TATA binding protein (TBP) is a key component of the eukaryotic transcription initiation machinery. It functions in several complexes involved in core promoter recognition and assembly of the pre-initiation complex. Through gene duplication eukaryotes have expanded their repertoire of TATA binding proteins, leading to a variable composition of the transcription machinery. In vertebrates this repertoire consists of TBP, TBP-like factor  and TBP2 . All three factors are essential, with TLF and TBP2 playing important roles in development and differentiation, in particular gametogenesis and early embryonic development, whereas TBP dominates somatic cell transcription. TBP-related factors may compete for promoters when co-expressed, but also show preferential interactions with subsets of promoters. Initiation factor switching occurs on account of differential expression of these proteins in gametes, embryos and somatic cells. Paralogs of TFIIA and TAF subunits account for additional variation in the transcription initiation complex. This variation in core promoter recognition accommodates the expanded regulatory capacity and specificity required for germ cells and embryonic development in higher eukaryotes. Transcription Factor II A (TFIIA), TFIIB, TFIID, TFIIE, TFIIF and TFIIH (reviewed in [pre initiation complex (PIC). PIC formation typically starts with the recognition of the core promoter by TFIID, which consists of TATA box binding protein (TBP) and up to 14 other polypeptides called TBP-associated factors (TAFs) [Eukaryotic cells have three RNA polymerases: RNA polymerase I (RNAP-I) transcribes the ribosomal RNA genes, RNAP-II mostly transcribes protein-coding genes and some non-coding genes, whereas RNAP-III generally transcribes small RNA genes. The RNA polymerases, however, do not have the ability to recognize the proper sites of transcription initiation. For the recruitment of each RNA polymerase to the site of initiation, an additional set of polypeptides is required, the basal transcription factors. These proteins recognize the core promoter, a sequence of approximately 100 bp around the transcription start site that is required for initiation of transcription and which contains specific sequence motifs, such as the TATA box, Initiator (Inr) and Downstream Promoter Element (DPE) [iewed in ) which ts (TAFs) . TFIID as (TAFs)  and refeTBP is the founding member of the family and is involved in transcription by all of the three major RNA polymerases  in eukaryotes . It is cHigh resolution crystal structures of the core domain of TBP from different species have been solved -12. Thesth and the 8th base pairs of the TATA box. The second kink restores the DNA back to its usual B-DNA conformation. In this way, while bound to TBP, the middle six base pairs of the TATA box are in a partially unwound right-handed double helical conformation with a widened minor groove which fits in an energetically favored manner to the concave surface. TBP binding to a canonical TATA box proceeds via a two-step pathway; first an unstable complex with unbent DNA is established. Second this complex is slowly converted into a stable complex in which the DNA is bent [TBP binds to the TATA box via an induced-fit mechanism of protein-DNA recognition -10,12,13 is bent . The secTBP is part of a number of complexes which function with different RNA polymerases. To function at RNAP-I promoters, TBP joins the Selectivity factor 1 (SL1) complex together with five RNAP-I specific TAFs (reviewed in ). TBP anTATA boxes and AT-rich sequences are scattered over the genome and TBP has a relatively low specificity for binding to the TATA box. The affinity for its DNA element is ~1000 times lower than the proteins that bind the major groove of DNA . In addiTwo distinct domains in the N-terminal region of TAF1, the largest subunit of TFIID, interact with TBP and negatively modulate its ability to bind TATA box . TAF N-tin vivo [TBP also forms a distinct complex involved in RNAP-II transcription called B-TFIID, in which TBP interacts with a unique TAF called BTAF1 (reviewed in ). It is in vivo . Interesin vivo ,38.In vitro studies have shown that this domain is important for TATA-containing RNAP-II and III promoters but dispensable for TATA-less transcription [in vitro findings, homozygous loss of most of the N-terminal domain has no apparent effect on either cell growth or transcription by RNAP-II and RNAP-III [TBPs from vertebrates contain a conserved N-terminal domain Figure . In vitrcription . Furthercription ,40. ContRNAP-III . HoweverRNAP-III . The mecRNAP-III ).Changes in TBP levels affect the transcription of genes involved in the cell cycle. TBP expression is often elevated in Ras oncogene-transformed cells. Concordantly, stable over-expression of TBP leads to transformation of cells ,45. InveXenopus mid-blastula transition, at the onset of zygotic transcription. Likewise, TBP protein only accumulates in 2-cell mouse zygote, at the onset of mouse zygotic gene activation [TBP mRNA is abundant in oocytes, but the encoded protein is not. The TBP messenger is translationally masked in oocytes and is only translated during early cleavage stages . TBP protivation ,51  requires the presence of TBP [Xenopus embryos [Not all transcription in early embryos  analysis, which shows that TRF1 is recruited to a majority of RNAP-III and some RNAP-II promoters in the fly genome [Drosophila cells, TFIIIB activity  is accommodated by Brf1 and TRF1, which is in clear contrast to the situation in yeast and human cells where Brf1 is in a complex with TBP [TBP-related factor  was the first TBP paralog to be discovered in animals . TRF1 was Figure . It is hin vitro , whereasromoters . Most oftor Brf1  and TRF1y genome . Thus, iwith TBP .neurofibromatosis type 1 gene [TBP-like factor  is a more distant paralog of TBP that is found in all metazoans Figure . It has e 1 gene . On a sie 1 gene .Drosophila embryos TLF has been reported to be a part of a large protein complex containing components of nucleosome remodeling factor (NURF) complex and the fly-specific DNA replication element binding factor (DREF) [Drosophila genome [Drosophila, however no differences in their expression pattern were observed indicating that they perform similar functions [Drosophila salivary glands causes severe disruption of chromatin structure in polytene chromosomes, again suggestive of a link between TLF expression and proper chromatin function [Drosophila TLF controls the transcription from one of the histone H1 genes, whereas the other histone genes in the cluster are TBP-dependent [Drosophila S2 cells revealed that TLF is bound to more than a thousand sites [Drosophila are controlled independently of TBP. Interestingly, TLF binding sites are enriched for DREs and are mostly lacking TATA boxes [The function of TLF has been studied in worm, frog and fish using knockdown and dominant negative strategies ,55,70,76r (DREF) . TLF proa genome ,81. Two unctions . Partialunctions ,82. Furtfunction . In Drosependent . A genomnd sites . About 8TA boxes .Taken together, TLF most likely provides an alternative mechanism of transcription initiation. The details of this alternative mechanism of initiation are not yet known but may involve chromatin remodeling. A requirement for TLF in germ cells is conserved from fly to mouse, whereas TLF is also essential for early embryogenesis in all non-mammalian metazoans examined  but not in mouse.in vitro [Xenopus oocytes [mespa gene, which encodes a transcription factor that initiates a pathway required for the commitment of ventral mesoderm to the hematopoietic lineage [-/- Tbp2mice sterile [TBP2  is the most recently identified member of TBP family. It has only been found in vertebrate genomes Figure , 2 84,8,884,85. in vitro ,84-88. T oocytes . TBP2 pr oocytes   when mouse myoblasts differentiate into myotubes in culture . Howeverin vivo  and TBP2ould not , highligThere is increasing evidence that exquisitely specialized mechanisms of gene expression operate in germ cells. These germ cell-specific programs of transcription are facilitated not only by specialized isoforms of sequence-specific transcription factors but also by variation in the core transcription machinery. As reviewed in previous sections female germ cells lack detectable levels of TBP protein with abundant TBP2, which is essential for their development. Parallel to TBP2 in oocytes, TLF is essential for sperm development. TBP2 knockout male mice and TLF-lacking female mice are fertile showing strict division of labor between the two factors in mammals. In lower vertebrates, on the contrary, both TLF and TBP2 are also important for proper embryogenesis as detailed above.Drosophila [This germ cell-restricted expression pattern of TBP-related factors is also accompanied by variations in the composition and expression of some other key components of core transcription machinery Figure . For exaosophila ,102.Presence of highly specialized versions of core transcription machinery components in germ cells raises this fundamental question why these cells need a different core transcription apparatus. One possibility is that meiosis requires a specialized environment for transcription. Meiotic cell division is unique to germ cells. The expression of both TBP2 and TLF is highest and that of TBP is the lowest during the meiotic phases of germ cell development. For instance TLF protein is detectable during early pachytene stages of spermatocytes where it is principally cytoplasmic . Later iTBP-related factors may support chromatin remodeling by controlling the expression of key chromatin regulators at important time points during the gametogenesis. A lack of TLF results in defective chromocentre formation , whereasThe transcription initiation machinery in not universal but variable in composition in higher eukaryotes, which is mainly on account of duplicated paralog subunits of the canonical TFIID and TFIIA complexes. The TBP family presents a prime and well-characterized example of how non-universal mechanisms of transcription initiation contribute to cell-specific developmental gene expression programs. This diversification most likely serves to expand the gene regulatory potential required for embryonic development in metazoans (TLF), and vertebrates in particular (TBP2). Initiation factor switching occurs on account of differential expression of these proteins in gametes (TBP2 and TLF), embryos  and somatic cells (transcription dominated by TBP). Mice however have lost the genetic requirement for TLF and TBP2 during embryogenesis. An important goal of future research is to decipher the molecular mechanisms underlying the specificity of factor-promoter interaction, both at the level of core promoter recognition and at the level of protein-protein interactions. TBP is known to work in several multi-subunit complexes. For other members of the family this area has not been fully explored. Germ cell and embryonic gene regulation involve gene regulatory networks that integrate intercellular signaling and epigenetic regulation to reprogram chromatin state and produce activators and repressors required for pluripotency and induction of lineage commitment. The transcription initiation machinery is adapted to mediate the versatility required for the transcription program of germ cell and embryonic development. Elucidation of the molecular mechanisms underlying the diverse biological roles of the TBP family factors, using classical biochemistry, molecular biology and state-of-the-art genomics approaches, will provide deeper insight in the role of transcriptional regulation in pluripotency and differentiation in higher eukaryotes.ALF: TFIIA-like factor; DPE: downstream promoter element; DRE: DNA replication element; DREF: DRE-binding factor; Inr: initiator element; NURF: nucleosome remodeling factor; PCNA: proliferating cell nuclear antigen; PIC: pre-initiation complex; SL1: selectivity factor 1; SNAPc: snRNA activating protein complex; TAF: TBP-associated factor; TBP: TATA box binding protein; TFIIA: transcription factor II A (RNAP-II initiation factor); TFIIB: transcription factor II B (RNAP-II initiation factor); TFIIIB: transcription factor III B (RNAP-III initiation factor); TFIID: transcription factor II D (RNAP-II initiation factor); TLF: TBP-like factor; MBT: mid-blastula transition; TRF: TBP-related factor; RNAP: RNA polymerase.The authors declare that they have no competing interests.WA wrote the manuscript and made figures. GJCV contributed to the writing and made figures. Both authors read and approved the final manuscript.


