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Intrinsically disordered proteins are abundant in the nucleus and are prime sites for posttranslational modifications that modulate transcriptional regulation. Lacking a defined three-dimensional structure, intrinsically disordered proteins populate an ensemble of several conformational states, which are dynamic and often altered by posttranslational modifications, or by binding to interaction partners. Although there is growing appreciation for the role that intrinsically disordered regions have in regulating protein-protein interactions, we still have a poor understanding of how to determine conformational population shifts, their causes under various conditions, and how to represent and model conformational ensembles. Here, we study the effects of serine phosphorylation in the nucleosome-binding domain of an intrinsically disordered protein - HMGN1 - using NMR spectroscopy, circular dichroism and modelling of protein complexes. We show that phosphorylation induces local conformational changes in the peptide backbone and decreases the helical propensity of the nucleosome binding domain. Modelling studies using AlphaFold3 suggest that phosphorylation disrupts the interface between HMGN1 and the nucleosome acidic patch, but that the models over-predict helicity in comparison to experimental data. These studies help us to build a picture of how posttranslational modifications might shift the conformational populations of disordered regions, alter access to histones, and regulate chromatin compaction., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

The structure of the nucleosome, the basic building block of the chromatin fiber, plays a key role in epigenetic regulatory processes that affect DNA-dependent processes in the context of chromatin. Members of the HMGN family of proteins bind specifically to nucleosomes and affect chromatin structure and function, including transcription and DNA repair. To better understand the mechanisms by which HMGN 1 and 2 alter chromatin, we analyzed their effect on the organization of histone tails and linker histone H1 in nucleosomes. We find that HMGNs counteract linker histone (H1)-dependent stabilization of higher order 'tertiary' chromatin structures but do not alter the intrinsic ability of nucleosome arrays to undergo salt-induced compaction and self-association. Surprisingly, HMGNs do not displace H1s from nucleosomes; rather these proteins bind nucleosomes simultaneously with H1s without disturbing specific contacts between the H1 globular domain and nucleosomal DNA. However, HMGNs do alter the nucleosome-dependent condensation of the linker histone C-terminal domain, which is critical for stabilizing higher-order chromatin structures. Moreover, HMGNs affect the interactions of the core histone tail domains with nucleosomal DNA, redirecting the tails to more interior positions within the nucleosome. Our studies provide new insights into the molecular mechanisms whereby HMGNs affect chromatin structure., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Throughout the cell cycle, the histones remain associated with DNA, but the repertoire of proteins associated with the chromatin fiber continuously changes. The chromatin interaction of HMGNs, a family of nucleosome binding proteins that modulates the structure and activity of chromatin, during the cell cycle is controversial. Immunofluorescence studies demonstrated that HMGNs are not associated with chromatin, whereas live cell imaging indicated that they are present in mitotic chromosomes. To resolve this controversy, we examined the organization of wild-type and mutated HMGN1 and HMGN2 proteins in the cell nucleus by using immunofluorescence studies, live cell imaging, gel mobility shift assays, and bimolecular fluorescence complementation (BiFC). We find that during interphase, HMGNs bind specifically to nucleosomes and form homodimeric complexes that yield distinct BiFC signals. In metaphase, the nucleosomal binding domain of the protein is inactivated, and the proteins associate with chromatin with low affinity as monomers, and they do not form specific complexes. Our studies demonstrate that the mode of binding of HMGNs to chromatin is cell cycle dependent.

Utilizing different computational methods; phosphorylation, O-GlcNAc modification and Yin Yang sites are predicted in HMGN-1. Prediction results suggest that interplay of phosphorylation and O-GlcNAc modification regulates binding and removal of HMGN-1 with the nucleosome and its translocation from nucleus to cytoplasm and back to nucleus, consequently modulating gene expression.

High mobility group (HMG) N1 protein, formerly known as HMG 14, is a member of the chromosomal HMG protein family. Protein kinase CK2 was previously reported to be able to phosphorylate bovine HMGN1 in vitro; Ser89 and Ser99, corresponding to Ser88 and Ser98 in human HMGN1, were shown to be major and minor recognition sites, respectively. In this report, we employed mass spectrometry and examined both the extent and the sites of phosphorylation in HMGN1 protein catalyzed by recombinant human protein kinase CK2. We found that five serine residues, i.e., Ser6, Ser7, Ser85, Ser88, and Ser98, in HMGN1 can be phosphorylated by the kinase in vitro. All five sites were previously shown to be phosphorylated in MCF-7 human breast cancer cells in vivo. Among these five sites, Ser6, Ser7, and Ser85 were new sites of phosphorylation induced by protein kinase CK2 in vitro.

We have demonstrated that levels of specific modification in histone H3 are modulated by members of the nucleosome-binding high mobility group N (HMGN) protein family in a variant-specific manner. HMGN1 (but not HMGN2) inhibits the phosphorylation of both H3S10 and H3S28, whereas HMGN2 enhances H3K14 acetylation more robustly than HMGN1. Two HMGN domains are necessary for modulating chromatin modifications, a non-modification-specific domain necessary for chromatin binding and a modification-specific domain localized in the C terminus of the HMGNs. Thus, chromatin-binding structural proteins such as HMGNs affect the levels of specific chromatin modifications and therefore may play a role in epigenetic regulation.

The dynamic modulation of chromatin structure is determined by many factors, including enzymes that modify the core histone proteins, enzymes that remodel the structure of chromatin, and factors that bind to genomic DNA to affect its structure. Previous work indicates that the nucleosome binding family of high mobility group proteins (HMGN) facilitates the formation of a chromatin structure that is more conducive for transcription. SWI/SNF complexes are ATP-dependent chromatin remodeling enzymes that alter nucleosome structure to facilitate the binding of various regulatory proteins to chromatin. Here we examine the structural consequences of reconstituting chromatin with HMGN1 and the resulting effects on hSWI/SNF function. We demonstrate that HMGN1 decreases the sedimentation velocity of nucleosomal arrays in low ionic strength buffers but has little effect on the structure of more highly folded arrays. We further demonstrate that HMGN1 does not affect SWI/SNF-dependent chromatin remodeling on either mononucleosomes or nucleosomal arrays, indicating that SWI/SNF functions independently of HMGN1.