DBC1, p300, HDAC3, and Siah1 coordinately regulate ELL stability and function for expression of its target genes

Significance Human RNA polymerase II (Pol II) transcribes messenger RNA from underlying DNA sequences for the purpose of generating proteins for proper biological functions. One of the key steps of Pol II-driven transcription is elongation, which is directly stimulated by transcription factors such as ELL and affects expression of vast number of genes. However, mechanisms by which the overall functions of ELL are regulated through its stabilization are completely unknown. In this study, we decipher a mechanism of regulation of ELL stability, and thus its functions through multiple factors involving DBC1, HDAC3, p300, and Siah1 for regulation of expression of diverse sets of genes that are required for several physiological processes, including maintenance of glucose homeostasis in human.

In presence of over-expressed SIRT6, we consistently observe the appearance of additional bands above the target SIRT6 band when immunoblotted with α-FLAG antibody. H. Western blotting showing interaction of ELL and HDAC3 in 293T cells by immunoprecipitation of ELL. I. Western blotting showing interaction of HDAC3 and ELL in 293T cells by immunoprecipitation of HDAC3.

Cell Culture and Transfection
All mammalian cells used in this study were cultured in DMEM (Invitrogen, USA), supplemented with 10% FBS (Gibco, USA) and 100 U/ml Penicillin-Streptomycin (Gibco, USA). Cells were maintained in a humidified incubator at 37:C and 5% CO 2 . Sf9 cells were cultured in Grace's Insect Media (Himedia, India), supplemented with 10% FBS (Gibco, USA) and 7 µg/mL gentamicin (Gold Biotechnology, USA). For transfection in mammalian cells, Lipofectamine2000 (Invitrogen, USA) was used as per manufacturer's protocol. Sf9 transfections were performed using Cellfectin II reagent (Invitrogen, USA) as per manufacturer's protocol. For all assays, unless otherwise mentioned, cells were harvested 48 hours post transfection.

Creation of different plasmid constructs
The cDNAs for ELL and DBC1 were purchased commercially (Open Biosystems, USA). The genes were cloned into different epitope tagged pcDNA5/FRT/TO plasmids for mammalian expression and generation of stable cell lines. Deletion constructs of ELL as well as DBC1 were also created in epitope tagged pcDNA5/FRT/TO vectors. For baculovirus-mediated expression in insect cells, genes were cloned in pFASTBAC vector as mentioned. For His-tagged and GST tagged protein expression in bacteria, genes were cloned in 6xHis pET-11d and pGEX vectors respectively. Site directed mutagenesis was performed using the QuikChange II Site-Directed Mutagenesis Kit (Agilent technologies, USA). Details of all clones are available upon request from the corresponding author.

Stable Cell line Generation
Generation of stable mammalian cell line for FLAG-HA-ELL has been described previously (1). For creation of stable lines expressing FLAG-HA-DBC1, essentially the same protocol was followed. Briefly, after transfection of Flp-In T-REx 293 cells with the respective plasmid, along with pOG44, cells were subjected to 200 µg/ml Hygromycin (Invitrogen, USA) selection for several weeks until individual colonies were obtained. Individual colonies were further amplified and then screened for the expression of the protein of interest. Cells were harvested in 1X Phosphate buffered saline (PBS, 137mMNaCl, 2.7mMKCl, 8mM Na 2 HPO 4 , and 2mM KH 2 PO 4 ) and lysed in RIPA Buffer (10mMTris-Cl (pH 8.0), 1mM EDTA, 1% Triton X-100, 0.1% SDS, 150mMNaCl). Expression of target proteins was checked via SDS-PAGE followed by probing with epitope tag-specific antibodies.