Gnrh1 gene transcription.Precise coordination of the hypothalamic-pituitary-gonadal axis orchestrates the normal reproductive function. As a central regulator, the appropriate synthesis and secretion of gonadotropin-releasing hormone I (GnRH-I) from the hypothalamus is essential for the coordination. Recently, emerging evidence indicates that histone deacetylases (HDACs) play an important role in maintaining normal reproductive function. In this study, we identify the potential effects of HDACs on Gnrh1 gene in the mouse immortalized mature GnRH neuronal cells GT1–7. The suppression was connected with a specific region of Gnrh1 gene promoter, which contains two consensus Otx2 binding sites. Otx2 has been known to activate the basal and also enhancer-driven transcription of Gnrh1 gene. The transcriptional activity of Otx2 is negatively modulated by Grg4, a member of the Groucho-related-gene (Grg) family. In the present study, the expression of Otx2 was downregulated by TSA and VPA in GT1–7 cells, accompanied with the opposite changes of Grg4 expression. Chromatin immunoprecipitation and electrophoretic mobility shift assays demonstrated that the DNA-binding activity of Otx2 to Gnrh1 gene was suppressed by TSA and VPA. Overexpression of Otx2 partly abolished the TSA- and VPA-induced downregulation of Gnrh1 gene expression.Inhibition of HDACs activities by trichostatin A (TSA) and valproic acid (VPA) promptly and dramatically repressed transcription of Gnrh1 gene expression via repressing Otx2-driven transcriptional activity. This study should provide an insight for our understanding on the effects of HDACs in the reproductive system and suggests that HDACs could be potential novel targets for the therapy of GnRH-related diseases.Our data indicate that HDAC inhibitors downregulate  Gnrh1 gene expression will improve our understanding on abnormal gonadotropin secretion in various GnRH-related disorders and provide new strategies for treatment of these diseases.Normal reproductive function requires the precise orchestration and integration of sex steroids secretion to effectively coordinate the hypothalamic-pituitary-gonadal axis Lhβ and Fshβ gene repression in immature gonadotropes Histone deacetylases (HDACs) have gained an increasing attention for their crucial roles in numerous physiological and pathological processes via dynamically regulating gene expression. According to the profiles of global gene expression, a range of 2–20% of genes in the genome is affected by HDAC inhibitors (HDACIs) Hdac1–10 genes were co-expressed in the mouse immortalized GnRH neuronal cells GT1–7, which are representative of mature postmigratory GnRH neurons. Two HDAC inhibitors (HDACIs), trichostatin A (TSA) and valproic acid (VPA), induced marked inhibition of Gnrh1 gene transcription, and the suppression was connected with a specific region of Gnrh1 gene promoter, which contains two consensus Otx2 binding sites. Otx2, a vertebrate homologue of Drosophila orthodenticle, has been demonstrated to activate basal and also enhancer-driven transcription of Gnrh1 gene in several vertebrate species Gnrh1 transcription. The analysis of transcript and protein levels showed that Otx2 expression was simultaneously inhibited by HDACIs treatments. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) demonstrated that TSA and VPA induced the suppression of the DNA-binding activity of Otx2 to Gnrh1 gene. Overexpression of Otx2 partly abolished the TSA- and VPA-induced downregulation of Gnrh1 gene expression. In addition, Grg4 (Groucho-related-gene 4), a co-repressor regulating Gnrh1 transcription, repressed the Otx2-directed activation of Gnrh1 transcription by direct interaction with Otx2. The increases in the expression of Grg4 and accumulation of Grg4 protein in nuclei followed by HDACIs treatments also supported our conclusion that HDACs regulate the Gnrh1 gene transcription partly via modulating Otx2-driven transcriptional activity.So far, at least 18 HDACs have been identified in mammals, in which they are divided into four classes based on their structure and functions. Both Class I (HDACs 1–3 and 8) and class II (HDACs 4–7 and 9–10) HDACs are Zn-dependent enzymes which usually combine with other proteins to form a large multi-protein complex that increases the stringency of HDACs recruitment to a particular locus in chromatin to deacetylate histones Hdac transcripts were detected in GT1–7 cells by real-time PCR analysis, showing that Hdac1–10 genes were co-expressed with Hdac1 and Hdac2 being the predominant transcripts , an RNA polymerase inhibitor, was used to investigate whether HDACIs influence  present . PretreaGnrh1 gene contains four exons and three introns, and simultaneously encodes the GnRH-I decapeptide and the initial amino acids of GnRH-associated peptide (GAP) Gnrh1 gene is located between −3446 bp to +28 bp Gnrh1 gene in the pGL3 reporter system to identify the corresponsive region  has been demonstrated to directly interact with Otx2 to repress Otx2-directed activation of f HDACIs . Twenty-n levels , especian levels  were obvp16Bcl-2VegfWt1Gnrh1 belongs to the class of genes that are downregulated by the TSA and VPA. TSA, a pan-HDAC inhibitor, inhibits both class I and II HDACs. VPA, a potent anti-convulsant and a short-chain fatty acid, acts as a selective class I HDAC inhibitor that especially inhibits HDACs 1, 2, 3 and 8 in a millimolar range without influencing the activities of HDACs 6, 7 and 9 Gnrh1 expression. Based on the rapid downregulation of Gnrh1 transcripts within 2 hours, we could presume that the suppression of Gnrh1 expression is an early event following HDACIs treatments.Conceptually, hyperacetylation of lysines in the core histones would enhance gene expression via opening the chromatin structure and easily recruiting transcription-associated proteins to the DNA template. Thus, it is counterintuitive that HDACIs would repress a gene expression rather than activate it. However, a small number of genes, including Gnrh1 expression: directly downregulating the Gnrh1 transcription, or influencing mRNA stability to promote mRNA degradation. To rule out the second possibility, the RNA polymerase inhibitor AcD is used to inhibit gene transcription. In our study, the results show that the half-life of Gnrh1 mRNA may be about 3 hours. TSA and VPA do not induce Gnrh1 mRNA degradation and Gnrh1 mRNA downregulation required ongoing RNA synthesis.There may be two possibilities for the HDACIs-induced repression of Gnrh1 have well been characterized Gnrh1 expression Gnrh1 transcription in GT1–7 cells is associated with a specific region (−1134∼+23 bp) in Gnrh1 gene promoter and the region between −356 bp and +23 bp mediates a half of suppression.The elements in the promoter driving tissue-specific expression of mouse Gnrh1 transcription is associated with the chromatin status of the regulatory region in Gnrh1 promoter. Larder and colleague believed that compared to the non-neuronal NIH3T3 cells and immature GnRH neuronal cells (GN11), the acetylation of histone H3 in Gnrh1 promoter in GT1–7 is responsible for the activation of Gnrh1 transcription via remodeling the chromatin and recruiting transcription factors Gnrh1 promoter is highly acetylated in the presence of TSA or VPA. Thus, it is counterintuitive that the expression of Gnrh1 is inhibited by HDACIs via the direct effects of HDACs on Gnrh1 promoter. In the previous studies, little progress has been achieved in clarifying the exact molecular mechanisms of HDACIs-induced attenuation of gene transcription. Some studies show that increased acetylation of histones in regulatory region would lead nucleosomes move and remodel in specific loci to mask critical cis-acting sites and repress gene expression Gnrh1 promoter in the presence of HDACIs, the mechanism mentioned above may be more crucial for the repression.It has been demonstrated that the Gnrh1 gene transcription Otx2 transcripts in explanted cultures of newborn murine retinae are dramatically repressed by TSA or VPA Otx2 transcripts in GT1–7 cells are suppressed by TSA or VPA in dose- and time-dependent manners within 2 hours, which is consistent with the changes of Gnrh1 expression. Meanwhile, western blot analysis and immunofluorescence staining assay demonstrate that TSA and VPA induce obvious decreases in the intracellular protein level and the nuclear translocation of Otx2. The results from ChIP and EMSA assays show that the affinity of Otx2 to Gnrh1 gene promoter is significantly inhibited by HDACIs, which may be due to the less Otx2 expression and less Otx2 protein accumulated in nuclei. The HDACIs-induced the decrease in Gnrh1 mRNA was partly reversed via overexpression of Otx2. The exact mechanisms of HDACIs-induced downregulation of Otx2 expression have remained unclear and need to be further delineated. Based on the above results, we reasonably conclude that Otx2-driven transcription activation of Gnrh1 gene is partly modulated by HDACs.There are two conserved binding sites for Otx2 in the certain region identified by reporter gene assay Gnrh1 transcription in GT1–7 cells via physically interacting with Otx2 rather than recruiting HDACs Grg4 expression is regulated by HDACs, showing that the expression of Grg4 is upregulated by TSA and VPA in dose- and time-dependent manners. Inconsistent with rapid attenuation of Gnrh1 and Otx2 transcription within 2 hours after HDACIs treatments, the Grg4 transcripts is slowly elevated, indicating the limited effect of Grg4 on the early inhibition of Gnrh1 transcription induced by HDACIs. The accumulation of Grg4 protein in nuclei induced by HDACIs may repression the Otx2-induced transcriptional activation, and the results may explain the phenomenon of a part reverse in Gnrh1 mRNA mediated by exogenous Otx2 with HDACIs treatments. All the results suggest that a potential modulation effect of HDACs on Otx2-induced activation of Gnrh1 transcription may be via Grg4-dependent repression. In addition, in the present study, EMSA show that the intensities of two top bands are obviously increased following HDACIs treatments, suggesting that the suppression of Gnrh1 expression might be mediated by proteins or complexes which bind to the regions of Gnrh1 promoter surrounding the confirmed Otx2 consensus sites. Therefore, further studies should be conducted to identify the potential trans-acting factors which are responsible