Cornelia Steinhauer

Histone Acetyltransferase PCAF Is Required for Hedgehog- Gli-Dependent Transcription and Cancer Cell Proliferation

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of aberrant Hh-Gli signaling has, thus, emerged as an attractive approach for anticancer therapy; however, the mechanisms that mediate Hh-Gli signaling in vertebrates remain poorly understood. Here, we show that the histone acetyltransferase PCAF/KAT2B is an important factor of the Hh pathway. Specifically, we show that PCAF depletion impairs Hh activity and reduces expression of Hh target genes. Consequently, PCAF downregulation in medulloblastoma and glioblastoma cells leads to decreased proliferation and increased apoptosis. In addition, we found that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Finally, we observed that PCAF silencing reduces the tumor-forming potential of neural stem cells in vivo. In summary, our study identified the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, leading us to propose PCAF as a candidate therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.

HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin

In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate UbG76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB+1. Precipitation experiments revealed that HUWE1 is associated with both the UbG76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.

A Screen Identifies the Oncogenic Micro-RNA miR-378a-5p as a Negative Regulator of Oncogene-Induced Senescence

Oncogene-induced senescence (OIS) can occur in response to hyperactive oncogenic signals and is believed to be a fail-safe mechanism protecting against tumorigenesis. To identify new factors involved in OIS, we performed a screen for microRNAs that can overcome or inhibit OIS in human diploid fibroblasts. This screen led to the identification of miR-378a-5p and in addition several other miRNAs that have previously been shown to play a role in senescence. We show that ectopic expression of miR-378a-5p reduces the expression of several senescence markers, including p16INK4A and senescence-associated β-galactosidase. Moreover, cells with ectopic expression of miR-378a-5p retain proliferative capacity even in the presence of an activated Braf oncogene. Finally, we identified several miR-378a-5p targets in diploid fibroblasts that might explain the mechanism by which the microRNA can delay OIS. We speculate that miR-378a-5p might positively influence tumor formation by delaying OIS, which is consistent with a known pro-oncogenic function of this microRNA.

Structure-Based Design of a New Scaffold for Cell-Penetrating Peptidic Inhibitors of the Histone Demethylase PHF8

The histone demethylase PHF8 catalyzes demethylation of mono‐ and di‐methylated Lys9 on histone H3 (H3K9me1/2), and is a transcriptional activator involved in the development and cancer. Affinity and specificity of PHF8 towards H3K9me2 is affected by interaction with both the catalytic domain and a PHD reader domain. The latter specifically recognizes tri‐methylated Ly4 on histone H3. A fragment of the histone H3 tail with tri‐methylated Lys4 was used as a template for the structure‐based design of a cyclic, cell‐penetrating peptide that exhibits micromolar binding affinity to PHF8 in biochemical assays. The inhibitor has significantly lower affinity towards KDM2 enzymes (the phylogenetically closest subfamily), and to KDM3 and KDM6 subfamilies. Selectivity is only marginal towards an enzyme from the KDM4 family, which shares histone tail specificity with PHF8. It is a substrate of KDM5B, thus implying that the free N terminus is not part of the KDM5 enzyme substrate recognition machinery. The cyclic peptides ability to penetrate cells is achieved by incorporation of a sequence derived from HIV Tat. The derived cyclic peptide can be used as a starting compound in the search for potent and selective PHF8 inhibitors.

High-Throughput siRNA Screening Applied to the Ubiquitin–Proteasome System

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Due to the large number of genes dedicated to the ubiquitin-proteasome system, mapping degradation pathways for short lived proteins is a daunting task, in particular in mammalian cells that are not genetically tractable as, for instance, a yeast model system. Here, we describe a method relying on high-throughput cellular imaging of cells transfected with a targeted siRNA library to screen for components involved in degradation of a protein of interest. This method is a rapid and cost-effective tool which is also highly applicable for other studies on gene function.

Abstract A19: The histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and proliferation

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of an aberrant Hh-Gli signaling has thus emerged as an attractive target for anticancer therapy, however the mechanisms that mediate Hh-Gli signaling in vertebrates remain still poorly understood. Here, we show that the histone acetyltransferase (HAT) PCAF/KAT2B is an important regulator of the Hh pathway. Specifically, we demonstrate that PCAF depletion leads to an impairment of Hh activity and reduced expression of Hh target genes. Consequently, downregulation of PCAF in medulloblastoma and glioblastoma cell lines leads to decreased cell proliferation and increased apoptosis. Furthermore, we show that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Our study identifies the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, and we propose PCAF as a potential interesting novel therapeutic target for the treatment of patients with medulloblastoma and glioblastoma. Citation Format: Martina Malatesta, Cornelia Steinhauer, Massimo Squatrito, Kristian Helin. The histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and proliferation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A19.

High-throughput proteomics on antibody-based microarrays: the importance of probe and surface design

Scandinavian Society for Immunology 35th Annual Meeting and 20th Summer School Aarhus, Denmark, June 13–16, 2004 The following workshops will be running at the 35th Annual meeting of The Scandinavian Society for Immunology in Aarhus, Denmark, June 13–16, 2004. The number given to the abstracts in each workshop does not reflect the order of presentation. Monday: Autoimmunity Infection and immunity Stimulus and response Tuesday: Hypersensitivity Commensals and immunity Complement Wednesday: Tumourimmunology Immunotechnology