Transcript dysregulated genes

Journal Club 5/14/15
Dysregulation of Gene Expression as a Cause of Cockayne
Syndrome Neurological Disease.
Wang Y, et al. PNAS. 2014.
Agenda
 Background
 Cockayne Syndrome
 Clinical manifestations
 Known pathophysiology
 Rationale for the study
 Experimental findings




Gene expression in fibroblasts
iPS reprogramming
Neuroblast differentiation
Cerebellar gene expression
 Discussion of results
 Importance
 Further Directions
Cockayne Syndrome (and me)
What causes Cockayne
Syndrome?
Cockayne Syndrome
 Autosomal Recessive
 Loss of function
 Mutations in ERCC6 (CSB) = 80%
 Mutations in ERCC8 (CSA) = 20%
What does this do?
 UV  big,
bulky DNA
damage
 CSA & B help
perform
nucleotide
excision repair
 Restore ability
of
transcription
Does this make sense?

Problem 1: CS ≠ XP
 XP is in fact a broader defect
than CS, but the symptoms
don’t overlap

Relatedly, Problem 2: Lack of skin
cancer
 Some aren’t sun sensitive at all

Problem 3: Neurologic disease
 How does this fit with UV
exposure?
 Non-UV damage requiring NER
< UV damage by 3 orders of
magnitude

Problem 4: Time course
 Pre/neonatal onset of
symptoms

Problem 5: sensitivity to other DNA
damage, oxidative stress
So, what else?
 Secondary mitochondrial
disease
 Free radical generation
 Mitochondrial DNA repair
 Transcription regulation
effects
 Evidence:
 Known to interact with
RNAPI, RNAPII
Brooks, PJ. DNA Repair. 2014
Hypothesis: Transcription
regulation is a major feature of
mutations in CSB
Goals
1. Demonstrate change in regulation
2. Determine affected tissues
3. Determine relationship between dysregulation and disease
Genes are dysregulated as a
result of CSB mutation
Hypothesis 1.
Cell lines
 Fibroblast line from patient with genetically proven
Cockayne syndrome (CS1AN)
 Immortalized with Sv40
 Rescued cell line
 BAC rescue (BAC-CSB)
 Conditional tetra/doxy promotor rescue (CSB-TetON)
Experiment 1: Comparison of
expression
1,200 “dysregulated genes”
 Statistically significantly
different between
 control v. Bac and
 control v. tetON
 >1.5 fold difference
What is in common?
 Noted mostly neuronal
genes
 Confirmed by RT-PCR
Why does this happen
 Genome wide CHiP-Seq
for
 CSB
 RNAPII
 Looked at genes that are
downregulated in CSB
 Loss of CSB binding
 Loss of RNAPII binding
Summary, experiment 1
 What have we shown?
 There is selective dysregulation of multiple genes
 Many of the downregulated genes are neuronal
 Downregulation happens because mutant CSB does not
bind to the gene target, and RNAPII subsequently doesn’t
bind
 What are the problems
 (immortalized with Sv40)
 Looking at neuronal genes in fibroblasts
 Next step: try to reprogram to NPC
Genes that are dysregulated in
fibroblasts are meaningful for
neuronal function
Hypothesis 2
Experiment 2: reprogramming FCL
•
•
•
•
shRNA knockdown of PTBP1 or
Overexpression of miR-9/124
And introduction of three neuronal transcription regulators
Key event: transition from PTB to nPTB
Unable to transduce mutant cells
32 Genes
Selected for neuronal
function
Consistently upregulated in
WT and not in CSB
Summary, experiment 2
 Unable to transduce cells with loss of function of CSB into
neurons
 Fibroblast to neuronal transduction is not normal
physiology
 Patients with CS obviously have neurons
 What meaning does this have for CSB-mutant neurons?
Experiment 3: neuroblast
differentiation
SH-SY5Y
CSB knockdown
Attempted differentiation
Pahlman, et al. Cell diff. 1984
Experiment 4: neuronal maintenance
Knocked down CSB in
differentiated SH-SY5Y
• Loss of long neurites
• Cell death
Summary of experiments 2 & 3
 Knockdown of CSB affects
 Neuroblast differentiation into neurons
 Neuronal maintenance of established neurons
 Remaining questions
 Why does this happen?
 Is this laboratory model applicable to patients with CS?
Defects in neuronal
differentiation and maintenance
are due to genetic dysregulation
Hypothesis 3
Transcriptome analysis of neuronal
differentiation
Is there a difference in CSB k.d.?
 Overall, no
 Selected by K-means
clustering specific
differences in expression
 Genes identified
 100 genes
 Different at every time
point
 P<0.01
 Did not do multiple
comparison
adjustment
 17 in neuronal ontology
Summary experiment 4
 Some evidence that neuronal problems are due to
transcriptional dysregulation
 These is pretty weak
 Their stats are even weaker
 Now what?
 Mice with CSB K.O. have no neuronal phenotype
 So, turn to human brain
Gene dysregulation will be
demonstrable in human brains
from CS patients
Hypothesis 4
Experiment 5:
 Tissue
 Human cerebellum
 Patients with molecularly
confirmed CS
 Does not specify gene
 Does not specify
mutation type
 “matched” controls
 Extracted RNA
 Hierarchical, nonsupervised
clustering
 Selected genes >2-fold
dysregulated
What are the functions of the
dysregulated genes?
 Exocytosis machinery
 Synaptotagmins
 Voltage dependent
calcium channels
 Maybe explains
 Brain calcifications
 Calcium-dependent
myelination
 Preservation of cerebellar
signature
Summary 1
 In models of CSB mutation there is genetic dysregulation
 Resulting from abnormal CSB binding
 Abnormal RNAPII recruitment
 Genetic dysregulation specifically targets
 Neuronal genes
 Important for neuronal secretion, synaptic density and
neuronal differentiation
 Evidence supports dysregulation as a major cause of
neuronal dysfunction in Cockayne Syndrome
Generalizing the results
Some side experiments
How similar are the models to each
other?
How similar is CSA?
 Does reduce overlap, but
does not change enriched
ontology
But what about mice?
Mice are interesting because they don’t have neuronal dysfunction
Genes differentially regulated between mice and humans with CSB mutations
may be interesting targets for understanding the neuronal disease
Conclusions
 There is compelling evidence that CSB is important for
transcriptional regulation
 Intriguing identification of models of neuronal dysfunction
in CS
 Large dataset with some overlap is best used for
hypothesis generation
 Further questions




Mechanism of CSB targeting to genes
CSA transcriptional analysis
Effects in additional tissue types
Understanding of specific genetic dysregulation important in
disease