for the HDACs-induced suppression of Gnrh1 expression or co-repressor which inhibit Otx2 function. Grg4 may be a candidate of these proteins.Moreover, emerging evidence has shown that interactions between Otx2 and cofactors regulate Otx2 transcriptional activity Gnrh1 gene transcription, (2) the HDACIs-induced repression is related to a specific region of the Gnrh1 gene promoter (−1134∼+23 bp), which contains two consensus Otx2 binding sites, (3) the Otx2-directed activation of Gnrh1 gene transcription is modulated by HDACs. This study has complemented the potential roles of HDACs in the regulation of the reproductive system and suggests a potential avenue for the anti-GnRH therapies in the future.In summary, to our knowledge, the present study provides evidence for the first time that (1) TSA and VPA repress Trichostatin A (TSA), valproic acid (VPA), and actinomycin D (AcD) were purchased from Sigma Chemical Co. TSA, actinomycin D, cycloheximide were diluted in dimethyl sulfoxide (DMSO), and VPA was diluted with phosphate buffered saline (PBS).The mouse immortalized GnRH neuronal cell line GT1–7 GAPDH in each sample was used as an internal control. Relative quantities were analyzed by the 2−ΔΔCt method. In most case, the samples with vehicle treatment were used as controls (set as 1.0). All reactions were carried out in duplicate.Total RNA was isolated and reverse transcribed to cDNA by M-MLV reverse transcriptase (Invitrogen). The real-time quantitative PCR reactions were carried out with SYBR® Premix Ex Taq™ II real-time PCR kit . The primers used are listed in The GT1–7 cells were incubated with serum-free DMEM for 24 h to synchronize the cell cycles. Subsequently, twenty four hours after co-incubation, supernatants (medium) and cells were harvested. The cells were lysed in 100 µl PBS containing 0.1% Triton X-100 at 0°C Twenty-four hours after HDACIs treatments, the cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 1% Triton X-100 for 30 min, and then blocked with 3% BSA and 1% Triton for 15 min. Following incubation with anti-GnRH-I  or anti-Otx2  for 2 h, the cells were washed twice with PBS and incubated with Alexa Fluor 488-conjugated antibody  for 1.5 h. The nuclei were stained by 4′, 6-diamidino-2-phenylindole  for 10 min. Images were collected by a digital camera  attached to a microscope.myc-His C vector (Invitrogen) to form the plasmid Otx2-myc-His/pcDNA3.1. 3446mGnRH-LUC and 2078mGnRH-LUC plasmids were kindly provided by Dr. Kim . The segments of the Gnrh1 promoter  were digested from 3446mGnRH-LUC and subcloned into the pGL3-Basic vector to form the plasmids pGL3-P3446, pGL3-P2087, pGL3-P1848, pGL3-P1134. The fragments of the Gnrh1 promoter , intron 1, intron 2 and intron 3 were amplified by PCR and subcloned into the pGL3-Basic vector or pGL3-Promoter vector to form the plasmids pGL3-P520, pGL3-P356, pGL3-P249 and pGL3-Int1–3. The details of the primers used in construction of plasmids were listed in The Otx2 cDNA fragment was obtained by RT-PCR and subcloned into the pcDNA™ 3.1/4 cells/well) overnight. The cells in each well were transfected with 4 µg Otx2-myc-His/pcDNA3.1 or the corresponding empty vector as a control. In reporter gene assay, the GT1–7 cells were plated on a 96-well plate (1.5×104 cells/well) overnight. The cells in each well were transfected with 0.4 µg of reporter plasmid or the corresponding empty vector (pGL3-Basic or pGL3-Promoter) as a control, along with 20 ng of pRL-SV40 as an internal control reporter. Twenty-four hours after transfection, HDACIs was added. The gene expression and the activities of firefly and renilla luciferases  were detected 24 h after co-incubation. All experiments were performed in triplicate and repeated a minimum of three times.The plasmids were transfected into cells using Lipofectamine 2000 (Invitrogen). In Otx2 overexpression experiment, the GT1–7 cells were plated on a 6-well plate  and sonicated  to shear DNA to lengths between 200 and 1000 bp at ice box. Ten microliters of anti-Otx2 (Abcam) and anti-Acetyl-H3  was used to immunoprecipitate the interacting DNA fragments. The no-antibody immunoprecipitation was performed as the negative control. Finally, the DNA sample was used in a PCR reaction. The primers were listed in ChIP analysis was performed with Chromatin Immunoprecipitation (ChIP) Assay Kit (Millipore) according to manufacturer's instructions. Briefly, a total of 3×106 GT1–7 cells in a 10-cm dish were treated with HDACIs for 24 h and then harvested. The nuclear protein was extracted by NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo). Biotin 5′ end-labeled and unlabeled probes containing the regions of Gnrh1 promoter surrounding the confirmed Otx2 consensus sites (−268 to −239 bp and −330 to −301 bp) A total of 3×10t-test mostly comparing all values vs. control group. P<0.05 was considered to represent a significant difference.All data were represented as mean ± SD with at least three independent experiments. The statistical significances among groups were analyzed by One-way ANOVA or Table S1Details of the primers used in the RT-PCR assay.(DOC)Click here for additional data file.Table S2Details of the primers used in construction of plasmids.(DOC)Click here for additional data file.Table S3Details of the primers used in ChIP assay.(DOC)Click here for additional data file.


Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system.A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. β-galactosidase production was used to examine translation from mRNA containing the bzpR 5′ UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no β-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5′ UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated.Cells disrupted in the Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5′ UTR.The findings indicate that BzpR regulates aspects of the development program in  One mechanism of translational control of gene expression utilizes the translational initiation factor eIF2. This multi-subunit protein, in what is referred to as the ternary complex, delivers initiator tRNAs to scanning ribosomes assembling on mRNAs. Phosphorylation of the subunit eIF2α at serine 51 reduces the ability to deliver initiator tRNAs and thus the efficiency of translation Many of the mechanistic insights of the eIF2 regulatory system and eIF2α specific kinases came from studies of the yeast GCN2 protein which, when activated by stresses such as general nutrient limitation and/or amino acid starvation, subsequently activates GCN4 translation Proteins that are encoded by mRNAs whose translation is upregulated upon eIF2α phosphorylation often are transcription factors within the basic leucine zipper (bZIP) family Dictyostelium cells have three GCN2 homologs that are termed initiation factor kinases: IfkA, IfkB, and IfkC. Three other uncharacterized genes have kinase domains similar to eIF2α kinases but lack obvious regulatory domains. All three of the ifk genes are expressed during growth and throughout development, with a high degree of spatially restricted expression for each gene during development ifk genes indicate that IfkA functions in modulating the timing of aggregation and early gene expression, that IfkB functions in maintaining proper prestalk specific gene expression, and that IfkA and IfkB function in maintaining proper cell-cell and cell-substrate adhesion and the equilibrium between different cell types for proper spatial patterning Dictyostelium cell proliferation via direct regulation of two chalone proteins Dictyostelium possesses 19 potential bZIP family members bzpR 5′ UTR.In the latter study, a strain was used (BS167) that possesses an eIF2α that cannot be phosphorylated due to an S51A mutation. This strain shows several developmental defects, including smaller size of the developing entities, various stalk defects, and production of non-viable spores. We wondered if one or more of these defects were the result of the lack of production of a bZIP transcription factor, given the inability to phosphorylate eIF2α in BS167. bzpR gene (covering the 5′ UTR and the first few codons) and a 900 bp 3′ region (corresponding to codons 370 to 670) that had been cloned into the pGEM T-easy vector (Promega). The 5′ fragment was generated by PCR using primers bzr-3 and bzr-10, while the 3′ fragment was made with bzr-7 and bzr-8  was used for the experiments in this paper.A disruption construct, pbzpR-5, was made as follows. The blasticidin resistance gene cassette from pBSR519 nd bzr-8 . DigestibzpR no-UTR control sequence was generated by annealing the bzr-16 and bzr-17 oligonucleotides  and the bzpR no-UTR (translation) (A leotides  and clones (bzpR . The expslation) .bzpR 5′ UTR and first 9 amino acid codons (bzpR 5′ UTR (translation) while the RFP gene is controlled by the actin 15 promoter and the bzpR no-UTR  and thus become fused to a rapid turnover version of β-galactosidase. The resulting plasmid was named pbzpR-9.PCR using bzr-12 and bzr-13  and genoKlebsiella pneumoniae on SM plates, harvested, and excess bacteria removed by differential centrifugation prior to plating cells on nitrocellulose filters for standard development Strains were maintained and grown in HL-5 medium RNA was isolated using Trizol (Invitrogen). RT-PCR was carried out as described http://www.dictybase.org/techniques/media/lowflo_medium.html) under appropriate selection to minimize cell auto-fluorescence. To minimize media fluorescence, the cells were transferred to fresh Loflo+ medium the night before the experiment and transferred to Loflo medium, which lacks yeast extract, six hours prior to the experiment. The cells were pelleted and suspended in fresh Loflo at a titer of 2×106 immediately prior to flow cytometry.Strains transformed with the 5′ UTR GFP/RFP fusion plasmids and strains with GFP and RFP expressed individually were maintained in HL-5 plus 10 µg/ml G418 or 50 µg/ml hygromycin. Prior to their use, the strains and the parental wild type Ax4 strain were grown for 72 hours in Loflo+ medium (www.cytobank.org) using the control data for compensation and gating.Flow cytometry was conducted on a 5-laser BD LSR Fortessa Special Order Research Product running BD FACSDiva Software Version 6.1.3. Prior to collecting data, the cytometer was calibrated using the non-fluorescing Ax4 strain and the strains individually transformed with either pRFP or pGFP as