BORIS poster

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BORIS and CTCF yin yang epigenetically regulated
in transcription factors regulating
proliferation Wang, Tony Jau Cheng Bar-Sela,
Gil Huang, Lei Sun, Lunching Nguyen,
Phuongmai Pennington, J. Daniel Gius,
DavidRadiation Oncology Branch, National Cancer
Institute, National Institutes of Health,
Bethesda, MD 20892
Introduction Redundancy of regulation is a common
theme in biology. Epigenetic regulation of the
genome includes commonly accepted mechanisms
including chromatin compaction, nucleoside
methylation, and covalent modification of
histones. The paralogous gene pair CTCF and BORIS
may act as an antagonistically regulated switch
to alter epigenetic regulation during
gametogenesis. BORIS is normally only expressed
in primary spermatocytes, and should not be
expressed at the same time as CTCF in normal
cells. These proteins share the same 11 zinc
finger DNA-binding motif which is used
combinatorially to bind varying DNA sequences to
regulate gene transcription in trans. This
flexibility imparts diverse functions to CTCF
including chromatin organization, enhancer
blocking, X-chromosome inactivation, and
imprinting. Various cancers express BORIS, and
this aberrant expression may indicate a role for
BORIS in triggering epigenetic dysregulation.
Here we explore how BORIS may participate in
epigenetic regulation.
Results A yeast two-hybrid screen yielded a
number of candidate interactions for BORIS. We
chose to further explore Scythe because of its
biologically plausible interaction with BORIS.
Scythe, like BORIS, is also highly expressed in
the testis (Fig. 3). Scythe (also known as
Bag-6, Bat-3) is known to have an important role
in apoptosis, so its interaction with BORIS could
prove highly relevant in the context of cancer
research. Since we are interested in exploring
interactions that cause BORIS to interact
differently with other proteins, Scythes known
ability to modulate chaperone function also makes
it highly relevant.

To measure the effect of Scythe on
transcriptional activity of a promoter with a
CTCF/BORIS-binding site, cells were transiently
transfected with Scythe, BORIS, or both (Fig. 7).
The presence of Scythe caused an increase in
luciferase activity reflective on increased
trans-activation of the Bag-1 promoter, which
contains a CTCF/BORIS binding site.
Fig. 4. Confirmation by immunoprecipitation of
Scythe-BORIS interaction. His-tagged Scythe was
precipitated with either Scythe or BORIS.
Fig. 5. Confirmation by immunoprecipitation of
Scythe-BORIS interaction. GFP- tagged BORIS was
immunoprecipitated and probed for Scythe. A
plasmid expressing antisense Scythe Scythe (R)
was used as a negative control.
Conclusions We have demonstrated that Scythe
binds to BORIS and that this interaction likely
alters the in trans regulatory role of BORIS.
Because the CTCF/BORIS regulatory switch acts
through epigenetic mechanisms, this has
implications broader than the regulation of the
BAG-1 gene we used as our model. BORIS protein
has been found in a wide variety of cancers. The
aberrant expression of this protein could
upregulate a number of pro-proliferative,
pro-survival, and development genes normally
suppressed by the enhancer-blocking insulator
protein CTCF. While our work does not address the
extent or timing of this activation, it is clear
that expression of genes normally activated only
in the developing fetal stages is a common
occurrence in cancer. As well as being a known
cancer/testis gene, BORIS may promote the
upregulation of other developmentally
inappropriate genes. By doing so, expression of
BORIS may accelerate the failure of normal
genetic regulation that is a hallmark of
malignancies.
Fig. 6. Co-localization of Scythe and BORIS in
HCT-116 human cancer cells. DAPI nuclear stain is
blue Scythe is red BORIS is green
Table 1. A high-throughput yeast two-hybrid
screen was used to search for protein-protein
interactions with BORIS. Scythe, an apoptotic
regulator, shows interaction with BORIS localized
to Scythe amino acids 398-488.
Scythe-BORIS interaction was confirmed through
immunoprecipitation using His-tagged Scythe (Fig.
4) and GFP-tagged BORIS (Fig. 5). Colocalization
in a human colon cancer cell line also helped
confirm biological plausibility of the
interaction (Fig. 6).
Fig. 1. CTCF and BORIS share homology at the 11
zinc finger motif and bind the same DNA
sequences. From Klenova et al. 2002 Cancer Biology
Acknowledgments This research was made possible
through the Clinical Research Training Program, a
public private partnership supported jointly by
the NIH and a grant to the Foundation for NIH
from Pfizer Pharmaceuticals Group.
Fig. 7. A plasmid encoding luciferase under the
control of the BAG-1 promoter, which contains a
CTCF/BORIS binding site, was transiently
transfected into CALU-6 human cancer cells with
plasmids expressing BORIS or Scythe under control
of the CMV promoter, or both. A plasmid with the
CMV promoter alone was used as a negative control.
Fig. 2. CTCF and BORIS bind the same DNA
sequences and antagonistically regulate gene
expression. Aberrant expression of BORIS may lead
to epigenetic dysregulation through displacement
of CTCF.
Fig. 3. Relative levels of Scythe (here denoted
BAT3) mRNA in various human tissues. Scythe is
most highly expressed in testis, with a 3.5-fold
increase over average. From Su et al. 2002 PNAS,
accessed online at http//expression.gnf.org May
03, 2006.
For further information Please contact
WangTo_at_mail.nih.gov or GiusD_at_mail.nih.gov. More
information on the Gius laboratory can be
obtained at http//ccr.cancer.gov/staff/staff.asp?
profileid5563.