Annals of Clinical & Laboratory Science 35:270-277 (2005)
© 2005 Association of Clinical Scientists
SMYD3-NY, a Novel SMYD3 mRNA Transcript Variant, May Have a Role in Human Spermatogenesis
Zuomin Zhou,
Xuan Ren,
Xiaoyan Huang,
Li Lu,
Min Xu,
LanLan Yin,
Jianmin Li and
Jiahao Sha
Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, People’s Republic of China
Address correspondence to Dr Jiahao Sha, Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, 210029, People’s Republic of China: tel and fax 86 25 8686 2908; e-mail shajh{at}njmu.edu.cn.
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Abstract
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Identification of genes specifically expressed in adult and fetal testis is important to further our understanding of testis development and function. In this study, a novel SMYD3 transcript variant, termed SMYD3-NY (GenBank Accession No. AY186742
[GenBank]
), was identified by hybridization of adult and fetal human testis cDNA probes with a human cDNA microarray. SMYD3-NY transcript was expressed at 2.3-fold higher levels in adult human testis than in fetal testis, with a low expression level in human spermatozoa. Bioinformatical analysis showed that SMYD3-NY protein has the SET domain that is involved in histone methyltransferase (HTMase) activity. Southern blotting showed that SMYD3-NY is distributed in several tissues, including testis. In summary, SMYD3-NY is a novel transcript variant of the SMYD3 gene, and SMYD3-NY protein may influence transcriptional regulation during spermatogenesis via HTMase activity.
(received 17 January 2005; accepted 9 March 2005)
Keywords: testis cDNA microarray, SET domain, histone methylation, spermatogenesis
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Introduction
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In nuclei of all eukaryotic cells, genomic DNA is highly folded, constrained, and compacted by histone and nonhistone proteins in a dynamic polymer called chromatin. Histones are small basic proteins consisting of a globular domain and a more flexible and charged NH2-terminus (histone tail). Histone tails are susceptible to covalent modification, including acetylation, phosphorylation, methylation, and ubiquitination [1–4]. A convincing body of evidence shows that such histone modifications regulate chromatin structure as well as transcriptional activation and repression. Among the modifications, histone lysine methylation on lysine residues 4, 9, 27, and 36 in H3, and on residue 20 in H4 are critical for transcriptional regulation [1,5,6]. Many histone lysine (K) methyltransferases (HMTases) have been characterized with a common SET domain (eg, SUV39H1, SUV39H2, SET7/SET9, SMYD3) [5,7,8].
Histone methylation was discovered 3 decades ago and its functional significance is now widely recognized, not only in mitosis, but also in meiosis. As we know, spermatogenesis is a unique process that comprises 3 phases: mitotic, meiotic, and postmeiotic. During spermatogenesis, a remarkable feature of gene expression is transcriptional silencing. The classical explanation of transcriptional silencing is the replacement of histones by protamines. Besides this, silencing has been shown to be coupled to heterochromatin condensation and recognition of methyated residues on histone tails by the protein HP1 (heterochromatin protein-1) [1,9]. After meiosis, the beginning of spermatogenesis is characterized by a massive wave of transcriptional activity. Histone methylation is highly related to transcriptional regulation during spermatogenesis [10], although the mechanism has not been thoroughly characterized. Therefore, the isolation and characterization of novel HMTases in testis may contribute to our understanding of the functions of histone methylation.
Using an adult testis cDNA microarray prepared in our laboratory, we compared the expression of genes in the fetal and adult human testis. A novel SMYD3 mRNA transcript variant was identified and named SMYD3-NY. To explore the function of SMYD3-NY, we studied its expression in various tissues. Based on these observations and bioinformatical analysis, we conclude that SMYD3-NY may be a novel gene with HMTase activity related to spermatogenesis.
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Materials and Methods
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Subjects and samples.
