Genentech Hall, N412-F, box 2240
PH: 514-0394; FX: 514-4242
Assistant: Felix Aburto, 514-4180
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OF CHROMATIN REMODELING
are interested in two different yet related questions:
(1) How is chromatin structure modulated to achieve the precise
regulation of nuclear processes such as transcription, DNA replication,
recombination and repair?
(2) What are the physical and chemical principles underlying the
action of large macromolecular machines?
We have begun addressing these questions by investigating the mechanisms
of a fascinating class of molecular machines: ATP-dependent nucleosome
of eukaryotic DNA in the form of chromatin represents a major organizational
feat allowing the packaging of approximately 2 meters of DNA into
a space of the order of 5 microns. This compaction, however also
makes the DNA less accessible for the central nuclear processes
of replication, transcription, recombination and repair. DNA accessibility
is greatly reduced even in the smallest unit of chromatin, the nucleosome,
which consists of roughly 150 bp of DNA wrapped around an octamer
of histone proteins. Modulation of chromatin structure is thus a
crucial component of the regulation of any nuclear process that
deals with DNA.
the last decade, several categories of multi-subunit complexes have
been identified that alter or "remodel" chromatin structure and
contribute to making it dynamic in vivo. A major class of remodeling
enzymes consists of complexes that expose nucleosomal DNA by using
ATP hydrolysis to alter nucleosome conformation. The SWI/SNF family
and the ISWI–family are two families of ATP-dependent remodelers
that play central roles in regulation of gene expression during
normal development. Another major class consists of complexes that
site-specifically modify histone residues by adding or removing
acetyl, methyl, ubiquityl or phosphoryl groups. It is thought that
specific combinations of modifications in one or more histones are
recognized by particular regulatory proteins, leading to additional
downstream events. This has been described as the "histone code"
hypothesis. Several of the subunits of both categories of complexes
have been identified as have many of their in vivo targets. The
molecular mechanisms by which these complexes catalyze nucleosome
remodeling however, are far from understood.
are using biochemical and biophysical approaches to quantitatively
dissect the mechanisms of ATP-dependent chromatin remodeling complexes.
Unraveling how these enzymes work is crucial for understanding their
biological impact and the levels at which they are regulated. At
the same time, these enzymes provide a new class of molecular machines
for studying how ATP hydrolysis is coupled to catalyzing a complex
structural change. Some specific areas of investigation are outlined
Framework for Remodeling
of the biochemical activities of the ~11-subunit human SWI/SNF complex
can be performed by the ATPase subunit, BRG1. As a starting point
for defining the remodeling process, we have established a minimal
reaction framework for BRG1 action on single nucleosomes. We find
that BRG1 as well as SWI/SNF generate multiple distinct remodeled
states and continuously interconvert them. SWI/SNF action can thus
create multiple opportunities for binding of distinct regulatory
factors. The reaction framework leads to several questions, which
we plan to address:
(1) How many distinct products are generated and how is the distribution
of products altered in the presence of other nuclear factors that
interact with chromatin?
(2) What is the role of the conserved SWI/SNF subunits? Do any of
them directly participate in the remodeling reaction and for example,
alter the product distribution, or final structure(s) of the product(s)?
(3) Can we identify additional steps in the remodeling reaction
that can shed light on how ATP-hydrolysis is coupled to remodeling?
Nature of the Remodeled Products
ISWI-family and SWI/SNF family of remodelers have distinct biological
roles. To fully comprehend the in vivo consequences these remodelers,
we need to know what the remodeled nucleosomes look like. The ISWI-family
appears to expose DNA sites by catalyzing sliding of the histone
octamer away from the sites. In this case the products have a canonical
nucleosome structure but altered nucleosome position. This strategy
provides an efficient way to alter and specify nucleosome position
in vivo but cannot explain how DNA is exposed in regions of closely
spaced nucleosomes that have little room to slide. Our recent work
with the SWI/SNF family suggests that these remodelers open up DNA
on the surface of the histone octamer, without a requirement for
sliding away the octamer. These results provide a biochemical basis
to explain the distinct biological roles of the two families. An
important unanswered question that we are currently addressing is
whether SWI/SNF products have an altered histone octamer structure
in addition to the alteration in DNA conformation as this can result
in exposure of specific histone regions for covalent modification.
of Higher Order Chromatin Structures
the nucleus, chromatin is organized in arrays of nucleosomes, which
fold into several levels of higher order structure. In vivo data
suggests that in addition to remodeling individual nucleosomes,
SWI/SNF plays an important role in modulating higher order chromatin
structures. We plan to use the reaction framework with mononucleosomes
as a foundation to tackle the more complex problem of understanding
SWI/SNF action on higher order chromatin structures.
Between ATP-dependent Remodeling and Histone Acetylation
and biochemical studies have suggested a strong functional link
between SWI/SNF and histone acetyl transferase (HAT) complexes such
as the SAGA complex in yeast and humans. Remodeling of the nucleosomes
by SWI/SNF may increase the accessibility of the histone N-termini
for acetylation or deacetylation. This may then facilitate recruitment
of activator and repressor proteins that specifically bind acetylated
and deacetylated nucleosomes, respectively. Alternatively, SWI/SNF
might simply bind more strongly to, or fall off more slowly from
nucleosomes having N-termini acetylated at specific positions as
suggested by recent biochemical experiments. This may keep the chromatin
in an accessible state longer for activator proteins. It is also
possible that direct physical interactions between ATP-dependent
remodeling proteins and HAT complexes modulate each other's activities.
We plan to address these questions by investigating how the in vitro
reaction parameters for SWI/SNF mediated remodeling are altered
in the presence of HAT complexes and vice-versa.
1. Phelan, M.L., Sif, S, Narlikar, G.J., Kingston, R.E., (1999)
Mol Cell 3, 247-53. "Reconstitution of a core chromatin remodeling
complex from SWI/SNF subunits."
2. Guyon, J.R., Narlikar, G.J., Sullivan, E.K., Kingston R.E. (2001)
Mol Cell Biol. 21, 1132-1144. "Stability of a Human SWI-SNF Remodeled
3. Narlikar, G.J., Phelan, M.L., Kingston, R.E. (2001) Mol Cell
8, 1219-30. "Generation and Interconversion of Multiple Distinct
Nucleosomal States as a Mechanism for Catalyzing Chromatin Fluidity."
4. Narlikar, G.J., Fan, H-Y., Kingston R.E. (2002) Cell. 108, 475-87.
"Cooperation between complexes that regulate chromatin structure