Saccharomyces cerevisiae genes that are located close to a telomere can become transcriptionally repressed by an epigenetic process known as telomere position effect. There is large variation in the level of the telomere position effect among telomeres, with many native ends exhibiting little repression.In Chromatin analysis, using microccocal nuclease and indirect end labelling, reveals distinct patterns for ends with different silencing states. Differences were observed in the promoter accessibility of a subtelomeric reporter gene and a characteristic array of phased nucleosomes was observed on the centromere proximal side of core X at a repressive end. The silent information regulator proteins 2 - 4, the yKu heterodimer and the subtelomeric core X element are all required for the maintenance of the chromatin structure of repressive ends. However, gene deletions of particular histone modification proteins can eliminate the silencing without the disruption of this chromatin structure.Our data identifies chromatin features that correlate with the silencing state and indicate that an array of phased nucleosomes is not sufficient for full repression. S. cerevisiae genome show similarities to the heterochromatin of higher organisms [HML and HMR, the tandem rDNA array and regions close to telomeres, in particular, exhibit heterochromatic properties, such as position effects on gene expression and chromatin that is less accessible to restriction enzymes and DNA methylases [Heterochromatin is defined as regions of DNA that remain highly condensed throughout the cell cycle. Although, yeast chromosomes are too small to visualize condensed chromatin, several regions of the rganisms ,2. The sthylases -5.HML and HMR the Sir complex is recruited by Rap1, Abf1 and the origin recognition complex (ORC) which bind to the HM silencers. At telomeres the Sir complex is recruited by Rap1 bound to the telomeric repeats. Once recruited, Sir2 is thought to deacetylate the histones of adjacent nucleosomes allowing the Sir complex to spread outwards from the site of assembly [Transcriptional silencing at telomeres and the silent mating-type loci is dependent on the silent information regulator proteins 2 - 4 (Sir2 - 4), which are integral components of the silenced chromatin. The Sir proteins interact with each other and with hypoacetylated histones H3 and H4 to form a repressive structure. At assembly ,7. The passembly -12.HM loci nucleosomes are arranged in regularly spaced arrays, in contrast to the less ordered structure at the expressed MAT locus [The multiple interactions formed among the Sir proteins and histones are thought to create an inaccessible chromatin structure resulting in silencing -5. At boAT locus ,14, and AT locus ,14. SimiAT locus . This stAT locus .The regular chromatin structure observed at truncated telomeres, cannot explain certain features of silencing at native ends. First, the silencing at the native ends is discontinuous, with the greatest degree of silencing observed immediately adjacent to the telomeric repeats and around the subtelomeric core X element . This isThis study examines the chromatin structures of truncation, native repressive and native non-repressive telomeres in order to establish a link between the underlying chromatin structure and the silencing state of the telomeres. We also examine the roles of core X, the yKu and Sir proteins and certain chromatin modifying proteins  in the formation of the chromatin structure at native telomeres.URA3-GFP reporter construct, either adjacent to the core X element at telomeres IIIR or XIL or adjacent to the telomeric repeats at the truncated telomere VIIL  of the truncated and native ends reveals several striking features  -25. In tyKU70 causes a substantial reduction of silencing at both the truncated VIIL and the native XIL telomeres [yku80Δ strain  for Sir protein associations with specific subtelomeres . The nontructure .The chromatin structure over a core X element and the X associated repeats has been described previously . We haveURA3 marker adjacent to core X. The phased array of nucleosomes proximal to the promoter is still intact, though they are not quite as sharply demarcated. This demonstrates that the phased nucleosomes are not sufficient for silencing but are probably necessary for the silencing to occur. A similar change over the promoter chromatin is seen when yKu is deleted. In this mutant, TPE is abrogated and the phased nucleosomes again remain intact. It is possible that, in both cases, there is sufficient recruitment of Sir proteins to the region to produce the phased nucleosomes but there are either not enough Sir proteins to produce full repression or another factor is missing. Both yKu and core X could influence Sir protein recruitment to the region. The ORC protein when bound to the ACS site at core X could recruit the Sir complex via an interaction with Sir1, similar to its role at the HM silencers. yKu has been shown to associate with core X elements [ku80 deletion with the core X mutations resulted in a loss of the phased nucleosomes, similar to that seen in the absence of Sir proteins. Recruitment of the Sir complex by yKu and by the factors binding to core X may, therefore, be independent of one another.Mutation of the Abf1 and ACS sites at core X also reduces TPE at particular ends  and we selements  and coulelements ,41. CombHistone methylation, acetylation, and H2A.Z incorporation have all been proposed to prevent the spread of the Sir proteins into euchromatin ,9,11,28.URA3 reporter gene was reduced without altering nucleosome positioning or removal of Sir proteins [Several previous studies have shown that, in particular conditions, Sir protein binding and spreading can occur without gene silencing -44. We eproteins . We suggURA3 reporter construct adjacent to the core X of XIL were created as described previously [URA3-yEGFP reporter construct adjacent to the core X of XIL and IIIR were obtained by transformation of FYBL1-8B [SacI. Strains containing the URA3-yEGFP reporter construct adjacent to a terminal truncation of VIIL were created by transformation of FYBL1-8B [EcoRI and SalI. PIY125 was created by transforming DS288C3-39c with a PCR fragment containing the URA3-yEGFP cassette amplified from pFEP43 (using primers AAGAAACATGAAATTGCCCAG and AATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTTATTTGTACAATTCATCCATAC). Gene deletions were created by replacement of coding sequences with the kanMX4 or hphMX4 cassettes [All yeast strains were derived from the isogenic S288c strains FY833, 1679-13B, DS288c3-1a, and DS288C3-39c  or FYBL1eviously . StrainsFYBL1-8B  with theFYBL1-8B  with pFEassettes ,49.AscI restriction site, by polymerase chain reaction mutagenesis, at the 3' end of the URA3 gene on plasmids pFEP24 [yEGFP3 gene was amplified from pUG35 (provided by J Hegemann and U Güldener), using primers 5' GGCgcGCcCGTCGACCTCGACATGTCTA 3' and 5' ggcgcgccTTTGTACAATTCATCCATAC 3' and cloned into the AscI site of pFEP37 and pFEP40 to create plasmids pFEP43 and pFEP41.Plasmids pFEP37 and pFEP40 were obtained by introducing an s pFEP24  and pADHs pFEP24 . The yEGURA3 gene were resuspended in water and serial dilutions spotted onto complete synthetic media and medium containing 5-FOA. The percentage of colonies resistant to 5-FOA after three days growth at 30°C was determined.Single colonies from strains marked with a subtelomeric 9 yeast cells using zymolyase 100T and permeabilized with the detergent NP-40. Chromatin from 2.0 × 108 permeabilized cells was digested with 1, 2.5 or 5 units/ml of MNase at 37°C for 4 min. An equivalent amount of purified DNA was digested with 5 units/ml of MNase for 35 s at 37°C, to yield the deproteinized DNA digestion patterns. Marker DNA was obtained by digesting purified DNA from the same cells with an appropriate restriction enzyme. All samples were purified and analysed by indirect end-labelling [StuI or BstXI. Digested samples were separated by agarose gel electrophoresis and transferred to nylon membranes. The MNase digestion pattern towards either the centromere or telomere was visualized with an appropriate 200 bp probe adjacent to the end-label digestion site. Probes were generated by radio-labelling gel-purified polymerase chain reaction fragments amplified from yeast genomic DNA.Chromatin analysis of yeast cells using micrococcal nuclease I was performed as previously described -52. Spheabelling  by digesAs MNase preferentially cuts the linker DNA between nucleosomes the presence of a protected region of sites (that are cut in the deproteinized DNA control), flanked by two hypersensitive sites ~150 bp apart, is consistent with the placement of a translationally positioned nucleosome. Other non-histone DNA-binding proteins may also protect regions of DNA, of varying size, from MNase digestion. However, in this study we interpret regions of protection of ~150 bp to imply the presence of bound nucleosomes.Relative band intensities of MNase cleavage products were also determined using KODAK 1D image analysis software (Kodak) in order to assist the interpretation of chromatin blots.ACS: ARS consensus consequence; ORC: origin recognition complex; MAT: mating type; TPE: telomere position effect.The authors declare that they have no competing interests.ERL and PWI contributed equally to this work. ERL, PWI and SS performed the MNase digestion and end-labelling experiments. FEP, PWI and ERL were involved in the yeast strain creation. ERL, PWI, NAK, JM and EJL all participated in the overall study design and planning. EJL and SS wrote the manuscript. All authors read and approved the final manuscript.Repressive and non-repressive ends have distinct subtelomeric chromatin structures. MNase digestion and indirect end labelling was used to analyse (A & C) the non-repressive telomeres IVL and XVR and (B) the repressive telomere IIR, as described in Figure URA3 marker or (C) downstream of the URA3 marker (towards the telomere). (A & B) Arrows adjacent to the blots indicate the most telomere-proximal open reading frames: YOR394W (e), YDL248W (f) and YBR302C (g). (C) A schematic of the XVR telomere is shown, with the core X ACS and Abf1p binding sites indicated by black bars. Telomere IVL is identical except that it lacks a Y' element.Click here for fileURA3 marker does not alter the chromatin structure of telomere XILIntegration of a . (A) Schematic of telomere XIL showing positions of URA3 marker insertion (1 - 5). Position 1, adjacent to the core X ACS site, is the site used in the majority of the strains described in this paper. (B&C) The subtelomeric chromatin structure of XIL was analyzed by MNase digestion and indirect end labelling, as described in Figure URA3 marker inserted into one of the locations shown in (A). Chromatin structure was analysed (B) upstream of the URA3 marker (towards the centromere) and (C) downstream of the URA3 marker (towards the telomere). Three hypersensitive sites associated with the URA3 promoter are indicated by black arrow heads and an array of evenly spaced hypersensitive sites by white arrow heads. MNase hypersensitive sites adjacent to the core X ACS and Abf1p binding sites are indicated by grey arrows.nsertion  - 5. PosClick here for fileDeletion of Sir proteins does not alter the chromatin structure of core X. Chromatin structures of the XIL and IIIR telomeres were analysed in Δsir1::KanMX, Δsir2::KanM, Δsir3::KanM and Δsir4::KanM strains, by MNase digestion and indirect end labelling, as described for Figure Click here for fileHistone modifiers are not required for nucleosome positioning at telomeres. The subtelomeric chromatin structure of the XIL telomere was analysed in the Δbre1::KanMX, Δdot1::KanMX and Δbdf1::KanMX strains, by MNase digestion and indirect end labelling, as for Figure Click here for fileYeast strains used in this study.Click here for file


