PCNA-binding Domain Research Articles

EP117 - IMAGe syndrome: further delineation of the clinical phenotype of PCNA binding domain CDKN1C variants

eP117 - IMAGe syndrome: further delineation of the clinical phenotype of PCNA binding domain CDKN1C variants

Analysis of CDKN1C in fetal growth restriction and pregnancy loss.

Background: Cyclin-dependent kinase inhibitor 1C (CDKN1C) is a key negative regulator of cell growth encoded by a paternally imprinted/maternally expressed gene in humans. Loss-of-function variants in CDKN1C are associated with an overgrowth condition (Beckwith-Wiedemann Syndrome) whereas "gain-of-function" variants in CDKN1C that increase protein stability cause growth restriction as part of IMAGe syndrome ( Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies). As three families have been reported with CDKN1C mutations who have fetal growth restriction (FGR)/Silver-Russell syndrome (SRS) without adrenal insufficiency, we investigated whether pathogenic variants in CDKN1C could be associated with isolated growth restriction or recurrent loss of pregnancy. Methods: Analysis of published literature was undertaken to review the localisation of variants in CDKN1C associated with IMAGe syndrome or fetal growth restriction. CDKN1C expression in different tissues was analysed in available RNA-Seq data (Human Protein Atlas). Targeted sequencing was used to investigate the critical region of CDKN1C for potential pathogenic variants in SRS (n=66), FGR (n=37), DNA from spontaneous loss of pregnancy (n= 22) and women with recurrent miscarriages (n=78) (total n=203). Results: All published single nucleotide variants associated with IMAGe syndrome are located in a highly-conserved "hot-spot" within the PCNA-binding domain of CDKN1C between codons 272-279. Variants associated with familial growth restriction but normal adrenal function currently affect codons 279 and 281. CDKN1C is highly expressed in the placenta compared to adult tissues, which may contribute to the FGR phenotype and supports a role in pregnancy maintenance. In the patient cohorts studied no pathogenic variants were identified in the PCNA-binding domain of CDKN1C. Conclusion: CDKN1C is a key negative regulator of growth. Variants in a very localised "hot-spot" cause growth restriction, with or without adrenal insufficiency. However, pathogenic variants in this region are not a common cause of isolated fetal growth restriction phenotypes or loss-of-pregnancy/recurrent miscarriages.

Analysis of CDKN1C in fetal growth restriction and pregnancy loss

Background: Cyclin-dependent kinase inhibitor 1C (CDKN1C) is a key negative regulator of cell growth encoded by a paternally imprinted/maternally expressed gene in humans. Loss-of-function variants in CDKN1C are associated with an overgrowth condition (Beckwith-Wiedemann Syndrome) whereas “gain-of-function” variants in CDKN1C that increase protein stability cause growth restriction as part of IMAGe syndrome ( Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies). As three families have been reported with CDKN1C mutations who have fetal growth restriction (FGR)/Silver-Russell syndrome (SRS) without adrenal insufficiency, we investigated whether pathogenic variants in CDKN1C could be associated with isolated growth restriction or recurrent loss of pregnancy. Methods: Analysis of published literature was undertaken to review the localisation of variants in CDKN1C associated with IMAGe syndrome or fetal growth restriction. CDKN1C expression in different tissues was analysed in available RNA-Seq data (Human Protein Atlas). Targeted sequencing was used to investigate the critical region of CDKN1C for potential pathogenic variants in SRS (n=66), FGR (n=37), DNA from spontaneous loss of pregnancy (n= 22) and women with recurrent miscarriages (n=78) (total n=203). Results: All published single nucleotide variants associated with IMAGe syndrome are located in a highly-conserved “hot-spot” within the PCNA-binding domain of CDKN1C between codons 272-279. Variants associated with familial growth restriction but normal adrenal function currently affect codons 279 and 281. CDKN1C is highly expressed in the placenta compared to adult tissues, which may contribute to the FGR phenotype and supports a role in pregnancy maintenance. In the patient cohorts studied no pathogenic variants were identified in the PCNA-binding domain of CDKN1C. Conclusion: CDKN1C is a key negative regulator of growth. Variants in a very localised “hot-spot” cause growth restriction, with or without adrenal insufficiency. However, pathogenic variants in this region are not a common cause of isolated fetal growth restriction phenotypes or loss-of-pregnancy/recurrent miscarriages.

Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets

BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.

Analysis of CDKN1C in fetal growth restriction and pregnancy loss.

Background: Cyclin-dependent kinase inhibitor 1C (CDKN1C) is a key negative regulator of cell growth encoded by a paternally imprinted/maternally expressed gene in humans. Loss-of-function variants in CDKN1C are associated with an overgrowth condition (Beckwith-Wiedemann Syndrome) whereas "gain-of-function" variants in CDKN1C that increase protein stability cause growth restriction as part of IMAGe syndrome ( Intrauterine growth restriction, Metaphyseal dysplasia, Adrenal hypoplasia and Genital anomalies). As two families have been reported with CDKN1C mutations who have fetal growth restriction (FGR)/Silver-Russell syndrome (SRS) without adrenal insufficiency, we investigated whether pathogenic variants in CDKN1C could be associated with isolated growth restriction or recurrent loss of pregnancy. Methods: Analysis of published literature was undertaken to review the localisation of variants in CDKN1C associated with IMAGe syndrome or fetal growth restriction. CDKN1C expression in different tissues was analysed in available RNA-Seq data (Human Protein Atlas). Targeted sequencing was used to investigate the critical region of CDKN1C for potential pathogenic variants in SRS (n=58), FGR (n=26), DNA from spontaneous loss of pregnancy (n= 21) and women with recurrent miscarriages (n=71) (total n=176). Results: All published single nucleotide variants associated with IMAGe syndrome are located in a highly-conserved "hot-spot" within the PCNA-binding domain of CDKN1C between codons 272-279. Variants associated with familial growth restriction but normal adrenal function currently affect codons 279 and 281. CDKN1C is highly expressed in the placenta compared to adult tissues, which may contribute to the FGR phenotype and supports a role in pregnancy maintenance. In the patient cohorts studied no pathogenic variants were identified in the PCNA-binding domain of CDKN1C. Conclusion: CDKN1C is a key negative regulator of growth. Variants in a very localised "hot-spot" cause growth restriction, with or without adrenal insufficiency. However, pathogenic variants in this region are not a common cause of isolated fetal growth restriction phenotypes or loss-of-pregnancy/recurrent miscarriages.

Abstract 2307: Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer

Abstract Introduction Approximately 15% of muscle invasive bladder cancers harbor mutations in the CDKN1A gene, which encodes the p53 target gene p21, with the majority of alterations truncating the peptide. We previously found that proximal truncation of p21 sensitized cells to cisplatin, while distal truncations made them more resistant. We hypothesized that truncations preserving the CDKi domain but ablating the PCNA binding domain would abrogate p21's ability to sequester PCNA but maintain cell cycle arrest capacities, enhancing the interaction between mono-ubiquitiniated-PCNA (ub-PCNA) and DNA polymerase eta (Pol η), resulting in increased TLS and DNA damage tolerance. Methods We used SW780 cell line for this study based on its resistance to cisplatin and p53 and p21 WT status. CRISPR sgRNAs designed to target amino acids 12 and 109 (SW780-sg12 and SW780-sg109) were used to introduce frameshifts and disrupt the majority of the peptide or only the PCNA the binding domain, respectively. sgRNA designed to target GFP was used as a control (sgGFP). We treated the cells with GI50 dose of cisplatin and analyzed post-cisplatin cell-cycle profile over 96 hours using PI-FACS and immunoblot. We treated all three cell types with increasing doses (0-40 μM) of cisplatin for 24 hours and measured the levels of ub-PCNA (K164) and Pol η using immunoblot. Results SW780-sgGFP arrested in S-phase at 48 and 72 hours but overcame this arrest at 96 hours and began to cycle again. SW780-sg12 were unable to arrest in S-phase, whereas SW780-sg109 were able to arrest in S-phase at 48 to 96 hours. This pattern of S-phase arrest was further confirmed by prolonged upregulation of cyclin D1 in SW780-sg109 and brief upregulation in SW780-sgGFP. ub-PCNA and Pol η accumulated in a dose responsive manner in all three cell types at 24 hours after cisplatin treatment. ub-PCNA increased abruptly at higher cisplatin doses in SW780-sgGFP, whereas the increase was gradual in CDKN1A-edited cells. Interestingly, in SW780-sg109, Pol η induction was strongest at all doses. Conclusion Cisplatin causes S-phase arrest in SW780-sgGFP cells, possibly by inducing replicative stress. However, this arrest is eventually bypassed and the cells progress through the cell cycle. S-phase arrest is not possible in the absence of a functioning p21, indicating that p21 plays a vital role in bringing about post-cisplatin cell cycle arrest. Interestingly, truncated p21 with intact CDKi domain is capable of causing stronger and more prolonged S-phase arrest compared to the full-length peptide. TLS may play a role in DNA damage tolerance in SW780 cells as indicated by elevated ub-PCNA and pol η. Stronger accumulation of Pol η in SW780-sg109, possibly enhances TLS and thus cisplatin resistance. Moving forward, we would like to confirm the anticipated competition between p21 and Pol η for PCNA and further verify this phenomenon in other bladder cancer cell lines. Citation Format: Rahmat K. Sikder, Wafik S. El-Deiry, Philip H. Abbosh. Differential effects of N-terminal vs C-terminal truncating CDKN1A mutations on cisplatin resistance in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2307.

