Michael Hendzel | University of Alberta (original) (raw)

Papers by Michael Hendzel

Nature cell biology, 2015

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous r... more DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.

Journal of Cellular Biochemistry, 2007

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synth... more Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Journal of Cellular Biochemistry, 1994

Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear fu... more Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear functions, including replication, transcription, and RNA splicing. It is becoming increasingly evident that nuclear processes are localized to distinct regions in the nucleus. For example, transcriptionally active genes and RNA transcripts are found in discrete transcription foci. Current evidence suggests that nuclear matrix-bound transcriptionally active DNA sequences are in nucleosomes with dynamically acetylated histones. Histone acetylation, which precedes transcription, alters nucleosome and chromatin structure, decondensing the chromatin fibre and making the nucleosomal DNA accessible to transcription factors. Histone acetyltransferase and histone deacetylase, which catalyze this rapid acetylation and deacetylation, are associated with the internal nuclear matrix. We hypothesize that these enzymes play a role in maintaining the association of the active chromatin domains with the internal nuclear matrix at sites of ongoing transcription. D 1994 ~i i e y -~i s s , Inc.

Journal of Biological Chemistry, 2008

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand... more Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNAdamaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Journal of Biological Chemistry, 2013

DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to ... more DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double-strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and dimethylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-enhanced GFP (EGFP) and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly(ADP-ribose) polymerase 1 activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at γ-irradiation-induced γH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of γH2AX foci 6 h after irradiation as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points after 2 gray of γ-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.

Journal of Biological Chemistry, 2006

Reduction in levels of sex hormones at menopause in women is associated with two common, major ou... more Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.

Journal of Biological Chemistry, 2008

Journal of Biological Chemistry, 2013

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in a... more Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

Development, 2011

Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the ... more Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear -actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear -actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear -actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular -actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and actin in developing mammary end buds. These data indicate a novel role for nuclear -actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.

Current Opinion in Cell Biology, 2006

Recent discoveries have resulted in significant breakthroughs in the understanding of PARPs and P... more Recent discoveries have resulted in significant breakthroughs in the understanding of PARPs and PARG functions within a broad range of cellular processes. The novel and sometimes unexpected pathways that are regulated by poly(ADPribosylation) bring new questions and hypotheses, some of them being contentious. In this review, we highlight current areas of investigation such as the clinical potential of PARP and PARG inhibitors and the important mitotic regulatory functions of poly(ADP-ribose) in cell-cycle progression, a recent discovery that has broadened our knowledge regarding poly(ADP-ribose) functions. A special emphasis is placed on recent advances in relation to PARG that are stimulating new directions in future research. Noticeably, the existence of various PARG isoforms characterized by distinct cellular localizations and nucleocytoplasmic shuttling properties challenges our current comprehension of pADPr metabolism. Observations and suppositions towards functionally important regulatory elements in the N-terminal portion of PARG are also discussed.

Chromosoma, 1997

We have generated and characterized a novel site-specific antibody highly specific for the phosph... more We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Chromosoma, 1997

We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluore... more We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci.

Chromosoma, 1995

The study of RNA pol II-mediated transcription regulation has been dominated by molecular biologi... more The study of RNA pol II-mediated transcription regulation has been dominated by molecular biological approaches. Although these methods continue to provide important insights, other approaches are required to insure against an oversimplified view of gene expression. Improvements in EM methods and the development of the confocal light microscope have provided alternative and complementary means of investigating gene regulation. Information on the "context" in which cis- and trans-acting factors operate can be achieved with these techniques. As a result, the spatial compartmentalization of nuclear processes involved in transcriptional and post-transcriptional processing has received considerable attention.

Cell Cycle, 2013

Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently ove... more Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating the DNA damage response. Here, we show that polycomb repressive complex 2 (PRC2) is recruited to sites of DNA damage. This recruitment is independent of histone 2A variant X (H2AX) and the PI-3-related kinases ATM and DNA-PKcs. We establish that PARP activity is required for retaining PRC2 at sites of DNA damage. Furthermore, depletion of EZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gamma-irradiation. These data unravel a crucial role of PRC2 in determining cancer cellular sensitivity following DNA damage and suggest that therapeutic targeting of EZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy.

Breast Cancer Research, 2009

Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleava... more Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γsecretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γsecretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.

Breast Cancer Research, 2011

BMC Biology, 2009

Background: Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear proc... more Background: Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues.

