Hongzhuang Peng

MAGE-A, mMage-b, and MAGE-C Proteins Form Complexes with KAP1 and Suppress p53-Dependent Apoptosis in MAGE-Positive Cell Lines

The MAGE-A, MAGE-B, and MAGE-C protein families comprise the class-I MAGE/cancer testes antigens, a group of highly homologous proteins whose expression is suppressed in all normal tissues except developing sperm. Aberrant expression of class I MAGE proteins occurs in melanomas and many other malignancies, and MAGE proteins have long been recognized as tumor-specific targets; however, their functions have largely been unknown. Here, we show that suppression of class I MAGE proteins induces apoptosis in the Hs-294T, A375, and S91 MAGE-positive melanoma cell lines and that members of all three families of MAGE class I proteins form complexes with KAP1, a scaffolding protein that is known as a corepressor of p53 expression and function. In addition to inducing apoptosis, MAGE suppression decreases KAP1 complexing with p53, increases immunoreactive and acetylated p53, and activates a p53 responsive reporter gene. Suppression of class I MAGE proteins also induces apoptosis in MAGE-A-positive, p53wt/wt parental HCT 116 colon cancer cells but not in a MAGE-A-positive HCT 116 p53-/- variant, indicating that MAGE suppression of apoptosis requires p53. Finally, treatment with MAGE-specific small interfering RNA suppresses S91 melanoma growth in vivo, in syngenic DBA2 mice. Thus, class I MAGE protein expression may suppress apoptosis by suppressing p53 and may actively contribute to the development of malignancies and by promoting tumor survival. Because the expression of class I MAGE proteins is limited in normal tissues, inhibition of MAGE antigen expression or function represents a novel and specific treatment for melanoma and diverse malignancies.

The LIM Protein AJUBA Recruits Protein Arginine Methyltransferase 5 To Mediate SNAIL-Dependent Transcriptional Repression

ABSTRACT The SNAIL transcription factor contains C-terminal tandem zinc finger motifs and an N-terminal SNAG repression domain. The members of the SNAIL family have recently emerged as major contributors to the processes of development and metastasis via the regulation of epithelial-mesenchymal transition events during embryonic development and tumor progression. However, the mechanisms by which SNAIL represses gene expression are largely undefined. Previously we demonstrated that the AJUBA family of LIM proteins function as corepressors for SNAIL and, as such, may serve as a platform for the assembly of chromatin-modifying factors. Here, we describe the identification of the protein arginine methyltransferase 5 (PRMT5) as an effector recruited to SNAIL through an interaction with AJUBA that functions to repress the SNAIL target gene, E-cadherin. PRMT5 binds to the non-LIM region of AJUBA and is translocated into the nucleus in a SNAIL- and AJUBA-dependent manner. The depletion of PRMT5 in p19 cells stimulates E-cadherin expression, and the SNAIL, AJUBA, and PRMT5 ternary complex can be found at the proximal promoter region of the E-cadherin gene, concomitant with increased arginine methylation of histones at the locus. Together, these data suggest that PRMT5 is an effector of SNAIL-dependent gene repression.

KAP1, a Novel Substrate for PIKK Family Members, Colocalizes with Numerous Damage Response Factors at DNA Lesions

The DNA damage response requires a coordinated nucleo-cytoplasmic cascade of events, which ultimately converge on damaged DNA packaged in chromatin. Few connections between the proteins that remodel chromatin and the proteins that mediate this damage response have been shown. We have investigated the DNA damage-induced phosphorylation of the KRAB-ZFP-associated protein 1 (KAP1), the dedicated corepressor for Krüppel-associated box (KRAB) zinc finger protein (ZFP) proteins. We show that KAP1 is rapidly phosphorylated following DNA damage by members of the phosphatidylinositol-3 kinase-like family of kinases. This phosphorylation occurs at a single amino acid residue that is conserved from mice to humans and is located adjacent to the bromodomain, suggesting that it may regulate chromatin recognition by that module. Phosphorylated KAP1 rapidly localizes to sites of DNA strand breaks in the nucleus in response to ionizing radiation. This discovery provides a novel link between chromatin-mediated transcriptional repression and the recognition/repair of DNA, which must be accomplished by the cellular DNA damage response.

Hetero-oligomerization Among the TIF Family of RBCC/TRIM Domain-containing Nuclear Cofactors: A Potential Mechanism for Regulating the Switch Between Coactivation and Corepression

