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1.
Microscopy (Oxf) ; 72(4): 299-309, 2023 Aug 04.
Article in English | MEDLINE | ID: mdl-37040437

ABSTRACT

Transmission electron microscopy (TEM) has been essential in defining the structural organization of the cell due to its ability to image cell structures at molecular resolution. However, the absence of colour has made it very difficult to compare the distributions and relationships of two or more types of biomolecules simultaneously if they lack clear morphological distinctions. Furthermore, single-channel information limits functional analysis, particularly in the nucleoplasm, where fibrillar material could be chromatin, ribonucleic acid or protein. Where specific stains exist to discriminate among these molecules, they cannot be combined because conventional TEM is a single-channel technology. A potential path around this barrier is through electron spectroscopic imaging (ESI). ESI can map the distributions of chemical elements within an ultrathin section. Here, we present methods to stain specific molecules with elements that ESI can visualize to enable multichannel electron microscopy.


Subject(s)
Cell Nucleus , Chromatin , Microscopy, Electron , Microscopy, Electron, Transmission , Staining and Labeling
2.
Front Genet ; 13: 876862, 2022.
Article in English | MEDLINE | ID: mdl-36092926

ABSTRACT

Chromatin is thought to regulate the accessibility of the underlying DNA sequence to machinery that transcribes and repairs the DNA. Heterochromatin is chromatin that maintains a sufficiently high density of DNA packing to be visible by light microscopy throughout the cell cycle and is thought to be most restrictive to transcription. Several studies have suggested that larger proteins and protein complexes are attenuated in their access to heterochromatin. In addition, heterochromatin domains may be associated with phase separated liquid condensates adding further complexity to the regulation of protein concentration within chromocenters. This provides a solvent environment distinct from the nucleoplasm, and proteins that are not size restricted in accessing this liquid environment may partition between the nucleoplasm and heterochromatin based on relative solubility. In this study, we assessed the accessibility of constitutive heterochromatin in mouse cells, which is organized into large and easily identifiable chromocenters, to fluorescently tagged DNA damage response proteins. We find that proteins larger than the expected 10 nm size limit can access the interior of heterochromatin. We find that the sensor proteins Ku70 and PARP1 enrich in mouse chromocenters. At the same time, MRE11 shows variability within an asynchronous population that ranges from depleted to enriched but is primarily homogeneously distribution between chromocenters and the nucleoplasm. While larger downstream proteins such as ATM, BRCA1, and 53BP1 are commonly depleted in chromocenters, they show a wide range of concentrations, with none being depleted beyond approximately 75%. Contradicting exclusively size-dependent accessibility, many smaller proteins, including EGFP, are also depleted in chromocenters. Our results are consistent with minimal size-dependent selectivity but a distinct solvent environment explaining reduced concentrations of diffusing nucleoplasmic proteins within the volume of the chromocenter.

3.
Nat Commun ; 12(1): 2939, 2021 05 19.
Article in English | MEDLINE | ID: mdl-34011960

ABSTRACT

Elucidation of non-canonical protein functions can identify novel tissue homeostasis pathways. Herein, we describe a role for the Bcl-2 family member BAD in postnatal mammary gland morphogenesis. In Bad3SA knock-in mice, where BAD cannot undergo phosphorylation at 3 key serine residues, pubertal gland development is delayed due to aberrant tubulogenesis of the ductal epithelium. Proteomic and RPPA analyses identify that BAD regulates focal adhesions and the mRNA translation repressor, 4E-BP1. These results suggest that BAD modulates localized translation that drives focal adhesion maturation and cell motility. Consistent with this, cells within Bad3SA organoids contain unstable protrusions with decreased compartmentalized mRNA translation and focal adhesions, and exhibit reduced cell migration and tubulogenesis. Critically, protrusion stability is rescued by 4E-BP1 depletion. Together our results confirm an unexpected role of BAD in controlling localized translation and cell migration during mammary gland development.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Mammary Glands, Animal/growth & development , Mammary Glands, Animal/metabolism , Mammary Glands, Human/growth & development , Mammary Glands, Human/metabolism , bcl-Associated Death Protein/metabolism , Amino Acid Substitution , Animals , Cell Line , Cell Movement/genetics , Female , Gene Knock-In Techniques , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Morphogenesis , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Organoids/growth & development , Organoids/metabolism , Phosphorylation , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Serine/chemistry , bcl-Associated Death Protein/deficiency , bcl-Associated Death Protein/genetics
4.
Cell ; 183(7): 1772-1784.e13, 2020 12 23.
Article in English | MEDLINE | ID: mdl-33326747

ABSTRACT

The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.