Abstract 2356: Functional characterization of CDKN1A loss in bladder cancer and effects on cisplatin sensitivity

Abstract Introduction Muscle-invasive bladder cancer (MIBC) is most optimally treated with cisplatin-containing combination chemotherapy, and if clinically localized, bladder removal. With recent discoveries delineating aberrant driver genes involved in MIBC, it is now possible to personalize treatment approaches. Development of a therapeutic rationale for personalized therapies which is based on mutational landscape or other characteristic of each tumor may prospectively identify patients prone to chemoresponse, thus maximizing therapeutic index. Inactivating mutations in CDKN1A, the gene encoding cyclin dependent kinase inhibitor p21, occur in about 14% of MIBCs, with a majority of these truncating the peptide. We hypothesized that after DNA-damaging events, cells deficient in p21 would be unable to halt the cell cycle to repair the damage, and subsequently proceed down an apoptotic pathway, potentially serving as a therapeutic vulnerability. Methods RT4 and SW780 cell lines were chosen for study based on chemoresistance to cisplatin and WT status of CDKN1A and TP53. Cell-cycle arrest and apoptotic pathways were evaluated with Western blot after treatment with IC50 doses. CRISPR sgRNAs were generated to introduce frameshifts 5’ to the regions encoding the cyclin binding domain (CDI), within the CDI, and 5’ to the PCNA binding domains. Knock-outs were confirmed by immunoblot. MTT was used to assess cipslatin response. Results p53 increased in both cell lines 24 hours after a 3h pulse of cisplatin exposure. However, p53 peaked at 24 hours and then diminished in RT4 but continued to rise in SW780. p21 was basally expressed in both cell lines, decreased at 24 hours, then increasing at 72 hours. In RT4, peak Rb phosphorylation (pRb) occurred at 24 hours, indicating cell cycle arrest, and gradually decreased at 72 hours. In SW780 pRb remained unchanged during the course of the treatment. In RT4, PARP cleavage was observed at 24 hours and increased at 48 hours indicating apoptosis. RT4 cells harbored extensive DNA damage as indicated by persistent phosphorylated γH2AX. In contrast a smaller fraction of SW780 cells underwent PARP cleavage at 48 and 72 hours and there was less phosphorylated γH2AX. Single cell CDKN1A knockout clones were generated and confirmed by immunoblotting. sgRNAs targeting amino acids 12 and 59 completely abolished p21 detection, and an sgRNA targeting amino acid 109 generated a truncated peptide, confirming p21’s apparent stability. CDKN1A disruption at any of the three sites did not reverse chemorensitance to cisplatin in either cell line. Conclusion Cisplatin resistant cell lines use a combination of arrest and repair to survive an apoptotic insult. Although p21 loss did not reverse cisplatin resistance, it may impact sensitivity to other agents. Other mechanisms of chemo- and radio-sensitization are being explored. Citation Format: Philip H. Abbosh, Rahmat K. Sikder, Wafik S. El-Deiry. Functional characterization of CDKN1A loss in bladder cancer and effects on cisplatin sensitivity [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 2356. doi:10.1158/1538-7445.AM2017-2356

