The document analyzes RNA sequencing data from human cells deficient and proficient in the XPA nucleotide excision repair protein. It finds that XPA deficiency impacts the expression of genes involved in metabolism, neurology and mitochondria. Specifically, it identifies several genes over 1.5 fold changed related to these functions. Comparing different cell lines, it shows patient lines are more similar to each other than to the HeLa line. The analysis suggests further studying how XPA interacts with impacted genes and pathways could help understand its role in cell physiology and potentially increase life expectancy for those with XPA deficiency.

1. Changes in Transcriptome in Human
Cells Deficient and Proficient in XPA
Usman Hyder
P.I.: Dr. Richard D. Wood
Mentor: Mandira Manandhar

2. Acknowledgements
Wood Lab:
– Dr. Richard Wood
– Mandira Manandhar (Mentor)
– Karen Boulware
– Megan Lowery
– Sara Martin
– Shelley Reh
– Dr. Kei-ichi Takata
– Dr. Junya Tomida
NGS Core Facility
Summer Program in Cancer
Research:
– Chris Contreras
– Dr. Johnson
– Dr. McBride

3. XPA
• Nucleotide Excision Repair (NER) Protein
• Deficiency in any NER proteins can lead to
Xeroderma Pigmentosum (XP)
• Skin Cancer and neurological disorders
• Looking at the extent to which XPA deficiency
influences overall genome expression, and to see
what those genes can tell us about the XP
phenotype

4. XPA
• Nucleotide Excision Repair (NER) Protein
• Deficiency in any NER proteins can lead
to Xeroderma Pigmentosum
• Skin Cancer and neurological disorders
• Looking at the extent to which XPA
deficiency influences overall genome
expression, and to see what those genes
can tell us about the XP phenotype
Halpern et al.,
2008, Cases
Journal

5. Nucleotide Excision Repair
• TFllH complex binds to
damage site and
unwinds the strand
• XPA verifies damage
XPC
Complex
TFIIH
Complex
UV
XPA
Excision
• XPC complex looks for
helix distortions
DNA
Pol
Ligase
• Specific
endonucleases
cut and excise
• Strand is
repaired
XPGERCC1-
XPF
Adapted from
Martejin et al.,
2014, Nature
5’
5’
3’ 5’
5’
3’ 5’
5’
5’
3’
3’
3’
3’
3’
3’

6. NER Proteins at Promoter Sites
Le May et al.,
2010, Molecular
Cell

7. GO term GM969 shXPA vs.
GM969 SCRM
XPA- vs. XPA +
Z-Ratio Z-Ratio
GO0005743: Mt. inner membrane > 100 > 100
GO0005743: Mitochondrion > 100 > 100
GO0005743: Mt. Respiratory Chain > 100 93.96
GO0005743: Mt. electron transport > 100 93.96
GO0005743: Cytochrome C oxidase > 100 > 100
GO0005743: NADH dehydrogenase > 100 85.54
XPA Deficiency impairs mitophagy
Fang et al.,
2014, Cell
XPA
Red = Mitochondrial Disease
Green = Nonmitochondrial Disease
Scheibye-Knudsen
et al., 2013, Aging

8. Three Cell Lines
HeLa Cell
Line
KO
WT
2OS Patient
Cell Line
2OS
XPA2OS
12RO
Patient Cell
Line
12RO
XPA12ROPositive
Cell Lines
Negative
Cell Lines
All cell lines
not subject
to UV
irradiation

9. RNA-Seq
1. Extraction of RNA
2. Isolation of mRNA
• Using the Poly A Library and Thymine beads
3. Sequencing
• Using the indirect method

10. Sequencing
Junction Reads
Exonic Reads
Poly(A) end Reads
Extracted mRNA
ssDNA
cDNAmRNA
Short Sequence Reads
Reverse Transcriptase
Adapted from
Wang et al., 2009
Nature Review

11. Pathway Analysis: Mitophagy GO Term
GO term related to Mitophagy Negative Vs.
Positive HeLa
Negative Vs.
Positive 12RO
Negative Vs.
Positive 2OS
Fold Enrichment Fold Enrichment Fold Enrichment
Response to mitochondrial depolarization (GO:0098780) > 100 > 100 > 100
Mitophagy in response to mitochondrial depolarization
(GO:0098779)
> 100 > 100 > 100
Mitophagy (GO:0000422) > 100 93.96 > 100
Mitochondrion disassembly (GO:0061726) > 100 93.96 > 100
Macromitophagy (GO:0000423) > 100 > 100 67.96
Organelle disassembly (GO:1903008) > 100 85.54 97.08
Macroautophagy (GO:0016236) 77.66 67.53 > 100
Autophagy (GO:0006914) 54.34 47.26 50.95
Mitochondrion organization (GO:0007005) 33.41 27.6 33.41

12. Metabolic
NeurologicalMitochondrial

13. Metabolic
• 143/685 genes
• 13 genes >1.5
fold change
Neurological
• 131/685 genes
• 12 genes >1.5
fold change
Mitochondrial
• 59/685 genes
• 7 genes >1.5
fold change
Analysis of Results
• 685 total genes have the same trend (7.09%-9.20% of genome)
• 79 of those genes are 1.5 fold change (FC) or greater (0.82%-1.06%)

14. Two-Fold Genes
Gene Function
AKR1C1 Enzyme that converts aldehydes to alcohols
AKR1C2 Enzyme that converts aldehydes to alcohols
AKR1C3 Enzyme that converts aldehydes to alcohols
GNAO1 G protein subunit, defects can lead to epilepsy
EGF Cell proliferation (Nervous system development)
TENM1 Cell signal transduction, nervous development
SERPINE2 Serine Protease inhibitor, Related to Alzheimer's/ALS
TLR4 Innate immunity, Apoptotic (microglia/macrophages)
MAGEA3
Ubiquitin ligase activity and p53 degradation, Apoptosis
(Multiple Myeloma)
Metabolic
functions
Mitochondrial
functions
Red = Up
Green = Down
when XPA present:
Neural
functions

15. Fibroblasts
• The patient cell lines
are more closely
related to each other
than either of them are
to the HeLa cell line
Negative vs.
Positive HeLa
Negative vs.
Positive 2OS
Negative vs.
Positive 12RO

16. Metabolic
• 527/3,044
genes
• 112 genes >1.5
fold change
Neurological
• 516/3,044
genes
• 147 genes >1.5
fold change
Mitochondrial
• 250/3,044
genes
• 50 genes >1.5
fold change
Analysis for Patient Cell Lines Only
• 3,044 genes have the same trend (31.50%-34.95%)
• 762 of those genes are 1.5 fold change or greater (7.88%-9.06%)

17. Summary/Outlook
• There are a number of genes and pathways that are XPA
dependent (with functions outside of DNA repair).
• Those genes and pathways are related to metabolic and
neurological functions as well as others.
• Studying those genes in coordination with XPA may be
helpful to understand XPA’s role in cell physiology.
• Potential mechanisms
• With time, we may be able to increase the life
expectancy of XP patients.