Focal structural variants revealed by whole genome sequencing disrupt the histone demethylase KDM4C in B-cell lymphomas.

Histone methylation-modifiers, such as EZH2 and KMT2D, are recurrently altered in B-cell lymphomas. To comprehensively describe the landscape of alterations affecting genes encoding histone methylation-modifiers in lymphomagenesis we investigated whole genome and transcriptome data of 186 mature B-cell lymphomas sequenced in the ICGC MMML-Seq project. Besides confirming common alterations of KMT2D (47% of cases), EZH2 (17%), SETD1B (5%), PRDM9 (4%), KMT2C (4%), and SETD2 (4%), also identified by prior exome or RNA-sequencing studies, we here found recurrent alterations to KDM4C in chromosome 9p24, encoding a histone demethylase. Focal structural variation was the main mechanism of KDM4C alterations, and was independent from 9p24 amplification. We also identified KDM4C alterations in lymphoma cell lines including a focal homozygous deletion in a classical Hodgkin lymphoma cell line. By integrating RNA-sequencing and genome sequencing data we predict that KDM4C structural variants result in loss-offunction. By functional reconstitution studies in cell lines, we provide evidence that KDM4C can act as a tumor suppressor. Thus, we show that identification of structural variants in whole genome sequencing data adds to the comprehensive description of the mutational landscape of lymphomas and, moreover, establish KDM4C as a putative tumor suppressive gene recurrently altered in subsets of B-cell derived lymphomas.

The histone acetyltransferase inhibitor Nir regulates epidermis development.

In addition to its function as an inhibitor of histone acetyltransferases, Nir (Noc2l) binds to p53 and TAp63 to regulate their activity. Here, we show that epidermis-specific ablation of Nir impairs epidermal stratification and barrier function, resulting in perinatal lethality. Nir-deficient epidermis lacks appendages and remains single layered during embryogenesis. Cell proliferation is inhibited, whereas apoptosis and p53 acetylation are increased, indicating that Nir is controlling cell proliferation by limiting p53 acetylation. Transcriptome analysis revealed that Nir regulates the expression of essential factors in epidermis development, such as keratins, integrins and laminins. Furthermore, Nir binds to and controls the expression of p63 and limits H3K18ac at the p63 promoter. Corroborating the stratification defects, asymmetric cell divisions were virtually absent in Nir-deficient mice, suggesting that Nir is required for correct mitotic spindle orientation. In summary, our data define Nir as a key regulator of skin development.

Phosphorylation of histone H3T6 by PKCbeta(I) controls demethylation at histone H3K4.

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression.

Posttranslational modifications of histones, such as methylation, regulate chromatin structure and gene expression. Recently, lysine-specific demethylase 1 (LSD1), the first histone demethylase, was identified. LSD1 interacts with the androgen receptor and promotes androgen-dependent transcription of target genes by ligand-induced demethylation of mono- and dimethylated histone H3 at Lys 9 (H3K9) only. Here, we identify the Jumonji C (JMJC) domain-containing protein JMJD2C as the first histone tridemethylase regulating androgen receptor function. JMJD2C interacts with androgen receptor in vitro and in vivo. Assembly of ligand-bound androgen receptor and JMJD2C on androgen receptor-target genes results in demethylation of trimethyl H3K9 and in stimulation of androgen receptor-dependent transcription. Conversely, knockdown of JMJD2C inhibits androgen-induced removal of trimethyl H3K9, transcriptional activation and tumour cell proliferation. Importantly, JMJD2C colocalizes with androgen receptor and LSD1 in normal prostate and in prostate carcinomas. JMJD2C and LSD1 interact and both demethylases cooperatively stimulate androgen receptor-dependent gene transcription. In addition, androgen receptor, JMJD2C and LSD1 assemble on chromatin to remove methyl groups from mono, di and trimethylated H3K9. Thus, our data suggest that specific gene regulation requires the assembly and coordinate action of demethylases with distinct substrate specificities.

Deficiency in the LIM-only protein Fhl2 impairs skin wound healing.

After skin wounding, the repair process is initiated by the release of growth factors, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as fibroblast invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359-369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. 21:736-748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of alpha-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription.

Gene regulation in eukaryotes requires the coordinate interaction of chromatin-modulating proteins with specific transcription factors such as the androgen receptor. Gene activation and repression is specifically regulated by histone methylation status at distinct lysine residues. Here we show that lysine-specific demethylase 1 (LSD1; also known as BHC110) co-localizes with the androgen receptor in normal human prostate and prostate tumour. LSD1 interacts with androgen receptor in vitro and in vivo, and stimulates androgen-receptor-dependent transcription. Conversely, knockdown of LSD1 protein levels abrogates androgen-induced transcriptional activation and cell proliferation. Chromatin immunoprecipitation analyses demonstrate that androgen receptor and LSD1 form chromatin-associated complexes in a ligand-dependent manner. LSD1 relieves repressive histone marks by demethylation of histone H3 at lysine 9 (H3-K9), thereby leading to de-repression of androgen receptor target genes. Furthermore, we identify pargyline as an inhibitor of LSD1. Pargyline blocks demethylation of H3-K9 by LSD1 and consequently androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions. Here, we link demethylation of a repressive histone mark with androgen-receptor-dependent gene activation, thus providing a mechanism by which demethylases control specific gene expression.

