Journal Club 5/14/15Dysregulation 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)

dysmorphic

Microcephalic Very small

Hypomyelination

deafRetinal disease

Abnormal liver enzymes

Developmental delay

Ataxia

Joint contracture

sSpine differences

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

UV

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

UV

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 CSBGoals1. Demonstrate change in regulation2. Determine affected tissues3. Determine relationship between dysregulation and disease

Genes are dysregulated as a result of CSB mutationHypothesis 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 functionHypothesis 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 GenesSelected for neuronal functionConsistently 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

RA

Pahlman, et al. Cell diff. 1984

Attempted differentiation

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 dysregulationHypothesis 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 patientsHypothesis 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 resultsSome 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