Tyrosyl-DNA phosphodiesterases are involved in mutagenic events at a ribonucleotide embedded into DNA in human cells.

Ribonucleoside triphosphates are often incorporated into genomic DNA during DNA replication. The accumulation of unrepaired ribonucleotides is associated with genomic instability, which is mediated by DNA topoisomerase 1 (Top1) processing of embedded ribonucleotides. The cleavage initiated by Top1 at the site of a ribonucleotide leads to the formation of a Top1-DNA cleavage complex (Top1cc), occasionally resulting in a DNA double-strand break (DSB). In humans, tyrosyl-DNA phosphodiesterases (TDPs) are essential repair enzymes that resolve the trapped Top1cc followed by downstream repair factors. However, there is limited cellular evidence of the involvement of TDPs in the processing of incorporated ribonucleotides in mammals. We assessed the role of TDPs in mutagenesis induced by a single ribonucleotide embedded into DNA. A supF shuttle vector site-specifically containing a single riboguanosine (rG) was introduced into the human lymphoblastoid TK6 cell line and its TDP1-, TDP2-, and TDP1/TDP2-deficient derivatives. TDP1 and TDP2 insufficiency remarkably decreased the mutant frequency caused by an embedded rG. The ratio of large deletion mutations induced by rG was also substantially lower in TDP1/TDP2-deficient cells than wild-type cells. Furthermore, the disruption of TDPs reduced the length of rG-mediated large deletion mutations. The recovery ratio of the propagated plasmid was also increased in TDP1/TDP2-deficient cells after the transfection of the shuttle vector containing rG. The results suggest that TDPs-mediated ribonucleotide processing cascade leads to unfavorable consequences, whereas in the absence of these repair factors, a more error-free processing pathway might function to suppress the ribonucleotide-induced mutagenesis. Furthermore, base substitution mutations at sites outside the position of rG were detected in the supF gene via a TDPs-independent mechanism. Overall, we provide new insights into the mechanism of mutagenesis induced by an embedded ribonucleotide in mammalian cells, which may lead to the fatal phenotype in the ribonucleotide excision repair deficiency.

Quantification of the effect of site-specific histone acetylation on chromatin transcription rate.

Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation ∼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.

Processing of a single ribonucleotide embedded into DNA by human nucleotide excision repair and DNA polymerase η.

DNA polymerases often incorporate non-canonical nucleotide, i.e., ribonucleoside triphosphates into the genomic DNA. Aberrant accumulation of ribonucleotides in the genome causes various cellular abnormalities. Here, we show the possible role of human nucleotide excision repair (NER) and DNA polymerase η (Pol η) in processing of a single ribonucleotide embedded into DNA. We found that the reconstituted NER system can excise the oxidized ribonucleotide on the plasmid DNA. Taken together with the evidence that Pol η accurately bypasses a ribonucleotide, i.e., riboguanosine (rG) or its oxidized derivative (8-oxo-rG) in vitro, we further assessed the mutagenic potential of the embedded ribonucleotide in human cells lacking NER or Pol η. A single rG on the supF reporter gene predominantly induced large deletion mutations. An embedded 8-oxo-rG caused base substitution mutations at the 3'-neighboring base rather than large deletions in wild-type cells. The disruption of XPA, an essential factor for NER, or Pol η leads to the increased mutant frequency of 8-oxo-rG. Furthermore, the frequency of 8-oxo-rG-mediated large deletions was increased by the loss of Pol η, but not XPA. Collectively, our results suggest that base oxidation of the embedded ribonucleotide enables processing of the ribonucleotide via alternative DNA repair and damage tolerance pathways.

Wolf-Hirschhorn Syndrome Candidate 1 Is Necessary for Correct Hematopoietic and B Cell Development.

Immunodeficiency is one of the most important causes of mortality associated with Wolf-Hirschhorn syndrome (WHS), a severe rare disease originated by a deletion in chromosome 4p. The WHS candidate 1 (WHSC1) gene has been proposed as one of the main genes responsible for many of the alterations in WHS, but its mechanism of action is still unknown. Here, we present in vivo genetic evidence showing that Whsc1 plays an important role at several points of hematopoietic development. Particularly, our results demonstrate that both differentiation and function of Whsc1-deficient B cells are impaired at several key developmental stages due to profound molecular defects affecting B cell lineage specification, commitment, fitness, and proliferation, demonstrating a causal role for WHSC1 in the immunodeficiency of WHS patients.

TBP-like Protein (TLP) Disrupts the p53-MDM2 Interaction and Induces Long-lasting p53 Activation.

Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions.

Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection.

