Alexey V. Ivanov

Ezh2 Requires PHF1 To Efficiently Catalyze H3 Lysine 27 Trimethylation In Vivo

ABSTRACT The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.

Methylation-Acetylation Interplay Activates p53 in Response to DNA Damage

ABSTRACT p53, an important tumor suppressor protein, exerts its function mostly as a sequence-specific transcription factor and is subjected to multiple posttranslational modifications in response to genotoxic stress. Recently, we discovered that lysine methylation of p53 at K372 by Set7/9 (also known as SET7 and Set9) is important for transcriptional activation and stabilization of p53. In this report we provide a molecular mechanism for the effect of p53 methylation on transcription. We demonstrate that Set7/9 activity toward p53, but not the nucleosomal histones, is modulated by DNA damage. Significantly, we show that lysine methylation of p53 is important for its subsequent acetylation, resulting in stabilization of the p53 protein. These p53 modification events can be observed on the promoter of p21 gene, a known transcriptional target of p53. Finally, we show that methylation-acetylation interplay in p53 augments acetylation of histone H4 in the promoter of p21 gene, resulting in its subsequent transcriptional activation and, hence, cell cycle arrest. Collectively, these results suggest that the cross talk between lysine methylation and acetylation is critical for p53 activation in response to DNA damage and that Set7/9 may play an important role in tumor suppression.

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.

Suppression of the Epithelial–Mesenchymal Transition by Grainyhead-like-2

Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell-like compartment of tumors, including TGF-β dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-β receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.

Structural insights into human KAP1 PHD finger–bromodomain and its role in gene silencing

The tandem PHD finger–bromodomain, found in many chromatin-associated proteins, has an important role in gene silencing by the human co-repressor KRAB-associated protein 1 (KAP1). Here we report the three-dimensional solution structure of the tandem PHD finger–bromodomain of KAP1. The structure reveals a distinct scaffold unifying the two protein modules, in which the first helix, αZ, of an atypical bromodomain forms the central hydrophobic core that anchors the other three helices of the bromodomain on one side and the zinc binding PHD finger on the other. A comprehensive mutation-based structure-function analysis correlating transcriptional repression, ubiquitin-conjugating enzyme 9 (UBC9) binding and SUMOylation shows that the PHD finger and the bromodomain of KAP1 cooperate as one functional unit to facilitate lysine SUMOylation, which is required for KAP1 co-repressor activity in gene silencing. These results demonstrate a previously unknown unified function for the tandem PHD finger–bromodomain as an intramolecular small ubiquitin-like modifier (SUMO) E3 ligase for transcriptional silencing.

Functional Analysis of KAP1 Genomic Recruitment

ABSTRACT TRIM28 (KAP1) is upregulated in many cancers and has been implicated in both transcriptional activation and repression. Using chromatin immunoprecipitation and sequencing, we show that KAP1 binding sites fall into several categories, specifically, the 3′ coding exons of zinc finger (ZNF) genes and promoter regions of ZNFs and other genes. The currently accepted model is that KAP1 is recruited to the genome via interaction of its N-terminal RBCC domain with KRAB ZNFs (KRAB domain containing ZNFs). To determine whether the interaction of KAP1 with KRAB ZNFs is the mechanism by which KAP1 is recruited to genomic binding sites, we analyzed stable cell lines that express tagged wild-type and mutant KAP1. Surprisingly, deletion of the RBCC domain abolished KAP1 binding to the 3′ exons of ZNF genes but KAP1 binding to promoter regions was unaffected. Using KAP1 knockdown cells, we showed that the genes most responsive to KAP1 were not ZNF genes but instead were either indirect targets or had KAP1 bound 10 to 100 kb from the transcription start site. Therefore, our studies suggest that KAP1 plays a role distinct from transcriptional regulation at the majority of its strongest binding sites.

A KLF4–miRNA-206 Autoregulatory Feedback Loop Can Promote or Inhibit Protein Translation Depending upon Cell Context

ABSTRACT Krüppel-like factor 4 (KLF4), a transcription factor that regulates cell fate in a context-dependent fashion, is normally induced upon growth arrest or differentiation. In many cancer cells there is dysregulation, with increased expression in proliferating cells. To identify sequence elements that mediate KLF4 suppression in normal epithelial cells, we utilized a luciferase reporter and RK3E cells, which undergo a proliferation-differentiation switch to form an epithelial sheet. A translational control element (TCE) within the KLF4 3′-untranslated region interacted with microRNAs (miRs) 206 and 344-1 to promote or inhibit KLF4 expression, respectively, in proliferating epithelial cells. Overall, the TCE suppressed expression in proliferating primary human mammary epithelial cells, but this suppressive effect was attenuated in immortalized mammary epithelial MCF10A cells, in which Dicer1 and miR-206 promoted KLF4 expression and TCE reporter activity. In contrast to MCF10A cells, in breast cancer cells the activity of miR-206 was switched, and it repressed KLF4 expression and TCE reporter activity. As miR-206 levels were KLF4 dependent, the results identify a KLF4–miR-206 feedback pathway that oppositely affects protein translation in normal cells and cancer cells. In addition, the results indicate that two distinct miRs can have opposite and competing effects on translation in proliferating cells.

The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells

Lung cancer remains the leading cause of cancer-related mortality for both men and women. Tumor recurrence and metastasis is the major cause of lung cancer treatment failure and death. The microRNA-200 (miR-200) family is a powerful regulator of the epithelial-mesenchymal transition (EMT) process, which is essential in tumor metastasis. Nevertheless, miR-200 family target genes that promote metastasis in non-small cell lung cancer (NSCLC) remain largely unknown. Here, we sought to investigate whether the microRNA-200 family regulates our previously identified NSCLC prognostic marker genes associated with metastasis, as potential molecular targets. Novel miRNA targets were predicted using bioinformatics tools based on correlation analyses of miRNA and mRNA expression in 57 squamous cell lung cancer tumor samples. The predicted target genes were validated with quantitative RT-PCR assays and western blot analysis following re-expression of miR-200a, -200b and -200c in the metastatic NSCLC H1299 cell line. The results show that restoring miR-200a or miR-200c in H1299 cells induces downregulation of DLC1, ATRX and HFE. Reinforced miR-200b expression results in downregulation of DLC1, HNRNPA3 and HFE. Additionally, miR-200 family downregulates HNRNPR3, HFE and ATRX in BEAS-2B immortalized lung epithelial cells in quantitative RT-PCR and western blot assays. The miR-200 family and these potential targets are functionally involved in canonical pathways of immune response, molecular mechanisms of cancer, metastasis signaling, cell-cell communication, proliferation and DNA repair in Ingenuity pathway analysis (IPA). These results indicate that re-expression of miR-200 downregulates our previously identified NSCLC prognostic biomarkers in metastatic NSCLC cells. These results provide new insights into miR-200 regulation in lung cancer metastasis and consequent clinical outcome, and may provide a potential basis for innovative therapeutic approaches for the treatment of this deadly disease.

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.

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.