PHASE CHANGES IN PLANT DEVELOPMENT WITH RESPECT TO FLORAL _ TRANSITION AND SECONDARY XYLEM FORMATION By Sookyung Oh During development, plants undergo a switch in the potential of merist
LITERATURE REV HE Y nh n0 304460665601609609695695699896 1 1 Phase change in plants 07 a
Transcriptional Regulation of Secondary Growth in Arabidopsis
Transcriptional Regulation of Secondary Growth in Arabidopsis thaliana
This work was published in Sookyung Oh, Sunchung Park, Kyung-Hwan Han (2003), J.Exp Bot 54:2709-2722, Transcriptional regulation of secondary growth inArabidopsis thaliana Computational analysis of cis-element in Table 2-2 was done bySunchung Park.
Despite its economic and environmental significance, understanding the molecular biology of secondary growth (i.e wood formation) in tree species has been lagging behind that of primary growth, primarily due to the inherent difficulties of tree biology In recent years, Arabidopsis has been shown to express all of the major components of secondary growth Arabidopsis was induced to undergo secondary growth and the transcriptome profile changes were surveyed during secondary growth using 8.3 K Arabidopsis Genome Arrays Twenty per cent of the ~8300 genes surveyed in this study were differentially regulated in the stems treated for wood formation Genes of unknown function made up the largest category of the differentially expressed genes, followed by transcription regulation-related genes Examination of the expression patterns of the genes involved in the sequential events of secondary growth (i.e cell division, cell expansion, cell wall biosynthesis, lignification, and programmed cell death) identified several key candidate genes for the genetic regulation of secondary growth In order to gain further insight into the transcriptional regulation of secondary growth, the expression patterns of the genes encoding transcription factors were documented in relation to secondary growth A computational biology approach was used to identify regulatory cis- elements from the promoter regions of the genes that were up-regulated in wood-forming stems The expression patterns of many previously unknown genes were established and various existing insights confirmed The findings described in this report should add new information that can lead to a greater understanding of the secondary xylem formation process.
IntroductionPlant growth by means of apical meristems results in the development of sets of primary tissues such as epidermis, vascular bundles, and leaves In addition to this primary growth, tree species undergo secondary growth and produce the secondary tissue ‘wood’(secondary xylem) from the vascular cambium (i.e secondary meristems) The vascular cambium originates from the procambium and normally consists of S—15 dividing cells It occurs as a continuous ring of meristem cells that are located between the xylem and the phloem (the so-called ‘cambial zone’) (Larson, 1994; Mauseth, 1998) The transition from procambium to cambium is not clearly understood On the xylem side of the cambium, the cells first go through stages of differentiation that involve cell division,expansion, maturation, lignification, secondary cell wall thickening, and programmed cell death, in which all cellular processes are terminated (Chaffey, 1999) The growth of vascular cambium increases the diameter (by periclinal divisions) and the circumference(by anticlinal divisions) of an axis Positional information appears to be required to co- ordinate this development of secondary xylem (Uggla et al., 1996, 1998) To achieve the patterned growth, each cell must express the appropriate sets of genes in a co-ordinated manner after receiving the necessary positional information In other words, the control of cambial growth and differentiation is accomplished by changing the activity of key genes involved in developmental pathways Recently, significant progress has been made in the study of the genes and signaling mechanisms responsible for secondary wall formation,lignin and cellulose biosynthesis (Arioli et al., 1998), and xylem development (Fukuda,1997; Ye, 2002).
Secondary growth is one of the most important biological processes on Earth Its product, wood, is of primary importance to humans as timber for construction, fuelwoods, and wood-pulp for paper manufacturing It is also the most environmentally cost-effective renewable source of energy However, despite its economic and environmental significance, secondary growth has received little research interest, mainly because most agricultural products are derived from seeds or roots Furthermore, the biology of wood formation is surprisingly understudied because of the inherent problems of tree species: long generation time, large size, and lack of genetically pure lines Study of wood formation at the molecular level using real trees has begun in recent years A genomics approach has been successfully used to examine global gene expression patterns in developing xylem tissues of black locust (Yang et a/., 2003), pine (Allona et al., 1998; Lorenz and Dean, 2002), and poplar (Sterky et al., 1998; Hertzberg et al., 2001). However, current understanding of the molecular mechanisms of wood formation in trees is still limited.