The MIA system for protein import into the mitochondrial intermembrane space.

When thinking of the mitochondrial intermembrane space we envisage a small compartment that is bordered by the mitochondrial outer and inner membranes. Despite this somewhat simplified perception the intermembrane space has remained a central focus in mitochondrial biology. This compartment accommodates many proteinaceous factors that play critical roles in mitochondrial and cellular metabolism, including the regulation of programmed cell death and energy conversion. The mechanism by which intermembrane space proteins are transported into the organelle and folded remained largely unknown until recently. In pursuit of the answer to this question a novel machinery, the Mitochondrial Intermembrane Space Assembly machinery, exploiting a unique regulated thiol-disulfide exchange mechanism has been revealed. This exciting discovery has not only put in place novel concepts for the biogenesis of intermembrane space precursors but also raises important implications on the mechanisms involved in the generation and transfer of disulfide bonds.

FHL2 inhibits the activated osteoclast in a TRAF6-dependent manner.

TNF receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-kappaB (RANK). This event is central to normal osteoclastogenesis. We discovered that TRAF6 also interacts with FHL2 (four and a half LIM domain 2), a LIM domain--only protein that functions as a transcriptional coactivator or corepressor in a cell-type--specific manner. FHL2 mRNA and protein are undetectable in marrow macrophages and increase pari passu with osteoclast differentiation in vitro. FHL2 inhibits TRAF6-induced NF-kappaB activity in wild-type osteoclast precursors and, in keeping with its role as a suppressor of TRAF6-mediated RANK signaling, TRAF6/RANK association is enhanced in FHL2-/- osteoclasts. FHL2 overexpression delays RANK ligand-induced (RANKL-induced) osteoclast formation and cytoskeletal organization. Interestingly, osteoclast-residing FHL2 is not detectable in naive wild-type mice, in vivo, but is abundant in those treated with RANKL and following induction of inflammatory arthritis. Reflecting increased RANKL sensitivity, osteoclasts generated from FHL2-/- mice reach maturation and optimally organize their cytoskeleton earlier than their wild-type counterparts. As a consequence, FHL2-/- osteoclasts are hyperresorptive, and mice lacking the protein undergo enhanced RANKL and inflammatory arthritis-stimulated bone loss. FHL2 is, therefore, an antiosteoclastogenic molecule exerting its effect by attenuating TRAF6-mediated RANK signaling.

Extracellular signal-regulated kinase 2 interacts with and is negatively regulated by the LIM-only protein FHL2 in cardiomyocytes.

The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling.

Lsd1 Ablation Triggers Metabolic Reprogramming of Brown Adipose Tissue.

Previous work indicated that lysine-specific demethylase 1 (Lsd1) can positively regulate the oxidative and thermogenic capacities of white and beige adipocytes. Here we investigate the role of Lsd1 in brown adipose tissue (BAT) and find that BAT-selective Lsd1 ablation induces a shift from oxidative to glycolytic metabolism. This shift is associated with downregulation of BAT-specific and upregulation of white adipose tissue (WAT)-selective gene expression. This results in the accumulation of di- and triacylglycerides and culminates in a profound whitening of BAT in aged Lsd1-deficient mice. Further studies show that Lsd1 maintains BAT properties via a dual role. It activates BAT-selective gene expression in concert with the transcription factor Nrf1 and represses WAT-selective genes through recruitment of the CoREST complex. In conclusion, our data uncover Lsd1 as a key regulator of gene expression and metabolic function in BAT.

Focal adhesion kinase interacts with the transcriptional coactivator FHL2 and both are overexpressed in epithelial ovarian cancer.

Abnormal signal transduction arising from integrins and protein tyrosine kinases has been implicated in the initiation and progression of a variety of human cancers. Integrin-mediated signal transduction pathways require regulated cytoplasmic protein-protein interactions. However, little is known about integrin-associated proteins and ovarian cancer. In our study we investigated the association of pp125FAK, a cytoplasmic tyrosine kinase, involved in anchorage-independent growth of tumor cells, and the Four and a Half LIM domain (FHL) protein FHL2, which was recently shown to interact with integrins. Our data demonstrated that pp125FAK and FHL2 form a protein complex in human ovarian carcinoma. Furthermore, we showed that pp125FAK is overexpressed in epithelial ovarian cancer, but virtually absent in normal ovary. Our immunohistochemistry data showed that FHL2 protein expression is also augmented in epithelial ovarian cancer. Taken together, our results demonstrated for the first time FHL2 expression in human ovarian cancer cells, suggesting an important functional role of pp125FAK and FHL2 complex in gynecologic malignancies.

PRK1/PKN1 controls migration and metastasis of androgen-independent prostate cancer cells.

The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer.

Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane.

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport-associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of ß-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import ß-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of ß-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane ß-barrel proteins.

The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins.

