.112,  Transcription is a key mechanism underlying the control of gene activities and cell identity during animal development and disease. Expression of genes underlying cell fate choices is coordinated by the binding of lineage-specific transcription factors to gene-proximal promoters or distal enhancers. As increased number of these pioneer and lineage specific key transcription factors have been isolated and identified, the underlying molecular mechanisms by which these factors establish regulatory networks to act on enhancers and promoters for gene expression remain poorly understood. One important way to understand the molecular mechanisms of these TFs in establishing their function is to identify TF interacting partners and to characterize protein co-regulatory complexes that these factors form , 3. TradRecently, a team of researchers led by Dr. Keji Zhao of the National Heart, Lung, and Blood Institute, National Institute of Health, has developed a novel strategy to identify protein interacting partners in a genome-wide scale . The metChromatin plays critical role in transcription by assembling genome into nucleosomes and modulating accessibility of transcription regulators and RNA polymerases. Whereas histone H2A is core structure component of nucleosome mainly involved in packaging DNA into chromatin during genome replication, H2A.Z replaces core H2A in promoter regions of genes during transcription cycle. H2A.Z containing nucleosomes facilitate promoter DNA accessibility and transcription activation , 7. ThusIn summary, the novel bPPI-seq provides a strategy for genome-wide identification of protein–protein interaction network in physiological condition and can be widely applied to characterize the molecular complexes by which transcription factor forms and establishes function.


CHD7 gene causes a severe congenital disease known as CHARGE syndrome. Most CHARGE syndrome patients have brain structural anomalies, implicating an important role of CHD7 during brain development. In this review, we summarize studies dissecting developmental functions of CHD7 in the brain and discuss pathogenic mechanisms behind neurodevelopmental defects caused by mutation of CHD7. As we discussed, CHD7 protein exhibits a remarkably specific and dynamic expression pattern in the brain. Studies in human and animal models have revealed that CHD7 is involved in multiple developmental lineages and processes in the brain. Mechanistically, CHD7 is essential for neural differentiation due to its transcriptional regulation in progenitor cells.CHD7 (Chromo-Helicase-DNA binding protein 7) protein is an ATP-dependent chromatin remodeler. Heterozygous mutation of the  CHD7 (Chromo-Helicase-DNA binding protein 7) is a long gene spanning approximately 189 kb at chromosome 8, containing 38 exons encoded for a large protein . CHD7 protein is a member of CHD family of ATP-dependent chromatin remodelers. These enzymes utilize the energy from ATP hydrolysis to mobilize or relocate nucleosomes, thereby control DNA accessibility of chromatin. Chromatin remodeling is crucial for DNA-related biological processes such as transcription, chromosome segregation, DNA replication, and DNA repair , a developmental disease characterized with IHH (idiopathic hypogonadotropic hypogonadism) and anosmia , initially described in 1979  and namepatients , which tHD7 gene . Moreove anosmia . Up to n protein . In contutations , which cCHD7 mutations, the common brain phenotype is hypoplasia of olfactory bulb and reduced number of GnRH (gonadotropin-releasing hormone) neurons in the hypothalamus  in mouse brain regions including forebrain and midbrain neural fold, neural tube, and neuroepithelial prominence  embryo, where the expression of β-galactosidase reporter closely mimics endogenous Chd7 . In adult mouse brain, Chd7 shows a very specific and dynamic expression pattern in the adult neurogenic region subgranular zone (SGZ) in hippocampal dentate gyrus (DG) . With immunostaining analysis, CHD7 is found to be expressed upon the activation of adult neural stem cells (NSCs), and is upregulated in transit progenitor cells and neuroblasts. In contrast, the expression of Chd7 is completely switched off in granule neurons in DG . Besides neuronal cells in the brain, while Chd7 is barely expressed in astrocytes, Chd7 is broadly expressed in oligodendrocyte (OL) lineage (Chd7 in differentiated oligodendrocytes is higher than oligodendrocyte precursor cells (OPCs) . With such a specific and dynamic expression pattern in the brain, the expression of Chd7 is expected to be tightly regulated. Mechanism behind is currently not known, however.In cerebellum, immunostaining results show that CHD7 is highly expressed in cerebellar granule cells throughout development and persists in adult cerebellum, in contrast to almost no expression in Purkinje neurons and Bergmann glia cells . In cont lineage . SimilarTable 1). Mice carrying germline loss-of-function mutation of Chd7 have been generated with both ethylnitrosourea (ENU)-induced mutations and the gene-trap approaches. While homozygous loss-of-function mutation of Chd7 is embryonic lethal at E10.5, heterozygous mutant mice are viable but show phenotypes closely mimicking CHARGE syndrome  mouse lines (flox/floxChd7) (Table 1). Two flox/floxChd7 mouse lines have been used, with either exon 2 or 3 being floxed  of lateral ventricle . Given nogenesis . In Chd7CKO mice . MutatioNSC pool . DissectChd7. Heterozygous gene-trap mutation of Chd7 leads to reduced expression of key signaling molecule Fgf8 (Fibroblast growth factor 8) in isthmus organizer (IsO), an organizer region that directs cerebellar development during early embryonic