Informed consent was received from all participants, and the Ethics Committee of Nanjing Medical University approved the research protocol. Human adult testes were obtained from the Body Donor Center (Nanjing Medical University). Fetal testes were obtained from fetuses aborted at 6-mo gestation. Human ejaculates were obtained from healthy volunteers of proven fertility and normal semen quality, as assessed by the WHO (1999) criteria (ie, >20 x 106 spermatozoa/ml; >50% active sperm; >25% sperm moving forcefully in one direction [rapid and linearly progressive motion]; and <1 lymphocyte per high-power field [40 x magnification]).
Construction of human testis cDNA microarray.
A total of 9,216 positive phage clones were picked randomly from Human Testis Insert 
phage cDNA library (Clontech, Hl5503U) and amplified by PCR. The PCR products were spotted onto a membrane to make a human testis cDNA microarray as previously described [11].
Screening of genes differentially expressed in fetal testis, adult testis, and human spermatozoa.
The adult testis cDNA microarray was hybridized with 32P-labeled fetal testis, adult testis, and human spermatozoa cDNA probes, respectively, and microarray hybridization and data analysis were performed [11,12]. All differentially-expressed cDNA plasmids were proliferated, extracted, and purified and the full insert lengths were sequenced as previous described [11,12].
Characterization of positive cDNA clones.
The generated sequences were subjected to BLAST analysis [http://www.ncbi.nlm.nih.gov/], which identified one sequence as SMYD3-NY, a novel human SMYD3 mRNA transcript variant in human testis (GenBank Accession AY186742
[GenBank]
). GenBank sequence analyses were performed to identify SMYD3-NY homologs and their chromosomal localization. The nucleic acid-deduced amino-acid sequences of SMYD3-NY were analyzed with Gene Runner (http://www.generunner.com) and SMART PROGRAM software (http://smart.embl-heidelberg.de). Analysis with Promoter 2.0 Prediction Server software (http://www.cbs.dtu.dk/services/Promoter) was performed to find promoter regions. In this analysis, scores >1.0 indicate highly likely predictions.
Validation of cDNA microarray.
To validate the cDNA microarray results, SMYD3-NY specific RT-PCR was carried out on cDNA from human adult testis, fetal testis, and spermatozoa. RNA was extracted from the tissues with Trizol reagent (Gibco BRL, Grand Island, NY, USA) and reverse-transcribed into cDNA with AMV reverse transcriptase (Promega). The various cDNAs were PCR amplified with SMYD3-NY primers (P1, 5'-TAAGCGCCTCCTAGAAGAC-3', forward, nt 117–135; P2, 5'-CAGATGGTGAAAGAGTTGC-3', reverse, nt 403–421). ß-actin was used as the positive control. PCR was performed according to the manufacturer’s instructions; the reaction conditions were as follows: denaturation at 94° for 30 sec, annealing at 60° for 30 sec, and extension at 72° for 60 sec. The first cycle had a denaturation period of 5 min; the last cycle had an extension period of 7 min. Thirty-five cycles were performed and the PCR products were analysed after electrophoresis.
Tissue distribution of SMYD3-NY transcript.
To determine the tissue distribution of the SMYD3-NY transcript, SMYD3-NY specific primers were used to amplify cDNAs of 16 human tissues using the Human MTC Panel I and II kit (Clontech). PCR products of the 16 tissues were resolved by electrophoresis on 1% agarose gel and transferred to Hybond-N+ nylon membrane. The SMYD3-NY cDNA probe was labeled with the same primers used for the PCR. The template was a SMYD3-NY clone plasmid, and digoxigenin (Dig)-labeled dNTPs were used (Dig DNA Labeling Mix; Roche Diagnostics, Indianapolis, IN). The reaction solution contained 2 µl of 10x reaction buffer, 1.5 µl 25 mmol/L MgCl2, 1.5 µl Dig-dNTPs (1 mM dATP, 1 mM dCTP, 1 mM dGTP, 0.65 mM dTTP, 0.35 mM Dig-11-dUTP, alkali-labile), 0.5U Taq DNA polymerase, 12 µl distilled water, 1 µl template, and 1 µl of each primer. The reaction protocol was as follows: 35 cycles of 94° for 30 sec, 60° for 30 sec, and 72° for 60 sec. The membrane was hybridized with Dig-labeled probe, incubated with anti-Dig antibody conjugated with alkaline phosphatase (1:10,000 dilution) and CSPD chromogenic substrate (disodium 3-(4-methoxyspiro-{1,2-dioxetane-3,2'-{5'-chloro}-tricydo-[3.3.1.13,7]-decan}-4-yl) phenylphosphate, Roche), and exposed to X-ray film. The integrity of tissue cDNA was tested using the primers of human ß-actin.
