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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.
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