Belmont Lab
 Unversity of Illinois at Urbana-Champaign
Alumni Labs
 
Tethering assays for analysis of transcription activators or repressors and other chromosomal proteins

We adapted the lac operator / lac repressor system to analysis of the effect of particular proteins on large-scale chromatin compaction using a lac repressor tethering assay. Using a heterochromatic engineered chromosome region carrying lac operator repeats, we first applied this methodology to examine the effects on chromatin structure of the VP16 acidic activation domain, demonstrating a dramatic unfolding of the heterochromatin chromosome region through the extension and unfolding of chromonema fibers. We demonstrated that acidic activators as a class appear to share this large-scale chromatin decondensation activity. Using this methodology we were able to analyze what coactivators were recruited by VP16 and the timing of their recruitment relative to the large-scale chromatin decondensation. This activity appeared to be shared by a subset of other RNA pol 2 transcriptional activators, as demonstrated by our laboratory and collaborators, as well as UBF, a RNA pol 1 transcriptional activator. With collaborators this approach was extended to analysis of large-scale chromatin condensation induced by transcriptional repressors.

Other laboratories have extended this approach to study DNA replication and repair, as well as to studies of nuclear body formation and RNA processing.

Selected Publications:

Balamotis MA, Pennella MA, Stevens JL, Wasylyk B, Belmont AS, Berk AJ. Complexity in transcription control at the activation domain-mediator interface. Sci Signal. 2009 May 5; 2 (69) :ra20. PubMed PMID:19417216; PubMed Central PMCID: PMC2774526.

Deng H, Bao X, Cai W, Blacketer MJ, Belmont AS, Girton J, Johansen J, Johansen KM. Ectopic histone H3S10 phosphorylation causes chromatin structure remodeling in Drosophila. Development. 2008 Feb; 135 (4) :699-705. PubMed PMID:18199578.

Brink MC, van der Velden Y, de Leeuw W, Mateos-Langerak J, Belmont AS, van Driel R, Verschure PJ. Truncated HP1 lacking a functional chromodomain induces heterochromatinization upon in vivo targeting. Histochem Cell Biol. 2006 Jan; 125 (1-2) :53-61. PubMed PMID:16283356.

Verschure PJ, van der Kraan I, de Leeuw W, van der Vlag J, Carpenter AE, Belmont AS, van Driel R. In vivo HP1 targeting causes large-scale chromatin condensation and enhanced histone lysine methylation. Mol Cell Biol. 2005 Jun; 25 (11) :4552-64. PubMed PMID:15899859; PubMed Central PMCID: PMC1140641.

Carpenter AE, Memedula S, Plutz MJ, Belmont AS. Common effects of acidic activators on large-scale chromatin structure and transcription. Mol Cell Biol. 2005 Feb; 25 (3) :958-68. PubMed PMID:15657424; PubMed Central PMCID: PMC544008.

Chen D, Belmont AS, Huang S. Upstream binding factor association induces large-scale chromatin decondensation. Proc Natl Acad Sci U S A. 2004 Oct 19; 101 (42) :15106-11. PubMed PMID:15477594; PubMed Central PMCID: PMC524054.

Carpenter AE, Ashouri A, Belmont AS. Automated microscopy identifies estrogen receptor subdomains with large-scale chromatin structure unfolding activity. Cytometry A. 2004 Apr; 58 (2) :157-66. PubMed PMID:15057969.

Carpenter AE, Belmont AS. Direct visualization of transcription factor-induced chromatin remodeling and cofactor recruitment in vivo. Methods Enzymol. 2004; 375:366-81. PubMed PMID:14870678.

Li Y, Danzer JR, Alvarez P, Belmont AS, Wallrath LL. Effects of tethering HP1 to euchromatic regions of the Drosophila genome. Development. 2003 May; 130 (9) :1817-24. PubMed PMID:12642487.

Memedula S, Belmont AS. Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16. Curr Biol. 2003 Feb 4; 13 (3) :241-6. PubMed PMID:12573221.

Nye AC, Rajendran RR, Stenoien DL, Mancini MA, Katzenellenbogen BS, Belmont AS. Alteration of large-scale chromatin structure by estrogen receptor. Mol Cell Biol. 2002 May; 22 (10) :3437-49. PubMed PMID:11971975; PubMed Central PMCID: PMC133805.

Ye Q, Hu YF, Zhong H, Nye AC, Belmont AS, Li R. BRCA1-induced large-scale chromatin unfolding and allele-specific effects of cancer-predisposing mutations. J Cell Biol. 2001 Dec 10; 155 (6) :911-21. PubMed PMID:11739404; PubMed Central PMCID: PMC2150890.

Stenoien DL, Nye AC, Mancini MG, Patel K, Dutertre M, O'Malley BW, Smith CL, Belmont AS, Mancini MA. Ligand-mediated assembly and real-time cellular dynamics of estrogen receptor alpha-coactivator complexes in living cells. Mol Cell Biol. 2001 Jul; 21 (13) :4404-12. PubMed PMID:11390668; PubMed Central PMCID: PMC87100.

Tumbar T, Sudlow G, Belmont AS. Large-scale chromatin unfolding and remodeling induced by VP16 acidic activation domain. J Cell Biol. 1999 Jun 28; 145 (7) :1341-54. PubMed PMID:10385516; PubMed Central PMCID: PMC2133171.

 

 

 

 

 

 

 

 

 

 

       
 
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