Bioinformatical Analysis Of BRD Family And Their Expression And Function In Nile Tilapia

Bromodomain(BRD)proteins can specifically recognize N-acetyllysine motifs which are highly conserved in evolution.BRD proteins play a key role in epigenetic regulation.Previous studies have shown that BRD proteins play an important role in cell proliferation,differentiation and apoptosis.In recent years,the whole genome identification of BRD family has been carried out in mammals(Homo sapiens and Bubalus bubalis).However,few studies have been carried out in many animal groups,especially in teleost fish.The origin and evolution of the BRD family in the animal kingdom is still unclear,and the relationship between the family expansion and the whole genome replication remains to be explored.The patterns of expression throughout the family also remain to be explored.The testis specific gene BRDT is expressed specifically in human testis and has been proved to play an important role in mouse(Mus musculus)spermatogenesis.Previous studies have shown that brdt is highly expressed in the testis and ovary of gilthead seabream(Sparus aurata)and European seabass(Dicentrarchus labrax),but the specific mechanism of brdt in gametogenesis is still unclear due to the lack of evidence of direct knockout of brdt.In this study,BRD family members were isolated and identified from 25 representative animals,the phylogenetic evolution of BRD family members,the relationship between BRD family members amplification and animal genome replication were analyzed systematically,including genome identification,phylogeny,gene mapping,syntenic analysis and protein domain prediction.In addition,based on the transcriptome of different tissues and gonads of Nile tilapia(Oreochromis niloticus)at different developmental stages,the expression pattern of BRD family in tilapia was analyzed and verified by real-time fluorescence quantitative PCR(RT-q PCR)and fluorescence in situ hybridization(FISH).In addition,we evaluated the role of BETs in gonad development by experiments of BETs inhibitor treatment and explored the role of brdt in gametogenesis by CRISPR/Cas9 technology.The main results were as follows:1)In this study,we identified 54 BRDs in Nile tilapia genome,and 16-120 BRDs were identified from the genomes of 25 representative animals.In addition,16-20 BRDs were isolated and identified in 1R species(fruit fly,vase tunicate and lancelet),37-43 BRDs were identified in 2R species(elephant shark,coelacanth,python,frog,chicken,mouse and human),26 members were identified in lamprey,54-62 BRDs were identified in teleost that experienced 3R events,and 101-120 BRDs were isolated in 4R animals(chum salmon and common carp),which indicates that the BRD family has significantly expanded with the 2R,3R and 4R events.2)Phylogenetic analysis showed that BRD family was divided into eight subfamilies(Ⅰ-Ⅷ)in vertebrates,each subfamily was divided into different branches.Most members were conservative in evolutionary process,while 10 members(brd2,brd3,brd4,baz2 b,ep300,crebbp,brpf3,kmt2 b,brd1,and brd8)showed divergence,there are two copies of them,indicating that these members have replicated in these vertebrates,and expanded in teleost,while most duplicated genes were lost secondarily under selection pressure.3)The 54 BRDs of tilapia were found to be unevenly distributed in the genome according to chromosome mapping.In total,7 BRDs were on LG4,and 5 BRDs were on LG6,LG20 and LG22,respectively.Only 1 BRD gene was on LG2,LG8,LG16,LG18,LG19 and LG23.Interestingly,paralogs of brd4 and crebbp were found on LG4 and LG6,and paralogs of brd2 and kmt2 b were found to be located the LG11 and LG22.The prediction of BRD protein domains in tilapia showed that all BRD proteins had at least one bromodomain.The BET subfamily members displayed two bromodomains.Members of the subfamily Ⅵ had only one bromodomain,while Pbrml and Pbrm had 5 and 6bromodomains,respectively.In addition to Kat2 a,Kat2b,Cecr2,Brd8 a,Brd8b,Brd7 and Brd9,BRD proteins had other domains besides the bromodomain,such as the ET domain,PHD domain,BAH and HMG domain.4)According to the transcriptome data of 8 different tissues(ovary,testis,skeletal muscle,liver,kidney,head kidney,heart and brain)of adult fish,we found that most BRD genes were expressed in at least one tissue.In total,53 out of 54 genes were expressed in testis,while kmt2 a showed background level.Among these genes,pbrm1 showed the highest expression in testis,while brd2 a showed the highest expression in ovary.Notably,brd2 a was also the highest expressed gene in muscle and heart.Brd8 a,brpf3b and smarca4 were the highest expressed gene in liver,kidney and brain,respectively.5)The expression patterns of BRDs in gonads of tilapia at 5,30,90 and 180 dah(days after hatching)indicated all 54 BRD genes were expressed in at least one of gonad samples.More than 55%(30/54)of those genes showed sexually dimorphic expression in at least one developmental stage.Among them,21 BRD genes(cecr2,brd4 a,brdt,phip,ep300 a,brd8a,brpf3 a,brd1a,atad2 l,trim66,baz2 b,kmt2a,kmt2 bb,zmynd11,zmynd8,pbrm1,smarca2,ash1 l,brd9,atad2 and sp100.1)were highly expressed in testis,while9 BRD members(bptf,kat2 a,kat2b,brd3 b,baz1b,ep300 b,brpf3b,baz2 l and zmynd8l)were dominantly expressed in ovary.6)By intraperitoneal injection inhibitor JQ1 of BET subfamily into tilapia at 5dah,it was found that the testis of male fish were abnormal,the number of spermatogonia,spermatocytes and spermatids decreased.The expression of the apoptosis-promoting genes baxa,caspase3 a,caspase3b,caspase8,and caspase9 was upregulated,while the expression of the apoptosis-inhibiting gene bcl2 was downregulated in the testis of treated fish.The RT-q PCR test revealed that the expression of germ cell-related genes oct4(spermatogonia),rec8a(spermatocytes),prm and spata18(spermatids)was significantly decreased in the testes of treated fish compared with that of control fish.the expression of Sertoli-cell marker genes(amh and gsdf)in the testes of treated fish was significantly decreased.Moreover,the expression of steroidogenic enzyme genes cyp11a1,cyp11c1,cyp17a1,and star1 was also decreased in the testes of treated fish.In addition,sperm motility decreased and sperm morphology was abnormal in male fish treated with JQ1.However,ovaries were not significantly different after JQ1 treatment compared to controls.Lastly,testis development was also abnormal in male fish with deletion of brdt,indicating that BETs might play an important role in gonadal development of Nile tilapia,especially in the process of spermatogenesis,and the specific mechanism of their role needs to be further clarified.In conclusion,the BRD family members expanded significantly after the four rounds of whole-genome replication.Most BRDs were ubiquitously expressed in multiple tissues,and nearly half of them displayed sexual dimorphic expression during gonadal development in tilapia.JQ1 treatment resulted in a reduced number of germ cells and abnormal spermatogenesis,decreased sperm motility and abnormal sperm morphology in tilapia.The absence of brdt also caused abnormal testicular development.This study enriched our understanding of the evolution,structure,and expression patterns of the BRD family,and provided a new perspective on the functions,particularly in gonadal development and gametogenesis.