The Prolyl Isomerase Pin1 Affects Che-1 Stability in Response to Apoptotic DNA Damage

We have previously demonstrated that DNA damage leads to stabilization and accumulation of Che-1, an RNA polymerase II-binding protein that plays an important role in transcriptional activation of p53 and in maintenance of the G(2)/M checkpoint. Here we show that Che-1 is down-regulated during the apoptotic process. We found that the E3 ligase HMD2 physically and functionally interacts with Che-1 and promotes its degradation via the ubiquitin-dependent proteasomal system. Furthermore, we found that in response to apoptotic stimuli Che-1 interacts with the peptidyl-prolyl isomerase Pin1 and that conformational changes generated by Pin1 are required for Che-1/HDM2 interaction. Notably, a Che-1 mutant lacking the capacity to bind Pin1 exhibits an increased half-life and this correlates with a diminished apoptosis in response to genotoxic stress. Our results establish Che-1 as a new Pin1 and HDM2 target and confirm its important role in the cellular response to DNA damage

Che-1 arrests human colon carcinoma cell proliferation by displacing HDAC1 from the p21WAF1/CIP1 promoter.

Che-1 is a recently identified human RNA polymerase II binding protein involved in the regulation of gene transcription and cell proliferation. We previously demonstrated that Che-1 inhibits the Rb growth-suppressing function by interfering with Rb-mediated HDAC1 recruitment on E2F target gene promoters. By hybridization of cancer profile arrays, we found that Che-1 expression is strongly down-regulated in several tumors, including colon and kidney carcinomas, compared with the relative normal tissues. Consistent with these data, Che-1 overexpression inhibits proliferation of HCT116 and LoVo human colon carcinoma cell lines by activation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 in a p53-independent manner and by promoting growth arrest at the G1 phase of the cell cycle. Che-1 activates p21WAF1/Cip1 by displacing histone deacetylase (HDAC)1 from the Sp1 binding sites of the p21WAF1/Cip1 gene promoter and accumulating acetylated histone H3 on these sites. Accordingly, Che-1-specific RNA interference negatively affects p21WAF1/Cip1 transactivation and increases cell proliferation in HCT116 cells. Taken together, our results indicate that Che-1 can be considered a general HDAC1 competitor and its down-regulation is involved in colon carcinoma cell proliferation

Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice

Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter “A”. Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics

The RNA polymerase II core subunit 11 interacts with keratin 19, a component of the intermediate filament proteins

AbstractWe have previously cloned the human RNA polymerase II subunit 11, as a doxorubicin sensitive gene product. We suggested multiple tasks for this subunit, including structural and regulatory roles. With the aim to clarify the human RNA polymerase II subunit 11 function, we have identified its interacting protein partners using the yeast two-hybrid system. Here, we show that human RNA polymerase II subunit 11 specifically binds keratin 19, a component of the intermediate filament protein family, which is expressed in a tissue and differentiation-specific manner. In particular, keratin 19 is a part of the nuclear matrix intermediate filaments. We provide evidence that human RNA polymerase II subunit 11 interacts with keratin 19 via its N-terminal α motif, the same motif necessary for its interaction with the human RNA polymerase II core subunit 3. We found that keratin 19 contains two putative leucine zipper domains sharing peculiar homology with the α motif of human RNA polymerase II subunit 3. Finally, we demonstrate that keratin 19 can compete for binding human RNA polymerase II subunit 11/human RNA polymerase II subunit 3 in vitro, suggesting a possible regulatory role for this molecule in RNA polymerase II assembly/activity

Functional interaction of the subunit 3 of RNA polymerase II (RPB3) with transcription factor-4 (ATF4)

AbstractRPB3 is a core subunit of RNA polymerase II (pol II) that, together with the RPB11 subunit, forms the heterodimer considered as a functional counterpart of the bacterial α subunit homodimer involved in promoter recognition. We previously employed the yeast two-hybrid system and identified an interaction between RPB3 and the myogenic transcription factor myogenin, demonstrating an involvement of this subunit in muscle differentiation. In this paper we report the interaction between RPB3 and another known transcription factor, ATF4. We found that the intensity of the interaction between RPB3 and ATF4 is similar to the one between RPB3 and myogenin. This interaction involves an RPB3 specific region not homologous to the prokaryotic α subunit. We demonstrated that RBP3 is able to enhance ATF4 transactivation, whereas the region of RPB3 (Sud) that contacts ATF4, when used as a dominant negative, markedly inhibits ATF4 transactivation activity. Interestingly, ATF4 protein level, as reported for its partner RPB3, increases during C2C7 cell line muscle differentiation

Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1α stabilization

BACKGROUND: Solid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia. METHODS: The human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses. RESULTS: Here we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism. CONCLUSIONS: We show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13046-017-0497-1) contains supplementary material, which is available to authorized users