TSPYL1 as a Critical Regulator of TGFß Signaling through Repression of TGFBR1 and TSPYL2.

Nucleosome assembly proteins (NAPs) have been identified as histone chaperons. Testis-Specific Protein, Y-Encoded-Like (TSPYL) is a newly arisen NAP family in mammals. TSPYL2 can be transcriptionally induced by DNA damage and TGFß causing proliferation arrest. TSPYL1, another TSPYL family member, has been poorly characterized and is the only TSPYL family member known to be causal of a lethal recessive disease in humans. This study shows that TSPYL1 and TSPYL2 play an opposite role in TGFß signaling. TSPYL1 partners with the transcription factor FOXA1 and histone methyltransferase EZH2, and at the same time represses TGFBR1 and epithelial-mesenchymal transition (EMT). Depletion of TSPYL1 increases TGFBR1 expression, upregulates TGFß signaling, and elevates the protein stability of TSPYL2. Intriguingly, TSPYL2 forms part of the SMAD2/3/4 signal transduction complex upon stimulation by TGFß to execute the transcriptional responses. Depletion of TSPYL2 rescues the EMT phenotype of TSPYL1 knockdown in A549 lung carcinoma cells. The data demonstrates the prime role of TSPYL2 in causing the dramatic defects in TSPYL1 deficiency. An intricate counter-balancing role of TSPYL1 and TSPYL2 in regulating TGFß signaling is also unraveled.

Sex specific regulation of TSPY-Like 2 in the DNA damage response of cancer cells.

Females have a lower probability to develop somatic cancers and a better response to chemotherapy than males. However, the reasons for these differences are still not well understood. The X-linked gene TSPY-Like 2 (TSPYL2) encodes for a putative tumor suppressor protein involved in cell cycle regulation and DNA damage response (DDR) pathways. Here, we demonstrate that in unstressed conditions TSPYL2 is maintained at low levels by MDM2-dependent ubiquitination and proteasome degradation. Upon genotoxic stress, E2F1 promotes TSPYL2 expression and protein accumulation in non-transformed cell lines. Conversely, in cancer cells, TSPYL2 accumulates only in females or in those male cancer cells that lost the Y-chromosome during the oncogenic process. Hence, we demonstrate that while TSPYL2 mRNA is induced in all the tested tumor cell lines after DNA damage, TSPYL2 protein stability is increased only in female cancer cells. Indeed, we found that TSPYL2 accumulation, in male cancer cells, is prevented by the Y-encoded protein SRY, which modulates MDM2 protein levels. In addition, we demonstrated that TSPYL2 accumulation is required to sustain cell growth arrest after DNA damage, possibly contributing to protect normal and female cancer cells from tumor progression. Accordingly, TSPYL2 has been found more frequently mutated in female-specific cancers. These findings demonstrate for the first time a sex-specific regulation of TSPYL2 in the DDR of cancer cells and confirm the existence of sexual dimorphism in DNA surveillance pathways.

The elevated transcription of ADAM19 by the oncohistone H2BE76K contributes to oncogenic properties in breast cancer.

The recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect.

TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage.

Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.

TSPYL2 Regulates the Expression of EZH2 Target Genes in Neurons.