ICS1 gene. Also AVRPPHB SUSCEPTIBLE 3 (PBS3) plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3.Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 promoter::β-glucuronidase (GUS) genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA.Expression studies with ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress signaling pathways.The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of  Because of their sessile nature, plants have evolved very sophisticated mechanisms to actively cope with different sorts of stresses. The various defense mechanisms are controlled by signaling molecules like salicylic acid (SA), jasmonic acid (JA), and ethylene, or by combinations of these signal compounds. SA accumulates locally in infected leaves, as well as in non-infected systemic leaves after infection with biotrophic pathogens and mediates the induced expression of defense genes, resulting in an enhanced state of defense known as systemic acquired resistance (SAR) -5. SAR iENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), which is probably activated after elicitor perception , commonly referred to as the W-box ,30,34-39ICS1 promoter that have a high affinity for WRKY28. The consensus WRKY28 binding site that emerged from this analysis has some characteristics that differ from the W-box consensus , suggesting that possibly these factors include endogenous WRKY28. Besides the direct activation of ICS1 gene expression, WRKY28 might also indirectly effect the ICS1 gene via transcriptional activation of genes encoding other transcription factors acting on the ICS1 promoter. Moreover, the residual GUS expression remaining with the m1, m2 and m1+2 mutant promoters could indicate that other sites in the ICS1 promoter are still able to bind WRKY28, although the existence of such sites was not supported by the results of the ChIP analysis.The transactivation experiments also showed that mutation of the sites at -460 (m1) and -445 (m2) did not completely knock out reporter gene expression. In comparison to the GUS activity obtained with the wild type construct, approximately 20% remained. Furthermore, the reduction in basal expression levels seen with the mutant WRKY28 gene -dCTP, after which unincorporated label was removed by Autoseq G-50 column chromatography (Amersham-Pharmacia Biotech). EMSA reaction mixtures contained 0.5 μg purified protein, 3 μL 5× gel shift binding buffer  in a total volume of 14 μL. After 10-min incubation at room temperature, 1 μL containing 30,000 cpm of labeled probe, representing approximately 0.01 pmol, was added and incubation was continued for 20 min at room temperature. Fifty- and 250-fold molar excess of unlabelled annealed oligonucleotides were added insome reactions as competitor. The total mixtures were loaded onto a 5% polyacrylamide gel in Tris-borate buffer and electrophoresed. After electrophoresis, the gel was dried, autoradiographed, and analyzed using X-ray film.EMSAs were performed essentially as described by Green et al. . DNA pro35S::WRKY28-HA or 35S::HA constructs in plasmid pRT101. After 24 h, protoplasts were harvested and ChIP assays were conducted as described by [ICS1 promoter as shown in Figure PR1 gene and the promoter of PDF1.2 gene of Arabidopsis were used as controls. The primers used for the ChIP assays are listed in Table For ChIP assays, protoplasts were prepared as described above and transfected with 6 μg of ribed by , with miMVV designed the study, carried out the analysis, helped in data interpretation, and made the draft of the manuscript. JFB and HL designed the study, helped in data interpretation, and edited the manuscript. All authors have given final approval for this version to be published.MIQE information for the qPCR experiment.Click here for file


Drosophila melanogaster, where homologous chromosomes remain aligned throughout most of development. However, our understanding of the mechanism of somatic homolog pairing remains unclear, as only a few genes have been implicated in this process. In this study, we introduce a novel high-throughput fluorescent in situ hybridization (FISH) technology that enabled us to conduct a genome-wide RNAi screen for factors involved in the robust somatic pairing observed in Drosophila. We identified both candidate “pairing promoting genes” and candidate “anti-pairing genes,” providing evidence that pairing is a dynamic process that can be both enhanced and antagonized. Many of the genes found to be important for promoting pairing are highly enriched for functions associated with mitotic cell division, suggesting a genetic framework for a long-standing link between chromosome dynamics during mitosis and nuclear organization during interphase. In contrast, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression, DNA replication, and chromatin compaction, including several components of the condensin II complex. In combination with a variety of secondary assays, these results provide insights into the mechanism and dynamics of somatic pairing.The pairing of homologous chromosomes is a fundamental feature of the meiotic cell. In addition, a number of species exhibit homolog pairing in nonmeiotic, somatic cells as well, with evidence for its impact on both gene regulation and double-strand break (DSB) repair. An extreme example of somatic pairing can be observed in  In addition to their number and structure, the position and spatial dynamics of chromosomes are under tight control, as direct interactions between chromosomes can contribute to the activation or repression of genes. Here, we focus on a particular type of interaction, known as somatic homolog pairing, which occurs between the maternal and paternal copies of chromosomes. While the role of somatic pairing on downstream homology-driven processes is well-established, there is much to be learned about how homologous chromosome segments find each other, physically align, and form stable pairing interactions within somatic cells. Taking advantage of a novel high-throughput FISH technology and the fact that homologous chromosomes are intimately paired along their lengths in the somatic cells of Drosophila, we have conducted a screen for factors that are important for the fidelity of somatic pairing. Ultimately, the characterization of these pairing genes will shed light on the mechanism of pairing, as well as pairing-mediated processes that have implications for development and disease. Finally, the efficacy of our screen for pairing genes suggests that the high-throughput FISH technology described here will prove useful for studying forms of nuclear organization and chromosome positioning beyond pairing. Drosophila melanogaster, where homologous chromosomes are intimately paired in virtually all cell types throughout development Pairing of homologous chromosomes is a fundamental aspect of nuclear organization. Although most well-known for its role in chromosome segregation during meiosis, studies have also documented homolog pairing in somatic tissues In contrast to Drosophila, the homologous pairing of any particular chromosome or chromosomal region in most organisms, if it occurs at all, is transient and localized. For example, short-lived homolog associations have been implicated in both mammalian X-inactivation One strategy to better understand the mechanistic and functional basis of somatic pairing and its downstream role in transcriptional regulation is to identify the genes involved. To date, only two proteins have been directly implicated in promoting somatic pairing in Drosophila: Suppressor of Hairy Wing (Su(Hw)) Searches for genes involved in somatic pairing have also taken advantage of transvection-associated phenotypes and, while not a direct measure of pairing, these phenotypes have enabled genetic studies to isolate additional candidates. These include genes encoding proteins that mediate long-range interactions, such as Zeste and the Polycomb group proteins Here, we present a genome-wide FISH-based screen in Drosophila cell culture to identify the factors involved in the somatic pairing of heterochromatic regions. This screen was made possible through the development of a high-throughput FISH technology that permits chromosomal positions to be directly visualized in a 384-well format. Combined with RNAi, this approach permitted us to screen two heterochromatic regions simultaneously for double-stranded RNAs (dsRNAs) that alter the fidelity and/or strength of somatic homolog pairing. Using an increased number of FISH signals per nucleus as a readout for decreased pairing, we report the identification of 40 candidate ‘pairing promoting genes,’ none of which had been previously associated with pairing functions. Importantly, many of these genes were also found to influence pairing at euchromatic regions, revealing a potentially strong mechanistic overlap between heterochromatic and euchromatic pairing. In addition, we identified 65 candidate ‘anti-pairing genes,’ which when knocked down enhance pairing, consistent with a wild-type function of antagonizing somatic pairing interactions. We propose a model in which interchromosomal associations are mediated by a dynamic interplay between groups of proteins with opposing functions: those that induce or augment pairing and others whose normal function is to disrupt pairing. This perspective suggests that the difference between Drosophila and other organisms may be a shift in the balance of gene function. Finally, in combination with a variety of secondary assays, the identification of these proteins has provided insights into the mechanism and dynamics of somatic pairing, pointing to an intriguing connection between the progression of the cell cycle and the control of somatic pairing.167 cell line due to its amenability to RNAi The design of our studies began with an earlier observation that the onset of pairing in the Drosophila embryo does not require the zygotic expression of any particular