Research Paper
Generation and characterization of antibody against paf1 complex in Drosophila melanogaster
WEI Wen-Xiang1,2, YANG Ji-Cheng1, ZHUANG Wen-Zhuo1, BAI Yan-Yan1, SHENG Wei-Hua1, MIAO Jing-Cheng1,*
1Department of Cellular and Molecular Biology, School of Medicine, Soochow University, Suzhou 215123, China; 2Howard Hughes Medical Institute, Division of Nucleic Acids Enzymology, Department of Biochemistry,University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA
Abstract: Paf1 complex was identified in yeast and characterized to function in transcription and its related events. We identified the Drosophila homological components of paf1, CDC73 and RTF1 of paf1 complex. The genes encoding Drosophila paf1, CDC73 and RTF1 were cloned and expressed. With the purified recombinant proteins of truncated components of paf1 complex, antibodies against the Drosophila paf1, CDC73 and RTF1 were generated. These antibodies have been shown to be able to detect the endogenous paf1 subunits as well as their human counterparts in the HeLa extract. On Drosophila polytene chromosomes, these antibodies have been demonstrated to locate the paf1 complex at actively transcribing sites, which co-localized with phosphorylated RNA polymerase II, indicating that paf1 complex in Drosophila is involved in transcription or the events coupling with transcription.
Key words: paf1 complex; paf1; CDC73; RTF1; transcription
抗果蝇 paf1 复合体抗体的制备及其特性
1苏州大学医学院细胞与分子生物学教研室，苏州 215123；2新泽西医学及齿科大学，罗伯特·伍得·约翰医学院，浩伍得 休斯医学研究所，生物化学系核酸酶研究室，新泽西，匹斯卡韦 08854，美国
摘 要：Paf1复合体已在酵母菌被发现，并阐明了其在转录及RNA加工过程中发挥功能。我们在果蝇基因组数据库中鉴定 出paf1的三个同源成分：paf1，CDC73和RTF1。应用纯化的重组蛋白，我们制备了抗果蝇paf1，CDC73和RTF1的抗体。这些抗体不但能够检测出果蝇内源性paf1成分，而且能检测出人类的paf1复合体的亚基。在多线染色体上，这些抗体 所检测到的paf1复合体均位于转录的活性位点，并且与磷酸化的RNA聚合酶重合，表明果蝇paf1复合体参与了转录及其相 关的分子活动。
关键词：paf1复合体；paf1；CDC73；RTF1；转录
中图分类号：R3
Transcription is a consecutive and concerted process with recruitment and release of various transcription factors to and from the transcription machinery as it assembles and elongates along a transcribing gene. To start transcription, activators bind to specific DNA elements in the promoter. Activator recruits coactivators, which in turn can influence the accessibility of chromatin structure and progression of the transcription machinery through initiation, promoter clearance, and the maturation of RNA polymerase II (Pol II) into a productive elongation and RNA processing complex[1-5].
The mechanisms by which some protein complexes participate broadly in Pol II transcription remain mysterious. Paf1 is such a complex, which is composed of five subunits of paf1, CDC73, RTF1, Leo1, Ctr9 in yeast[6]. Initially paf1 was identified as a complex associated with elongating Pol II[7-9] and hence was principally taken as an elongation factor[10]. Actually some evidence supports the role of paf1 as an elongation factor. The mutations in the paf1 components conferred sensitivity to the 6-arauracil, suggesting a function in elongation[11] . The expression of a subset gene was altered in the mutants of disruption of paf1 components[2]. Importantly Aguilera group confirmed an elongation function of paf1 by in vitro transcription. They compared the cell extract of paf1 mutants with that of wild type for elongation assay and found that the elongation efficiency of paf1 mutants were greatly reduced compared with that of the wild type, suggesting an important role of paf1 in the elongation[13]. Paf1 complex has also been connected to histone methylation, chromatin structure, signal transduction and tumorigenesis[14-17].
However some other observations do not support a transcription function of paf1. The disruption of paf1 components only interferes with the expression of small proportion of yeast genes in contrast to the majority of genes affected in the expression in the SRB mutants, indicating that paf1 is not a general and critical factor for the gene expression. The knockout of paf1 components, even those which dissociate the paf1 complex from the Pol II complex, did not affect the Pol II abundance on the chromatin template, nor affect other elongation factors such as Spt5 and FACT[18].
