Research:
Keypapers

Hansen, J.C. and Wolffe, A.P. (1992) Influence of Chromatin Folding on Transcription Initiation and Elongation by RNA Polymerase III. Biochemistry 31, 7977-7988.

This collaborative paper with Alan Wolffe directly compared the extent of chromatin folding and extent of transcription by Pol III in vitro using a defined chromatin model system. The primary conclusion from this work was that increased levels of chromatin folding led to decreased elongation by Pol III. Also, the chromatin studied in these experiments contained no H1, indicating that loss of H1 per se is insufficient to activate transcription in a fiber context. An important ramification of these studies is that the standard ionic conditions used in all in vitro transcription assays induces inhibitory chromatin condensation. Hence, how much of the inhibition by chromatin is due to condensation and how much is due to the nucleosome proper? This question remains as relevant now as it was in 1992.

Schwarz, P. M. and Hansen, J.C. (1994) Formation and Stability of Higher Order Chromatin Structures. Contribution of the Histone Octamer. J. Biol. Chem. 269, 16284-16289.

This paper demonstrated that a regular array of nucleosomes lacking linker histones will compact into a terminally folded “30 nm diameter” conformation [link] in the presence of physiologically relevant concentrations of divalent cations. The extensively folded conformation of a nucleosomal array is unstable, and can only form if the array is regular, i.e., if there are no nucleosome-free gaps. The primary conclusions from this work were that the macromolecular determinants required for folding into the highly condensed 30 nm fiber reside in the histone octamer, and linker histones stabilize rather than induce folded chromatin fibers.

Fletcher, T.M. and Hansen, J.C. (1995) Core Histone Tail Domains Mediate Oligonucleosome Folding and Nucleosomal DNA Organization Through Distinct Molecular Mechanisms. J. Biol. Chem. 270, 25359-25362.

This paper used the agarose multigel electrophoresis technique developed by Tracy Fletcher to show that in low salt, where nucleosomal arrays are in an unfolded “beads-on-a-string conformation [link to pic], the core histone N-terminal “tail” domains (NTDs) are bound to nucleosomal DNA. However, under ionic conditions that induce chromatin folding, the NTDs are no longer bound to nucleosomal DNA; they “rearrange” to mediate types of macromolecular interactions involved chromatin condensation. Together with work from the lab of Juan Ausio, this paper firmly established that the core histone tail domains are required for salt-dependent condensation of nucleosomal arrays.

Tse, C. and Hansen, J.C. (1997) Hybrid Trypsinized Nucleosomal Arrays: Identification of Multiple Functional Roles of the H2A/H2B and H3/H4 N-termini in Chromatin Fiber Compaction. Biochemistry 36, 11381-11388.

Chris Tse performed a technical tour-de-force extended by assembling “hybrid” model nucleosomal arrays lacking only the H3/H4 or H2A/H2B NTDs. The hybrid histone octamers were obtained from selectively trypsinized chicken erythrocyte histones. The primary conclusion from this work was that the core histone NTDs mediate chromatin condensation acting through multiple molecular mechanisms, some of which involve protein-protein rather than protein-DNA interactions.

Tse, C., Sera, T., Wolffe, A.W., and Hansen, J.C. (1998) Acetylation-induced Decondensation of Nucleosomal Arrays Dramatically Facilitates Transcription by RNA Polymerase III. Mol. Cell. Biol. 18, 4629-4638.

Chris Tse went on to assemble model nucleosomal arrays with differentially acetylated HeLa histone octamers. He observed that a threshold level of acetylation (~40% of all available sites) led to a dramatic destabilization of folded nucleosomal arrays. In collaboration experiments with Alan Wolffe, he further showed that the decondensed acetylated arrays were more transcriptionally active. The assay used in these studies primarily measured for elongation. The fundamental conclusion from this paper was that under specific circumstances acetylation can lead to unfolding of nucleosomal arrays, and subsequent increased transcriptional elongation.

Carruthers, L.M.., Bednar, J., Woodcock, C. and Hansen, J.C. (1998) Linker Histones Stabilize the Intrinsic Salt-dependent Folding of Nucleosomal Arrays: Mechanistic Ramifications for Higher Order Chromatin Folding. Biochemistry, 37, 14776-14787.

Lenny Carruthers took our model system studies to the next level by binding chicken erythrocyte linker histone H5 to model nucleosomal arrays at physiological stoichiometries. Collaborative cyo-EM experiments with Chris Woodcock showed that 3-D structures of the reconstituted chromatin model systems were identical to isolated native chicken erythrocyte chromatin fragments. These studies definitely established that linker histones are required to stabilize the 30 nm folded conformation intrinsically formed by nucleosomal arrays.