Blood, 2005

characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients Overexpression of transcrip... more characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients Overexpression of transcripts originating from the MMSET locus http://bloodjournal.hematologylibrary.org/content/105/10/4060.full.html Updated information and services can be found at: (4217 articles) Neoplasia (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Multiple myeloma (MM) is a B-lineage malignancy characterized by diverse genetic subtypes and clinical outcomes. The recurrent immunoglobulin heavy chain (IgH) switch translocation, t(4;14)(p16;q32), is associated with poor outcome, though the mechanism is unclear. Quantitative reverse-transcription-polymerase chain reaction (RT-PCR) for proposed target genes on a panel of myeloma cell lines and purified plasma cells showed that only transcripts originating from the WHSC1/ MMSET/NSD2 gene are uniformly dysregulated in all t(4;14) POS patients. The different transcripts detected, multiple myeloma SET domain containing protein (MMSET I), MMSET II, Exon 4a/MMSET III, and response element II binding protein (RE-IIBP), are produced by alternative splicing and alternative transcription initiation events. Translation of the various transcripts, including those from major breakpoint region 4-2 (MB4-2) and MB4-3 breakpoint variants, was confirmed by transient transfection and immunoblotting. Green fluorescent protein (GFP)tagged MMSET I and II, corresponding to proteins expressed in MB4-1 patients, localized to the nucleus but not nucleoli, whereas the MB4-2 and MB4-3 proteins concentrate in nucleoli. Cloning and localization of the Exon 4a/MMSET III splice variant, which contains the protein segment lost in the MB4-2 variant, identified a novel protein domain that prevents nucleolar localization. Kinetic studies using photobleaching suggest that the breakpoint variants are functionally distinct from wild-type proteins. In contrast, RE-IIBP is universally dysregulated and also potentially functional in all t(4;14) POS patients irrespective of fibroblast growth factor receptor 3 (FGFR3) expression or breakpoint type. (Blood. 2005;105: 4060-4069)

Bioorganic & Medicinal Chemistry, 2011

We present a combination of database screening, synthesis and in vitro testing to identify novel ... more We present a combination of database screening, synthesis and in vitro testing to identify novel histone acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors. Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for new anticancer agents directed against HATs as new targets have been identified.

Biochemistry and Cell Biology, 1999

Nature cell biology, 2015

DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous r... more DNA double-strand breaks (DSBs) are repaired mainly by non-homologous end joining or homologous recombination (HR). Cell cycle stage and DNA end resection are believed to regulate the commitment to HR repair. Here we identify RNF138 as a ubiquitin E3 ligase that regulates the HR pathway. RNF138 is recruited to DNA damage sites through zinc fingers that have a strong preference for DNA with 5'- or 3'-single-stranded overhangs. RNF138 stimulates DNA end resection and promotes ATR-dependent signalling and DSB repair by HR, thereby contributing to cell survival on exposure to DSB-inducing agents. Finally, we establish that RNF138-dependent Ku removal from DNA breaks is one mechanism whereby RNF138 can promote HR. These results establish RNF138 as an important regulator of DSB repair pathway choice.

Journal of Cellular Biochemistry, 2007

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synth... more Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage.

Journal of Cellular Biochemistry, 1994

Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear fu... more Besides its role in organizing nuclear DNA, the nuclear matrix is involved in specific nuclear functions, including replication, transcription, and RNA splicing. It is becoming increasingly evident that nuclear processes are localized to distinct regions in the nucleus. For example, transcriptionally active genes and RNA transcripts are found in discrete transcription foci. Current evidence suggests that nuclear matrix-bound transcriptionally active DNA sequences are in nucleosomes with dynamically acetylated histones. Histone acetylation, which precedes transcription, alters nucleosome and chromatin structure, decondensing the chromatin fibre and making the nucleosomal DNA accessible to transcription factors. Histone acetyltransferase and histone deacetylase, which catalyze this rapid acetylation and deacetylation, are associated with the internal nuclear matrix. We hypothesize that these enzymes play a role in maintaining the association of the active chromatin domains with the internal nuclear matrix at sites of ongoing transcription. D 1994 ~i i e y -~i s s , Inc.