Although paf1 was found to be involved in the expression of a subset of genes, it has been identified in all active genes tested in yeast. In the purified elongation factors, the amount of paf1 was comparable to Pol II and other elongation factor such as FACT, Spt5, etc[11,18,19]. Actually the abundance and distribution of paf1 is similar to Pol II on the chromatin transcribing template. It seems that paf1 is not a local factor regulating some genes, but likely a factor playing a global role in gene expression. To test this hypothesis, we examined paf1's distribution on Drosophila polytene chromosome under normal conditions. To correlate the paf1 complex with the transcription in Drosophila, we started to generate and characterize the antibodies against Drosophila paf1 complex. With the antibodies against paf1 complex, it is possible to characterize the localization and distribution of paf1 on polytene chromosome and further to demonstrate the function of paf1 complex.
1 MATERIALS AND METHODS
1.1 Plasmid constructions
Drosophila genes of paf1 (CG2503), CDC73 (CG11990) and RTF1 (CG10955) were identified by searching the Drosophila genome database[20] for the homologs with the counterparts of Saccharomyces cerevisiae. The Drosophila cDNA clones were purchased from Open Biosystems. A DNA fragment encoding N-terminal of paf1 amino acid residuals from 1 to 238 (paf1-N) was amplified by PCR with primer pair of 5'TCCGAATTCATGCCACCCA-CGATCAAC 3'and 5' ATGCGGCCGCCTACAGCTGGG-CGGGCACGTT 3' DNA fragment encoding N-terminal of CDC73 amino acid residuals from 1 to 176 (CDC73-N) was amplified with primer pair of 5?TCCGAATTCAT-GGCAGATCCGCTCAGC 3' and 5' ATGCGGCCGCCTA-CGTCTCGGACAGCGACTT 3' DNA fragment encoding C-terminal of RTF1 amino acid residuals from 639 to 767 (RTF1-C) were amplified by PCR with primer pair of 5'GCAAGCTTAACGACAGGAACAGGAAGA 3'and 5' TAGCGGCCGCATCCTCTAGATTCAGCGAACG 3' The PCR products for paf1-N and CDC73-N were digested with EcoR I and Not I, while the PCR product for RTF1-C was digested with Hind III and Not I. The vector pET30a (Novagen) was digested with EcoR I and Not I for paf1-N and CDC73-N, or digested with Hind III and Not I for RTF1-C. The digested vectors and inserted sequences were purified and ligated to transform the competent Escherichia coli. The positive clones were selected and sequenced.
1.2 Preparation of recombinant proteins, generation and characterization of antibody
The bacterial expression vectors of Drosophila paf1-N, CDC73-N and RTF1-C in pET30a were used to transform the Escherichia coli BL21.The His-tagged truncated proteins were expressed in Escherichia coli by induction with 0.4 mmol/L isopropyl-D-thio-galactopyranoside (IPTG) at 30 ℃ for 3 h. The cells were harvested and sonicated in phosphate-buffered saline containing 1% Triton X-100 (PBST)[4,5]. After centrifugation, the extracts (supernatants) were collected and stored at ?0 ℃. Recombinant protein in the cell lysate was purified using Ni-NTA magnetic agarose beads (Qiagen) and eluted from the resin according to manufacturer's recommendation. Protein was quantified either by Bradford assay or direct comparison with BSA standard. Purified recombinant proteins were used as antigens for the generation of rabbit antibodies according to the protocol of Antibodies: a laboratory manual (Ed Harlow and David Lane). 100 μg aliquots of each recombinant protein were sent to Pocono Rabbit Farm and Laboratory for rat antibody production.
To characterize the antibody, 50 μg of Drosophila or HeLa extracts were applied in the SDS-PAGE together with bacterial lysate expressing recombinant paf1-N or bacterial lysate which serves as a negative control. Samples were fractionated by 12.5% SDS-PAGE, transferred onto nitrocellulose membranes, and subjected to Western blot analysis with the antibody. The proteins were visualized by enhanced chemiluminescence (ECL), according to the manufacturer's instructions (Amersham Pharmacia Biotech).
