ELK1 uses different DNA binding modes to regulate functionally distinct classes of target genes.

Eukaryotic transcription factors are grouped into families and, due to their similar DNA binding domains, often have the potential to bind to the same genomic regions. This can lead to redundancy at the level of DNA binding, and mechanisms are required to generate specific functional outcomes that enable distinct gene expression programmes to be controlled by a particular transcription factor. Here we used ChIP-seq to uncover two distinct binding modes for the ETS transcription factor ELK1. In one mode, other ETS transcription factors can bind regulatory regions in a redundant fashion; in the second, ELK1 binds in a unique fashion to another set of genomic targets. Each binding mode is associated with different binding site features and also distinct regulatory outcomes. Furthermore, the type of binding mode also determines the control of functionally distinct subclasses of genes and hence the phenotypic response elicited. This is demonstrated for the unique binding mode where a novel role for ELK1 in controlling cell migration is revealed. We have therefore uncovered an unexpected link between the type of binding mode employed by a transcription factor, the subsequent gene regulatory mechanisms used, and the functional categories of target genes controlled.

Elucidation of the ELK1 target gene network reveals a role in the coordinate regulation of core components of the gene regulation machinery.

Transcription factors play an important role in orchestrating the activation of specific networks of genes through targeting their proximal promoter and distal enhancer regions. However, it is unclear how the specificity of downstream responses is maintained by individual members of transcription-factor families and, in most cases, what their target repertoire is. We have used ChIP-chip analysis to identify the target genes of the ETS-domain transcription factor ELK1. Two distinct modes of ELK1 target gene selection are identified; the first involves redundant promoter binding with other ETS-domain family members; the second occurs through combinatorial binding with a second transcription factor SRF, which specifies a unique group of target genes. One of the most prominent groups of genes forming the ELK1 target network includes classes involved in core gene expression control, namely, components of the basal transcriptional machinery, the spliceosome and the ribosome. Amongst the set of genes encoding the basal transcription machinery components, are a functionally linked subset of GTFs and TAFs. Our study, therefore, reveals an unsuspected level of coordinate regulation of components of the core gene expression control machinery and also identifies two different modes of promoter targeting through binding with a second transcription factor or redundant binding with other ETS-domain family members.

Immediate-early gene activation by the MAPK pathways: what do and don't we know?

The study of IE (immediate-early) gene activation mechanisms has provided numerous paradigms for how transcription is controlled in response to extracellular signalling. Many of the findings have been derived from investigating one of the IE genes, FOS, and the models extrapolated to regulatory mechanisms for other IE genes. However, whereas the overall principles of activation appear similar, recent evidence suggests that the underlying mechanistic details may differ depending on cell type, cellular stimulus and IE gene under investigation. In the present paper, we review recent advances in our understanding of IE gene transcription, chiefly focusing on FOS and its activation by ERK (extracellular-signal-regulated kinase) MAPK (mitogen-activated protein kinase) pathway signalling. We highlight important fundamental regulatory principles, but also illustrate the gaps in our current knowledge and the potential danger in making assumptions based on extrapolation from disparate studies.

SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence.

Pericentric heterochromatin silencing at mammalian centromeres is essential for mitotic fidelity and genomic stability. Defective pericentric silencing has been observed in senescent cells, aging tissues, and mammalian tumors, but the underlying mechanisms and functional consequences of these defects are unclear. Here, we uncover an essential role of the human SIRT6 enzyme in pericentric transcriptional silencing, and we show that this function protects against mitotic defects, genomic instability, and cellular senescence. At pericentric heterochromatin, SIRT6 promotes deacetylation of a new substrate, residue K18 of histone H3 (H3K18), and inactivation of SIRT6 in cells leads to H3K18 hyperacetylation and aberrant accumulation of pericentric transcripts. Strikingly, depletion of these transcripts through RNA interference rescues the mitotic and senescence phenotypes of SIRT6-deficient cells. Together, our findings reveal a new function for SIRT6 and regulation of acetylated H3K18 at heterochromatin, and demonstrate the pathogenic role of deregulated pericentric transcription in aging- and cancer-related cellular dysfunction.

The ETS transcription factors ELK1 and GABPA regulate different gene networks to control MCF10A breast epithelial cell migration.

Members of the ETS transcription factor family often target the same binding regions and hence have the potential to regulate the same genes and downstream biological processes. However, individual family members also preferentially bind to other genomic regions, thus providing the potential for controlling distinct transcriptional programmes and generating specific biological effects. The ETS transcription factor ELK1 controls cell migration in breast epithelial cells through targeting a cohort of genes, independently from another family member GABPA, and therefore achieves biological specificity. Here, we demonstrate that GABPA also controls cell migration in breast epithelial cells. However, GABPA controls the expression of a different network of target genes to ELK1. Both direct and indirect target genes for GABPA are identified and amongst the direct targets we confirm the importance of RAC1 and KIF20A for cell migration. Therefore, although ELK1 and GABPA ultimately control the same biological process, they do so by regulating different cohorts of target genes associated with cytoskeletal functions and cell migration control.

The GD2-specific 14G2a monoclonal antibody induces apoptosis and enhances cytotoxicity of chemotherapeutic drugs in IMR-32 human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood. The majority of children suffers from high risk neuroblastoma and has disseminated disease at the time of diagnosis. Despite recent advances in chemotherapy, the prognoses for children with high risk NB remain poor. Therefore, new treatment modalities are urgently needed. GD2 ganglioside is an antigen that is highly expressed on NB cells with only limited distribution on healthy tissues. Consequently, it appears to be an ideal target for both active and passive immunotherapy. The immunological effector mechanisms mediated by anti-GD2 monoclonal antibodies (mAbs) have been already well characterized. However, a growing number of reports suggest that GD2-specific antibodies may exhibit anti-proliferative effects without the immune system involvement. Here, we have shown that anti-GD2 14G2a mAb is capable of decreasing survival of IMR-32 human neuroblastoma cells in a dose-dependent manner. Death induced by this antibody exhibited several characteristics typical for apoptosis such as increased number of Annexin V- and propidium iodide-positive cells, cleavage of caspase 3 and prominent rise in caspase activity. The use of a pan caspase inhibitor Z-VAD-fmk suggested that the killing potential of this mAb is partially caspase-dependent. 14G2a mAb was rapidly endocytosed upon antigen binding. Employment of chloroquine, an inhibitor of lysosomal degradation, did not rescue IMR-32 cells from antibody-induced cell death suggesting lack of ceramide involvement in the observed effect. Most importantly, our studies showed that at particular drug concentrations 14G2a mAb exerts a synergistic effect with doxorubicin and topotecan, as well as an additive effect with carboplatin in killing IMR-32 cells in vitro. Our results provide guidance regarding how to best combine GD2-specific 14G2a antibody with existing cancer therapeutic agents to improve available treatment modalities for neuroblastoma.