Journal of Biological Chemistry, 2008

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand... more Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNAdamaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Journal of Biological Chemistry, 2013

DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to ... more DNA damage evokes a complex and highly coordinated DNA damage response (DDR) that is integral to the suppression of genomic instability. Double-strand breaks (DSBs) are considered the most deleterious form damage. Evidence suggests that trimethylation of histone H3 lysine 9 (H3K9me3) presents a barrier to DSB repair. Also, global levels of histone methylation are clinically predictive for several tumor types. Therefore, demethylation of H3K9 may be an important step in the repair of DSBs. The KDM4 subfamily of demethylases removes H3K9 tri- and dimethylation and contributes to the regulation of cellular differentiation and proliferation; mutation or aberrant expression of KDM4 proteins has been identified in several human tumors. We hypothesize that members of the KDM4 subfamily may be components of the DDR. We found that Kdm4b-enhanced GFP (EGFP) and KDM4D-EGFP were recruited rapidly to DNA damage induced by laser micro-irradiation. Focusing on the clinically relevant Kdm4b, we found that recruitment was dependent on poly(ADP-ribose) polymerase 1 activity as well as Kdm4b demethylase activity. The Kdm4 proteins did not measurably accumulate at γ-irradiation-induced γH2AX foci. Nevertheless, increased levels of Kdm4b were associated with decreased numbers of γH2AX foci 6 h after irradiation as well as increased cell survival. Finally, we found that levels of H3K9me2 and H3K9me3 were decreased at early time points after 2 gray of γ-irradiation. Taken together, these data demonstrate that Kdm4b is a DDR protein and that overexpression of Kdm4b may contribute to the failure of anti-cancer therapy that relies on the induction of DNA damage.

Journal of Biological Chemistry, 2006

Reduction in levels of sex hormones at menopause in women is associated with two common, major ou... more Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.

Journal of Biological Chemistry, 2008

Journal of Biological Chemistry, 2013

Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in a... more Polycomb-repressive complex 1 (PRC1)-mediated histone ubiquitylation plays an important role in aberrant gene silencing in human cancers and is a potential target for cancer therapy. Here we show that 2-pyridine-3-yl-methylene-indan-1,3-dione (PRT4165) is a potent inhibitor of PRC1-mediated H2A ubiquitylation in vivo and in vitro. The drug also inhibits the accumulation of all detectable ubiquitin at sites of DNA double-strand breaks (DSBs), the retention of several DNA damage response proteins in foci that form around DSBs, and the repair of the DSBs. In vitro E3 ubiquitin ligase activity assays revealed that PRT4165 inhibits both RNF2 and RING 1A, which are partially redundant paralogues that together account for the E3 ubiquitin ligase activity found in PRC1 complexes, but not RNF8 nor RNF168. Because ubiquitylation is completely inhibited despite the efficient recruitment of RNF8 to DSBs, our results suggest that PRC1-mediated monoubiquitylation is required for subsequent RNF8- and/or RNF168-mediated polyubiquitylation. Our results demonstrate the unique feature of PRT4165 as a novel chromatin-remodeling compound and provide a new tool for the inhibition of ubiquitylation signaling at DNA double-strand breaks.

Development, 2011

Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the ... more Functional differentiation is orchestrated by precise growth-regulatory controls conveyed by the tissue microenvironment. Cues from laminin 111 (LN1) lower transcription and suppress mammary epithelial cell growth in culture, but how LN1 induces quiescence is unknown. Recent literature points to involvement of nuclear -actin in transcriptional regulation. Here, we show that quiescence induced by growth factor withdrawal, or LN1 addition, rapidly decreases nuclear -actin. LN1, but not other extracellular matrix (ECM) molecules, decreases the levels of nuclear -actin and destabilizes RNA polymerase (RNA Pol) II and III binding to transcription sites, leading to a dramatic drop in transcription and DNA synthesis. Constitutive overexpression of globular -actin in the nucleus reverses the effect of LN1 on transcription and RNA Pol II association and prevents the cells from becoming quiescent in the presence of LN1. The physiological relevance of our findings was verified by identifying a clear spatial separation of LN1 and actin in developing mammary end buds. These data indicate a novel role for nuclear -actin in growth arrest of epithelial cells and underscore the importance of the integrity of the basement membrane in homeostasis.

Current Opinion in Cell Biology, 2006

Recent discoveries have resulted in significant breakthroughs in the understanding of PARPs and P... more Recent discoveries have resulted in significant breakthroughs in the understanding of PARPs and PARG functions within a broad range of cellular processes. The novel and sometimes unexpected pathways that are regulated by poly(ADPribosylation) bring new questions and hypotheses, some of them being contentious. In this review, we highlight current areas of investigation such as the clinical potential of PARP and PARG inhibitors and the important mitotic regulatory functions of poly(ADP-ribose) in cell-cycle progression, a recent discovery that has broadened our knowledge regarding poly(ADP-ribose) functions. A special emphasis is placed on recent advances in relation to PARG that are stimulating new directions in future research. Noticeably, the existence of various PARG isoforms characterized by distinct cellular localizations and nucleocytoplasmic shuttling properties challenges our current comprehension of pADPr metabolism. Observations and suppositions towards functionally important regulatory elements in the N-terminal portion of PARG are also discussed.

