Hironobu Morinaga

A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts

Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, δ that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, δ induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. δ treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching “” the complex transcriptional gene network.

Synthetic small molecules for epigenetic activation of pluripotency genes in mouse embryonic fibroblasts.

Considering the essential role of chromatin remodeling in gene regulation, their directed modulation is of increasing importance. To achieve gene activation by epigenetic modification, we synthesized a series of pyrrole–imidazole polyamide conjugates (PIPs) that can bind to predetermined DNA sequences, and attached them with suberoylanilide hydroxamic acid (SAHA), a potent histone deacetylase inhibitor. As histone modification is associated with pluripotency, these new types of conjugates, termed SAHA–PIPs, were screened for their effect on the expression of induced pluripotent stem cell (iPSC) factors. We found certain SAHA–PIPs that could differentially up‐regulate the endogenous expression of Oct‐3/4, Nanog, Sox2, Klf4 and c‐Myc. SAHA and other SAHA–PIPs did not show such induction; this implies a role for PIPs and their sequence specificity in this differential gene activation. Chromatin immunoprecipitation analysis suggested that SAHA–PIP‐mediated gene induction proceeds by histone H3 Lys9 and Lys14 acetylation and Lys4 trimethylation, which are epigenetic features associated with transcriptionally active chromatin.

Purification and characterization of Caenorhabditis elegans NTH, a homolog of human endonuclease III: essential role of N-terminal region.

Oxidatively damaged bases in DNA cause many types of deleterious effects. The main enzyme that removes such lesions is DNA glycosylase, and accordingly, DNA glycosylase plays an important role in genome stability. Recently, a relationship between DNA glycosylases and aging has been suggested, but it remains controversial. Here, we investigated DNA glycosylases of C. elegans, which is a useful model organism for studying aging. We firstly identified a C. elegans homolog of endonuclease III (NTH), which is a well-conserved DNA glycosylase for oxidatively damaged pyrimidine bases, based on the activity and homology. Blast searching of the Wormbase database retrieved a sequence R10E4.5, highly homologous to the human NTH1. However, the R10E4.5-encoded protein did not have NTH activity, and this was considered to be due to lack of the N-terminal region crucial for the activity. Therefore, we purified the protein encoded by the sequence containing both R10E4.5 and the 117-bp region upstream from it, and found that the protein had the NTH activity. The endogenous CeNTH in the extract of C. elegans showed the same DNA glycosylase activity. Therefore, we concluded that the genuine C. elegans NTH gene is not the R10E4.5 but the sequence containing both R10E4.5 and the 117-bp upstream region. NTH-deficient C. elegans showed no difference from the wild-type in lifespan and was not more sensitive to two oxidizing agents, H2O2 and methyl viologen. This suggests that C. elegans has an alternative DNA glycosylase that repairs pyrimidine bases damaged by these agents. Indeed, DNA glycosylase activity that cleaved thymine glycol containing oligonucleotides was detected in the extract of the NTH-deficient C. elegans.

Cysteine cyclic pyrrole-imidazole polyamide for sequence-specific recognition in the DNA minor groove.

Pyrrole-imidazole (PI) polyamides are small DNA-binding molecules that can recognize predetermined DNA sequences with high affinity and specificity. Hairpin PI polyamides have been studied intensively; however, cyclic PI polyamides have received less attention, mainly because of difficulties with their synthesis. Here, we describe a novel cyclization method for producing PI polyamides using cysteine and a chloroacetyl residue. The cyclization reaction is complete within 1 h and has a high conversion efficiency. The method can be used to produce long cyclic PI polyamides that can recognize 7 bp DNA sequences. A cyclic PI polyamide containing two β-alanine molecules had higher affinity and specificity than the corresponding hairpin PI polyamide, demonstrating that the cyclic PI polyamides can be used as a new type of DNA-binding molecule.

Cloning and characterization of uracil-DNA glycosylase and the biological consequences of the loss of its function in the nematode Caenorhabditis elegans

Uracil arises in DNA from spontaneous deamination of cytosine and through incorporation of dUMP by DNA polymerase during DNA replication. Excision of uracil by the action of uracil-DNA glycosylase (Ung) initiates the base excision repair pathway to counter the promutagenic base modification. In this study, we cloned a cDNA-encoding Caenorhabditis elegans homologue (CeUng-1) of Escherichia coli Ung. There was 49% identity in amino acid sequence between E.coli Ung and CeUng-1. Purified CeUng-1 removed uracil from both U:G and U:A base pairs in DNA. It also removed uracil from single-stranded oligonucleotide substrate less efficiently than double-stranded oligonucleotide. The CeUng-1 activity was inhibited by Bacillus subtilis Ung inhibitor, indicating that CeUng-1 is a member of the family-1 Ung group. The mutation in the ung-1 gene did not affect development, fertility and lifespan in C.elegans, suggesting the existence of backup enzyme. However, we could not detect residual uracil excision activity in the extract derived from the ung-1 mutant. The present experiments also showed that the ung-1 mutant of C.elegans was more resistant to NaHSO(3)-inducing cytosine deamination than wild-type strain.

