In the previous section I focused on the purification and analysis of cryptochrome
associated proteins possibly involved in ubiquitination and deubiquitination. In Part III, I shall present preliminary results obtained for a transcriptional corepressor detected in CRY1 complexes.
CRY1 associated factors included KRIP1, also called KAP1 or TIF1β. KRIP1 is a universal corepressor of Krüppel associated box zinc finger proteins (KRAB-ZFP)
(Friedman et al., 1996; Kim et al., 1996; Moosmann et al., 1996) and functions by recruiting a set of factors involved in gene silencing, such as the histone deacetylase complexes NuRD and N-coR1, histone methyltransferase SETDB1, and heterochromatin protein 1 (HP1) (Lechner et al., 2000; Schultz et al., 2002; Schultz et al., 2001; Underhill et al., 2000). To determine whether KRIP1 regulates circadian transcription I generated two short hairpin RNAs (shRNA) against the KRIP1 transcript. In order to determine the efficiency of KRIP1 depletion we generated NIH-3T3 cell lines stably expressing the shRNAs. Extracts from these cell lines were analyzed by western blotting using anti- KRIP1 antibody. Efficient depletion was achieved in both shRNA1 and shRNA2 expressing cell lines, indicated by the markedly reduced protein levels of KRIP1
compared to the parental NIH-3T3 cells (Fig 1A). I then transfected these cell lines with Bmal1-luc and measured bioluminescence in real time for 68 hours. The depletion of KRIP1 by shRNA1 resulted in a 2-3 fold increase in Bmal1-luc expression (Fig 1B).
Similarly, I observed a 7-10 fold increase in Bmal1-luc expression in the stable cell line expressing shRNA2. In contrast, neither of the two shRNAs changed the expression of CMV-driven luciferase reporter (CMV-luc) (Fig 1C). These results suggested that KRIP1 might be involved in the negative regulation of Bmal1 expression.
Figure 1.Circadian reporter gene expression in SKP1-depleted cells
(A) Whole cell extracts from NIH-3T3 cells and NIH-3T3 cell stably expressing shRNAs against KRIP1 were analyzed by western blotting using anti-KRIP1 antibody and anti-U2AF65 antibody as a loading control. (B) NIH-3T3 cells stably expressing shRNAs against KRIP1 were transfected with Bmal1 promoter driven luciferase reporter (Bmal1-luc).Cells were synchronized with a 100 nM dexamethasone treatment for an hour and luciferase expressions were measured in live-cell luminometer for 68 hours..(C) NIH-3T3 cells were transfected with KRIP1 shRNAs together with CMV-driven luciferase reporter. Cells were synchronized with 100 nM dexamethasone treatment for one hour, and luciferase expression was measured in a live-cell luminometer for 60 hours.
To elucidate the association of clock proteins and KRIP1, I size-fractionated cellular extracts from NIH-3T3 by using gel filtration. The majority of CRY1 and CRY2 proteins migrated with complexes with sizes between 2 MDa and 660 kDa at this time point (Fig 2A). CLOCK also elutes in these fractions but within a much narrower range; complexes with sizes from 1 MDa to 660 kDa were observed. KRIP1 was present in complexes of sizes almost indistinguishable from those of CLOCK. Because the majority of KRIP1 protein was present in complexes with sizes close to those containing CRYs and CLOCK,
wb: αKRIP1
it was possible that they existed in same complex. We also examined the size distribution of SKP1 complexes. SKP1 was present in complexes of varying sizes, and also in the fractions where CRYs, CLOCK and KRIP1 were recovered. Next I examined the
temporal accumulation of KRIP1 protein. Mice were sacrificed at four hour intervals, and liver nuclear proteins were analyzed by western blotting. The results showed that KRIP1 accumulation was constant over the circadian day (Fig 2B).
Figure 2. Characterization of KRIP1, SKP1 and circadian protein complexes
(A) NIH-3T3 cells were harvested four hours after one hour treatment with 50 % horse serum. Whole cell extract was fractionated by Superose 6 gel filtration. Fractions were analyzed by western blotting with the indicated antibodies. Approximate size estimates were obtained by comparing the elution profiles of Blue Dextran (MW 2-3 MDa excluded), thyroglobulin (660 kDa) and β-galactosidase (118 kDa, below column resolution) (B) Liver nuclear extracts from mice sacrificed every four hours were analyzed by western blotting using anti-KRIP1 antibody and anti-U2AF65 antibody as a loading control.
In order to verify the interaction of KRIP1 and CRY1, I cloned full length KRIP1 cDNA into hemagglutinin–tag containing expression vector (pCI-HA-KRIP1) and expressed HA-KRIP1 in the His-CRY1-Flag cell line. While PER1 readily coimmunoprecipitated with His-CRY1-Flag, I failed to observe any coimmunoprecipitation of KRIP1 under these experimental conditions (Fig 3A). Since it was possible that the HA-tag in my KRIP1 construct interfered with the interaction of KRIP1 and CRY1, I
wb: αKRIP1
immunoprecipitated endogenous CRY1 from rat nuclear extracts using anti-CRY1 antibody. PER1 coimmunoprecipitated with CRY1 at both time points; ZT02 and ZT14 (Fig 3B). Coimmunoprecipitated CLOCK and BMAL1 were also observed at both time points, although a stronger interaction was seen at ZT02. This was consistent with earlier observations (Asher et al., 2008). Disappointingly, I did not observe
coimmunoprecipitated KRIP1 at either ZT02 or at ZT14. Perhaps, the interaction between KRIP1 and CRY1 might have been transient and only visible at very specific timepoint. Hence, a more rigorous analysis of CRY1 immunoprecipitates around the clock might be required to validate the association of CRY proteins with KRIP1.
