Karen T. Smith

Rere controls retinoic acid signalling and somite bilateral symmetry

One of the most notable features of the vertebrate body plan organization is its bilateral symmetry, evident at the level of vertebrae and skeletal muscles. Here we show that a mutation in Rere (also known as atrophin2) leads to the formation of asymmetrical somites in mouse embryos, similar to embryos deprived of retinoic acid. Furthermore, we also demonstrate that Rere controls retinoic acid signalling, which is required to maintain somite symmetry by interacting with Fgf8 in the left–right signalling pathway. Rere forms a complex with Nr2f2, p300 (also known as Ep300) and a retinoic acid receptor, which is recruited to the retinoic acid regulatory element of retinoic acid targets, such as the Rarb promoter. Furthermore, the knockdown of Nr2f2 and/or Rere decreases retinoic acid signalling, suggesting that this complex is required to promote transcriptional activation of retinoic acid targets. The asymmetrical expression of Nr2f2 in the presomitic mesoderm overlaps with the asymmetry of the retinoic acid signalling response, supporting its implication in the control of somitic symmetry. Misregulation of this mechanism could be involved in symmetry defects of the human spine, such as those observed in patients with scoliosis.

Histone deacetylase inhibitors: Anticancer compounds

The reversible acetylation of proteins is mediated by histone acetyltransferases which acetylate proteins and histone deacetylases that remove the acetyl groups. High levels of histone acetylation are correlated with active genes, while hypoacetylation of histones corresponds with gene repression. Importantly, acetylation also occurs on non-histone proteins and this can affect the activity and stability of these proteins. Aberrant epigenetic changes are a common hallmark of tumors and imbalances in the activities of deacetylases have been associated with cancers. Accordingly, inhibitors to the histone deacetylases are in clinical trials for the treatment of several cancer types. These drugs mediate a number of molecular changes and in turn can induce cell cycle arrest, apoptosis or differentiation of cancer cells while displaying limited toxicity in normal cells.

Introducing the acetylome

Abstract Mass spectrometry reveals protein acetylation to be as widespread as phosphorylation.

Deacetylase Inhibitors Dissociate the Histone-Targeting ING2 Subunit from the Sin3 Complex

Histone deacetylase (HDAC) inhibitors are in clinical development for several diseases, including cancers and neurodegenerative disorders. HDACs 1 and 2 are among the targets of these inhibitors and are part of multisubunit protein complexes. HDAC inhibitors (HDACis) block the activity of HDACs by chelating a zinc molecule in their catalytic sites. It is not known if the inhibitors have any additional functional effects on the multisubunit HDAC complexes. Here, we find that suberoylanilide hydroxamic acid (SAHA), the first FDA-approved HDACi for cancer, causes the dissociation of the PHD-finger-containing ING2 subunit from the Sin3 deacetylase complex. Loss of ING2 disrupts the in vivo binding of the Sin3 complex to the p21 promoter, an important target gene for cell growth inhibition by SAHA. Our findings reveal a molecular mechanism by which HDAC inhibitors disrupt deacetylase function.

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.

Human Family with Sequence Similarity 60 Member A (FAM60A) Protein: a New Subunit of the Sin3 Deacetylase Complex

Here we describe the function of a previously uncharacterized protein, named family with sequence similarity 60 member A (FAM60A) that maps to chromosome 12p11 in humans. We use quantitative proteomics to determine that the main biochemical partners of FAM60A are subunits of the Sin3 deacetylase complex and show that FAM60A resides in active HDAC complexes. In addition, we conduct gene expression pathway analysis and find that FAM60A regulates expression of genes that encode components of the TGF-beta signaling pathway. Moreover, our studies reveal that loss of FAM60A or another component of the Sin3 complex, SDS3, leads to a change in cell morphology and an increase in cell migration. These studies reveal the function of a previously uncharacterized protein and implicate the Sin3 complex in suppressing cell migration.

Chromatin Proteins: Key Responders to Stress

Environments can be ever-changing and stresses are commonplace. In order for organisms to survive, they need to be able to respond to change and adapt to new conditions. Fortunately, many organisms have systems in place that enable dynamic adaptation to immediate stresses and changes within the environment. Much of this cellular response is coordinated by modulating the structure and accessibility of the genome. In eukaryotic cells, the genome is packaged and rolled up by histone proteins to create a series of DNA/histone core structures known as nucleosomes; these are further condensed into chromatin. The degree and nature of the condensation can in turn determine which genes are transcribed. Histones can be modified chemically by a large number of proteins that are thereby responsible for dynamic changes in gene expression. In this Primer we discuss findings from a study published in this issue of PLoS Biology by Weiner et al. that highlight how chromatin structure and chromatin binding proteins alter transcription in response to environmental changes and stresses. Their study reveals the importance of chromatin in mediating the speed and amplitude of stress responses in cells and suggests that chromatin is a critically important component of the cellular response to stress.

