Retinitis pigmentosa (RP), the most frequent form of inherited retinal dystrophy is characterized by progressive photoreceptor degeneration. Many genes have been implicated in RP development, but several others remain to be identified. Using a combination of homozygosity mapping, whole-exome and targeted next-generation sequencing, we found a novel homozygous nonsense mutation in SAMD11 in five individuals diagnosed with adult-onset RP from two unrelated consanguineous Spanish families. SAMD11 is ortholog to the mouse major retinal SAM domain (mr-s) protein that is implicated in CRX-mediated transcriptional regulation in the retina. Accordingly, protein-protein network analysis revealed a significant interaction of SAMD11 with CRX. Immunoblotting analysis confirmed strong expression of SAMD11 in human retina. Immunolocalization studies revealed SAMD11 was detected in the three nuclear layers of the human retina and interestingly differential expression between cone and rod photoreceptors was observed. Our study strongly implicates SAMD11 as novel cause of RP playing an important role in the pathogenesis of human degeneration of photoreceptors.

The transcription factor Myocyte enhancer factor 2C (MEF2C) plays a key role in the late differentiation of skeletal muscle progenitor cells, the so-called myoblasts. During myoblast differentiation, both MEF2C expression and transcriptional activity are regulated. We have reported that nTRIP6, the nuclear isoform of the focal adhesion LIM domain protein TRIP6, acts as an adaptor transcriptional co-activator for several transcription factors. It interacts with the promoter-bound transcription factors and consequently mediates the recruitment of other co-activators. Based on a described interaction between MEF2C and TRIP6 in a yeast-two-hybrid screen, we hypothesised a co-regulatory function of nTRIP6 for MEF2C. In proliferating myoblasts, nTRIP6 interacted with MEF2C and was recruited together with MEF2C to the MEF2-binding regions of the MEF2C target genes Myom2, Mb, Tnni2 and Des. Silencing nTRIP6 or preventing its interaction with MEF2C increased MEF2C transcriptional activity and increased the expression of these MEF2C target genes. Thus, nTRIP6 acts as a co-repressor for MEF2C. Mechanistically, nTRIP6 mediated the recruitment of the class IIa histone deacetylase HDAC5 to the MEF2C-bound promoters. In conclusion, our results unravel a transcriptional co-repressor function for nTRIP6. This adaptor co-regulator can thus exert either co-activator or co-repressor functions, depending on the transcription factor it interacts with.

PCGF1 encodes one of six human Polycomb RING finger homologs that are linked to transcriptional repression and developmental gene regulation. Individual PCGF proteins define discrete Polycomb Repressor Complex 1 (PRC1) multi-protein complexes with diverse subunit composition whose functions are incompletely understood. PCGF1 is a component of a variant PRC1 complex that also contains the BCL6 co-repressor BCOR and the histone demethylase KDM2B. To further investigate the role of PCGF1, we mapped the physical interactions of the protein under endogenous conditions in a cell model of neuronal differentiation. Using stringent statistical cut-offs, 83 highly enriched interacting proteins were identified, including all previously reported members of the variant PRC1 complex containing PCGF1, as well as proteins linked to diverse cellular pathways such as chromatin and cell cycle regulation. Notably, a sub-network of proteins associated with the establishment and maintenance of pluripotency (NANOG, OCT4, PATZ1, and the developmental regulator DPPA4) were found to independently interact with PCGF1 in a subsequent round of physical interaction mapping experiments. Furthermore, knockdown of PCGF1 results in reduced expression of DPPA4 and other subunits of the variant PRC1 complex at both mRNA and protein levels. Thus, PCGF1 represents a physical and functional link between Polycomb function and pluripotency.