Chromosoma, 1997

We have generated and characterized a novel site-specific antibody highly specific for the phosph... more We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Chromosoma, 1997

We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluore... more We have evaluated the effects of different DNA denaturation protocols commonly used in DNA fluorescent in situ hybridization (FISH) experiments on chromatin structure using indirect immunofluorescence. The use of antibodies to acetylated histones H3 and H4 demonstrates that the different procedures differ considerably in their extent of histone displacement. Procedures involving paraformaldehyde fixation were found to be compatible with the structural preservation of acetylated chromatin organization by indirect immunofluorescence. These results provide a basis for interpreting DNA FISH experiments aimed at determining chromatin organization of individual loci.

Chromosoma, 1995

The study of RNA pol II-mediated transcription regulation has been dominated by molecular biologi... more The study of RNA pol II-mediated transcription regulation has been dominated by molecular biological approaches. Although these methods continue to provide important insights, other approaches are required to insure against an oversimplified view of gene expression. Improvements in EM methods and the development of the confocal light microscope have provided alternative and complementary means of investigating gene regulation. Information on the "context" in which cis- and trans-acting factors operate can be achieved with these techniques. As a result, the spatial compartmentalization of nuclear processes involved in transcriptional and post-transcriptional processing has received considerable attention.

Cell Cycle, 2013

Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently ove... more Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating the DNA damage response. Here, we show that polycomb repressive complex 2 (PRC2) is recruited to sites of DNA damage. This recruitment is independent of histone 2A variant X (H2AX) and the PI-3-related kinases ATM and DNA-PKcs. We establish that PARP activity is required for retaining PRC2 at sites of DNA damage. Furthermore, depletion of EZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gamma-irradiation. These data unravel a crucial role of PRC2 in determining cancer cellular sensitivity following DNA damage and suggest that therapeutic targeting of EZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy.

Breast Cancer Research, 2009

Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleava... more Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by γsecretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting γ-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that γsecretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells.

Breast Cancer Research, 2011

BMC Biology, 2009

Background: Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear proc... more Background: Within chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues.

Blood, 2005

characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients Overexpression of transcrip... more characterizes all t(4;14)(p16;q32)-positive multiple myeloma patients Overexpression of transcripts originating from the MMSET locus http://bloodjournal.hematologylibrary.org/content/105/10/4060.full.html Updated information and services can be found at: (4217 articles) Neoplasia (5019 articles) Immunobiology Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at: http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at: http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at: Multiple myeloma (MM) is a B-lineage malignancy characterized by diverse genetic subtypes and clinical outcomes. The recurrent immunoglobulin heavy chain (IgH) switch translocation, t(4;14)(p16;q32), is associated with poor outcome, though the mechanism is unclear. Quantitative reverse-transcription-polymerase chain reaction (RT-PCR) for proposed target genes on a panel of myeloma cell lines and purified plasma cells showed that only transcripts originating from the WHSC1/ MMSET/NSD2 gene are uniformly dysregulated in all t(4;14) POS patients. The different transcripts detected, multiple myeloma SET domain containing protein (MMSET I), MMSET II, Exon 4a/MMSET III, and response element II binding protein (RE-IIBP), are produced by alternative splicing and alternative transcription initiation events. Translation of the various transcripts, including those from major breakpoint region 4-2 (MB4-2) and MB4-3 breakpoint variants, was confirmed by transient transfection and immunoblotting. Green fluorescent protein (GFP)tagged MMSET I and II, corresponding to proteins expressed in MB4-1 patients, localized to the nucleus but not nucleoli, whereas the MB4-2 and MB4-3 proteins concentrate in nucleoli. Cloning and localization of the Exon 4a/MMSET III splice variant, which contains the protein segment lost in the MB4-2 variant, identified a novel protein domain that prevents nucleolar localization. Kinetic studies using photobleaching suggest that the breakpoint variants are functionally distinct from wild-type proteins. In contrast, RE-IIBP is universally dysregulated and also potentially functional in all t(4;14) POS patients irrespective of fibroblast growth factor receptor 3 (FGFR3) expression or breakpoint type. (Blood. 2005;105: 4060-4069)

Bioorganic & Medicinal Chemistry, 2011

We present a combination of database screening, synthesis and in vitro testing to identify novel ... more We present a combination of database screening, synthesis and in vitro testing to identify novel histone acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors. Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for new anticancer agents directed against HATs as new targets have been identified.

Biochemistry and Cell Biology, 1999