controls. Five thousand events were collected for the controls and approximately 12,000 events were collected for each experimental strain. The resulting FCS files were analyzed with Cytobank (2Cl). After two washes in PDF, spores were titered and suspended in 10 mM EDTA, pH 7.5, 0.1% NP40. The samples were incubated at 42°C for 45 minutes followed by two washes with PDF. Spores were suspended with Klebsiella pneumoniae and were plated at 100 spores per plate onto SM plates Spore filled sori from Ax4 and BS168 cells growing on bacteria were picked and suspended in PDF . While several of the genes possess uORFs, only a few had distributions somewhat analogous to that of GCN4 of yeast, the prototypical gene regulated by eIF2α phosphorylation Dictyostelium gene, bzpR, whose 5′ upstream sequence is shown in bzpR. Typically for Dictyostelium genes, transcription start sites are located much closer to the ATG of the coding region. Thus, we determined if these uORFs were present within the bzpR mRNA in what would be a lengthy 5′ UTR.The upstream regions of the 19 bzpR mRNA has a lengthy 5′ UTR covering all of the 7 uORFs, with the transcriptional start site most likely located 30 to 50 nucleotides upstream of uORF 1.Oligonucleotides corresponding to various positions within the upstream sequence, paired with a 3′ primer just within the coding region, were used in RT-PCR reactions on RNA and PCR reactions on genomic DNA. The results using three such primers are shown in bzpR mRNA presence during growth and development. Expression of bzpR mRNA was found during growth and throughout the developmental program, with some increase in levels from 8 to 12 hours evident in some experiments. Recent RNA-seq data give a similar pattern but show a more pronounced increase around 8 hours (http://dictyexpress.biolab.si/).bzpR gene was carried out, and the null cells (BS168) were developed to compare their phenotype to that of wild type cells (Ax4). The developing entities were small  cells, transformants were plated for development, and the lacZ mRNA and protein produced in each strain was compared. The same pattern and importantly the same levels of lacZ mRNA were produced in both the Ax4 and BS167 strains  and the start codon of  strains .lacZ mRNA levels were the same as those in Ax4 cells. If the staining process was carried out at 37°C and for extended times, transformed populations of BS167 did show faint scattered speckling during culmination.In Ax4 cells the production of the LacZ protein varied temporally and spatially, but in striking contrast little to no protein was observed in BS167 cells . For thebzpR 5′ UTR was substantiated by finding minimal staining and no upper cup expression in strains that lacked either IfkA (BS153) or IfkB (BS160) , another eIF2α kinase  . IfkA anα kinase .bzpR uORFs mediate the translation suppression demonstrated above for the bzpR 5′ UTR fused to the lacZ gene, either the bzpR upstream sequences containing the 7 uORFs plus the first nine codons (5′ UTR) or a sequence corresponding only to the ribosome binding site and the first nine codons (no-UTR) was fused to a GFP gene that was transcriptionally controlled by the actin 15 promoter. As an internal control within both of the resulting plasmids, the ribosome binding site and the first nine codons (no-UTR) was fused to an RFP gene under the control of another copy of the actin 15 promoter bzpR 5′ UTR and a no-UTR RFP . In each case, similar suppression was seen to that of the unmutated 5′ UTR. A number of pair wise combinations of start site mutations were also examined and similarly found not to relieve the suppression. We thus made a more drastic alteration in the 5′ UTR by deleting the internal 5 uORFs and fusing the beginning of the first and end of the seventh uORFs to give a 5′ UTR with a single uORF  within the prespore region bzpR null cells, as upper cup cells have been shown to function in lifting the spore mass up the stalk Fusion of the bzpR 5′ UTR was seen in the BS167 strain, i.e., in cells that possess an eIF2α that cannot be phosphorylated. Lack of protein production was found even though levels of lacZ mRNA containing the bzpR 5′ UTR were comparable in BS167 and wild type cells, and the levels were unchanging during growth and development in both strains. These results indicate that protein production from mRNA containing the bzpR 5′ UTR was dependent on eIF2α phosphorylation. This dependency was confirmed by the finding that little to no β-galactosidase was produced in strains that lacked either of the eIF2α kinases IfkA or IfkB. Significantly, IfkA and IfkB are expressed in ALCs in fingers and slugs, and in culminants, IfkA is expressed only in upper cup cells while IfkB is expressed in both upper and lower cup cells bzpR 5′ UTR.Little to no production of β-galactosidase protein from mRNA containing the bzpR 5′ UTR and first nine codons to the GFP gene resulted in severe suppression (12 fold) of translation of the GFP protein when compared to a GFP gene fused only to the ribosome binding site and first 9 codons (no-UTR). The bzpR 5′ UTR is complicated by having 7 uORFs. The prototypical UTR regulated by eIF2α phosphorylation is the GCN4 gene, which has four uORFs Fusing the bzpR 5′ UTR. Deleting uORFs 2 through 6 and fusing the first and last uORF to give a 5′ UTR with a single uORF resulted in even greater suppression of translation than the intact 5′ UTR, with little to no GFP being detected. However, when the start site of this single uORF was destroyed, translation was increased by more than 90 fold. Comparison of typical Dictyostelium start codon contexts bzpR uORFs indicates that uORF 2 and 3 possess a somewhat rare context while the sequence contexts of 6 and 7 are quite poor. Thus, it is unclear if all seven of the uORFs are translated by ribosomes.A mutation destroying any one of the uORF start codons and pair wise combinations of these mutations did not relieve the repression of translation by the bzpR 5′ UTR has 7 uORFs, a single uORF is clearly sufficient for repression. Recent work with CHOP mRNA also demonstrated translational repression by eIF2α phosphorylation through a single uORF bzpR 5′ UTR suggest that the various uORFs contribute in subtle ways to a variable repression by the bzp 5′ UTR. It may be that the 7 uORFs allow variation in the levels of de-repression in the presence of varying amounts of phosphorylated eIF2α. A more systematic mutational analysis will be required to tease out the mechanistic subtleties of the 7 uORFs.While the


Schizosaccharomyces pombe, the transcriptional-regulatory network that governs flocculation remains poorly understood. Here, we systematically screened an array of transcription factor deletion and overexpression strains for flocculation and performed microarray expression profiling and ChIP–chip analysis to identify the flocculin target genes. We identified five transcription factors that displayed novel roles in the activation or inhibition of flocculation , in addition to the previously-known Mbx2, Cbf11, and Cbf12 regulators. Overexpression of +mbx2 and deletion of +rfl1 resulted in strong flocculation and transcriptional upregulation of gsf2+/+pfl1 and several other putative flocculin genes (+–pfl9+pfl2). Overexpression of the +pfl genes singly was sufficient to trigger flocculation, and enhanced flocculation was observed in several combinations of double +pfl overexpression. Among the +pfl1 genes, only loss of +gsf2 abrogated the flocculent phenotype of all the transcription factor mutants and prevented flocculation when cells were grown in inducing medium containing glycerol and ethanol as the carbon source, thereby indicating that Gsf2 is the dominant flocculin. In contrast, the mild flocculation of +adn2 or +adn3 overexpression was likely mediated by the transcriptional activation of cell wall–remodeling genes including +gas2, +psu1, and SPAC4H3.03c. We also discovered that Mbx2 and Cbf12 displayed transcriptional autoregulation, and Rfl1 repressed +gsf2 expression in an inhibitory feed-forward loop involving +mbx2. These results reveal that flocculation in S. pombe is regulated by a complex network of multiple transcription factors and target genes encoding flocculins and cell wall–remodeling enzymes. Moreover, comparisons between the flocculation transcriptional-regulatory networks of Saccharomyces cerevisiae and S. pombe indicate substantial rewiring of transcription factors and cis-regulatory sequences.In the fission yeast  S. pombe, many of the flocculins and transcription factors that function in flocculation are not known. To address this gap in knowledge, we have employed molecular genetics and functional genomic approaches to uncover transcription factors and their target genes that play a role in flocculation. We discover that flocculation in S. pombe is regulated by a complex network of transcription factors that activate and repress themselves, as well as multiple target genes that encode for flocculins and cell wall–remodeling enzymes. The comparison of the flocculation regulatory networks between fission and budding yeasts indicates that they mainly differ in the types of transcription factors and their binding sequences.Flocculation is a process that involves yeast cells adhering to one another to form clumps called flocs. This trait is important for industrial yeast applications as it provides a cost-effective and efficient method to remove yeast cells. The adherence between cells occurs by the binding of glycoproteins known as flocculins and carbohydrate molecules located on the cell surface. To better understand how flocculation works, the genes that encode for flocculins and the transcription factors that regulate their expression need to be identified. In the fission yeast  Flocculation is an inherent characteristic of yeasts involving asexual aggregation of cells into flocs that separate rapidly from the medium  in addition to our independent finding of Mbx2, Cbf11, and Cbf12. The strongest flocculation was observed upon overexpression of +mbx2 and deletion of +rfl1 (SPBC15D4.02) which encodes an uncharacterized fungal Zn(2)-Cys(6) transcription factor. Microarray expression profiling of the mbx2OE and rfl1Δ strains revealed good overlap in the upregulation of several flocculin genes, while ChIP-chip analysis of HA-tagged Mbx2 and Rfl1 under control of the nmt41 promoter indicated that