Recently, Arabidopsis, the most well-studied herbaceous model species, has been used as a model for the study of wood and fiber production in trees (Lev-Yadun, 1994;Zhao et al., 2000) When kept from flowering by repeated removal of inflorescences (i.e.decapitation) and grown at a low density, Arabidopsis produces a significant quantity of secondary xylem (i.e wood) that is sufficient for various developmental studies (Lev-Yadun, 1994; Beers and Zhao, 2001) Zhao et al (2000) characterized xylem-specific proteases in Arabidopsis Chaffey et al (2002) reported wood formation in the hypocotyls of short-day-grown Arabidopsis plants and demonstrated that the secondary xylem tissues produced in their study were structurally similar to those of an angiosperm tree (poplar).
The primary objectives of this research were to identify xylogenesis-associated genes and to determine how they are regulated in Arabidopsis thaliana Wood formation was induced as described by Lev-Yadun (1994) Then, using Arabidopsis Genome GeneChip (8.3K) Arrays, the global gene expression patterns were examined by comparing treatment versus control stems and xylem versus bark tissues, the genes were identified that are differentially regulated for wood formation, and the differentially expressed genes were clustered into several groups based on their expression patterns. The expression profiles of three types of transcription factors (AUX/IAA, R2R3-MYB, and HD-containing) that have previously been shown to regulate the developmental processes involved in secondary growth were also documented A computational biology approach was then used to identify several cis-regulatory elements from the promoter regions of genes whose expression patterns could be associated with wood formation. Elucidation of any commonality found in those regulatory elements frequently presented in wood formation-associated genes might provide some insight into the genetic regulation of secondary growth.
Materials and Methods Plant growth and treatment for wood formation in Arabidopsis
Arabidopsis thaliana (L.) Heynh Columbia plants were grown in a greenhouse using Baccto soil under 16/8 h light/dark conditions at 2343 °C The plants in the wood formation treatment were grown at the density of one plant per 100 cm” pot Four weeks after germination, the inflorescence was removed as previously described (Lev-Yadun,
1994) At that time, most of the plants had about ten rosette leaves and 4-5 cm long inflorescences The removal all newly emerging inflorescences was continued for an additional 5 weeks In addition, a 4-month-old poplar (Populus deltoides) stem was prepared as sample material to be used for an anatomical comparison of xylem structures found in Arabidopsis plant samples For use in the control stem, 25 Arabidopsis plants were grown in a 100 cm’ pot After 3 weeks without any treatment, 3-4 cm long young inflorescence samples were harvested and used as control stems Xylem and bark samples were also isolated from the treatment plant as described by Zhao et al (2000) Briefly, about 1 cm of the root—hypocoty! junction region was excised from treatment plants and the lateral roots were trimmed from the primary root using a razor blade (VWR Co., West Chester, PA) Xylem and bark portions were separated by forceps and razor blade, quenched with liquid N; and stored at -80 °C until use Cross-section samples were prepared by fixation in 3% paraformaldehyde and 1.25% gluteraldehyde solution After fixation, the samples were dehydrated in a series of ethanol solutions (25, 50, 75, 95, and 100%), embedded in paraffin (Sigma-Aldrich Co., St Louis, MO), cut using razor blade (VWR) and stained with 0.025% toluidene blue O The sliced samples were observed under the microscope (American Optical Instruments, Buffalo, NY).
RNA extraction and cDNA synthesis
For treatment stem, control stem, xylem tissue, and bark tissue samplings, two biologically duplicate sets were prepared At least 150 individual plants were harvested for each set Total RNA was isolated using Qiagen RNeasy columns (Qiagen Co.,Valencia, CA) and mRNA was isolated using Qiagen mRNA Midi kit (Qiagne Co.) The first strand cDNA was synthesized from 800 ng of mRNA, in the reaction mixture using
100 pmol of an oligo dT (24) primer, containing a 5'-T7 RNA polymerase promoter sequence, and 200 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) in 75 mM KCl, 3 mM MgCh, 50 mM Tris-HCI (pH 8.3), 10 mM dithiothreitol (DTT), and 0.5 mM dNTP The second cDNA synthesis was performed in a reaction mixture with 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCh, 10 mM (NH4)2SO4, 1 mM dNTP, 40 units E coli DNA polymerase, 10 units E coli ligase, and two units of RNase
H Double-stranded cDNA products were purified by phenol/chloroform extraction and ethanol precipitation. cRNA synthesis
Biotinylated cRNA were in vitro transcribed from synthesized cDNA by T7 RNA polymerase (BioArray high yield RNA Transcript Labeling Kit, Enzo Diagnostics Inc., Farmingdale, NY) The cRNAs were purified using Qiagen RNeasy Spin Columns 20 ug were then randomly fragmented via incubation at 94 °C for 35 min, in a buffer containing
30 mM magnesium acetate, 100 mM potassium acetate and 40 mM Tris-acetate (pH 8.1), in order to produce molecules of approximately 35—200 base long cRNA.