1.3 Indirect immunofluorescence
Polytene squashes were performed as previously described with minor modifications to the heat shock procedure[21]. Squashes were performed immediately thereafter, as was immunofluorescence staining. Briefly, slides were washed in phosphate buffer saline (PBS) and were blocked for 60 min in PBS with 5% non-fat skim milk. Antibodies were added, in TBS with 1% BSA, to the following dilutions: H14, 1:50 (Covance, Inc.); and paf1, 1:50. Chromosomes were incubated with primary antibodies overnight at 4 ℃, and then the slides were washed in PBS and were treated with the appropriate secondary antibody at 1:500 (Jackson Laboratory, Inc., or Molecular Probes, Inc.) in PBS with 1% BSA and 2% donkey serum for 1 h at 25 ℃. Slides were then washed in PBS, stained with Hoechst 33258 (Sigma), mounted in antifade solution, and analyzed. Analysis was performed by using a Zeiss Axioplan 2 microscope and OpenLab 3.0.7 imaging software. Images were taken by using the OpenLab z-series capture sequence and three-dimensional restoration.
2 RESULTS
2.1 Expression and purification of truncated proteins of paf1 subunits
As the paf1, CDC73 and RTF1 are core subunits of paf1 complex, we generated antibodies against Drosophila paf1, CDC73 and RTF1 to characterize the function of paf1 complex in Drosophila melanogaster. Drosophila genes of paf1, CDC73 and RTF1 were identified by searching the Drosophila genome database[20] for the homolog with yeast counterparts. The cDNA clones were purchased from Open Biosystems. To express and purify the recombinant proteins as antigens for the generation of antibodies against the subunits of Drosophila paf1 complex, we constructed the expression vectors encoding paf1-N, CDC73-N) and RTF1-C, respectively, with His-tag in the N-terminal of pET30a (Fig.1). The expression vectors were introduced into Escherichia coli and the truncated proteins were induced by IPTG. Bacterial lysate without transfection by expression vectors was also incubated with Ni-NTA magnetic agarose beads and subjected to the same extensive washing and elusion as a negative control. The eluted proteins were titrated and fractionated by 15% SDS-PAGE. As shown in Fig.2, there were considerable amount of expressed and purified paf1-N and CDC73-N and RTF1-C. However there was no eluted protein from the beads of the negative control with bacterial lysate, as was expected, indicating the specificity of purification of His-tagged recombinant protein.
2.2 Generation and characterization of rabbit antibody against Drosophila paf1 subunits
After the purification of His-tagged recombinant proteins, the truncated Drosophila paf1-N, CDC73-N and RTF1-C were applied as antigens to immune the rabbit for the generation of antibodies against paf1, CDC73 and RTF1. The bleedings from rabbits were tested and the serum was collected until both recombinant antigen and endogenous components of Drosophila paf1 complex were detected.
The immunoglobulin was purified from serum with affinity column conjugated with antigen of recombinant paf1-N. For Western blot analysis of the antibody against paf1, Drosophila extracts from both adult and larva were checked with recombinant paf1-N as a positive control. Bacterial lysate of non-transfection as well as HeLa cell extract were applied as additional controls (Fig.3). The rabbit antibody against paf1 (Fig.3A) detected the recombinant paf1-N as well as a band about 72 kDa in both larva and adult extracts (lanes 2 and 3), which was the Drosophila endogenous paf1. Interestingly a band about 75 kDa slightly higher than Drosophila endogenous paf1 was detected in HeLa cell extract (lane 4), which has been identified as the endogenous human paf1.
The rabbit antibody against Drosophila CDC73 was also tested for its specific recognition of recombinant and endogenous CDC73. The purified CDC73 antibody identified the same size bands in Drosophila extracts of both larva and adult (Fig.3B), which were the expected size of 73 kDa for the endogenous CDC73 protein. A band lower than the endogenous Drosophila CDC73, about 71 kDa, was also detected in HeLa cell extract (Fig.3B, lane 8), which was the expected endogenous human CDC73 homolog. In the negative control there was no detectable band in bacterial lysate by the rabbit antibody against Drosophila CDC73 (Fig.3B, lane 5). The identification of human homologs by rabbit antibodies against both Dosophila paf1 and CDC73 was further confirmed by the detection of human paf1 and CDC73 subunits in the purified human paf1 complex with the Drosophila antibodies (data not shown).