The RING-B box-coiled-coil (RBCC) motif (also re-named recently as the tripartite motif (TRIM)) is a widely distributed motif that is hypothesized to be a protein-protein interface. The RBCC/TRIM domain of the corepressor KAP-1 is both necessary and sufficient to interact directly with the transcription repressor KRAB domain. Each subdomain of the KAP-1-RBCC contributes directly to the oligomerization and/or ligand recognition. Little is known about the function or the natural binding ligands for the RBCC/TRIM domain of the other TIF family members. In order to investigate whether hetero-oligomerization might be a biological regulatory mechanism, we have evaluated the hetero-oligomerization potential of the TIF family members including KAP-1, TIF1alpha, TIF1gamma, and the RBCC/TRIM family members including PML1, and MID1. We have reconstituted and characterized the oligomerization for these proteins using baculovirus and mammalian expression systems by biochemical approaches. Our data indicate that the RBCC/TRIM domains of KAP-1, TIF1alpha and TIF1gamma exist in a homo-oligomeric state. However, there is little cross-talk between KAP-1 and other TIF family members, suggesting that a high degree of specificity for oligomerization interface and ligand recognition is intrinsically built into the RBCC/TRIM domain of KAP-1. Finally, we demonstrate that TIF1alpha interacts with TIF1gamma and the coiled-coil region of TIF1gamma is necessary for this interaction. The hetero-oligomerization between TIF1alpha and TIF1gamma implies a potential regulatory mechanism for transcriptional regulation.

Drosophila SETDB1 Is Required for Chromosome 4 Silencing

Histone H3 lysine 9 (H3K9) methylation is associated with gene repression and heterochromatin formation. In Drosophila, SU(VAR)3–9 is responsible for H3K9 methylation mainly at pericentric heterochromatin. However, the histone methyltransferases responsible for H3K9 methylation at euchromatic sites, telomeres, and at the peculiar Chromosome 4 have not yet been identified. Here, we show that DmSETDB1 is involved in nonpericentric H3K9 methylation. Analysis of two DmSetdb1 alleles generated by homologous recombination, a deletion, and an allele where the 3HA tag is fused to the endogenous DmSetdb1, reveals that this gene is essential for fly viability and that DmSETDB1 localizes mainly at Chromosome 4. It also shows that DmSETDB1 is responsible for some of the H3K9 mono- and dimethyl marks in euchromatin and for H3K9 dimethylation on Chromosome 4. Moreover, DmSETDB1 is required for variegated repression of transgenes inserted on Chromosome 4. This study defines DmSETDB1 as a H3K9 methyltransferase that specifically targets euchromatin and the autosomal Chromosome 4 and shows that it is an essential factor for Chromosome 4 silencing.

Tripartite motif-containing protein 28 is a small ubiquitin-related modifier E3 ligase and negative regulator of IFN regulatory factor 7.

IFN regulatory factor 7 (IRF7) is a potent transcription factor of type I IFNs and IFN-stimulated genes and is known as the master regulator of type I IFN-dependent immune responses. Because excessive responses could harm the host, IRF7 itself is delicately regulated at the transcriptional, translational, and posttranslational levels. Modification of IRF7 by small ubiquitin-related modifiers (SUMOs) has been shown to regulate IFN expression and antiviral responses negatively, but the specific E3 ligase needed for IRF7 SUMOylation has remained unknown. As reported in this article, we have identified the tripartite motif-containing protein 28 (TRIM28) as a binding partner of IRF7. We have demonstrated that TRIM28 also interacts with the SUMO E2 enzyme and increases SUMOylation of IRF7 both in vivo and in vitro, suggesting it acts as a SUMO E3 ligase of IRF7. Unlike the common SUMO E3 ligase, protein inhibitor of activated STAT1, the E3 activity of TRIM28 is specific to IRF7, because it has little effect on IRF7’s close relative IRF3. TRIM28 is therefore, so far as we know, the first IRF7-specific SUMO E3 reported. TRIM28-mediated SUMOylation of IRF7 is increased during viral infection, and SUMOylation of transcription factors usually results in transcriptional repression. Overexpression of TRIM28 therefore inhibits IRF7 transactivation activity, whereas knockdown of TRIM28 has the opposite effect and potentiates IFN production and antiviral responses. Collectively, our results suggest that TRIM28 is a specific SUMO E3 ligase and negative regulator of IRF7.

Germline mutation of Bap1 accelerates development of asbestos-induced malignant mesothelioma

Malignant mesotheliomas are highly aggressive tumors usually caused by exposure to asbestos. Germline-inactivating mutations of BAP1 predispose to mesothelioma and certain other cancers. However, why mesothelioma is the predominate malignancy in some BAP1 families and not others, and whether exposure to asbestos is required for development of mesothelioma in BAP1 mutation carriers are not known. To address these questions experimentally, we generated a Bap1(+/-) knockout mouse model to assess its susceptibility to mesothelioma upon chronic exposure to asbestos. Bap1(+/-) mice exhibited a significantly higher incidence of asbestos-induced mesothelioma than wild-type (WT) littermates (73% vs. 32%, respectively). Furthermore, mesotheliomas arose at an accelerated rate in Bap1(+/-) mice than in WT animals (median survival, 43 weeks vs. 55 weeks after initial exposure, respectively) and showed increased invasiveness and proliferation. No spontaneous mesotheliomas were seen in unexposed Bap1(+/-) mice followed for up to 87 weeks of age. Mesothelioma cells from Bap1(+/-) mice showed biallelic inactivation of Bap1, consistent with its proposed role as a recessive cancer susceptibility gene. Unlike in WT mice, mesotheliomas from Bap1(+/-) mice did not require homozygous loss of Cdkn2a. However, normal mesothelial cells and mesothelioma cells from Bap1(+/-) mice showed downregulation of Rb through a p16(Ink4a)-independent mechanism, suggesting that predisposition of Bap1(+/-) mice to mesothelioma may be facilitated, in part, by cooperation between Bap1 and Rb. Drawing parallels to human disease, these unbiased genetic findings indicate that BAP1 mutation carriers are predisposed to the tumorigenic effects of asbestos and suggest that high penetrance of mesothelioma requires such environmental exposure.

