Sequential chromatin immunoprecipitation to detect SUMOylated
MeCP2 in neurons
Tao Wu
a,b, Mary E. Donohoe
a,b,n aBurke Medical Research Institute, White Plains, NY 10605, USA
bDepartment of Neuroscience, Brain Mind Research Institute, Department of Cell & Development, Weill Cornell Medical College, New York, NY 10065, USA
a r t i c l e i n f o
Article history: Received 29 June 2015 Received in revised form 4 January 2016 Accepted 22 January 2016 Available online 23 January 2016 Keywords:
Small ubiquitin-like modifier SUMO
MeCP2 Neurons
Sequential quantitative chromatin im-munoprecipitation
Seq-ChIP
Chromatin biochemistry
a b s t r a c t
The small ubiquitin-like modifier (SUMO) is a short peptide that can be covalently linked to proteins altering their function. SUMOylation is an essential post-translational modification (PTM). Because of its dynamic nature, low abundance levels, and technical limitations, the occupation of endogenous SU-MOylated transcription factors at genomic loci is challenging to detect. The chromatin regulator Methyl CpG binding protein 2 (MeCP2) is subjected to PTMs including SUMO. Mutations in MeCP2 lead to Rett syndrome, a severe neurodevelopmental disorder. Here, we present an efficient method to perform sequential chromatin immunoprecipitation (Seq-ChIP) for detecting SUMOylated MeCP2 in neurons. This Seq-ChIP technique is a useful tool to determine the occupancy of SUMOylated transcription and chro-matin factors at specific genomic regions.
& 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
The covalent addition of Small Ubiquitin Modifier, SUMOyla-tion, is an essential post-translational modification (PTM) that regulates diverse cellular processes including gene transcription, chromatin remodeling, replication, chromosome segregation, and DNA repair[1–4]. In mammals, the conjugation of SUMO to a ly-sine residue in a target protein is mediated by three paralogs: SUMO1–3[1]. Most substrate proteins are rapidly deSUMOylated by one of six Sentrin/SUMO-specific proteases (SENPs)[5]. Defects in SUMOylation are associated with multiple diseases such as cancer, heart failure, and neurodegeneration[6–8].
In the nervous system, SUMOylation plays a crucial function with roles in synapse formation, axonal transport, and neuronal excitability [9–12]. One way that neurons employ a rapid mod-ulation of their transcription program in response to neuronal activity is by regulating the PTMs of transcription factors. Tran-scription factors can be modified by multiple PTMs, such as phosphorylation, acetylation, ubiquitination, and SUMOylation [13]. These PTMs modulate either the target factors binding ability to DNA or interaction with protein co-factors[2].
One neuronal factor that is regulated by PTMs is the Methyl CpG binding protein 2(MeCP2)[8]. Although it wasfirst described in the early 1990s as a transcriptional repressor binding methy-lated DNA, it is now appreciated that MeCP2 can also function as an activator and chromatin modifier[8,14,15]. The importance of MeCP2 is underscored as its mutations lead to a severe neurode-velopmental disorder known as Rett syndrome (RTT)[16,8]. SU-MOylation modifies MeCP2, modulates its transcriptional activity, and plays a crucial role in synaptic formation[17]. However, our knowledge of the occupancy of endogenous SUMOylated MeCP2 at specific neuronal regulatory regions is lacking. The identification of neuronal promoters harboring SUMOylated MeCP2 will define its biological function during normal development. But detection of endogenous SUMOylated chromatin and transcription factors at genomic regions is problematic. Many SUMOylated proteins such as MeCP2 and other chromatin and transcription factors are low in abundance[18]. SUMOylation is very dynamic; therefore, identi-fication of factors occupying specific genomic regions is difficult [18,19]. Furthermore, no antibodies exist that specifically re-cognize a SUMOylated protein at particular genomic sites. Here, we describe an efficient sequential chromatin immunoprecipita-tion (Seq-ChIP) method to identify SUMOylated MeCP2 at a given neuronal genomic region in vivo.
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Biochemistry and Biophysics Reports
http://dx.doi.org/10.1016/j.bbrep.2016.01.014
2405-5808/& 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
nCorresponding author.
2. Materials and methods 2.1. Neuronal cell culture
Mouse hippocampal neuronal cells (HT22) were maintained in DMEM (Life Technologies) supplemented with 10% fetal calf ser-um, 2 mM L-Glutamine, and 1 Penicillin/Streptomycin.
