Role of the M-loop and Reactive Center Loop Domains in the Folding and Bridging of Nucleosome Arrays by MENT

Evelyn M. Springhetti, Natalia E. Istomina, James C. Whisstock, Tatiana Nikitina, Chris L. Woodcock, Sergei A. Grigoryev

Research output: Contribution to journalArticle

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Abstract

MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wild-type MENT with nucleosome arrays caused their tight folding and self-association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, w,hereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL.

Original languageEnglish (US)
Pages (from-to)43384-43393
Number of pages10
JournalJournal of Biological Chemistry
Volume278
Issue number44
DOIs
StatePublished - Oct 31 2003

Fingerprint

Nucleosomes
Chromatin
Oligomerization
DNA
Serpins
Heterochromatin
Fibers
Association reactions
Proteins
Blood Cells
Blood
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Springhetti, Evelyn M. ; Istomina, Natalia E. ; Whisstock, James C. ; Nikitina, Tatiana ; Woodcock, Chris L. ; Grigoryev, Sergei A. / Role of the M-loop and Reactive Center Loop Domains in the Folding and Bridging of Nucleosome Arrays by MENT. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 44. pp. 43384-43393.
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Role of the M-loop and Reactive Center Loop Domains in the Folding and Bridging of Nucleosome Arrays by MENT. / Springhetti, Evelyn M.; Istomina, Natalia E.; Whisstock, James C.; Nikitina, Tatiana; Woodcock, Chris L.; Grigoryev, Sergei A.

In: Journal of Biological Chemistry, Vol. 278, No. 44, 31.10.2003, p. 43384-43393.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Role of the M-loop and Reactive Center Loop Domains in the Folding and Bridging of Nucleosome Arrays by MENT

AU - Springhetti, Evelyn M.

AU - Istomina, Natalia E.

AU - Whisstock, James C.

AU - Nikitina, Tatiana

AU - Woodcock, Chris L.

AU - Grigoryev, Sergei A.

PY - 2003/10/31

Y1 - 2003/10/31

N2 - MENT is a developmentally regulated heterochromatin-associated protein that condenses chromatin in terminally differentiated avian blood cells. Its homology to the serpin protein family suggests that the conserved serpin reactive center loop (RCL) and the unique M-loop are important for its function. To examine the role of these domains, we studied the interaction of wild-type and mutant MENT with naked DNA and biochemically defined nucleosome arrays reconstituted from 12-mer repeats containing nucleosome positioning sequences. Wild-type MENT folded the naked DNA duplexes into closely juxtaposed parallel structures ("tramlines"). Deletion of the M-loop, but not inactivation of the RCL, prevented tramline formation and the cooperative interaction of MENT with DNA. Reconstitution of wild-type MENT with nucleosome arrays caused their tight folding and self-association. M-loop deletion inhibited nucleosome array folding, whereas the inactive RCL mutant was competent to fold the nucleosome arrays, but had a significantly impaired ability to cause their self-association. Bifunctional chemical cross-linking of MENT revealed oligomerization of wild-type MENT in the presence of chromatin and DNA. This oligomerization was severely reduced in the RCL mutant. We propose that the mechanism of MENT-induced heterochromatin formation involves two independent events: bringing together nucleosome linkers within a chromatin fiber and formation of protein bridges between chromatin fibers. Ordered binding of MENT to linker DNA via its unique M-loop domain promotes the folding of chromatin, w,hereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL.

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