stage (between E8 and E9). Importantly, heterozygous loss-of-function mutations of both Chd7 and Fgf8 show a synergistic effect in cerebellar development, resulting in severe vermis aplasia . Using shRNA-mediated gene knock down approach in cultured mouse NSCs, Jag1, Gli2, Gli3, and MycN. In adult mouse NSCs, CHD7 is required for the activation of two SoxC family of transcription factors SOX4 and SOX11, and overexpression of SOX4 and SOX11 could largely rescue the neuronal differentiation defect in Chd7 mutant NSCs  analysis in differentiating OLs, CHD7 was found to regulate genes like Osterx and Creb3l2, both of them are required for both OL differentiation and bone formation , NPCs, and GNPs . In partuse GNPs , consist of Chd7 . In diff of Chd7 . The reqCHD7 mutations in CHARGE syndrome patients result in truncated CHD7 protein that apparently lost its ATPase and chromatin remodeling activities. Importantly, CHD7 mutations lead to reduction or loss of its ATPase and remodeling activity in vitro. Intriguingly, CHD7 interacts with other chromatin remodelers. Multiple evidences suggest a possible functional interaction between CHD7 and the BAF (Brg1/Brahma-associated factors) complex. First, in human neural crest cells, CHD7 is associated with multiple subunits of the PBAF (Polybromo- and Brg1/Brahma-associated factors) complex  from patients. The full reservoir of differentiation capacity enables iPSCs as an excellent cell model for disease modeling. Recent development of genome editing tools such as CRISPR-Cas technology has in principle enabled the genome editing at anywhere of the genome in any cell. In particular, genome editing in human iPSCs has enormous application in biomedical research . MoreoveAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The cellular transcriptome is shaped by both the rates of mRNA synthesis in the nucleus and mRNA degradation in the cytoplasm under a specified condition. The last decade witnessed an exciting development in the field of post-transcriptional regulation of gene expression which underscored a strong functional coupling between the transcription and mRNA degradation. The functional integration is principally mediated by a group of specialized promoters and transcription factors that govern the stability of their cognate transcripts by “marking” them with a specific factor termed “coordinator.” The “mark” carried by the message is later decoded in the cytoplasm which involves the stimulation of one or more mRNA-decay factors, either directly by the “coordinator” itself or in an indirect manner. Activation of the decay factor(s), in turn, leads to the alteration of the stability of the marked message in a selective fashion. Thus, the integration between mRNA synthesis and decay plays a potentially significant role to shape appropriate gene expression profiles during cell cycle progression, cell division, cellular differentiation and proliferation, stress, immune and inflammatory responses, and may enhance the rate of biological evolution. Saccharomyces cerevisiae revealed that transcription of a specific subset of genes and the stability of their corresponding mRNAs in the cytoplasm is intimately connected through various mechanisms 8910111213cis-regulatory elements and special transcription factors, which bind to these elements, collectively affect the stability and decay rates of their corresponding messages and, thereby, functionally integrate the nuclear phase of mRNA life with their cytoplasmic fate 8910111213Gene expression in the eukaryotes is a highly complex process, involving a multitude of activities at different cellular compartments. Each of these processes was discovered independently and was subsequently investigated in an isolated fashion. Several current studies, however, revealed complex networks among them with additional layers of control as exemplified by the fascinating connections among various nuclear events, such as transcription elongation, capping, splicing, 3’-end processing, and nuclear export 123456In eukaryotes, productive expression of a gene involves transcription, maturation of the transcripts in the nucleus, their export into the cytoplasm, subsequent translation of the translation competent messages, and their destruction in specific cytoplasmic location. During this long journey, every transcript undergoes extensive covalent modification and structural remodeling events. Nuclear events of mRNA biogenesis involve the transcription by RNA polymerase II (RNAPII), capping of the primary transcript at the 5’-end, splicing, and maturation at the 3’-end of the message, involving a site-specific cleavage and polyadenylation 115161718192021222325115161718192021282930S. cerevisiae, transcription and nuclear pre-mRNA processing events are physically and functionally coupled via the C-terminal domain (CTD) of the Rpb1p (largest subunit of RNAPII) 2432333427293436373827293436373824041424344454647484950In In the cytoplasm, the mRNPs undergo another remodeling event to shed off some of the mRNA-binding proteins carried from the nucleus. A unique pioneer round of translation immediately follows this remodeling event while they still carry the nuclear CBC 525255S. cerevisiae. This degradation-committed message is subsequently destroyed by the default degradation pathway within the P-bodies 40574059604062636465666724041424344454647484950After predestined rounds of translation a translating mRNA is transformed into a degradation-committed message and eventually disengages from translating polysomes to associate with the cytoplasmic P-bodies or stress granules, which are considered the cellular sites of mRNA decay in in vivo by Pat1p, Edc1-3p, Scd6p, the