Figure 3. Immunoprecipitation of CRYs and western analysis of associated proteins
(A) Whole cell extracts from CRY1 (left) and CRY2 (right) transgenic cell lines transfected with a HA-tagged KRIP1 were immunoprecipitated with anti-FLAG antibody. 5% of the input and the
immunoprecipitates were analyzed by western blotting using the indicated antibodies. (B) Liver nuclear extracts from rats harvested at indicated time points were subjected to immunoprecipitation using anti-CRY1 antibody. The inputs and immunoprecipitates were analyzed by western blotting using the indicated antibodies.
Materials and methods
Antibodies. Monoclonal antibodies against Flag and U2AF were purchased from Sigma and monoclonal antibodies against SKP1 and KRIP1 (KAP1) from Abcam. Anti-HA antibody 12CA5 was a kind gift from R.Loewith. Polyclonal antibodies against clock proteins were produced from rabbits by Charles River Laboratories, using E.coli-derived recombinant proteins. Antibodies were purified as described in (Brown et al., 2005).
input ip αCRY1
Plasmids. pCI-HA is a modified version of pCI and has hemagglutinin coding sequence inserted into site. pCI-HA-KRIP1 was constructed by PCR amplification of full length KRIP1 cDNA from IMAGE clone 6811294 with forward primer:
GAATTCGCCACCATGGCGGCCTCGGCGGCAGCGACT-3’ and reverse primer: 5’-GCGGCCGCTCAGGGGCCATCACCAGGGCCAGAG-3’ and insertion into the EcoRI/NotI site of pCI-HA. pBmal1-lucis described in (Brown et al., 2005). shRNAs were cloned into pSUPER-neo-gfp (Oligoengine) according to the supplier’s instructions.
KRIP1 shRNA1 and shRNA2 have the sequences 5’-AACAGTCTACTGTAATGT-3’
and 5’-TGCTCTGAACCACTTTGT-3’, respectively.
Nuclear extracts, immunoprecipitation, size fractionation and western blotting.
Liver nuclei (mouse and rat) were prepared at different circadian time points according to the method described in (Lavery and Schibler, 1993). Nuclear proteins were extracted in NUN buffer (0.3 M NaCl, 1 M urea, and 1% Nonidet P-40) for 20 min on ice.
Centrifugation for 10 min at 4°C left the core histones and histone H1 in the pellet and soluble nonhistone proteins in the supernatant. For immunoprecipitation and size
fractionation, whole cell extracts were prepared as follows. NIH-3T3 cells were harvested in PBS, suspended in an equal volume of NLB (100 mM KCl, 100 nM EDTA, 10%
glycerol, 150 nM spermine, 500 nM spermidine, 10 mM HEPES pH 7.6), and proteins were extracted by adding 3 volumes of NUN buffer (0.3 M NaCl, 1 M urea, and 1%
Nonidet P-40, 0.5 mM DTT, 0.5mM PMSF, 0.1% trasylol, 1µM pepstatin A, 1µM leupeptin, 5mM NaF) for 20 min on ice. Centrifugation for 10 mins at 4°C separated the non-soluble and soluble fraction. Size fractionations of proteins were conducted on 100 ml columns of Superose 6 (Amersham), equilibrated, and eluted in Buffer BC100 (20mM HEPES pH 7.9, 0.2mM EDTA, 20% glycerol, 100mM KCl, 0.5 mM DTT, 0.5mM
PMSF, 0.1% trasylol, 1µM pepstatin A, 1µM leupeptin, 5mM NaF). 500 µg of whole cell extract was loaded per run, and the resulting fractions were precipitated with TCA and used for Western analysis as described below. Immunoprecipitations and western blotting were performed using standard techniques.
Transient transfections and real-time bioluminescence monitoring.
For transient overexpression and knock-down assays, 3.5 cm dishes of NIH-3T3 cells were transfected with 2µg total DNA using FUGENE 6 (Roche) according to the
instructions provided by the manufacturer. Reactions contained 200 ng of the reporter pBMAL-luc, pDBP-luc, pRev-erbα-luc, or pPer2-luc, and 1µg pSUPER-neo-gfp hairpin vector, and where appropriate, 1µg of pCDNA-His-CRY1-Flag or pCDNA-His-CRY2-Flag. The remaining quantity of DNA was composed of pcDNA3.1 without insert. After three days, cells were stimulated with 100 nM dexamethasone as described in (Nagoshi et al., 2004), and cells were incubated in the presence of 0.1mM luciferin (Promega) and maintained at 37°C in a light–tight incubator. Bioluminescence was monitored
continuously using Hamamatsu photomultiplier tube detector assemblies. Photon counts were integrated over 1-min intervals.
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