1. Journal Club 5/14/15 Dysregulation of Gene Expression as a Cause of Cockayne Syndrome Neurological Disease. Wang Y, et al. PNAS. 2014.

2. 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

3. Cockayne Syndrome (and me)

4. What causes Cockayne Syndrome?

5. Cockayne Syndrome  Autosomal Recessive  Loss of function  Mutations in ERCC6 (CSB) = 80%  Mutations in ERCC8 (CSA) = 20%

6. What does this do?  UV  big, bulky DNA damage  CSA & B help perform nucleotide excision repair  Restore ability of transcription

7. 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

8. 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

9. 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

10. Genes are dysregulated as a result of CSB mutation Hypothesis 1.

11. 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)

12. Experiment 1: Comparison of expression 1,200 “dysregulated genes”  Statistically significantly different between  control v. Bac and  control v. tetON  >1.5 fold difference

13. What is in common?  Noted mostly neuronal genes  Confirmed by RT-PCR

14. 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

15. 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

16. Genes that are dysregulated in fibroblasts are meaningful for neuronal function Hypothesis 2

17. 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

18. Unable to transduce mutant cells 32 Genes Selected for neuronal function Consistently upregulated in WT and not in CSB

19. 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?

20. Experiment 3: neuroblast differentiation SH-SY5Y CSB knockdown Pahlman, et al. Cell diff. 1984 Attempted differentiation

22. Experiment 4: neuronal maintenance Knocked down CSB in differentiated SH-SY5Y Loss of long neurites Cell death

23. 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?

24. Defects in neuronal differentiation and maintenance are due to genetic dysregulation Hypothesis 3

25. Transcriptome analysis of neuronal differentiation

26. 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

27. 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

28. Gene dysregulation will be demonstrable in human brains from CS patients Hypothesis 4

29. 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

30. 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

31. 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

32. Generalizing the results Some side experiments

33. How similar are the models to each other?

34. How similar is CSA?  Does reduce overlap, but does not change enriched ontology

35. 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

36. 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