El Bachir Affar

Université de Montréal | UdeM · Department of Medicine

Aberrant Receptor Tyrosine Kinase (RTK) signaling allows cancer cells to modulate survival, proliferation and death, leading to tumorigenesis and chemoresistance. In leukemia, the RTK FMS-Related Tyrosine Kinase 4 (FLT4) (also known as VEGFR-3, Vascular Endothelial Growth Factor- 3) is deregulated and correlates with cancer progression. However, th...

Ubiquitination is an important post-translational modification (PTM) that regulates a large spectrum of cellular processes in eukaryotes. Abnormalities in ubiquitin signaling underlie numerous human pathologies including cancer and neurodegeneration. Much progress has been made during the last three decades in understanding how ubiquitin ligases re...

Deubiquitinases (DUBs) are required for the reverse reaction of ubiquitination and act as major regulators of ubiquitin signaling processes. Emerging evidence suggests that these enzymes are regulated at multiple levels in order to ensure proper and timely substrate targeting, and to prevent the adverse consequences of promiscuous deubiquitination....

Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage of RNAi or chemical inhibitors to modulate OGT an...

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA doublestrand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prog...

Germline mutations in BRCA1 and BRCA2 (BRCA1/2) genes considerably increase breast and ovarian cancer risk. Given that tumors with these mutations have elevated genomic instability, they exhibit relative vulnerability to certain chemotherapies and targeted treatments based on poly (ADP-ribose) polymerase (PARP) inhibition. However, the molecular me...

The BAP1 gene has emerged as a major tumor suppressor mutated with various frequencies in numerous human malignancies, including uveal melanoma, malignant pleural mesothelioma, clear cell renal cell carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, and thymic epithelial tumors. BAP1 mutations are also observed at low frequency i...

Histone posttranslational modifications are key regulators of chromatin-associated processes including gene expression, DNA replication and DNA repair. Monoubiquitinated histone H2A, H2Aub (K118 in Drosophila or K119 in vertebrates) is catalyzed by the Polycomb group (PcG) repressive complex 1 (PRC1) and reversed by the PcG-repressive deubiquitinas...

DNA double-strand break (DSB) resection, once thought to be a simple enzymatic process, is emerging as a highly complex series of coordinated activities required to maintain genome integrity. Progress in cell biology, biochemistry, and genetics has deciphered the precise resecting activities, the regulatory components, and their ability to properly...

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has...

Nucleophosmin (NPM1) is frequently mutated or subjected to chromosomal translocation in acute myeloid leukemia (AML). NPM protein is primarily located in the nucleus, but the recurrent NPMc+ mutation, which creates a nuclear export signal, is characterized by cytoplasmic localization and leukemogenic properties. Similarly, the NPM-MLF1 translocatio...

Pink red-like pigments of crud extracts produced by Streptomyces coelicoflavus MFB11, MFB20, MFB21, MFB23 and MFB24 strains and variants from two spontaneous mutants (MFB11-V and MFB11-Y) as well as prepared fractions (FA and FB) from MFB21 and MFB24 strain pigments were screened for antiproliferative effect by MTT. Cancer cell targets used in this...

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using t...

On-chip high-throughput phenotyping of single cells has gained a lot of interest recently due to the discrimination capability of label-free biomarkers such as whole-cell deformability and refractive index. Here we present on-chip Refractive Index Cytometry (RIC) for whole-cell deformability at a high measurement rate. We have further exploited a p...

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitin...

The relative contribution of intrinsic genetic factors and extrinsic environmental ones to cancer aetiology and natural history is a lengthy and debated issue. Gene–environment interactions (G x E) arise when the combined presence of both a germline genetic variant and a known environmental factor modulates the risk of disease more than either one...

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor pro...

BRCA1-associated protein 1 (BAP1) is a potent tumour suppressor gene that modulates environmental carcinogenesis. All carriers of inherited heterozygous germline BAP1-inactivating mutations (BAP1(+/-)) developed one and often several BAP1(-/-) malignancies in their lifetime, mostly malignant mesothelioma, uveal melanoma, and so on. Moreover, BAP1-a...

APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts wi...

The ability to conduct image-based, non-invasive cell tagging, independent of genetic engineering, is key to cell biology applications. Here we introduce cell labelling via photobleaching (CLaP), a method that enables instant, specific tagging of individual cells based on a wide array of criteria such as shape, behaviour or positional information....

