Abstract
Lysine specific demethylase 1 (LSD1) regulates gene expression as part of the CoREST complex, along with co-repressor of REST (CoREST) and histone deacetylase 1 (HDAC1). CoREST is recruited to specific genomic loci by core components and numerous transient interactions with chromatin-associated factors and transcription factors. We hypothesise that many of these weaker and transient associations may be difficult to identify using traditional co-immunoprecipitation methods. We have therefore employed proximity-dependent biotin-identification (BioID) with four different members of the CoREST complex, in three different cell types, to identify a comprehensive network of LSD1/CoREST associated proteins. In HEK293T cells, we identified 302 CoREST-associated proteins. Among this group were 16 of 18 known CoREST components and numerous novel associations, including readers (CHD3, 4, 6, 7 and 8), writers (KMT2B and KMT2D) and erasers (KDM2B) of histone methylation. However, components of other HDAC1 containing complexes (e.g. Sin3) were largely absent. To examine the dynamic nature of the CoREST interactome in a primary cell type, we replaced endogenous LSD1 with BirA*-LSD1 in embryonic stem (ES) cells and performed BioID in pluripotent, early- and late-differentiating environments. We identified 156 LSD1-associated proteins of which 67 were constitutively associated across all three time-points (43%), including novel associations with the MMB and ChAHP complexes, implying that the majority of interactors are both dynamic and cell type dependent. In total, we have performed 16 independent BioID experiments for LSD1 in three different cell types, producing a definitive network of LSD1-assoicated proteins that should provide a major resource for the field.
Highlights
The BirA* protein was tagged with a nucleoporin nuclear localisation signal (NLS, BirA*-NLS), to identify proteins incidentally biotinylated by BirA* alone
Due to the presence of unstructured N-terminal regions in Lysine specific demethylase 1 (LSD1), PHF21A and CoREST1, the BirA* tag was added to their N-termini, whereas histone deacetylase 1 (HDAC1) was C-terminally fused to BirA* because the N-terminus forms part of the histone deacetylase domain.[18]
Transient transfection of HEK293T cells followed by western blotting revealed that each of the constructs was expressed (Fig. 1C) and that BirA*-LSD1 and HDAC1-BirA* levels approximated those of the endogenous protein (Fig. S1A, Electronic supplementary information (ESI)†)
Summary
The core CoREST complex is comprised of three components: lysine specific demethylase 1 (LSD1/KDM1A), co-repressor of RE1silencing transcription factor (CoREST) and HDAC1. The combination of demethylase/deacetylase activities within the core LSD1/CoREST/ HDAC1 complex allows for the coordinated demethylation of H3K4me/me[2] and deacetylation of the histone H3 tail, promoting transcriptional repression.[9,16,17,20,21] in vitro studies with purified LSD1/CoREST and cellular experiments suggest that the deacetylated H3 tail may be the preferred LSD1 substrate for demethylation.[17,20] In addition to the core components, the CoREST complex contains a number of additional chromatin associated subunits, such as PHF21A, HMG20A/B and CTBP1/2 which enable recruitment to specific genomic loci.[7,9]
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