these transcription factors bound to some of the flocculin gene promoters. Nine flocculin gene targets (pfl1+–pfl9+) including gsf2+/+pfl1 were identified. The single overexpression of these genes triggered flocculation to varying degrees and cumulative effects on flocculation were observed in double overexpression experiments. Only loss of +gsf2 could abrogate the flocculent phenotype of all the transcription factor mutants indicating that gsf2+ encodes the dominant flocculin in S. pombe. Interestingly, we discovered that certain cell wall-remodeling enzymes can also function in flocculation, and some of these genes are likely regulated by the LisH transcription factors Adn2 and Adn3. In addition to the identification of target genes within the transcriptional-regulatory network, autoregulatory and inhibitory feed-forward loops involving several transcription factors were also detected. These results provide a significant insight into the transcriptional control of flocculation in S. pombe.In this study, we have initiated an extensive characterization of the transcriptional-regulatory network of S. pombe remains limited. To further decipher this network, we sought to systematically identify transcription factors that play a role in flocculation. A list of 101 genes encoding sequence–specific transcription factors containing a bona-fide DNA-binding domain was assembled from nmt1-driven overexpression strains and 92 nonessential deletions in which the entire ORF was replaced with the KanMX6/NatMX6 cassette. A detailed description of the construction and phenotypic characterization of this transcription factor mutant collection will be described elsewhere (unpublished data). The transcription factor array of overexpression and deletion strains were screened for flocculation in EMM lacking thiamine and YES media, respectively. We recovered a total of eight transcription factors in which four overexpression strains  and four deletions  exhibited flocculation. These transcription factors represent positive and negative regulators of flocculation, respectively. Among these transcription factors, only the overexpression of +cbf12 and +mbx2 and deletion of +cbf11 have been reported to cause flocculation Our understanding of the transcriptional-regulatory network that governs flocculation in mbx2OE and rfl1Δ strains. The flocs of the rfl1Δ strain in YES medium were larger and sedimented faster than the flocs produced in the mbx2OE strain after 48 hour induction  gene encodes an uncharacterized fungal Zn(2)-Cys(6) transcription factor.The strongest flocculation was observed in the nduction . The mbxmbx2OE strain could be deflocculated with 2% galactose while rfl1Δ strain required 5–10 times more galactose to completely deflocculate. Reflocculation of these strains was achieved in CaCl2 or in YES medium (data not shown).The flocculation exhibited by these overexpression and deletion transcription factor mutants recovered from our screens could be abolished with the addition of galactose, but not mannose or glucose (data not shown). The amount of galactose required to completely deflocculate cells depended on the degree of flocculation. For example, S. pombe are not well known. To identify the inducing conditions, −972 h and +975 h cells were tested on different carbon sources at different cell densities for flocculation. We determined that heterothallic wild-type cells were able to flocculate when cultured for five days at an initial concentration of 1×106 cells/ml in medium containing 1% yeast extract, 3% glycerol and, 4% ethanol . Similarly, +adn2 and +adn3 which were previously observed to have defects in invasive growth when deleted were recovered in our screens as flocculent when overexpressed Fungal genes that function in flocculation are usually associated with filamentous invasive growth ild type . Under tmbx2OE and rfl1Δ strains indicated that these two genes encode the major regulators of flocculation. Therefore, we initially focused on the characterization of these two transcription factors and proceeded to identify their target genes involved in flocculation. The nmt41-driven mbx2-HA strain was subjected to microarray expression profiling with a custom-designed S. pombe 8×15 K Agilent expression microarray . These induced genes were highly enriched in cell wall components with p-values of 9.0e-9 and 6.3e-6 for the mbx2OE and rfl1Δ strains, respectively. Strikingly, the most-induced genes in the mbx2OE strain encoded cell surface glycoproteins. The cell surface glycoprotein genes up-regulated above two-fold were SPAC186.01, +gsf2, SPAC977.07c/SPBC1348.08c, SPCC188.09c, +fta5, SPBC947.04, SPBC359.04c, SPBC1289.15, SPAPB2C8.01, SPAC1F8.02c, SPAPB18E9.04c, SPCC553.10, and SPBPJ4664.02, which all but gsf2+ and the last 4 genes were predicted to be pombe adhesins based on BLAST sequence analysis  encoding cell wall-remodeling enzymes such as glucan glucosidases and a betaglucanosyltransferase were induced up to 91-fold compared to the empty vector control when +mbx2 was overexpressed . Moreover, mbx2OE flocculation was completely abrogated in the gsf2Δ pfl9Δ double mutant indicating that the reduction of mbx2OE flocculation in these mutants were additive in some cases -3′5′-(C/T)TA(T/A)++ closely resembled this known consensus sequence -Cys(6) transcription factor family , +pfl2/SPAPB15E9.01c, +pfl3/SPBC947.04, +pfl4/SPCC188.09c, +pfl5/SPBC1289.15, +pfl6/SPAC977.07c, +pfl7/SPBC359.04c, +pfl8/+fta5 (referred as fta5+ hereafter) and +pfl9/SPAC186.01. Furthermore, we overexpressed some double combinations of the weaker flocculin genes to determine whether flocculation could be additive. Indeed, the + pfl9+pfl4, + pfl9+pfl6, and + pfl9+fta5 double overexpression strains flocculated earlier and formed larger flocs than their corresponding single overexpressors, thus, demonstrating the additive effect of these flocculins (+pfl genes for their ability to flocculate in flocculation-inducing medium. No visible flocculation was observed in the gsf2Δ strain while wild type was flocculent (pfl2Δ–pfl9Δ strains in the inducing medium indicating that +gsf2 encodes the dominant flocculin and the other flocculin genes are dispensable for flocculation (data not shown).Besides  one day . Weaker occulins . We nextocculent . In cont+pfl genes varied and certain combinations of +pfl were additive. The strength of flocculation by the single overexpression of +pfl genes was directly correlated with the reduction of mbx2OE flocculation in the corresponding deletion strains . However, the GFP-tagged strain resulted in a hypermorphic allele that displayed constitutive flocculation and nuclear localization of Mbx2-GFP (see below). We speculated that the removal of the 3′-untranslated region of mbx2+ during the C-terminal tagging may be the cause of the hypermorphic allele. To bypass this potential problem, we created an N-terminal GFP-tagged allele (GFP-mbx2) with an intact 5′-untranslated region and approximately 1 kb of native promoter sequence. In contrast to the C-terminal tagged hypermorphic allele, the N-terminal tagged GFP-Mbx2 expression was comparable to background levels and the strain did not exhibit constitutive flocculation . The positive autoregulation of mbx2+ is likely to be direct as several putative MEF2-binding sequences (e.g. 5′-TTAAAAATAG-3′) are located within 1000 bp upstream from the mbx2+ start codon (data not shown).Interestingly, ChIP-chip analysis also detected binding of Mbx2 and Rfl1 to their own promoters, as well as Rfl1 binding to the promoter , indicathIP-chip . To inveculation . Moreovenctional . When nmpression . As experfl1+, a C-terminal GFP-tagged strain under native control was generated (rfl1-GFP). The localization of Rfl1-GFP was nuclear in the rfl1-GFP strain . In contrast to our observations with the Rfl1-GFP protein expression, we found that there was no decrease of the Rfl1-GFP transcript when +rfl1 was overexpressed . These results support the hypothesis that +mbx2 expression is repressed by Rfl1 in non-flocculent cells.We next utilized the C-terminal and N-terminal GFP-tagged P signal  and flocpression  and floccbf12+ in flocculation, we took a similar approach to identify its direct target genes by concurrent expression microarray profiling and ChIP-chip analysis of the nmt41-driven cbf12-HA strain . To reduce the toxic effects of +cbf12 overexpression, an nmt41-driven cbf12-HA strain was used for concurrent expression profiling and ChIP-chip analysis.When bolished . In contculation  and prodgsf2+ and pfl7+ were among the five highest induced genes  in the cbf12OE strain and were also detected by ChIP-chip . The much weaker flocculation observed in the cbf12OE strain in comparison to the mbx2OE and gsf2OE strains may be attributed to additional defects in cell and nuclear division, which would cause early growth arrest before the full flocculation potential could be reached Gene ontology analysis was carried out separately on the top 50 most highly-induced genes and all 160 promoter-occupied genes by Cbf12 with the Princeton GO Term Finder. Functional enrichment of genes in cell surface (p = 1.8e-7) and plasma membrane (p = 5.7e-4) was detected for the highly-induced and promoter-occupied genes, respectively. These genes included several flocculin genes, . Both gshIP-chip  suggestie effect . This wanmt1-driven cbf12+ was ectopically expressed in logarithmically growing cells  by gel mobility shift assay cbf12OE datasets by RankMotif++ and MEME. RankMotif++ and MEME analyses of the expression microarray and ChIP-chip data, respectively, did not identify a binding specificity for Cbf12. However, when the promoters of up-regulated genes in the cbf12OE strain belonging to the cell surface GO category were subjected to MEME analysis, a motif closely matching the canonical CSL binding motif (6/7 nucleotide match) was recovered  and mitochondrial membrane transporters . These findings did not intuitively answer our questions as to how these two transcription factors might be related or associated with the flocculation pathway. We next examined whether any of the flocculin genes and their putative regulators were induced in the yox1Δ and sre2Δ strains. In the sre2Δ strain, gsf2+, pfl3+ and fta5+ transcripts were upregulated 3.7, 2.5 and 3.1-fold, respectively, indicating that the expression of these genes could be contributing to the flocculent phenotype  