Suberoylanilide Hydroxamic Acid (SAHA)-Induced Dynamics of a Human Histone Deacetylase Protein Interaction Network

Histone deacetylases (HDACs) are targets for cancer therapy. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor approved by the U.S. Food and Drug Administration for the treatment of cutaneous T-cell lymphoma. To obtain a better mechanistic understanding of the Sin3/HDAC complex in cancer, we extended its protein–protein interaction network and identified a mutually exclusive pair within the complex. We then assessed the effects of SAHA on the disruption of the complex network through six homologous baits. SAHA perturbs multiple protein interactions and therefore compromises the composition of large parts of the Sin3/HDAC network. A comparison of the effect of SAHA treatment on gene expression in breast cancer cells to a knockdown of the ING2 subunit indicated that a portion of the anticancer effects of SAHA may be attributed to the disruption of ING2s association with the complex. Our dynamic protein interaction network resource provides novel insights into the molecular mechanism of SAHA action and demonstrates the potential for drugs to rewire networks.

Abstract LB-267: A role for the Sin3 histone deacetylase complex in cell migration

Histone deacetylases (HDACs) remove acetyl groups from histones and other proteins, leading to alterations in protein activity and gene expression. Drugs that target these enzymes (HDAC inhibitors or HDACis) are currently approved for the treatment of specific cancers and are under study in numerous additional clinical trials. These drugs work by causing cell cycle arrest, differentiation or apoptosis. However, less is known about their potential effects on cancer cell migration and invasion, processes implicated in metastasis. There are eleven human zinc dependent histone deacetylases and many HDACis target several of these structurally related proteins in cells. Our studies are focused on HDACs1 and 2 which reside in large protein complexes, one of which is the Sin3 complex. This complex is important for promoter-mediated histone deacetylation, transcriptional repression and cell cycle control. We previously found that specific HDACis disrupt the function of the Sin3 complex in distinct ways. Here we describe the function of a novel subunit of the Sin3 complex, a previously uncharacterized protein, named “Family with sequence similarity 60, member A” (FAM60A). We find that FAM60A represses expression of genes linked to cancer cell migration and invasion. Moreover, we show that loss of FAM60A, or a core subunit of the Sin3 complex can increase the migration of lung cancer cells. This suggests that not all HDAC-related functions are cancer promoting, and adds to data suggesting that some HDAC-related functions may act in steps in metastasis suppression pathways. This suggests that some HDACis could increase the potential for metastasis. Current studies are focused on delineating the cancer-promoting functions of the complex from those that are tumor or metastasis suppressive and testing the effects of specific HDACis on cancer cell migration and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-267. doi:1538-7445.AM2012-LB-267

13-P031 Rere (Atrophin2) controls retinoic acid signaling and somite bilateral symmetry

The insulin like growth factors (IGFs) first appeared early in phylogeny about 600 million years ago and have increased in number through gene duplication. In mammals IGFs express in all tissues and are found in many biological fluids. IGF-1 has an important role during central nervous system (CNS) development. IGF-1 promotes differentiation, proliferation and prevents apoptosis of neuronal and brain derived cells. In this study CSF was obtained from the lateral ventricle by puncture using micromanipulation. IGF-1 concentration in CSF samples from chick on embryonic days 8 to 21 (E8–E21) were measured. We have used Western blot and enzyme linked immunosorbent assay (ELISA) to study IGF-1 expression and concentrations. It was shown that IGF-1 levels in the CSF decreased from days E8 to E14. There was a rapid increase in IGF-1 contents on days E15 to E16, after that the concentrations decreased from days E17 to E21. Days E15 to E16 coincide with the onset of neuron proliferation, migration and organization of the cytoarchetecture of the developing chick cortex. Thereafter the IGF-1 concentration decreases until hatching. Since CSF is in contact with cerebral cortical germinal epithelium, changes in the IGF-1 concentration in the CSF might affect on the activity of germinal neuroepithelium and IGF-1 may also be involved in the cerebral cortical development.