The mitochondrial outer membrane contains translocase complexes for the import of precursor proteins. The translocase of the outer membrane complex functions as a general preprotein entry gate, whereas the sorting and assembly machinery complex mediates membrane insertion of ß-barrel proteins of the outer membrane. Several α-helical outer membrane proteins are known to carry multiple transmembrane segments; however, only limited information is available on the biogenesis of these proteins. We report that mitochondria lacking the mitochondrial import protein 1 (Mim1) are impaired in the biogenesis of multispanning outer membrane proteins, whereas overexpression of Mim1 stimulates their import. The Mim1 complex cooperates with the receptor Tom70 in binding of precursor proteins and promotes their insertion and assembly into the outer membrane. We conclude that the Mim1 complex plays a central role in the import of α-helical outer membrane proteins with multiple transmembrane segments.

Dual role of mitofilin in mitochondrial membrane organization and protein biogenesis.

The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We report that mitofilin plays a dual role. Mitofilin is part of a large inner membrane complex, and we identify five partner proteins as constituents of the mitochondrial inner membrane organizing system (MINOS) that is required for keeping cristae membranes connected to the inner boundary membrane. Additionally, mitofilin is coupled to the outer membrane and promotes protein import via the mitochondrial intermembrane space assembly pathway. Our findings indicate that mitofilin is a central component of MINOS and functions as a multifunctional regulator of mitochondrial architecture and protein biogenesis.

Identification of the signal directing Tim9 and Tim10 into the intermembrane space of mitochondria.

The intermembrane space of mitochondria contains the specific mitochondrial intermembrane space assembly (MIA) machinery that operates in the biogenesis pathway of precursor proteins destined to this compartment. The Mia40 component of the MIA pathway functions as a receptor and binds incoming precursors, forming an essential early intermediate in the biogenesis of intermembrane space proteins. The elements that are crucial for the association of the intermembrane space precursors with Mia40 have not been determined. In this study, we found that a region within the Tim9 and Tim10 precursors, consisting of only nine amino acid residues, functions as a signal for the engagement of substrate proteins with the Mia40 receptor. Furthermore, the signal contains sufficient information to facilitate the transfer of proteins across the outer membrane to the intermembrane space. Thus, here we have identified the mitochondrial intermembrane space sorting signal required for delivery of proteins to the mitochondrial intermembrane space.

Precursor oxidation by Mia40 and Erv1 promotes vectorial transport of proteins into the mitochondrial intermembrane space.

The mitochondrial intermembrane space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential intermembrane space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of intermembrane space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. Whereas the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the intermembrane space. Thus, oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial intermembrane space.

Mitochondrial biogenesis, switching the sorting pathway of the intermembrane space receptor Mia40.

Mitochondrial precursor proteins are directed into the intermembrane space via two different routes, the presequence pathway and the redox-dependent MIA pathway. The pathways were assumed to be independent and transport different proteins. We report that the intermembrane space receptor Mia40 can switch between both pathways. In fungi, Mia40 is synthesized as large protein with an N-terminal presequence, whereas in metazoans and plants, Mia40 consists only of the conserved C-terminal domain. Human MIA40 and the C-terminal domain of yeast Mia40 (termed Mia40(core)) rescued the viability of Mia40-deficient yeast independently of the presence of a presequence. Purified Mia40(core) was imported into mitochondria via the MIA pathway. With cells expressing both full-length Mia40 and Mia40(core), we demonstrate that yeast Mia40 contains dual targeting information, directing the large precursor onto the presequence pathway and the smaller Mia40(core) onto the MIA pathway, raising interesting implications for the evolution of mitochondrial protein sorting.

Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase.

The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.

Expression of the transcriptional coregulator FHL2 in human breast cancer: a clinicopathologic study.

OBJECTIVE: Although the Four and a Half LIM domain protein 2 (FHL2) has been suggested to play an important role in tumor development, this has not been investigated in breast cancer. METHODS: Paraffin-embedded tissues from patients (n = 85) with primary breast cancer were submitted to immunohistochemical investigation of FHL2 expression and subsequent correlation with clinicopathologic parameters and patient survival. RESULTS: The expression of FHL2 was confined to the cytoplasm of the tumor cells. Forty (47%) of 85 samples showed weak expression of FHL2, whereas high expression was found in 45 tumors (53%). A statistically significant positive correlation was observed between FHL2 and androgen receptor expression (P = .029). Patients with tumors expressing low amounts of FHL2 were characterized by a significantly better survival compared to those with high intratumoral FHL2 expression (P = .0215, log-rank test). The additional stratification according to adjuvant tamoxifen treatment revealed a significantly improved survival rate for patients receiving tamoxifen and being diagnosed with a tumor expressing high amounts of FHL2. This might indicate that tamoxifen is at least partially capable of reversing the negative prognostic impact of high FHL2 expression. Multivariate Cox regression analysis revealed FHL2 expression as a significant independent predictor of survival. CONCLUSION: The specific expression in tumor tissue points to an important functional role of FHL2 in human breast cancer. Our survival data indicate that the expression of FHL2 in primary breast cancer is a potentially relevant prognostic factor. Further studies are warranted to elucidate whether analysis of FHL2 expression is suitable to predict response to antihormonal treatment with tamoxifen.