Richard A. Katz's research while affiliated with Fox Chase Cancer Center and other places
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The eukaryotic genome is organized in three dimensions within the nucleus. Transcriptionally active chromatin is spatially separated from silent heterochromatin, a large fraction of which is located at the nuclear periphery. However, the mechanisms by which chromatin is localized at the nuclear periphery remain poorly understood. Here we demonstrate that Proline Rich 14 (PRR14) protein organizes H3K9me3-modified heterochromatin at the nuclear lamina. We show that PRR14 dynamically associates with both the nuclear lamina and heterochromatin, and is able to reorganize heterochromatin in the nucleus of interphase cells independent of mitosis. We characterize two functional HP1-binding sites within PRR14 that contribute to its association with heterochromatin. We also demonstrate that PPR14 forms an anchoring surface for heterochromatin at the nuclear lamina where it interacts dynamically with HP1-associated chromatin. Our study proposes a model of dynamic heterochromatin organization at the nuclear lamina via the PRR14 tethering protein.
The eukaryotic genome is organized in three dimensions within the nucleus. Transcriptionally active chromatin is spatially separated from silent heterochromatin, a large fraction of which is located at the nuclear periphery. However, the mechanisms by which chromatin is localized at the nuclear periphery remain poorly understood. Here we demonstrate that Proline Rich 14 (PRR14) protein specifically organizes H3K9me3-modified heterochromatin at the nuclear lamina. We show that PRR14 dynamically associates with both the nuclear lamina and heterochromatin, and is able to reorganize heterochromatin in the nucleus of interphase cells independent of mitosis. We demonstrate that PRR14 can bind all isoforms of heterochromatin protein 1 (HP1). We characterize two functional HP1-binding sites within PRR14 that contribute to its association with heterochromatin. Results of fluorescent recovery after photobleaching (FRAP) and super-resolution imaging, indicate that PPR14 forms an anchoring surface for heterochromatin at the nuclear lamina where it interacts dynamically with HP1-associated, H3K9me3-modified chromatin. Our study reveals the mechanism through which PRR14 tethers heterochromatin to the nuclear lamina and we propose a model of dynamic heterochromatin organization at the nuclear periphery.
A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via "tethering proteins" that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We identified previously a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We also show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation-dephosphorylation of the LBD. Furthermore, we identified a PP2A phosphatase recognition motif within the evolutionarily conserved PRR14 C-terminal Tantalus domain. Disruption of this motif affected PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable, modular domains. The findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.
A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via tethering proteins that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We identified previously a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify a centrally located, evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We also show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation of the LBD. We also show that the evolutionary conserved PRR14 C-terminal Tantalus domain encodes a PP2A phosphatase recognition site that regulates PRR14 nuclear lamina association. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two modular domains. Furthermore, the identification of a modular LBD may provide an engineering strategy for delivery of cargo to the nuclear lamina.
Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.
Background: Breast cancer (BCa) is a genetically heterogeneous disease and many genes contributing to BCa risk remain to be identified. Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (>50) have strongly implicated genetic alterations at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of BCa. ESR1, encoding the estrogen receptor, might be a more obvious candidate for influencing risk. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for BCa susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, accounts for GWAS-associated BCa risk at this locus. CCDC170 is a coiled-coiled domain-containing protein of poorly understood function. BCa-specific truncation and missense mutations in CCDC170 also have been detected, with the truncations being implicated in driving Luminal B subtype BCa. Together these findings demonstrate that the CCDC170 gene is involved in BCa, but the underlying molecular mechanisms for its role in tumorigenesis are unknown.
Experimental designs and results: By using the approaches of confocal microscopy and cell imaging analysis, here we report for the first time that CCDC170 is associated with the Golgi apparatus and perinuclear microtubules (MTs), and support a role for CCDC170 in the Golgi-associated microtubule network. We have shown that overexpression of CCDC170 triggers Golgi reorganization and stabilizes Golgi-associated MTs, accompanied by dramatically increased acetylation of α-tubulin that is driven by the acetyltransferase ATAT1. The Golgi-associated MT network has been proposed to regulate cell polarity and migration. In support of this concept, we have shown that CRISPR knockout increases, and overexpression of CCDC170 decreases, BCa directional cell migration in vitro. We also found that the BCa-specific truncations result in mislocalization of CCDC170 and/or diminished stability of Golgi-associated MTs. Lastly, we identified candidate CCDC170 functional binding partners (e.g. MAP4) that are consistent with its localization and proposed function. These partners may serve to mediate the acetylation and stabilization of MTs.
Conclusions: Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and provide a mechanism for how perturbations in CCDC170 could alter Golgi-mediated cell polarity, and thereby drive BCa and other abnormalities. This work was partially supported by the Susan G. Komen for the Cure (KG100274), NCI (CA186853), and Eileen Stein Jacoby Fund.
Citation Format: Pengtao Jiang, Yueran Li, Andrey Poleshko, Valentina Medvedeva, Natalia Baulina, Yongchao Zhang, Yan Zhou, Carolyn M. Slater, Trinity Pellegrin, Jason Wasserman, Michael Lindy, Mary Daly, Richard A. Katz, Xiaowei Chen. The breast cancer gene CCDC170 regulates the Golgi-associated microtubule network and directional cell migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1884. doi:10.1158/1538-7445.AM2017-1884
Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (>50) have implicated single nucleotide polymorphisms (SNPs) located at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of breast cancer. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for breast cancer susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, account for GWAS-associated breast cancer risk at this locus. Breast cancer-associated CCDC170 nonsense mutations and rearrangements have also been detected, with the latter being specifically implicated in driving breast cancer. Here we report that the wild type CCDC170 protein localizes to the region of the Golgi apparatus and binds Golgi-associated microtubules (MTs), and that breast cancer-linked truncations of CCDC170 result in loss of Golgi localization. Overexpression of wild type CCDC170 triggers Golgi reorganization, and enhances Golgi-associated MT stabilization and acetyltransferase ATAT1-dependent α-tubulin acetylation. Golgi-derived MTs regulate cellular polarity and motility, and we provide evidence that dysregulation of CCDC170 affects polarized cell migration. Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and implicate a mechanism for how perturbations in the CCDC170 gene may contribute to the hallmark changes in cell polarity and motility seen in breast cancer.