gene but relies instead on parental contributions nd, and 3rd chromosomes, respectively , confirming efficient knockdown of Top2.We next conducted a pilot screen using dsRNA to knock down transcript levels of genes that had been previously shown to be important for the pairing of euchromatic regions. For example, RNAi inhibition of either Top2 167 cells should reveal genes involved in the somatic pairing of Drosophila heterochromatic regions. To this end, we developed a high-throughput FISH technology, allowing FISH assays to be performed in a 384-well format using a protocol that can be carried out, from fixation to imaging, within five hours. This strategy also enabled us to target two different heterochromatic regions simultaneously using probes against the 359 and dodeca repeated elements  to each well. Rank-order analysis of the primary genome-wide screening results demonstrated that the majority of dsRNAs had no effect on the number of FISH signals per nucleus, suggesting that pairing is not commonly disrupted by RNAi (z-score ≥2.0), as would be expected for those that target genes important for somatic pairing. A second group of 63 dsRNAs resulted in a significantly increased number of single-signal nuclei (z-score ≤−2.0), as expected for those that target genes required for suppressing somatic pairing levels  of the percentage of single-signal nuclei compared to untreated control wells were considered “hits”  encode proteins with known or expected roles in cell division , the larThese data suggest that somatic pairing of heterochromatic regions requires a complex network of genes that can promote as well as antagonize interchromosomal interactions. Below we describe our candidate pairing promoting and anti-pairing genes, and address their relationships to both heterochromatic and euchromatic pairing, aneuploidy, heterochromatin clustering, cell cycle progression, and each other. Taken together, these candidates point to an intriguing connection between the progression of the cell cycle and the control of somatic pairing as well as reveal an extensive overlap between heterochromatic pairing factors with those important for pairing at euchromatic regions.slmb and lin19 were the two strongest hits identifying candidate pairing promoters , 49% (P = 0.0075), and 56% (P = 0.002), respectively  compared to that of controls . Thus, these data point to a novel role for the SCF complex in controlling the organization and structure of interphase nuclei.We reasoned unpairing of homologs would also increase the distance between FISH signals in addition to increasing their number per nucleus. Of those nuclei with multiple dodeca FISH signals in SLMB, LIN19, and SKPA-depleted cells, 99% exhibited two signals >1 µm apart and were therefore considered unpaired. In fact, 25% of SLMB-depleted cells had two signals >4 µm apart, a distance rarely observed in control nuclei  and paired nuclei  at dodeca (P<0.05), suggesting that, similar to SCF, proper APC function is important for pairing at both heterochromatic and euchromatic regions (The Anaphase-Promoting Complex (APC) contains 11–13 proteins that, similar to SCF, function together as an E3 ubiquitin ligase, which targets cell cycle proteins for degradation. Our validation screen identified three components of the APC as candidate pairing promoters; RNAi depletion of SHTD (Drosophila APC1), CDC16, and IDA (Drosophila APC5) each reduced the percentage of single-signal nuclei  were identified in our primary screen, with depletion of each causing a significant increase in the number of FISH signals per nucleus for both 359 and dodeca , furtherP = 0.0002) (P = 0.0097)  and CDH1 (interphase)  0.0002) , whereas 0.0097) .mcph1, the Dynein motor protein encoding gene dhc64C, and the Dynein regulator Lis-1. Knockdown of each of these genes reduced the percentage of single-signal and paired nuclei at dodeca to 51–58% (P<0.004), while not significantly affecting the number of signals at 359 , which is required for proper microtubule nucleation and establishment of a bipolar spindle: s at 359 . Signifi P<0.02; . Interes64C RNAi , suggestial (Drosophila Aurora B), borr, and Incenp all reduced the percentage of single-signal and paired nuclei at dodeca to 51–66%  and cytokinesis  each increased the number of FISH signals per nucleus as well as the distance between signals (P<0.05) following borr, pav, polo, mars, chb, cal1, and sti RNAi, suggesting these genes are important for pairing at both heterochromatic and euchromatic regions , which is involved in various aspects of mitosis, including chromosome alignment, spindle assembly, and the completion of cytokinesis  P<0.05; . These r signals . FISH ta regions .borr, ial, polo, mars, chb, cal1, sti, as well as for mcph1, dhc64C, ida, and slmb  to identify and exclude nuclei undergoing mitosis and slmb . These r167 cells used in this study are already tetraploid , and no correlation was found between the frequency of large nuclei and levels of pairing , 66±8 (P = 0.4359), and 61±9% (P = 0.1693) of nuclei with a single dodeca FISH signal; none of these pairing levels differs significantly from the 70±3% observed in controls. As these findings may reflect incomplete knockdown of cohesins, we treated cells with dsRNA for longer time periods (5 and 6 days) and while simultaneously targeting two cohesin subunits. We observed no change in pairing levels in interphase nuclei (data not shown). Furthermore, depletion of Mei-S332/Shugoshin, required to protect pericentromeric cohesion from premature separation P = 0.6709). We pursued this unexpected finding by assessing knockdown of rad21 in mitotic nuclei and observed nearly complete chromatid separation in the majority of nuclei  and directly interrogated pairing levels in G1, early S, late S, and G2. In unsorted populations, probes against the 359, AACAC, and dodeca repeats showed pairing frequencies of 80, 42, and 58%, respectively . In the control, we found that ∼40% of nuclei that were paired for 359 as well as for dodeca exhibited complete or partial colocalization of the two signals, indicating that the pericentromeric regions of the X and 3rd chromosomes were indeed nonhomologously clustered in a subpopulation of the cells  in nlp cap-H2 double knockdowns, the latter being similar to levels found following cap-H2 RNAi alone  were suppressed by Cap-H2 co-depletion . Thus, these results reveal a novel genetic interaction between the SCF ubiquitin-ligase and condensin II complexes that is important for nuclear organization.Strikingly, we found that 13 (33%) of the pairing hits were completely suppressed by co-depletion of Cap-H2 , suggestP<0.005; , Table 1pav, scra, and feo) and mitotic spindle organization   , Table 1eriments . Our exaeriments . This secap-H2 RNAi. Proteins corresponding to these hits are involved in chromosome organization or alignment (e(bx) and tlk) and spindle organization (αTub84B and mars) and include members of the APC (shtd and cdc16). We cannot rule out the possibility that some wells experienced inefficient Cap-H2 depletion, although we consider this to be unlikely given the low variability between three replicate tests  or completely independent  of Cap-H2 co-depletion , perhapste tests .cap-H2 RNAi targeted genes that encode components of the CPC  and genes associated with MTOC function (mcph1 and dhc64C). dsRNAs targeting genes necessary for cytokinesis  and chromosome alignment  were also all found to elicit their pairing effects independent of Cap-H2. Thus, these data argue that pairing can be influenced by a complex network of genes, including those that function through condensin II and those that do not.Those dsRNAs whose effects were unchanged in the presence of In this report, we introduce a high-throughput FISH technology that enabled a genome-wide RNAi screen for factors involved in somatic homolog pairing. We identified both candidate pairing promoting genes as well as candidate anti-pairing genes, supporting the idea that homologous pairing is mediated by a balance of factors with opposing functions . As discslmb, although, of course, this result could also be a consequence of incomplete RNAi. However, it is important to note that of the 11 representative heterochromatic pairing promoting genes we tested, 9 are important for homologous pairing of euchromatic regions as well. Therefore, the pairing of heterochromatic and euchromatic regions may be established by the same mechanism(s) and maintained independently, vice versa, or, perhaps, achieved through overlapping forces, with the potential of each contributing in cis to the proximity of the other.Our data are consistent with different regions of the Drosophila genome exhibiting different levels and stabilities of pairing Our work further indicates that even amongst heterochromatic regions, different loci may exhibit different stabilities of pairing. For example, pairing frequencies at the 359-bp repeat locus on the X-chromosome are higher compared to that of the autosomal heterochromatic repeated sequences AACAC and dodeca (∼80% compared to ∼50%), suggesting that pairing of 359 may be more difficult to disrupt. Indeed, only 16% of the pairing hits significantly affected pairing at this locus, and 359 was the only heterochromatic region tested that did not reveal a drop in pairing levels during S-phase. We reason the more robust pairing of 359 could be due to structural features, such as the large nature of the repeated region, estimated to be ∼11 Mb in length, and/or the acrocentric nature of the X-chromosome. Alternatively, the proximity of the X-linked rDNA gene cluster to 359 may contribute to pairing via independent forces, as the rDNA locus is associated with pairing of the X and Y chromosomes during male meiosis Our most striking result was the abundance of candidate anti-pairers which, similar to Cap-H2 e.g. e2f and cdk8), suggesting that homolog unpairing is functionally coupled to the progression of the cell cycle.The advantages of preventing heterochromatic pairing might include safeguarding the genome against aberrant repair or mitotic recombination. Specifically, the unpairing of similar heterochromatic sequences between homologous and heterologous chromosomes would preclude either from being used as templates for repair and therefore prevent loss-of-heterozygosity or chromosome rearrangements, respectively. The anti-pairing activity of HP1a is particularly interesting in this light, as HP1a has been proposed to contribute to the prevention of aberrant repair between nonhomologous chromosomes in heterochromatin by relocalizing broken sites outside the heterochromatic domain Anti-pairing may also be a potent means to globally, locally, and/or transiently control gene expression through the cell cycle. For example, it may be used to inhibit cross-communication between alleles that are physically paired Finally, our screen suggests that the