Loss of ATM causes R-loop-associated transcriptional dysregulation and attenuates the related response to DNA damage Open Access

Abstract

An early childhood onset neurodegenerative disorder, Ataxia Telangiectasia (AT), affects 1 in 40,000 to 100,000 individuals worldwide and is caused by mutations in the ataxia telangiectasia mutated (ATM) threonine-serine kinase, which regulates the DNA damage response (DDR). While the genetic cause of AT has been known for years, the exact molecular mechanisms, particularly at the transcriptomic level, underlying disease progression within neurons, which are susceptible to cellular apoptosis due to increased levels of unrepaired DNA damage, remain poorly understood. Three stranded structures, known as R-loops, have recently emerged as important players in DDR via regulating key gene expression. This thesis utilizes neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells reprogrammed from AT patient-derived somatic cells to answer three main questions: 1) How does loss of ATM impact R-loop and transcriptional regulation, 2) how does loss of ATM impact cellular R-loop and transcriptional response to DNA damage, and 3) what is the hierarchical order and mechanistic causality between R-loops and transcription in response to DNA damage. AT-derived NPCs (AT-NPCs) exhibit elevated spontaneous R-loop levels compared to controls, as well as a strong positive correlation between R-loop accumulation and bidirectional transcriptional changes on a subset of dysregulated genes. Upon acute damage, loss of ATM results in an attenuated response, characterized by the impaired R-loop and transcriptional response to irradiation. Both control and AT-NPCs undergo a similar cell cycle arrest, but AT-NPCs display an attenuated R-loop and transcriptional response, failing to activate proper DDR. Importantly, R-loop formation is required for many key genes to properly respond to DNA damage, supporting a direct and causal role in this process. Overall, our data reveal an underappreciated mechanistic link between ATM, R-loop regulation, and transcription, the disruption of which may contribute to the impaired DDR observed in AT-NPCs.

Table of Contents

Chapter 1: Introduction to Dissertation 1

1.1: Ataxia Telangiectasia 2

1.1.1: AT diagnosis 3

1.1.2: Models of AT 4

1.2: DNA damage and repair 5

1.2.1: DNA damage 5

1.2.2: DNA repair 6

1.2.3: The brain is vulnerable to DNA damage 8

1.3: R-loops 9

1.3.1: R-loop formation 9

1.3.2: R-loop resolution 10

1.4: R-loops are involved in multiple biological processes 11

1.5: R-loops in DNA damage and response 12

1.5.1: R-loops cause DNA damage 12

1.5.2: R-loops contribute to DDR 16

1.6: R-loops in disease 18

1.6.1: R-loops in trinucleotide repeat disorders 19

1.6.2: Monogenic disorders caused by mutations in R-loop factors 22

1.6.3: Several ATM substrates can regulate R-loops 24

1.7: Concluding remarks 26

Chapter 2: Loss of ATM causes R-loop-associated transcriptional dysregulation and attenuates the related response to DNA damage 27

2.1: Summary 28

2.2: Introduction 28

2.3: Results 31

2.3.1: Loss of ATM protein in AT-NPCs results in a global increase in DNA damage and R-loops 31

2.3.2: AT-NPCs demonstrate a higher accumulation of R-loops, which correlate with upregulation of genes involved in neuronal function and DDR 32

2.3.3: Irradiation induces accumulation of DNA damage and activation of ATM 34

2.3.4: AT-NPCs demonstrate an impaired R-loop response to irradiation 35

2.3.5: AT-NPCs lack R-loop-associated changes in transcription in DDR-related genes 37

2.3.6: R-loops play broad and causal roles in responding to DNA damage induced from diverse sources, by regulating the expression of a subset of key DDR genes 40

2.4: Discussion 43

2.5: Figures and Figure Legends 48

2.6: Materials and Methods 72

Chapter 3: Conclusions and Future Directions 79

3.1: Conclusions 80

3.2: Future directions 80

3.2.1: Models for AT 81

3.2.2: Methodology 82

3.2.3: Mechanisms 84

3.2.4: Translational 86

3.3: Closing remarks 87

References: 89

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Genetics Biology, Molecular
Keyword	gene transcription DNA damage response R-loops neuronal progenitor cells Ataxia Telangiectasia
Committee Chair / Thesis Advisor	Yao, Bing, Emory University
Committee Members	Corbett, Anita, Emory University Wen, Zhexing, Emory University Escayg, Andrew, Emory University Jin, Peng, Emory University

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