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1、Vol.:(0123456789)1 3Cell.Mol.Life Sci.DOI 10.1007/s00018-017-2684-9REVIEWEpigenetic regulators:multifunctional proteins modulating hypoxiainducible factor protein stability andactivityWeiboLuo1,2 YingfeiWang1,3Received:24 July 2017/Revised:26 September 2017/Accepted:9 October 2017 Springer Internati

2、onal Publishing AG 2017constitutively expressed subunit in response to low oxygen in metazoans 1.Three subunits(HIF-1,HIF-2,and HIF-3)and two subunits(HIF-1,also known as ARNT,and ARNT2)have been cloned thus far 25.Three HIF-1 mRNA transcripts are encoded by human HIF1A gene,whereas only one HIF-2 m

3、RNA transcript is transcribed from human EPAS1 gene(Fig.1).HIF-1 and HIF-2 share about 48%overall amino acid sequence identity and con-tain the same functional domains:basic helixloophelix(bHLH)domain,Per-Arnt-Sim(PAS)domain,oxygen-dependent degradation(ODD)domain,N-terminal trans-activation domain(

4、N-TAD),inhibitory domain(ID),and C-terminal transactivation domain(C-TAD)(Fig.1).A total of 19 distinct HIF-3 transcripts exist in the human genome database due to alternative mRNA splicing but only 8 variants may encode HIF-3 proteins.Human HIF-3 iso-forms 1 and 9 contain a N-TAD and a unique leuci

5、ne zipper(LZIP)domain and are shown to activate gene transcription in human cells(Fig.1)6.However,HIF-3 isoform 4 lacks the transactivation domain and LZIP domain,and func-tions as a transcriptional inhibitor for HIF-1 and HIF-2 7.HIF-1 is ubiquitously expressed,but ARNT2 expression is restricted to

6、 the brain and kidney in rat and mouse 8,9.Hundreds of genes have been discovered to be regulated by HIF in various cell types and their protein products regulate erythropoiesis,angiogenesis,metabolism,pH homeostasis,stem cell maintenance,autophagy,immune evasion,and cell migration/invasion 10.There

7、fore,HIF-1 and HIF-2 play an important role in development,physiology,and diseases,such as cancer,heart disease,sleep apnea,and trauma 10.Recent studies showed that HIF-3 overexpression causes aberrant late branching morphogenesis,alveolar forma-tion,and epithelial differentiation during lung develo

8、pment in transgenic mice 11.HIF-3 knockout impairs pulmo-nary endothelial cell proliferation and angiogenic potential Abstract The hypoxia-inducible factor(HIF)is a heter-odimeric transcription factor governing a transcriptional program in response to reduced O2 availability in metazo-ans.It contrib

9、utes to physiology and pathogenesis of many human diseases through its downstream target genes.Emerg-ing studies have shown that the transcriptional activity of HIF is highly regulated at multiple levels and the epigenetic regulators are essential for HIF-mediated transactivation.In this review,we w

10、ill discuss the comprehensive regulation of HIF transcriptional activity by different types of epigenetic regulators.Keywords Hypoxia-inducible factor Epigenetic writer Epigenetic eraser Epigenetic reader ATP-dependent chromatin remodeler Chromatin reprogramming Gene regulationIntroductionThe hypoxi

11、a-inducible factor(HIF)is a master transcrip-tional factor consisting of an inducible subunit and a Cellular and Molecular Life Sciences*Weibo Luo Weibo.LuoUTSouthwestern.edu*Yingfei Wang Yingfei.WangUTSouthwestern.edu1 Department ofPathology,UT Southwestern Medical Center,5323 Harry Hines Blvd.,Dal

12、las,TX75390,USA2 Department ofPharmacology,UT Southwestern Medical Center,5323 Harry Hines Blvd.,Dallas,TX75390,USA3 Department ofNeurology andNeurotherapeutics,UT Southwestern Medical Center,5323 Harry Hines Blvd.,Dallas,TX75390,USA W.Luo,Y.Wang 1 312,suggesting a critical role of HIF-3 in physiolo

13、gy and pathology.The transcriptional activity of HIF,particularly HIF-1 and HIF-2,is regulated at both mRNA and protein levels.Under normoxic conditions,HIF-is hydroxylated on pro-line(Pro)residues(Pro 402 and Pro 564 for human HIF-1;Pro 405 and Pro 531 for human HIF-2;Pro 406 and Pro 492 for human