Despite the pivotal role of jasmonic acid in the outcome of plant-microorganism interactions, JA-signaling components in roots of perennial trees like western balsam poplar (Populus trichocarpa) are poorly characterized. Here we decipher the poplar-root JA-perception complex centered on PtJAZ6, a co-repressor of JA-signaling targeted by the effector protein MiSSP7 from the ectomycorrhizal basidiomycete Laccaria bicolor during symbiotic development. Through protein-protein interaction studies in yeast we determined the poplar root proteins interacting with PtJAZ6. Moreover, we assessed via yeast triple-hybrid how the mutualistic effector MiSSP7 reshapes the association between PtJAZ6 and its partner proteins. In the absence of the symbiotic effector, PtJAZ6 interacts with the transcription factors PtMYC2s and PtJAM1.1. In addition, PtJAZ6 interacts with it-self and with other Populus JAZ proteins. Finally, MiSSP7 strengthens the binding of PtJAZ6 to PtMYC2.1 and antagonizes PtJAZ6 homo-/heterodimerization. We conclude that a symbiotic effector secreted by a mutualistic fungus may promote the symbiotic interaction through altered dynamics of a JA-signaling-associated protein-protein interaction network, maintaining the repression of PtMYC2.1-regulated genes.

Suppressor of Hairy-wing [Su(Hw)] is one of the best characterized architectural proteins in Drosophila and recruits the CP190 and Mod(mdg4)-67.2 proteins to chromatin, where they form a well-known insulator complex. Recently, HP1 and insulator partner protein 1 (HIPP1), a homolog of the human co-repressor Chromodomain Y-Like (CDYL), was identified as a new partner for Su(Hw). Here, we performed a detailed analysis of the domains involved in the HIPP1 interactions with Su(Hw)-dependent complexes. HIPP1 was found to directly interact with the Su(Hw) C-terminal region (aa 720-892) and with CP190, but not with Mod(mdg4)-67.2. We have generated Hipp1 null mutants (HippΔ1) and found that the loss of Hipp1 does not affect the enhancer-blocking or repression activities of the Su(Hw)-dependent complex. However, the simultaneous inactivation of both HIPP1 and Mod(mdg4)-67.2 proteins resulted in reduced CP190 binding with Su(Hw) sites and significantly altered gypsy insulator activity. Taken together, these results suggested that the HIPP1 protein stabilized the interaction between CP190 and the Su(Hw)-dependent complex.

Forkhead Box P (FOXP) transcriptional repressors play a major role in brain development and their dysfunction leads to human cognitive disorders. However, little is known about how the activity of these proteins is regulated. Here, we show that FOXP1 SUMOylation at lysine 670 is required for recruiting the co-repressor CtBP1 and transcriptional repression. FOXP1 SUMOylation is tightly controlled by neuronal activity, in which synapse to nucleus signalling, mediated via NMDAR and L-type calcium channels, results in rapid FOXP1 deSUMOylation. Knockdown of FOXP1 in cultured cortical neurons stunts dendritic outgrowth and this phenotype cannot be rescued by replacement with a non-SUMOylatable FOXP1-K670R mutant, indicating that SUMOylation of FOXP1 is essential for regulation of proper neuronal morphogenesis. These results suggest that activity-dependent SUMOylation of FOXP1 may be an important mediator of early cortical development and neuronal network formation in the brain.

SIN3/HDAC is a multi-protein complex that acts as a regulatory unit and functions as a co-repressor/co-activator and a general transcription factor. SIN3 acts as a scaffold in the complex, binding directly to HDAC1/2 and other proteins and plays crucial roles in regulating apoptosis, differentiation, cell proliferation, development, and cell cycle. However, its exact mechanism of action remains elusive. Using the Caenorhabditis elegans (C. elegans) model, we can surpass the challenges posed by the functional redundancy of SIN3 isoforms. In this regard, we have previously demonstrated the role of SIN-3 in uncoupling autophagy and longevity in C. elegans. In order to understand the mechanism of action of SIN3 in these processes, we carried out a comparative analysis of the SIN3 protein interactome from model organisms of different phyla. We identified conserved, expanded, and contracted gene classes. The C. elegans SIN-3 interactome -revealed the presence of  well-known proteins, such as DAF-16, SIR-2.1, SGK-1, and AKT-1/2, involved in autophagy, apoptosis, and longevity. Overall, our analyses propose  potential mechanisms by which SIN3 participates in multiple biological processes and their conservation across species and identifies candidate genes for further experimental analysis.