The following hybridization was performed at the Genomics Technology Support Facility
(GTSF) on the campus of Michigan State University Briefly, fragmented cRNAs were denatured at 99 °C for 5 min in the mixture of 0.1 mg ml” sonicated herring sperm DNA and hybridization buffer containing 100 mM 2-N-morpholino-ethanesulphonic acid(MES), 1 M NaCl, 20 mM EDTA, and 0.01% (w/v) Tween 20 Then, the hybridization mix was hybridized with GeneChip® Arabidopsis Genome Arrays (Affymetrix, Santa
Clara, CA) at 45 °C for 16 h on a rotisserie at 60 rpm After hybridization, the array cartridge was rinsed and stained in a fluidics station (Affymetrix) The hybridized arrays were first rinsed with wash buffer A (6x SSPE [0.9 M NaCl, 0.06 M NaH;POa, 0.006 M EDTA], 0.01% [w/v] Tween 20, and 0.005% [w/v] Antifoam) at 25 °C for 10 min and then incubated with wash buffer B (100 mM MES, 0.1 M NaCl, and 0.01% [w/v] Tween
20) at 50 °C for 20 min Next, the arrays were incubated with streptavidin phycoerythrin (SAPE) solution containing 100 mM MES, 1 M NaCl, 0.05% [w/v] Tween 20, 0.005%
[w/v] Antiform, 10 mg ml! SAPE, and2 mg ml bovine serum albumin at 25 °C for 10 min, washed with wash buffer A at 25 °C for 20 min, and stained with biotinylated antistreptavidin antibody at 25 °C for 10 min in a fluidics station (Affymetrix) The probe array was scanned twice, and then the intensities were averaged with a Hewlett-Packard
Gene expression levels were measured by the calculated signal value which assigns a relative measure of abundance to the transcript, and the reliability of those data were evaluated using the P-value system as described in the Microarray Suite 5.0 (Affymetrix).
A Mechanism Related to the Yeast Transcriptional Regulator
Global and Locus-Specific Roles for Arabidopsis PafÍC Homologs
Global and Locus-Specific Roles for Arabidopsis PaflC Homologs in Transcription and Chromatin Modifications
RNA Polymerase II-Associated Factor 1 Complex (Paf1C) in budding yeast plays a key role in reinforcing transcriptional activity by mediating the establishment and/or maintenance of specific chromatin modifications, promoting elongation and linking Pol II with elements of pre-mRNA processing machinery This transcription factor is associated with chromatin at all canonical transcriptional units yet investigated and therefore probably plays a general transcriptional role Although components of PaflC are conserved in higher eukaryotes, their potential mechanism in transcription has not been explored In Arabidopsis, PaflC subunit homologs are encoded by the VERNALIZATIONINDEPENDENCE (VIP) genes We found that loss of VIP gene function affects a substantial portion of the transcriptome, but strongly silences only a small subset of genes, including the FLC/MAF family of MADS-domain flowering regulators To better understand the mechanism of VIP proteins in transcription, we characterized genome- wide and locus-specific effects of loss of VIP3 on histone modifications, Pol II distribution, and phosphorylation of the carboxyl-terminal domain (CTD) of Pol II We analyzed methylation (Lys-4, Lys-36) and acetylation (Lys-9, Lys-14) sites on the canonical and variant histone H3 proteins, and found that VIP3 does not play a significant role in establishing these modifications when evaluated on a bulk-chromatin level, but is important for establishing these modifications within a subset of VIP- dependent genes Loss of VIP3 resulted in a decrease of Pol II occupancy throughoutFLC chromatin, including the promoter regions, suggesting that the major influence onFLC expression is effected through Pol II recruitment and/or transcriptional initiation.Loss of VIP2, VIP3, VIP4, VIP5, or VIP6 gene function resulted in CTD hyperphosphorylation, suggesting a significant role of VIP complex in regulating the activity of the CTD.