Hydrochlorothiazide Enhances UVA-Induced DNA Damage

The UVA is currently thought to be carcinogenic because, similar to UVB, it induces the formation of cyclobutane pyrimidine dimers (CPDs). Various drugs have been reported to cause photosensitive drug eruptions as an adverse effect. Although the precise mechanism of photosensitive drug eruption remains to be elucidated, it is generally accepted that free radicals and other reactive molecules generated via UV‐irradiated drugs play important roles in the pathogenesis of photosensitive drug eruptions. The waveband of concern for photo‐reactive drugs is UVA‐visible light, but some extend into the UVB region. We tested whether photosensitive drugs could enhance CPD formation after UVA exposure by using isolated DNA in the presence of several reported photosensitive drugs using high‐performance liquid chromatography. We found that the diuretic agent hydrochlorothiazide (HCT) significantly enhanced the production of TT dimers over a wide range of UVA. Furthermore, we investigated whether UVA plus HCT could enhance CPD production in xeroderma pigmentosum model mice defective in nucleotide excision repair. Immunofluorescence studies showed that CPD formation in the skin significantly increased after 365 nm narrow‐band UVA irradiation in the presence of HCT, compared with that in wild‐type mice. HCT could be used with caution because of its enhancement of UVA‐induced DNA damage.

Sequence-specific electron injection into DNA from an intermolecular electron donor

Electron transfer in DNA has been intensively studied to elucidate its biological roles and for applications in bottom-up DNA nanotechnology. Recently, mechanisms of electron transfer to DNA have been investigated; however, most of the systems designed are intramolecular. Here, we synthesized pyrene-conjugated pyrrole-imidazole polyamides (PPIs) to achieve sequence-specific electron injection into DNA in an intermolecular fashion. Electron injection from PPIs into DNA was detected using 5-bromouracil as an electron acceptor. Twelve different 5-bromouracil-containing oligomers were synthesized to examine the electron-injection ability of PPI. Product analysis demonstrated that the electron transfer from PPIs was localized in a range of 8 bp from the binding site of the PPIs. These results demonstrate that PPIs can be a useful tool for sequence-specific electron injection.

KsgA, a 16S rRNA adenine methyltransferase, has a novel DNA glycosylase/ AP lyase activity to prevent mutations in Escherichia coli

The 5-formyluracil (5-foU), a major mutagenic oxidative damage of thymine, is removed from DNA by Nth, Nei and MutM in Escherichia coli. However, DNA polymerases can also replicate past the 5-foU by incorporating C and G opposite the lesion, although the mechanism of correction of the incorporated bases is still unknown. In this study, using a borohydride-trapping assay, we identified a protein trapped by a 5-foU/C-containing oligonucleotide in an extract from E. coli mutM nth nei mutant. The protein was subsequently purified from the E. coli mutM nth nei mutant and was identified as KsgA, a 16S rRNA adenine methyltransferase. Recombinant KsgA also formed the trapped complex with 5-foU/C- and thymine glycol (Tg)/C-containing oligonucleotides. Furthermore, KsgA excised C opposite 5-foU, Tg and 5-hydroxymethyluracil (5-hmU) from duplex oligonucleotides via a β-elimination reaction, whereas it could not remove the damaged base. In contrast, KsgA did not remove C opposite normal bases, 7,8-dihydro-8-oxoguanine and 2-hydroxyadenine. Finally, the introduction of the ksgA mutation increased spontaneous mutations in E. coli mutM mutY and nth nei mutants. These results demonstrate that KsgA has a novel DNA glycosylase/AP lyase activity for C mispaired with oxidized T that prevents the formation of mutations, which is in addition to its known rRNA adenine methyltransferase activity essential for ribosome biogenesis.

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole-imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence-specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence-specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β-alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.

Construction and characterization of Cy3- or Cy5-conjugated hairpin pyrrole–imidazole polyamides binding to DNA in the nucleosome

Sequence-specific DNA-binding modules, N-methylpyrrole (Py)-N-methylimidazole-(Im) polyamides have been recently conjugated with fluorophores, and some of these conjugates could be used for the detection of specific DNA sequences. In this study, we synthesized two Py-Im polyamides 1 and 2, which interact with the 145-bp nucleosome positioning sequence 601. We conjugated the cyanine dye Cy3 or Cy5 with 1 or 2. In the absence of target DNA, the fluorescent conjugate of a Py-Im polyamide had lower fluorescence intensity compared with Cy3 or Cy5 alone. In the presence of either the target DNA or the nucleosome, the fluorescence intensity of the conjugates increased. Furthermore, we observed a Förster resonance energy transfer between the Cy3-Py-Im polyamide and the Cy5-Py-Im polyamide on the nucleosome. These results open up the possibilities that fluorescent conjugates of Py-Im polyamides can be used for characterization of the dynamic interactions within protein-DNA complexes.