Abstract
Upon integration into the host cell genome, the nucleosomal organization and epigenetic control of the HIV-1 provirus play an active role in its transcriptional regulation. Therefore, characterization of the chromatin changes that occur in the viral promoter region in response to different cellular stimuli or drug treatments represents an important aspect of our understanding of HIV-1 transcription. Moreover, the viral transactivator Tat protein potently activates HIV-1 transcription by recruiting the cellular positive transcription elongation factor p-TEFb to the TAR element located at the 5′ end of all nascent viral transcripts, thereby promoting efficient elongation. This chapter describes two complementary techniques for analyzing chromatin structure. The first technique is called indirect end-labeling and uses DNase I, micrococcal nuclease (MNase) or specific restriction enzymes to provide a view of nucleosome positions and of nucleosome-free regions within genes that are usually associated with transcriptional regulatory elements. The second technique, called chromatin immunoprecipitation (ChIP), provides a detailed analysis of chromatin structure by determining the pattern of histone modification marks in the DNA region of interest and by identifying the transcription factors as well as the components of the transcriptional initiation and elongation machineries that are recruited in vivo to this chromosomal region.
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References
Colin L, Van Lint C (2009) Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies. Retrovirology 6:111
van Opijnen T, Kamoschinski J, Jeeninga RE, Berkhout B (2004) The human immunodeficiency virus type 1 promoter contains a CATA box instead of a TATA box for optimal transcription and replication. J Virol 78:6883–6890
Rittner K, Churcher MJ, Gait MJ, Karn J (1995) The human immunodeficiency virus long terminal repeat includes a specialised initiator element which is required for Tat-responsive transcription. J Mol Biol 248:562–580
Harrich D, Garcia J, Wu F, Mitsuyasu R, Gonazalez J, Gaynor R (1989) Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat. J Virol 63:2585–2591
Van Lint C, Amella CA, Emiliani S, John M, Jie T, Verdin E (1997) Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity. J Virol 71:6113–6127
Ping YH, Rana TM (2001) DSIF and NELF interact with RNA polymerase II elongation complex and HIV-1 Tat stimulates P-TEFb-mediated phosphorylation of RNA polymerase II and DSIF during transcription elongation. J Biol Chem 276:12951–12958
Yamaguchi Y, Takagi T, Wada T, Yano K, Furuya A, Sugimoto S, Hasegawa J, Handa H (1999) NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation. Cell 97:41–51
Parada CA, Roeder RG (1996) Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature 384:375–378
Barboric M, Peterlin BM (2005) A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation. PLoS Biol 3:e76
Benkirane M, Chun RF, Xiao H, Ogryzko VV, Howard BH, Nakatani Y, Jeang KT (1998) Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat. J Biol Chem 273:24898–24905
Henderson A, Holloway A, Reeves R, Tremethick DJ (2004) Recruitment of SWI/SNF to the human immunodeficiency virus type 1 promoter. Mol Cell Biol 24:389–397
Treand C, du Chene I, Bres V, Kiernan R, Benarous R, Benkirane M, Emiliani S (2006) Requirement for SWI/SNF chromatin-remodeling complex in Tat-mediated activation of the HIV-1 promoter. EMBO J 25:1690–1699
Mahmoudi T, Parra M, Vries RG, Kauder SE, Verrijzer CP, Ott M, Verdin E (2006) The SWI/SNF chromatin-remodeling complex is a cofactor for Tat transactivation of the HIV promoter. J Biol Chem 281:19960–19968
Sobhian B, Laguette N, Yatim A, Nakamura M, Levy Y, Kiernan R, Benkirane M (2010) HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP. Mol Cell 38:439–451
Nakatani Y, Ogryzko V (2003) Immunoaffinity purification of mammalian protein complexes. Methods Enzymol 370:430–444
Ott M, Geyer M, Zhou Q (2011) The control of HIV transcription: keeping RNA polymerase II on track. Cell Host Microbe 10:426–435
Kiernan RE, Vanhulle C, Schiltz L, Adam E, Xiao H, Maudoux F, Calomme C, Burny A, Nakatani Y, Jeang KT, Benkirane M, Van Lint C (1999) HIV-1 tat transcriptional activity is regulated by acetylation. EMBO J 18:6106–6118
Chiu YL, Ho CK, Saha N, Schwer B, Shuman S, Rana TM (2002) Tat stimulates cotranscriptional capping of HIV mRNA. Mol Cell 10:585–597
Berro R, Kehn K, de la Fuente C, Pumfery A, Adair R, Wade J, Colberg-Poley AM, Hiscott J, Kashanchi F (2006) Acetylated Tat regulates human immunodeficiency virus type 1 splicing through its interaction with the splicing regulator p32. J Virol 80:3189–3204