Lsm1-7p complex, and the DEAD box helicase Dhh1p 40707172737677The default decay process is initiated with the shortening of the poly(A) tail from 60-90 residues long adenylate tail  to a 10-15 residue oligo-A state 4059606S. cerevisiae implicated various mechanisms that functionally couple nuclear transcription and cytoplasmic mRNA stability/decay. The majority of these mechanisms are mediated by a variety of trans-acting protein factors (collectively termed "coordinators") that are selectively recruited onto specific subsets of transcripts by distinct cis-acting sequences (such as gene promoters). Recruitment of the coordinator "marks" the specific subset of transcripts and thereby alters their stability in response to a physiologic cue. These "marks" on the transcript are later decoded in the cytoplasm by a variety of mechanisms, which ultimately stimulates specific mRNA decay factors and thereby triggers the kinetics of decay of a specific or a cluster of transcripts in response to various cues. Functional coupling modulated by different classes of coordinators are discussed below.Emerging studies carried out in Two subunits of the core RNAPII, Rpb4p and Rpb7p, were implicated in the functional coupling between transcription and mRNA degradation 12131482121314Several pieces of information provided evidence that the Rpb4/7 heterodimer promotes the decay of a specific class of cellular mRNAs encoding ribosomal proteins and translation factors  121212871412An early instance of the influence of the specific promoter on the decay of its cognate message was exemplified by a study in mammalian cells in which the abundance of the β-globin mRNA was demonstrated to be affected by its own promoter S. cerevisiae demonstrated that promoters and associated cis-regulatory elements of certain genes and their cognate transcription factors play crucial roles in coupling their transcription and the decay rates of their corresponding transcripts 891011RPL30 mRNA, encoding the large ribosomal protein 30 in S. cerevisiae. Promoter swapping experiments showed that exchanging the native UAS of the RPL30 gene with that of the ACT1 gene, without altering its coding sequence, has a remarkable influence on the stability of the RPL30 mRNA RPL30 transcript expressed from its native promoter (harboring RPL30 UAS) vs. the ACT1 promoter (harboring ACT1 UAS) displayed a dramatic difference RPL30 promoter harbors two binding sites for the specific transcription activator Rap1, and eliminating them led to the dramatic stabilization of the RPL30 mRNA. Thus, recruitment of Rap1p to the RPL30 UAS appears critical for the stimulation of the decay of the corresponding message . Therefore, this observation directly connected the transcription factor Rap1p to the promoter-influenced decay kinetics of the RPL30 mRNA. Consequently, Rap1p has been termed a "synthegradase" to underscore its effect in coupling transcription with mRNA decay, presumably by marking the RPL30 message 3RPL30 message to affect its decay. An extension of this work by Dori-Bachash et al. cis-regulatory sequences of orthologous genes from two related yeast species affects both, the mRNA transcription and the decay for some genes More recent studies in the baker’s yeast SWI5 and CLB2 genes in S. cerevisiae are playing a vital role in modulating the stability of their corresponding messages in a cell cycle-dependent manner SWI5 is a transcription regulator associated with late mitosis genes, and CLB2 is a G2 phase cyclin that promotes the entry of yeast cells into mitosis. Replacing the SWI5 and CLB2 promoters with the ACT1 promoter altered the native decay rates of these two mRNAs. This regulation involves the mitotic exit network (MEN) kinase Dbf2p and its interacting partner polo kinase Cdc5p SWI5 and CLB2 promoter, subsequently loaded onto these messages in a transcription-dependent manner and eventually carried to the cytoplasm . Once in the cytoplasm, Dbf2p is associated with the ancillary factor Dbf20p  GAL7 transcripts is unclear.Expression of the reg1 yeast mutant), the same sets of transcripts were found to undergo diminished mRNA decay 96104et al. demonstrated that fusing nonglucose-responsive genes MAP2 and IDP2 to the ADH1 promoter caused a dramatic destabilization of these corresponding non-cognate transcripts, thereby showing that the ADH1 promoter alone could modulate glucose-induced mRNA decay 103ADH1 promoter appeared to be a crucial factor for the glucose-induced mRNA decay. Although the exact mechanism how Snf1p brings about the decay of the glucose-induced messages remained unclear, an involvement of additional RNA binding proteins, such as Vts1p, was postulated. In this respect, it is very interesting to note that four mRNA decay factors, Eap1p and Ccr4p (both interacting with Vts1p), Dhh1p and Xrn1p were found to be targets for Snf1p-dependent phosphorylation (note that Snf1p is AMP-activated protein kinase) as revealed by a previous phosphoproteomic study XRN1, DHH1 or CCR4 led to the enhanced stability of Snf1-dependent transcripts. Moreover, Puf5p, another target of Snf1p, was found to promote mRNA degradation by recruiting the Ccr4p/Pop2p/Not complex along with the helicase Dhh1p and the decapping enzyme Dcp1p to promote deadenylation, decapping, and decay 108109In a recent study, Snf1p, the yeast ortholog of mammalian/human AMP-activated protein kinase involved in multiple and diverse stress conditions 9899100101103105S. cerevisiae constitutes the predominant deadenylase which catalyzes the initial deadenylation step of polyadenylated mRNAs prior to their decapping by the Dcp1p/Pat1p/Lsm complex (see the previous section) 111112113112111Apart from