scholarly journals Nde1 is Required for Heterochromatin Compaction and Stability in Neocortical Neurons

2021 ◽  
Author(s):  
Alison A Chomiak ◽  
Clara C Lowe ◽  
Yan Guo ◽  
Dennis Mcdaniel ◽  
Hongna Pan ◽  
...  
Keyword(s):  
Nuclear Architecture ◽  
Brain Dysfunction ◽  
Copy Number Variations ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Neocortical Neurons ◽  
Satellite Repeats ◽  
Wide Range

The NDE1 gene encodes a scaffold protein essential for brain development. While biallelic NDE1 loss of function (LOF) causes microcephaly with profound mental retardation, NDE1 missense mutations and copy number variations are associated with multiple neuropsychiatric disorders. However, the etiology of the diverse phenotypes resulting from NDE1 aberrations remains elusive. Here we show Nde1 controls neurogenesis through heterochromatin compaction via histone H4K20 trimethylation. This mechanism patterns diverse chromatin landscapes and stabilizes constitutive heterochromatin of neocortical neurons. We show NDE1 undergoes dynamic liquid-liquid phase separation, partitioning to the nucleus and interacting with pericentromeric and centromeric satellite repeats. Nde1 LOF results in nuclear architecture aberrations, DNA double strand breaks, as well as instability and derepression of pericentromeric satellite repeats in neocortical neurons. These findings uncover a pivotal role of NDE1/Nde1 in establishing and maintaining neuronal heterochromatin. They suggest that heterochromatin impairments underlie a wide range of brain dysfunction.

scholarly journals Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair

2021 ◽  
Author(s):  
Yasunori Horikoshi ◽  
Hiroki Shima ◽  
Wataru Kobayashi ◽  
Jiying Sun ◽  
Volker J Schmid ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Super Resolution ◽  
Double Strand Breaks ◽  
Genome Integrity ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Sister Chromatids ◽  
Super Resolution Microscopy ◽  
Damaged Dna

Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.

scholarly journals Spatiotemporal dynamics of 53BP1 dimer recruitment to a DNA double strand break

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jieqiong Lou ◽  
David G. Priest ◽  
Ashleigh Solano ◽  
Adèle Kerjouan ◽  
Elizabeth Hinde
Keyword(s):  
Nuclear Architecture ◽  
Strand Break ◽  
Cell System ◽  
Spatiotemporal Dynamics ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Dna Double Strand Break ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
Self Association

AbstractTumor suppressor p53-binding protein 1 (53BP1) is a DNA repair protein essential for the detection, assessment, and resolution of DNA double strand breaks (DSBs). The presence of a DSB is signaled to 53BP1 via a local histone modification cascade that triggers the binding of 53BP1 dimers to chromatin flanking this type of lesion. While biochemical studies have established that 53BP1 exists as a dimer, it has never been shown in a living cell when or where 53BP1 dimerizes upon recruitment to a DSB site, or upon arrival at this nuclear location, how the DSB histone code to which 53BP1 dimers bind regulates retention and self-association into higher-order oligomers. Thus, here in live-cell nuclear architecture we quantify the spatiotemporal dynamics of 53BP1 oligomerization during a DSB DNA damage response by coupling fluorescence fluctuation spectroscopy (FFS) with the DSB inducible via AsiSI cell system (DIvA). From adopting this multiplexed approach, we find that preformed 53BP1 dimers relocate from the nucleoplasm to DSB sites, where consecutive recognition of ubiquitinated lysine 15 of histone 2A (H2AK15ub) and di-methylated lysine 20 of histone 4 (H4K20me2), leads to the assembly of 53BP1 oligomers and a mature 53BP1 foci structure.

scholarly journals Impact of DNA Double-Strand Breaks on Pancreaticobiliary Maljunction Carcinogenesis

2021 ◽  
Author(s):  
Yasuhiro Kuraishi ◽  
Takeshi Uehara ◽  
Takashi Muraki ◽  
Mai Iwaya ◽  
Yasuhiro Kinugawa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Biliary Tract ◽  
Cell Damage ◽  
Pancreaticobiliary Maljunction ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Positive Proportion ◽  
Wide Range ◽  
The Impact

Abstract Background: Pancreaticobiliary maljunction (PBM) is a condition characterized by chronic inflammation due to refluxed pancreatic juice into the biliary tract that is associated with an elevated risk of biliary tract cancer. DNA double-strand breaks (DSBs) are considered the most serious form of DNA damage. DSBs are provoked by inflammatory cell damage and are recognized as an important oncogenic event in several cancers. This study used γ-H2AX, an established marker of DSB formation, to evaluate the impact of DNA damage on carcinogenesis in PBM. Methods: We investigated γ-H2AX expression immunohistochemically in gallbladder epithelium samples obtained from 71 PBM cases and 19 control cases. Results: Fourteen PBM cases with gallbladder adenocarcinoma were evaluated at non-neoplastic regions. A wide range of nuclear γ-H2AX staining was detected in all PBM and control specimens. γ-H2AX expression was significantly higher in PBM cases versus controls (median γ-H2AX-positive proportion: 14.4% vs. 4.4%, p = 0.001). Among the PBM cases, γ-H2AX expression was significantly higher in patients with carcinoma than in those without (median γ-H2AX-positive proportion: 21.4% vs. 11.0%, p = 0.031). Conclusion: DSBs occurred significantly more abundantly in the PBM gallbladder mucosa, especially in the context of cancer, indicating an involvement in PBM-related carcinogenesis.