Mbonye U, Karn J (2011) Control of HIV latency by epigenetic and non-epigenetic mechanisms. Curr HIV Res 9:554–567
Verdin E (1991) DNase I-hypersensitive sites are associated with both long terminal repeats and with the intragenic enhancer of integrated human immunodeficiency virus type 1. J Virol 65:6790–6799
Verdin E, Paras P Jr, Van Lint C (1993) Chromatin disruption in the promoter of human immunodeficiency virus type 1 during transcriptional activation. EMBO J 12:3249–3259
Van Lint C, Emiliani S, Ott M, Verdin E (1996) Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO J 15:1112–1120
Van Lint C, Ghysdael J, Paras P Jr, Burny A, Verdin E (1994) A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1. J Virol 68:2632–2648
Verdin E, Becker N, Bex F, Droogmans L, Burny A (1990) Identification and characterization of an enhancer in the coding region of the genome of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A 87:4874–4878
Goffin V, Demonte D, Vanhulle C, de Walque S, de Launoit Y, Burny A, Collette Y, Van Lint C (2005) Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity. Nucleic Acids Res 33:4285–4310
Colin L, Vandenhoudt N, de Walque S, Van Driessche B, Bergamaschi A, Martinelli V, Cherrier T, Vanhulle C, Guiguen A, David A, Burny A, Herbein G, Pancino G, Rohr O, Van Lint C (2011) The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for viral replication. PLoS One 6:e19084
Trono D, Van Lint C, Rouzioux C, Verdin E, Barre-Sinoussi F, Chun TW, Chomont N (2010) HIV persistence and the prospect of long-term drug-free remissions for HIV-infected individuals. Science 329:174–180
Lewin SR, Evans VA, Elliott JH, Spire B, Chomont N (2011) Finding a cure for HIV: will it ever be achievable? J Int AIDS Soc 14:4
Chavez L, Kauder S, Verdin E (2011) In vivo, in vitro, and in silico analysis of methylation of the HIV-1 provirus. Methods 53:47–53
Tyagi M, Karn J (2007) CBF-1 promotes transcriptional silencing during the establishment of HIV-1 latency. EMBO J 26:4985–4995
Williams SA, Chen LF, Kwon H, Ruiz-Jarabo CM, Verdin E, Greene WC (2006) NF-kappaB p50 promotes HIV latency through HDAC recruitment and repression of transcriptional initiation. EMBO J 25:139–149
He G, Margolis DM (2002) Counterregulation of chromatin deacetylation and histone deacetylase occupancy at the integrated promoter of human immunodeficiency virus type 1 (HIV-1) by the HIV-1 repressor YY1 and HIV-1 activator Tat. Mol Cell Biol 22:2965–2973
Lusic M, Marcello A, Cereseto A, Giacca M (2003) Regulation of HIV-1 gene expression by histone acetylation and factor recruitment at the LTR promoter. EMBO J 22:6550–6561
Marban C, Suzanne S, Dequiedt F, de Walque S, Redel L, Van Lint C, Aunis D, Rohr O (2007) Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J 26:412–423
du Chene I, Basyuk E, Lin YL, Triboulet R, Knezevich A, Chable-Bessia C, Mettling C, Baillat V, Reynes J, Corbeau P, Bertrand E, Marcello A, Emiliani S, Kiernan R, Benkirane M (2007) Suv39H1 and HP1gamma are responsible for chromatin-mediated HIV-1 transcriptional silencing and post-integration latency. EMBO J 26:424–435
Imai K, Togami H, Okamoto T (2010) Involvement of histone H3 lysine 9 (H3K9) methyltransferase G9a in the maintenance of HIV-1 latency and its reactivation by BIX01294. J Biol Chem 285:16538–16545
Marban C, Redel L, Suzanne S, Van Lint C, Lecestre D, Chasserot-Golaz S, Leid M, Aunis D, Schaeffer E, Rohr O (2005) COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells. Nucleic Acids Res 33:2318–2331
Friedman J, Cho WK, Chu CK, Keedy KS, Archin NM, Margolis DM, Karn J (2011) Epigenetic silencing of HIV-1 by the histone H3 lysine 27 methyltransferase enhancer of zeste 2 (EZH2). J Virol 85:9078–9089
Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E (2009) Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog 5:e1000495
Blazkova J, Trejbalova K, Gondois-Rey F, Halfon P, Philibert P, Guiguen A, Verdin E, Olive D, Van Lint C, Hejnar J, Hirsch I (2009) CpG methylation controls reactivation of HIV from latency. PLoS Pathog 5:e1000554
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Colin, L., Verdin, E., Van Lint, C. (2014). HIV-1 Chromatin, Transcription, and the Regulatory Protein Tat. In: Vicenzi, E., Poli, G. (eds) Human Retroviruses. Methods in Molecular Biology, vol 1087. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-670-2_8
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DOI: https://doi.org/10.1007/978-1-62703-670-2_8
Publisher Name: Humana Press, Totowa, NJ
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