A specific 90 kDa band of endogenous RTF1 in the Drosophila larva extract was detected with the rabbit antibody against Drosophila RTF1 of paf1 complex (Fig.3C, lane 10). With clean background, there was no any detectable band in bacterial lysate, Drosophila adult extract and HeLa extract in the Western blot (Fig.3C, lanes 9, 11 and 12).
2.3 Generation and characterization of rat antibody against Drosophila paf1 subunits
With recombinant proteins not only rabbit antibodies, but also rat antibodies against paf1 complex were generated. Antibodies to the same antigen and from different sources are able to verify the specificity of each other and are also useful for the identification of co-localization of different antigens on Drosophila polytene chromosome.
To confirm the specificity of rabbit antibody, we further tested the antibody to Drosophila paf1 from rat. The affinity-purified antibody from rat was applied in the Western blot analysis as shown in Fig.4A. The result detected by rat antibody to Drosophila paf1 was similar to the result with rabbit antibody in Fig.3A. Rat antibody against Drosophila paf1 recognized recombinant paf1-N as well as the endogenous paf1 in both adult and larva extracts (Fig.4A, lanes 1~3). Strikingly similar to the case by rabbit antibody, rat antibody to paf1 detected a band about 75 kDa in HeLa extract, which was supposed to be the human paf1. We further confirmed the human paf1 component of 75 kDa by application of antibodies against Drosophila paf1 from both rabbit and rat in purified human paf1 complex (data not shown).
The rat antibody against Drosophila CDC73 was also checked for its specificity to endogenous CDC73. The result demonstrated the CDC73 antibody from rat could also detect a 73 kDa band of endogenous CDC73 protein in Drosophila extracts of both larva and adult (Fig.4B). However in the negative control, there was no detectable band in bacterial lysate by the same antiboy (Fig.4B, lane 5).
2.4 Co-localization of paf1 with phosphorylated RNA Pol II on polytene chromosome
After characterization of antibodies against Drosophila paf1 complex with recombinant paf1 proteins and in Drosophila extract, we wonder if these antibodies could detect paf1 complex on polytene chromosome. As shown in Fig.5A, the immunostaining of the chromosome with the antibody against paf1 subunit demonstrated the distribution of paf1 along all chromosomes with strong signal. The H14 antibody which targets the Pol II with CTD phosphorylated at serine 5 (Ser5-P) was co-stained with antibody to paf1 and Hoechest 33258 which stains the DNA for the mapping of chromosome. Interestingly the paf1 (red) strikingly co-localized with the phosphorylated Pol II (green) along the chromosomes (merged). Then we checked the localiazation of two other subunits of CDC73 and RTF1, which were shown to be important components in the yeast paf1 complex. The co-staining of H14 antibody with antibody to either CDC73 or RTF1 also demonstrated close co-localization of the two subunits with the phosphorylated Pol II on the chromosome, which was similar to the co-localization of paf1 subunit with the phosphorylated Pol II (Fig.5B, C).
To systematically investigate the localization of paf1 complex on the polytene chromosomes, we did a fine mapping on the chromosomes co-stained with antibody against both paf1 and H14 antibody. The chromosome spreads immunostained with the antibodies were also stained by Hoechest 33258 to demonstrate consistent and clear banding on polytene chromosome. Interestingly phosphorylated Pol II co-localizes with paf1 complex at transcription-active sites. For example both the paf1 complex and phosphorylated Pol II are abundant at the ecdysone puffs (21F, 71DE, 78D and 98F, etc.), which are highly active transcription sites at developmental stage. However there were barely detectable paf1 at the sites which contain endogenous heat shock genes such as hsp70 at 87 A&C, hsp83 at 63B, and minor heat shock genes (hsp22, hsp23, hsp26 and hsp27) at 67B. There was either no paf1 detected at the 59B which harbors a transgenic hsp70-LacZ gene. These heat shock genes were inactive under non-heat shock condition. The close co-localization of paf1 with phosphorylated Pol II suggests that paf1 may be involved in or related to the gene transcription.