Mass Spectrometry Analysis
Mass spectrometry analyses of DBC1 associated proteins was performed as described previously (1). Purified DBC1.com was resolved on a 4-12% SDS-PAGE gel and proteins were stained using Coomassie Brilliant Blue-G. Protein bands were excised carefully and destained in 20% methanol for 6 hours. Cysteines were reduced and alkylated with IAA. Proteins were then digested overnight with modified Trypsin (sequencing grade). The peptides were resolved on a nanocapillary reverse phase column using a 1% acetic acid/acetonitrile gradient at 400nl/min rate flow. The separated peptides were injected into a linear ion-trap mass spectrometer (LTQ XL, ThermoFisher). MS/MS spectra of the most were collected and proteins identified by comparing the data against Human IPI database (v 3.41) using X!Tandem/Trans-Proteomic Pipeline (TPP) software suite.

Nuclear Extract Preparation
Nuclear extract preparation from cells was done as described earlier (1). Briefly, cells were harvested in PBS and centrifuged at 800xg for 5 minutes. The packed cell volume (PCV) was estimated and cells were resuspended in 2xPCV of Buffer NE1 (10mM Tris-Cl pH 7.3, 1.5mM MgCl 2 , 10mMNaCl, 0.7 µl/ml β-Mercaptoethanol. After leaving on ice for 15 minutes, the cells were drawn into a syringe with a 23 gauge needle, and repeatedly injected and aspirated 8 times in order to shear the cell membrane. The lysate was centrifuged at 6000 rpm for 5 minutes at 4:C to obtain the nuclear pellet. After estimation of the nuclear pellet volume (NPV), the pellet was resuspended in 0.5xNPV of Buffer NE2 (20mM Tris-Cl pH 7.3, 1.5mM MgCl 2 , 20mM NaCl, 0.2mM EDTA, 25% Glycerol, 0.7 µl/ml β-Mercaptoethanol and Protease inhibitor Cocktails (PIC) (Roche, USA). An equal volume of NE3 Buffer (20mM Tris-Cl pH 7.3, 1.5mM MgCl 2 , 1.2M NaCl, 0.2mM EDTA, 25% Glycerol, 0.7 µl/ml β-Mercaptoethanol and PIC) was then added and the cells were vortexed immediately to ensure uniform mixing. The resulting homogenate was incubated on ice for 45 minutes and vortexed every 3 minutes for mixing and subsequent extraction from nuclei. Finally, the nuclear lysate was centrifuged at 12000xg for 20 minutes at 4:C to obtain the supernatant containing the nuclear extract. The extract was either immediately utilized for experimental procedures or flash frozen and stored at -80:C for future use.

Immunoprecipitation analyses
For exogenously expressed epitope tagged proteins, cells were harvested 48 hours post transfection and lysed in BC300 buffer (20mM Tris-Cl, pH 8.0, 300mM KCl, 2mM EDTA, 20% Glycerol). The cell supernatant containing the total protein was subjected to immunoprecipitation using anti-epitope antibody-tagged magnetic/agarose beads. After incubation, the beads were washed thrice with BC300 + 0.1% NP40 to remove unbound proteins and non specific interactors. The beads were then boiled in 1X SDS loading dye at 95:C for 10 minutes to elute bead bound proteins, which were subsequently analyzed by Western blotting using tag/protein-specific antibodies.
For endogenous immunoprecipitation experiments, nuclear extract was prepared from 293T cells as described previously. The extract, containing approximately 500µg of proteins was subjected to preclearing using Protein-A agarose beads for 2hours at 4:C. The beads were then discarded and the precleared cell lysate was incubated with 2µg of the indicated primary antibody overnight at 4:C. The antibody bound proteins were then immunoprecipitated using Protein G magnetic beads (BioRad, USA) at 4:C for 1 hour. The supernatant was discarded and the beads were washed 3X using the buffer used for nuclear extract preparation. Subsequently, the beads were boiled in 1X SDS loading dye to at 95:C for 10 minutes to elute bound proteins, which were analyzed by SDS-PAGE followed by Western blotting.

DBC1.com complex purification from nuclear extract
Flag-HA tagged DBC1 expressing stable cells were grown in a large volume (~ 6 litres) in a spinner flask. Nuclear extract was prepared as per the method described earlier. The extract was pre-cleared using Protein-Aagarose beads for 2 hours at 4:C. The pre-cleared extract was incubated with Anti-FLAG M2 Magnetic Beads (Sigma, USA) overnight at 4:C. Bead bound proteins were subjected to extensive washing with BC300 buffer. Finally, bound proteins were eluted by incubating with 3X FLAG peptide (250ng/µl) for 1 hour at 4:C. The purified complexes were separated on a 4-12% gel and stained using ProteoSilver Silver Stain Kit(Sigma, USA).