2.2. Chromatin immunoprecipitation (ChIP) antibodies and primer selection
ChIP-grade antibodies were used for the Seq-ChIP. If an anti-body has not been tested for ChIP, the efficiency of the antibody can be tested by co-immunoprecipitation of the target protein as previously described [20]. Western blotting can be performed following the immunoprecipitation of the protein-DNA complexes to check whether the SUMO PTM is enriched within the im-munoprecipitated complex. This co-immunoprecipitation test should be utilized to test other SUMOylated transcription factors or chromatin modulators prior to the sequential ChIP. To design primers for quantitative PCR, we selected an amplicons with a size range of 75–200 base pair (bp) flanking the regulatory regions in genes of interest. We designed several sets of PCR primers located upstream and downstream of each target genomic site and com-pared the ChIP signals from these primer sets to observe protein enrichment at a particular locus. PCR primers were designed with the aid of several complimentary software applications such as OLIGO Primer Analysis Software (http://www.oligo.net/), Primer 3 (www.bioinfo.ut.ee/primer3/), or Primer Premier (http://www. premierbiosoft.com/primerdesign/). Prior to oligo synthesis, we checked the specificity of the primers using the National Center for Biotechnology (NCBI) Basic Local Alignment Search Tool (BLAST). In addition, we obtained a melt curve analysis following the real-time PCR analysis, to determine the specificity of the PCR primers. A suitable set of PCR primers exhibits a single peak in the melt curve. An agarose gel analysis of the PCR reaction product can be also used to test the adequacy of the primers with the demon-stration of a single band.
2.3. Sequential ChIP and quantitative Polymerase chain reaction (PCR)
To detect SUMOylated MeCP2 at neuronal chromatin, we es-tablished a Sequential-Chromatin immunoprecipitation (Seq-ChIP) method:
(1) 2.5 108HT22 cells were crosslinked with 1% formaldehyde for 10 min at room temperature with gentle agitation then quenched by 125 mM glycine with agitation for an additional 10 min. The crosslinked cells were washed twice each with ice cold phosphate buffered saline (PBS). The cells were pelleted by centrifugation (Eppendorf 5424 centrifuge) for 5 min at 2000 rpm and resuspended in Lysis Buffer (1% SDS, 5 mM EDTA, 50 mM Tris–HCl (pH 8.0), 150 mM NaCl) supplemented with protease inhibitors (phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail (Roche Diagnostics) prior to use. The chromatin fraction was sheared using a mi-crotip sonicator (Branson) on an output 7 setting for 22 cycles of 10 s sonication followed by rest on ice for 50 s. A small aliquot of the chromatin was removed and checked for soni-cation efficiency (o500 bp) by agarose gel electrophoresis. The sheared chromatin was then centrifuged for 10 min at 4°C maximum speed. The supernatant containing the chromatin was diluted 1:10 with ChIP Dilution Buffer (20 mM Tris (pH 8.0), 1% Triton-X 100, 150 mM NaCl, 2 mM EDTA. Supple-mented with protease inhibitor before use). Next, the
chromatin was pre-cleared by adding 100
μ
l agarose A/G beads (Santa Cruz) at 4°C for 2 h with constant rotation. The mixture was centrifuged at 4°C 2000 rpm for 5 min. The su-pernatant was removed, retaining 1% as an input. The re-mainder of the diluted chromatin was then aliquoted into 1.5 ml tubes with an aliquot per ChIP antibody.(2) To perform thefirst ChIP, 6
μ
g of antibody (IgG control (Santa Cruz sc-2027), SUMO1 (Santa Cruz sc-9060), and anti-MeCP2 (Millipore 07013)) was added to each tube of the di-luted chromatin and rocked overnight at 4°C. Following the overnight incubation, agarose A/G beads (150μ
l) were added to each sample and rotated for an additional 2–4 h at 4 °C. (3) The beads were pelleted and washed once each with Buffer 1(0.1% SDS, 1%Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.0), 150 mM NaCl), Buffer 2 (0.1%SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl (pH 8.0), 500 mM NaCl), Buffer 3 (1% NP40, 1% Deoxycholic acid, 1 mM EDTA, 10 mM Tris–HCl (pH 8.0), 250 mM LiCl), and Buffer 4 (2 mM EDTA, 10 mM Tris–HCl (pH 8.0)). After the final wash, the DNA-protein complexes were eluted twice with 100
