shrnas for mad2l2  (PeproTech)

Journal: Cell Cycle

Article Title: H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

doi: 10.1080/15384101.2017.1404210

Figure Lengend Snippet: CDK activity regulates end resection downstream of RIF1 and MAD2L2. a) Analysis of RIF1 foci formation upon inhibition of CDK activity. HeLa cells were irradiated with 5Gy, fixed and stained for Cyclin A and RIF1 after 2h of recovery. 2 different thresholds of cyclin A levels were used to distinguish between G1/early S-phase (low cyclin A), S-phase (intermediate cyclin A), late S/G2 (high cyclin A). CDK inhibitor RO-3306 (10µM) was added to the medium 15 min before cell irradiation. The bracket highlights a subpopulation of cells in late S/G2 with a high number of foci, **** indicates p-value ≤ 0.0001. Statistical analysis is included in Material and Methods. b) Representative images of the experiment in a). c) Inhibition of CDK activity greatly reduced end-resection in MAD2L2 depleted U2OS cells. Cells were treated with 10µM RO-3306 or 20µM roscovitine for 15 minutes prior to adding neocarzinostatin (NCS) at 250ng/ml for 1 hour and western blotting of RPA phosphorylation (Ser-4/8) in whole cell extracts was used as a measure of end-resection. d) U2OS cells were treated with RO-3306 (10µM) or roscovitine (25µM) and irradiated with 5Gy. After 3 h, to allow for end-resection to take place, cells were pre-extracted to remove all chromatin-unbound RPA, fixed and stained for RPA foci. For each condition > 100 cells were quantified. Corresponding immunoblot shows the knockdown of MAD2L2 achieved.

Article Snippet: To obtain MAD2L2 depleted cells, U2OS or HeLa cells were infected with lentivirus containing an PLKO-puro shRNA targeting human MAD2L2 or a PLKO-puro scrambled control (Human MAD2L2 sh Sigma Mission library clone, TRCN0000006570: 5′-CCCGGAGCTGAATCAGTATAT-3′ and Scrambled control shRNA: 5′-CAACAAGATGAAGAGCACCAA​-3′) and selected with puromycin.

Techniques: Activity Assay, Inhibition, Irradiation, Staining, Western Blot

Journal: Cell Cycle

Article Title: H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

doi: 10.1080/15384101.2017.1404210

Figure Lengend Snippet: MAD2L2 is recruited at DSBs by protein interaction with the 53BP1-RIF1 complex and suppresses BRCA1 accumulation. a) HeLa cells were irradiated with 10 Gy and 53BP1 was immuno-precipitated after 2 h recovery. H4K20me2 and MAD2L2 were co-purified with 53BP1 exclusively upon irradiation. b) Cells expressing FLAG-tagged MAD2L2 were irradiated and FLAG immunoprecipitation was performed after 2 h recovery. Western blot analysis of inputs, supernatant and elution shows that FLAG-MAD2L2 is efficiently and equally purified from both non-irradiated and irradiated cells. c) 53BP1, RIF1 and ATM were identified by mass spectrometry analysis as novel protein interactors of MAD2L2 together with 7 previously known MAD2L2 protein interactors. PSM (peptide spectrum matches) counts are shown for 2 independent experiments. Specificity of the interaction is addressed in a smaller scale FLAG-MAD2L2 immunoprecipitation (Supplementary Figure 1). d) U2OS cells transfected with control GFP vector (EV) or GFP-MAD2L2 where irradiated with 10 Gy at 48 h after transfection, fixed at 1 h post IR and stained for Cyclin A (serving as S/G2 marker) and BRCA1 to assess BRCA1 accumulation to DSBs in S/G2 (n = 2 independent replicates).

Article Snippet: To obtain MAD2L2 depleted cells, U2OS or HeLa cells were infected with lentivirus containing an PLKO-puro shRNA targeting human MAD2L2 or a PLKO-puro scrambled control (Human MAD2L2 sh Sigma Mission library clone, TRCN0000006570: 5′-CCCGGAGCTGAATCAGTATAT-3′ and Scrambled control shRNA: 5′-CAACAAGATGAAGAGCACCAA​-3′) and selected with puromycin.

Techniques: Irradiation, Purification, Expressing, Immunoprecipitation, Western Blot, Mass Spectrometry, Transfection, Plasmid Preparation, Staining, Marker

Journal: Cell Cycle

Article Title: H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2

doi: 10.1080/15384101.2017.1404210

Figure Lengend Snippet: H4K20me2 levels control DNA repair pathway choice. 53BP1 forms foci at DSBs by simultaneous binding to ubiquitinated lysine 15 of histone H2A (H2AK15ub; not represented in the model) and di-methylated lysine of histone H4 (H4K20me2), using respectively a ubiquitin dependent recruitment domain (UDR) and a tandem tudor domain. The single disruption of one of the 2 binding sites completely abolishes 53BP1 foci formation. H2AK15ub is specifically induced at DSBs, ensuring the binding of 53BP1 exclusively at sites of damage, while H4K20me2 is present in more than 90% of the nucleosomes of non-replicated DNA, which indicates that it is not induced for the formation of 53BP1 foci. However, H4K20me2 plays a critical role for the choice of the correct DNA repair pathway depending on the replication state of DNA. In non-replicated DNA, all the nucleosomes bear H4K20me2 and thereby present the binding site for 53BP1, which in complex with RIF1 and MAD2L2, leaves no access points for BRCA1. FRAP experiments show that 80% of 53BP1 molecules at foci dynamically exchange with the nucleoplasmic pool within 30 min after photo-bleaching. In replicated DNA, H4K20me2 nucleosomes are flanked by newly synthesized nucleosomes with unmodified lysine 20 of histone H4 (H4K20me0). This opens breaches for the access of BRCA1 to the chromatin that by ubiquitination of lysine 125, 127, and 129 of histone H2A modifies the binding site for 53BP1, inhibiting the rebinding of 53BP1 from the nucleoplasmic pool and, thus, leading to its consequent release.

Article Snippet: To obtain MAD2L2 depleted cells, U2OS or HeLa cells were infected with lentivirus containing an PLKO-puro shRNA targeting human MAD2L2 or a PLKO-puro scrambled control (Human MAD2L2 sh Sigma Mission library clone, TRCN0000006570: 5′-CCCGGAGCTGAATCAGTATAT-3′ and Scrambled control shRNA: 5′-CAACAAGATGAAGAGCACCAA​-3′) and selected with puromycin.

Techniques: Binding Assay, Methylation, Synthesized