promoters and transcription factors, three mRNA decay components, the major deadenylase, Ccr4p/Pop2p/Not complex, the principal decapping factor, Dcp2p, and the major cellular exoribonuclease Xrn1p, are also implicated in the process of transcription. Ccr4p/ Pop2/Not, a nine subunit protein complex in Remarkably, for a long time the involvement of Ccr4p in the deadenylation process remained unnoticed, and the protein was initially discovered as a transcription activator of alcohol dehydrogenase II and some glucose-regulated genes 116111112113118119121122124125The second example of functional influence on the process of mRNA transcription by a decay component is provided by Dcp2p, which is the major catalytic subunit of yeast decapping complex and belongs to the nudix family of pyrophosphatase 127xrn1 mutants display multiple phenotypic defects, such as sensitivity to Li2+ ions 132et al. proposed a stimulatory role of Xrn1p in transcription by showing that deletion of XRN1 led to a reduced transcription rate as measured by nuclear run-on assay, FISH (to show that a decreased number of mRNA in the transcription sites of the affected gene is a consequence of the loss of Xrn1p) and global gene expression analyses et al., in contrast, inferred that depletion of Xrn1p caused a global enhancement of mRNA synthesis rates as measured by incorporation of 4-thiouracil Xrn1p, the major cytoplasmic 5’→3‘ exoribonuclease, presents another instance of the influence of an mRNA decay factor on the nuclear transcription 132DCP1 gene (Δdcp1 strains) were observed to have decreased decay rates of specific reporter mRNAs without a similar enhancement in their cellular abundance. This finding thus indicated that the cells possess the capability of counteracting the decline in mRNA decay rates by appropriate alterations in their transcription rate The functional influence of mRNA decay factors on transcription is further supported by the following additional observations. First, null strains of yeast in the et al. in mammalian cells, which demonstrated the dependence of the stability of a β-globin mRNA harboring a PTC on its native promoter (see above) et al. Transcription and mRNA degradation also appears to be functionally coupled in mammalian cells. The very first instance of interconnection between mRNA synthesis and turnover was reported in the studies by Enssle Additional instances for the interplay between synthesis and decay of mRNA in mammalian cells are exemplified by several families of mammalian proteins, BTG/Tob, TIS11, and KSRP. Many members of these protein families play pivotal roles in both, nuclear transcription and cytoplasmic mRNA turnover 137138139139140141143144145139141142147148149151153149155156157158159140161161The second protein family, TIS11, consisting of BRF-1, BRF-2, and TTP, are known for a long time as the effector of degradation of mRNAs harboring AU-rich elements (ARE) in their 3‘-UTR region, such as those encoding growth factors, inflammatory cytokines, and proto-oncogenes 164166166170171170171173174173176Remarkably, a recent study strongly suggests the existence of a functional coupling between the transcription and mRNA degradation in the mammalian cell, which is harnessed by the gamma-herpes virus during its infection cycle The collective evidence, therefore, indicates the functional involvement of several mammalian protein families in the synthesis and degradation of a diverse set of specific mRNAs controlling cell differentiation, cell-cycle progression, cell proliferation, immune and inflammatory response and is crucial during viral infection. Although a direct functional connection between the transcription and decay is currently lacking, available data strongly suggest the existence of such interplay which may play a vital role in diverse physiological processes.S. cerevisiae, where a small change in the osmolarity in the medium brings about a dramatic up-regulation of a large subset of messages 182183The functional connection between the synthesis and degradation of mRNAs in eukaryotic cells play a vital physiological role in shaping the characteristic gene expression patterns during diverse cellular processes, such as cell division/cell cycle, developmental program, cell proliferation, cell cycle progression, inflammatory response, cellular response to stress and other environmental cues, and in biological evolution. This interplay is vital to coordinate the gene expression pattern into a rapid and sharp oscillation in the levels of hundreds to thousands of transcripts simultaneously within a very narrow window of time which is essential to respond to such cues. Under such circumstances, the sharp rise in the steady state levels of the mRNA(s) can be more efficiently achieved if the decreased rate of decay kinetics of such transcripts renders a simultaneous and positive feedback to enhance their rate of transcription. Examples for such functional coupling between these two processes shaping appropriate gene expression profile include the regulation of specific and global mRNA steady-state levels during osmotic stress in S. cerevisiae revealed that the cells perfectly and stably maintain a steady and continuous level of diverse transcripts by appropriately modulating their decay rates when the synthesis of the global transcriptome is attenuated by introducing a mutation in the RNAPII S. cerevisiae, a mutual feedback between these two antagonistic processes is critical for buffering the global level of transcripts dcp1 mutation on enhancing the rate of transcription to maintain an optimal level of cellular transcripts (see above in the previous section) Furthermore, the functional linkage between