DNMT1 mutations found in HSANIE patients affect interaction with UHRF1 and neuronal differentiation

DNMT1 is recruited to substrate sites by PCNA and UHRF1 to maintain DNA methylation after replication. The cell cycle dependent recruitment of DNMT1 is mediated by the PCNA-binding domain (PBD) and the targeting sequence (TS) within the N-terminal regulatory domain. The TS domain was found to be mutated in patients suffering from hereditary sensory and autonomic neuropathies with dementia and hearing loss (HSANIE) and autosomal dominant cerebellar ataxia deafness and narcolepsy (ADCA-DN) and is associated with global hypomethylation and site specific hypermethylation. With functional complementation assays in mouse embryonic stem cells, we showed that DNMT1 mutations P496Y and Y500C identified in HSANIE patients not only impair DNMT1 heterochromatin association, but also UHRF1 interaction resulting in hypomethylation. Similar DNA methylation defects were observed when DNMT1 interacting domains in UHRF1, the UBL and the SRA domain, were deleted. With cell-based assays, we could show that HSANIE associated mutations perturb DNMT1 heterochromatin association and catalytic complex formation at methylation sites and decrease protein stability in late S and G2 phase. To investigate the neuronal phenotype of HSANIE mutations, we performed DNMT1 rescue assays and could show that cells expressing mutated DNMT1 were prone to apoptosis and failed to differentiate into neuronal lineage. Our results provide insights into the molecular basis of DNMT1 dysfunction in HSANIE patients and emphasize the importance of the TS domain in the regulation of DNA methylation in pluripotent and differentiating cells.

Distinct and overlapping DNMT1 interactions with multiple transcription factors in erythroid cells: Evidence for co-repressor functions

DNMT1 is the maintenance DNA methyltransferase shown to be essential for embryonic development and cellular growth and differentiation in many somatic tissues in mammals. Increasing evidence has also suggested a role for DNMT1 in repressing gene expression through interactions with specific transcription factors. Previously, we identified DNMT1 as an interacting partner of the TR2/TR4 nuclear receptor heterodimer in erythroid cells, implicated in the developmental silencing of fetal β-type globin genes in the adult stage of human erythropoiesis. Here, we extended this work by using a biotinylation tagging approach to characterize DNMT1 protein complexes in mouse erythroleukemic cells. We identified novel DNMT1 interactions with several hematopoietic transcription factors with essential roles in erythroid differentiation, including GATA1, GFI-1b and FOG-1. We provide evidence for DNMT1 forming distinct protein subcomplexes with specific transcription factors and propose the existence of a “core” DNMT1 complex with the transcription factors ZBP-89 and ZNF143, which is also present in non-hematopoietic cells. Furthermore, we identified the short (17a.a.) PCNA Binding Domain (PBD) located near the N-terminus of DNMT1 as being necessary for mediating interactions with the transcription factors described herein. Lastly, we provide evidence for DNMT1 serving as a co-repressor of ZBP-89 and GATA1 acting through upstream regulatory elements of the PU.1 and GATA1 gene loci.

Dual Functions of the RFTS Domain of Dnmt1 in Replication-Coupled DNA Methylation and in Protection of the Genome from Aberrant Methylation

In mammals, DNA methylation plays important roles in embryogenesis and terminal differentiation via regulation of the transcription-competent chromatin state. The methylation patterns are propagated to the next generation during replication by maintenance DNA methyltransferase, Dnmt1, in co-operation with Uhrf1. In the N-terminal regulatory region, Dnmt1 contains proliferating cell nuclear antigen (PCNA)-binding and replication foci targeting sequence (RFTS) domains, which are thought to contribute to maintenance methylation during replication. To determine the contributions of the N-terminal regulatory domains to the DNA methylation during replication, Dnmt1 lacking the RFTS and/or PCNA-binding domains was ectopically expressed in embryonic stem cells, and then the effects were analyzed. Deletion of both the PCNA-binding and RFTS domains did not significantly affect the global DNA methylation level. However, replication-dependent DNA methylation of the differentially methylated regions of three imprinted genes, Kcnq1ot1/Lit1, Peg3, and Rasgrf1, was impaired in cells expressing the Dnmt1 with not the PCNA-binding domain alone but both the PCNA-binding and RFTS domains deleted. Even in the absence of Uhrf1, which is a prerequisite factor for maintenance DNA methylation, Dnmt1 with both the domains deleted apparently maintained the global DNA methylation level, whilst the wild type and the forms containing the RFTS domain could not perform global DNA methylation under the conditions used. This apparent maintenance of the global DNA methylation level by the Dnmt1 lacking the RFTS domain was dependent on its own DNA methylation activity as well as the presence of de novo-type DNA methyltransferases. We concluded that the RFTS domain, not the PCNA-binding domain, is solely responsible for the replication-coupled DNA methylation. Furthermore, the RFTS domain acts as a safety lock by protecting the genome from replication-independent DNA methylation.