Subject(s)
Chromatin/metabolism , Acetylation/drug effects , Animals , Cell Line , Cell Survival/drug effects , Chromatin/drug effects , DNA Damage , Euchromatin/metabolism , Fluorescence , Heterochromatin/metabolism , Histone Deacetylase Inhibitors/pharmacology , Lasers , Mice , Models, Biological , Osmolar Concentration , Photobleaching
5.
Trauma Case Rep ; 30: 100373, 2020 Dec.
Article in English | MEDLINE | ID: mdl-33299923

ABSTRACT

Cardiac injury secondary to non-penetrating trauma is more common than thought, albeit, the injury is usually minor and goes undiagnosed without significant sequelae in most cases. Blunt cardiac rupture is much rarer accounting for <0.05% of all trauma cases but lethal in most circumstances. We present a case report of a young trauma victim who presented with both right atrial rupture and traumatic atrial septal disruption (ASD) requiring extra-corporeal life support (ECLS) and surgical repair. Blunt cardiac trauma with chamber rupture and septal disruption is a devastating injury. Stopping the hemorrhage and using ECLS gave our patient time to stabilize before definitive management of her traumatic ASD.

6.
CMAJ Open ; 5(2): E308-E314, 2017 Apr 25.
Article in English | MEDLINE | ID: mdl-28442493

ABSTRACT

BACKGROUND: Combined MD/PhD programs provide a structured path for physician-scientist training, but assessment of their success within Canada is limited by a lack of quantitative data. We collected outcomes data for graduates of Canadian MD/PhD programs. METHODS: We developed and implemented a Web-based survey consisting of 41 questions designed to collect outcomes data for Canadian MD/PhD program alumni from 8 Canadian universities who had graduated before September 2015. Respondents were categorized into 2 groups according to whether they had or had not completed all training. RESULTS: Of the 186 eligible alumni of MD/PhD programs, 139 (74.7%) completed the survey. A total of 136/138 respondents (98.6%) had completed or were currently completing residency training, and 66/80 (82%) had completed at least 1 postgraduate fellowship. Most (58 [83%]) of the 70 respondents who had completed all training were appointed as faculty at academic institutions, and 37 (53%) had been principal investigators on at least 1 recent funded project. Among the 58 respondents appointed at academic institutions, 44/57 (77%) dedicated at least 20% of their time to research, and 25/57 (44%) dedicated at least 50% to research. During their combined degree, 102/136 respondents (75.0%) published 3 or more first-author papers, and 133/136 (97.8%) matched with their first choice of specialty. The median length of physician-scientist training was 13.5 years. Most respondents graduated with debt despite having been supported by Canadian Institutes of Health Research MD/PhD studentships. INTERPRETATION: Most Canadian MD/PhD program alumni pursued careers consistent with their physician-scientist training, which indicates that these programs are meeting their primary objective. Nevertheless, our findings highlight that a minority of these positions are research intensive; this finding warrants further study. Our data provide a baseline for future monitoring of the output of Canadian MD/PhD programs.

7.
Neurotherapeutics ; 14(2): 274-283, 2017 04.
Article in English | MEDLINE | ID: mdl-28233220

ABSTRACT

Progress in the treatment of adult high-grade gliomas (HGG), including chemoradiation with concurrent and adjuvant temozolomide for glioblastoma, has not translated into significant therapeutic advances for pediatric HGG, where overall survival has plateaued at 15% to 20%, especially when considering specialized pediatric treatment in tertiary care centers, maximal safe neurosurgical resection, optimized delivery of involved field radiation, and improvements in supportive care. However, recent advances in our understanding of pediatric HGG, including the application of next-generation sequencing and DNA methylation profiling, have identified mutations in the histone variant H3.3 and canonical H3.1 genes, respectively. These mutations are relatively specific to neuroanatomic compartments (cortex, midline structures, thalamus, brainstem) and are often associated with other mutations, especially in specific growth factor receptor tyrosine kinases. Targeting epigenetic pathways affected by these histone mutations, alone or in combination with small molecule inhibitors of growth factor receptor signaling pathways, will inform new treatment strategies for pediatric HGG and should be incorporated into novel cooperative group clinical trial designs.


Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/therapy , Epigenesis, Genetic , Glioma/genetics , Glioma/therapy , Histones/metabolism , Adolescent , Animals , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Child , Child, Preschool , DNA Methylation , Glioma/metabolism , Glioma/pathology , Humans , Mutation , Neoplasm Grading
8.
Science ; 351(6279): 1324-9, 2016 Mar 18.
Article in English | MEDLINE | ID: mdl-26989253

ABSTRACT

Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting that loss of function may play a role in disease. We found that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and the loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS human patient tissue. Thus, C9orf72 is required for the normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.


Subject(s)
Amyotrophic Lateral Sclerosis/immunology , Frontotemporal Dementia/immunology , Guanine Nucleotide Exchange Factors/physiology , Macrophages/immunology , Microglia/immunology , Myeloid Cells/immunology , Proteins/physiology , Aging/immunology , Amyotrophic Lateral Sclerosis/genetics , Animals , C9orf72 Protein , Frontotemporal Dementia/genetics , Gene Knockdown Techniques , Guanine Nucleotide Exchange Factors/genetics , Heterozygote , Humans , Lymphatic Diseases/genetics , Lymphatic Diseases/immunology , Mice , Mice, Knockout , Proteins/genetics , Rats , Splenomegaly/genetics , Splenomegaly/immunology
9.
J Biol Chem ; 291(4): 1789-1802, 2016 Jan 22.
Article in English | MEDLINE | ID: mdl-26559976

ABSTRACT

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.


Subject(s)
Chromatin Assembly and Disassembly/radiation effects , Chromatin/metabolism , Chromatin/radiation effects , Histones/metabolism , Animals , Cell Line , DNA Damage/radiation effects , DNA Repair , Fibroblasts/metabolism , Fibroblasts/radiation effects , Humans , Lasers , Mice , Poly Adenosine Diphosphate Ribose/metabolism , Poly(ADP-ribose) Polymerases/metabolism
10.
Mucosal Immunol ; 9(2): 492-502, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26349660

ABSTRACT

Dectin-1 is an innate antifungal C-type lectin receptor necessary for protective antifungal immunity. We recently discovered that Dectin-1 is involved in controlling fungal infections of the gastrointestinal (GI) tract, but how this C-type lectin receptor mediates these activities is unknown. Here, we show that Dectin-1 is essential for driving fungal-specific CD4(+) T-cell responses in the GI tract. Loss of Dectin-1 resulted in abrogated dendritic cell responses in the mesenteric lymph nodes (mLNs) and defective T-cell co-stimulation, causing substantial increases in CD4(+) T-cell apoptosis and reductions in the cellularity of GI-associated lymphoid tissues. CD8(+) T-cell responses were unaffected by Dectin-1 deficiency. These functions of Dectin-1 have significant implications for our understanding of intestinal immunity and susceptibility to fungal infections.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Candida albicans/immunology , Candidiasis/immunology , Gastrointestinal Tract/immunology , Lectins, C-Type/immunology , Adoptive Transfer , Animals , Apoptosis/immunology , CD4-Positive T-Lymphocytes/microbiology , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/transplantation , CD8-Positive T-Lymphocytes/microbiology , CD8-Positive T-Lymphocytes/pathology , Candidiasis/genetics , Candidiasis/microbiology , Candidiasis/pathology , Cell Survival/immunology , Female , Gastrointestinal Tract/microbiology , Gastrointestinal Tract/pathology , Gene Expression , Lectins, C-Type/deficiency , Lectins, C-Type/genetics , Lymph Nodes/immunology , Lymph Nodes/microbiology , Lymph Nodes/pathology , Lymphocyte Activation , Mesentery/immunology , Mesentery/microbiology , Mesentery/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout
11.
Anticancer Res ; 34(9): 4717-22, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25202049

ABSTRACT

BACKGROUND: Proper re-establishment of heterochromatin after each round of DNA replication is critical to the preservation of cell identity. Paired box 3 (PAX3), a transcription factor important in embryonic development, was found to mediate the formation of pericentromeric heterochromatin. However, how PAX3 recognizes the heterochromatic environment and re-establishes it after DNA replication remains unclear. MATERIALS AND METHODS: Cell-cycle synchronization, fluorescence microscopic analyses, and co-immunoprecipitation were used to analyze the heterochromatic localization of PAX3 in HEK 293 cells and NIH 3T3 cells. RESULTS: We found that PAX3 binds pericentromeric heterochromatin during middle-to-late S phase. Loading of PAX3 onto pericentromeric heterochromatin requires poly(ADP-ribose) polymerase 1 (PARP1). Furthermore, loss of PAX3 or PARP1 delays cell-cycle progression through the S phase. CONCLUSION: Our results reveal how PAX3 recognizes and maintains pericentromeric heterochromatin at the S phase of the cell cycle.


