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Anti-Fibrillarin Antibody [38F3] (A85370) - Identical to Abcam (ab4566)

Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization.

Cites: Anti-Fibrillarin Antibody [38F3] [38F3] (ab4566)

Abstract:

There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including γH2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of aberrant mRNA and protein products encoded by damaged genes.

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Acta Neuropathol Commun. 2016 Apr 22; 4:41. doi: 10.1186 / s40478-016-0312-9.

Neuronal accumulation of unrepaired DNA in a novel specific chromatin domain: structural, molecular and transcriptional characterization.

Mata-Garrido J.1, Casafont I.1, Tapia O1, Berciano MT1, Lafarga M2.
1
Departamento de Anatomía y Biología Celular and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", Universidad de Cantabria-IDIVAL, Avda. Cardenal Herrera Oria s / n, Santander, Spain.
2
Departamento de Anatomía y Biología Celular and "Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)", Universidad de Cantabria-IDIVAL, Avda. Cardenal Herrera Oria s / n, Santander, Spain. [email protected]

Abstract:

There is growing evidence that defective DNA repair in neurons with accumulation of DNA lesions and loss of genome integrity underlies aging and many neurodegenerative disorders. An important challenge is to understand how neurons can tolerate the accumulation of persistent DNA lesions without triggering the apoptotic pathway. Here we study the impact of the accumulation of unrepaired DNA on the chromatin architecture, kinetics of the DNA damage response and transcriptional activity in rat sensory ganglion neurons exposed to 1-to-3 doses of ionizing radiation (IR). In particular, we have characterized the structural, molecular and transcriptional compartmentalization of unrepaired DNA in persistent DNA damaged foci (PDDF). IR induced the formation of numerous transient foci, which repaired DNA within the 24 h post-IR, and a 1-to-3 PDDF. The latter concentrate DNA damage signaling and repair factors, including γH2AX, pATM, WRAP53 and 53BP1. The number and size of PDDF was dependent on the doses of IR administered. The proportion of neurons carrying PDDF decreased over time of post-IR, indicating that a slow DNA repair occurs in some foci. The fine structure of PDDF consisted of a loose network of unfolded 30 nm chromatin fiber intermediates, which may provide a structural scaffold accessible for DNA repair factors. Furthermore, the transcription assay demonstrated that PDDF are transcriptionally silent, although transcription occurred in flanking euchromatin. Therefore, the expression of γH2AX can be used as a reliable marker of gene silencing in DNA damaged neurons. Moreover, PDDF were located in repressive nuclear environments, preferentially in the perinucleolar domain where they were frequently associated with Cajal bodies or heterochromatin clumps forming a structural triad. We propose that the sequestration of unrepaired DNA in discrete PDDF and the transcriptional silencing can be essential to preserve genome stability and prevent the synthesis of aberrant mRNA and protein products encoded by damaged genes.

Acta Neuropathol Commun. 2016 Apr 22; 4:41. doi: 10.1186 / s40478-016-0312-9.

Fig. 6

a-e Confocal microscopy images of SGN nuclei co-stained for γH2AX in combination with fibrillarin (a, b), coilin (c), histone H4K20me3 (d) and HP1γ (e). They illustrate the direct association of PDDF with a triad of structures: the nucleolus (a, c, e), Cajal bodies, immunolabeled for fibrillarin (b) or coilin (c), and perinucleolar heterochromatin clumps immunolabled for the tri-methylated histone H4K20me3 (d) or HP1γ (e). f Double immunolabeling for 53BP1 and TMG-cap illustrate the distribution of the nuclear speckles of splicing factors labeled with the anti-TMG-cap antibody and the close association of a PDDF with a speckle in the vicinity of the nucleolus. 45d post-IR. Scale bar = 5 μm

Acta Neuropathol Commun. 2016 Apr 22; 4:41. doi: 10.1186 / s40478-016-0312-9.

Fig. 7

a, b Immunogold electron microscopy illustrates the direct association of PDDF with the nucleolus and a Cajal body labeled with the anti-fibrillarin antibody (a), and the nucleolus and a coilin-positive Cajal body (b). c A 53BP1-positive PDDF appears associated with a cluster of interchromatin granules (IGC) and a heterochromatin mass (hc). Scale bar: a = 500 nm, b = 400 nm and c = 100 nm