Abstract
Of critical importance to many of the events underlying transcriptional control of gene expression are modifications to core and linker histones that regulate the accessibility of trans-acting factors to the DNA substrate within the context of chromatin. Likewise, control over the initiation of DNA replication, as well as the ability of the replication machinery to proceed during elongation through the multiple levels of chromatin condensation that are likely to be encountered, is known to involve the creation of chromatin accessibility. In the latter case, chromatin access will likely need to be a transient event so as to prevent total genomic unraveling of the chromatin that would be deleterious to cells. While there are many molecular and biochemical approaches in use to study histone changes and their relationship to transcription and chromatin accessibility, few techniques exist that allow a molecular dissection of the events underlying DNA replication control as it pertains to chromatin changes and accessibility. Here, we outline a novel experimental strategy for addressing the ability of specific proteins to induce large-scale chromatin unfolding (decondensation) in vivo upon site-specific targeting to an engineered locus. Our laboratory has used this powerful system in novel ways to directly address the ability of DNA replication proteins to create chromatin accessibility, and have incorporated modifications to the basic approach that allow for a molecular genetic analysis of the mechanisms and associated factors involved in causing chromatin decondensation by a protein of interest. Alternative approaches involving co-expression of other proteins (competitors or stimulators), concurrent drug treatments, and analysis of co-localizing histone modifications are also addressed, all of which are illustrative of the utility of this experimental system for extending basic findings to physiologically relevant mechanisms. Although used by our group to analyze mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.
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References
Belmont AS, Li G, Sudlow G et al (1999) Visualization of large-scale chromatin structure and dynamics using the lac operator/lac repressor reporter system. Methods Cell Biol 58:203–222
Li G, Sudlow G, Belmont AS (1998) Interphase cell cycle dynamics of a late-replicating, heterochromatic homogeneously staining region: precise choreography of condensation/decondensation and nuclear positioning. J Cell Biol 140:975–989
Tumbar T, Sudlow G, Belmont AS (1999) Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J Cell Biol 145:1341–1354
Alexandrow MG, Hamlin JL (2005) Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation. J Cell Biol 168:875–886
Ye Q, Hu Y-F, Zhong H et al (2001) BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations. J Cell Biol 155:911–921
Wolffe AP (1997) Histones, nucleosomes and the roles of chromatin structure in transcriptional control. Biochem Soc Trans 25:354–358
Wolffe AP, Khochbin S, Dimitrov S (1997) What do linker histones do in chromatin? Bioessays 19:249–255
Dou Y, Bowen J, Liu Y et al (2002) Phosphorylation and an ATP-dependent process increase the dynamic exchange of H1 in chromatin. J Cell Biol 158:1161–1170
Dou Y, Gorovsky MA (2000) Phosphorylation of linker histone H1 regulates gene expression in vivo by creating a charge patch. Mol Cell 6:225–231
Dou Y, Mizzen CA, Abrams M et al (1999) Phosphorylation of linker histone H1 regulates gene expression in vivo by mimicking H1 removal. Mol Cell 4:641–647
Shen X, Yu L, Weir JW et al (1995) Linker histones are not essential and affect chromatin condensation in vivo. Cell 82:47–56
Nye AC, Rajendran RR, Stenoien DL et al (2002) Alteration of large-scale chromatin structure by estrogen receptor. Mol Cell Biol 22:3437–3449
Takeda DY, Wohlschlegel JA, Dutta A (2001) A bipartite substrate recognition motif for cyclin-dependent kinases. J Biol Chem 276:1993–1997
Liu P, Barkley LR, Day T et al (2006) The Chk1-mediated S-phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2-independent mechanism. J Biol Chem 281:30631–30644
Verschure PJ, van der Kraan I, de Leeuw W et al (2005) In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol Cell Biol 25:4552–4564
Wong PG, Glozak MA, Cao TV et al (2010) Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin. Cell Cycle 9:4351–4363
Miotto B (2011) Regulation of DNA licensing by targeted chromatin remodeling. Cell Cycle 10:1522
Miotto B, Struhl K (2008) HBO1 histone acetylase is a coactivator of the replication licensing factor Cdt1. Genes Dev 22:2633–2638
Miotto B, Struhl K (2010) HBO1 histone acetylase activity is essential for DNA replication licensing and inhibited by Geminin. Mol Cell 37:57–66
Belmont AS, Straight AF (1998) In vivo visualization of chromosomes using lac operator-repressor binding. Trends Cell Biol 8:121–124
Belmont AS (2001) Visualizing chromosome dynamics with GFP. Trends Cell Biol 11:250–257
Tumbar T, Belmont AS (2001) Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator. Nat Cell Biol 3:134–139
Tsukamoto T, Hashiguchi N, Janicki SM et al (2000) Visualization of gene activity in living cells. Nat Cell Biol 2:871–878
Janicki SM, Tsukamoto T, Salghetti SE et al (2004) From silencing to gene expression: real-time analysis in single cells. Cell 116:683–698
Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
Kennedy BK, Barbie DA, Classon M et al (2000) Nuclear organization of DNA replication in primary mammalian cells. Genes Dev 14:2855–2868
Acknowledgements
We are extremely grateful to Andrew Belmont (University of Illinois, Urbana-Champaign) for providing us with the A03_1 cell line, the LacI-VP16 vector. The Alexandrow lab is supported by funds from the National Institutes of Health (R01-CA130865 and R21-CA155393).
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Borysov, S., Bryant, V.L., Alexandrow, M.G. (2015). Analysis of DNA Replication Associated Chromatin Decondensation: In Vivo Assay for Understanding Chromatin Remodeling Mechanisms of Selected Proteins. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 1288. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2474-5_16
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DOI: https://doi.org/10.1007/978-1-4939-2474-5_16
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