Precise control of transcription is crucial for growth and development in eukaryotes The formation of mRNA by RNA Polymerase II (Pol II) involves a complex, multistep pathway wherein each step provides an opportunity for regulation (Shilatifard et al.,
2003) The primary phases of transcript generation form a so-called transcription cycle and include preinitiation, initiation, promoter clearance, elongation, and termination An immense amount of research has been performed to identify and characterize transcription factors that are involved in the regulation of the transcription cycle Recent evidence indicates that there is a dynamic interplay between the protein complexes that carry out mRNA transcription, processing, and export, such that the efficiency of one step can have significant consequences for other steps in the pathway Thus, understanding the mechanisms of transcriptional regulation requires the identification of both the direct and indirect activities of the numerous factors implicated in RNA production.
The budding yeast Pafl complex (PaflC) containing at least five polypeptides (Pafl, Ctr9, Cdc73, Rtfl, and Leol) was first identified as a Pol II-associated factor (Wade et al., 1996) Paf1C interacts with TATA-box binding protein (TBP) (Stolinski et al, 1997), elongation factors Spt4-5 and FACT (FAcilitates Chromatin Transcription) (Squazzo et al., 2002) and can stimulate transcription elongation in vitro (Rondon et al.,
2004) PaflC has been shown to be present at both promoter and coding regions of transcriptionally active genes (Pokholok et al., 2002) Thus, Paf1C was initially thought to be a factor involved in both Pol H-mediated transcription initiation and elongation.
Recent evidence has revealed that PaflC plays an important role in cotranscriptional histone modification and mRNA 3’ end processing Paf1C assists in the ubiquitination of histone H2B at lysine (K) 123 (H2B K123) in the promoter-proximal region of activated genes (Ng et al., 2003; Wood et al., 2003) PaflC-dependent H2B ubiquitination is required for subsequent methylation of histone H3 K4 and H3 K79, catalyzed by Setl and Dotl, respectively (Ng et al., 2003) Cells lacking functional Rtfl show global defects in di- and trimethylation of H3 K4 and dimethylation of H3 K79 (Ng et al., 2003). These histone modifications have most often been associated with actively transcribed genes (Hampsey and Reinberg, 2003) Histone lysine methylation exists in the mono-, di- , or trimethylated state and each distinct methylation might have unique biological relevance For example, in budding yeast, dimethylation of H3 K4 occurs on a genome- wide scale, whereas trimethylation of H3 K4 strictly corresponds to actively transcribed genes (Santos-Rosa et al., 2002) Paf1C is also required for methylation of H3 K36 and recruitment of yeast Set2 (a histone H3 K36 methylase) at specific loci (Krogan et al.,
2003) Paf1C also assists in modification of the carboxyl-terminal domain (CTD) of Pol
TI (Mueller et al., 2004) The CTD consists of multiple repeats of the amino acid sequence Y;S2P3T4SsP6S7 and the phosphorylation state of serine (Ser) on the CTD is important not only for proper initiation/elongation of transcription but also pre-mRNA processing Ser-5 of CTD is phosphorylated by a cyclin-dependent kinase associated with the general transcription machinery at the 5' region of the gene during initiation; whenPol II travels toward the 3' end of the gene, Ser-2 of CTD becomes preferentially phosphorylated (Cho et al., 2001) In yeast, dephosphorylation of Ser-5 or Ser-2 residues within the CTD is catalyzed by FcpI and Ssu72 (Sims et al., 2004) Loss of Paf] or R7 gene function results in a reduction of Ser-2 phosphorylation of CTD of Pol II (Mueller et al., 2004), but the mechanism of Paf1C in modification of CTD is not known Pre-mRNA cleavage/polyadenylation specificity factor complex (CPSF) is recruited to elongating Pol
II near the poly(A) site in a process that requires Ser-2 phosphorylation of CTD (Ahn et al., 2004) Loss of Pafl or Rtfl gene function also causes alternative poly(A) site utilization within Paf1C target genes, and shortened poly(A) tails globally (Mueller et al., 2004; Penheiter et al., 2005).