Luciferase Reporter Gene Assay
Luciferase assays were carried out using the G-293T cell line (2). G-293T is a HEK-293T-based cell line containing a chromosomally integrated luciferase gene downstream of Gal4 binding sites. For the assay, cells were transfected with the mentioned amount of the respective constructs along with Gal4-VP16 activator. 48 hours post transfection, cells were harvested as per the protocol mentioned in Dual-Glo® Luciferase Assay System (Promega, USA) and luciferase activity measured using the GloMax 20/20 Luminometer (Promega, USA).

Cycloheximide Chase Assay
Cells were transfected with the indicated constructs in the combinations mentioned. 24 hours post transfection, cells were treated to 100 µg/ml cycloheximide (Sigma, USA). At the indicated time points, cells were harvested in ice-cold PBS and lysed using RIPA buffer. Lysates were analysed by western blotting with respective antibodies.

Recombinant protein purification
For the purpose of in vitro reactions and interaction analyses, proteins were purified either from bacterial or mammalian cells. For purification of proteins from bacteria, E. coli BL21-DE3 cells expressing the protein of interest were grown for 4 hours after induction with 1mM IPTG (Goldbio, USA). Harvested cells were lysed in appropriate buffer by sonication [60% amplitude with 10 cycles of alternating on/off phases]. The cell supernatant containing the protein of interest was incubated with Ni-NTA beads for His-tagged proteins and GST agarose beads for GST-tagged proteins. The beads were then washed with appropriate buffers, and eluted using GST elution buffer or His elution buffer.

In vitro acetylation assay
Purified ELL was incubated with purified p300 HAT domain and 0.5mM acetyl-CoA in HAT buffer (75mMTris-Cl (pH 8.0), 1.25mM EDTA, 12.5mM DTT, 0.25% Tween20, 25% glycerol). The reaction mix was incubated at 30°C for 2 hours with intermittent tapping. The reaction was stopped by adding SDS loading dye, followed by boiling at 95°C for 10 minutes. The products were analysed by SDS PAGE followed by immunoblotting using pan acetyl lysine-specific antibody.

In vitro deacetylation assay
Flag tagged HDAC3 was purified by immunoprecipitation from 293T cells. Purified acetylated ELL was incubated with HDAC3 in HDAC buffer (50mMTris [pH 8.0], 4mM MgCl 2 , 0.2mM DTT) on a rotor for 3 hours at 37°C. The reaction was stopped by adding SDS loading dye and boiling the samples for 10 minutes at 95°C. Proteins were resolved by SDS-PAGE and analyzed by western blotting.

NGS data mining and GO analysis
Identification of target genes for both DBC1 and ELL was done using the publicly available datasets GSE35480 (3) and GSE34104 (4). Genes were sorted in MS Excel and overlapping genes identified using Venny. Initial gene ontology of overlapping genes was performed using Protein Annotation Through Evolutionary Relationship (PANTHER) software (5). Further functional classification and disease annotation of common genes was done using Database for Annotation, Visualization and Integrated Discovery (DAVID) (6).

Generation of stable knockdown cells
For the purpose of generating cells with DBC1 stably knocked down, DBC1 specific shRNAs were cloned into pLKO.1 vector. 375ng psPAX2 packaging plasmid, 125ng pMD2.G VSV-G envelope plasmid and 500ng of the respective shRNAs were co-transfected into 5x10 5 cells in a 6 well plate. 72 hours post transfection, the supernatant containing virus particles was collected, centrifuged briefly and used to transduce fresh cells. Polybrene was added at a concentration of 8 µg/ml to aid viral transduction. Cells were then subjected to selection with 3 µg/ml puromycin. Puromycin resistant cells were amplified and screened to identify knock down cell lines via immunoblotting and qRT-PCR analysis.