Mechanistic insights into the transcriptional arrest in the presence of Double Strand Breaks

Double-strand breaks (DSBs) occur frequently in the genome during genome replication or by DNA damaging agents. DNA lesions affect fundamental DNA-dependent nuclear processes, such as replication and transcription. We have developed an experimental system where DSBs are induced at coding regions of RNA polymerase II transcribing genes. We have started to study the kinetics of RNA polymerase II transcription inhibition in the presence of DNA breaks. We observed that induction of the break led to transcription inhibition and the restoration of transcription closely followed the dynamics of the repair of breaks. We confirmed by chromatinimmunoprecipitation that the break induction led to displacement of RNA polymerase II affecting both the elongation and the initiation of transcription. Our results show that this is dependent on one of the major kinase in DNA damage repair called DNAPKcs. We also investigated the downstream steps of RNA polymerase II removal and we claimed that it was a multistep process involving additional kinases and ubiquitin ligases NEDD4 and CUL3. At the last step of break dependent transcriptional silencing the RNA polymerase II is targeted for proteasome dependent degradation. These data demonstrate that the DNA damage repair complexes and proteasomal system have a synergistic and active role in transcriptional silencing during the DSB repair by removing the RNA pol II from the transcribing region. We show here that DNA lesions occurring at transcribed regions cause a transient repression until the lesion is repaired. This is probably a cell defense mechanism to avoid production of truncated or mutated transcripts in essential genes whose alterations in their gene expression would endanger cell viability. Understudying the role of DNAPKcs, in preventing RNA pol II bypassing a DSB might be a key in avoiding the production of mutated transcripts that could lead to cancerous phenotypes

Determination of the anti-inflammatory and cytoprotective effects of l-glutamine and l-alanine, or dipeptide, supplementation in rats submitted to resistance exercise

We evaluated the effects of chronic oral supplementation with l-glutamine and l-alanine in their free form or as the dipeptide l-alanyl-l-glutamine (DIP) on muscle damage, inflammation and cytoprotection, in rats submitted to progressive resistance exercise (RE). Wistar rats (n 8/group) were submitted to 8-week RE, which consisted of climbing a ladder with progressive loads. In the final 21 d before euthanasia, supplements were delivered in a 4 % solution in drinking water. Glutamine, creatine kinase (CK), lactate dehydrogenase (LDH), TNF-α, specific IL (IL-1β, IL-6 and IL-10) and monocyte chemoattractant protein-1 (MCP-1) levels were evaluated in plasma. The concentrations of glutamine, TNF-α, IL-6 and IL-10, as well as NF-κB activation, were determined in extensor digitorum longus (EDL) skeletal muscle. HSP70 level was assayed in EDL and peripheral blood mononuclear cells (PBMC). RE reduced glutamine concentration in plasma and EDL (P<0·05 v. sedentary group). However, l-glutamine supplements (l-alanine plus l-glutamine (GLN+ALA) and DIP groups) restored glutamine levels in plasma (by 40 and 58 %, respectively) and muscle (by 93 and 105 %, respectively). GLN+ALA and DIP groups also exhibited increased level of HSP70 in EDL and PBMC, consistent with the reduction of NF-κB p65 activation and cytokines in EDL. Muscle protection was also indicated by attenuation in plasma levels of CK, LDH, TNF-α and IL-1β, as well as an increase in IL-6, IL-10 and MCP-1. Our study demonstrates that chronic oral l-glutamine treatment (given with l-alanine or as dipeptide) following progressive RE induces cyprotective effects mediated by HSP70-associated responses to muscle damage and inflammation

Nuclear position dictates DNA repair pathway choice

Faithful DNA repair is essential to avoid chromosomal rearrangements and promote genome integrity. Nuclear organization has emerged as a key parameter in the formation of chromosomal translocations, yet little is known as to whether DNA repair can efficiently occur throughout the nucleus and whether it is affected by the location of the lesion. Here, we induce DNA double-strand breaks (DSBs) at different nuclear compartments and follow their fate. We demonstrate that DSBs induced at the nuclear membrane (but not at nuclear pores or nuclear interior) fail to rapidly activate the DNA damage response (DDR) and repair by homologous recombination (HR). Real-time and superresolution imaging reveal that DNA DSBs within lamina-associated domains do not migrate to more permissive environments for HR, like the nuclear pores or the nuclear interior, but instead are repaired in situ by alternative end-joining. Our results are consistent with a model in which nuclear position dictates the choice of DNA repair pathway, thus revealing a new level of regulation in DSB repair controlled by spatial organization of DNA within the nucleus

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

International audienceDNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation

Activation of homologous recombination in G1 preserves centromeric integrity

Centromeric integrity is key for proper chromosome segregation during cell division1. Centromeres have unique chromatin features that are essential for centromere maintenance2. Although they are intrinsically fragile and represent hotspots for chromosomal rearrangements3, little is known about how centromere integrity in response to DNA damage is preserved. DNA repair by homologous recombination requires the presence of the sister chromatid and is suppressed in the G1 phase of the cell cycle4. Here we demonstrate that DNA breaks that occur at centromeres in G1 recruit the homologous recombination machinery, despite the absence of a sister chromatid. Mechanistically, we show that the centromere-specific histone H3 variant CENP-A and its chaperone HJURP, together with dimethylation of lysine 4 in histone 3 (H3K4me2), enable a succession of events leading to the licensing of homologous recombination in G1. H3K4me2 promotes DNA-end resection by allowing DNA damage-induced centromeric transcription and increased formation of DNA-RNA hybrids. CENP-A and HJURP interact with the deubiquitinase USP11, enabling formation of the RAD51-BRCA1-BRCA2 complex5 and rendering the centromeres accessible to RAD51 recruitment and homologous recombination in G1. Finally, we show that inhibition of homologous recombination in G1 leads to centromeric instability and chromosomal translocations. Our results support a model in which licensing of homologous recombination at centromeric breaks occurs throughout the cell cycle to prevent the activation of mutagenic DNA repair pathways and preserve centromeric integrity

Temporal and spatial uncoupling of DNA double strand break repair pathways within mammalian heterochromatin

Repetitive DNA is packaged into heterochromatin to maintain its integrity. We use CRISPR/Cas9 to induce DSBs in different mammalian heterochromatin structures. We demonstrate that in pericentric heterochromatin, DSBs are positionally stable in G1 and recruit NHEJ factors. In S/G2, DSBs are resected and relocate to the periphery of heterochromatin, where they are retained by RAD51. This is independent of chromatin relaxation but requires end resection and RAD51 exclusion from the core. DSBs that fail to relocate are engaged by NHEJ or SSA proteins. We propose that the spatial disconnection between end resection and RAD51 binding prevents the activation of mutagenic pathways and illegitimate recombination. Interestingly, in centromeric heterochromatin, DSBs recruit both NHEJ and HR proteins throughout the cell cycle. Our results highlight striking differences in the recruitment of DNA repair factors between pericentric and centromeric heterochromatin and suggest a model in which the commitment to specific DNA repair pathways regulates DSB position

TNKS loads the BRCA1A complex on chromatin.

<p><b>(A)</b> TNKS tethering to the lacO array leads to MERIT40 and RAP80 binding in a PARP activity independent manner even in the absence of DSBs. U2OS17 cells were transfected with GFP-lacR, GFP-lacR-TNKS1 or the PARP activity mutant version: GFP-lacR-TNKS1mut. ISce-I was co-transfected to induce pure DSBs at the lacO array. Immunofluorescence staining was performed to visualize the localization pattern of MERIT40 or RAP80 in the cells. The values represent the results of three independent experiments with SD (N = 100). On the right representative confocal microscopy pictures are shown. <b>(B)</b> The recruitment of MERIT40 and RAP80 is affected in TNKS1/2 knock down cells. U2OS17 cells were transfected with the indicated siRNAs. After depletion, cells were transfected with mCherry-lacR and ISce-I where indicated. Immunofluorescence staining was performed to visualize Merit40 or RAP80 in the cells. Relative values compared to the control are the results of three independent experiments with SD (N = 100 cells in each condition). <b>(C)</b> TNKS depleted cells are deficient for RAP80 foci formation. U2OS cells were transfected with siRNAs as shown and treated with NCS 48 hours later. The frequency of foci-positive cells was determined in three independent experiments (N = 100) and is shown as relative to the control <b>(D)</b> MDC1-mediated TNKS recruitment is necessary for efficient binding of the BRCA1 complex to DSBs <i>in vivo</i>. U2OS17 cells were transfected with mCherry-lacR-MDC1 wt or its TBD mutant version and ISce-I. Percent of cells harboring MERIT40 or RAP80 signal on the array was determined after immunofuorescence staining. Results from three independent experiments are shown with SD (N = 100) as relative frequencies compared to control. <b>(E)</b> MERIT40 stabilizes BRCA1 on the TNKS-bound chromatin. Control or MERIT40 depleted U2OS17 cells were transfected with GFP-lacR, GFP-lacR-TNKS1, GFP-lacR-TNKS1mut and ISce-I. Percent of cells harboring BRCA1 signal on the array was determined. Results from three independent experiments are shown with SD (N = 100).</p