the rate of synthesis and decay provides an efficient way to maintain an appropriate dosage of global transcriptome under a specified physiological condition. Consistent with this view, a recent investigation in S. cerevisiae, more than 10% of the protein-coding genes are regulated in a cell cycle-dependent manner 186SWI5 and CLB2 mRNAs as mentioned above. Entry into mitosis is associated with the reduction of the abundance of SWI5 and CLB2 messages, which is presumed to be the result of a reduced transcription and an increased decay 185190191192The characteristic and integrated pattern of gene expression achieved via the interplay between the transcription and the decay kinetics of mRNAs appears to shape the expression profiles of genes during the cell cycle. In et al., the coordination between the rates of synthesis and decay of messenger RNAs calls for relatively fewer numbers of mutations required to create a unique and desired profile of gene expression to respond to a changing environment Finally, a functional coupling between transcription and mRNA decay was implicated in promoting the rate of evolution of the organisms. As suggested by Haimovich Possible crosstalk between the synthesis of specific sets mRNAs in the nucleus and their regulated degradation appears to shape the expression profiles of the relevant transcriptome in the mammalian system as well during cell differentiation and proliferation, cell-cycle progression, immune and inflammatory response. Strikingly, gamma herpes virus seems to exploit the feedback loop between transcription and mRNA decay to take control of the cellular machinery to shape their gene expression pattern. Future research should unveil (i) more direct evidence of functional crosstalk between transcription and mRNA decay in mammals as well as (ii) the molecular insight of such interplay between these two counterintuitive processes in mammals.S. cerevisiae, clusters of transcripts encoding functionally related proteins, which are co-regulated in a collective fashion during cell growth and differentiation 194195196Drosophila and mammals 910198Emerging evidence strongly suggests that a functional coupling exists between the synthesis and decay of mRNAs in yeast, and possibly in mammals. Their coordinated control appears to play key roles in the underlying mechanisms involved in achieving the optimum expression patterns of gene clusters during the cell cycle, cell proliferation, cell cycle progression, immune and inflammatory response, cellular response to stress, drugs, diverse environmental signals, and developmental cues. Such cross-talk between these two opposing physiological processes is thought to contribute to the origin of mRNA regulons in This functional interplay between mRNA synthesis and degradation, however, leave us with quite a few interesting, yet unresolved, issues with unanswered questions. Despite a substantial amount of progress in this field, it is still unclear how promoters and transcription factors bring about the decay rates of a specific subset of transcripts. For example, how the specific RNAPII core proteins, Rpb4/7, stimulate the deadenylation of a specific set of transcripts is unclear. Although Rpb4/7 was found to interact with the decay factors  and was also found to localize to P-bodies together with them 12First, Rpb4/7p was implicated in the deadenylation event 121312Second, how Rpb4/7p is selectively deposited onto the specific sets of messages in a transcription-dependent manner (presumably via Rpb1p-CTD) is not known. Whether this deposition involves modifications of specific residues of Rpb1p-CTD or requires another new coordinator is not known.Third, comprehensive knowledge about the functional connections between diverse transcriptional aspects and the mRNA decay linked via the Ccr4p/Pop2/Not complex are also incomplete. The principal reason for this lack of information could be accounted by the fact that the roles of the deadenylase complex in transcription and mRNA decay were investigated independently and separately. This gap could be successfully bridged if they are studied together.et al., who provided strong evidence that recruitment of Dbf2p, the MEN kinase onto the promoter of SWI5 and CLB2 promoter sequence is critical for the stability of these transcripts SWI5 and CLB2 messages and interaction of Dbf2p with its cytosolic partner, Dbf20p, plays a vital role in forming a cell cycle specific "mark" on the SWI5 and CLB2 mRNA. However, how the recruitment of Dbf2p to the promoter sequence influences the stability of these messages is not understood. Interestingly, Dbf2p was found to be part of a larger interactome consisting of the Ccr4p/Pop2p/Not complex, implicating this factor in recruiting the decay components onto the marked message. Thus, Dbf2p appears to link the processes of initial marking of the message with the coordinator and later recruitment of decay factors onto the marked message in a selective fashion. However, the factor(s) critical for marking the SWI5 and CLB2 messages is yet to be identified, which also leaves the molecular mechanism of Dbf2/Dbf20 dependent decay of these mRNAs an unresolved issue. Further research is certainly required to answer these questions.Fourth, the exact molecular mechanism of "marking" specific messages or subsets of messages with a given coordinator remains poorly understood. In other words, it is uncertain how the specific transcription factors/coordinators, which influence mRNA decay rates, leave their marks on the specific transcripts for the rest of their life. Nonetheless, a clue to this question was partially addressed by Trcek et al., who suggested that promoter mediated decay