Structural basis of transcription regulation by CNC family transcription factor, Nrf2.

Several basic leucine zipper (bZIP) transcription factors have accessory motifs in their DNA-binding domains, such as the CNC motif of CNC family or the EHR motif of small Maf (sMaf) proteins. CNC family proteins heterodimerize with sMaf proteins to recognize CNC-sMaf binding DNA elements (CsMBEs) in competition with sMaf homodimers, but the functional role of the CNC motif remains elusive. In this study, we report the crystal structures of Nrf2/NFE2L2, a CNC family protein regulating anti-stress transcriptional responses, in a complex with MafG and CsMBE. The CNC motif restricts the conformations of crucial Arg residues in the basic region, which form extensive contact with the DNA backbone phosphates. Accordingly, the Nrf2-MafG heterodimer has approximately a 200-fold stronger affinity for CsMBE than canonical bZIP proteins, such as AP-1 proteins. The high DNA affinity of the CNC-sMaf heterodimer may allow it to compete with the sMaf homodimer on target genes without being perturbed by other low-affinity bZIP proteins with similar sequence specificity.

Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2.

Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

Knockdown of E2F5 induces cell death via the TP53­dependent pathway in breast cancer cells carrying wild­type TP53.

E2F transcription factor 5 (E2F5) is a member of the E2F family of transcription factors, which are involved in regulation of various cellular processes, including cellular proliferation, apoptosis, differentiation and DNA damage response. Previously, we reported that E2F5 was aberrantly overexpressed in estrogen receptor (ER)­negative breast cancer, especially in triple­negative breast cancer (TNBC). In the present study, it was revealed that E2F5 gene silencing caused a significant reduction in the proliferation rate of breast cancer MCF7 (ER­positive luminal­type) and MDA­MB­231 (TNBC­type) cells. Additional experiments demonstrated that E2F5 knockdown triggered cell death of MCF7 cells but not MDA­MB­231 cells. As MCF7 and MDA­MB­231 cells carry wild­type and mutant TP53, respectively, and BT474 (ER­negative, HER2­positive type) carrying mutant TP53 exhibited similar results to MDA­MB­231, the possible effects of E2F5 gene depletion on cell death­related TP53­target gene expression were examined. Real­time RT­qPCR analysis revealed that knockdown of E2F5 in MCF7 cells stimulated cell death­related transcription of TP53­target genes such as BAX, NOXA and PUMA. For MDA­MB­231 and BT474 cells, E2F5 gene silencing revealed marginal effects on the expression of TP53 target genes. In addition, silencing of TP53 abrogated the effect of E2F5 silencing in MCF7 cells. Collectively, the present results indicated that E2F5 participated in the carcinogenesis of breast cancer carrying wild­type TP53 through suppression of TP53, while E2F5 had a pro­proliferative but not anti­apoptotic effect on breast cancer with TP53 mutation.

Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole-imidazole polyamide, which targets an E-box motif.

Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors. In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.