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

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 wildtype 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.In eukaryotic chromatin, DNA is coiled around histone octamers to form nucleosome arrays, which are further packed into compact higher order structures (for review, see Refs. 1-4). Two major types of chromatin are distinguished by their packing density: open, less compact euchromatin and highly condensed heterochromatin (5, 6). Heterochromatin contains predominantly repressed genes and spreads to the bulk of nuclear chromatin during terminal cell differentiation when most of the genome becomes inactive (7,8).Heterochromatin spreading in terminally differentiated cells is associated with profound remodeling of higher order chromatin structure. Linker DNA at the entry/exit site of the nucleosome becomes more condensed, and extensive lateral interactions form between chromatin fibers (reviewed in Ref. 9). Such changes are driven by developmentally regulated factors. Surprisingly, the expression of major non-histone proteins that promote heterochromatin-specific repression in proliferating cells, such as Ikaros and heterochromatin protein 1, declines in terminally differentiated cells (10, 11) and thus cannot account for heterochromatin spreading.As developing avian blood cells enter the final maturation stage and RNA and protein synthesis declines, few proteins capable of carrying out chromatin condensation are up-regulated. Nucleated erythrocytes in verteb...

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“…Our in vitro experiments revealed that the RCL is directly involved in MENT's ability to condense chromatin (58). The RCL is also indispensable for direct interaction with and specific inhibition of the protease cathepsin L in vitro (22).…”

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The Silent Information Regulator 3 Protein, SIR3p, Binds to Chromatin Fibers and Assembles a Hypercondensed Chromatin Architecture in the Presence of Salt

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