of RPL30 mRNA involves the deadenylase Ccr4p/Pop2p/Not complex and cytoplasmic exoribonuclease Xrn1p via a 5’→3‘ decay mechanism Fifth, how a "mark" present on the specific transcript or a set of messages is later decoded in the cytoplasm is another unresolved issue. A clue to this question came from the studies by Bregman Sixth, it would be quite exciting to uncover the entire plethora of "synthegradases", class of transcription factors which might be involved in coordinating transcription with mRNA decay of other classes of transcripts and also participate in the regulation of this event. Identification of the novel synthegradases can be accomplished by exploring the status of the post-translational modifications (if any) of these transcription factors following their initial identification. Also, the epigenetic requirement/consequences (if any) of the functional coupling between the synthesis and decay of mRNA should be addressed and explored. Particularly, the role of chromatin and other DNA binding proteins in this event (which in turn may govern the regulation of larger subsets of genes) in connection to this question would be very relevant.S. cerevisiae exercise this kind of connected circuitry to respond to a wide range of stress, environmental, cell-cycle specific and developmental cues. Furthermore, input from research using other model organisms would also help this field to evaluate the relevance of the interplay between the transcription and mRNA decay, concerning the overall regulation of eukaryotic gene expression with a possible connection to human diseases (if any).While the molecular mechanism involved in the interplay between mRNA synthesis and decay is currently unknown, these studies pointed out that coupling the transcription to decay certainly increases the efficacy of the fine-tuning of the expression of environmentally induced genes 910901828


The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers. The cohesin protein complex binds chromosomes to facilitate their proper division into two daughter cells when a cell divides. Cohesin and the Nipped-B protein that loads cohesin onto chromosomes also bind to genes and regions that regulate genes to ensure that genes produce proper amounts of RNA. Nipped-B and cohesin preferentially bind a subset of genes important for growth and development, and small changes in cohesin activity cause severe birth defects. Why cohesin binds some genes and not others is unknown. We tested the idea that proteins that specifically bind the RNA sequences being produced by genes may help determine which genes bind cohesin. We find that the Drosophila TBPH and Lark RNA-binding proteins bind RNA produced by genes that bind Nipped-B and cohesin. They also bind to these genes on chromosomes and the chromosomal regions that regulate them. TBPH ensures that Nipped-B and cohesin are present at high levels on regulatory regions and genes. Lark inhibits Nipped-B and cohesin binding to some genes and increases their binding to some regulatory regions. Nipped-B interacts with both RNA-binding proteins and promotes their binding to genes. These findings show that RNA-binding proteins help determine which genes bind Nipped-B and cohesin. The cohesin complex plays crucial roles in sister chromatid cohesion, chromosome segregation, DNA repair, and gene transcription –3. CohesNIPBL ortholog of Nipped-B . Cells were suspended in three times the cell pellet volume of hypotonic buffer and incubated on ice 5 min prior to homogenization with 60 strokes in a ground glass homogenizer. Nuclei were collected by centrifugation at 2,000g for 5 min at 4°. The nuclear pellet was washed with an equal volume of hypotonic, and then suspended in two volumes of extraction buffer . The NaCl concentration was adjusted to 0.35 M, and nuclei were incubated for 30 min on ice. Insoluble material was removed by centrifugation at 16,000g for 60 min at 4°. Nuclear extract was adjusted to 20% glycerol (v/v) and rapidly frozen on dry ice and stored at -80°.To prepare nuclear extract, BG3 cells were grown to a density of approximately 5 x 102, 0.8 mM magnesium acetate, 10% (v/v) glycerol, 1 mM dithiothreitol (DTT), 0.1 mg per mL tRNA, 0.5 mg per mL heparin, 75 mM NaCl, cOmplete protease inhibitor cocktail (Roche), RNasin (Promega)] in a final volume of 0.8 mL at 30°C for 40 min. After binding, the beads were washed with RNA wash buffer  4 times at room temperature. Washed beads were boiled in SDS-PAGE loading buffer and the proteins were separated by SDS-PAGE and visualized by silver staining (Thermo Scientific Pierce Silver Stain for Mass Spectrometry). Protein bands present only in the lanes using leaders from the cohesin-binding genes  and the equivalent region in the lanes from the leaders from genes that don’t bind cohesin  were excised from the gel for identification by mass spectrometry in the Proteomics and Microchemistry Core Facility at Memorial Sloan-Kettering Center. Lark was identified with a 99% probability in the proteins binding CG8177 and was not detected in control leader lanes.To identify proteins that bind RNA leader sequences, 1.2 mg of nuclear extract was incubated with 80 pmol of RNA leader bound to 50 microliters of magnetic beads in binding buffer  and eluted with stripping buffer . Eluted protein was dialyzed in PBS containing 0.01% (v/v) Tween, 100 micromolar ZnCl2, and 0.5 mM PMSF and was used to immunize a guinea pig . Anti-Lark antibodies were prepared by expressing amino acids 3 to 235 of the Lark protein as a His6 fusion in bacteria using the pET-16b vector and immunizing a rabbit with purified protein.TBPH protein bound to NTA-Zny w crawling 3rd instar larvae as described