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Results
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cDNA microarray hybridization and validation.
Hybridization intensities of SMYD3-NY in adult testis and fetal testis were 59.93 and 24.85, respectively (Fig. 1
), indicating 2.33-fold greater expression in the adult vs the fetal testis; the expression level in human spermatozoa was low. As shown in Fig. 2, RT-PCR confirmed that SMYD3-NY was expressed at a high level in adult testis and at low levels in fetal testis and human spermatozoa.
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Fig. 1. Partial cDNA hybridization images showing differential expression of SMYD3-NY in the fetal testis, adult testis, and spermatazoa. White rings indicate SMYD3-NY cDNA. The intensities of the hybridization images in adult testis and fetal testis were 59.93 and 24.85 units, respectively.
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Fig. 2. Validation of microarray hybridation results. RT-PCR indicates that SMYD3-NY expression was high in adult human testis, with lower levels in fetal testis and spermatozoa. The left panel shows SMYD3-NY expression and the right panel shows ß-actin expression as a control. M: marker; P: plasmid; A: adult testis; F: fetal testis; S: sperm; B: blank.
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Features of cDNA and deduced protein.
SMYD3-NY was 1387 bp in length and encoded a 258 amino acid protein with predicted molecular weight of 29kDa and an isoelectric point of 4.79. A Blast search in the human genome database localized SMYD3-NY to 1q44. Analysis of the amino acid sequence using SMART software revealed that the protein includes a SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain ranging from amino acids 1 to 76 of SMYD3-NY (Fig. 3).
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Fig. 3. Nucleotide and deduced amino acid sequences of the cDNA for SMYD3-NY. The initiation and stop codon are bold and in italic type. The SET domain is boxed (76 aa). The specific primers with which the Southern blot and RT-PCR procedures were performed are shadowed.
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Homologous comparison of SMYD3-NY gene.
Blast search showed that SMYD3-NY gene bears a high similarity to SMYD3 gene (GenBank accession AB057595
[GenBank]
), which is also localized on human chromosome 1. Comparisons showed that they both have 7 identical exons in the 3'of cDNA. However, SMYD3-NY gene lacks the initial 5 exons of SMYD3; the 2 exons at the 5' of the cDNA are specific in SMYD3-NY, and are not found in SMYD3 (Fig. 4). The cDNA of SMYD3-NY is shorter than SMYD3, and contains many highly likely predicted promoter regions (Fig. 4), indicating that the two genes are transcribed from the same DNA via different promoter regions. Two motifs responsible for the HMTase activity in the SET domains of methyltransferases are also conserved in the sequences of SMYD3-NY (Fig. 5).
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Fig. 4. Transcript comparison of SMYD3-NY with SMYD3. Seven identical exons are shown in black; the differences are shown with gray (SMYD3) and empty rectangles (SMYD3-NY). Between the 2 arrows, the black triangles indicate the multiple promoter regions in the genome sequences.
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Fig. 5. Sequence similarity of SET domains of SMYD3-NY with those of SMYD3, human SUV39H1, human SUV39H2, and SET7/9 proteins. Conserved sequences that may be involved in the activity in the SET domains are boxed and discussed in the text.
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Tissue distribution of SMYD3-NY transcript.
