Yoh-ichi Kawabe

A specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory

DNA demethylation plays a critical role in transcriptional regulation in differentiated somatic cells. However, there is no experimental evidence that CpG methylation in a small region of a genome restricts gene expression. Here, we show that the anti‐CD3ε/CD28 antibody stimulation of human CD4+ T cells induces IL2 expression following epigenetic changes, including active demethylation of a specific CpG site, recruitment of Oct‐1, and changes in histone modifications. When the stimulatory signal is withdrawn, Oct‐1 remains on the enhancer region as a stable marker of the stimulation, causing the second induction to be faster and stronger. Our observations indicate that Oct‐1‐binding followed by CpG demethylation are key events in the epigenetic regulation of IL2 expression and may act as a memory of the regulatory event.

The ubiquitin ligase CHIP acts as an upstream regulator of oncogenic pathways.

CHIP is a U-box-type ubiquitin ligase that induces ubiquitylation and degradation of its substrates, which include several oncogenic proteins. The relationship between CHIP and tumour progression, however, has not been elucidated. Here, we show that CHIP suppresses tumour progression in human breast cancer by inhibiting oncogenic pathways. CHIP levels were negatively correlated with the malignancy of human breast tumour tissues. In a nude mouse xenograft model, tumour growth and metastasis were significantly inhibited by CHIP expression. In contrast, knockdown of CHIP (shCHIP) in breast cancer cells resulted in rapid tumour growth and metastastic phenotypes in mice. In cell-based experiments, anchorage-independent growth and invasiveness of shCHIP cells was significantly elevated due to increased expression of Bcl2, Akt1, Smad and Twist. Proteomic analysis identified the transcriptional co-activator SRC-3 (refs 13, 14, 15, 16, 17, 18, 19) as a direct target for ubiquitylation and degradation by CHIP. Knocking down SRC-3 in shCHIP cells reduced the expression of Smad and Twist, and suppressed tumour metastasis in vivo. Conversely, SRC-3 co-expression prevented CHIP-induced suppression of metastasis formation. These observations demonstrate that CHIP inhibits anchorage-independent cell growth and metastatic potential by degrading oncogenic proteins including SRC-3.

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor.

Recent evidence indicates that the transactivation of estrogen receptor α (ERα) requires estrogen‐dependent receptor ubiquitination and degradation. Here we show that estrogen‐unbound (unliganded) ERα is also ubiquitinated and degraded through a ubiquitin–proteasome pathway. To investigate this ubiquitin–proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERα and identified a protein complex containing the carboxyl terminus of Hsc70‐interacting protein (CHIP). CHIP preferentially bound to misfolded ERα and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERα. In CHIP−/− cells, the degradation of unliganded ERα was abrogated; however, estrogen‐induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERα is regulated by two independent ubiquitin–proteasome pathways, which are switched by ligand binding to ERα. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.

Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation.

ABSTRACT Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1−/− , RECQL5−/− , RECQL1−/− /RECQL5−/− , RECQL1−/− /BLM−/− , and RECQL5−/− /BLM−/− cells from chicken B-lymphocyte line DT40 cells. Although BLM−/− DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an ∼10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1−/− , RECQL5−/− , and RECQL1−/− /RECQL5−/− cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1−/− /BLM−/− and RECQL5−/− /BLM−/− cells grew more slowly than BLM−/− cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5−/− /BLM−/− cells showed a higher frequency of SCE than BLM−/− cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.

Turning Off Estrogen Receptor β-Mediated Transcription Requires Estrogen-Dependent Receptor Proteolysis

ABSTRACT Recent studies have shed light on the ligand-dependent transactivation mechanisms of nuclear receptors (NRs). When the ligand dose is reduced, the transcriptional activity of NRs should be downregulated. Here we show that a ubiquitin-proteasome pathway plays a key role in turning off transcription mediated by estrogen receptor β (ERβ). ERβ shows estrogen-dependent proteolysis, and its degradation is regulated by two regions in the receptor. The N-terminal 37-amino acid-region is necessary for the recruitment of the ubiquitin ligase, i.e., the carboxyl terminus of HSC70-interacting protein (CHIP), to degrade ERβ. In contrast, the C-terminal F domain protects ligand-unbound ERβ from proteolysis to abrogate proteasome association. Suppression of CHIP by interfering RNA inhibited this switching off of receptor-mediated transcription when the ligand dose was reduced. Our results suggest that after ligand withdrawal, the active form of the NR is selectively eliminated via ligand-dependent proteolysis to downregulate receptor-mediated transcription.

A Novel Protein Interacts with the Werner's Syndrome Gene Product Physically and Functionally

Werners syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Wernerhelicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells andsgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1disruptants, respectively. In contrast, disruption of theyWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.

Characterization of the slow-growth phenotype of S. cerevisiae whip/mgs1 sgs1 double deletion mutants

RecQ DNA helicases from many organisms have been indicated to function in the maintenance of genomic stability. In human cells, mutation in the WRN helicase, a RecQ-like DNA helicase, results in the Werner syndrome (WS), a genetic disorder characterized by genomic instability and premature ageing. Similarly, mutation in SGS1, the RECQ homologue in budding yeast, results in genomic instability and accelerated ageing. We previously demonstrated that mouse WRN interacts physically with a novel, highly conserved protein that we named WHIP, and that in budding yeast cells, simultaneous deletion of WHIP/MGS1 and SGS1 results in slow growth and shortened life span. Here we show by using genetic analysis in Saccharomyces cerevisiae that mgs1Delta sgs1Delta cells have increased rates of terminal G2/M arrest, and show elevated rates of spontaneous sister chromatid recombination (SCR) and rDNA array recombination. Finally, we report that complementation of the synthetic relationship between SGS1 and WHIP/MGS1 requires both the helicase and Top3-binding activities of Sgs1, as well as the ATPase activity of Mgs1. Our results suggest that Whip/Mgs1 is implicated in DNA metabolism, and is required for normal growth and cell cycle progression in the absence of Sgs1.

The E3 Ubiquitin Ligase Activity of Trip12 Is Essential for Mouse Embryogenesis

Protein ubiquitination is a post-translational protein modification that regulates many biological conditions [1], [2], [3], [4]. Trip12 is a HECT-type E3 ubiquitin ligase that ubiquitinates ARF and APP-BP1 [5], [6]. However, the significance of Trip12 in vivo is largely unknown. Here we show that the ubiquitin ligase activity of Trip12 is indispensable for mouse embryogenesis. A homozygous mutation in Trip12 (Trip12mt/mt) that disrupts the ubiquitin ligase activity resulted in embryonic lethality in the middle stage of development. Trip12mt/mt embryos exhibited growth arrest and increased expression of the negative cell cycle regulator p16 [7], [8], [9], [10]. In contrast, Trip12mt/mt ES cells were viable. They had decreased proliferation, but maintained both the undifferentiated state and the ability to differentiate. Trip12mt/mt ES cells had increased levels of the BAF57 protein (a component of the SWI/SNF chromatin remodeling complex) and altered gene expression patterns. These data suggest that Trip12 is involved in global gene expression and plays an important role in mouse development.