and exhibit defects in invasive growth and cell-to-surface adhesion when deleted during nitrogen starvation +adn2 and +adn3 triggered minor flocculation while loss of +adn2 and +adn3 prevented flocculation in flocculation-inducing medium . To identify the target genes of Adn2 and Adn3 that are involved in flocculation, expression microarray profiling was performed on nmt1-driven adn2OE and adn3OE strains  were also highly upregulated in both adn2OE and adn3OE strains  and also appeared within the top 20 most induced genes in the adn3OE strain. These genes were also induced in the mbx2OE strain except for psu1+ , as well as detected by ChIP-chip in the mbx2OE strain. Indeed, the single overexpression of these four genes resulted in flocculation after 5-days (including 3rd day sub-culturing into fresh medium) in medium lacking thiamine, implicating the involvement of these cell wall-remodeling enzymes in flocculation . These results suggest that Adn2 and Adn3 may regulate cell separation and flocculation independently through different sets of target genes. Our microarray expression data suggests that Adn2 and Adn3 may control cell separation through +ace2, which encodes a major transcriptional activator of this process . Overexpression of +adn2 and +adn3 resulted in the down-regulation of +ace2 and many of its known target genes such as adg1+, adg2+, adg3+, cfh4+, agn1+, eng1+, and mid2+ by 1.5 to 3.4-fold  and cell wall-remodeling enzymes . These target genes were sufficient to trigger flocculation when overexpressed. Moreover, instances of regulation between transcription factors (Rfl1 repression of +mbx2), as well as positive (+mbx2 and +cbf12) autoregulation were detected within the flocculation network.In this study, we have deciphered a significant portion of the transcriptional-regulatory network governing flocculation in mbx2OE and rfl1Δ strains compared to the other flocculent mutants in this study. Our initial efforts to identify the target genes of Mbx2 and Rfl1 revealed several putative flocculin genes that were strikingly upregulated in the mbx2OE and rfl1Δ flocculent mutants. Previously, Gsf2 was the only S. pombe flocculin demonstrated to be directly involved in flocculation, and its transcription was influenced by the activity of Mbx2 +gsf2 triggers flocculation while loss of +gsf2 abrogates the flocculent phenotype of several mutants including mbx2OE. Here, we identified an additional eight flocculin genes (pfl2+–+pfl9) as putative target genes of Mbx2. Seven of these target genes (pfl3+–+pfl9) were reported to contain tandem repeats found in fungal adhesins, while pfl2+ is a sequence orphan predicted to encode a GPI-anchored protein +pfl genes  have been reported to be upregulated in loss-of-function flocculent mutants of Cdk8 module genes  suggesting that the transcriptional repression of these putative flocculin genes may be controlled by Mediator +mbx2 since its expression is highly upregulated in the cdk8 kinase-mutant and med12Δ strain . This result indicates that +gsf2 encodes the most dominant flocculin compared to the other +pfl genes. In agreement are the observations that only deletion of +gsf2 and not the other +pfl genes prevented flocculation in flocculation-inducing medium, and reduced the constitutive flocculent phenotype to the greatest extent of all the transcription factor mutants examined in this study. Moreover, the strength of the flocculins was directly correlated with the amount of reduction in mbx2OE flocculation observed in the various pfl deletion backgrounds  within the transcriptional-regulatory network -Cys(6) transcription factor as a novel repressor of flocculation in fission yeast. The repression of flocculation by Rfl1 appears to be primarily mediated by the inhibition of  network . These radh1+, hxk2+, pfk1+, tpi1+, adh4+, pgi1+, gpd3+, tdh1+, pgk1+, fba1+, eno101+, pyr1+, SPCC794.01c (predicted glucose-6-phosphate 1-dehydrogenase), SPBC2G5.05 (predicted transketolase) and SPBC660.16 (phosphogluconate dehydrogenase). Most of these genes with the exception of fba1+, eno101+, SPCC794.01, and SPBC660.16 were upregulated 1.2 to 26-fold in the rfl1Δ strain  including Δ strain . From th+cbf12 or loss of +cbf11 triggers flocculation. However, none of their target genes have been identified. We present supportive evidence that Cbf12 induces flocculation by directly activating the transcription of +gsf2. In addition, +gsf2 expression is up-regulated approximately 2.4-fold in the cbf11Δ strain suggesting that the repressive flocculation function of Cbf11 may also be directly mediated through +gsf2. The activation and repression of +gsf2 transcription by Cbf12 and Cbf11, respectively, may occur by competitive binding to promoter sites since both transcription factors have been shown to interact with a canonical CSL consensus sequence in vitro+gsf2 promoter (data not shown). Further experimentation would be required to verify this proposed mechanism of +gsf2 transcriptional regulation by Cbf11 and Cbf12. It is likely that cbf12+ plays a lesser role in activating flocculation compared to mbx2+ since the floc size resulting from cbf12+ overexpression is considerably smaller than the mbx2OE strain. Also unlike +mbx2, deletion of cbf12+ is not sufficient to abrogate the flocculation of the rfl1Δ strain (data not shown). These data suggest that the flocculent phenotype of the cbf12Δ rfl1Δ double mutant is probably caused by the presence of mbx2+ activity.Previously, the CSL proteins Cbf11 and Cbf12 were shown to exhibit antagonistic roles in flocculation CSL transcription factors are components of the conserved Notch signaling pathways in metazoans which primarily function in cell-to-cell communication during development +yox1 or sre2+ results in a mild flocculent phenotype. The Yox1 homeodomain transcription factor functions as a repressor of MBF (Mlu1 binding factor) target genes to prevent their inappropriate expression at the end of S-phase +yox1 causes a cell cycle delay and results in elevated constitutive expression of MBF gene targets cdc18+, cdc22+, cdc10+, cdt1+, cdt2+, cig2+ and nrm1+) were also found to be upregulated 2.2 to 6.4-fold in our yox1Δ microarray expression data    which mayox1Δ and sre2Δ strains displayed similar differential gene expression despite the supposedly different functions of these transcription factors  were highly induced when +mbx2, +adn2 or +adn3 was overexpressed. In the adn2OE strain, gas2+ and SPAC4H3.03c were the most highly induced genes   encode for glycoside hydrolases while the fourth (gas2+) encodes for a glycosyltransferase. Agn2 is an endo--α-glucanase that hydrolyzes -α-glucans of the ascus wall for ascospore release agn2+ function appears only specific for sporulation, its ectopic expression could alter the cell wall structure during vegetative growth by inappropriate hydrolysis of -α-glucan. Similarly, inappropriate glucan hydrolysis of the cell wall could be occurring as a result of ectopic expression of SPAC4H3.03c which encodes a putative -α-glucanase . Psu1, which exhibits close homology to the members of the SUN family in Sacc. cerevisiae and C. albicans, as well as the BglA beta glucosidase of C. wickerhamii, has an essential function in cell wall synthesis psu1+ activity conferred resistance to -β-glucanase suggesting that Psu1 may influence the amount or structure of -β-glucan in the cell wall gas1Δ cells S. pombe cells? The expression of flocculin genes during vegetative growth is not well characterized in yeasts, but studies in Sacc. cerevisiae indicate that flocculin synthesis and insertion into the cell wall initiate in early exponential phase prior to the onset of flocculation during stationary phase Sacc. cerevisiae support this hypothesis. First, alteration of cell wall structure by disruption of PKC1 activity results in flocculation agn2+, psu1+, SPAC4H3.03c and gas2+ are the only cell wall remodeling enzymes that can trigger flocculation when overexpressed. Other genes with potential functions in cell wall modification and integrity such as gas4+, gma12+, meu7+, agl1+, meu10+ and mde5+ were also detected as putative target genes of Mbx2   while ined genes . Similar of Mbx2 . In cont process . Altogetadn2OE and adn3OE strains. A possible explanation for the different degrees of flocculation between the transcription factor and its target genes could be that overexpression of +adn2 and +adn3 causes reduced fitness due to toxicity effects associated with a greater misregulation of genes compared to the aberrant production of a single enzyme. Consistent with this theory is that adn2OE and adn3OE strains exhibited additional phenotypes including septation defects . Furthermore, a systematic overexpression analysis of 5280 genes in Sacc. cerevisiae revealed that genes encoding for transcription factors, signalling molecules and cell cycle regulators were more likely to cause reduced fitness The single overexpression of the cell wall-remodeling genes triggered flocculation to a greater extent than the  defects  which weS. pombe, cell separation involves the transcriptional activation of adg1+, adg2+, adg3+, agn1+, eng1+, cfh4+ and +mid2 by the Ace2 transcription factor, which is in turn regulated by the Sep1 forkhead transcription factor adn2OE or adn3OE strains displayed multisepta and forkhead phenotypes similar to loss-of-function mutations of these cell separation genes. The cell separation defect in adn2OE and adn3OE strains is likely due to the downregulation of ace2+ transcription since +ace2 and its target genes were substantially downregulated in these strains . Moreover, overexpression of +cbf12 has been reported to produce multisepta phenotypes albeit at a low frequency Interestingly, we also found some evidence that supports a role of Mbx2 and Cbf12 in cell separation perhaps through repression of control.  