Testis-specific protein, Y-encoded-like 2 (TSPYL2) is an X-linked gene in the locus for several neurodevelopmental disorders. We have previously shown that Tspyl2 knockout mice had impaired learning and sensorimotor gating, and TSPYL2 facilitates the expression of Grin2a and Grin2b through interaction with CREB-binding protein. To identify other genes regulated by TSPYL2, here, we showed that Tspyl2 knockout mice had an increased level of H3K27 trimethylation (H3K27me3) in the hippocampus, and TSPYL2 interacted with the H3K27 methyltransferase enhancer of zeste 2 (EZH2). We performed chromatin immunoprecipitation (ChIP)-sequencing in primary hippocampal neurons and divided all Refseq genes by k-mean clustering into four clusters from highest level of H3K27me3 to unmarked. We confirmed that mutant neurons had an increased level of H3K27me3 in cluster 1 genes, which consist of known EZH2 target genes important in development. We detected significantly reduced expression of genes including Gbx2 and Prss16 from cluster 1 and Acvrl1, Bdnf, Egr3, Grin2c, and Igf1 from cluster 2 in the mutant. In support of a dynamic role of EZH2 in repressing marked synaptic genes, the specific EZH2 inhibitor GSK126 significantly upregulated, while the demethylase inhibitor GSKJ4 downregulated the expression of Egr3 and Grin2c. GSK126 also upregulated the expression of Bdnf in mutant primary neurons. Finally, ChIP showed that hemagglutinin-tagged TSPYL2 co-existed with EZH2 in target promoters in neuroblastoma cells. Taken together, our data suggest that TSPYL2 is recruited to promoters of specific EZH2 target genes in neurons, and enhances their expression for proper neuronal maturation and function.

Cysteine (C)-x-C receptor 4 undergoes transportin 1-dependent nuclear localization and remains functional at the nucleus of metastatic prostate cancer cells.

The G-protein coupled receptor (GPCR), Cysteine (C)-X-C Receptor 4 (CXCR4), plays an important role in prostate cancer metastasis. CXCR4 is generally regarded as a plasma membrane receptor where it transmits signals that support transformation, progression and eventual metastasis. Due to the central role of CXCR4 in tumorigenesis, therapeutics approaches such as antagonist and monoclonal antibodies have focused on receptors that exist on the plasma membrane. An emerging concept for G-protein coupled receptors is that they may localize to and associate with the nucleus where they retain function and mediate nuclear signaling. Herein, we demonstrate that CXCR4 associated with the nucleus of malignant prostate cancer tissues. Likewise, expression of CXCR4 was detected in nuclear fractions among several prostate cancer cell lines, compared to normal prostate epithelial cells. Our studies identified a nuclear pool of CXCR4 and we defined a nuclear transport pathway for CXCR4. We reveal a putative nuclear localization sequence (NLS), 'RPRK', within CXCR4 that contributed to nuclear localization. Additionally, nuclear CXCR4 interacted with Transportinß1 and Transportinß1-binding to CXCR4 promoted its nuclear translocation. Importantly, Gαi immunoprecipitation and calcium mobilization studies indicated that nuclear CXCR4 was functional and participated in G-protein signaling, revealing that the nuclear pool of CXCR4 retained function. Given the suggestion that functional, nuclear CXCR4 may be a mechanism underlying prostate cancer recurrence, increased metastatic ability and poorer prognosis after tumors have been treated with therapy that targets plasma membrane CXCR4, these studies addresses a novel mechanism of nuclear signaling for CXCR4, a novel mechanism of clinical targeting, and demonstrate an active nuclear pool that provides important new information to illuminate what has been primarily clinical reports of nuclear CXCR4.

TSPYL2 is important for G1 checkpoint maintenance upon DNA damage.

Nucleosome assembly proteins play important roles in chromatin remodeling, which determines gene expression, cell proliferation and terminal differentiation. Testis specific protein, Y-encoded-like 2 (TSPYL2) is a nucleosome assembly protein expressed in neuronal precursors and mature neurons. Previous studies have shown that TSPYL2 binds cyclin B and inhibits cell proliferation in cultured cells suggesting a role in cell cycle regulation. To investigate the physiological significance of TSPYL2 in the control of cell cycle, we generated mice with targeted disruption of Tspyl2. These mutant mice appear grossly normal, have normal life span and do not exhibit increased tumor incidence. To define the role of TSPYL2 in DNA repair, checkpoint arrest and apoptosis, primary embryonic fibroblasts and thymocytes from Tspyl2 deficient mice were isolated and examined under unperturbed and stressed conditions. We show that mutant fibroblasts are impaired in G1 arrest under the situation of DNA damage induced by gamma irradiation. This is mainly attributed to the defective activation of p21 transcription despite proper p53 protein accumulation, suggesting that TSPYL2 is additionally required for p21 induction. TSPYL2 serves a biological role in maintaining the G1 checkpoint under stress condition.

