Role of the M-loop and reactive center loop domains in the folding and bridging of nucleosome arrays by MENT.
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, whereas bridging of chromatin fibers is facilitated by MENT oligomerization mediated by the RCL.
Springhetti
Evelyn M
EM
Department of Biochemistry and Molecular Biology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Istomina
Natalia E
NE
Whisstock
James C
JC
Nikitina
Tatiana
T
Woodcock
Chris L
CL
Grigoryev
Sergei A
SA
D000818:Animals; D030161:Avian Proteins; D001717:Birds; D015153:Blotting, Western; D002234:Carbodiimides / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D008854:Microscopy, Electron; D008954:Models, Biological; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012617:Sea Urchins
D030161:Avian Proteins; D002234:Carbodiimides; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D003432:Cross-Linking Reagents; D006657:Histones; C081441:MENT protein, Gallus gallus; D009707:Nucleosomes; C074139:1-ethyl-3-(3-dimethylaminoethyl)carbodiimide; D004247:DNA