and S6. Sacc. cerevisiae, the positive and negative transcriptional controls of the dominant flocculin gene FLO1 by Flo8p or Mss11p, and Sfl1p, respectively, draw parallel to +gsf2 regulation by the Mbx2-Rfl1 and Cbf12-Cbf11 opposing transcription factors in S. pombeFLO1, FLO9 and FLO11), while the latter may also regulate genes encoding cell wall enzymes (STA1 and SGA1 which both encode glycoside hydrolases) FLO1 and FLO11Sacc. cerevisiae. These observations would imply that the cis-regulatory elements controlling transcription of the flocculin genes have likely undergone considerable rewiring within the transcriptional-regulatory network between the two yeasts. However, it was recently demonstrated that heterologous expression of Flo8p and Mbx2 could induce +gsf2 and FLO1 transcription in fission and budding yeast, respectively A comparison between the flocculation network of budding and fission yeast revealed both conserved and divergent features within the transcriptional circuitry. In S. pombe and Sacc. cerevisiaeFLO1 and FLO11 transcription through physical interactions with Ssn6p and Srb proteins  FLO8 in Saccharomyces diastaticus. The observation that Sfl1p can repress FLO1 transcription directly and indirectly through FLO8 seems very similar to the inhibitory feed-forward regulation of +gsf2 by Rfl1 in S. pombe. If these connections are truly analogous, then there is a possibility that Rfl1 repression could also be mediated through physical interactions with the Cdk8 module proteins. In the −srb10 mutant, +gsf2 and +mbx2 expression are upregulated suggesting that its flocculent phenotype could be caused by a failure to repress +mbx2 transcription tup11+/tup12+ mutants lkh1Δ flocculation in the absence of mbx2++gsf2 and +mbx2. Future experiments focusing on the interactions between Rfl1, Tup11/12 and Srb8-10 in relation to flocculation would provide clarification to our speculation.Although the transcription factors regulating flocculation appear to be quite different between the two yeasts, the downstream transcriptional events involved in the repression of flocculin genes are likely to be conserved. Disruption of genes encoding the Ssn6p-Tup1p general corepressor or the Cdk8 module of Srb/Mediator complex have been shown to cause upregulation of flocculin genes and constitutive flocculation in S. pombe revealed the possible existence of several network motifs including positive autoregulation of +mbx2 and +cbf12 and regulation of +gsf2 by a inhibitory feed-forward loop (coherent type 2). The latter involves the Rfl1 transcriptional repression of +gsf2 directly and indirectly by inhibition of +mbx2 expression. Autoregulatory motifs have not been detected so far for FLO8, MSS11 and SFL1. The discovery of these network motifs in S. pombe suggests that the transcriptional inhibition of +gsf2 could occur more rapidly than its transcriptional activation. Experimental and modeling studies have proposed that positive and negative autoregulation of transcription factors generate slow and fast response times, respectively, within a transcriptional-regulatory network +gsf2 expression likely happens in a shorter period compared to its activation. Altogether, these data suggest that the onset of flocculation may occur gradually while repression of the flocculation pathway is a much faster process. Consistent with this speculation is the observation that it requires several days for wild-type S. pombe cells to undergo flocculation when grown in inducing medium.Our analyses of the transcription factors implicated in flocculation of S. pombe. Found within this network are the master regulators Mbx2, Cbf12, Adn2 and Adn3, which are able to trigger flocculation when overexpressed by the activation of their target genes encoding for flocculins and cell wall-remodeling enzymes. In addition, several repressors including Rfl1 were uncovered that play a major role in the regulation of these target genes. However, significant gaps of knowledge surrounding the transcriptional-regulatory network still remain. The environmental cues that impinge upon the activity of the positive and negative regulators, as well as the dynamics of transcription factor binding and regulation of target genes during the onset of flocculation remain to be elucidated. Also, although +gsf2 encodes the dominant flocculin, it is currently unclear whether the other flocculins have nonessential or more specialized roles during flocculation. Detailed analyses of the temporal and spatial expression of the +pfl genes would be required to address these questions. Moreover, the exact mechanism of how other biological processes such as cell wall restructuring and mitochondrial function influence flocculation is unknown. Further studies to expand our knowledge of this transcriptional-regulatory network would provide a more comprehensive understanding of flocculation control and contribute to a valuable resource for the improvement of industrial yeast applications.In summary, we have provided an initial and substantial view of the transcriptional-regulatory network governing flocculation in nmt1 or nmt41 promoter were grown in EMM minus thiamine medium. Standard genetics and molecular biology techniques were performed as described in All strains used in this study are listed in mbx2+ start codon was amplified along with four other fragments for PCR stitching: (1) ∼500 bp upstream of the aforementioned 1 kb fragment; (2) ∼500 bp downstream of the mbx2+ start codon; (3) KanMX6 cassette and; (4) the GFP ORF with its stop codon removed and a GDGAGL linker added  of each PCR fragment were combined and stitched together in a 20 µl PCR reaction (0.2 mM dNTPs and 0.4 units of Phusion HF DNA polymerase (New England Biolabs), and subjected to one cycle of 98°C (30 sec), 5 cycles of 98°C (15 sec), 60°C (1 min), and 72°C (1–2 min) and a final extension at 72°C (5 min). The stitched product was then amplified in a 50 µl PCR reaction by combining the entire stitched reaction with 6 nmol dNTPs, 0.6 units of Phusion HF DNA polymerase and 20 pmol each of the outer pair of primers and then subjected to one cycle of 98°C (30 sec), 30 cycles of 98°C (10 sec), 60°C (30 sec) and 72°C (2 min), and a final extension at 72°C (5 min). The amplified product was gel-purified and transformed into the appropriate strain by lithium acetate transformation. A similar strategy was used to construct GFP-tagged transcription factors under the control of the native promoter. To tag the transcription factor with GFP at the C-terminus, ∼500 bp upstream and downstream fragments flanking the stop codon and the GFP-KanMX6 cassette , followed by immediate freezing of the cell pellets in liquid nitrogen. Culturing of adn2OE and adn3OE strains were performed similarly except that these genes were driven by the nmt1 promoter and were not epitope-tagged. For transcription factor deletion strains , the mutant and an isogenic wild-type strain were concurrently grown in YES medium and harvested at a similar cell density as described above. Total RNA extraction, mRNA isolation, reverse transcription with aminoallyl-dUTP , and Cy™3/Cy™5  dye coupling of cDNA were performed with dye reversal as previously described S. pombe ORFs in 2–3 times coverage per gene. The hybridization procedure was carried out according to the manufacturer's instructions  with the exception for the use of Human Cot-1 DNA. The microarrays were washed in 6× SSPE/0.005% sodium N-lauroylsarcosine at room temperature for 5 min followed by a second wash in pre-heated 42°C 0.6× SSPE for 2 min.Strains overexpressing the triple HA-tagged Mbx2, Rfl1, and Cbf12 were grown in 200 ml of EMM medium containing appropriate supplements without thiamine for 18-20 hr to induce the 2 ratios with the corresponding t-test p values The microarrays were scanned with a GenePix4200A scanner . The raw microarray data was lowess normalized 2HPO4, 1.8 mM KH2PO4 pH 7.4) and washed once with 2 ml ice-cold lysis buffer ). The cell pellet was resuspended in 1.6 ml lysis buffer and stored at −80°C.Culturing of the HA-tagged transcription factor strains are described above. The culture was fixed by the addition of a final concentration of 1% formaldehyde and agitation for 30 min at room temperature. The formaldehyde was quenched by the addition of 2.5 M glycine to a final concentration of 125 mM and agitation for 5 min at room temperature. The cells were then centrifuged , washed twice in 25 ml 1× ice-cold PBS . For the input DNA, 200 µl of the cell lysate was added to 300 µl TES. Both the immunoprecipitated and input cell lysates were incubated at 65°C overnight to reverse the DNA-protein cross-linking. Western blotting with anti-HA antibody was performed to confirm proper pull-down of the transcription factor.The cell suspension was transferred to two 2 ml bead beating vials containing 800 µl of 0.5 mm Zirconia/Silica beads  and subjected to 3 cycles of alternating 2 min beating and 2 min incubation on ice with a Mini Beadbeater 16 . The lysed cells were collected by puncturing the bottom of the bead-beating vial with a flame-heated inoculating needle and placing the vial on a sonication tube nested in 10 ml disposable culture tubes prior to centrifugation . The cell pellet was resuspended, transferred to chilled microcentrifuge tubes, centrifuged  to remove unbound soluble proteins, and the resulting pellet resuspended in 800 µl of fresh lysis buffer in a sonication tube. Total cell lysate volume was adjusted to 2.2 ml with lysis buffer and subjected to 4 cycles of sonication and 1 min on ice incubation at 30% amplitude, 30 sec setting using a Sonic Dismembrator with a 1/8 tapered microtip probe . The sonicated cell lysate was centrifuged  and the supernatant stored at −80°C. The supernatant was tested to ensure that greater than 90% of the sonicated DNA was in the size range of 100 bp–1 kb by subjecting a sample (∼50 µl) of the supernatant to overnight reverse-crosslinking at 65°C and phenol-chloroform extraction, followed by gel electrophoresis of 3–5 µg of DNA. To immunoprecipitate the chromatin-bound transcription factor, 100–200 µl of Dynabeads conjugated with sheep anti-mouse IgG  were washed twice in 800 µl ice cold 1× PBS-BSA , resuspended in 800 µl cold 1× PBS-BSA with 5 µg of anti-HA F-7 antibody  and shaken gently for 2 hr at 4°C on a Labquake Tube Shaker . The beads were washed twice in 1 ml cold deoxycholate buffer  sodium deoxycholate, 0.5% (v/v) NP-40, 250 mM LiCl) and twice in 1 ml cold lysis buffer. The beads were resuspended in 200 µl 1× PBS-BSA, combined with 400 µl of sonicated cell lysate, and shaken gently for 2 hr at 4°C. Four washes of 5 minutes each were next carried out: (1) 1.4 ml cold lysis buffer at 4°C; (2) 1.4 ml cold lysis buffer with 400 mM NaCl at 4°C; (3) 1.4 ml deoxycholate buffer at room temperature and; (4) 1.4 ml TE (pH 8) at room temperature. The transcription factor and bound DNA were eluted twice from the Dynabeads by