mechanism of anti-pairing involves the maintenance of higher order chromosome structure, as it recovered several components of the condensin II complex and ORC1, in addition to HP1a, as anti-pairers. The connection between anti-pairing and condensin II is particularly intriguing, given the role of the latter in chromosome compaction. How might anti-pairing and compaction be mechanistically linked? One possibility is that the forces of compaction drive the formation of unpaired loops Interestingly, the human ORC complex has been implicated in HP1a recruitment to heterochromatin slmb, lin19, and skpA. Considering that SCF functions as an E3 ubiquitin-ligase slmb RNAi also resulted in smaller nuclear volumes, a phenotype dependent on Cap-H2 and characteristic of hypercondensation. Alternatively, the role of SCF in pairing may be linked to its role in cell cycle regulation, with knockdown causing an enrichment of stages where pairing is normally reduced.Given the role of condensin II in anti-pairing, it is perhaps not surprising that many of the candidate pairing promoters we identified had RNAi phenotypes that were dependent on Cap-H2 activity. This ‘condensin-dependent’ subset of pairing promoters may facilitate pairing indirectly through controlling condensin II activity . For exapolo and pav. Interestingly, while Cap-H2 co-depletion suppressed the pairing phenotype following polo and pav knockdown, only a mild reduction in the frequency of large nuclei was observed. Thus, the increased nuclear volumes and multi-nucleated cells (both characteristic of cytokinesis defects) in the double knockdowns confirms that pairing can accommodate larger nuclear volumes and, likely, extra chromosomal copies. Although the relationship between cap-H2 and these pairing promoters remains to be elucidated, our findings argue that these factors may lead to a disruption in pairing by modulating condensin II activity and/or inhibiting the activity of proteins necessary for decondensation at the end of mitosis.Additional dsRNAs whose FISH phenotypes were dependent on Cap-H2 included those targeting genes known to be involved in mitotic spindle organization or cytokinesis, such as cap-H2 RNAi may suppress unpairing simply by precluding paired homologs from decatenating. Considering that mitotic spindle forces are required for the resolution of DNA catenations e.g. pav RNAi) could create new antagonizing forces against homologous chromosomes and thus aberrantly remove any residual catenations or pairing interactions. In the absence of condensin II, however, the accumulation of DNA catenations between homologous chromosomes may enhance pairing and prevent homolog separation.We note an alternative model in which the consequences of depleting pairing promoters can be countered by the loss of condensin II. For example, the role of condensin II in resolving DNA catenations suggests that pairing may involve DNA catentation and, if so, Of the candidate pairing promoters, 27 had RNAi phenotypes that were not dependent or only partially dependent on Cap-H2 activity . This ‘cdhc64C, lis-1, and mcph1; dhc64C and mcph1, these genes were also found to be important for euchromatic pairing, suggesting the pairing of chromosome arms could be facilitated and/or stabilized by a similar mitosis-driven mechanism, possibly extending, although not necessarily linearly, from both pericentromeric and interstitial heterochromatin. A complete understanding of pairing, however, will require a screen for pairing factors wherein the FISH assay targets euchromatic loci directly to determine whether there are pairing factors that are essential only at euchromatin.Our identification of mitotic regulators is consistent with a critical step of pairing occurring during mitosis, with anaphase and/or telophase being of particular import. Although pairing may be disrupted at the onset of anaphase An additional, yet not mutually exclusive, model proposes that at least some pairing promoters function directly in the maintenance of homolog pairing during interphase. For example, kinesin Klp61F, Dynein motor protein Dhc64C, and microtubule binding protein CHB also localize to cytoplasmic interphase microtubule arrays Lastly, as we identified CPC components INCENP and IAL (Drosophila Aurora B) as condensin-independent pairing promoters, an intriguing parallel to our work may be the discovery of DNA tethers, coated with INCENP and Aurora B, that connect and mediate the accurate segregation of chromosome fragments at mitosis In sum, this study indicates that somatic homolog pairing may be mediated by antagonistic mechanisms, possibly involving >100 genes, many of which are highly conserved throughout higher eukaryotes. Excitingly, a number of these genes have also been identified as pairing factors through a whole-genome screen examining localization of the MSL dosage compensation machinery in Drosophila . Our work also brings support to long-standing observations correlating stages of the cell cycle to differing degrees of homolog pairing 167 cells 6 cells/mL prior to the analyses.KcGGGATCGTTAGCACTGGTAATTAGCTGC, Ty3-AACAC: Tye3-AACACAACACAACACAACACAACACAACACAACAC, and FAM488-dodeca: FAM488-ACGGGACCAGTACGG. Oligo probes were resuspended in 1×TE at 100 µM concentration and stored at −20°.Oligo probes for the 359, AACAC, and dodeca heterochromatic repeats DNA probes to 16E and 28B were synthesized according to standard protocols. Bacterial artificial chromosome BACR17D02 RP98-17D2  corresponding to 16E1–16E2 (abbreviated as 16E) and P1 plasmids (Berkeley Drosophila Genome Project) containing cloned Drosophila genomic DNA corresponding to chromosomal regions 28B1–28B2  were centrifuged (1200 RPM for 5 minutes), counted, and diluted in FBS-free media to 1×106 cells/mL. Sterile, autoclaved Wellmate tubing was purged with sterile PBS in a tissue culture hood. After the Wellmate was primed with the diluted cells, 10 µL were added to each well. The plate was then spun  and incubated in a 25°C incubator for 30 minutes. With freshly primed sterile tubing, regular FBS-containing Schneider's media was added to each well (30 µL/well) and the plate was then spun  before being transferred to a humidifying chamber in a 25°C cell culture incubator. Four (primary screen) or five  days after dsRNA treatment, the plates were removed from the incubator. The cell media was aspirated and, with a primed Wellmate, wells were quickly washed with PBS (60 µL/well), which was then immediately aspirated. Plates were incubated with 4% Formaldehyde (30 µL/well) for 5 minutes, aspirated, and quickly rinsed (30 µL PBS/well), then washed with 2×SSCT (80 µL/well) for 5 minutes. Then, plates were washed with 50% formamide/2×SSCT (80 µL/well) for 5 minutes. The plates were double-sealed with adhesive aluminum seals, pre-denatured by being floated in a 91°C waterbath for 3 minutes, 60°C waterbath for 20 minutes, and allowed to cool to room temperature.A 384-well plate containing dsRNA was centrifuged at 1200 RPM for 2 minutes. Log-phase KcProbes were prepared in 10 mL of Hybridization Buffer (20% dextran sulfate/2× SSCT/50% formamide) per plate . The plate was aspirated, after which probe mix was added to each well (20 µL/well). The plates were again double-sealed with aluminum adhesive seals, centrifuged (1200 RPM for 2 minutes), and denatured in a 91°C waterbath for 20 minutes. Hybridization was conducted for 30 minutes at 45°C by floating the plate in prewarmed 45°C wash buffer (50% formamide in 2×SSCT) in a Tupperware within an incubator. The plate was washed by being submerged in 45°C wash buffer while having its seal removed, allowing buffer to wash immediately into the wells. The plates, still submerged in 45°C wash buffer, were then placed on a slow moving shaker. Buffer was vigorously “flicked” out of the wells after 5 minutes and again after 20 minutes, being quickly resubmerged after each. The plate was aspirated and washed with room temperature 50% formamide/2×SSCT (80 µL/well) for 5 minutes. Hoechst was diluted 1∶1,500 in 2×SSCT and added to each well (30 µL), after which the plate was incubated for 5 minutes. The plate was then washed twice for 10 minutes each with 2×SSCT (60 µL/well). The plate was then sealed with a clear adhesive seal and centrifuged (1200 RPM for 2 minutes). To ensure optimal imaging, all plates were prepped and imaged in the same day. For automated microscopy, the cells were imaged with an Evotec Opera Confocal Screening Microscope (Perkin-Elmer) with a 63× water immersion lens. 10 images per well were acquired, each of which were autofocused prior to taking a single optical section through the nuclei. Note that the Opera system had limits as to how many images could be taken per well. Considering the short depth of nuclei within adhered Drosophila cells and the brightness of our FISH signals, we chose to take 10 autofocused images per well without Z-slices to maximize the number of cells being assayed.th percentile cell sizes of control wells (data not shown).All the images acquired in this screen were analyzed automatically using a customized algorithm developed in MATLAB. The analysis was carried out in three main steps: nuclear segmentation, FISH foci identification, and cell classification/scoring. In the nuclear segmentation step, the DAPI image was first smoothed and background corrected. The corrected image was then segmented by applying a threshold determined by Ridler-Calvard method z-score of ≤−2.0 or increased the percentage of nuclei containing two, three, or four foci to a z-score of ≥2.0. 