Construction and Expression Analysis of Eukaryotic Fusion Expression Vector Carrying Flag Tag of Ash2l Gene in Chicken (Gallus gallus)
ZHANG Chen, ZUO Qi-Sheng, ZOU Yi-Chen, ZHAO Juan-Juan, ZHANG Ya-Ni, LI Bi-Chun*
Institutes of Agricultural Science and Technology Development/Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China/College of Animal Science and Technology, Yangzhou University Yangzhou, 225009, China
Abstract:Absent, small, or homeotic-like (ASH2L) is an important histone methylation modification enzyme involved in a variety of cell development. However, its function and molecular mechanism in the process of male germ cell development are not clear. The purpose of this study was to study the expression of Ash2l in the formation of chicken (Gallus gallus) male reproductive stem cells and to construct Ash2l eukaryotic fusion expression vector NCBI conserved domain analysis, UniPort, TMHMM Serverv.2.0 and SignalP online websites were used to analyze the conserved domain, hydrophilic and hydrophobic domain, transmembrane domain and signal peptide of ASH2L. qRT-PCR was used to detect the expression of Ash2l in different cells (embryonic stem cells (ESC), primordial germ cell (PGC) and spermatogonial stem cell (SSC)) and tissues of 18.5 d chicken embryo. The recombinant vector pcDNA3.1-Ash2l-Flag was identified by double restriction endonuclease digestion and sequencing. The expression of recombinant protein in PGC was detected by qRT-PCR and Western blot.The binding of recombinant protein and interaction protein was verified by Co-immunoprecipitation (Co-IP). The results showed that ASH2L protein contained 2 conserved domains, SPIa and ryanodine receptor (SPRY) and plant homeodomain (PHD) had histone methylase characteristic domain, and had high hydrophilicity, no transmembrane domain and no signal peptide expression. The results of qRT-PCR showed that the expression of Ash2l in testis was the highest, which was significantly higher than that in other tissues (P<0.01), and that in PGC was significantly higher than that in other cells (P<0.01). The results of double restriction endonuclease digestion showed that Ash2l was correctly inserted into pcDNA3.1 vector without frameshift and mutation. qRT-PCR showed that the recombinant vector in PGC could overexpress Ash2l gene, and Western blot indicated that the recombinant protein was successfully expressed in PGC. Co-IP results showed that the recombinant protein could bind to the interacting proteins Dpy-30 histone methyltransferase complex regulatory subunit (DPY30) and WD repeat containing protein 5 (WDR5) to form a complex. These results provide a reference basis and technical means for the study of the function and mechanism of Ash2l in the formation of male germ cells in chickens.
[1] 何娜娜. 2018. 组蛋白H3K4me2对鸡SSCs形成的作用机制研究[D]. 硕士学位论文, 扬州大学, 导师: 李碧春, pp. 54-78. (He N N.2018. Study on the function and regulation mechanism of H3K4me2 in the formation of chicken SSCs[D]. Thesis for M.S., Yangzhou University, Supervisor: Li B C, pp. 54-78.) [2] 谢晶. 2018. Ash2l-1/Ash2l-2在小鼠胚胎干细胞中的表达特异性及互补效应[D]. 硕士学位论文, 西北农林科技大学, 导师: 张仕强, pp. 1-45. (Xie J, 2018. Expression specificity and compensation effect of Ash2l-1/Ash2l-2 in mouse embryonic stem cells[D]. Thesis for M.S. Northwest A& F University, Supervisor: Zhang S Q, pp. 1-45.) [3] Adriana R, Hugo S, Berta H, et al.2018. Mll‐COMPASS complexes mediate H3K4me3 enrichment and transcription of the osteoblast master gene Runx2/p57 in osteoblasts[J]. Journal of Cellular Physiology, 234(5): 6244-6253. [4] Bienz M.2006. The PHD finger, a nuclear protein-interaction domain[J]. Trends in Biochemical Sciences, 31(1):35-40. [5] Chen Y, Wan B, Wang K C, et al.2011. Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding[J]. EMBO Reports, 12(8): 797-803. [6] Douillet D, Sze C C, Ryan C, et al.2020. Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, polycomb and DNA methylation[J]. Nature Genetics, 52(6): 615-625. [7] Einhauer A, Jungbauer A.2001. The FLAG peptide, a versatile fusion tag for the purification of recombinant proteins[J]. Journal of Biochemical and Biophysical Methods, 49(1-3): 455-465. [8] Filippakopoulos P, Low A, Sharpe T D, et al.2010. Structural basis for Par-4 recognition by the SPRY domain-and SOCS box-containing proteins SPSB1, SPSB2, and SPSB4[J]. Journal of Molecular Biology, 401(3): 389-402. [9] Hsu P L, Li H, Lau H T, et al.2018. Crystal Structure of the compass H3K4 methyltransferase catalytic module[J]. Cell, 174(5): 1106-1116.e9. [10] Ikegawa S, Isomura M, Koshizuka Y, et al.1999. Cloning and characterization of ASH2L and Ash2l, human and mouse homologs of the Drosophila ash2 gene[J]. Cytogenetics and Cell Genetics, 84(3-4): 167-172. [11] Kasinath V, Faini M, Poepsel S, et al.2018. Structures of human PRC2 with its cofactors AEBP2 and JARID2[J]. Science, 359(6378): 940-944. [12] Kleiber M L, Singh S M.2009. Divergence of the vertebrate sp1A/ryanodine receptor domain and SOCS box-containing (Spsb) gene family and its expression and regulation within the mouse brain[J]. Genomics, 93(4): 358-366. [13] Kuang Z, Yao S, Xu Y, et al.2008. SPRY domain-containing SOCS box protein 2: Crystal structure and residues critical for protein binding[J]. Journal of Molecular Biology, 386(3): 662-674. [14] Li T, Kelly W G.2011. A role for Set1/MLL-related components in epigenetic regulation of the Caenorhabditis elegans germ line[J]. PLoS Genetics, 7(3): e1001349. [15] Li Y, Han J, Zhang Y, et al.2016. Structural basis for activity regulation of MLL family methyltransferases[J]. Nature, 530(7591): 447-452. [16] Liang W, Lüscher-Firzlaff J, Ullius A, et al.2016. Loss of the epigenetic regulator Ash2l results in desintegration of hepatocytes and liver failure[J]. International Journal of Clinical and Experimental Pathology, 9(5): 5167-5175. [17] Qu Q, Takahashi Y H, Yang Y, et al.2018. Structure and Conformational dynamics of a COMPASS histone H3K4 methyltransferase complex[J]. Cell, 174(5): 1117-1126. [18] Rampalli S, Li L, Mak E, et al.2007. p38 MAPK signaling regulates recruitment of Ash2L-containing methyltransferase complexes to specific genes during differentiation[J]. Nature Structural & Molecular Biology, 14(12): 1150-1156. [19] Sarvan S, Avdic V, Tremblay V, et al.2011. Crystal structure of the trithorax group protein ASH2L reveals a forkhead-like DNA binding domain[J]. Nature Structural & Molecular Biology, 18(7): 857-859. [20] Shilatifard A.2012. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis[J]. Annual Review of Biochemistry, 81(1): 65-95. [21] Simonet T, Dulermo R, Schott S, et al.2007. Antagonistic functions of SET-2/SET1 and HPL/HP1 proteins in C. elegans development[J]. Developmental Biology, 312(1): 367-383. [22] South P F, Fingerman I M, Mersman D P, et al.2010. A conserved interaction between the SDI domain of Bre2 and the Dpy-30 domain of Sdc1 is required for histone methylation and gene expression[J]. Journal of Biological Chemistry, 285(1): 595-607. [23] Steward M M, Lee J S, O' Donovan A, et al.2006. Molecular regulation of H3K4 trimethylation by ASH2L, a shared subunit of MLL complexes[J]. Nature Structural & Molecular Biology, 13(9): 852-854. [24] Tan C C, Walsh M J, Gelb B D.2009. Fgfr3 is a transcriptional target of Ap2delta and Ash2l-containing histone methyltransferase complexes[J]. PLOS ONE, 4(12): e8535. [25] Wan M, Liang J, Xiong Y, et al.2013. The trithorax group protein Ash2l is essential for pluripotency and maintaining open chromatin in embryonic stem cells[J]. Journal of Biological Chemistry, 288(7): 5039-5048. [26] Wang J, Zhou Y, Yin B, et al.2001. ASH2L: Alternative splicing and downregulation during induced megakaryocytic differentiation of multipotential leukemia cell lines[J]. Journal of Molecular Medicine (Berlin, Germany), 79(7): 399-405. [27] Wang M, Liu C, Wang W, et al.2019. A SPRY domain-containing SOCS box protein 3 (SPSB3) involved in the regulation of cytokine production in granulocytes of Crassostrea gigas[J]. Developmental and Comparative Immunology, 95: 28-37.