Collaborations between the Wlodawer and Skalka laboratories have covered a period of almost 30 years. During that time our groups have co-authored 18 publications, including several much cited journal articles, book chapters, and scholarly reviews. It has therefore been most rewarding for us to share enthusiasm, insights, and expertise with our Frederick colleagues over the years, and also to enjoy lasting friendships.
... To further understand the mechanisms by which peripheral heterochromatin may govern nuclear shape, we altered the interactions between heterochromatin and the lamina in our simulations. Experiments have shown that heterochromatinlamina interactions are critical in regulating nuclear morphology [50,[82][83][84]. We modeled these interactions through a nonspecific (nonbonded) attractive interaction with strength u HS between heterochromatin and lamina (shell) beads. ...
... MchSLE-1 is a novel LTR-RT (4.76 Kbp) recently described in the genome of M. chilensis [36]. From the sense strand, we identified a single ORF encoding a polyprotein of 1376 amino acids where the most conserved residues correspond to the retroviral Pol regions, including similarities with the retroviral protease with a diagnosed DSG active site motif [44], a reverse transcriptase (RT) with a polymerase domain containing a YxDD box [45], an RNAse H domain with a diagnostic DEDD catalytic core [46] and an integrase (IN) with an HHCC zinc finger and a conserved D,D(35)E motif [47] (Figure 1). In the amino terminal of the polyprotein, the only Gag similarity is a nucleocapsid domain with two putative zinc fingers containing CCCC and CCHC motifs. ...
... It has been speculated that the highly mutated HERV genome may disrupt canonical SD/SA signals and allow cryptic splicing sites to emerge. In fact, many of such sites have been identified throughout the HERV genome (123,124). ...
... As for the nervous system, the roles of chromatin organization in neurodevelopment, specifically via transcriptional regulation, have recently been actively explored [11][12][13][14] , whereas the roles of subnuclear positioning of chromatin involving transcriptional regulation have remained relatively less understood. The anchoring of heterochromatin to the inner nuclear membrane (INM) is crucial for subnuclear positioning of chromatin 15,16 , which is mediated by chromatinbinding proteins/chromatin-anchoring proteins, such as the evolutionally conserved barrier-to-autointegration factor (Baf) [17][18][19] , mammalian proline-rich-protein (PRR14) 20 , and C. elegans-specific chromodomain-containing protein (cec-4) 21,22 . These chromatin-binding proteins/chromatin-anchoring proteins interact with lamin-associated proteins, such as proteins with LEM (LAP2, Emerin, MAN1) domains that reside in the INM [23][24][25] , to form physical interactions between the chromosome and the nuclear lamina. ...
... Wortmannin is a specific PI3K inhibitor. In the presence of wortmannin, the efficiency of transduction of primary B-cell precursors that express normal DNA-PKcs by RSV-or HIV-1-based pseudoviruses decreased tenfold [37]. However, these effects may result from inhibition of not only DNA-PKcs but also other PI3K members. ...
... However, it is overexpressed in human colorectal cancer (CRC) patients, and knocking down TDG in several CRC cell lines inhibited cell growth [59]. Moreover, TDG knockdown suppressed tumor formation of melanoma cell lines in xenograft models, indicating that TDG activity is important for tumorigenesis [60]. Our data evince its potential oncogenic function in breast cancer. ...
... Although little is known about the role of CCDC170 in UF, it is recurrently fused with the ESR1 gene in~14 % of ER+ breast cancers, and patients with such fusion have worse clinical outcomes 53 . Studies in breast cancer cell lines have implicated CCDC170 in cell migration through the alteration of the Golgi-associated microtubule network 54 . Given the lack of evidence for the role of CCDC170 in fibroid pathogenesis, we used CRISPR to knock out this gene and study its phenotype (Fig. 4e). ...
... Subsequently, an siRNA screen by Carolian D'Alesio et al. identified CHD4 as a therapeutic target in colon cancer. In this study, CHD4 depletion was found to sensitize colon cancer cells to DNMT inhibitors in reactivating hypermethylated genes [52]. Later, depletion of CHD4 in acute myeloid leukemia blasts was found to sensitize cells both in vitro and in vivo to genotoxic agents daunorubicin and cytarabine [37]. ...
... In fact, ID4 has been proposed as a regulatory factor of the expression of the remaining ID proteins [36]. Increased expression of ID4 has been observed in some tumor types such as hepatocellular carcinoma [37], glioblastoma [38], or melanoma [39], indicating a possible role in tumor development. On the contrary, other authors have reported that loss of ID4 by hypermethylation increases tumor progression and metastasis [40,41], supporting the theory of ID4 being a tumor suppressor gene. ...
... Epigenetic repression of GFP in HeLa TI cells is driven by more than 15 factors, including chromatin-modifying enzymes and histone chaperones [50]. HDAC enzyme that contributes most to the GFP epigenetic silencing in HeLa TI cells is HDAC1 [55]. Previously, HeLa TI cells were shown to be sensitive to a wide range of epigenetic modulators, including HDAC inhibitors, as well as inhibitors of DNMTs, HMTs, and BRD (bromodomain)-containing proteins. ...