μ
l 10 mM DTT at 37°C for 30 min with gentle shaking twice and combined. The beads were pelleted, the supernatant was saved, and the wash steps with Buffers 1–4 (iii) were repeated once as described above. The supernatants were combined from both steps. One tenth of the eluate was saved as a template for the primary ChIP for qPCR analysis. The remaining supernatant was diluted with 20 times ChIP dilution buffer and the secondary ChIP was per-formed beginning with the addition of the second ChIP anti-body at the above step (ii).(4) After thefinal wash, as listed above in step (iii), the DNA was eluted from the beads with 250
μ
l Elution Buffer (1 mM EDTA, 10 mM Tris–HCl (pH 7.5), 1% SDS) twice for 30 min at room temperature with constant rotation. Proteinase K (final con-centration 10μ
g/ul) and NaCl (final concentration 200 mM) were added to the eluted chromatin from the primary ChIP, secondary ChIP, and input. The crosslinks were reversed at 65°C for 4 h to overnight. The DNA was purified using phenol/ chloroform extraction. The purified DNA was resuspended in 60μ
l double-distilled H20 (ddH20).2.4. Seq-ChIP quantitative PCR analysis
PCR reactions were set up in triplicate using the primer sets shown inTable 1. Each PCR was assembled as described inTable 2 using SYBRsGreen PCR Master Mix (Life Technologies). The ana-lysis was performed using the real-time PCR ABI7500 system (Applied Biosystem). For data analysis 2–(CtAbCtinput). Ct¼cycle
threshold. Statistical analysis was done using the Student's t-test. A positive result showed a significant enrichment of the MeCP2 protein at genomic regions compared with either IgG or the ne-gative control regions.
3. Results
3.1. Scheme of sequential ChIP to detect SUMOylated MeCP2 in neurons
MeCP2 is an important regulator in the brain[8]. However, the precise mechanism of action for MeCP2 is unknown. Although MeCP2 is subjected to numerous PTMs, it is not known where along the genome SUMOylated MeCP2 binds. SUMO modifications can alter its substrate protein–protein interactions, nuclear-cyto-plasmic location, ubiquitination status, and transcriptional activity [7]. Thus, the identification of regulatory regions harboring SU-MOylated MeCP2 is crucial for understanding the role for this PTM
in neurons. In order to detect SUMOylated MeCP2 at particular neuronal genomic regions, we established a Sequential-ChIP (Seq-ChIP) system. The scheme of our method is shown inFig. 1. The order of thefirst and second ChIP antibodies can be reversed in this Seq-ChIP technique, to further validate the presence of SU-MOylated MeCP2 at regulatory regions.
3.2. MeCP2 and SUMO1 occupy several genomic regions targeted by MeCP2
First, we checked several known MeCP2 targets for individual MeCP2 and SUMO1 binding using quantitative ChIP (qChIP). Mouse hippocampal neurons were subjected to qChIP using anti-SUMO1, anti-MeCP2, and control IgG antibodies. We then tested the guanidinoacetate methyltransferase (Gamt), the myocyte en-hancer factor 2C (Mef2c), and the opioid receptor kappa 1 (Oprk1) promoters for SUMO1 and MeCP2 occupancy. MeCP2 is an acti-vator at both the Gamt and Oprk1 genes[14]. In contrast, MeCP2 is a repressor for neuronal Mef2c expression [14,21]. As shown in Fig. 2, the Gamt, Mef2c, and Oprk1 promoters demonstrate occu-pancy for both the SUMO1 and MeCP2 proteins.
3.3. Identification of SUMOylated MeCP2 at the Gamt promoter Next, we used our Seq-ChIP technique to determine the pro-moters occupied by SUMOylated MeCP2 in HT22 cells. We per-formed Seq-ChIP using anti-MeCP2 as the first ChIP antibody, followed by anti-SUMO1 as the second ChIP antibody. As shown in Fig. 3A, only the Gamt promoter is occupied by SUMOylated MeCP2. The Mef2c and Oprk1 promoters do not show an enrich-ment of SUMOylated MeCP2. We then used Seq-ChIP with anti-SUMO1 as thefirst and anti-MeCP2 as the second ChIP antibody. In agreement with what we observed above, SUMOylated MeCP2 is detected at the Gamt promoter (Fig. 3B). Taken together, SUMOy-lated MeCP2 occupancy at the Gamt promoter is verified using Seq-ChIP with two different antibody schemes. These data suggest that SUMOylated MeCP2 is present at specific genomic regions in neurons.
