Page 92 - 2014 Printable Abstract Book
P. 92
mutated in some cancers. SETX appears to be specifically involved in the response to oxidative stress and
is suggested to resolve R loops formed at transcription termination sites or at sites of collisions between
the transcription and replication machineries. R loops are hybrids between RNA and DNA that can lead to
DNA damage and genomic instability. We previously discovered that Rrp45, a core component of the
exosome, is a SETX-interacting protein and upon replication stress induction, SETX and Rrp45 co-localize
in nuclear foci that constitute sites of R-loop formation generated by transcription and replication
machinery collisions. This data implies that the exosome might also be involved in the DNA damage
response. Indeed, knockdown (KD) of the exonuclease Rrp6 resulted in an increase in γH2AX by both
immunofluorescence and western blotting. In order to further dissect SETX functions, we analyzed the
effect on transcription after SETX KD in the glioblastoma-astrocytoma U87 cells using an exon array.
Among the 4000 genes affected by SETX KD, 70% of them are downregulated and have a vast range of
biological functions. Based on the identification of these putative SETX target genes, we show that SETX
KD affects several unexpected functions important for cell homeostasis and cell migration. All together,
these data suggest that SETX is involved in gene expression regulation and raise the intriguing possibility
that SETX might act as a tumor suppressor in addition to its role linked to neurological disorders. Further
characterization of these new cellular function(s) of SETX will provide important insights into the
understanding of mechanisms that lead to diseases.
(S2504) Kub5/Hera (K-H) plays dual roles in RNA transcription termination and DNA double strand break
1
1
1
1
repair. Julio Morales, PhD ; Amy Rommel, PhD ; Edward Motea, PhD ; Praveen Patidar, PhD ; Farjana
2
1
1
2
Fattah, PhD ; Patricia Richards, PhD ; James Manley, PhD ; David A. Boothman, PhD
1
UT Southwestern Medical Center at Dallas, Dallas, TX and Columbia University, New York, NY
2
RNA-DNA hybrids are formed as a result of the natural processivity of RNA polymerase II (RNAPol
II)-mediated transcription. RNAPol II requires a series of factors that bind to its differentially
phosphorylated C-terminal domain (CTD) for smooth processing and for termination. A deficiency in one
or more of these factors can lead to the formation of persistent RNA-DNA hybrids, known as R-loops.
Persistent R-loops result in the formation of complex DNA double strand breaks (DSBs), chromosomal
aberrations and genetic instability. Studies examining the roles of these factors in basal as well as damage-
induced genomic instability are in their infancy. Using hKu70 as bait in a yeast two-hybrid screen, Kub5-
Hera (K-H), the human homolog of the yeast transcription termination factor Rtt103, was isolated. We
show that K-H forms two higher order complexes by gel filtration and proteomics and plays two separate
functions in RNA transcription termination as well as DSB repair. K-H forms distinct protein complexes
with factors that repair DSBs (e.g., Ku70, Ku86, and Artemis) and terminate transcription (e.g., RNA
polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, an activated DNA damage response
(DDR), and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-
H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H is a highly conserved
gene with only one base-pair change from mouse to humans. Yeast deficient in Rtt103 were specifically
defective in repair of complex (blunt or over-hang DNA), but not easily repairable DSBs, in ligation assays,
different from hdfr1 (yKu70-deficient) cells. K-H deficient cells were hypersensitive to cytotoxic agents
that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed [[Unsupported
Character - Symbol Font ]]-H2AX and 53BP1 repair-related foci regression in response to ionizing
radiation (IR). Artemis re-expression in K-H-deficient cells restored DNA repair function and resistance to
DSB-inducing agents. However, R-loops persisted consistent with dual roles of K-H in transcription
90 | P a g e
is suggested to resolve R loops formed at transcription termination sites or at sites of collisions between
the transcription and replication machineries. R loops are hybrids between RNA and DNA that can lead to
DNA damage and genomic instability. We previously discovered that Rrp45, a core component of the
exosome, is a SETX-interacting protein and upon replication stress induction, SETX and Rrp45 co-localize
in nuclear foci that constitute sites of R-loop formation generated by transcription and replication
machinery collisions. This data implies that the exosome might also be involved in the DNA damage
response. Indeed, knockdown (KD) of the exonuclease Rrp6 resulted in an increase in γH2AX by both
immunofluorescence and western blotting. In order to further dissect SETX functions, we analyzed the
effect on transcription after SETX KD in the glioblastoma-astrocytoma U87 cells using an exon array.
Among the 4000 genes affected by SETX KD, 70% of them are downregulated and have a vast range of
biological functions. Based on the identification of these putative SETX target genes, we show that SETX
KD affects several unexpected functions important for cell homeostasis and cell migration. All together,
these data suggest that SETX is involved in gene expression regulation and raise the intriguing possibility
that SETX might act as a tumor suppressor in addition to its role linked to neurological disorders. Further
characterization of these new cellular function(s) of SETX will provide important insights into the
understanding of mechanisms that lead to diseases.
(S2504) Kub5/Hera (K-H) plays dual roles in RNA transcription termination and DNA double strand break
1
1
1
1
repair. Julio Morales, PhD ; Amy Rommel, PhD ; Edward Motea, PhD ; Praveen Patidar, PhD ; Farjana
2
1
1
2
Fattah, PhD ; Patricia Richards, PhD ; James Manley, PhD ; David A. Boothman, PhD
1
UT Southwestern Medical Center at Dallas, Dallas, TX and Columbia University, New York, NY
2
RNA-DNA hybrids are formed as a result of the natural processivity of RNA polymerase II (RNAPol
II)-mediated transcription. RNAPol II requires a series of factors that bind to its differentially
phosphorylated C-terminal domain (CTD) for smooth processing and for termination. A deficiency in one
or more of these factors can lead to the formation of persistent RNA-DNA hybrids, known as R-loops.
Persistent R-loops result in the formation of complex DNA double strand breaks (DSBs), chromosomal
aberrations and genetic instability. Studies examining the roles of these factors in basal as well as damage-
induced genomic instability are in their infancy. Using hKu70 as bait in a yeast two-hybrid screen, Kub5-
Hera (K-H), the human homolog of the yeast transcription termination factor Rtt103, was isolated. We
show that K-H forms two higher order complexes by gel filtration and proteomics and plays two separate
functions in RNA transcription termination as well as DSB repair. K-H forms distinct protein complexes
with factors that repair DSBs (e.g., Ku70, Ku86, and Artemis) and terminate transcription (e.g., RNA
polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, an activated DNA damage response
(DDR), and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-
H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H is a highly conserved
gene with only one base-pair change from mouse to humans. Yeast deficient in Rtt103 were specifically
defective in repair of complex (blunt or over-hang DNA), but not easily repairable DSBs, in ligation assays,
different from hdfr1 (yKu70-deficient) cells. K-H deficient cells were hypersensitive to cytotoxic agents
that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed [[Unsupported
Character - Symbol Font ]]-H2AX and 53BP1 repair-related foci regression in response to ionizing
radiation (IR). Artemis re-expression in K-H-deficient cells restored DNA repair function and resistance to
DSB-inducing agents. However, R-loops persisted consistent with dual roles of K-H in transcription
90 | P a g e