14、HIF-3 isoform 9)by a family of prolyl hydroxylases(PHDs)in the presence of the cofactors(iron,-ketoglutarate,and ascorbate)and the substrate O2 13.Prolyl hydroxylated HIF-is recognized by von Hippel-Lindau(VHL),which recruits the Cullin-2/Elongin-B/C ubiquitin E3 ligase complex to induce HIF-protein

15、 degra-dation in the 26S proteasome(Fig.2)14.Under hypoxic conditions,prolyl hydroxylation of HIF-is impaired,lead-ing to stabilization of HIF-protein(Fig.2).HIF-is then translocated into the nucleus and dimerized with HIF-1.The heterodimer binds to the hypoxia response element Fig.1 The structure s

16、cheme of active human HIF-isoforms and human HIF-.The func-tional domains of each isoform are indicated.bHLH basic helix-loop-helix,PAS Per-Arnt-Sim,ODD oxygen-dependent degradation,N-TAD N-terminal transactivation domain,ID inhibitory domain,C-TAD C-terminal transactivation domain,LZIP leucine zipp

17、erFig.2 O2-dependent regulation of HIF-1 activity.Under normoxia,human HIF-1 is hydroxylated on proline(P)residues 402 and 564 by a family of prolyl hydroxylases(PHDs).Prolyl hydroxylated HIF-1 is recognized by von Hippel-Lindau(VHL),which recruits the Cullin-2/Elongin-B/C ubiquitin E3 ligase comple

18、x to induce HIF-1 protein degradation in the 26S proteasome.On the other hand,human HIF-1 is also hydroxylated on asparagine(N)803 residue by another dioxygenase factor inhibiting HIF-1(FIH-1),leading to blockade of p300 recruitment to the C-terminal transacti-vation domain of HIF-1,thereby preventi

19、ng HIF-1-dependent gene transcription.Under hypoxia,PHDs and FIH-1 lose their enzymatic activity.As such,HIF-1 protein is stabilized and the epigenetic regulators(e.g.,p300/CBP,JMJD2C)are recruited to promote HIF-1-mediated transactivationEpigenetic regulators:multifunctional proteins modulating hyp

20、oxiainducible factor1 3(5-A/GCGTG-3)across the genome to enhance gene tran-scription 15.Emerging studies have shown that epigenetic regulators,including epigenetic writers,erasers,and readers,and ATP-dependent chromatin remodelers,are essential for HIF-mediated transactivation(Table1).In this review

21、,we will discuss the comprehensive regulation of HIF transcrip-tional activity by different types of epigenetic regulators.Regulation ofHIF transcriptional activity byepigenetic writersAcetyltransferases,methyltransferases,protein kinases,and ubiquitin E3 ligases function as epigenetic writers by ad

22、d-ing epigenetic marks onto histones,DNA,or RNA.p300/CBP possess the intrinsic histone acetyltransferase activ-ity that induces histone acetylation to relax the chromatin 16.They bind to the transactivation domain of HIF-to coactivate HIF-mediated transactivation,and are responsi-ble for expression

23、of about 3050%of global HIF-1 down-stream target genes 17.Post-translational modifications of HIF-are known to modulate p300 coactivator functions.Asparagine 803 of HIF-1 is hydroxylated by factor inhib-iting HIF-1(FIH-1)under nonhypoxic conditions,thereby blocking p300 binding to HIF-1 to inhibit H

24、IF-1 transcrip-tional activity(Fig.2)18,19.In contrast,S-nitrosylation of cysteine 800 enhances p300 recruitment to HIF-1 to increase HIF-1-mediated transactivation 20.Our previous study showed that the recruitment of p300 to the hypoxia response element is enhanced by pyruvate kinase M2,a HIF coact

25、ivator 21.Several other HIF-1-interacting proteins,including CITED2,EAF2,protein kinase C zeta,FOXO3a,ORF3,p65,histone deacetylase 4(HDAC4),HDAC5,and FHL1,are also shown to regulate HIF-1-p300 interaction,leading to altered HIF-1 transcriptional activity 2229.Similarly,the histone acetyltransferase

26、TIP60 was recently reported to enhance HIF-1 transcriptional activity and activate about 25%of HIF-1 downstream target genes in colorectal cancer HCT116 cells 30.TIP60 is recruited by HIF-1 to the hypoxia response element of the HIF-1 target gene ANKRD37.Knockdown of TIP60 decreases RNA polymerase I

