Basic HTML Version
Full Abstracts – CELL CYCLE AND CELL DEATH
142
63
A role for the Drosophila histone variant H2Av in mitotic chromosome segregation.
Giovanni Cenci
1,2
, Fiammetta Vernì
3
. 1) Dip. Biologia di Base ed
Applicata, Università dell'Aquila, Via Vetoio, 67100 L'Aquila, Italy; 2) Sbarro Institute for Cancer Research and Molecular Medicine, Dept. of Biology,
Temple University, PA 19122, USA; 3) Dip. di Biologia e Biotecnologie "C. Darwin", Sapienza Università di Roma, Roma 00185, Italy.
We found that mutations in the Drosophila
H2Av
gene, which encodes both H2A.X and H2A.Z variants, impair compaction of pericentric chromosome
regions and lead to irregular chromosome segregation during larval mitotic divisions. Analysis of spindle assembly revealed that ~23% of H2Av-depleted
mutant cells displayed apparent anaphase-looking bipolar spindles with chromosomes not connected to the spindle poles by bundles of kinetochore
microtubules (MTs). In addition, we found that
H2Av
mutant chromosomes showed defective sister chromatid separation and were not able to congress to
the equator of the cell. Moreover, ~85% of irregular mitotic figures exhibited high levels of Cyclin B with respect to control anaphases (~3%), suggesting
that the anaphase-like figures are indeed in a metaphase status. Consistently, the checkpoint proteins ZW10 and BubR1 remained strongly localized at
centromeres of mutant chromosomes. Interestingly, ZW10 failed to stream towards the poles suggesting that loss of H2Av caused defective MT attachment
to the kinetochore. Indeed, MT regrowth experiments after cold exposure revealed that loss of H2Av impaired MT capture by kinetochores. All phenotypes
were rescued by a
H2Av
transgene with a C-terminal truncation that lacks of H2A.X function, suggesting that it is the loss of H2A.Z activity that affected
chromosome behavior. Our Co-Ip experiments showed that H2Av interacts with Hp1. Furthermore, Hp1 immunostaining revealed that ~50% of mutant
neuroblasts displayed reduced pericentric Hp1 localization on mitotic chromosomes, although Hp1 levels in interphase cells remained normal. Altogether,
our results suggest that H2Av is required for the regulation of mitotic chromosome segregation in
D. melanogaster
highlighting an unanticipated role of this
histone variant for the Hp1 localization on mitotic chromosomes.
64
The Tumor Suppressor APC2 and the Chk2 DNA Damage Checkpoint Promote Genomic Stability in the Early Embryo.
John Poulton, Frank Mu,
Mark Peifer. Dept of Biology, Linberger Comprehensive Cancer Center, Univ North Carolina, Chapel Hill, NC.
APC proteins regulate Wnt signaling, cytoskeletal processes, and genomic stability, though their precise roles in these processes are controversial. One of
APC’s best characterized cytoskeletal roles is in the early fly embryo, where it helps maintain nuclei at the embryonic cortex. Loss of APC2 leads to
detachment of nuclei from the cortex (nuclear fallout), but its mechanism of action is unclear. To better understand APC2’s cytoskeletal function we sought
to identify mechanisms underlying nuclear fallout in
APC2
-
embryos. From live imaging, we observed that nuclear fallout is frequently preceded by defects
in chromosome segregation. Previous work showed that DNA damage activates the Chk2 pathway, leading to centrosome inactivation and nuclear fallout.
We hypothesized that nuclear fallout in
APC2
-
embryos is due to Chk2 activation. Consistent with this, we found that nuclei undergoing fallout in
APC2
-
embryos accumulated high levels of the DNA damage marker γH2Av, and underwent centrosome inactivation prior to fallout. Importantly, embryos double
mutant for
chk2
and
APC2
displayed almost no nuclear fallout, indicating fallout in
APC2
-
embryos requires Chk2. Surprisingly however, these double
mutant embryos had many more mitotic defects than either single mutant, suggesting Chk2 can correct some defects caused by loss of APC2. We also
elucidated the upstream defects that lead to increased DNA damage in
APC2
-
embryos.
APC2
-
embryos had a high frequency defect in centrosome
separation; this appears to lead to ectopic pseudocleavage furrows which disrupted mitotic spindles. We propose a model where initial errors in centrosome
separation lead to cytoskeletal misregulation, including formation of ectopic pseudocleavage furrows. These cytoskeletal defects disrupt some mitotic nuclei,
causing chromosome segregation defects that activate the Chk2 DNA damage pathway. This can either promote damage correction or nuclear fallout. Our
findings provide in vivo evidence that the cytoskeletal function of an APC protein serves an important role in promoting mitosis and genomic stability.
65
A FISH-based RNAi screen identifies genes involved in somatic homolog pairing of heterochromatic regions.
Eric Joyce, Ting Wu. Genetics, Harvard
Medical School, Boston, MA.
Pairing of homologous chromosomes is generally considered to be a special property of the meiotic cell. However, reports have also implicated pairing
during the somatic cell cycle, with evidence for its impact in both double-strand break (DSB) repair and gene regulation. In Drosophila, homologous
chromosomes are intimately paired in virtually all cell types throughout development; however, our understanding of the underlying mechanism remains
unclear and only a few genes have been implicated in this process. Here, we introduce a novel high-throughput FISH (fluorescent in-situ hybridization)
technique that enabled us to rapidly screen for factors involved in this robust level of somatic pairing. Using a genome-wide RNAi library, we identified both
candidate ‘pairing promoting genes,’ as well as candidate ‘anti-pairing genes,’ providing evidence that pairing may be a dynamic process that can be both
enhanced and antagonized. Many of the genes found to be important for pairing are especially enriched for functions associated with mitotic cell division,
providing a genetic framework to understand a long-standing link between chromosome dynamics during mitosis and homologous pairing in interphase. In
contrast to the pairing promoting genes, several of the candidate anti-pairing genes have known interphase functions associated with S-phase progression,
replication, and chromatin compaction, including several components of the condensin II complex. These findings complement studies conducted over
several decades regarding pairing mechanics from many labs including that of Henikoff, Sedat, Hawley, Bosco and others. These results, in combination
with a variety of secondary assays, has led to new insights into the mechanism and dynamics of somatic pairing that have implications for gene regulation,
DSB repair, and cell cycle progression. This work is supported by grants from the National Institutes of Health to E.F.J. (F32CA157188) and T.W.
(RO1GM085169).