Significance Understanding how cell cycle and cell differentiation are coordinated during normal hematopoiesis will reveal molecular insights in leukemogenesis. LIM-only 2 (LMO2) is a transcriptional regulator that controls the erythroid lineage via activation of an erythroid-specific gene expression program. Here, we uncover an unexpected function...

Oncogenic transcription factors are major drivers in acute leukemias. These oncogenes are believed to subvert normal cell identity via the establishment of gene expression programs that dictate cell differentiation and growth. The LMO2 oncogene, which is commonly activated in T-cell acute lymphoblastic leukemia (T-ALL), has a well-established funct...

The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A K119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for...

O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is cen...

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associat...

The tumor suppressor BAP1 interacts with chromatin-associated proteins and regulates cell proliferation, but its mechanism of action and regulation remain poorly defined. We show that the ubiquitin-conjugating enzyme UBE2O multi-monoubiquitinates the nuclear localization signal of BAP1, thereby inducing its cytoplasmic sequestration. This activity...

Significance BAP1 is a deubiquitinase of histone H2A involved in chromatin remodeling. Several studies identified BAP1 as major tumor suppressor inactivated in various cancers. Nonetheless, the manner in which BAP1 protects against cancer development remains enigmatic. We now show that BAP1 is recruited to double-strand DNA break sites and promotes...

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-...

The development of electron-based, unimolecular dissociation mass spectrometry (MS), i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile post-translational modification (PTM) site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine)...

Rivaling or cooperating with other post-translational modifications, ubiquitination plays central roles in regulating numerous cellular processes. Not surprisingly, gain- or loss-of-function mutations in several components of the ubiquitin system are causally linked to human pathologies including cancer. The covalent attachment of ubiquitin to targ...

An accurate and sensitive detection of catalytic activation of poly(ADP-ribose) polymerase-1 (PARP-1) is required to be performed in a wide variety of samples because this activity plays a role in various cellular responses to DNA damage ranging from DNA repair to cell death, as well as in housekeeping functions, such as transcription. Since PARP-1...

Host Cell Factor 1 (HCF-1) plays critical roles in regulating gene expression in a plethora of physiological processes. HCF-1 is first synthesized as a precursor, and subsequently specifically proteolytically cleaved within a large middle region termed the proteolytic processing domain (PPD). Although the underlying mechanism remains enigmatic, pro...

Immunostaining for BRCA1, Rad51, RPA or γH2AX following MMS treatment. HeLa cells were treated with 200 µM MMS and harvested for immunostaining. (1.44 MB TIF)

Immunostaining for BRCA1, Rad51 and γH2AX following IR and proteasome inhibition. HeLa cells were treated for 6 hrs with IR (10 Gy) (with or without pretreatment with MG132) and harvested for immunostaining. (1.44 MB TIF)

Immunodetection of ATM or DNA-PK in the cell lines used. Left, HeLa or ATM-deficient fibroblasts were used for immunodetection with anti-ATM antibody. Right, Immunostaining detection of DNA-PKcs in glioblastoma cell lines, proficient (MO59K) or deficient (MO59J). (0.68 MB TIF)

The proteasome mediates BRCA1 downregulation in response to DNA damage. (A) HCT116 cells were pre-treated with 20 µM of the proteasome inhibitor MG132 for 30 min and then treated with 200 µM MMS in the presence of the inhibitor, and then harvested at the indicated times for immunoblotting. (B) HeLa cells were pre-treated with 20 µM of another prote...

Downregulation of BRCA1 with low dose of UVC. Immunoblotting detection of BRCA1 in HeLa cells following treatment with UVC (10 J/m2). Immunodetection of PARP-1 was used as loading control. (0.52 MB TIF)

Dose-dependent downregulation of BRCA1 following MMS treatment. HEK293 Cells were harvested at the indicated time points for immunoblotting with anti-BRCA1 and anti-β-actin antibodies (left panel). The band signals were directly acquired with a LAS-3000 LCD camera (Fuji, Stamford, CT, USA) coupled to MultiGauge software (Fuji). The protein levels a...

Antibodies used in this study. (1.23 MB TIF)

Immunoblotting detection of BRCA1 at various times post-UV using additional specific antibodies. (A–B) HeLa cells were treated with UVC (30 J/m2) and harvested at the indicated times for immunodetection with anti-BRCA1 antibodies. The monoclonal SD118 antibody which recognizes the C-terminus (A), or the polyclonal rabbit specific for the middle reg...