374 dsRNAs were considered hits using these criteria. Importantly, using these cut-offs, greater than 90% of positive control wells seeded with dsRNA targeting pav or cap-H2 resulted in the expected increase or decrease in FISH signals per nucleus. In the validation screen, however, we sought to identify the strongest hits and, therefore, only listed dsRNAs that significantly (P≤0.05) decreased the percentage of nuclei with a single FISH signal as compared to control cells. This created a much more stringent cut-off and reduced the number of gene hits to 40.In the primary genome-wide screen, a dsRNA was considered a ‘pairing promoting’ hit if, in both replicate plates, the dsRNA either decreased the percentage of nuclei with a single FISH signal to a z-score of ≥2.0. Similarly, the criterion used for hits in the validation screen was those dsRNAs that significantly (P≤0.05) increased the percentage of nuclei with a single FISH signal as compared to control cells.For dsRNAs that produced an ‘anti-pairing’ phenotype in the primary screen, each replicate plate produced an increase in the percentage of nuclei with a single FISH signal to a Primary antibody against phosphohistone H3  was used for immunofluorescence in a PBS buffer following FISH reactions. A Cy3-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories) was used at 1∶165 according to the manufacturer's instruction.167 cells were grown in six-well plates, pipetted onto slides, exposed to hypotonic solution (1% Sodium Citrate) for 30–45 minutes and then fixed in 3 Methanol: 1 Acetic acid. Cells were then dried for a few minutes and DNA was stained with DAPI.KcFixed cells were RNAsed (1 mg/mL), incubated with a 2 µM solution of Propidium Iodide (PI), and sorted on a Becton Dickinson FACSAria. The cells were then adhered to lysine-coated slides for 2–3 hours, and then subjected to FISH. We confirmed that cells had been successfully sorted into G1, early S, late S, and G2 subpopulations by assessing nuclear DNA content as determined by DAPI staining (data not shown).Figure S12R = 0.3586 represents significant fit of the data to a linear regression, suggesting that pairing levels between the two chromosomal regions are correlative. A minimum number of 250 nuclei were scored for each dsRNA.Correlation between 359 and dodeca pairing following RNAi knockdown of pairing promoters. The results from RNAi of all 40 pairing promoters are plotted. X-axis is the percentage of single-signal nuclei at 359. The Y-axis is the percentage of single-signal nuclei at dodeca. The coefficient of determination (TIF)Click here for additional data file.Figure S2P<0.05). Error bars denote SD. A minimum number of 100 nuclei were scored for each dsRNA.Pairing promoters are important for pairing in interphase nuclei. a, Representative image showing both interphase (PH-3-minus) and mitotic (PH-3-positive) nuclei with dodeca FISH. b, Following depletion of 11 representative pairing promoters, the percentage of single-signal nuclei was significantly decreased compared to control ((TIF)Click here for additional data file.Figure S3RAD21 depletion leads to premature sister chromatid separation during mitosis. Chromosomes from control metaphase cell with paired sister chromatids and from a RAD21 RNAi metaphase cell clearly showing separated sister chromatids.(TIF)Click here for additional data file.Figure S4P<0.05). A minimum number of 100 nuclei were scored for each subpopulation.Heterochromatic pairing through the cell cycle in S2R+ cells. FACS plot of S2R+ cells with four gates for G1, early S (S1), late S (S2), and G2 phases of the cell cycle. The frequency ± SD of paired nuclei when targeting 359, AACAC, and dodeca in the G1, S1, S2, and G2 subpopulations. Asterisks denote a significant reduction in paired nuclei at each locus compared to that of G1 cells ((TIF)Click here for additional data file.Table S1Comparison of pairing levels under different control conditions.(XLSX)Click here for additional data file.Table S2z-score in the primary screen. Plate and well location are noted along with dsRNA amplicon and target gene (if one is annotated). %1 359 and %1 dod denote the percentage of nuclei with a single 359 or dodeca focus, respectively. Corresponding z-scores are noted.Pairing candidates isolated in primary screen. dsRNAs listed are those that produced a significant (XLSX)Click here for additional data file.Table S3Validated candidate pairing promoters. dsRNA amplicon, target gene, and well location are noted along with total cell count (Cell_cnt), the percentage of large nuclei (Large_nuclei), and the percentage of nuclei with one, two, three, four, and five or more FISH foci for 359 and dodeca. The percentage of nuclei with a single focus for both 359 and dodeca (1R1G_nuclei) as well as the subset of these that exhibit colocalization (1R1G_touch_nuclei) are also noted. Standard deviations (stdev) from three replicate tests are presented to the right of each parameter.(XLSX)Click here for additional data file.Table S4Validated candidate anti-pairers. dsRNA amplicon, target gene, and well location are noted along with total cell count (Cell_cnt), the percentage of large nuclei (Large_nuclei), and the percentage of nuclei with one, two, three, four, and five or more FISH foci for 359 and dodeca. The percentage of nuclei with a single focus for both 359 and dodeca (1R1G_nuclei) as well as the subset of these that exhibit colocalization (1R1G_touch_nuclei) are also noted. Standard deviations (stdev) from three replicate tests are presented to the right of each parameter.(XLSX)Click here for additional data file.Table S5Cap-H2–independent pairing promoters. Data are presented for the pairing promoters whose RNAi phenotypes were either partially or completely independent of Cap-H2 co-depletion. dsRNA target genes and thier putative human orthologs are noted as well as the percentage of nuclei with a single dodeca FISH signal in the presence and absence of Cap-H2 RNAi.(XLSX)Click here for additional data file.


Recent discoveries of new modifications, kinases, and binding proteins have challenged previously established paradigms. In this paper, we examine results and implications of recent studies related to modifications of the CTD and the respective enzymes; we also survey characterizations of new CTD-binding proteins and their associated processes and new information regarding known CTD-binding proteins. Finally, we bring into focus new results that identify two additional CTD-associated processes: nucleocytoplasmic transport of mRNA and DNA damage and repair.Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA but also coordinates transcription-related processes via its unique C-terminal repeat domain (CTD). The CTD is an RNAPII-specific protein segment consisting of repeating heptads with the consensus sequence Y The number of these heptad repeats varies from organism to organism and appears to correlate with genomic complexity; there are 26 repeats in yeast, 44 in Drosophila, and 52 in humans [Since its discovery by Fischer and Krebs in 1955 . Distingn humans ). The plThe CTD's unique structure, functional characteristics, and fundamental role in transcription have generated a substantial amount of interest and have made it the subject of considerable study. This research has greatly expanded our understanding of the CTD, its interacting factors, and the process of transcription in general, but as demonstrated by recent discoveries of hitherto uncharacterized CTD modifications, kinases, and binding factors/modes, there is still much that remains to be learned. As the study of gene expression and transcriptional control mechanisms expands from preinitiation into elongation, a deeper and more nuanced understanding of the CTD will likely become essential in order to deconvolute the relationships between various aspects of gene control. This is especially pertinent in terms of understanding the crosstalk between transcriptional elongation and co/post-transcriptional events, such as splicing, export, and translation. In this review, we will discuss recent developments and emerging paradigms in the study of the CTD, its modifications, binding partners, and associated processes. To further expand on an especially relevant  and newly emerging CTD-associated process, we will also present an in-depth discussion of the nuclear export of RNA, with a particular focus on the interactions between the nuclear export machinery, the CTD, and transcription.The binding specificity of the CTD, and therefore the recruitment of particular factors, is determined by the CTD's phosphorylation state, which undergoes a series of alterations throughout the transcription cycle. Serine 2, serine 5, and more recently serine 7 of the heptad repeat have been identified as the primary targets for this transcriptionally regulated phosphorylation. In the general model of the “phosphoCTD cycle,” RNAPII is recruited for assembly at the promoter with an unphosphorylated CTD; moreover, it appears that CTD phosphorylation prior to preinitiation complex (PIC) formation has an inhibitory effect on transcription . Upon PIDespite being highly intuitive, this gradient model of the “phosphoCTD cycle”  is oversimplified for several reasons. First, although the model accounts for variations in the general phosphorylation pattern of the CTD , it fails to fully capture the highly dynamic nature of the process. The phosphorylation state of the CTD is likely to be in continuous flux throughout the entire transcription cycle, with multiple kinases and phosphatases working together to maintain specific phosphorylation patterns on particular subsets of heptads. For example, it has been recently shown that the Ser7 phosphates at the 3′ end of the gene are actually placed anew, after being removed by an unidentified phosphatase  . It is worth reiterating that the Ser5P to Ser2P gradient model presented above is almost entirely based on the reactivity of these antibodies . The mosThe general model of the phospho-CTD cycle also fails to account for exceptions to the canonical patterns of phosphorylation. The recent publication of three genome-wide studies of phosphoCTD RNAPII occupancies in yeast , 19, 20 Lastly (in terms of complications), it has not escaped notice that modification of the CTD is not theoretically limited to the phosphorylation of Ser2, Ser5, and Ser7. Many other post-translational modifications have been observed, including phosphorylation of Tyr1  and Thr4cis/trans isomerases (Ess1 in yeast and Pin1 in humans) in transcription and CTD phosphorylation [trans state. This changed last year when two structural studies found that the Ser5-specific CTD phosphatase Ssu72 bound to the cis conformation of an Ser5-Pro6 motif within the heptad repeat [in vitro, suggesting that this cis/trans interconversion plays a role in the fine-tuning of the phosphorylation state of the CTD [Yet another important CTD modification, which until recently had not been directly observed to play a direct role in factor binding, is the enzymatic isomerization of the heptad repeat's peptidyl-proline bonds. Although multiple studies have suggested a role for the CTD-interacting peptidyl-prolyl rylation –26, all d repeat , 28. Con the CTD . These f the CTD , a distiA good example of a transcript class-specific CTD modification is the newly discovered methylation of an arginine (R1810) in heptad 31 of the human CTD . As an aThus, the general “phosphoCTD cycle” has given way to a “CTD code” of staggering complexity, one that we are just beginning to explore in detail. This complexity reflects the vast number of different genes, processing events, and transcriptional programs that RNAPII must coordinate. Although the segmented gradient model has proven to be very useful for conveying the CTD's principal function during RNAPII transcription, as our understanding of the CTD and associated processes improves, it is likely to undergo drastic changes in the near future. Understanding the nuances of this CTD code will be imperative to understanding the link between transcription and cotranscriptional events and to perhaps eventually unlock the therapeutic potential of the CTD.in vitro  and stages of transcription. Thus, the various CTD kinases are most