27、I loading and activation at the promoter of ANKRD37.Acetylation of histone H3 at lysine(K)9 and histone H4 at the promoter of ANKRD37 is also reduced in TIP60 knockdown HCT116 cells.TIP60 is known to acetylate the N-terminal lysine residues of histone H4 31,and thus TIP60 may regulate chromatin repr

28、ogramming to enhance HIF-1 transcriptional activity.In addition,histone acetyltransferases directly acetylate HIF-to modulate HIF transcriptional activity.p300 acety-lates HIF-1 at K709 and reduces HIF-1 ubiquitination,leading to increased HIF-1 protein stability 32.p300/CBP-associated factor(PCAF)a

29、lso acetylates HIF-1 at K674 and increases transcription of the HIF target genes BID,CA9 and VEGFA in human osteosarcoma cells 33,34.Jeong etal.showed that the acetyltransferase arrest-defective-1(ARD1)induces acetylation of K532 of HIF-1 to increase HIF-1 ubiquitination and subsequent protein degra

30、dation by the PHD/VHL pathway,which diminishes HIF-1 transcriptional activity 35.However,ARD1 fails to acetylate HIF-1 invitro 36,37.It was recently suggested that prolyl hydroxylation and K391 methylation are prereq-uisite for ARD1-mediated K532 acetylation of HIF-1 38.Histone methyltransferases al

31、so play a critical role in HIF transcriptional activity.An inhibitor of the lysine methyl-transferase G9a,BIX01294,increases prolyl hydroxylation of HIF-1 under hypoxic conditions,leading to increased HIF-1 ubiquitination and decreased HIF-1 protein stabil-ity in human hepatocellular carcinoma HepG2

32、 cells 39.Treatment of BIX01294 blocks expression of the HIF target gene VEGFA.Pharmacological or genetic inhibition of G9a prevents tumorigenesis in mice 40,41.G9a is induced by hypoxia at both transcriptional and post-translational levels in embryonic stem cells and cancer cells and may mediate hy

33、poxia-induced gene repression through increasing dime-thyl K9 of histone H3 in cancer cells 4143.However,the direct effect of G9a on HIF transcriptional activity remains unknown.Regulation of HIF activity by the lysine methyltransferase SET7/9 has been the focus of recent studies,but its role is con

34、troversial.Liu etal.found that SET7/9 interacts with HIF-1 and blocks the ubiquitin E3 ligase CHIP-mediated degradation of HIF-1 protein in human cancer cells 44.SET7/9 is also enriched at the hypoxia response elements of a subset of HIF-1 regulated genes LDHA,HK2,and PDK1,and increases transcriptio

35、n of these glycolytic genes in hypoxic cells 44.In contrast,Xiao and his colleagues recently showed that SET7/9 directly induces monometh-ylation of HIF-1 at K32 and of HIF-2 at K29,leading to suppression of HIF transcriptional activity without affecting their protein levels 45.Knockdown of SET7/9 i

36、ncreases HIF-1 binding to the hypoxia response element to promote HIF-1 transcriptional activity and expression of the glyco-lytic genes in human clear cell renal carcinoma RCC4 cells,thereby increasing glucose uptake and ATP production.SET7/9-mediated monomethylation of HIF-1 at K32 was later confi

37、rmed by Kim etal.invitro and in human cervical carcinoma HeLa cells 46.SET7/9 also catalyzes dimeth-ylation of HIF-1 at K391 in HEK293T cells 38.How-ever,these two recent studies showed that SET7/9-mediated HIF-1 methylation decreases HIF-1 protein levels through increasing VHL-HIF-1 interaction and

38、 HIF-1 ubiquit-ination,thereby inhibiting HIF-1 transcriptional activity in transfected cells 38,46.Upregulated HIF-1 protein and the HIF-1 target genes Epo,Vegfa,and Slc2a1 are observed in knockin mice bearing mutant Hif1aK32A/K32A 46.These W.Luo,Y.Wang 1 3Table 1 The functions of epigenetic regula

39、tors in HIF activityEpigenetic regulators FunctionsDisease phenotypesReferencesEpigenetic writersp300/CBPHistone acetylation at the HIF target genes and enhanced gene transcription;ND16HIF-1 acetylation at K709 and protein stabilityND32TIP60Acetylation of histones H3K9 and H4 at the HIF target genes