Carruthers, L.M. and Hansen, J.C. (2000) The Core Histone N-termini Function Independently of Linker Histones During Chromatin Condensation J. Biol. Chem. 275, 37285-37290.

At this point we asked ourselves whether the functions of the core histone NTDs and linker histones were independent or linked. Lenny answered this question by assembling H5 onto model nucleosomal arrays lacking their core histone NTDs. ThE key finding from these studies was that the core histone NTDs were required for formation of condensed chromatin fibers, even when linker histones were bound to the tailless nucleosomal arrays. Thus, the answer to our question is that they function independently. Of note, this paper was the last in a long series that investigated core histone NTD and linker histone function using chicken erythrocyte histone octamers. The recombinant era had dawned in the lab.

Georgel, P.T., Palacios DeBeer, M.A., Pietz, G., Fox, C.A. and Hansen, J.C. (2001) Sir3-dependent Assembly of Supramolecular Chromatin Structures in vitro. Proc. Natl. Acad. Sci., 98, 8584-8589.

After a decade of studying the intrinsic properties of model nucleosomal arrays and linker histone containing chromatin fibers, we became very interested in determining the properties of arrays bound to specific functional nucleosome binding proteins. Catherine Fox’s lab had recently purified recombinant yeast Sir3p, a nucleosome binding protein known to be involved in transcriptional silencing. Using Catherine’s protein, Philippe Georgel bound Sir3p to model nucleosomal arrays and examined the resulting nucleoprotein complexes using agarose gel electrophoresis. Rather than observing local changes in chromatin structure, Sir3p mediated formation of large “supramolecular” assemblages composed of multiple individual Sir3p–nucleosomal array complexes. These studies opened up a new area of study in the lab: nucleosome binding proteins that influence global chromatin architecture.

Georgel, P.T., Horowitz-Scherer, R.A., Adkins, N., Woodcock, C.L., Wade, P.W. and Hansen, J.C. (2003) Chromatin Compaction by Human MeCP2: Assembly of Novel Secondary Chromatin Structures in the Absence of DNA Methylation. J. Biol. Chem., 278, 32181-32188.

The next chromosomal protein we examined in an entirely pure chromatin model system was recombinant human MeCP2 (provided by Paul Wade), a methyl CpG binding protein thought to be a specific local acting transcriptional repressor. However, we found that MeCP2 in vitro was a potent chromatin condensing protein capable of mediating both local and global changes in chromatin architecture. This paper contains striking EM images and reconstructions by Chris Woodcock and Rachel Horowitz-Scherer of the unique secondary chromatin structure formed by MeCP2-bound nucleosomal arrays [links]. Although we were not expecting these results, they firmly cemented our growing interests in chromatin architectural proteins.

Georgel, P.T., Fletcher, T.M., Hager G., and Hansen, J.C. (2003) Formation of Condensed Secondary and Tertiary Chromatin Structures by Genomic MMTV Promoters. Genes Dev., 17, 1617-1629.

After a decade of model system studies strongly implicating chromatin fiber architecture as a major player in regulation of nuclear function, we felt it was imperative to test this idea by directly characterizing the structural properties of inactive and active genomic promoters. In collaboration with Gordon Hager’s lab, Philippe adapted the agarose multigel electrophoresis technique to characterize structural properties of inactive, basically active, and hormonally activated MMTV promoter fragments that were assembled into chromatin in vivo in the intact genome. The major observation from this work was that activation of the MMTV promoter did not involve complete unfolding into beads-on-a-string chromatin. Rather, gene activation was associated with a shift in the equilibrium between different types of folded secondary chromatin structure [link]. This paper challenges the perception that active chromatin exists in a primary beads-on-a-string chromatin structure, as is universally depicted in functional models.

Lu, X. and Hansen, J.C. (2004) Identification of Specific Functional Sub-domains Within the Linker Histone H1º C-terminal Domain. J. Biol. Chem. Epub ahead of print: 2003 Dec 10.

What is the mechanism through which linker histones stabilize condensed chromatin? Xu Lu used recombinant mouse histone H1-0 C-terminal domain (CTD) truncation mutants to show that distinct “subdomains” spread throughout the H1 CTD mediate different functions of H1 within the chromatin fiber. This paper pointed out that unique the amino acid composition of the linker histone CTDs is conserved among subtypes, and is characteristic of “intrinsically disordered proteins” [link]. This work has led to a working model of CTD function in which the CTD binds to linker DNA through electrostatic interactions, and these interactions induce formation of specific types of secondary structure that mediate many downstream functions of linker histones in the chromatin fiber.

Gordon, F., Luger, K., and Hansen, J.C. (2005) The Core Histone N-Terminal Tail Domains Function Independently and Additively During Salt-Dependent Oligomerization of Nucleosomal Arrays. J. Biol. Chem. 280, 33701-33706.