To confirm the band specificity generated by antibodies against paf1 components, competition assay was carried out in which CDC73 polypeptide (CDC73-N purified as antigen) was applied as a competitor for the immunostaining by the rabbit antibody to CDC73. Increasing amount of CDC73 polypeptide used as antigen was added to reaction mixture to neutralize the rabbit antibody to CDC73 mixed together with rat antibody to paf1. After an incubation of 10 min, the mixture was applied for polytene chromosome staining overnight. As shown in Fig.6, CDC73 polypeptide completely blocked the immunostaining by rabbit antibody to CDC73 and the CDC73 bands disappeared from the chromosome spread. Meanwhile on the same chromosome spread confirmed by the DNA staining (blue), the bands generated by rat antibody against paf1 (red) remained intact after the treatment of CDC73 polypeptide. In the merged picture of two subunits staining by paf1 and CDC73 antibodies, only bands detected by paf1 (red) remained. This observation strongly suggest the banding specificity generated by the antibodies to paf1 and CDC73 and hence supports our conclusion that paf1 complex is required for the process of gene transcription.
3 DISCUSSION
Paf1 is a transcription complex identified recently and its roles in both transcription and RNA processing are becoming more and more important. However the paf1 complex has been identified and characterized mainly in yeast. To identify and characterize the paf1 complex in high eukaryote, we generated antibodies against three core subunits of paf1 complex in Drosophila. With the antibodies to paf1 complex, it is possible to locate the paf1 complex on the Drosophila polytene chromosome with its correlation with the actively transcribing sites where the phosphorylated Pol II locates. If paf1 complex is a factor of globe transcription, then it should be observed in transcriptionally active sites on polytene chromosomes.
From the Drosophila genome database we identified the homolog genes encoding paf1, CDC73 and RTF1 components. And with the recombinant proteins, we generated and characterized the antibodies against paf1 complex both in Drosophila extract and on polytene chromosome. The data from our observation support a function of paf1 complex in the actively transcribing genes. Polytene chromosome staining demonstrates that paf1 complex does not exist in the non-transcribing gene, such as hsp70 in the non-heat shock status (Fig.5). However at developmental sites, such as ecdysone puffs (21F, 71DE, 78D and 98F, etc) which are highly active transcription sites at developmental stage, there were abundant paf1 complex at the locations.
Although it has been shown that only a subset gene expression was affected in the paf1 mutant in yeast, our high resolution mapping of genome-wide distribution of paf1 on polytene chromosome demonstrates that most paf1 co-localizes with the phosphorylated Pol II at the active sites under the physiological condition of non-heat shock status. This observation strongly suggests that paf1 might be a general factor coupling to the transcription events, and functions in the transcription or posttranscription or both events.
Not only paf1 co-localizes with phosphorylated Pol II, but also the abundance of paf1 components is in proportional to the Pol II. The distribution of paf1 components and phosphorylated Pol II is also similar. Although it is difficult to quantify the immunostaining by different antibodies, we can compare the staining pattern and distribution of factors stained by different antibodies on the polytene chromosome. The components of paf1, CDC73 and RTF1 co-localized with phosphorylated Pol II at transcription active sites. We observed the distribution and abundance of paf1 components along the polytene chromosomes were similar to that of Pol II as well as to each other subunit of paf1 complex. For example, the sites of 21F, 71DE, 78D and 98F of the development puffs on the non-heat shock chromosomes were of a few locations with most abundance of paf1, CDC73 and RTF1. And actually these sites were also the locations of highest intensive staining with antibody against phosphorylated Pol II.
Although it has been identified that paf1 correlates to globe transcribing genes, the detail function of paf1 remains unclear. Paf1 is dispensable for the normal distribution and abundance of transcribing Pol II and elongation factors[18]. Paf1 does not help in recruiting the elongation factor to the transcription machinery. The major function of paf1 may still remain to be identified.
ACKNOWLEDGEMENTS: We thank Drs. Danny Reinberg and Bing Zhu in the University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School for the creative ideas and assistance in the generation of Drosophila paf1 antibodies. We also thank Drs. John T. Lis, Janis Werner and Karen Adelman in Cornell University for the assistance in immunofluorescence of polytene chromosome.
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