Expression of SMYD3-NY in 16 tissues was tested by hybridization. The results indicated that SMYD3-NY mRNA is expressed highly in the testis, ovary, placenta, kidney, spleen, and skeletal muscle; weakly in the heart, brain, lung, liver, prostate gland, small intestine, and leukocytes; and almost imperceptibly in the colon, thymus, and pancreas (Fig. 6).
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Fig. 6. Southern blot containing cDNA from various human tissues (a multiple-tissue cDNA, Clontech) was hybridized with Dig-labeled SMYD3-NY (304 bp) and ß-actin (247 bp), respectively. Upper line: SMYD3-NY mRNA is expressed highly in the testis, ovary, placenta, kidney, spleen, and skeletal muscle; weakly in the heart, brain, lung, liver, prostate gland, small intestine, and leukocytes; and almost imperceptibly in colon, thymus, and pancreas. Lower line: Expression of ß-actin in these tissues was used as a positive control. He: heart; Br: brain; Pl: placenta; Lu: lung; Li: liver; Sm: skeletal muscle; Ki: kidney; Pa: pancreas; Po: positive plasmid; Sp: spleen; Th: thymus; Pr: prostate gland; Te: testis; Ov: ovary; Si: small intestine; Co: colon; Le: leukocytes; Nc: negtive control.
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Discussion
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In eukaryotes, organization of higher order chromatin structure is believed to be essential for epigenetic gene control and proper chromosome condensation in mitosis [13]. Targeted covalent modification of the amino-terminal tails of the core histones in nucleosomes has emerged as an important mechanism in this process [14]. Various modifications of the N-terminal of the histone tail are well documented and have been functionally characterized as changes in acetylation, phosphorylation, and most recently, methylation [15]. Much evidence shows that histone methylation plays an important role in transcriptional regulation and that during this process, histones are methylated only on arginine and lysine residues [7]. There are 2 prime families of histone methyltransferases (HMTases): protein arginine methyltransferases (PRMTs) and protein lysine methyltransferases. Recent findings indicate that a nuclear receptor co-activator-related protein, CARM1, which is an arginine-specific histone methyltransferase (HTMase), contributes to transcriptional activation [16,17]. The human homolog of the Drosophila heterochromatic protein Su(var)3-9, a H3-specific lysine methyltransferase, is believed to contribute to the organization of repressive chromatin regions [14,18].
Histone lysine (K) methylation occurs on lysine residue 4, 9, 27, and 36 in H3, and on position 20 in H4 [5]. A hallmark signature of this class of HMTase is the presence of the 130-amino-acid SET domain, which is crucial for catalytic activity [18]. The SET domain was named after 3 proteins in which it was originally identified in Drosophila: (i) suppressor of variegation SU(VAR)3-9, (ii) enhancer of zeste E(Z), and (iii) trithorax (TRX) [19]. SU(VAR)3-9 is a suppressor of PEV (position effect variegation), which is heterochromatin-induced gene silencing [20]; enhancer of zeste and trithorax are members of the polycomb group (pcG) and trithorax group (trG) of proteins, respectively; PcG proteins are involved in stable repression of homeotic genes, whereas TrG proteins are needed for the stable expression of homeotic genes in appropriate segments, during the development of Drosophlia [21]. SET domain genes are widely represented in eukaryotic genomes. In the current database, there are around 300 real and hypothetical SET-domain genes [22]. Identification of the SET-domain gene in the SU(var)3-9 family shows that they all have the activity of histone methyltransferase (HMTase). In addition to its role in somatic cells, histone methylation is important in meiosis [9,23].
In the present study, a human testis cDNA microarray was constructed and used to identify genes related to spermatogenesis. As a result a human cDNA representing a novel gene containing the SET domain was isolated and characterized. After bioinformatical analysis, we concluded that it is a novel transcript variant of the SMYD3 gene, and therefore named it SMYD3-NY. The 2 genes are transcribed from the same genome sequence via different promoter regions (Fig. 4
). In the SET domain of other proteins, such as SMYD3, SUV39H1, SUV39H2, and SET7/9, 2 conserved amino-acid sequences (NHSCXPN and GEELXXXY) are considered responsible for HMTase activity and these 2 conserved motifs are also conserved in the SET domain of SMYD3-NY (Fig. 5).