Subject(s)
Heterochromatin/metabolism , Paired Box Transcription Factors/metabolism , Poly(ADP-ribose) Polymerases/metabolism , S Phase , Animals , Cell Cycle , HEK293 Cells , Heterochromatin/genetics , Humans , Mice , NIH 3T3 Cells , Paired Box Transcription Factors/genetics , Protein Binding , Protein Transport , Recombinant Fusion Proteins/metabolism
12.
Oncogene ; 32(6): 736-46, 2013 Feb 07.
Article in English | MEDLINE | ID: mdl-22430212

ABSTRACT

Anti-mitotic agents such as paclitaxel and docetaxel are widely used for the treatment of breast, ovarian and lung cancers. Although paclitaxel induces apoptosis, this drug also modulates autophagy. How autophagy affects paclitaxel activity, is unclear. We discovered that paclitaxel inhibited autophagy through two distinct mechanisms dependent on cell cycle stage. In mitotic cells, paclitaxel blocked activation of the class III phosphatidyl inositol 3 kinase, Vps34, a critical initiator of autophagosome formation. In non-mitotic paclitaxel-treated cells, autophagosomes were generated but their movement and maturation was inhibited. Chemically or genetically blocking autophagosome formation diminished paclitaxel-induced cell death suggesting that autophagosome accumulation sensitized cells to paclitaxel toxicity. In line with these observations, we identified that primary breast tumors that expressed diminished levels of autophagy-initiating genes were resistant to taxane therapy, identifying possible mechanisms and prognostic markers of clinical chemotherapeutic resistance.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Breast Neoplasms/drug therapy , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Paclitaxel/pharmacology , Breast Neoplasms/pathology , Cell Cycle , Cell Line, Tumor , Female , Humans
13.
J Otolaryngol Head Neck Surg ; 41 Suppl 1: S65-70, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22569052

ABSTRACT

BACKGROUND: Epigenetic modifications are defined as heritable changes in gene expression that are not encoded in deoxyribonucleic acid (DNA). Despite the importance of epigenetics in tumorigenesis, there is a paucity of information regarding the epigenetic profiles of oropharyngeal squamous cell carcinoma (OPSCC). OBJECTIVE: The objective of this study was to identify epigenetic signatures associated with human papillomavirus (HPV)-positive and -negative OPSCC. METHODS: We collected demographic, pathologic, and survival data from 44 patients with advanced-stage OPSCC treated with surgery and chemoradiation at the University of Alberta between January 2006 and December 2008. Tumour specimen from these patients were retreived and sectioned for immunohistochemical analysis. Double immunofluorescence staining was performed with p16 (HPV surrogate) and a panel of epigenetic markers, namely, histone methyl-lysines 4, 9, and 27 and H4 methyl-lysine 20. Correlation between p16 and epigenetic markers was measured using Metamorph and Image J software. RESULTS: Forty-one percent of patients were p16 positive. No statistically significant differences were found between p16-positive and -negative patients in terms of age at diagnosis, tumour subsite, or smoking history. We found significant differences in histone methylation between p16-positive and -negative tumours. OPSCC tumours positive for p16 had global elevations of histone H4 monomethylated lysine 20 (H4K20me1) and H3 trimethylated lysine 27 (H3K27me3) with depletions of H4 trimethylated lysine 20 (H4K20me3). In contrast, p16-negative tumours had depleted levels of H4K20me1 and H3K27me3 with high levels of H4K20me3. CONCLUSIONS: HPV-positive and -negative OPSCCs have distinct epigenetic profiles representing broad gene expression differences between these tumours.