conveniently presented in the context of a segmented transcription cycle; however, it should be made clear that in reality the various kinase activities lack the clearly delineated boundaries that such a presentation suggests.The specific phosphorylation events within the heptad repeat are mediated through the activity of a transcription-associated subset of cyclin-dependent kinases (CDKs) known as the CTD kinases. Unlike their cell cycle counterparts, the CTD kinases form complexes with members of the noncycling “transcription cyclin” family and are active throughout the cell cycle. Nevertheless, CTD kinase activity is tightly regulated through a variety of mechanisms, including selective recruitment, binding by kinase-associated factors, and sequestration by inhibitory factors. Although somewhat promiscuous in vitro ), in vivAlthough we will not be discussing them in detail, it should be kept in mind that the CTD kinases function in conjunction with the more enigmatic CTD phosphatases  and coincides with RNAPII entry into productive elongation. Coupled with the activity of Ser5P-specific phosphatases (Rtr1 in yeast ), this lSaccharomyces cerevisiae (Sc), the phosphorylation of Ser2 is primarily mediated by CTDK-I, a three subunit enzyme  [in vivo, Ctk1 is not essential for viability or for transcriptional elongation. The CTD kinase activity of Ctk1 has been linked to several transcription-associated processes, including the recruitment of the Set2 histone methyltransferase [Saccharomyces pombe (Sp), where Lsk1, the Sp orthologue of Ctk1, has been shown to be responsible for the bulk of Ser2 phosphorylation, while Sp CDK9, the Sp orthologue of Bur1, is able to phosphorylate both the CTD and Spt5 [In unknown) , 52. Altnsferase , 54, 3′ nsferase , 55, 56,nsferase ). In addnsferase . Despitensferase . While insferase , 60, ratnsferase , 61. As nsferase . Anotherand Spt5 , 62.Sc Bur1 is the closest Sc relative of metazoan CDK9 proteins, Sc Ctk1 is actually more closely related to another set of relatively little-studied metazoan CDK proteins [Drosophila CDK12 (dCDK12) and little-studied human CDK12 (hCDK12) as elongation phase CTD kinases and the metazoan orthologues of yeast Ctk1 [in vitro kinase assays, only CDK12 seems to have an effect on global CTD phosphorylation in vivo; thus CDK13's role in transcription (assuming one exists) remains elusive [Until recently, higher eukaryotes appeared to have only one Ser2 CTD kinase: P-TEFb (which is composed of CDK9 and cyclinT). P-TEFb is able to phosphorylate both the Ser2 position of the CTD and the elongation factor Spt5 and is essential for transcriptional elongation –65  capping, splicing, 3′ end processing, termination, and more recently nuclear export (discussed below). In terms of non-RNA processing-associated events, the CTD has been shown to play roles in transcriptional activation, cotranscriptional chromatin modification, chromatin remodeling, and genome stability. Of course, the analysis of the CTD's role in each specific function is very challenging, as many of the CTD-mediated transcriptional processes are interlinked. For example, capping has been shown to influence both the splicing of the first intron and 3′ end processing –87; spliin vivo, which correlates with the enzyme's function, and that this association is dependent on phosphorylation of Ser5 of the CTD [Candida albicans guanylyltransferase Cgt1 complexed with a synthetic Ser5P four heptad repeat peptide revealed that the CTD binds within an extended docking site on the enzymes surface using two nonadjacent heptads, and a full-heptad repeat was looped out away from the interaction site [As mentioned previously, one of the most clearly recognized functions of the CTD is its involvement in the 5′ end capping of mRNA through the recruitment of the capping machinery. The modification of the 5′ end of the RNA with the 5′ 7-methyl guanosine cap is unique to RNAPII transcripts and occurs just after the transcript clears the polymerases exit channel , 95. Tra the CTD , 37, 46. the CTD . The intion site . This loion site .trans prolines within a mixed population of cis-trans isomers [Another well-recognized function of the CTD is its role in 3′ end processing and termination . A isomers . It is lOne of the relatively more recently discovered functions of the CTD is its role in the transcription and 3′ end processing of snRNAs , 115. EvTying together two important aspects of gene expression and transcriptional coordination, the CTD has also been shown to be involved in the cotranscriptional modification of histones and remodeling of chromatin structure. Although a full discussion of histone modifications and the histone code hypothesis is beyond the scope of this paper, the significance of these processes cannot be understated: they play integral functions in almost every aspect of gene expression and regulation . Here, in vivo. Several other recently characterized chromatin-related CTD interactions include the recruitment of the chromatin-remodeling factor CHD8 [Providing the first clear link between the CTD and histone modification, the yeast Set1 and Set2 methyltransferases were found to be recruited to actively transcribed genes at specific stages of the transcription cycle  through interactions with the Ser5P (via the PAF complex for Set1) and Ser2/5P CTD –123 . Aextracts ), experiextracts , and in extracts –140. Con[in vivo , and the[in vivo . In whatin vitro , 143. Inin vitro , severalin vitro  and the in vitro , PSF, anin vitro . A recenubstrate . Althougubstrate , the facubstrate , 151 hasubstrate . Surprisubstrate ). In addubstrate , it has in vivo splicing assays using reporter genes have demonstrated that the ectopic overexpression or depletion of CDK12 and CDK13 modulates alternative splice site selection [in vitro assays, make it tempting to speculate that the two kinases may serve as a central link between the processes of transcription, CTD phosphorylation, and splicing; however, whether such a link exists is still undetermined.Although it is not the only CDK kinase to have been implicated in the regulation of both transcription and alternative splicing , Celection , 75, 77.election . These selection , but allelection . ComplicWhile the connection between mRNA processing and the CTD has been established, recent studies have begun to investigate CTD involvement with the last step in mRNA production: formation of an export competent messenger ribonucleoprotein particle (mRNP). While this area of research has yet to mature, it is known that mRNAs are exported through a Ran-GTP-independent pathway that involves a specific set of conserved export receptor and adaptor proteins. There appears to be one universal receptor for mRNA export, Mex67/Mtr2 in yeast and TAP/p15 in mammals, which interacts with the mature mRNP and the nuclear pore complex (NPC) to facilitate export. The export receptor functions in conjunction with the export adaptor proteins, which cotranscriptionally associate with the nascent mRNA. There are two main mRNA export adaptor proteins, Yra1/ALY in the transcription and mRNA export (TREX) complex and Sac3 in the TREX-2 complex. While export mediated by TREX-2 and Sac3 has been shown to be coupled to chromatin modification through Sus1, a common factor in both TREX-2 and the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex , wΔhpr1 yeast displays a decrease in the levels of both Sub2 and Yra1 occupancy on certain genes [HPR1 has been shown to affect transcription elongation, this decrease in Yra1 and Sub2 occupancy may be due to an elongation defect rather than a direct effect of Hpr1 on the export proteins [TREX was the first characterized transcription export complex and consists of the THO complex of elongation and hyperrecombination-related factors, including Hpr1, Tho2, Thp1, and Mft1; the export adaptor protein Yra1/ALY; the RNA helicase Sub2/UAP56; a protein of unknown function, Tex1 . Yra1/ALin genes ; howeverproteins .via ChIP) that partial deletion of its phospho-CTD interaction domain (PCID) leads to a near loss of Yra1 association with active genes ). We therefore think that either Yra1 is responsible for recruiting the rest of TREX to active genes via the CTD or Yra1 is recruited to active genes independently of THO. Further chromatin immunoprecipitation studies using additional Yra1 variants that do not bind the CTD are needed to further clarify the role of the CTD in Yra1 function and the dependence of the rest of TREX on Yra1.In a previous study from our lab, a proteomics screen in yeast identified Yra1 as a putative phospho-CTD-associated protein (PCAP) . We havevia PCTD binding and then for associating with the nascent transcript through RNA binding. Structural studies on Yra1 in complex with the PCTD are necessary to examine the role and binding mode of the RRM in this interaction.We also found that the PCID of Yra1 contains an RNA recognition motif (RRM). While RRMs are known to be versatile-binding domains that mediate both protein-nucleic acid and protein-protein interactions . For exin prep). A different kind of damage repair, as signaled by mitotic recombination, also appears PCTD linked since diploid yeast deleted for CTK1 (ctk1Δ/ctk1Δ strains) displays reduced rates of spontaneous mitotic recombination at several loci . Thus, the PCTD appears to be involved in processes that maintain genome stability. For example, Bennett and colleagues used the diploid yeast deletion strain collection to identify a large number of genes whose homozygous deletion leads to ionizing radiation (IR) sensitivity , 179. WeIn vitro experiments showed that RecQ5 binds directly to the elongation-associated phospho-CTD via a Set2 Rpb1-interaction (SRI) domain; moreover, deletion of the SRI domain resulted in loss of RecQ5 protein at multiple loci [In a related vein, it was shown recently that mammalian RecQ5 protein, a putative “antirecombinase,” is associated with RNAPII on active genes , 181. Inple loci . While tple loci –179). Itin vivo. Even so, the list of proteins known to interact with a specifically modified form of the CTD also continues to grow, expanding our knowledge of the roles played by the CTD in coordinating transcription-related processes. As our knowledge of CTD modifications and interactions expands and becomes more refined, our understanding of the “phospho-CTD cycle” and the manner in which the PCTD orchestrates the numerous events connected to the process of DNA-dependent RNA synthesis will continue to evolve.After 25 years of research, much is still unknown about the CTD of RNAPII and its role in coordinating a surprising number of nuclear events with transcription. In addition to transcript elongation , mRNA processing, and chromatin modification, the collection of CTD-interacting processes is now thought to also include mRNA export and DNA repair; future investigations into the links between these events and the CTD should be remarkably informative. New information on CTD phosphorylation patterns, which modulate its interactions with nuclear factors, has recently been generated through genome-wide ChIP experiments, and multiple new insights into global CTD phosphorylation have emerged. On the other hand, the complexity and nuance of the patterns of post-translational modifications are such that the actual distribution of phosphate groups along the CTD is not known for even one transcription elongation complex. Thus, while the inventory of CTD-modifying enzymes continues to expand, it is clear that we have much to learn about the ways in which they collaborate to produce modification patterns as found