Homologous components of PaflC have been found in higher eukaryotes, including plants, humans and fruit flies (Zhang and van Nocker, 2002; Oh et al., 2004; Zhu et al., 2005; Adelman et al., 2006) In Arabidopsis, Paf1C subunit homologs are encoded by VERNALIZATION INDEPENDENCE (VIP) genes; VIP2 (also known as EARLY FLOWERING 7 (ELF7) [He et al., 2004]), VIP4, VIP5, and VIP6 (also known as ELF8 [He et al., 2004]) are closely related to Pafl, Leol, Rtfl, and Ctr9 from yeast, respectively (Zhang and van Nocker, 2002; Oh et al., 2004) VIP3 is homologous with hSki8, a higher eukaryotic-specific subunit of human Paf1C hSki8 is initially identified as a component of the human Superkiller (SKI) complex, interacting with the exosome, a protein complex required for 3’-5’ mRNA decay (Andrulis et al., 2002; Zhu et al., 2005).VIP3 physically interacts with VIP4 and VIP6 in vivo, and abundance of VIP6 protein is dependent on functional VIP3, VIP4, and VIPS, suggesting that these proteins comprise a protein complex analogous to PaflC (Oh et al., 2004) VIP complex is required for expression of only a restricted subset of genes, including the FLC/MAF family of MADS- box floral repressors (Zhang and van Nocker, 2002; Zhang et al., 2003; Oh et al., 2004).VIP complex from Arabidopsis may perform a conserved functions but in a different biochemical mechanism For example, in vip3, vip4, vip5, or vip6 mutants, there is no discernible reduction in global abundance of di- or trimethylated H3 K4, or dimethylated H3 K79 (Oh et al., 2004), although trimethylated H3 K4 at the 5” region of FLC is decreased in vip2 (elf7) or vip6 (elf8) mutants (our unpublished results; He et al., 2004).
Interestingly, unlike budding yeast, which does not carry variant H3 genes (Ahmad and Henikoff, 2002), the genome of higher eukaryotes including Arabidopsis, fly and human encodes for both canonical and variant H3 proteins (Waterborg, 1992; Hake and Allis, 2006) Canonical H3 proteins (named H3.1 in plants) are the major class of histones and their expression is coupled to the S-phase of the cell cycle, when histones assemble with the newly replicated DNA to form a duplicate set of chromatin (Schumperli, 1986) Variant H3 proteins (named H3.3 in humans and flies, H3.2 in plants) are synthesized outside the S-phase throughout the cell cycle in a replication- independent manner (Schumperli, 1986; Waterborg, 1992; McKittrick et al., 2004) In human and fly, chromatin associated with transcriptionally active loci becomes enriched for H3.3 di-or trimethylated at K4 (McKittrick et al., 2004; Mito et al., 2005).
To investigate the role of Arabidopsis Paf1C homologs in transcription, we first questioned whether the function of VIP complex in establishment and/or maintaining gene activity might be associated with modification of a subset of H3 histones,specifically H3.2 To this end, we analyzed the consequence of loss of V7P3 function for global modification of H3.2 and could not detect any difference in methylation or acetylation levels of either H3.1 or H3.2 between vip3 mutant and wild-type plants.However we found that VIP3 is required for H3 K4 methylation of a subset of VIP- dependent genes and H3 K36 methylation of FLC To further elucidate the regulatory role of PafIC for Pol II activity, we have measured the abundance of Pol II within the FLC gene, and the phosphorylation of Pol II in vip3 mutants We found that loss of VIP3 function resulted in a decrease of Pol II occupancy throughout FLC chromatin and overall increase of Ser-2 and Ser-5 phosphorylation of CTD of Pol II, suggesting an important role for VIP proteins in recruitment/retention and modification of Pol II.
Materials and Methods Plant Materials
The /7c-3 null mutant, winter annual FR/-introgressed line Col:FRAF, vip mutants, and transgenic line expressing FLAG-epitope-tagged VIP3 protein were previously described (Zhang et al., 2003) Transgenic line expressing FLAG-epitope-tagged VIP3 protein was as described previously (Zhang et al., 2003; Oh et al., 2004) Plant growth conditions and vernalizing cold treatments were previously described (Zhang and van Nocker, 2002).