Subject(s)
Carcinoma, Squamous Cell/virology , Cyclin-Dependent Kinase Inhibitor p16/genetics , DNA, Viral/analysis , Epigenomics/methods , Oropharyngeal Neoplasms/virology , Papillomaviridae/genetics , Papillomavirus Infections/virology , Biomarkers, Tumor/analysis , Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Female , Genetic Markers , Humans , Immunohistochemistry , Male , Middle Aged , Neoplasm Staging , Oropharyngeal Neoplasms/genetics , Oropharyngeal Neoplasms/pathology , Papillomaviridae/enzymology , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Prognosis , Retrospective Studies
14.
J Mol Biol ; 402(1): 178-93, 2010 Sep 10.
Article in English | MEDLINE | ID: mdl-20643146

ABSTRACT

The transcription factor PAX3 is essential for myogenesis and neural crest development, and is one of several genes mutated in human Waardenburg syndrome. Analysis of disease-causing missense mutations in PAX3 has established the interdependence of its two DNA-binding domains, the paired domain (PD) and the homeodomain (HD), as well as defects in localization and mobility. Paradoxically, mutants that retained DNA binding activity exhibited the greatest defects in localization and mobility, regardless of the domain in which they reside. In the present study, structure-function analyses were used to determine the mechanistic basis of this effect. In the context of the isolated DNA-binding domains, HD mutants adopted an increase in mobility proportional to their loss in DNA binding, while PD mutants continued to display the inverse relationship observed in the full-length protein. At the structural level, this reflected an unexpected dependence on base-specific contacts in the PD, whereas HD mobility was more severely affected by loss of backbone contacts, as has been observed with other DNA-binding proteins. This requires that the HD switch to a base-specific mode in the full-length protein. Moreover, both domains underwent substantial reduction in mobility and altered localization when in a contiguous polypeptide with the endogenous linker segment. Notably, although the HD conferred localization to heterochromatin, this activity was masked when linked to the PD, despite the absence of determinants for subnuclear compartmentalization in the PD or linker. Last, the propensity for PAX3 heterochromatin localization was modulated by sequences at the amino and carboxy termini, supporting a model in which alternate conformations lead to unmasking of the HD. These data indicate that the PD and the HD functionally interact in vivo and behave as a single binding module whose mobility and localization are dependent on sequence-specific contacts.


Subject(s)
DNA/metabolism , Paired Box Transcription Factors/metabolism , Animals , Cell Nucleus/metabolism , Cells, Cultured , DNA/genetics , Embryo, Mammalian , Fibroblasts , Homeodomain Proteins , Humans , Mice , Mutation/genetics , Nuclear Localization Signals , PAX3 Transcription Factor , Paired Box Transcription Factors/chemistry , Paired Box Transcription Factors/genetics , Protein Structure, Tertiary , Subcellular Fractions
15.
Oncogene ; 29(39): 5381-91, 2010 Sep 30.
Article in English | MEDLINE | ID: mdl-20603619

ABSTRACT

Antimitotic agents such as taxanes (paclitaxel and docetaxel) have greatly advanced the treatment of breast cancer, although variable patient response and drug toxicity are major limitations. Lack of validated predictive markers for taxane responsiveness precludes a priori identification of patients who are most likely to respond to treatment; thus, a subset of patients endure toxic side effects with marginal benefit. Mechanistic insights into taxane therapeutic activity may lead to rational therapeutic improvements. In this paper we report that the proapoptotic BH3-only protein Bad has a major role in taxane-induced cell death in vitro, and clinically is a prognostic indicator for overall survival of breast cancer patients after adjuvant taxane chemotherapy. Unexpectedly, Bad did not induce the mitochondrial apoptotic machinery in response to taxane treatment. Instead, Bad indirectly facilitated cell death by stimulating cellular proliferation. As dividing cells are the targets of taxane therapy, Bad-stimulated proliferation may be a marker of taxane sensitivity. Our studies indicate that quantification of Bad protein levels may have value as a diagnostic tool. They also suggest that cells expressing Bad are more sensitive to taxanes because of their altered cell cycle dynamics and reveal a clinically relevant proliferative role of Bad in breast cancer.