Beckwith-Wiedemann and IMAGe syndromes: two very different diseases caused by mutations on the same gene.

Genomic imprinting is an epigenetically regulated mechanism leading to parental-origin allele-specific expression. Beckwith–Wiedemann syndrome (BWS) is an imprinting disease related to 11p15.5 genetic and epigenetic alterations, among them loss-of-function CDKN1C mutations. Intriguing is that CDKN1C gain-of-function variations were recently found in patients with IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, congenital adrenal hypoplasia, and genital anomalies). BWS and IMAGe share an imprinted mode of inheritance; familial analysis demonstrated the presence of the phenotype exclusively when the mutant CDKN1C allele is inherited from the mother. Interestingly, both IMAGe and BWS are characterized by growth disturbances, although with opposite clinical phenotypes; IMAGe patients display growth restriction whereas BWS patients display overgrowth. CDKN1C codifies for CDKN1C/KIP2, a nuclear protein and potent tight-binding inhibitor of several cyclin/Cdk complexes, playing a role in maintenance of the nonproliferative state of cells. The mirror phenotype of BWS and IMAGe can be, at least in part, explained by the effect of mutations on protein functions. All the IMAGe-associated mutations are clustered in the proliferating cell nuclear antigen-binding domain of CDKN1C and cause a dramatic increase in the stability of the protein, which probably results in a functional gain of growth inhibition properties. In contrast, BWS mutations are not clustered within a single domain, are loss-of-function, and promote cell proliferation. CDKN1C is an example of allelic heterogeneity associated with opposite syndromes.

IMAGe syndrome in the era of genetic testing: clues to diagnosis

Abstract IMAGe syndrome (Intrauterine growth restriction (IUGR), Metaphyseal dysplasia, Adrenal hypoplasia congenita, and Genital anomalies) is a rare, multisystem disorder caused by mutations in the PCNA-binding domain of CDKN1C. Reported here is a male infant diagnosed with IMAGe syndrome by CDKN1C sequencing at 3 months of age. He presented with IUGR, primary adrenal insufficiency with adrenal crisis in the neonatal period, dysmorphic facies, and bilateral cryptorchidism. Interestingly, he demonstrates several additional clinical findings not previously reported with IMAGe syndrome including congenital hypothyroidism, recurrent bacterial infections, and severe eczema.

Identification of two functional PCNA-binding domains in human DNA polymerase κ.

Previously, we have shown that human DNA polymerase (Pol) η has two functional PCNA-binding motifs, PIP1 and PIP2, and that a C-terminal deletion of Polη that lacks the ubiquitin-binding UBZ domain and the PIP2 domain but retains the PIP1 domain promotes normal levels of translesion synthesis (TLS) opposite a cis-syn TT dimer in human cells. Here, we identify two PIP domains in Polκ and show that TLS occurs normally in human fibroblast cells in which the pip1 or pip2 mutant Polκ is expressed, but mutational inactivation of both PIP domains renders Polκ nonfunctional in TLS opposite the thymine glycol lesion. Thus, the two PIP domains of Polκ function redundantly in TLS opposite this DNA lesion in human cells. However, and surprisingly, whereas mutational inactivation of the PIP1 domain completely inhibits the stimulation of DNA synthesis by Polκ in the presence of proliferating cell nuclear antigen (PCNA), replication factor C, and replication protein A, mutations in PIP2 have no adverse effect on PCNA-dependent DNA synthesis. This raises the possibility that activation of Polκ PIP2 as a PCNA-binding domain occurs during TLS in human cells and that protein-protein interactions and post-transcriptional modifications are involved in such activation.