SMYD3 was isolated from colorectal carcinoma and hepatocellular carcinoma by Hamamoto et al [8] in 2004. They found a 1.7 kb transcript that is expressed specifically in testis and skeletal muscle and encodes a putative 428-amino-acid protein containing a SET domain (codons 148–239) and a zf-MYND domain (codons 49–87); the authors named this gene SMYD3 (SET- and MYND-domain-3). Because of the SET domain, SMYD3 has HMTase activity. Knowledge of histone methylation suggests that methylation of lysine 9 in histone H3 (H3-K9) is involved in transcriptionally-repressive heterochromatin formation [18]; and furthermore, that lysine-4 methylation of histone H3 (H3-K4) is important for transcriptional activation [24]. Hamamoto et al [8] confirmed that SMYD3 has H3-K4-specific HMTase activity. Due to the zf-MYND domain, SMYD3 can recognize and bind particular sequences of genomic DNA and produce transcriptional activation as well as HMTase activity [8].
The present study shows that SMYD3-NY is a novel transcript variant of SMYD3 and that it contains the SET domain. Since the zf-MYND domain cannot be found, we speculate that SMYD3-NY protein may have H3-K4 HMTase activity that is responsible for transcriptional activation. SMYD3-NY was isolated from a testis library and was highly expressed in human adult testis. Although it is expressed in several tissues (Fig. 6), we focused on its function during spermatogenesis. Spermatogenesis is a cyclic developmental process that is characterized by modifications in chromatin organization, basically during 2 periods, meiosis–which includes the synapsis and dysynapsis of the chromosomes–and the histone-protamine transition [9]. The replacement of histones by protamines results in silencing of transcription. Silencing has been shown to be coupled to heterochromatin condensation and recognition of methylated residues on histone tails by the protein HP1 [1]. After meiosis, the beginning of spermatogenesis is characterized by a massive wave of transcriptional activity. Evidence suggests that histone methylation may both positively and negatively regulate transcription [13]. Therefore, the histone methyltransferase gene may be involved in the process of spermatogenesis. Recently, a SUV39/clr4 family gene, SUVR39h2, expressed specifically in the testis, was reported to influence chromosomal alignments during meiotic divisions [10]. We suggest that SMYD3-NY may be a new SET-domain gene, found in testis, that affects the histone methylation involved in transcriptional activation.
In summary, SMYD3-NY, which is highly expressed in human adult testis, may play an important role in spermatogenesis through methylating the N-terminal tail of histones. Further study is required to gain information about the exact role of SMYD3-NY in spermatogenesis.
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Acknowledgements
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This study was supported by grants from National Project 973, China (#G1999055901), and the Chinese Natural Science Fund (#30170485).