BACKGROUND: Targeted gene modification by homologous recombination provides a powerful tool for studying gene function in cells and animals. In higher eukaryotes, non-homologous integration of targeting vectors occurs several orders of magnitude more frequently than does targeted integration, making the gene-targeting technology highly inefficient. For this reason, negative-selection strategies have been employed to reduce the number of drug-resistant clones associated with non-homologous vector integration, particularly when artificial nucleases to introduce a DNA break at the target site are unavailable or undesirable. As such, an exon-trap strategy using a promoterless drug-resistance marker gene provides an effective way to counterselect non-homologous integrants. However, constructing exon-trapping targeting vectors has been a time-consuming and complicated process. RESULTS: By virtue of highly efficient att-mediated recombination, we successfully developed a simple and rapid method to construct plasmid-based vectors that allow for exon-trapping gene targeting. These exon-trap vectors were useful in obtaining correctly targeted clones in mouse embryonic stem cells and human HT1080 cells. Most importantly, with the use of a conditionally cytotoxic gene, we further developed a novel strategy for negative selection, thereby enhancing the efficiency of counterselection for non-homologous integration of exon-trap vectors. CONCLUSIONS: Our methods will greatly facilitate exon-trapping gene-targeting technologies in mammalian cells, particularly when combined with the novel negative selection strategy.

TBP-like protein (TLP) interferes with Taspase1-mediated processing of TFIIA and represses TATA box gene expression.

TBP-TFIIA interaction is involved in the potentiation of TATA box-driven promoters. TFIIA activates transcription through stabilization of TATA box-bound TBP. The precursor of TFIIA is subjected to Taspase1-directed processing to generate α and ß subunits. Although this processing has been assumed to be required for the promoter activation function of TFIIA, little is known about how the processing is regulated. In this study, we found that TBP-like protein (TLP), which has the highest affinity to TFIIA among known proteins, affects Taspase1-driven processing of TFIIA. TLP interfered with TFIIA processing in vivo and in vitro, and direct binding of TLP to TFIIA was essential for inhibition of the processing. We also showed that TATA box promoters are specifically potentiated by processed TFIIA. Processed TFIIA, but not unprocessed TFIIA, associated with the TATA box. In a TLP-knocked-down condition, not only the amounts of TATA box-bound TFIIA but also those of chromatin-bound TBP were significantly increased, resulting in the stimulation of TATA box-mediated gene expression. Consequently, we suggest that TLP works as a negative regulator of the TFIIA processing and represses TFIIA-governed and TATA-dependent gene expression through preventing TFIIA maturation.

Auditory hair cell defects as potential cause for sensorineural deafness in Wolf-Hirschhorn syndrome.

WHSC1 is a histone methyltransferase (HMT) that catalyses the addition of methyl groups to lysine 36 on histone 3. In humans, WHSC1 haploinsufficiency is associated with all known cases of Wolf-Hirschhorn syndrome (WHS). The cardinal feature of WHS is a craniofacial dysmorphism, which is accompanied by sensorineural hearing loss in 15% of individuals with WHS. Here, we show that WHSC1-deficient mice display craniofacial defects that overlap with WHS, including cochlea anomalies. Although auditory hair cells are specified normally, their stereocilia hair bundles required for sound perception fail to develop the appropriate morphology. Furthermore, the orientation and cellular organisation of cochlear hair cells and their innervation are defective. These findings identify, for the first time, the likely cause of sensorineural hearing loss in individuals with WHS.

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

An epigenetic switch is crucial for spermatogonia to exit the undifferentiated state toward a Kit-positive identity.

Epigenetic modifications influence gene expression and chromatin remodeling. In embryonic pluripotent stem cells, these epigenetic modifications have been extensively characterized; by contrast, the epigenetic events of tissue-specific stem cells are poorly understood. Here, we define a new epigenetic shift that is crucial for differentiation of murine spermatogonia toward meiosis. We have exploited a property of incomplete cytokinesis, which causes male germ cells to form aligned chains of characteristic lengths, as they divide and differentiate. These chains revealed the stage of spermatogenesis, so the epigenetic differences of various stages could be characterized. Single, paired and medium chain-length spermatogonia not expressing Kit (a marker of differentiating spermatogonia) showed no expression of Dnmt3a2 and Dnmt3b (two de novo DNA methyltransferases); they also lacked the transcriptionally repressive histone modification H3K9me2. By contrast, spermatogonia consisting of ~8-16 chained cells with Kit expression dramatically upregulated Dnmt3a2/3b expression and also displayed increased H3K9me2 modification. To explore the function of these epigenetic changes in spermatogonia in vivo, the DNA methylation machinery was destabilized by ectopic Dnmt3b expression or Np95 ablation. Forced Dnmt3b expression induced expression of Kit; whereas ablation of Np95, which is essential for maintaining DNA methylation, interfered with differentiation and viability only after spermatogonia become Kit positive. These data suggest that the epigenetic status of spermatogonia shifts dramatically during the Kit-negative to Kit-positive transition. This shift might serve as a switch that determines whether spermatogonia self-renew or differentiate.

WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition.

Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1(-/-) cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways.

Impact of histone H4 lysine 20 methylation on 53BP1 responses to chromosomal double strand breaks.

Recruitment of 53BP1 to chromatin flanking double strand breaks (DSBs) requires γH2AX/MDC1/RNF8-dependent ubiquitination of chromatin and interaction of 53BP1 with histone H4 methylated on lysine 20 (H4K20me). Several histone methyltransferases have been implicated in 53BP1 recruitment, but their quantitative contributions to the 53BP1 response are unclear. We have developed a multi-photon laser (MPL) system to target DSBs to subfemtoliter nuclear volumes and used this to mathematically model DSB response kinetics of MDC1 and of 53BP1. In contrast to MDC1, which revealed first order kinetics, the 53BP1 MPL-DSB response is best fitted by a Gompertz growth function. The 53BP1 MPL response shows the expected dependency on MDC1 and RNF8. We determined the impact of altered H4K20 methylation on 53BP1 MPL response kinetics in mouse embryonic fibroblasts (MEFs) lacking key H4K20 histone methyltransferases. This revealed no major requirement for the known H4K20 dimethylases Suv4-20h1 and Suv4-20h2 in 53BP1 recruitment or DSB repair function, but a key role for the H4K20 monomethylase, PR-SET7. The histone methyltransferase MMSET/WHSC1 has recently been implicated in 53BP1 DSB recruitment. We found that WHSC1 homozygous mutant MEFs reveal an alteration in balance of H4K20 methylation patterns; however, 53BP1 DSB responses in these cells appear normal.

DNA methyltransferase 3b preferentially associates with condensed chromatin.

In mammals, DNA methylation is catalyzed by DNA methyltransferases (DNMTs) encoded by Dnmt1, Dnmt3a and Dnmt3b. Since, the mechanisms of regulation of Dnmts are still largely unknown, the physical interaction between Dnmt3b and chromatin was investigated in vivo and in vitro. In embryonic stem cell nuclei, Dnmt3b preferentially associated with histone H1-containing heterochromatin without any significant enrichment of silent-specific histone methylation. Recombinant Dnmt3b preferentially associated with nucleosomal DNA rather than naked DNA. Incorporation of histone H1 into nucleosomal arrays promoted the association of Dnmt3b with chromatin, whereas histone acetylation reduced Dnmt3b binding in vitro. In addition, Dnmt3b associated with histone deacetylase SirT1 in the nuclease resistant chromatin. These findings suggest that Dnmt3b is preferentially recruited into hypoacetylated and condensed chromatin. We propose that Dnmt3b is a 'reader' of higher-order chromatin structure leading to gene silencing through DNA methylation.

Histone methyltransferases: regulation of transcription and contribution to human disease.

Histone modifications contribute to the precise regulation of transcription by recruiting non-histone proteins and controlling chromatin conformation. These covalent modifications are dynamically regulated by many enzymes that modify histones at specific residues in different ways. Histone modifiers contribute to development as well as cellular responses to extracellular stimuli. Mutations in the genes encoding them cause various diseases, including developmental disorders and certain malignancies. Haploinsufficiency for some histone methyltransferases, one of the principal modifiers of the histone modification network, are associated with particular congenital diseases, including Sotos syndrome, Wolf-Hirschhorn syndrome, and 9q syndrome. In this review, we discuss the molecular function of the histone methyltransferases and the human diseases associated with their dysfunction.

A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syndrome.

Diverse histone modifications are catalysed and recognized by various specific proteins, establishing unique modification patterns that act as transcription signals. In particular, histone H3 trimethylation at lysine 36 (H3K36me3) is associated with actively transcribed regions and has been proposed to provide landmarks for continuing transcription; however, the control mechanisms and functions of H3K36me3 in higher eukaryotes are unknown. Here we show that the H3K36me3-specific histone methyltransferase (HMTase) Wolf-Hirschhorn syndrome candidate 1 (WHSC1, also known as NSD2 or MMSET) functions in transcriptional regulation together with developmental transcription factors whose defects overlap with the human disease Wolf-Hirschhorn syndrome (WHS). We found that mouse Whsc1, one of five putative Set2 homologues, governed H3K36me3 along euchromatin by associating with the cell-type-specific transcription factors Sall1, Sall4 and Nanog in embryonic stem cells, and Nkx2-5 in embryonic hearts, regulating the expression of their target genes. Whsc1-deficient mice showed growth retardation and various WHS-like midline defects, including congenital cardiovascular anomalies. The effects of Whsc1 haploinsufficiency were increased in Nkx2-5 heterozygous mutant hearts, indicating their functional link. We propose that WHSC1 functions together with developmental transcription factors to prevent the inappropriate transcription that can lead to various pathophysiologies.

Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression.

Sall4 is a mouse homolog of a causative gene of the autosomal dominant disorder Okihiro syndrome. We previously showed that the absence of Sall4 leads to lethality during peri-implantation and that Sall4-null embryonic stem (ES) cells proliferate poorly with intact pluripotency when cultured on feeder cells. Here, we report that, in the absence of feeder cells, Sall4-null ES cells express the trophectoderm marker Cdx2, but are maintained for a long period in an undifferentiated state with minimally affected Oct3/4 expression. Feeder-free Sall4-null ES cells contribute solely to the inner cell mass and epiblast in vivo, indicating that these cells still retain pluripotency and do not fully commit to the trophectoderm. These phenotypes could arise from derepression of the Cdx2 promoter, which is normally suppressed by Sall4 and the Mi2/NuRD HDAC complex. However, proliferation was impaired and G1 phase prolonged in the absence of Sall4, suggesting another role for Sall4 in cell cycle control. Although Sall1, also a Sall family gene, is known to genetically interact with Sall4 in vivo, Sall1-null ES cells have no apparent defects and no exacerbation is observed in ES cells lacking both Sall1 and Sall4, compared with Sall4-null cells. This suggests a unique role for Sall4 in ES cells. Thus, though Sall4 does not contribute to the central machinery of the pluripotency, it stabilizes ES cells by repressing aberrant trophectoderm gene expression.

Dynamic alterations of linker histone variants during development.

The process of development can be viewed as a series of linker histone replacements which take place throughout spermatogenesis and oogenesis, as well as following fertilization or somatic nuclear transfer (SNT). Although few of the histone H1 variants in question have been shown to be essential for viability, the timing of their appearance as well as the affinity with which they are able to bind to chromatin seem to be important factors in their developmental role. A looser binding of linker histones to chromatin seems to correlate with the meiotic phases of gametogenesis and the establishment of a totipotent, as well as the maintenance of a pluripotent, state in early embryos, while tighter binding of linker histones to chromatin appears to be associated with the mitotic phases, as well as the increased levels of condensation that are required for the packaging of DNA into sperm. This latter process also involves the binding of certain basic non-histone proteins to DNA. While all proteins involved in chromatin compaction during development are highly basic in nature, in general they can be seen to change from lysine-rich variants to arginine-rich ones, and back again. The fact that linker histone transitions are conserved across diverse metazoan species speaks of their importance in packaging DNA in a variety of ways during this crucial period.

Cracking the enigmatic linker histone code.

Recently, the existence of a 'histone code' has been proposed to explain the link between the covalent chemical modification of histone proteins and the epigenetic regulation of gene activity. Although the role of the four 'core' histones has been extensively studied, little is known about the involvement of the linker histone, histone H1 and its variants, in this code. For many years, few sites of chemical modification had been mapped in linker histones, but this has changed recently with the use of functional proteomic techniques, principally mass spectrometry, to characterize these modifications. The functionality of many of these sites, however, remains to be determined.

Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation.

DNA methylation is involved in fundamental cellular processes such as silencing of genes and transposable elements, but the underlying mechanism of regulation of DNA methylation is largely unknown. DNA methyltransferase 3-like protein (Dnmt3L), a member of the Dnmt3 family of proteins, is required during the establishment of DNA methylation patterns in germ cells. Dnmt3L does not possess enzymatic activity. Rather, in vitro analysis indicates that Dnmt3L stimulates DNA methylation by both Dnmt3a and Dnmt3b through direct binding to these proteins. In the current study, we demonstrated that in vivo, Dnmt3L physically and functionally interacted with the Dnmt3 isoform Dnmt3a2. In wild-type embryonic stem (ES) cells, but not in cells lacking Dnmt3a, endogenous Dnmt3L was concentrated in chromatin foci. In ES cells deficient in both Dnmt3a and Dnmt3b, Dnmt3L was distributed diffusely throughout the nucleus and cytoplasm, and ectopic expression of Dnmt3a2, but not Dnmt3a or Dnmt3b, restored wild-type Dnmt3L localization. We showed that endogenous Dnmt3L physically interacted with Dnmt3a2, but not Dnmt3a or Dnmt3b, in ES cells and embryonic testes. We also found that specific CpG sites were demethylated upon depletion of either Dnmt3a or Dnmt3L, but not Dnmt3b, in ES cells. These results provide evidence for a physical and functional interaction between Dnmt3L and Dnmt3a2 in the nucleus. We propose that Dnmt3a2 recruits Dnmt3L to chromatin, and induces regional DNA methylation in germ cells.