Histone-enriched extracts were prepared as described by Waterborg et al (1987) from floral tissues Briefly, total proteins were extracted from 1 g of liquid Na-ground tissues using 2 ml of Lysis Buffer [40% w/v guanidine hydrochloride (GuCl), 10 mM phosphate buffer (pH 8.0 at 22 °C), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 pg/ml Pepstatin A (Sigma)] Cell lysates were incubated with 100 mg of cation exchange resin (BioRex -70; Bio-Rad, Hercules, CA) per 1 g of plant samples at room temperature overnight Resin was washed five times with 1 ml ice-cold washing buffer (5% GuCl in
10 mM phosphate buffer, pH 8.0 at 22 °C), and subjected to elution with Lysis Buffer.
The anti-VIP3 and -VIP6 antisera were as described previously (Zhang et al., 2003; Oh et al., 2004) Anti-FLAG M2 monoclonal antibody was obtained from Sigma (St Louis,
MO, catalog no F-3165); Anti-H3 antibodies including anti-di-M-K4 (catalog no 07-
030), anti-di-M-K36 (catalog no 07-369), anti-Ac-K9/14 (catalog no 06-599), and anti- H3-CT (catalog no 06-866) were obtained from Upstate (Lake Placid, NY); Anti-H3 tri- M-K4 antibody was obtained from Abcam (Cambridge, MA) The 8WG16 antibody recognizing non-phosphorylated CTD of Pol II , H5 antibody recognizing Ser-2 phosphorylated CTD of Pol II, and H14 antibody recognizing Ser-5 phosphorylated CTD of Pol II were obtained from Covance (Denver, PA).
Proteins were extracted from floral tissues using a protocol described previously (Zhang et al., 2003) As a result, approximately 500 yg of protein was obtained in 500 ul of Extraction Buffer (50 mM Tris-HCl [pH 8.0 at 22 °C], 150 mM NaCl, 1 mM EDTA, 0.2% Triton X-100, containing 1 mM PMSF and phosphatase inhibitors [10 mM sodium fluoride, 5 mM sodium phosphate, and 1% phosphatase inhibitor cocktail I; Sigma]) The extract was incubated with 1 pg/ml of anti-FLAG antibody, and mixed continuously for 2 h Protein A-agarose beads (30 pl; Roche Biochemical, Indianapolis, IN) were then added, and the mixture was incubated for a further 1 h Protein A-agarose beads were collected by centrifugation and washed five times consecutively with 1 ml ice-cold washing buffer (Extraction Buffer lacking Triton X-100) After the final wash, the beads were resuspended in 50 yl of SDS-PAGE sample buffer and boiled All procedures described here were carried out at 4 °C.
Proteins were resolved on SDS-polyacrylamide gels or acetic acid/urea/triton X-100 (AUT)-polyacrylamide gels as described in Harlow and Lane (1988) and Bonner et al.
Perspectives and Future DireCfẽOTS .cscsĂcsĂSSSSSSASessessesee 156 - VỊP complex is required for histone H3 methylation in a locus-specific manner
VIP complex is required for histone H3 methylation in a locus-specific manner
We found that VZP3 is required for H3 methylation in a locus-specific manner As shown in the ChIP analysis, trimethylation of H3 K4 within FLC and At]g31690 was reduced in vip3 mutants However, unlike yeast (Ng et al., 2003), Arabidopsis vip mutants did not exhibit a global defect in the trimethylation of H3 K4, indicating that the VIP complex from Arabidopsis may perform a conserved functions but in a different biochemical mechanism In human or fruit fly, loss of function of Cir9 or Pafl-related genes causes loss of trimethylation of H3 K4 when assayed using single cell lines (Zhu et al., 2005; Adelman et al., 2006), suggesting that the locus-specific role of VIP complex for trimethylation of histone H3 K4 could be specific to plants Molecular cloning of several other VIP-like loci identified by our group (Zhang et al., 2003) would provide an insight into the functional divergence of VIP complex.