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References
|
|---|
- Jenuwein T, Allis CD. Translating the histone code. Science 2001;293:1074–1080.[Abstract/Free Full Text]
- Brownell JE, Zhou J, Ranalli T, Kobayashi R, Edmondson DG, Roth SY, Allis CD. Tetrahymena histone acetyltransferase A: A homolog to yeast Gcn5p linking histone acetylation to gene activation. Cell 1996;84:843–851.[Medline]
- Turner JM, Aprelikova O, Xu X, Wang R, Kim S, Chandramouli GV, Barrett JC, Burgoyne PS, Deng CX. BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation. Curr Biol 2004;14:2135–2142.[Medline]
- Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000;403:41–45.[Medline]
- Lachner M, Jenuwein T. The many faces of histone lysine methylation. Curr Opin Cell Biol 2002;14:286–298.[Medline]
- Kouzarides T. Histone methylation in transcritptional control. Curr Opin Genet Dev 2002;12:198–209.[Medline]
- Zhang Y, Reinberg D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails. Genes Dev 2001; 15:2343–2360.[Free Full Text]
- Hamamoto R, Furukawa Y, Morita M, Iimura Y, Silva FP, Li M, Yagyu R, Nakamura Y. SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat Cell Biol 2004;6:731–740.[Medline]
- Sassone-Corsi P. Unique chromatin remodeling and transcriptional regulation in spermatogenesis. Science 2002;296:2176–2178.[Abstract/Free Full Text]
- O’Carroll D, Scherthan H, Peters AH, Opravil S, Haynes AR, Laible G, Rea S, Schmid M, Lebersorger A, Jerratsch M, Sattler L, Mattei MG, Denny P, Brown SD, Schweizer D, Jenuwein T. Isolation and characterization of Suv39h2, a second histone H3 methyltransferase gene that displays testis-specific expression. Mol Cell Biol 2000;20:9423–9433.[Abstract/Free Full Text]
- Sha JH, Zhou ZM, Li JM, Yin LL, Yang HM, Hu GX, Luo M, Chan HC, Zhou KY. Spermatogenesis study group: Identification of testis development and spermatogenesis-related genes in human and mouse testis using cDNA microarray. Mol Hum Reprod 2002;8:511–517.[Abstract/Free Full Text]
- Wang H, Zhou ZM, Xu M, Li JM, Xiao JH, Xu ZY, Sha JH. A spermatogenesis-related gene expression profile in human spermatozoa and its potential clinical significance. J Mol Med 2004;82:317–324.[Medline]
- Tachibana M, Sugimoto K, Fukushima T, Shinkai Y. Set domain-containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem 2001;276:25309–25317.[Abstract/Free Full Text]
- Cheung P, Allis CD, Sassone-Corsi P. Signaling to chromatin through histone modifications. Cell 2000; 103:263–271.[Medline]
- Jenuwein T, Laible G, Dorn R, Reuter G. SET domain proteins modulate chromatin domains in eu- and heterochromatin. Cell Mol Life Sci 1998;54:80–93.[Medline]
- Nakajima T, Fukamizu A, Takahashi J, Gage FH, Fisher T, Blenis J, Montminy MR. The signal-dependent coactivator CBP is a nuclear target for pp90RSK. Cell 1996;86:465–474.[Medline]
- Chen D, Huang SM, Stallcup MR. Synergistic, p160 coactivator-dependent enhancement of estrogen receptor function by CARM1 and p300. J Biol Chem 2000;275:40810–40816.[Abstract/Free Full Text]
- Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T. Regulation of chromatin structure by site specific histone H3 methyltransferase. Nature 2000;406:593–599.[Medline]
- Tschiersch B, Hofmann A, Krauss V, Dorn R, Korge G, Reuter G. The protein encoded by the Drosophilia position-effect variegation suppressor gene su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J 1994;13:3822–3831.[Medline]
- Reuter G, Spierer P. Position effect variegation and chromatin proteins. Bioessays 1992;14:605–612.[Medline]
- Van Lohuizen M, Tijms M, Voncken JW, Schumacher A, Magnuson T, Wientjens E. Interaction of mouse polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes. Mol Cell Biol 1998;18:3572–3579.[Abstract/Free Full Text]
- Alvarez-Venegas R, Avramova Z. SET-domain proteins of the Su(var)3-9, E(z) and trithorax families. Gene 2002;285:25–37.[Medline]
- Dernburg AF, Sedat JW, Hawley RS. Direct evidence of a role for heterochromatin in meiotic chromosome segregation. Cell 1996;86:135–146.[Medline]
- Schneider R, Bannister AJ, Myers FA, Thorne AW, Crane-Robinson C, Kouzarides T. Histone H3 lysine 4 methylation patterns in higher eukaryotic genes. Nat Cell Biol 2004;6:73–77.[Medline]
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Abstract
Introduction
