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CoREST family of transcriptional co-repressors regulates gene expression and cell fate determination during development. CoREST co-repressors recruit with different affinity the histone demethylase LSD1 (KDM1A) and the deacetylases HDAC1/2 to repress with variable strength the expression of target genes. CoREST protein levels are differentially regulated during cell fate determination and in mature tissues. However, regulatory mechanisms of CoREST co-repressors at the protein level have not been studied. Here, we report that CoREST (CoREST1, RCOR1) and its homologs CoREST2 (RCOR2) and CoREST3 (RCOR3) interact with PIASγ (protein inhibitor of activated STAT), a SUMO (small ubiquitin-like modifier)-E3-ligase. PIASγ increases the stability of CoREST proteins and facilitates their SUMOylation by SUMO-2. Interestingly, the SUMO-conjugating enzyme, Ubc9 also facilitates the SUMOylation of CoREST proteins. However, it does not change their protein levels. Specificity was shown using the null enzymatic form of PIASγ (PIASγ-C342A) and the SUMO protease SENP-1, which reversed SUMOylation and the increment of CoREST protein levels induced by PIASγ. The major SUMO acceptor lysines are different and are localized in nonconserved sequences among CoREST proteins. SUMOylation-deficient CoREST1 and CoREST3 mutants maintain a similar interaction profile with LSD1 and HDAC1/2, and consequently maintain similar repressor capacity compared with wild-type counterparts. In conclusion, CoREST co-repressors form protein complexes with PIASγ, which acts both as SUMO E3-ligase and as a protein stabilizer for CoREST proteins. This novel regulation of CoREST by PIASγ interaction and SUMOylation may serve to control cell fate determination during development., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Epigenetic targeting of cancer is a recent effort to manipulate the gene without destroying the genetic material. Lysine-specific demethylase 1 (LSD1) is one of the enzymes associated with the chromatin for post-translational modifications, where it demethylates lysine amino acid in the chromatin H3 tail. Many studies showed that inhibiting LSD1 could potentially be used to treat cancer epigenetically. LSD1 is associated with its corepressor protein CoREST, and it uses tetrahydrofolate as a co-factor to accept CH 2 from the demethylation process. In this study, the co-crystallized co-factor tetrahydrofolate was utilized to determine possible binding regions in the active center of the LSD1/CoREST complex. Also, the flexibility of the complex has been investigated by molecular dynamics simulation and subsequent analysis by clustering and principal component analysis. This research supported other studies and showed that LSD1/CoREST complex exists in two main conformational structures: open and closed. Furthermore, this study showed that tetrahydrofolate stably binds to the LSD1/CoREST complex, in its open conformation, at its entrance. It then binds to the core of the complex, inducing the closed conformation. Furthermore, the interactions of tetrahydrofolate to these two binding regions and the corresponding binding mode of tetrahydrofolate were investigated to be used in structure-based drug design.

The promoter regions of active genes in the eukaryotic genome typically contain nucleosomes post-translationally modified with a trimethyl mark on histone H3 lysine 4 (H3K4), while transcriptional enhancers are marked with monomethylated H3K4. The flavin-dependent monoamine oxidase LSD1 (lysine-specific demethylase 1, also known as KDM1) demethylates mono- and dimethylated H3K4 in peptide substrates, but requires the corepressor protein, CoREST, to demethylate nucleosome substrates. The molecular basis for how the LSD1/CoREST complex interacts with its physiological nucleosome substrate remains largely unknown. We examine here the role of extranucleosomal DNA beyond the nucleosome core particle for LSD1/CoREST function. Our studies of LSD1/CoREST's enzyme activity and nucleosome binding show that extranucleosomal DNA dramatically enhances the activity of LSD1/CoREST, and that LSD1/CoREST binds to the nucleosome as a 1:1 complex. Our photocrosslinking experiments further indicate both LSD1 and CoREST subunits are in close contact with DNA around the nucleosome dyad as well as extranucleosomal DNA. Our results suggest that the LSD1/CoREST interacts with extranucleosomal DNA when it productively engages its nucleosome substrate., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

With its noncatalytic domains, DNA-binding regions, and a catalytic core targeting the histone tails, LSD1-CoREST (lysine-specific demethylase 1; REST corepressor) is an ideal model system to study the interplay between DNA binding and histone modification in nucleosome recognition. To this end, we covalently associated LSD1-CoREST to semisynthetic nucleosomal particles. This enabled biochemical and biophysical characterizations of nucleosome binding and structural elucidation by small-angle X-ray scattering, which was extensively validated through binding assays and site-directed mutagenesis of functional interfaces. Our results suggest that LSD1-CoREST functions as an ergonomic clamp that induces the detachment of the H3 histone tail from the nucleosomal DNA to make it available for capture by the enzyme active site. The key notion emerging from these studies is the inherently competitive nature of the binding interactions because nucleosome tails, chromatin modifiers, transcription factors, and DNA represent sites for multiple and often mutually exclusive interactions.

Mammalian genomes harbor three CoREST genes. rcor1 encodes CoREST (CoREST1), and the paralogues rcor2 and rcor3 encode CoREST2 and CoREST3, respectively. Here, we describe specific properties of transcriptional complexes formed by CoREST proteins with the histone demethylase LSD1/KDM1A and histone deacetylases 1 and 2 (HDAC1/2) and the finding that all three CoRESTs are expressed in the adult rat brain. CoRESTs interact equally strongly with LSD1/KDM1A. Structural analysis shows that the overall conformation of CoREST3 is similar to that of CoREST1 complexed with LSD1/KDM1A. Nonetheless, transcriptional repressive capacity of CoREST3 is lower than that of CoREST1, which correlates with the observation that CoREST3 leads to a reduced LSD1/KDM1A catalytic efficiency. Also, CoREST2 shows a lower transcriptional repression than CoREST1, which is resistant to HDAC inhibitors. CoREST2 displays lower interaction with HDAC1/2, which is barely present in LSD1/KDM1A-CoREST2 complexes. A nonconserved leucine in the first SANT domain of CoREST2 severely weakens its association with HDAC1/2. Furthermore, CoREST2 mutants with increased HDAC1/2 interaction and those without HDAC1/2 interaction exhibit equivalent transcriptional repression capacities, indicating that CoREST2 represses in an HDAC-independent manner. In conclusion, differences among CoREST proteins are instrumental in the modulation of protein-protein interactions and catalytic activities of LSD1/KDM1A-CoREST-HDAC complexes, fine-tuning gene expression regulation.