The ATM substrate KAP1 controls DNA repair in heterochromatin: regulation by HP1 proteins and serine 473/824 phosphorylation.

The repair of DNA damage in highly compact, transcriptionally silent heterochromatin requires that repair and chromatin packaging machineries be tightly coupled and regulated. KAP1 is a heterochromatin protein and co-repressor that binds to HP1 during gene silencing but is also robustly phosphorylated by Ataxia telangiectasia mutated (ATM) at serine 824 in response to DNA damage. The interplay between HP1-KAP1 binding/ATM phosphorylation during DNA repair is not known. We show that HP1α and unmodified KAP1 are enriched in endogenous heterochromatic loci and at a silent transgene prior to damage. Following damage, γH2AX and pKAP1-s824 rapidly increase and persist at these loci. Cells that lack HP1 fail to form discreet pKAP1-s824 foci after damage but levels are higher and more persistent. KAP1 is phosphorylated at serine 473 in response to DNA damage and its levels are also modulated by HP1. Unlike pKAP1-s824, pKAP1-s473 does not accumulate at damage foci but is diffusely localized in the nucleus. While HP1 association tempers KAP1 phosphorylation, this interaction also slows the resolution of γH2AX foci. Thus, HP1-dependent regulation of KAP1 influences DNA repair in heterochromatin. Mol Cancer Res; 10(3); 401–14. ©2011 AACR.

14-3-3 Binding Sites in the Snail Protein Are Essential for Snail-Mediated Transcriptional Repression and Epithelial-Mesenchymal Differentiation

The Snail transcription factor is a repressor and a master regulator of epithelial-mesenchymal transition (EMT) events in normal embryonic development and during tumor metastases. Snail directly regulates genes affecting cell adhesion, motility, and polarity. Invasive tumor cells express high levels of Snail, which is a marker for aggressive disease and poor prognosis. Transcriptional repression and EMT induction by Snail requires binding to its obligate corepressor, the LIM protein Ajuba. It is unclear how this complex is assembled and maintained on Snail target genes. Here we define functional 14-3-3 binding motifs in Snail and Ajuba, which selectively bind 14-3-3 protein isoforms. In Snail, an NH(2)-terminal motif in the repression domain cooperates with a COOH-terminal, high-affinity motif for binding to 14-3-3 proteins. Coordinate mutation of both motifs abolishes 14-3-3 binding and inhibits Snail-mediated gene repression and EMT differentiation. Snail, 14-3-3 proteins, and Ajuba form a ternary complex that is readily detected through chromatin immunoprecipitation at the endogenous E-cadherin promoter. Collectively, these data show that 14-3-3 proteins are new components of the Snail transcriptional repression machinery and mediate its important biological functions.

Epigenetic Gene Silencing by the SRY Protein Is Mediated by a KRAB-O Protein That Recruits the KAP1 Co-repressor Machinery

The sex determination transcription factor SRY is a cell fate-determining transcription factor that mediates testis differentiation during embryogenesis. It may function by repressing the ovarian determinant gene, RSPO1, action in the ovarian developmental pathway and activates genes, such as SOX9, important for testis differentiation at the onset of gonadogenesis. Further, altered expression of SRY and related SOX genes contribute to oncogenesis in many human cancers. Little is known of the mechanisms by which SRY regulates its target genes. Recently a KRAB domain protein (KRAB-O) that lacks a zinc finger motif has been demonstrated to interact with SRY and hypothesized to function as an adaptor molecule for SRY by tethering the KAP1-NuRD-SETDB1-HP1 silencing machinery to repress SRY targets. We have critically examined this hypothesis by reconstituting and characterizing SRY-KRAB-O-KAP1 interactions. These recombinant molecules can form a ternary complex by direct and high affinity interactions. The KRAB-O protein can simultaneously bind KAP1 and SRY in a noncompetitive but also noncooperative manner. An extensive mutagenesis analysis suggests that different surfaces on KRAB-O are utilized for these independent interactions. Transcriptional repression by SRY requires binding to KRAB-O, thus bridging to the KAP1 repression machinery. This repression machinery is recruited to SRY target promoters in chromatin templates via SRY. These results suggest that SRY has co-opted the KRAB-O protein to recruit the KAP1 repression machinery to sex determination target genes. Other KRAB domain proteins, which lack a zinc finger DNA-binding motif, may function in similar roles as adaptor proteins for epigenetic gene silencing.