scholarly journals Impact of DNA double-strand breaks on pancreaticobiliary maljunction carcinogenesis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Yasuhiro Kuraishi ◽  
Takeshi Uehara ◽  
Takashi Muraki ◽  
Mai Iwaya ◽  
Yasuhiro Kinugawa ◽  
...  
Keyword(s):  
Dna Damage ◽  
Biliary Tract ◽  
Cell Damage ◽  
Pancreaticobiliary Maljunction ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Positive Proportion ◽  
Wide Range ◽  
The Impact

Abstract Background Pancreaticobiliary maljunction (PBM) is a condition characterized by chronic inflammation due to refluxed pancreatic juice into the biliary tract that is associated with an elevated risk of biliary tract cancer. DNA double-strand breaks (DSBs) are considered the most serious form of DNA damage. DSBs are provoked by inflammatory cell damage and are recognized as an important oncogenic event in several cancers. This study used γ-H2AX, an established marker of DSB formation, to evaluate the impact of DNA damage on carcinogenesis in PBM. Methods We investigated γ-H2AX expression immunohistochemically in gallbladder epithelium samples obtained from 71 PBM cases and 19 control cases. Results Fourteen PBM cases with gallbladder adenocarcinoma were evaluated at non-neoplastic regions. A wide range of nuclear γ-H2AX staining was detected in all PBM and control specimens. γ-H2AX expression was significantly higher in PBM cases versus controls (median γ-H2AX-positive proportion: 14.4 % vs. 4.4 %, p = 0.001). Among the PBM cases, γ-H2AX expression was significantly higher in patients with carcinoma than in those without (median γ-H2AX-positive proportion: 21.4 % vs. 11.0 %, p = 0.031). Conclusions DSBs occurred significantly more abundantly in the PBM gallbladder mucosa, especially in the context of cancer, indicating an involvement in PBM-related carcinogenesis.

scholarly journals CRISPR FokI Dead Cas9 System: Principles and Applications in Genome Engineering

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2518
Author(s):  
Maryam Saifaldeen ◽  
Dana E. Al-Ansari ◽  
Dindial Ramotar ◽  
Mustapha Aouida
Keyword(s):  
Gene Editing ◽  
Genome Engineering ◽  
Catalytic Domain ◽  
Zinc Finger Nucleases ◽  
Dna Double Strand Breaks ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Protospacer Adjacent Motif ◽  
Cas9 Protein ◽  
Wide Range

The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering.

scholarly journals Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration

2020 ◽  
Author(s):  
Yan Guo ◽  
Alison A Chomiak ◽  
Ye Hong ◽  
Clara C Lowe ◽  
Wen-Ching Chan ◽  
...  
Keyword(s):  
Cellular Senescence ◽  
Brain Aging ◽  
Genome Instability ◽  
Cellular Level ◽  
Double Strand Breaks ◽  
Protein Accumulation ◽  
Histone H2a ◽  
Loss Of Function ◽  
Dna Double Strand Breaks ◽  
Strand Breaks

Aging is an intricate process that is characterized by multiple hallmarks including stem cell exhaustion, genome instability, epigenome alteration, impaired proteostasis, and cellular senescence. While each of these is detrimental at the cellular level, it remains unclear how they are interconnected to cause systemic organ deterioration. Here we show that abrogating Brap, a BRCA1 associated protein, results in cellular senescence with persistent DNA double-strand breaks and elevation of histone H2A mono- and poly-ubiquitination (H2Aub). The high H2Aub initiates proteasome-dependent histone proteolysis, leading to global epigenetic alteration, ubiquitinated protein accumulation, and senescence reinforcement. When these defects occur in mice carrying Brap deletions in cerebral cortical neural progenitors or postnatal neurons, they accelerate brain aging, induce neurodegeneration, and shorten lifespan. As we show H2Aub is also increased in human brain tissues of Alzheimer disease, these data together suggest that chromatin aberrations mediated by H2Aub act as a nexus of multiple aging hallmarks.

Repair pathway choice for double-strand breaks

2020 ◽  
Vol 64 (5) ◽  
pp. 765-777 ◽  
Author(s):  
Yixi Xu ◽  
Dongyi Xu
Keyword(s):  
Cell Cycle ◽  
Double Strand Breaks ◽  
Homologous Region ◽  
Gene Repair ◽  
Repair Pathway ◽  
Dna Double Strand Breaks ◽  
Environmental Radiation ◽  
Strand Breaks ◽  
Dna End Resection ◽  
End Resection

Abstract Deoxyribonucleic acid (DNA) is at a constant risk of damage from endogenous substances, environmental radiation, and chemical stressors. DNA double-strand breaks (DSBs) pose a significant threat to genomic integrity and cell survival. There are two major pathways for DSB repair: nonhomologous end-joining (NHEJ) and homologous recombination (HR). The extent of DNA end resection, which determines the length of the 3′ single-stranded DNA (ssDNA) overhang, is the primary factor that determines whether repair is carried out via NHEJ or HR. NHEJ, which does not require a 3′ ssDNA tail, occurs throughout the cell cycle. 53BP1 and the cofactors PTIP or RIF1-shieldin protect the broken DNA end, inhibit long-range end resection and thus promote NHEJ. In contrast, HR mainly occurs during the S/G2 phase and requires DNA end processing to create a 3′ tail that can invade a homologous region, ensuring faithful gene repair. BRCA1 and the cofactors CtIP, EXO1, BLM/DNA2, and the MRE11–RAD50–NBS1 (MRN) complex promote DNA end resection and thus HR. DNA resection is influenced by the cell cycle, the chromatin environment, and the complexity of the DNA end break. Herein, we summarize the key factors involved in repair pathway selection for DSBs and discuss recent related publications.

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