previously . Cells were flash frozen in liquid nitrogen, thawed, and 10 U DNase I (Epicentre) added to the lysate. Lysate was incubated for 1 hr at room temperature and sonicated for up to 6 cycles of 30 seconds, and debris were removed by centrifugation at 16,000g for 15 min. The supernatant was incubated with NTA- Zn2+ agarose beads (Qiagen) for 1 hr at 4°, collected by gentle centrifugation and washed five times with wash buffer . Beads were stored at 4° for up to two days in wash buffer before use. Examples of the expressed proteins are shown in Lark and TBPH full length and truncated proteins were cloned into the pCR-Blunt II-TOPO vector (Life Technologies). Inserts were released by digestion with 3 vector  in Rosetin vitro transcription of the templates using T7 RNA polymerase (Promega), and purified by chloroform extraction and ethanol precipitation. Equimolar RNA mixes (20 micrograms) were denatured by incubation at 65° for 3 min, and incubated with Lark or TBPH protein beads , 15 micrograms per mL heparin]. Beads were washed several times with binding buffer at room temperature, and the bound RNAs eluted by boiling in STE . Eluted RNA was subjected to chloroform extraction, collected by ethanol precipitation, and dissolved in 10 microliters H2O. cDNA was prepared from the RNA using SuperScript III Reverse Transcriptase using an oligo B2 primer and quantified by real-time PCR using RNA-specific primers as described elsewhere  for 2 hours. Nuclear extracts (approximately 400 micrograms) prepared as described above were diluted 1:6 into Binding Buffer  for a final volume of 0.85 mL and cleared of insoluble material by centrifugation at 16,000g for 10 min at 4°. The final concentrations in the diluted extract are: 1.67 mM Hepes KOH pH 7.9, 8.3 mM Na2HPO4, 1.5 mM KHPO4, 172 mM NaCl, 2.25 mM KCl, 1.95 mM MgCl2, 16.7 mM imidazole, 0.83% Triton X-100, 3.33% glycerol, and 2% BSA, giving an ionic strength greater than 190 mM, and a significant detergent concentration to decrease non-specific binding. Imidazole prevents non-specific binding of proteins to the NTA-Zn2+ beads. Cleared extracts were incubated with blocked TBPH or Lark beads for 2 hours 4°. Beads were washed twice with Binding Buffer , and twice with PBS only, before boiling in SDS-PAGE buffer and separating bound proteins by SDS-PAGE. Proteins were detected by Western blot. For ribonuclease treatment, nuclear extracts were treated with 0.2 mg per mL RNase A and 4 U per mL RNase T1 for 30 min at 37° prior to binding to TBPH or Lark beads.TBPH or Lark proteins (approximately 5 micrograms protein) bound to NTA-Zn2, 0.2% NP-40, 1 mM MnCl2, 1 mM DTT, 1 mM PMSF, cOmplete protease inhibitor cocktail]. The final concentrations in the diluted extract were: 36.7 mM Hepes KOH pH 7.9, 116.7 mM NaCl, 66.7 mM KCl, 2.5 mM MgCl2, 0.67 mM MnCl2, 0.133% NP-40, and 6.67% glycerol, giving a nominal ionic strength of 183.4 mM. The diluted extract was pre-cleared by incubation with Protein A agarose beads (Thermo Scientific) for 2 hours at 4°. Antibodies were added to pre-cleared nuclear extract and the reactions were incubated overnight at 4°. Immune complexes were collected by incubation with Protein A agarose beads for 4 hours at 4°, and the beads were washed five times with Buffer 2 before suspending in SDS-PAGE loading buffer, denaturing at 100° for 5 min and separating the proteins by SDS-PAGE. Precipitated proteins were detected by western blotting. For some experiments 50 micrograms per mL ethidium bromide was added to the nuclear extract prior to immunoprecipitation.Approximately 300 micrograms of nuclear extract (see above) was diluted 1:3 with Buffer 2 [50 mM Hepes KOH pH 7.9, 100 mM KCl, 2.5 mM MgClS1 FigBG3 cells were depleted for the indicated proteins for four to five days and 15 μg of total cell extracts were analyzed by western blotting. The blots shown are representative of at least three independent experiments. The panels on the left compares mock-depleted control cells (Mock) versus cells depleted for TBPH (iTBPH) using double-stranded RNA prepared using the primers in the table at the bottom. The blot was probed with antibodies against Nipped-B, Rad21, SA, TBPH (this study), Lark (this study) and actin antibodies. The panels in the middle compare control cells (Mock) to cells depleted for Lark (iLark) using dsRNA prepared using the primers shown in the table. The right panels compare control cells to cells depleted for Nipped-B (iNipped-B). Western blots with lanes loaded with 7.5 μg or 30 μg of extract gave equivalent results.(TIF)Click here for additional data file.S2 Figvestigial gene (transcribed left to right) and the PREs (orange boxes) that silence it. The top ChIP-chip track (gray) shows H3K27me3 (histone H3 lysine 27 trimethyl) mark made by the PRC2 silencing complex in BG3 cells [th percentile for ≥150 bp.The genome browser view shows the G3 cells  with enrG3 cells  score. T(TIF)Click here for additional data file.S3 FigA) Affinity chromatography strategy to identify RNA binding proteins in nuclear extracts that recognize a nascent RNA leader from a cohesin-binding gene. Nuclear extracts were prepared from cultured ML-DmBG3 (BG3) cells derived from larval central nervous system. The N terminal nascent RNA sequence from the CG8177 gene contains a region with four UG repeats, and one with eight. CG8177 is transcribed and binds Nipped-B and cohesin in both Sg4 (Schneider Line 2 derivative) and BG3 cells [CG13089 gene was used as a control. CG13089 is transcribed in both Sg4 and BG3 cells, but does not bind cohesin in either cell type. (B) Silver-stained SDS-PAGE gel separation of proteins retained by leader RNA from the CG8177 cohesin-binding