4. Discussion
In this study, we developed a Seq-ChIP method to detect in vivo SUMOylated MeCP2 at neuronal regulatory regions (Fig. 1). We tested several known MeCP2 gene targets (Gamt, Mef2c, and Oprk1) in our assay. Gamt encodes the guanidinoacetate N-me-thyltransferase enzyme, important for creatine production, a
Table 2
Composition of Seq-ChIP reaction.
DNA template 3ml (of the 60 ml) (Input or ChIP'ed samples)
10ml 2 PCR Master Mix 2ml 0.2 mM Forward Primer 2ml 0.2 mM Reverse Primer ddH2O variable volume (μl)
20μl Total volume
Fig. 1. Scheme of Sequential-Chromatin immunopreciptation (Seq-ChIP) proce-dure. Chromatin is prepared from neurons. After chromatin shearing, thefirst ChIP antibody (α-MeCP2) is immunoprecipitated followed by the addition of the second ChIP antibody (α-SUMO1). The order of the antibody addition can be reversed. The crosslinks are reversed and the target genomic regions are isolated and char-acterized using PCR for the presence of the SUMOylated MeCP2.
Table 1
Sequences of PCR primers.
Gamt Chip Forward: 5′-ACGGCTCAGCCACGGAG-3′ Gamt Chip Reverse: 5′-TTGCACTCAAGTGGGTGGG-3′ Mef2c Chip Forward: 5′-CACTTGAGCACACGCGTACA-3′ Mef2c Chip Reverse: 5′-ACCCACACAGAACCTTCAAAGTC-3′ Oprk1 Chip Forward: 5′-GCACCTTGCTGATCCCAAAC-3′ Oprk1 Chip Reverse: 5′-CCACGTTCCTGATGCTCAATT-3′
crucial component for the production and storage of energy. MeCP2 has been shown to activate the Gamt gene[14]. Mutations in the Gamt gene are associated with severe intellectual disability, an absence or impaired speech, and epilepsy exemplifying the need for large amounts of energy in the brain[22]. Indeed, each of these symptoms are present in RTT patients. MeCP2 has been shown to repress the Mef2c gene[14,21]. Mef2c is a member of the myocyte enhancer factor 2 (MEF2) transcription factor family and is a mediator of epigenetic regulatory mechanisms[23]. Mef2c is essential for neurodevelopment and function including neuro-genesis, neuronal migration, and synaptic plasticity [23,24]. Mef2c-related disorders include severe intellectual disability with an absence of speech, limited mobility, hypotonia, and seizures [25]. All of these clinical presentations resemble those observed in RTT patients. MeCP2 activates the Oprk1 in neurons [14]. The
Oprk1 gene encodes a
κ
-Opioid receptors, known to modulate neurotransmission in brain circuits. Mutations of Oprk1 are im-plicated in depression and anxiety [26]. Importantly, anxiety be-haviors have long been observed in RTT patients[27].Our data reveals that although MeCP2 occupies the Gamt, Mef2c, and Oprk1 promoters; only SUMOylated MeCP2 binds the Gamt promoter. These results suggest that SUMOylated MeCP2 is targeted to certain promoters in a context-dependent manner. MeCP2 has previously been shown to activate the Gamt promoter. Our results suggest that SUMOylated MeCP2 at Gamt is an acti-vation mark for this regulatory region. In summary, the technique described here can be utilized to detect SUMOylated MeCP2 at specific loci. It can be modified to isolate other also SUMOylated transcription and chromatin factors.
Acknowledgment
We thank Drs. Yasunao Kamikawa and Wilfredo Mellado for critically reading this manuscript. Work in the Donohoe lab is supported by National Institutes of Health (1R01MH090267), the Burke Foundation, the Thomas and Agnes Carvel Foundation, and the Eisenberg Ahsen Foundation.
Appendix A. Transparency Document
Transparency Document associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bbrep. 2016.01.014.
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