Page 123 - 2014 Printable Abstract Book
P. 123
Upon DNA double strand breaks (DSBs) damage, activated DNA damage response and DNA repair
proteins accumulate into microscopic sub-micrometer foci at the sites of DSBs. Although the DSB foci have
been widely used in the study of DNA repair pathways and repair kinetics, little is known on the inner
structure of the DSB focus: Does it exist substructure inside the protein focus? How different proteins are
spatially distributed in the focus connected to each other? These information reflect the function and
interaction of the DNA damage response and DNA repair proteins, but are kept unknown up to now, due
to the resolution limit of the conventional optical microscopy. Using STORM reconstruction microscopy
we revealed the substructure of g-H2AX and 53BP1 protein foci at the DSB sites with tens of nanometer
resolution for the first time. The reconstructed foci image gave in-situ view of non-homogeneous, histone
structure-related 53BP1 distribution and the nanoscopic localization of 53BP1 and g-H2AX protein at the
DSB sites.
(PS1-37) C/ebpδ-deficiency promotes radiation injury by an impaired DNA damage response. Snehalata
A. Pawar, PhD; Xiaoyan Zhu, MS; Wenze Wang, MD, PhD; and Martin Hauer-Jensen, MD, PhD, FACS,
University of Arkansas for Medical Sciences, Little Rock, AR
Background: CCAAT enhancer binding protein delta (C/EBPδ) is a basic leucine -zipper
transcription factor implicated in the regulation of oxidative stress, DNA damage response, genomic
stability, and inflammation, however its role in the ionizing radiation (IR) response has not been
elucidated. Our study is the first to demonstrate that Cebpd-knockout (KO) mice display increased lethality
to total body irradiation (TBI). Cebpd-KO mice displayed increased apoptosis of hematopoietic stem and
progenitors and intestinal stem cells. In this study we further investigate the mechanism via which C/EBPδ
protects from IR-induced injury. Methods: Proximal jejunums were harvested from Cebpd-KO and Cebpd-
wild type (WT) mice at 0, 1h, 4h and 24h after exposure to sublethal TBI (7.4 Gy). For γ-H2AX staining,
mean fluorescence intensity of 4 animals per timepoint per genotype was measured in crypts and villi.
Phospho-Histone H3 positive cells were quantified in at least 100 crypts, and the average mitotic index
was calculated as a mean of 4 animals per group derived from the number of mitotic cells per crypt. Early
passage WT and KO mouse embryonic fibroblasts (MEFs) (passage#3-6) generated from day 13.5 embryos
were exposed to ionizing radiation (2Gy) and harvested at 0, 30 min, 1h and 2h post-IR exposure. Whole
cell extracts were immunoblotted for the expression of Atm, Atr, and their substrates p53, Chk1, Chk2
and their phosphorylated forms, and γ-H2AX. Results & Conclusions: While both genotypes induced γ-
H2AX expression 1 h post-IR, Cebpd-WT mice expressed higher levels of γ-H2AX in intestinal crypts and
villi than Cebpd-KO mice. This correlated with increased phospho-histone H3 expression in Cebpd-KO
intestinal crypts compared to that in crypts of Cebpd-WT mice 24h post-IR. Post-IR expression of γ-H2AX
and Chk1 (Ser345) was severely impaired in KO MEFs, although there were no significant differences in
expression and activation of Atm and p53 between WT and KO MEFs. In conclusion, our studies point to
a role of C/EBPδ in the DNA damage response by promoting γ-H2AX and Chk1 activation to induce cell
cycle arrest. Further studies are underway to examine Atr and the putative phosphatase that may be
involved in the dephosphorylation of γ-H2AX and Chk1 in Cebpd-deficient cells to prevent IR-induced cell
cycle arrest.
121 | P a g e
proteins accumulate into microscopic sub-micrometer foci at the sites of DSBs. Although the DSB foci have
been widely used in the study of DNA repair pathways and repair kinetics, little is known on the inner
structure of the DSB focus: Does it exist substructure inside the protein focus? How different proteins are
spatially distributed in the focus connected to each other? These information reflect the function and
interaction of the DNA damage response and DNA repair proteins, but are kept unknown up to now, due
to the resolution limit of the conventional optical microscopy. Using STORM reconstruction microscopy
we revealed the substructure of g-H2AX and 53BP1 protein foci at the DSB sites with tens of nanometer
resolution for the first time. The reconstructed foci image gave in-situ view of non-homogeneous, histone
structure-related 53BP1 distribution and the nanoscopic localization of 53BP1 and g-H2AX protein at the
DSB sites.
(PS1-37) C/ebpδ-deficiency promotes radiation injury by an impaired DNA damage response. Snehalata
A. Pawar, PhD; Xiaoyan Zhu, MS; Wenze Wang, MD, PhD; and Martin Hauer-Jensen, MD, PhD, FACS,
University of Arkansas for Medical Sciences, Little Rock, AR
Background: CCAAT enhancer binding protein delta (C/EBPδ) is a basic leucine -zipper
transcription factor implicated in the regulation of oxidative stress, DNA damage response, genomic
stability, and inflammation, however its role in the ionizing radiation (IR) response has not been
elucidated. Our study is the first to demonstrate that Cebpd-knockout (KO) mice display increased lethality
to total body irradiation (TBI). Cebpd-KO mice displayed increased apoptosis of hematopoietic stem and
progenitors and intestinal stem cells. In this study we further investigate the mechanism via which C/EBPδ
protects from IR-induced injury. Methods: Proximal jejunums were harvested from Cebpd-KO and Cebpd-
wild type (WT) mice at 0, 1h, 4h and 24h after exposure to sublethal TBI (7.4 Gy). For γ-H2AX staining,
mean fluorescence intensity of 4 animals per timepoint per genotype was measured in crypts and villi.
Phospho-Histone H3 positive cells were quantified in at least 100 crypts, and the average mitotic index
was calculated as a mean of 4 animals per group derived from the number of mitotic cells per crypt. Early
passage WT and KO mouse embryonic fibroblasts (MEFs) (passage#3-6) generated from day 13.5 embryos
were exposed to ionizing radiation (2Gy) and harvested at 0, 30 min, 1h and 2h post-IR exposure. Whole
cell extracts were immunoblotted for the expression of Atm, Atr, and their substrates p53, Chk1, Chk2
and their phosphorylated forms, and γ-H2AX. Results & Conclusions: While both genotypes induced γ-
H2AX expression 1 h post-IR, Cebpd-WT mice expressed higher levels of γ-H2AX in intestinal crypts and
villi than Cebpd-KO mice. This correlated with increased phospho-histone H3 expression in Cebpd-KO
intestinal crypts compared to that in crypts of Cebpd-WT mice 24h post-IR. Post-IR expression of γ-H2AX
and Chk1 (Ser345) was severely impaired in KO MEFs, although there were no significant differences in
expression and activation of Atm and p53 between WT and KO MEFs. In conclusion, our studies point to
a role of C/EBPδ in the DNA damage response by promoting γ-H2AX and Chk1 activation to induce cell
cycle arrest. Further studies are underway to examine Atr and the putative phosphatase that may be
involved in the dephosphorylation of γ-H2AX and Chk1 in Cebpd-deficient cells to prevent IR-induced cell
cycle arrest.
121 | P a g e