Subject(s)
Breast Neoplasms/drug therapy , bcl-Associated Death Protein/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/therapeutic use , Apoptosis , Breast Neoplasms/chemically induced , Cell Proliferation , Female , Humans , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , bcl-X Protein/metabolism
16.
PLoS One ; 5(12): e14447, 2010 Dec 29.
Article in English | MEDLINE | ID: mdl-21206904

ABSTRACT

BACKGROUND: Methylation of histone H4 on lysine 20 plays critical roles in chromatin structure and function via mono- (H4K20me1), di- (H4K20me2), and trimethyl (H4K20me3) derivatives. In previous analyses of histone methylation dynamics in mid-gestation mouse embryos, we documented marked changes in H4K20 methylation during cell differentiation. These changes were particularly robust during myogenesis, both in vivo and in cell culture, where we observed a transition from H4K20me1 to H4K20me3. To assess the significance of this change, we used a gain-of-function strategy involving the lysine methyltransferases SUV420H1 and SUV420H2, which catalyze H4K20me2 and H4K20me3. At the same time, we characterized a second isoform of SUV420H1 (designated SUV420H1_i2) and compared the activity of all three SUV420H proteins with regard to localization and H4K20 methylation. PRINCIPAL FINDINGS: Immunofluorescence revealed that exogenous SUV420H1_i2 was distributed throughout the cell, while a substantial portion of SUV420H1_i1 and SUV420H2 displayed the expected association with constitutive heterochromatin. Moreover, SUV420H1_i2 distribution was unaffected by co-expression of heterochromatin protein-1α, which increased the targeting of SUV420H1_i1 and SUV420H2 to regions of pericentromeric heterochromatin. Consistent with their distributions, SUV420H1_i2 caused an increase in H4K20me3 levels throughout the nucleus, whereas SUV420H1_i1 and SUV420H2 facilitated an increase in pericentric H4K20me3. Striking differences continued when the SUV420H proteins were tested in the C2C12 myogenic model system. Specifically, although SUV420H1_i2 induced precocious appearance of the differentiation marker Myogenin in the presence of mitogens, only SUV420H2 maintained a Myogenin-enriched population over the course of differentiation. Paradoxically, SUV420H1_i1 could not be expressed in C2C12 cells, which suggests it is under post-transcriptional or post-translational control. CONCLUSIONS: These data indicate that SUV420H proteins differ substantially in their localization and activity. Importantly, SUV420H2 can induce a transition from H4K20me1 to H4K20me3 in regions of constitutive heterochromatin that is sufficient to enhance myogenic differentiation, suggesting it can act an as epigenetic 'switch' in this process.


Subject(s)
Histone-Lysine N-Methyltransferase/metabolism , Animals , Cell Differentiation , Cell Line , Chromatin/metabolism , Epigenesis, Genetic , Heterochromatin/metabolism , Humans , Mice , Mice, Inbred C3H , Microscopy, Fluorescence/methods , Muscle Development , Protein Isoforms
17.
Hum Mol Genet ; 17(12): 1825-37, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18325909

ABSTRACT

Mutations in the transcription factor PAX3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch mutant, which display similar neural crest-derived defects. Previous characterization of disease-causing mutations revealed pleiotropic effects on PAX3 DNA binding and transcriptional activity. In this study, we evaluated the impact of disease alleles on PAX3 localization and mobility. Immunofluorescence analyses indicated that the majority of PAX3 occupies the interchromatin space, with only sporadic colocalization with sites of transcription. Interestingly, PAX3 disease alleles fell into two distinct categories when localization and dynamics in fluorescence recovery after photobleaching (FRAP) were assessed. The first group (class I), comprising N47H, G81A and V265F exhibit a diffuse distribution and markedly increased mobility when compared with wild-type PAX3. In contrast, the G42R, F45L, S84F, Y90H and R271G mutants (class II) display evidence of subnuclear compartmentalization and mobility intermediate between wild-type PAX3 and class I proteins. However, unlike class I mutants, which retain DNA binding, class II proteins are deficient for this activity, indicating that DNA binding is not a primary determinant of PAX3 distribution and movement. Importantly, class I properties prevail when combined with a class II mutation, which taken with the proximity of the two mutant classes within the PAX3 protein, suggests class I mutants act by perturbing PAX3 conformation. Together, these results establish that altered localization and dynamics play a key role in PAX3 dysfunction and that loss of the underlying determinants represents the principal defect for a subset of Waardenburg mutations.