Differential effects of glial cell line-derived neurotrophic factor and neurturin in RET/GFRalpha1-expressing cells.

The c-ret protooncogene, RET, encodes a receptor tyrosine kinase. RET is activated by members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands, which include GDNF, neurturin, artemin, and persephin. The ligands bind RET through GDNF family receptor alpha, termed GFRalpha1-4. Despite the importance of RET signaling in the development of the enteric nervous system and the kidney, the differential signaling mechanisms between RET ligands are poorly established. It has been suggested that signal specificity is achieved through binding of the ligand to its preferred GFRalpha. To compare the signaling profiles of GDNF and neurturin, we have identified a cell line, NG108-15, which endogenously expresses RET and GFRalpha1 but not GFRalpha2-4. Immunoblot data showed that GDNF caused a transient activation, whereas neurturin caused a sustained activation, of both p44/p42 MAP kinases and PLCgamma. Under serum starvation, NG108-15 cells differentiate and form neurites. Neurturin but not GDNF stimulated neurite outgrowth, which could be blocked by the selective PLC inhibitor U73122. On the other hand, GDNF but not neurturin promoted cell survival, and this could be blocked by the p44/p42 MAP kinase inhibitor PD98059. Our findings not only show the differential signaling of GDNF and neurturin but also suggest that this can be achieved through binding to the same GFRalpha subtype, leading to distinct biological responses.

Epidermal growth factor as a biologic switch in hair growth cycle.

The hair growth cycle consists of three stages known as the anagen (growing), catagen (involution), and telogen (resting) phases. This cyclical growth of hair is regulated by a diversity of growth factors. Although normal expression of both epidermal growth factor and its receptor (EGFR) in the outer root sheath is down-regulated with the completion of follicular growth, here we show that continuous expression of epidermal growth factor in hair follicles of transgenic mice arrested follicular development at the final stage of morphogenesis. Data from immunoprecipitation and immunoblotting showed that epidermal growth factor signals through EGFR/ErbB2 heterodimers in skin. Furthermore, topical application of tyrphostin AG1478 or AG825, specific inhibitors of EGFR and ErbB2, respectively, completely inhibited new hair growth in wild type mice but not in transgenic mice. When the transgenic mice were crossed with waved-2 mice, which possess a lower kinase activity of EGFR, the hair phenotype was rescued in the offspring. Taken together, these data suggest that EGFR signaling is indispensable for the initiation of hair growth. On the other hand, continuous expression of epidermal growth factor prevents entry into the catagen phase. We propose that epidermal growth factor functions as a biologic switch that is turned on and off in hair follicles at the beginning and end of the anagen phase of the hair cycle, guarding the entry to and exit from the anagen phase.

RET receptor tyrosine kinase isoforms in kidney function and disease.

The RET proto-oncogene encodes two major isoforms, RET9 and RET51, which differ at the carboxyl-terminal. Loss-of-function mutations in RET result in gut aganglionosis while gain of function mutations result in cancer syndromes. From studies on transgenic mice, RET9 is important for early development of the kidney and the enteric nervous system. Little is known about the function of RET isoforms in later life. Here we report the expression of RET isoforms and its signalling complex, GDNF and GFRalpha1, in foetal and adult human kidneys. We found their expression in both the developing and the adult renal collecting system. We further show that only RET51 but not RET9 could promote the survival and tubulogenesis of mIMCD3 (mouse inner medullary collecting duct) cells in collagen gel. Our results agree with the hypothesis that RET51 signalling is related to differentiation events in later kidney organogenesis. In addition, it may also have a function in the adult kidney. We further extend our study by showing increased RET and GDNF expression in collecting duct cysts of polycystic kidney patients. This suggests that GDNF/RET signalling may contribute to proliferation of the collecting duct epithelium in an autocrine/paracrine manner.