Drosophila neuronal subtypes (Tv1 and Tv4), we test whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, we find that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply “locked-in,” but must be actively maintained by persistent developmental transcription factor networks. We also examined the cross-regulatory relationships between all transcription factors that persisted in adult Tv1/Tv4 neurons. We show that certain critical cross-regulatory relationships that had existed between these transcription factors during development were no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, our results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain.During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two  For neurons to function properly, they must establish and then maintain their unique, subtype-specific gene expression profiles. These unique gene expression profiles are established during development by networks of DNA–binding proteins, termed transcription factors (TFs). However, how neurons maintain their unique gene expression profiles in the mature and aging brain is largely unknown. Recent advances in inducible genetic technologies now allow us to manipulate gene expression in adult neurons, after normal development. Applying such techniques, we examined the effect of knocking down TF expression in two adult neuronal subtypes. We show that the TF networks that establish unique gene expression profiles during development are then required to maintain them thereafter. Thus, gene expression profiles are not simply “locked-in,” but must be actively maintained by persistent developmental TF networks. However, we found that critical cross-regulatory relationships that had existed between TFs during development were not present in the adult, even between persisting TFs. This highlights important differences between developmental and maintenance transcriptional networks in individual neurons. The dependence of subtype gene expression on active mechanisms represents a potential Achilles heel for long-lived cells, as deterioration of those active mechanisms could lead to functional degeneration of neurons with advancing age. Tremendous progress has been made in delineating the transcriptional mechanisms that diversify neuronal subtype identities during development. Spatiotemporally-patterned transcription factor cascades act within increasingly diversified progenitor populations to specify postmitotic neuron subtype fate. Within those postmitotic neurons, subtype-specific sets of transcription factors act combinatorially to differentiate subtype identity by initiating expression of the genes that define subtype form and function Drosophila neuronal subtypes to address the following largely unanswered questions: Do developmental subtype transcription networks persist in adult neurons or are they dispensed with? Are they required to maintain the expression of subtype-specific sets of terminal differentiation genes? If they are required, does maintenance of terminal differentiation genes require the same complex combinatorial codes of transcription factors as for their initiation, or a simplified code involving fewer transcription factors? Finally, do persisting developmental transcription factors retain the same cross-regulatory relationships that regulated their expression during development?After subtype-specific gene expression profiles are established by differentiation, continued neuronal function throughout life depends upon their maintenance of subtype gene expression profiles. However, we currently have only a rudimentary understanding of the mechanisms of long-term, subtype-specific gene maintenance. Two extreme models would posit that subtype identity is either actively maintained by persistent subtype transcription network activity or passively maintained by for example stabilized chromatin structure, independent of a subtype transcription network. Here, we test the active model in two Drosophila Tv1 and Tv4 neurons. There are six clusters of Tv neurons in the Drosophila ventral nerve cord, each comprising four distinct subtypes (Tv1–Tv4). Tv1 and Tv4 express subtype-specific terminal differentiation genes, the neuropeptides Nplp1 (Tv1) and FMRFa (Tv4) and the neuropeptide amidase PHM . Using these genes as markers for subtype-specific differentiation, previous work had revealed the elaborate subtype transcription networks that direct Tv1/4 subtype specification and differentiation Drosophila life. Thus, our detailed understanding of their subtype-specific initiation in the embryo provides an ideal background to investigate how such terminal differentiation genes, and hence subtype identity, are maintained in the adult. We previously established that persistent retrograde BMP signaling is required to initiate and maintain FMRFa in Tv4 neurons We model these questions in castor (cas) and grainy head (grh) collier (col), squeeze (sqz) and nabap, eya, dimmed (dimm) and col then differentiate Tv1 identity in part by initiating Nplp1 expression ap, eya, dimm, dachshund (dac), sqz and grh act combinatorially with target-derived BMP signaling to differentiate Tv4 identity, in part by initiating FMRFa expression dimm acts independently of other regulators to induce expression of the neuropeptide amidase, PHM cas or col mutants, whereas eya, ap, col and dimm all act combinatorially thereafter to initiate Nplp1 expression cas, col or grh mutants. In sqz and nab mutants, Tv4 neurons are not specified in a segment-specific manner eya, ap, sqz, dimm, dac, grh and BMP signaling all appear to combinatorially initiate FMRFa expression.Genetic analysis has defined cascades of transcription factors that specify Tv1 and Tv4 neuron fates and then differentiates their subtype-specific terminal differentiation gene expression profiles  [6], [8]ap and eya expression, as well as retrograde BMP-signaling. We also demonstrated that FMRFa maintenance in adult Tv4 neurons requires persistent retrograde BMP-signaling ap, eya, dimm, dac and sqz  or Tv4  neurons are not retained in the adult. The exceptions to this are col and sqz. Both are implicated in Tv subtype specification, but it is notable that both transcription factors have also been implicated, by loss and gain of function genetics, as part of the combinatorial transcription factor codes that initiate Nplp1 or FMRFa expression Which transcription factors persist in adult neurons is intriguing. All those previously implicated in the postmitotic differentiation of Tv1 and/or Tv4 neurons persist. In contrast, transcription factors that act within the neuroblast and newborn postmitotic neuron to specify the fate of Tv1 (GAL4ap (except where noted) to express UAS-dsRNAi transgenes (abbreviated to dsRNAi) targeted to each transcription factor. We also overexpressed UAS-Dicer2 in all experiments to enhance dsRNAi efficacy dsRNAi in adults, we utilized the TARGET system wherein a temperature-sensitive GAL4-repressor GAL80 (GAL80TS) controls the activity of GAL4 TS to repress GAL4 activity throughout development. Then at adult day 1 (A1), flies were switched to 29°C and kept at that temperature for the remainder of each experiment. At this temperature, GAL80TS becomes dysfunctional and thus GAL4 is allowed to induce dsRNAi expression  and quantified FMRFa transcript levels by fluorescent in situ hybridization, relative to the mean of controls. In adults, dsRNAieya dramatically reduced FMRFa transcript to 19.7±4.4% of control (p<0.0001) by adult day A15   . Eya immy alone) . Previouapterous (ap). As GAL4ap is a strong hypomorphic ap allele GAL4dac to express dsRNAiap in wildtype and heterozygous ap backgrounds. We observed a significant downregulation of FMRFa transcript when expressing dsRNAiap in ap heterozygotes, falling to 66.8±4.2% of control . No loss of FMRFa was observed in either ap heterozygotes or dsRNAiap alone . Finally, we also confirmed that an dsRNAiap targeting different ap sequences also significantly downregulated FMRFa   . Similari alone) . PrevioudsRNAidimm at A1 and found that immunoreactivity to the mature amidated FMRFa peptide was rapidly and profoundly reduced by dsRNAidimm to 24.0±3.2% of control by A10 (p<0.0001), and this was enhanced to 9.8±1.6% of control in dimm heterozygotes  (dimm heterozygotes to 67.1±2.9% of control at A20 (p<0.0001) ). Thus, we conclude that dimm retains its role in the initiation and maintenance of both FMRFa transcript and mature peptide. Why is the mature peptide more responsive to dsRNAidimm than is the transcript? We postulated that this was due to dimm's regulation of proprotein convertases and peptide amidases in secretory neurons, both of which are required to process the FMRFa prepropeptide into amidated neuropeptides dsRNAidimm induction (PHM). Confirming our hypothesis, dsRNAidimm entirely eliminated PHM immunoreactivity in Tv4 neurons  . Immunori alone) . In cont<0.0001) . Similarequences . It is n neurons . Thus, tdac mutants during development dac function in adults and found that dsRNAidac dramatically downregulated FMRFa immunoreactivity in adults to 14.8±2.5% of controls, as early as A10 (p<0.0001)   . Correspi alone) . Notablyi alone) . Moreoveequences . Thus, dgrh, dac, sqz and col requires transient cas activity ap and eya requires transient col expression dimm then requires eya, ap and grheyaap, eya, dimm, dac, sqz, grh and BMP signaling are all required for FMRFa initiation grh, cas and col are lost by early L1  and found that Dimm immunoreactivity was significantly downregulated to 58.5%±6.6 of control   . We veri  in either dsRNAiap or dsRNAieya backgrounds. UAS-dimm expression was found to only partially rescue Nplp1 expression in an dsRNAiap background, from 22.7±2.7% to 57.0±5.4%  (UAS-dimm expression failed to rescue Nplp1 expression in an dsRNAieya background (Genetic studies in the embryo had established that control) . Howeverckground . Thus, acol in all Tv neurons ectopically activated Nplp1 in Tv4, presumably reconstituting the col/ap/eya/dimm Tv1 subtype transcription network . Interestingly, this did not disrupt native FMRFa expression in Tv4 neurons, nor its known subtype transcription network profile. Here, we verify these data in embryos  (data not shown). However, co-expression of col and dimm in adult Tv neurons was sufficient to trigger ectopic Nplp1 expression in 100% of Tv4 neurons (n = 42)  . These dOur data provide novel insight supporting the view of Blau and Baltimore col, eya, ap and dimm are required for Nplp1initiation during development  and FMRFa/Nplp1 expression .In this study, all transcription factors implicated in the initiation of subtype-specific neuropeptide expression in Tv1 and Tv4 neurons were found to maintain subtype terminal differentiation gene expression in adults  is expressed normally in ap mutants dsRNAiap was not found to alter GAL4ap reporter activity  and the loss of cas, col and grh in Tv4 . However, we were surprised to find that the cross-regulatory relationships between persistently-expressed transcription factors were also significantly altered in adults. Notably, eya initiated but did not maintain dimm in Tv4. In Tv1, col initiated but did not maintain eya, ap or dimm. This was particularly unexpected as eya remained critical for FMRFa maintenance and col remained critical for Nplp1 maintenance. Indeed, although we tested for cross-regulatory interactions between all transcription factors in both the Tv1 and Tv4 subtype transcription networks, only Dimm was found to remain dependent upon its developmental input; Eya and Ap in Tv1 as well as Ap in Tv4. However, even in this case, the regulation of Dimm was changed; it no longer required eya in Tv4, and in Tv1 it no longer required col, in spite of the fact that both col and eya are retained in these neurons. We anticipate that such changes in transcription factor cross-regulatory relationships will be found in other Drosophila and vertebrate neurons, which exhibit high complexity in their subtype transcription networks In contrast, all available evidence in Tv1 and Tv4 neurons fails to support such an autoregulatory mechanism. An activity . MoreoveC. elegansWe are pursuing the potential role of autoregulation for the other factors in the Tv1/Tv4 subtype transcription networks. However, we consider there to be three additional, potentially overlapping, models for subtype transcription network maintenance. First, regulators may act increasingly redundantly upon one another. Second, unknown regulators may become increasingly sufficient for transcription factor maintenance. Third, transcription factors may be maintained by dedicated maintenance mechanisms, as has been shown for the role of trithorax group genes in the maintenance of Hox genes and Engrailed dimm. On the other, most transcription factors appear to no longer require regulatory input from their initiating transcription factor(s). Further work will be required to better understand whether these differences represent truly distinct modes of gene maintenance or reflect the existence of yet unidentified regulatory inputs onto these transcription factors. One issue to consider here is that the expression of certain terminal differentiation genes in neurons, but perhaps not subtype transcription factors, can be plastic throughout life, with changes commonly occurring in response to a developmental switch or physiological stimulus Taken together, our studies have identified two apparent types of maintenance mechanism that are operational in adult neurons. On one hand, there are sets of genes that are maintained by their initiating set of transcription factors. These include the terminal differentiation genes and the transcription factor foxa2 heterozygotes, another regulator of mDA neuron differentiation that is maintained in adult mDA neurons Pdx1 leads to a rare monogenic form of non-immune diabetes, MODY4 NeuroD1 haploinsufficiency is linked to MODY6 Finally, we propose that the active mechanisms utilized for maintenance of subtype differentiation represent an Achilles heel that renders long-lived neurons susceptible to degenerative disorders. Nurr1 ablation in adult mDA neurons reduced dopaminergic markers and promoted cell death Flies were maintained on standard cornmeal food and maintained at stable temperatures in environment rooms set at 70% humidity at 18°C, 25°C or 29°C.md544apterous (referred to as GAL4ap); P44; sqzIE; sqzGAL4; UAS-thickveins activatedap (AUAS-tkv); UAS-saxophone activated (AUAS-sax) GAL4dacrev4; UAS-dimm3dacCliIIDeyaGAL4grhTStubP>GAL80 (temperature-sensitive GAL80 under the control of the Drosophila tubulin 84B promoter) ; UAS-nEGFP ; UAS-dicer2UAS-col#24EdsRNAi 8376R-2; UAS-dacdsRNAi 4952R-2UAS-ap; dsRNAi 5557R-2UAS-sqz (NIG-FLY); dsRNAi GD44470UAS-dimm; dsRNAi GD43911UAS-eya (VDRC). Strains used secondarily to verify data: dsRNAi KK103356; UAS-eyadsRNAi 108071KKUAS-dimm (VDRC); dsRNAi JF03160; UAS-dacdsRNAi JF02322UAS-eya (TRiP).Strains used for primary data: Gal4; tubP>GAL80TS, UAS-nEGFP/SM6-TM6,TbUAS-dicer2/UAS-dicer2; ap) or  to UAS-dsRNAi  or 1118w flies (control). Experiments were performed on resulting progeny bearing appropriate genotypes . All experimental and control flies were raised at 18°C until eclosion . On adult day 1 (A1), flies were switched to 29°C for the duration of the induction period indicated. Throughout the text, we present dsRNAi data for the induction period at which we observe the maximal phenotype of Nplp1 and FMRFa expression. Further details for each dsRNAi are provided in the text.Flies for TARGET-mediated transgene induction were generated by crossing utility flies ; rabbit anti-GFP ; rabbit anti-FMRFa ; mouse anti-β-Galactosidase ; chicken anti-Nplp1 (1∶1000), guinea pig anti-Dimm (1∶1000) and rat anti-Cas (1∶1000) ; guinea pig anti-Collier ; rabbit anti-pMad ; rabbit anti-nab ; mouse anti-Eya  and mouse anti-Dac  .Drosophila Genomic Resource Centre, Indiana, USA) containing a 1584 bp FMRFa cDNA (using the Roche DIG-U-RNA Labelling Kit). Probe synthesis was confirmed using gel electrophoresis.Donkey anti-sheep Alexa 555 ; donkey anti-mouse Cy5 and donkey anti-rabbit Cy2 . Antisense DIG-RNA Probe: DIG-Uracil tagged RNA probes were generated using T3 RNA polymerase from clone RH03963 .All images acquired on an Olympus FV1000 confocal microscope. Fluorescent intensity of individual neurons was measured using Image J . The mean pixel intensity for each neuron was measured from compressed Z-slices, and corrected for background fluorescence. Analysis was performed on every identifiable Tv1 and Tv4 neuron in segments T1 and T3. The resulting value for each Tv neuron was then incorporated as a single datum point towards the mean intensity for each experiment. Each datum point is represented as a percentage of the mean of the Figure S1grh, cas and nab are lost in Tv4 neurons by early L1 stages. (A–C) Representative confocal images of adult Th1 and Th3 Tv clusters (green) in larval stage. Tv4 neurons (arrows) express FMRFa (white), Tv1 neurons (arrowheads) express Nplp1 (blue). Tv1 and Tv4 do not express transcription factors grh , cas  or nab  in larval stages. Flies were maintained at 25°C. Genotype: (A–F) Gal4; UAS-nEGFP+/+;FMRFa-LacZ ap.Expression of (PDF)Click here for additional data file.Figure S2dsRNAiUAS-ap phenocopies strong ap hypomorphs. (A–F) Strong ap hypomorphs Gal4/apRK568ap (B) and Gal4,/UAS-apdsRNAiap (C) flies did not develop wings (A–C), and develop the same thoracic dorsal mid line defects (arrows) (D–F). Genotypes:  Gal4/+ap;  Gal4/apRK568ap;  Gal4/+; UAS-ap-dsRNAi 8376R-2/+ap. Flies were incubated at 25°C.(PDF)Click here for additional data file.Figure S3dsRNAidac and dsRNAidimm lines is enhanced in a heterozygous background for the pertinent transcription factor. Experimental results compare relative pixel intensity of FMRFa peptide (A) and FMRFa transcript (B) of individual Tv4 neurons. Each datum point was normalized to the percentage of the mean of the 1118w control. Data for each genotype is presented as mean ± SEM. * FMRFa levels are significantly different from 1118w control p<0.0001. ** FMRFa levels are significantly different from dsRNAi only p<0.001. Genotypes:  1118w . (A) rev4 ; dsRNAidimm ; dsRNAirev4,dimm . (B) dac4 ; dsRNAidac ; dac4, dsRNAidac .Downregulation of FMRFa by (PDF)Click here for additional data file.Table S1eya, ap and sqz has the same effect on FMRFa peptide as on FMRFa transcript. To verify that downregulation of eya, ap and sqz equally affects FMRFa transcript and peptide levels, we measured the fluorescence intensity of FMRFa peptide following experimental conditions outlined in dsRNAi line for each TF; Fluorescent intensity for FMRFa in Control and Experimental groups normalized as a percentage of the mean of the control, and presented as mean ± SEM; Sample size where n = number of neurons.Downregulation of (PDF)Click here for additional data file.Table S2UAS-dsRNAi lines targeting different regions of transcription factors downregulate FMRFa immunoreactivity in Tv4. Utility flies  were crossed to various UAS-dsRNAi fly lines  and 1118w flies (control). F1 generation was raised at 18°C until they eclosed as adults, then kept at 29°C for specified time (Induction time). Table columns: UAS-dsRNAi line for each transcription factor; Fluorescence intensity for FMRFa in Control and Experimental groups normalized as a percentage of the mean of the control, and presented as mean ± SEM; Sample size where n = number of neurons; Induction time: duration of time adult flies were maintained at 29°C prior to sampling.Multiple (PDF)Click here for additional data file.Table S3UAS-dsRNAi of other transcription factors. Table columns: headings for UAS-dsRNAi line for each TF; Fluorescent intensity for each TF in Control and Experimental groups normalized as a percentage of the mean of the control, and presented as mean ± SEM; Sample size where n = number of neurons. Genotypes: 1118w . dsRNAieya . dsRNAisqz . dsRNAiapP44; ap . dsRNAidimm . dsRNAidac . dsRNAicol .Configuration of transcription factor cross-regulation within Tv1 and Tv4 subtype transcription networks. To test the potential effect of the downregulation of each transcription factor on every other transcription factor, we measured transcription factor immunoreactivity or reporter activity following induction of (PDF)Click here for additional data file.Table S4Gal4; tub-Gal80TS, UAS-nEGFP/SM6-TM6,TbUAS-dicer2/UAS-dicer2; ap) were crossed to various UAS-dsRNAi fly lines ) and 1118w flies (control). F1 generation was raised at 18°C until they eclosed as adults, then kept at 29°C for specified time (Induction time). Table columns: UAS-dsRNAi line for each transcription factor; Cell counts for the presence of immunoreactivity of the transcription factor being targeted by dsRNAi lines in 1118w flies and Experimental groups in Tv1 and Tv4 neurons. Presented as a fraction . Induction time: duration of time adult flies were maintained at 29°C prior to sampling.Configuration of transcription factor cross-regulation within Tv1 and Tv4 terminal selector networks. Utility flies ((PDF)Click here for additional data file.