Subject(s)
Cell Nucleus/chemistry , Paired Box Transcription Factors/analysis , Paired Box Transcription Factors/genetics , Waardenburg Syndrome/genetics , Waardenburg Syndrome/metabolism , Animals , Cell Line , Cell Nucleus/metabolism , Electrophoretic Mobility Shift Assay , Fluorescence Recovery After Photobleaching , Histones/metabolism , Humans , Mice , Models, Molecular , Mutation , PAX3 Transcription Factor , Paired Box Transcription Factors/metabolism , Protein Processing, Post-Translational
18.
Biochem Biophys Res Commun ; 368(2): 357-63, 2008 Apr 04.
Article in English | MEDLINE | ID: mdl-18222115

ABSTRACT

The granzyme B gene is activated upon cytotoxic T cell stimulation and the protein is a key inducer of apoptosis in target cells. Previous studies have identified important proximal regulatory regions but these proved insufficient to drive expression in vivo. We identified a DNase1 hypersensitive site (HS2) 3.9kb upstream of the transcription start site that was present in stimulated but not resting CD8+ cells. The CTL line CTLL R8 was stably transfected with GFP reporter constructs and showed consistently higher fluorescence values when HS2 was included. In transgenic mice the presence of the relevant region of DNA resulted in inducible, CTL-specific transcription of the transgene in all transgenic founder lines analyzed. Deletion of HS2 resulted in a 10-fold reduction in expression. This is the first report of a major distal regulatory element in the control of granzyme B transcription.


Subject(s)
CD8-Positive T-Lymphocytes/physiology , Gene Expression Regulation, Enzymologic/genetics , Granzymes/genetics , Mice, Transgenic/genetics , Promoter Regions, Genetic/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcriptional Activation/genetics , Animals , Cells, Cultured , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
19.
Biochem Cell Biol ; 84(4): 605-18, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16936832

ABSTRACT

In the midst of an increasingly detailed understanding of the molecular basis of genome regulation, we still only vaguely understand the relationship between molecular biochemistry and the structure of the chromatin inside of cells. The centromere is a structurally and functionally unique region of each chromosome and provides an example in which the molecular understanding far exceeds the understanding of the structure and function relationships that emerge on the chromosomal scale. The centromere is located at the primary constriction of the chromosome. During entry into mitosis, the centromere specifies the assembly site of the kinetochore, the structure that binds to microtubules to enable transport of the chromosomes into daughter cells. The epigenetic contributions to the molecular organization and function of the centromere are reviewed in the context of structural mechanisms of chromatin function.


Subject(s)
Centromere/physiology , Chromatin/physiology , Epigenesis, Genetic , Kinetochores/physiology , Amino Acid Sequence , Animals , Autoantigens/genetics , Autoantigens/metabolism , Cell Cycle , Centromere/genetics , Centromere Protein A , Chromatin/chemistry , Chromatin/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , Genomic Instability , Heterochromatin/physiology , Histones/genetics , Humans , Mice , Mitosis , Molecular Sequence Data , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Sequence Homology, Amino Acid
20.
J Cell Sci ; 119(Pt 5): 858-65, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16495481

ABSTRACT

Cytotoxic lymphocytes induce target cell apoptosis via two major pathways: Fas/FasL and granule exocytosis. The latter pathway has largely been defined by the roles of the pore-forming protein perforin and by the serine proteinases granzymes A and B. Upon entry into target cells, the granzymes cleave substrates that ultimately result in cell death. To gain further insight into granzyme B function, we have identified novel substrates. SDS-PAGE analysis of S100 cell lysates identified a 51 kDa protein that was cleaved by granzyme B. Mass spectrometry analysis revealed that this fragment was the microtubule protein, alpha-tubulin, which was confirmed by western blotting. In addition, two-dimensional gel analysis showed that the truncated form of alpha-tubulin had a more basic isoelectric point than the full-length molecule, suggesting that granzyme B removed the acidic C-terminus. Site-directed mutagenesis within this region of alpha-tubulin revealed the granzyme B recognition site, which is conserved in a subset of alpha-tubulin isoforms. Significantly, we showed that alpha-tubulin was cleaved in target cells undergoing apoptosis as induced by cytotoxic T lymphocytes. Therefore, in addition to its role in the activation of mitochondria during apoptosis, these results suggest a role for granzyme B in the dismantling of the cytoskeleton.


Subject(s)
Apoptosis/physiology , Serine Endopeptidases/metabolism , T-Lymphocytes, Cytotoxic/metabolism , Tubulin/chemistry , Tubulin/metabolism , Amino Acid Sequence , Cell Line , Electrophoresis, Polyacrylamide Gel , Granzymes , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence Alignment , Serine Endopeptidases/chemistry , Tubulin/genetics
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