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CONTROL OF CHROMOSOME TRANSMISSION FIDELITY
BY THE SMC PROTEIN COMPLEXES
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Alexander
Strunnikov, PhD, Head,
Unit on Chromosome Structure and Function Yoshimitsu
Takahashi, PhD, Visiting Fellow Bi-Dar
Wang, PhD, Visiting Fellow Vladimir
Yong-Gonzales, PhD, Visiting Fellow Ilia
Ouspenski, PhD, Research Fellow Kei Ko, BS, Postbaccalaureate Fellow |
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The
eukaryotic ATP-ases of SMC family (structural maintenance of chromosomes)
form several essential eukaryotic protein complexes. Two of them, cohesin and
condensin, are the focus of our studies, with special emphasis on the
condensin complex and its function and regulation during cell cycle
progression. The condensin complex is the molecular machine of chromosome
condensation, which is indispensable for proper untangling and separation of
chromatids during anaphase. We are investigating the regulatory mechanisms
that ensure specificity of condensin targeting to the natural chromatin sites
in budding yeast and human cells. Condensin is composed of five essential
subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4, and Brn1. Our previous studies
established that several specific pathways determine proper condensin localization
to the defined chromatin domains and thus regulate chromosome condensation.
We directed our research to (1) an investigation of the role of genome
organization in chromosomal distribution of condensin; (2) analysis of
posttranslational modifications (Smt3 deconjugation pathway) in condensin
regulation; and (3) screening for novel molecular mechanisms determining the
specificity of mitotic condensin targeting to the nucleolus. Whole-genome
analysis of condensin-binding sites Wang, Strunnikov Previously,
our work led to the discovery of the essential role of condensin in
compartmentalization of sister chromatids before anaphase. Currently,
condensin’s role is considered intrinsic to chromosome condensation
while associated compaction of chromatin is thought to be a consequence.
Despite substantial recent progress in understanding the structure and
enzymology of condensin, the molecular mechanisms controlling
condensin’s activity and chromosome distribution remain obscure. Our
studies in S. cerevisiae identified the rDNA locus (and corresponding
nucleolar chromatin) as the major binding site for condensin. However, the
use of this site to elucidate the molecular mechanisms of condensin placement
onto specific chromatin sites proved technically challenging owing to the
tandem-repeat makeup of the rDNA cluster. To find the unique condensin-bound
chromosomal sites, we conducted a whole-genome study (in a microarray format)
to identify DNA fragments extracted from immunoprecipitates of chromatin-bound
condensin (ChIP-on-Chip approach). As a result of the study, we were able to
elucidate some rules governing placement of condensin-binding sites in
vivo. In particular, we established that condensin is uniformly
distributed over chromosomal arms, with strong binding peaks every 8 to 9 kb.
Comparison of condensin-binding modes in the specialized chromatin regions
demonstrated that condensin sites exhibit either cell cycle–independent
or mitosis-specific binding. Knowledge of the genomic distribution of condensin-binding
sites should facilitate our understanding of condensin mechanics in vivo, help
elucidate genetic and epigenetic determinants of genomic condensin
distribution, and allow us to use the identified unique DNA sequences in
developing a specific quantitative assay for condensin function. Kagansky A, Freeman L, Lukyanov D, Strunnikov A. Histone-tail
independent chromatin-binding activity of recombinant cohesin holocomplex. J
Biol Chem 2004;279:3382-3388. Functional
interface between sumoation machinery and condensin Takahashi,
Yong-Gonzales, Ouspenski In
higher eukaryotes, condensin is known to be regulated primarily via
phosphorylation. However, our previous studies have established that another
pathway, SUMO (Smt3) deconjugation, is indispensable for proper condensin
targeting to chromatin in budding yeast. In particular, we showed that
mutations in Smt4, the sole nuclear Smt3p-isopeptidase, are detrimental to
proper condensin function in mitosis. It is therefore reasonable to envisage
two possible feedback pathways between a multifunctional sumoation machinery
and the specific condensin function. First, condensin could be directly
modified (and inhibited) by Smt3. Second, some other proteins important for
condensin function may be Smt3-modified and thus transmit a signal from the
sumoation machinery to condensin. To determine conclusively whether condensin
is directly regulated by Smt3 conjugation, we developed a recombinant in
vitro SUMO modification system and a comprehensive test for in vivo
Smt3 analysis. These two approaches demonstrated that condensin subunits are
not bona fide Smt3p substrates. Thus, control of condensin binding to
chromatin must be mediated by other proteins that probably interact
functionally with condensin. We showed that one such protein is topoisomerase
II (Top2). Our investigation demonstrated that Top2 is one of the most potent
sumoation substrates both in vitro and in vivo. Moreover, the
E3 step in the SUMO conjugation pathway is essential for Top2 modification.
We are currently analyzing the target sumoation sites in Top2p, particularly
their role in chromosome transmission fidelity. Cdc14
phospatase–mediated control of condensin function Strunnikov, Wang,
Yong-Gonzales Use
of the mitotic nucleolar accumulation of Smc4-GFP as an in vivo assay
for chromosome condensation led to our discovery of a novel regulatory
pathway that controls mitotic condensin targeting to the proper chromatin
domains, particularly to nucleolar chromatin (rDNA). We isolated several
mutants as a result of a screen for trans-mutations, which impair
mitotic condensin localization to the nucleolus. Among them were the cdc14,
esp1, and cdc5 mutants, disrupting the same genetic pathway,
namely, the Cdc14 early anaphase release (FEAR). We showed that the
phosphatase activity of Cdc14 released by the FEAR pathway is required for
proper condensin-to-rDNA targeting in anaphase. The late-mitosis pathway of
Cdc14 activation (MEN) was dispensable for condensin-to-rDNA targeting;
however, the MEN network was able to rescue both condensin targeting to rDNA
and successful segregation of nucleolus in the FEAR mutants. Analysis of
biochemical properties of Cdc14p inactivation showed that condensin was
physically removed from rDNA in the cdc14 mutant; however, it was
properly assembled and bound to chromatin elsewhere, suggesting that
condensin was specifically mistargeted by Cdc14 inactivation. The study
identified a novel pathway promoting condensin targeting to a specific
chromosomal domain. We are investigating the molecular mechanism of
interaction between the Cdc14 activity and condensin targeting to chromatin. Wang BD, Yong-Gonzalez V,
Strunnikov AV. Cdc14p/FEAR pathway controls segregation of nucleolus in S.
cerevisiae by facilitating condensin targeting to rDNA chromatin in
anaphase. Cell Cycle 2004;3:960-967. collaborators Munira Busrai, PhD, Cancer Genetics Branch,
NCI, Michael Lichten, PhD, Division of Basic
Sciences, NCI,
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