Salt-dependent oligomerization of nucleosomal arrays in vitro is related to fiber-fiber interactions and global chromosome structure. However, because of technical barriers, the function(s) of the individual core histone NTDs to this point has not been investigated. To address this question, all possible combinations of ‘tailless’ nucleosomal arrays were assembled from native and NTD-deleted recombinant Xenopus core histones and tandemly repeated 5S rDNA. The recombinant arrays were characterized over the range of 0-50 mM MgCl2 to determine how each NTD affects salt-dependent oligomerization. Results indicate that all core histone NTDs participate in the oligomerization process, and that the NTDs function additively and independently. These observations provided direct biochemical evidence linking all four core histone NTDs to the assembly and maintenance of global chromatin structures. Importantly, this work represented the first time that our model systems had been reconstituted with recombinant histones, opening the doorway for future mutagenesis-based studies of chromatin condensation.

Adams, V. A., McBryant, S.M., Wade. P.A., Woodcock, C.L. and Hansen, J.C. (2007) Intrinsic Disorder and Autonomous Domain Function in the Multifunctional Nuclear Protein, MeCP2. J. Biol. Chem. 282, 15057-64.

Although functional domains of chromatin-associated proteins are widely studied by x-ray crystallography and NMR, full-length chromatin proteins are rarely purified and characterized in solution. Given the potent ability of MeCP2 to compact chromatin, we decided it was essential to better understand the structural features of the protein. Analytical ultracentrifugation, circular dichroism (CD), and protease digestion were used to characterize recombinant human MeCP2 under a range of solution conditions. Quite surprisingly, results indicated that MeCP2 was monomeric and that the MeCP2 monomer was nearly 60% unstructured under conditions where it could preferentially recognize CpG dinucleotides and condense chromatin. Protease digestion experiments demonstrated that MeCP2 is composed of at least six structurally distinct domains. These domains collectively are organized into a tertiary structure with coil-like hydrodynamic properties, reflecting the extensive disorder in the MeCP2 sequence. The unusual structural features of MeCP2 are both fascinating per se, and have stimulated our growing interests in protein intrinsic disorder.

Lu X., Hamkalo B., Parseghian M.H., Hansen J.C. (2009) Chromatin Condensing Functions of the Linker Histone C-Terminal Domain Are Mediated by Specific Amino Acid Composition and Intrinsic Protein Disorder. Biochemistry 48, 164-172.

Given the results of Lu et al., (2004) and our emergent interest in intrinsic disorder, we attempted to understand the molecular basis of the function of the unstructured C-terminal domain of linker histone H1. Extensive mutagenesis was used to characterize chromatin model systems assembled with endogenous mouse somatic H1 isoforms, or recombinant H1 CTD mutants in which the primary sequence has been scrambled, the amino acid composition mutated, or the location of various CTD regions swapped. Results indicated that specific amino acid composition plays a fundamental role in molecular recognition and function by the H1 CTD. Additionally, these experiments support a new molecular model for H1 function based on DNA-dependent disorder to order transitions in specific locations of the CTD.

McBryant, S.M., Klonoski, J., Sorensen, T, Norskog, S., Williams, S., Resch, M., Toombs, T., Hobdey, S. and Hansen, J.C. (2009) Functional Determinants of Histone H4 N-Terminal Domain Action During Nucleosomal Array Oligomerization: Role of Amino Acid Sequence, Domain Length, and Charge Density. J. Biol. Chem. 284, 16716-22.

As a follow up to the Gordon et al. (2005) paper, we aimed to understand the mechanism through which the core histone H4 NTD mediates nucleosomal array oligomerization. To do so, we employed a novel mutagenesis-based approach. Core histones were engineered in which 1) the H2A, H2B and H3 NTDs were swapped onto the H4 histone fold, 2) the length of the H4 NTD, and the H2A NTD on the H4 histone fold, were increased, 3) the charge density of the NTDs on the H4 histone fold was increased or decreased, and 4) the H4 NTD was placed on the H2B histone fold. Results demonstrated that the H2B and H3 NTDs could replace the H4 NTD, as could the H2A NTD if it was duplicated to the length of the native H4 NTD. Arrays oligomerized at lower salt concentrations as the length of the NTD on the H4 histone fold was increased. Mutations that decreased the NTD charge density required more Mg2+ to oligomerize while mutants that increased the charge density required less salt. Finally, the H4 NTD functioned differently when attached to the H2B histone fold than the H4 histone fold. These studies have revealed new insights into the biochemical basis for H4 NTD effects on genome architecture, and underscored the power of the mutational approach to study chromatin dynamics.