Besides trimethylation of H3 K4, the reduced level of dimethylation of H3 K36 within FLC chromatin in vip3 mutants suggests that, similar to PaflC, VIP3 may be required for recruitment of a H3 K36 dimethylating activity to FLC Arabidopsis EARLYFLOWERING IN SHORT DAYS (EFS), homologous to yeast Set2 is required for FLC activation, and loss of function of EFS gene results in globally reduced levels of H3 K36 dimethylation (Zhao et al., 2005), suggesting that EFS is a plausible candidate for H3K36 dimethylation activity Thus, we speculate that VIP complex may assist in the recruitment of EFS at particular genes to maintain or reinforcing the transcriptional activity through a mechanism related to dimethylation of H3 K36.
VIP3 may be a higher eukaryote-specific component of Paf{C
Human PafiC shares five subunits with yeast PaflC and additionally contains a WD- repeat protein, hSki8 (Zhu et al., 2005) WD-repeat proteins are common constituents of large chromatin-associated complexes (van Nocker and Ludwig, 2003), and our phylogenetic analysis (data not shown) revealed that VIP3 is the closest Arabidopsis WD-repeat protein to hSki8 (35% identity with 2e-2 E-value at amino acid level) hSki8 is also a component of the human SKI complex, interacting with the exosome, which is required for 3’-5’ mRNA decay (Araki et al., 2001; Andrulis et al., 2002) The exosome in eukaryotes is known to serve as an RNA surveillance mechanism, which ensures high fidelity of gene expression by degrading aberrantly processed mRNAs For example, alternative splicing requires RNA surveillance to ensure high fidelity of gene expression in higher eukaryotes Higher eukaryotes developed the process of alternative splicing to generate multiple mRNAs from one gene: 45% of human genes or 22% of Arabidopsis genes are suggested to be alternatively spliced (Gupta et al., 2004; Wang and Brendel,
2006) However alternative splicing appears to be rare or non-existent in budding yeast because of the rare number of introns (i.e budding yeast has introns in only ~3% of its genes) (Ast, 2004) Since hSki8 and exosome co-localize to transcriptionally active genes in a human PaflC-dependent manner (Mitchell and Tollervey, 2003; Zhu et al., 2005), it is tempting to speculate that VIP3 coordinates surveillance of RNA quality required for alternative splicing Northern blot analysis of genes misregulated in vip mutants relative to wild-type plants or comparison of cDNA and genomic DNA from these genes may be a plausible approach to test if VIP complex is involved in alternative splicing In addition,ChIP-on-chip analysis (combining conventional ChIP and microarray analysis) could be performed to determine whether the VIP proteins participate directly in the regulation of these genes through alternative splicing.
VIP complex is required for Ser-2 and Ser-5 phosphorylation of CTD of Pol H CTD phosphorylation and dephosphorylation are crucial for the progression of the transcriptional cycle as they regulate the association of Pol II with the pre-initiation complex and the factors responsible for elongation, pre-mRNA 5’ capping, splicing, and 3’-end processing (Proudfoot et al., 2002; Sims et al., 2004) CTD phosphorylation during transcription is coordinated by various kinases (Kin28, Ctk1, Burl, and Srb10/11 in budding yeast; p-TEFb in fruit fly), and phosphatases (Fcpl and Ssu72 in budding yeast) (Ganem et al., 2003) The phosphorylation cycle of the CTD of Pol II mainly studied in yeast has been summarized (Sims et al., 2004; see Figure 5-1) The observation that loss of function of VIP genes was associated with global increase of Ser-2 and Ser-5 phosphorylation of CTD of Pol II suggests a requirement for the VIP complex to regulate modification of CTD One possibility is that loss of function of VIP genes causes misregulation of genes encoding cyclin-dependent kinases or phosphatases of CTD. However we could not detect any striking change of expression of these genes in our microarray analysis.