In this study, we demonstrate that the lysine methyltransferase G9a inhibits sarcomere organization through regulation of the MEF2C-HDAC5 regulatory axis. Sarcomeres are essential for muscle contractile function. Presently, skeletal muscle disease and dysfunction at the sarcomere level has been associated with mutations of sarcomere proteins. This study provides evidence that G9a represses expression of several sarcomere genes and its over-expression disrupts sarcomere integrity of skeletal muscle cells. G9a inhibits MEF2C transcriptional activity that is essential for expression of sarcomere genes. Through protein interaction assays, we demonstrate that G9a interacts with MEF2C and its co-repressor HDAC5. In the presence of G9a, calcium signaling-dependent phosphorylation and export of HDAC5 to the cytoplasm is blocked which likely results in enhanced MEF2C-HDAC5 association. Activation of calcium signaling or expression of constitutively active CaMK rescues G9a-mediated repression of HDAC5 shuttling as well as sarcomere gene expression. Our results demonstrate a novel epigenetic control of sarcomere assembly and identifies new therapeutic avenues to treat skeletal and cardiac myopathies arising from compromised muscle function.

Understanding the molecular pathology of neurodevelopmental disorders should aid the development of therapies for these conditions. In MeCP2 duplication syndrome (MDS)-a severe autism spectrum disorder-neuronal dysfunction is caused by increased levels of MeCP2. MeCP2 is a nuclear protein that binds to methylated DNA and recruits the nuclear co-repressor (NCoR) complex to chromatin via an interaction with the WD repeat-containing proteins TBL1 and TBLR1. The peptide motif in MeCP2 that binds to TBL1/TBLR1 is essential for the toxicity of excess MeCP2 in animal models of MDS, suggesting that small molecules capable of disrupting this interaction might be useful therapeutically. To facilitate the search for such compounds, we devised a simple and scalable NanoLuc luciferase complementation assay for measuring the interaction of MeCP2 with TBL1/TBLR1. The assay allowed excellent separation between positive and negative controls, and had low signal variance (Z-factor = 0.85). We interrogated compound libraries using this assay in combination with a counter-screen based on luciferase complementation by the two subunits of protein kinase A (PKA). Using this dual screening approach, we identified candidate inhibitors of the interaction between MeCP2 and TBL1/TBLR1. This work demonstrates the feasibility of future screens of large compound collections, which we anticipate will enable the development of small molecule therapeutics to ameliorate MDS.

The AML1/ETO fusion protein found in acute myeloid leukemias functions as a transcriptional regulator by recruiting co-repressor complexes to its DNA binding site. In order to extend the understanding of its role in preleukemia, we expressed AML1/ETO in a murine immortalized pluripotent hematopoietic stem/progenitor cell line, EML C1, and found that genes involved in functions such as cell-to-cell adhesion and cell motility were among the most significantly regulated as determined by RNA sequencing. In functional assays, AML1/ETO-expressing cells showed a decrease in adhesion to stromal cells, an increase of cell migration rate in vitro, and displayed an impairment in homing and engraftment in vivo upon transplantation into recipient mice. Our results suggest that AML1/ETO expression determines a more mobile and less adherent phenotype in preleukemic cells, therefore altering the interaction with the hematopoietic niche, potentially leading to the migration across the bone marrow barrier and to disease progression.

Proteomic analysis of histones has shown that they are subject to a superabundance of acylations, which extend far beyond acetylation, to include: crotonylation, propionylation, butyrylation, malonylation, succinylation, β-hydroxybutyrylation and 2-hydroxyisobutyrylation. To date, much of the functional data has focussed on histone crotonylation which, similar to acetylation, has been associated with positive gene regulation and is added by the acyltransferase, p300. Although Sirtuins 1-3, along with HDAC3, have been shown to possess decrotonylase activity in vitro, there is relatively little known about the regulation of histone crotonylation in vivo. Here we show that Histone Deacetylase 1 and 2 (HDAC1/2), the catalytic core of numerous co-repressor complexes, are important histone decrotonylase enzymes. A ternary complex of HDAC1/CoREST1/LSD1 is able to hydrolyse both histone H3 Lys18-acetyl (H3K18ac) and H3 Lys18-crotonyl (H3K18cr) peptide substrates. Genetic deletion of HDAC1/2 in ES cells increases global levels of histone crotonylation and causes an 85% reduction in total decrotonylase activity. Furthermore, we mapped H3K18cr in cells using ChIP-seq, with and without HDAC1/2, and observed increased levels of crotonylation, which largely overlaps with H3K18ac in the vicinity of transcriptional start sites. Collectively, our data indicate that HDAC1/2 containing complexes are critical regulators of histone crotonylation in vivo.