The PCNA binding domain of Rad2p plays a role in mutagenesis by modulating the cell cycle in response to DNA damage

The xeroderma pigmentosum group G (XPG) gene, encoding an essential element in nucleotide excision repair (NER), has a proliferating cell nuclear antigen-binding domain (PCNA-BD) at its C-terminal region. However, the role of this domain is controversial because its presence does not affect NER. Using yeast RAD2, a homolog of human XPG, we show that Rad2p interacts with PCNA through its PCNA-BD and the PCNA-BD of Rad2p plays a role in UV-induced mutagenesis. While a mutation of Rad2p endonuclease activity alone causes dramatically increased mutation rates and UV sensitivity, as well as growth retardation after UV irradiation, a mutation of the Rad2p PCNA-BD in the same mutant causes dramatically decreased mutation rates, reduced UV sensitivity and increased growth rate after UV irradiation. After UV irradiation, large-budded cells of Rad2p endonuclease defective mutants wane due to a mutation of the Rad2p PCNA-BD. Besides, the Rad2p PCNA-BD mutant protein exhibits alleviated PCNA-binding efficiency. These results show a hitherto unsuspected role of the Rad2p PCNA-BD that controls mutagenesis via cell cycle modulation together with PCNA. Furthermore, the high mutation rate of cells with other NER gene mutations was also decreased by the mutation of the Rad2p PCNA-BD, which indicates that the Rad2p-PCNA interaction might be responsible for mutagenesis control in the general NER pathway. Our results suggest that the drastically increased incidence of skin cancer in xeroderma pigmentosum patients could arise from the synergistic effects between cell cycle arrest due to the XPG-PCNA interaction and the accumulation of damaged DNA via defects in DNA damage repair.

CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome

BackgroundRussell Silver syndrome (RSS) leads to prenatal and postnatal growth retardation. About 55% of RSS patients present a loss-of-methylation of the paternal ICR1 domain on chromosome 11p15. CDKN1C is a...

Abstract 1281: POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells.

Abstract Methyl methanesulfonate (MMS) induces mutations in a nonlinear (hockey-stick-shaped) dose-response curve in DT40 cells, about 55% of which are located at A:T sites with a high frequency of A to T mutations. We hypothesized that these transversion mutations are due to base excision repair (BER) intermediates, such as abasic sites and single strand breaks derived from N3-methyladenine. One candidate polymerase to bypass abasic sites is polymerase delta (POLD). POLD is responsible for the lagging strand during DNA replication. POLD is referred to as a high fidelity DNA polymerase because of its ability to discriminate deoxynucleotides and its 3’->5’ exo-nuclease activity. One of the POLD subunits, POLD3, is important to recruit PCNA binding to the POLD core enzyme and is implied to have critical roles for translesion DNA synthesis. In yeast, cells deficient in POLD3 homologous gene POL32 are sensitive to DNA damaging agents and resistant to mutations caused by mutagens, such as MMS and UV. Here we generated POLD3 knock-down (KD) cells via lentivirus based shRNA in three different human cell lines. All POLD3 KD cells grow slower than the control cells, suggesting POLD3 function tolerates endogenous DNA lesions. Furthermore, we found that POLD3 KD cells were sensitive to various genotoxic agents including MMS and potassium chromate. To further investigate the role of POLD3 in the DNA damage response, we introduced a series of deletion mutations of POLD3 in POLD3 deficient cells. We found that both the POLD2 binding domain and the middle linker domain are critical for the activity of POLD3 while the C-terminal PCNA binding domain is dispensable. While there is little homology between human POLD3 and yeast POL32, function of these two proteins appears to be quite similar in structure biology analysis. Our results also suggest that POLD3 plays important roles to bypass DNA lesions caused by endogenous and various exogenous genotoxic agents. Citation Format: Xu Tian, James Swenberg, Jun Nakamura. POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1281. doi:10.1158/1538-7445.AM2013-1281

Dissection of cell cycle–dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling

DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle–dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.

Molecular Cloning, Expression, and Characterization of Starfish DNA (Cytosine-5)-methyltransferases

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.