incubating with 250 µl TES each  SDS) at 65°C for 6 min. Dynabead washing and the supernatant collection were performed using DynaMag™For protein removal, both immunoprecipitated and input samples were incubated with 200 µg Proteinase K  and 20 µg glycogen  at 56°C for 2 hr. The DNA was then extracted by phenol-chloroform extraction, ethanol-precipitated overnight, washed once with 70% EtOH, resuspended in 42 µl TE containing 0.1 µg DNAse-free RNaseA , and incubated for 30 min at 37°C.5′-GCGGTGACCCGGGAGATCTGAATTC-3′ and 15 µM Oligo #2 5′-GAATTCAGATC-3′ in 250 mM Tris) at 16°C overnight. The annealed linker and the ligation mix were kept on ice at all times prior to overnight incubation. The DNA was ethanol-precipitated, washed, and resuspended in 25 µl water as described above.Blunt ends were generated in the entire immunoprecipitate and input DNA samples with 1 unit of T4 DNA Polymerase , 1× NEB Buffer #2 , 5 µg NEB BSA, and 10 nmol dNTPs in a 110 µl reaction by incubation at 12°C for 20 min, followed by phenol-chloroform extraction and ethanol precipitation with 10 µg glycogen and 1/10 volume 3 M NaOAc. The DNA pellets were washed in 70% EtOH and resuspended in 25 µl water. Approximately 1/5 of precipitated input DNA was used in the subsequent ligation reaction as input DNA concentration was >100 times greater than that of immunoprecipitated DNA. For ligation of linkers to blunt ends, the resuspended DNA was incubated with 1000 units of concentrated T4 DNA Ligase , 1× T4 DNA Ligase Buffer , and 200 pmol annealed linker (15 µM Oligo #1 5′-GCGGTGACCCGGGAGATCTGAATTC-3′), 4 µl 10× ThermoPol Buffer  and 7.75 µl water) in a PCR cycler paused at 55°C. A 10 µl enzyme mix containing 5 units of GoTaq DNA polymerase , 0.001 units of Pfu Turbo DNA polymerase , and 1× ThermoPol Buffer  was added and the PCR proceeded with one cycle of 55°C (4 min); 72°C (5 min); 95°C (2 min) and 30 cycles of 95°C (30 sec); 55°C (30 sec); 72°C (1 min), followed by a final extension at 72°C (4 min). The PCR products were purified  with a few modifications: (1) buffer PE was replaced with phosphate wash buffer  and (2) buffer EB was replaced with phosphate elution buffer (4 mM KPO4 pH 8.5). A sample of the purified PCR product was run on an agarose gel to check for fragment sizes ranging between 100 bp and 1 kb. The purified PCR products were quantified, and equal amounts of immunoprecipitated samples and corresponding input samples were coupled to Cy™3 and Cy™5 dyes as described above.The ligated DNA was PCR-amplified by adding 15 µl of labeling mix ), 1.25 µl 40 µM Oligo #1  except for the use of Human Cot-1 DNA. The washing and scanning of the microarrays were performed as described above. The ChIP-chip data was normalized by scaling in Limma 2 ratio cut-off of 1. Peaks located within 3 kb upstream of a start codon and 2 kb downstream of a start codon within a coding region or 3′-UTR, in the case of short ORFs, were assigned to the gene. ChIP-chip data sets are found in The labelled samples  were hybridized onto an Agilent 4×44 K ++S. pombe promoter sequences 1000 bp upstream of the translational start site were used for these motif-finding algorithms. For MEME, promoter sequences of genes with various log ratio thresholds from expression microarray and ChIP-chip experiments were input into the MEME online server. RankMotif++ was applied to the entire expression microarray data since its motif-searching algorithm is threshold independent. The consensus sequences of the transcription factor binding sites were displayed by submitting the position weight matrices obtained from RankMotif++ analysis into the enoLOGOS online server The transcription factor binding specificities were determined by RankMotif2 and allowing the culture to sit for 30 min at room temperature.Strains were grown in flasks at 30°C for the appropriate time, and 10 ml of culture was transferred to culture tubes for strains with larger floc sizes. Images were acquired immediately after vigorous shaking in glass culture tubes with a Canon G10 digital camera. For strains with mild flocculation, flocs were harder to visualize in culture tubes, and therefore, were observed in 90 mm plastic petri dishes. 10–15 ml of culture was transferred to petri dishes, followed by gentle shaking +pfl genes, the strains were inoculated at a concentration of 107 cells in 100 ml of EMM without thiamine and cultured for 3 days at 30°C. For the weaker flocculent strains (pfl2+–pfl9+), 5 ml of the 3-day culture was then inoculated into 100 ml of fresh EMM without thiamine and incubated for another 3–4 days at 30°C followed by the petri dish flocculation assay as described above. Fresh EMM medium was added on the third day to prevent cells from remaining in stationary phase. Flocculation assays for the more flocculent overexpression strains were similarly carried out except the induction times were less than three days and did not require refeeding with fresh EMM medium. It should be noted that the empty vector control cells also eventually flocculate after refeeding with fresh EMM medium, but the onset of flocculation and flocs were delayed for several days and less pronounced, respectively, compared to the weakest flocculent overexpression strains. Wild-type strain and deletion mutants  were induced to flocculate by inoculating cells at a concentration of 108 cells in 100 ml of YEGlycEtOH medium and culturing for 5 days at 30°C followed by the petri dish flocculation assay as described above.For the overexpression of A patch of cells approximately 1/6 of a 90 mm petri dish was grown on YES medium for two days at 30°C and transferred as described in t-test. To view nuclei and cell wall material, cells were methanol-fixed and stained with DAPI (1 µg/ml) and calcofluor white (50 µg/ml), respectively.Images of GFP-tagged cells were acquired with a Zeiss AxioScope 2 microscope  and Scion CFW Monochrome CCD Firewire Camera . Fluorescence intensity was quantitated using the open source software ImageJ (version 1.44) . First, the background signal for each image was subtracted using the “Subtract Background” function . Individual cells were then selected as regions of interest using the freehand or polygon selection tools. Using the “Set Measurements” function both the area and integrated density were determined for each selected cell (n ranged between 27 and 50). Corrected GFP intensity was determined for each cell and was defined as the quotient of integrated density/area in background subtracted images. The averaged integrated density/area measurements for a given number cells is presented as the mean corrected GFP intensity with standard deviation. Significant differences between means were calculated by the Student Figure S1Overexpression of cell wall-remodeling genes enhances cell-to-surface adhesion, but not invasive growth. The assays for adhesion and invasive growth were carried out as described in the (TIF)Click here for additional data file.Table S1Schizosaccharomyces pombe strains used in this study.(DOC)Click here for additional data file.Table S2mbx2-HA OE strain versus empty vector strain.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S3mbx2-HA ChIP-chip data.(XLSX)Click here for additional data file.Table S4rfl1 deletion strain versus isogenic wild type.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S5rfl1-HA ChIP-chip data.(XLSX)Click here for additional data file.Table S6cbf12-HA OE strain versus empty vector strain.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S7cbf12-HA ChIP-chip data.(XLSX)Click here for additional data file.Table S8yox1 deletion strain versus isogenic wild type.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S9sre2 deletion strain versus isogenic wild type.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S10cbf11 deletion strain versus isogenic wild type.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S11adn2OE strain versus empty vector strain.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S12adn3OE strain versus empty vector strain.Expression Microarray Profiling of (XLSX)Click here for additional data file.Table S132 ratios determined from expression microarrays are shown for comparison. Culturing, RNA extraction and reverse transcription for each strain were performed independently from the microarray experiments. Primer sets were checked for specificity by the presence of a single amplicon of the correct size using their melting curves and gel electrophoresis. The +act1 gene was used as a reference for determining the relative expression of putative targets and overexpressed genes. Quantitative PCR was performed on a StepOne Real-Time PCR System with SYBR® green master mix  and the following program: 95°C for 10 min, 40 cycles of 95°C for 15 sec and 58°C for 1 min, followed by a melting curve program of 58°C to 95°C with a heating rate of 0.3°C per second. Three replicates were carried out for each combination of query gene and strain. The relative expression of each query gene was compared between the mutant and the corresponding wild type or empty vector strain. Fold changes were determined by ΔΔCt method according to manufacturer's recommendation (Life Technologies).Validation of putative targets and overexpressed genes by qPCR. The log(DOC)Click here for additional data file.Table S147 cells/ml. Reflocculation was carried out by the addition of CaCl2 at a final concentration of 20 mM to 2.7 ml of resuspended culture in a 60 mm petri dish shaken on an orbital low-speed shaker  at maximum speed for 30 min in room temperature. The entire culture was pipetted carefully into a 3.0 ml cuvette and an OD600 reading close to the top of the cuvette was obtained with a Spectramax Plus microplate reader . A control culture was carried out similarly except no CaCl2 was added. The degree of flocculation was determined by subtracting the difference of the optical density of the reflocculation culture and the control culture from 1 as described in Kobayashi O, Hayashi N, Kuroki R, Sone H, (1998) J. Bacteriol. 180: 6503–6510. The values of the degree of flocculation were derived from at least two technical replicates.Degree of flocculation observed in flocculent strains. Strains were grown in EMM minus thiamine medium unless indicated. Cultures were refed with fresh medium on the third day to prevent entry into stationary phase see . To semi(DOC)Click here for additional data file.