gene and the CG13089 gene that doesn’t bind cohesin. Three protein bands that appeared to be bound by the CG8177 leader RNA but not by the CG13089 leader were cut out of the gel to be analyzed by mass spectrometry. One of the bands did not contain RNA-binding proteins in either lane. One contained Rm62 in both the CG8177 and CG13089 lanes and Rb97D only in the CG8177 lane. Rb97D is only essential for spermatogenesis [CG8177 lane. (C) Lark preferentially binds RNAs produced by cohesin-binding genes. The left pie chart shows the fraction of all active genes that bind cohesin (red) and those that don’t (blue) in BG3 cells. Active genes were identified by precision run-on sequencing (PRO-seq [th percentile for ≥150 bp overlapping the gene. The pie chart on the right shows those genes whose RNAs co-precipitate with Lark [D) Lark is located primarily in the nucleus in BG3 cells. Fluorescent immunostaining in Mock and Lark-depleted BG3 cells with HP1 heterochromatin protein and Lark [(G3 cells . The leaogenesis  and was ogenesis  only in ith Lark  and are and Lark  antibodi(TIF)Click here for additional data file.S4 Figstring  gene. The bottom track shows the minus strand PRO-seq used to define the active promoters. The two tracks just above show the coverage at each base pair  from sequencing input chromatin and the Ser5P Pol II ChIP-seq for one biological replicate. The three tracks above the base pair coverage tracks show the ChIP-seq enrichment of sequences relative to input at 50 bp intervals calculated using sliding 250 bp windows as described elsewhere [All gene regulatory sequences were defined as 500 bp elements as illustrated in lsewhere  for two  stg, cdc5 gene. T(TIF)Click here for additional data file.S5 Figstring and Wrinkled genes. The table shows the genome-wide Pearson correlation coefficients for ChIP-seq enrichment between Nipped-B, TBPH and Lark in 3rd instar wing discs.The genome browser tracks show the log2 ChIP-seq enrichment for Lark (brown) TBPH (orange) and Nipped-B (purple) at the (TIF)Click here for additional data file.S6 FigEach dot is an individual promoter, enhancer or PRE, with its average log2 enrichment in control cells  plotted against its average log2 enrichment in cells depleted for TBPH (iTBPH) or Lark (iLark) . The red line has a slope of 1 and an intercept of 0. Dots above the line show an increase in enrichment with protein depletion and dots below the line show a decrease.(TIF)Click here for additional data file.S7 Figheadcase gene has several enhancers (red boxes) upstream of the promoter (pink arrow). The tracks show log2 ChIP-seq enrichment for Nipped-B (purple) and SA (green) in control cells and cells depleted for Lark (iLark). The Lark log2 ChIP-seq enrichment track in control cells is shown in brown. The heat maps below the Nipped-B and SA ChIP-seq tracks show where enrichment decreases (blue) and increases (red) are in the 5th and 95th percentile over regions of 300 bp.The (TIF)Click here for additional data file.S8 FigThe two western blots show the levels of the Nipped-B, TBPH and Lark proteins after treatment of BG3 cells with 10 μM triptolide for up to 4 hours.(TIF)Click here for additional data file.S9 Figstring promoters. The bottom graph shows the TBPH and Lark enrichment relative to the starting level over the same time course.The top graph shows the change in Ser5P Pol II and Nipped-B ChIP-seq enrichment relative to the starting level over a 4 hour time course of triptolide treatment at the two (TIF)Click here for additional data file.S10 FigA) Coomassie-stained SDS-PAGE separation of the His6-SUMO-TBPH proteins bound to NTA-Zn2+ agarose beads used for in vitro RNA (B) Coomassie stained SDS-PAGE separation of the His6-SUMO-Lark proteins used for in vitro RNA and protein binding experiments. Diagrams below show the amino acid residues contained in each fragment.(itro RNA  protein-itro RNA  and DNA-(TIF)Click here for additional data file.S11 Figcut and Wrinkled in BG3 cells (red boxes) and DNA fragments (blue boxes) tested for binding to TBPH and Lark on beads Click here for additional data file.S12 FigA) Western blots of protein co-immunoprecipitation of TBPH from BG3 cell nuclear extract with Nipped-B antibodies, and Nipped-B with Lark antibodies. The blot on the left shows TBPH precipitated by immunoprecipitation of Nipped-B in the presence and absence of ethidium bromide, and is representative of three independent experiments. The blot on the right shows precipitation of Nipped-B from BG3 nuclear extract with both Lark and Nipped-B antiserum, and is representative of two independent experiments. (B) Western blots of immunoprecipitation of BG3 cell nuclear extract with TBPH and Lark antibodies. The blot on the left shows Lark precipitation by Lark, but not by TBPH antibody. The blot on the right shows precipitation of TBPH but not Lark with TBPH antibody. Both blots are representative of two independent experiments. (C) Western blots showing Nipped-B binding to TBPH and Lark beads ((rk beads  after pr(TIF)Click here for additional data file.S1 TableUG repeats are indicated in red and CGUUC sequences are in bold and underlined. The ability of the RNA to bind (>10-fold enrichment) Lark and TBPH is indicated (Yes or No) and whether or not the RNA is produced by a cohesin-binding gene (Yes or No).(PDF)Click here for additional data file.S1 File(TXT)Click here for additional data file.S2 File(TXT)Click here for additional data file.S3 File(TXT)Click here for additional data file.S4 File(TXT)Click here for additional data file.S5 File(TXT)Click here for additional data file.S6 File(TXT)Click here for additional data file.S7 File(TXT)Click here for additional data file.