40、 and enhanced gene transcriptionND30PCAFHIF-1 acetylation at K674 and protein stability;ND33,34Upregulation of the HIF target genes BID,CA9,and VEGFAND33ARD1HIF-1 acetylation at K532 and protein destabilityND35SET7/9HIF-1 methylation at K32 and K391,protein destability,and inhibition of the HIF-1 ta

41、rget genes SLC2A1,PGK1,VEGFA,LDHA,PDK1,and EPO;K32 methylation-resistant HIF-1 knockin mice exhibit a haematologic abnormality,enhanced retinal angiogenesis,and increased lung tumor growth and angiogenesis38,45,46HIF-2 methylation at K29 and inhibition of the HIF-2 target genes PAI1,CITED2,and POU5F

42、1;ND45Blockade of CHIP-mediated HIF-1 protein degradation and upregulation of HIF-1 target genes LDHA,HK2,and PDK1ND44PRMT1HIF-1 mRNA downregulation;ND47Inhibition of the HIF-1 target genes PFKFB4,VEGFA,HIG2,and BNIP3ND47PRMT5Increased HIF-1 mRNA translation and de novo HIF-1 protein synthesis;ND50U

43、pregulation of the HIF-1 target genes VEGFA,LOX,and PDK1ND50PRMT9Decreased de novo synthesis of HIF-1 protein;ND49Inhibition of the HIF-1 target genes CA9,PDK1,and BNIP3ND49DNMT3aDNA methylation of the EPAS1 gene and decreased HIF-2 levelsOverexpression of DNMT3a reduces renal tumor and glioblastoma

44、 growth in mice54Epigenetic erasersHDAC1HIF-1 protein stability;ND59HIF-1 deacetylation at K532 and K709ND32,106HDAC2HIF-1 protein stability;HIF-1 deacetylation at K532HDAC2 knockdown decreases oral squamous cell carcinoma growth in mice5757HDAC3HIF-1 protein stabilityND59HDAC4HIF-1 protein stabilit

45、y;ND58HIF-1 deacetylation at K10/11/12/19/21;ND58Enhanced p300 recruitment to HIF-1;ND29Decreased FIH-1 binding to HIF-1;ND29Upregulation of the HIF-1 target genes VEGFA,SLC2A1,and LDHAND29,58HDAC5Enhanced p300 recruitment to HIF-1;ND29Decreased FIH-1 binding to HIF-1;ND29Upregulation of the HIF-1 t

46、arget gene VEGFAND29Epigenetic regulators:multifunctional proteins modulating hypoxiainducible factor1 3Table 1 (continued)Epigenetic regulators FunctionsDisease phenotypesReferencesHDAC6HIF-1 protein stabilityND56HDAC7Interaction with HIF-1,p300,and CBP;ND60Increased HIF-1 transcriptional activity

47、and expression of VEGFA and GLUT1ND60SIRT1HIF-2 deacetylation at K385/685/741 and increased expression of HIF-2-dependent EPO;ND65HIF-1 deacetylation at K674,blockade of p300 recruitment,and inhibition of the HIF-1 target genes PDK1,VEGFA,and CA9;Overexpression of SIRT1 reduces fibrosarcoma growth a

48、nd angiogenesis34,66HIF-1 protein stability and upregulation of the HIF-1 target genes SLC2A1,VEGFA,MMP2,CA9,and EPO;An SIRT1 inhibitor decreases hepatocellular carcinoma growth and angio-genesis in mice63,64HIF-1 protein downregulation in primary vascular smooth muscle cells and inhibition of VEGFA

49、;SIRT1 attenuates neointima formation,but increases blood flow in wire-injured femoral arteries of mice67Histone H3K14 deacetylation at the HIF1A gene and downregulation of HIF-1 mRNA in MPP+-treated cellsND68SIRT2HIF-1 deacetylation at K709 and protein degradation;SIRT2 knockdown increases cervical

50、 tumor growth and angiogenesis in mice 69Inhibition of the HIF-1 target genes SLC2A1,VEGFA,and LDHA69SIRT3Decreased ROS levels and HIF-1 protein stability;SIRT3 knockdown increases colon cancer growth in mice72Inhibition of the HIF-1 target genes PGK1 and PDK172SIRT6Decreased de novo synthesis and s