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DNA fragility in the context of neural stem cell fate : a multi-method integrative exploration of genome dynamics

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posted on 2024-09-03, 00:31 authored by Roberto BallarinoRoberto Ballarino

Recent advances in mapping the complex genetic architecture underlying various debilitating brain disorders have enabled identification of several genetic risk variants. However, these risk variants only explain part of the heritability and vulnerability to these disorders in early development. Moreover, de novo somatic mutations have been detected in subsets of brain cells, which might account for a significant portion of the missing heritability. However, it remains unclear where these mutations come from and at what developmental stage they might occur.

Genome fragility is subject to the functional activity and spatial chromatin organization characteristic of a distinct cell identity. Under physiological conditions, cells regulate their chromatin structure and organization to express necessary genes. DNA topoisomerases are a key player in all of these processes and in replication. Through generation of transient breaks in the DNA, topoisomerases are able to resolve topological problems and thereby activation of particular sections of the genome. Beyond topoisomerases, the genome is subject to perpetual challenges with DNA double-strand breaks (DSBs) being among the most deleterious. Each cell is estimated to suffer numerous transient DSBs per day, most of which are repaired. Incorrectly repaired DSBs however, pose a major threat to genome stability through formation of mutations or potential genomic rearrangements. Although the exact relationship of DNA damage to differentiation is still unclear, a recent investigation into neural specification demonstrated that loss of DNA repair sensors leads to centrosome amplification, thereby resulting in defective mitosis and chromosomal instability. Ensuing excessive stem cell proliferation and replication stress also happen to be a hallmark of neurodevelopmental disorders (NDDs). Despite the emerging evidence linking endogenous DSBs to NDDs, there has been a lack of genome-wide maps of DSBs spontaneously arising at different stages of human neurogenesis. This thesis brings together (I) a correlative genomics study describing endogenous DSBs genome-wide during neural differentiation in a cell-type specific manner, and (II) a mechanistic study into the regulatory role of Topoisomerase 1 (TOP1) in transcription and proliferation.

In paper I, we mapped the genomic DSB landscape of cells at various stages of neural differentiation and correlated our maps with genomic and epigenomic features. In so doing, we provide clues on how DSB formation and their incorrect repair might contribute to the pathogenesis of NDDs. The current view is that transcription-associated DSBs seem to be the main driver of de novo mutations. Indeed, we found that DSBs preferentially form around the transcription start site (TSS) of transcriptionally active genes, as well as at chromatin loop anchors in proximity of highly transcribed genes. This follows from the accumulation of DNA torsional stress and topoisomerase activity in these regions. Interestingly, hotspots of endogenous DSBs were detected around the TSS of highly transcribed genes involved in general cellular processes and along the gene body of long, neural-specific genes whose human orthologues had been previously implicated in NDDs. Through our integrative multimethod approach we corroborate previous findings regarding DSB-fragile loci at TSSs and loop anchors, and find a unique distribution pattern for this fragility in post-mitotic neurons. We show a cell type-specific preference for DSB accumulation in specific NDD genes and begin to describe the relation of DSB fragility and chromatin conformation.

In paper II, we investigated the role of Topoisomerase I (TOP1) in relation to transcription in the context of replication stress across mitosis and as subject of interruption of interphase chromatin conformation. In particular, we investigated different stages of the cell cycle for transcription patterns and transcriptional spiking by RNA polymerase II (RNAPII) in human colon carcinoma cells. TOP1 relieves torsional stress in actively transcribed DNA and facilitates the expression of long genes, many of which are important for neural functions. However, TOP1 also plays a direct role in transcriptional control through interaction with RNAPII Carboxy-Terminal Domain (CTD). We investigated control cells and a knock-in (KI) clone lacking TOP1 exon4, the phosphor-CTD-binding site for RNAPII. We found that in early mitosis TOP1 clears RNAPII during transcriptional elongation. When the TOP1 CTD-binding domain is disrupted, we detected replication stress and delay in mitotic exit. In this case, chromatin becomes topologically stressed, increasing the need for TOP2A cleavage resulting in DSBs. However, we did not detect substantial changes in DSB markers gamma- H2AX and 53BP1 when comparing WT and KI cells across different stages of the cell cycle. Therefore, we conclude that the observed delay in mitotic exit is most likely due to the deregulation of gene expression, rather than to the activation of DNA repair pathways. Acute depletion of TOP1 through the auxin-degron system resulted in absence of RNAPII spiking at the TSS. Efficient removal of RNAPII from chromosomes by TOP1 in early mitosis is both a prerequisite for the timely spike of RNAPII at TSSs in mid mitosis and might affect cellular memory. Indeed, we found that when mitotic transcription is poorly regulated, individual proliferating cells have a greater variance in transcriptional levels and thus could lead to loss of cell identity.

Concluding from these findings, we demonstrate that endogenous DSBs are distributed differentially in a cell type-specific manner. Through our integrative multi-method approach, we corroborate previous findings regarding DSB-fragile loci and discovered a unique distribution pattern for DSBs in post-mitotic neurons. We show a preference for specific NDDs genes and begin to describe the relation of DSB fragility and chromatin conformation in a developmental context. We assessed the role of TOP1 in a model for replication stress and found that outside of its canonical torsional stress function, the direct interaction with RNAPII across the cell cycle is crucial in maintaining transcriptional memory and could feed into loss of cell identity. While not exhaustive, the findings described in these papers begin to elucidate a complex mystery of human NDDs and provide valuable datasets for further investigation of genome fragility. Taken together, these findings contribute to a better understanding of how neural genome dynamics affect high transcriptional or replicative burden during neurodevelopment.

List of scientific papers

I. Ballarino R., Bouwman B.A.M., Agostini F., Harbers L., Diekmann C., Wernersson E., Bienko M., Crosetto N. (2022). An atlas of endogenous DNA double-strand breaks arising during human neural cell fate determination. Scientific Data. 9:400, 1-19.
https://doi.org/10.1038/s41597-022-01508-x

II. Wiegard A., Kuzin V., Cameron D.P., Grosser J., Ceribelli M., Mehmood R., Ballarino R., Valant F., Grochowski R., Karabogdan I., Crosetto N., Bizard A.H., Kouzine F., Natsume T., Baranello L. (2021). Topoisomerase 1 activity during mitotic transcription favors the transition from mitosis to G1. Molecular Cell. 81, 5007–5024.
https://doi.org/10.1016/j.molcel.2021.10.015

History

Defence date

2022-11-08

Department

  • Department of Medical Biochemistry and Biophysics

Publisher/Institution

Karolinska Institutet

Main supervisor

Crosetto, Nicola

Co-supervisors

Carlén, Marie; Falk, Anna; Bienko, Magda; Agostini, Federico

Publication year

2022

Thesis type

  • Doctoral thesis

ISBN

978-91-8016-773-4

Number of supporting papers

2

Language

  • eng

Original publication date

2022-10-18

Author name in thesis

Ballarino, Roberto

Original department name

Department of Medical Biochemistry and Biophysics

Place of publication

Stockholm

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