Targeting of proliferation-regulating genes in cancer cells
Many cancers including osteosarcomas arise from oncogenic transformation of cells of mesodermal origin. A protein that integrates multiple signaling pathways controlling cell cycle and cell motility is the nuclear A-kinase anchoring protein AKAP95. The aim of the project was to test the hypothesis that AKAP95 constitutes a relay platform processing information from signaling pathways involved in cell proliferation and gene expression control. AKAP95 harbours two Zn-fingers in regions involved in chromatin binding and chromatin condensation. We verified that AKAP95 binds DNA and an in vitro selection approach reveals that DNA bound to AKAP95 is enriched in CG dinucleotides reminiscent of CpG islands promoters. Therefore, we determined using a chromatin immunoprecipitation (ChIP) and genome-wide promoter array (ChIP-Chip) approach, promoters occupied by AKAP95 in U2OS cells. We tested the ability of several anti-AKAP95 antibodies to immunoprecipitate AKAP95 and found two antibodies showing efficient pulldown. Specificity of promoters targeted by AKAP95 was supported by the strong overlap of promoters bound by AKAP95 detected by ChIP with both antibodies. We found a total 1212 promoters bound by AKAP95, associated with genes involved in protein-DNA complex formation and chromatin assembly. We bioinformatically investigated the AKAP95-bound chromatin landscape using available ChIP-seq data sets for post-translational histone modifications. We find that AKAP95-bound genes are enriched in histone modifications of active promoters and transcribed genes (notably H3K4me3 and H3K36me3); supporting this finding, we find that AKAP95-bound genes are expressed. AKAP95 may thus play a role in the regulation of these genes perhaps by favouring anchoring of a transcription complex. Indeed, using a protein identification assay (BioID) based on the labeling of neighboring proteins by a biotinylating enzyme fused to AKAP95, we identified the FUS/TLS protein as a new AKAP95-interactor. FUS is a multifunctional protein involved in chromatin metabolism, including gene regulation. Among promoters co-occupied by AKAP95 and FUS, we identify a FUS binding pattern which strongly overlaps with AKAP95, suggesting that AKAP95 is implicated in FUS anchoring at these sites. Since FUS mutations trigger familial cases of major neurodegenerative diseases (amyotrophic lateral sclerosis; ALS) and frontal temporal degeneration; FTD), further work will be done in a neuronal system in order to elucidate the role of AKAP95 in FUS anchoring to its target promoters, and in regulation of expression of FUS target genes, with implication on neural differentiation. The BioID proteomic screen also revealed strong association of AKAP95 with nuclear pore complex protein TPR, an interaction which we independently confirmed. TPR (translocated promoter region), originally characterized in an osteosarcoma cell line as part of a TPR-MET oncogenic gene fusion product, was shown to be involved in other oncogenic fusion events. AKAP95 knockdowns showed an increased number of cells with micronuclei or abnormal nuclear morphology. Live cell microscopy in HeLa cells show that micronuclei result from lagging chromosomes in anaphase. Strikingly, a similar chromatin lagging phenotype has been reported for TPR-depleted cells, suggesting a functional interaction between AKAP95 and TPR. We further show that AKAP95 targets a fraction of TPR to the mitotic spindle during mitosis, and that depletion of AKAP95 interferes with the mitotic spindle checkpoint. Our results point to a role of AKAP95 in regulation of genetic material integrity during chromosome segregation.
In summary, our results demonstrate an implication of AKAP95 in chromosomal instability and thereby contribute to the molecular identification of components involved in this fundamental mitotic event of importance for normal cell division but which remains largely uncharacterized mechanistically. In several retrospective studies concerning a wide variety and including oral cancers, chromosomal instability has been significantly associated with poor prognosis. Despite the prevalence and clinical relevance of chromosome instability, a consistent basis for how chromosome instability is generated at the molecular level is lacking. Yet, frequency and clinical significance of chromosome instability and its restriction to neoplastic tissue suggests the chromosome instability phenotype could represent an attractive therapeutic target. It is therefore of high relevance and benefit to explore mechanisms leading to chromosome instability in order to design new strategies in anti-cancer therapeutics. We have also provided indications on the role of AKAP95 in the organization of signalling complexes at several human gene promoter areas involved in neuronal fate. Given the general emerging role of AKAPs as integrators of multiple signalling pathways implicated in cell communication, motility, proliferation and differentiation, we expect to unravel a novel function of AKAP95 in the regulation of the balance between neuronal differentiation and degeneration. Various human diseases, including cancer and neurological disorders, have been linked to mutations or altered levels of chromatin-modifying enzymes or of some of their regulators, leading to changed expression patterns of key regulatory genes. Therefore, chromatin regulators have recently emerged as successful therapeutic targets, mainly for the treatment of cancer. Inhibitors of DNA-methyltransferases and histone deacetylases are already clinically used. The study outlined here of AKAP95 will define the roles in regulation of chromatin dynamics of this anchoring platform for important signalling molecules (PKA and PP1) involved in differentiation. We expect to identify subgroups of genes critical neuronal differentiation and degeneration that are regulated by AKAP95 and detail the mechanisms of this regulation. This in turn will provide a more selective approach than global inhibitors to target these genes, for example by interference with the recruitment of signalling molecules or other components of the signalling platform assembled by AKAP95. Therefore, the study of AKAP95 has considerable therapeutic potential.