Downregulation of BRCA1 occurs in G2 phase. HeLa cells were synchronized in G2/M after 16 hrs exposure to nocodazole. Mitotic cells were removed by shake off and the purified G2 population was treated with 200 µM MMS for 3 hrs at various time points post-removal of nocodazole. Cell cycle analysis (left panel) and immunoblotting (right panel) were c...

DNA damage-activated MAPKs are not required for downregulation of BRCA1. Cells were pre-treated with 20 µM U0126, 30 µM SP600125, or 20 µM SB202190 for 30 min to inhibit signaling pathways involving ERK1/2, JNK1/2, or p38α/β, respectively (Rouget et al. 2008). Cells were then treated with 200 µM MMS and harvested after 3 hrs. Abrogation of MAPK sig...

Immunostaining for BRCA1, Rad51 and γH2AX in various conditions. HeLa cells were treated for 6 hrs with IR (10 Gy) or 200 µM MMS (with or without pretreatment with MG132) and harvested for immunostaining with (top panel) or without (bottom panel) a permeabilization step. (2.22 MB TIF)

Immunostaining for BAP1 following permeabilization. HeLa cells were harvested for immunostaining with (botton panel) or without (top panel) a permeabilization step. (0.74 MB TIF)

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 funct...

The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assemble...

Activation-induced deaminase (AID) is the mutator enzyme that initiates somatic hypermutation and isotype switching of the antibody genes in B lymphocytes. Undesired byproducts of AID function are oncogenic mutations. AID expression levels seem to correlate with the extent of its physiological and pathological functions. In this study, we identify...

Abrogation of stable RNAi but persistence of transient RNAi in same cells during UVB-induced apoptosis. The shGFP-234 clone #64 was transiently transfected with 3 µg of shGFP-234 DNA vector for 48 h. The CHO-GFP parental cells and shGFP-234 cells with or without additional transient RNAi by shGFP-234 or unrelated shRNA-generating DNA vector (contro...

Detailed methods for creation of clones, transfections and RT-PCR. (0.04 MB DOC)

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction...

During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human β-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling...

RNA interference (RNAi) is used as a reverse-genetic tool to examine functions of a gene in different cellular processes including apoptosis. As key cellular proteins are inactivated during apoptosis, and as RNAi requires cooperation of many cellular proteins, we examined whether DNA vector-based RNAi would continue to function during apoptosis. Th...

Global-genomic nucleotide excision repair (GG-NER) is the only pathway available to humans for removal, from the genome overall, of highly genotoxic helix-distorting DNA adducts generated by many environmental mutagens and certain chemotherapeutic agents, e.g., UV-induced 6–4 photoproducts (6–4PPs) and cyclobutane pyrimidine dimers (CPDs). The atax...

In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/...

Loss of function of the tumor suppressor protein BRCA1 is responsible for a high percentage of familial and also sporadic breast cancers. Early work identified a stimulatory transcriptional coactivator function for the BRCA1 protein, and more recently, BRCA1 has been implicated in transcriptional repression, although few examples of repressed genes...

To reach the lysosomes, down-regulated receptors such as the epidermal growth factor receptor must first be sorted into internal vesicles of late endosomes (multivesicular bodies), a ubiquitin-dependent event that requires the coordinated function of the endosome sorting complex required for transport (ESCRT) proteins. Here we report that CHMP3, an...

Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing ∼75%, ∼50%, and ∼25% of the normal complement of YY1...

The prostate apoptosis response-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli, and down-regulation of this protein has been suggested to be a key event during tumorigenesis. Several studies suggest an essential function for the COOH-terminal leucine repeats/death domain of Par-4 i...

Prostate apoptosis response 4 (Par-4) is a leucine zipper containing protein that plays a role in apoptosis. Although Par-4 is expressed in neurons, its physiological role in the nervous system is unknown. Here we identify Par-4 as a regulatory component in dopamine signaling. Par-4 directly interacts with the dopamine D2 receptor (D2DR) via the ca...

RNA-mediated interference (RNAi) is a powerful technique that is now being used in mammalian cells to specifically silence a gene. Some recent studies have used this technique to achieve variable extent of depletion of a nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These studies reported either transient silencing of PARP-1 using double-s...

Yin Yang 1 (YY1) is a transcription factor that plays an essential role in development. However, the full spectrum of YY1's functions and mechanism of action remains unclear. We find that YY1 ablation results in p53 accumulation due to a reduction of p53 ubiquitination in vivo. Conversely, YY1 overexpression stimulates p53 ubiquitination and degrad...