An alternative possibility is that VIP complex assists the activity of CTD phosphatases Ser-5 phosphorylation level of CTD is high at the promoter and then decreases towards the 3’-end of the gene; presumably, protein phosphatases target Ser-5 residues (Sims et al., 2004) Ser-2 phosphorylation level reaches a peak near the poly(A) site and then drops beyond the site (Cho et al., 2001), and complete dephosphorylation occurs prior to Pol II recycling (Sims et al., 2004) Budding yeast cells lacking Fep/ function cease transcription of majority of genes (Kobor et al., 1999) and accumulate Ser-
2 phosphorylation of CTD at the coding region of specific genes (Cho et al., 2001). Similarly, human Fcp1 dephosphorylates Ser-2 and Ser-5 of CTD to an equal degree (Lin et al., 2002), and Arabidopsis Fcp1 dephosphorylates Ser-5 of CTD specifically (Koiwa et al., 2004) Fcpl purified from human HeLa cells exhibits elongation stimulatory activity in vitro and participates in Pol II recycling (Cho et al., 1999; Mandal et al.,
2002) Fcpl genetically interacts with PaflC in yeast; rtf] mutants are lethal in combination with fcp/ mutants (Costa and Arndt, 2000) Fcp1 also genetically interacts with a Ser-5 CTD phosphatase, Ssu72 in yeast; Ssu72 mutation can be suppressed by Fep1 overexpression (Ganem et al., 2003; Krishnamurthy et al., 2004) Like Fep1, Ssu72 is involved in recycling the Pol II for reinitiation and subsequent rounds of transcription, although the specifics of recycling are currently unknown (Sims et al., 2004) Ssu72 is a component of the cleavage/polyadenylation specificity factor complex (CPSF) (He et al.,
2003) and required for mRNA 3’ end processing (Ganem et al., 2003) Based on the genetic interaction of Fcpl with PaflC and Ssu72, it is an interesting assumption that they may function in the same pathways Since hyperphosphorylation of CTD has been observed in vip mutants, we hypothesize that the VIP complex assists in recruitment of Arabidopsis Fcpl and/or Ssu72 homologs on target genes, resulting in proper mRNA 3’ end processing To begin to investigate the role of the VIP complex in mRNA 3’ end processing, it is necessary to test if loss of function of ƒ7P genes can alter the recognition of poly(A) site of specific genes misregulated in vip mutants Previously we identified eleven genes, the mRNA of which reproducibly and strongly increased (>3-fold) in both vip and vip6 mutants through microarray analysis (Oh et al., 2004) These genes could be used for hybridization probes in northern blot analyses to detect an extended 3” end of mRNA in vip mutants If extended forms of mRNA were detected in vip mutants, ChIP analysis could be performed to determine whether the VIP complex is required for the localization of the Arabidopsis Fcpl or Ssu72 homolog at poly(A) recognition site.
AtCPL1 and AtCPL2 are Arabidopsis Fcp1 homologs (Koiwa et al., 2004) Both have been characterized as negative transcriptional regulators of cold and drought stress- related genes (Koiwa et al., 2002) and loss of function of AtCPL2 causes early flowering and reduced fertility (Koiwa et al., 2004) The Arabidopsis genome encodes one Ssu72 homolog that has not been characterized.
Although the phosphorylation status of CTD of Pol II just after transcriptional termination is unknown, complete dephosphorylation occurs prior to polymerase recycling (Ejkova and Tansey, 2002) As mentioned above, both Fcp1 and Ssu72 appear to be required for the complete dephosphorylation of Pol II to possibly ensure rapid Pol II recycling It is tempting to speculate that the VIP complex coordinates Pol II recycling through dephosphorylation of CTD of Pol II To explore the role of VIP complex in recycling of Pol II, ChIP analysis or ChIP-on-chip analysis could be used to test if loss of function of VIP genes causes altered localization of phosphorylated CTD of Pol II at termination site If phosphorylated Pol II is not released at the termination site in vip mutants, the role of VIP complex is to prevent the stalling of Pol II at termination site by assisting dephosphorylation of CTD of Pol II.
Figure 5-1 The phosphorylation cycle of the CTD of Pol H.
The unphosphorylated CTD of Pol II is targeted on Ser-5 by the kinase activity of the Kin28 subunit of TFHH Ctkl kinase in yeast targets Ser-2 on the CTD During elongation, phosphatases dephosphorylate Ser-5 residues within the CTD The precise details surrounding the identity of the phosphatases and the specific residues that are targeted are not clear However, the Ser-5-specific phosphatase Ssu72 is involved in allowing the correct transcript cleavage necessary for efficient termination Fepl, a CTD- phosphatase is involved in recycling the Pol II for reinitiation and subsequent rounds of transcription, although the specifics of recycling are currently unknown In the diagram, the arrow indicates a promotive effect, while the “L” indicates an inhibitory effect (this figure is adapted from Sims et al., 2004).
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