Unravelling chromatin regulation by investigating chromatin-associated scaffold proteins
We are exploring the role of a nuclear scaffold protein, AKAP95, in directing signalling pathways involved in proliferation and/or differentiation to regulators of gene expression. AKAP95 belongs to PKA-anchoring protein family which is characterized by recruiting PKA through its regulatory (R)-binding domain, to specific subcellular localizations.
We have employed chromatin immunoprecipitation (ChIP) techniques coupled with DNA microarrays to investigate AKAP95 occupancy sites (promoter regions) in U2OS human osteosarcoma cells. The AKAP95-bound chromatin landscape was investigated by bioinformatics analysis and compared to publically available histone modification marks and gene expression datasets. These results, collectively, showed that AKAP95 is present in approximately 1000 human gene promoter areas which are preferentially enriched in active transcriptional histone marks (H3K4me3, H3K36me3) and display enhanced gene expression levels. We have employed a novel proximity-based protein identification assay (BioID), based on the in vivo (in situ?) targeting of AKAP95 vicinal proteins by a biotinylating enzyme fused to AKAP95. We have identified FUS as a new AKAP95-interacting partner. FUS/TLS is a multifunctional nuclear protein involved in several aspects of chromatin metabolism, including transcriptional regulation. We have verified this interaction by co-immunoprecipitation of AKAP95 and FUS, and partial colocalization using immunofluorescence microscopy. Our current work and future perspectives include the elucidation of AKAP95 and FUS functional role as well as its interacting chromatin environment. To do so, we are employing siRNA targeting strategies, where AKAP95 levels will be depleted and FUS genome-wide localization (ChIPseq) will be assessed and compared to the endogenous scenario. Also, gene expression levels will be quantified on AKAP95 depleted samples by RNA sequencing. We expect to map and characterize the genomic sites where AKAP95 and FUS are co-enriched and pinpoint those where a functional relation among the two exists.
Targeting of proliferation-regulating genes in cancer cells
A-kinase anchoring proteins (AKAPs) are scaffold proteins that bind and regulate PKA action in a spatio-temporal manner. AKAPs also bind other signaling molecules such as PKC and PP1. We aim here to identify genomic loci bound by AKAP95 in U2OS cells, and using a BioID approach, novel binding partners of AKAP95.
We have established several U2OS clones expressing AKAP95 fused to BirA, able to biotinylate interacting proteins. All controls behave as expected. Thus we have set up a large-scale BioID labeling coupled to a Streptavidin pull-down and proteomic analysis of the pulled-down proteins. The data reveal ~400 proteins uniquely associated with AKAP95 in this assay. We notably found strong association of AKAP95 with the nuclear pore complex (NPC) protein TPR. NPCs act as scaffolds for recruitment of transcriptional machinery components to the nuclear periphery, such as proteins coupling transcription and mRNA export. TPR is required for establishment of NPC-associated zones of heterochromatin exclusion. In addition, we have shown association of AKAP95 with 1000 promoters, 95% of which are marked by H3K4me3 and expressed. Altogether, our results suggest so far a model whereby AKAP95 and TPR form an anchoring platform at the nuclear periphery, that locally organizes peripheral chromatin (usually repressed) in the vicinity of NPCs in a more open conformation compatible with gene expression.
We now aim to identify co-occupying chromatin regulatory factors at distinct genomic sites and infer novel functions for AKAP95 in normal and cancer cells. We will also establish a link between anchoring proteins, cellular signaling and chromatin function regulation. To support this idea, our proteomic screen has also identified several components of the DNA damage response as binding partners of AKAP95.
Targeting of proliferation-regulating genes in cancer cells
Oncogenic transformation of mesenchymal stem cells (MSCs) is a serious risk factor in the expansion of stem cells in culture for use in therapy. This project examines the function of a nuclear protein recently implicated in cancer development.
A number of cancers (including osteosarcomas) also arise from the oncogenic transformation of cells of mesodermal origin. A central protein that integrates multiple signaling pathways controlling the cell cycle and cell motility, and therefore important in cancer, is the nuclear A-kinase anchoring protein AKAP95. We aim to understand the role of this protein in oncogenic transformation. The project started 01.09.2012 and is being carried out by a PhD student. Using an osteoblastic (from bone cancer) transformed MSC line called U2OS, we have, after setting up the procedure for this protein, identified all gene promoters bound by AKAP95 throughout the genome. We find several thousands of genes bound by AKAP95 in their regulatory region, which is consistent with our findings that AKAP95 can regulate gene expression. We detect strong enrichment of AKAP95 on subset of promoters. Binding often occurs on a DNA sequence motif similar to that bind by a regulator of chromatin organization and gene expression, suggesting that both proteins co-occupy these target genes. This is being investigated further. We are currently characterizing these genes and identifying interesting candidates to investigate further in light of their relevance for cancer.
We have also started to examine the underlying organization of chromatin, at gene promoters bond by AKAP95, to start evaluating any putative function in chromatin organization in cancer cells.
We have also set up a novel procedure for identifying new binding partners of AKAP95 in the osteosarcoma cell nucleus – a recently published procedure called Bio-ID. All the technical steps are now in place and we are ready to start pulling down AKAP95 and its binding partners.
These studies contribute to testing the hypothesis that AKAP95 constitutes a relay of information from signaling pathways involved in cancer cell proliferation and regulation of gene expression.
DAXX-dependent supply of soluble (H3.3-H4) dimers into PML bodies pending deposition into chromatin.
Genome Res (Epub ahead of print)
Epigenetic Regulation of Nestin Expression During Neurogenic Differentiation of Adipose Tissue Stem Cells
Stem Cells Dev (Epub ahead of print)