This is a protocol preprint. Preprints are preliminary reports that have not undergone peer review. They should not be considered conclusive, used to inform clinical practice, or referenced by the media as validated information.
Method Article
Ubp-M knockdown, RT-PCR and ChIP assays
Hengbin Wang
The University of Alabama at Birmingham
Corresponding Author
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Posttranslational histone modifications play important roles in regulating chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. In order to identify the deubiquitinase for histone H2A, we developed an in vitro deubiquitination assay employing uH2A (ubiquitinated H2A)-containing mononucleosomes as substrates.
Hela cells
Effectene (Qiagen)
puromycin
siRNA oligonucleotides
lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A]
dilution buffer [1 % Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A]
Ubp-M knock-down cell lines
Ubp-M knock-down cell lines were generated using a vector-based knockdown strategy as previously described (Wang et al., 2004).
- Transfect vectors expressing the hairpin RNAs as listed below into HeLa cells with Effectene (Qiagen).
Select transfected cells in the presence of 2 ug/ml puromycin. 3. Amplify selected clones and analyse the efficiency of Ubp-M knockdown as well as its effect on cellular uH2A levels were analyzed as described (Wang et al., 2004).
The DNA sequence encoding the stem hairpin RNA for Ubp-M: 5’-ACCAGTGCTTAGAGAACTATCTCTTGAATAGTTCTCTAAGCACTG
GT-3’ and 5’-ACCAGTGCTTAGAGAACTATTCAAGAGATAGTTCTCTAAGCA
CTGGT-3’.
For transient knockdown, purchase siRNA oligonucleotides against Ubp-M from Invitrogen in a purified, annealed duplex form and transfect into cells as described (Wei et al., 2006). The sequences for these siRNA are as follows:
siRNA1
5’-CCUCCUGUUCUUACUCUUCAUUUAA-3’
5’-UUAAAUGAAGAGUAAGAACAGGAGG-3’ and
siRNA2
5’-CCGGAAAUCUUAGAUUUGGCUCCUU-3’
5’-AAGGAGCCAAAUCUAAGAUUUCCGG-3’
RNAi resistant Ubp-M constructs
- To construct RNAi resistant Ubp-M constructs, introduce a point mutation into targeting siRNA nucleotide acid sequence but without changing the encoded amino acid.
- Perform site-directed mutagenesis as described (Wang et al., 2004).
- Use Primers F1: 5’-AAGGATCCGGAAAGAAACGGACAAAGGGAAAAACTGTTCC-3’ and R1: 5’-TTCTTTTAGGAGCTCCCTGAGTACAGGAGTTTGTGACAA-3’ to amplify the N-terminal portion.
- Use Primers F2: 5’-CAGAACTTGTCACAAACTCCTGTACTCAGGGAGCTCCTAAA-3’ and R2: 5’-GATCTAGATTACAGTATTCTCTCATAAAATAGGAGGTACG-3’ to amplify the C-terminal portion.
- Amplify the full length Ubp-M construct using primers F1 and R2 and inserted into pcDNA3 vector (Invitrogen).
- Verify the mutation by DNA sequencing.
- Perform transfection with Effectene (Qiagen) following the manufacturer’s instruction.
- 72 hrs after transfection, seed and transfect cells with the same plasmid again.
- Collect cells six days after the first transfection and analyze by RT-PCR as described (Wei et al., 2006).
Primers for RT-PCR amplification:
HoxA9: 5’-ACGTGGACTCGTTCCTGCTG-3' and
5’-AGGTTTAATGCCATAAGGCCG-3’HoxB1: 5’-TCAGGCGGTTGACAGCTATG-3’ and
5’-ATGCTGCGGAGGATATGGC-3’HoxC5: 5’-TGTGGGAACTATGGATCGGC-3’ and
5’-ACGGGTAAATCTGTGGCGG-3’HoxD10: 5’-GATTCCTTGATCAGTGCCTGC-3’ and
5’-GCCGAAATGAGTTTGTTGCG-3’GAPDH: 5’-ACCACAGTCCATGCCATCAC-3’ and
5’-TCCACCACCCTGTTGCTGTA-3’ChIP and double ChIP
Perform ChIP and double ChIP as described with modifications as the following 5.
- Lyse cells in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A] and dilute 10 times with dilution buffer [1 % Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A] for immunoprecipitation.
- Perform PCR reactions with the following primers: A: 5'-CTTCATTCAGCTTTGGGCACGCTT-3' and
5'-GGCCTGGTTGAAACAAGCGTTGAA-3'B: 5’-CCCAGAATGCTGAGGCGCTTTAAT-3’ and
5’-ACCACCACTACCACCATGGAAACT-3’C: 5’-TGGTTCTTTAATGAGCCGGACCAC-3’ and
5’-TGTTGCCACACACGGGAAGATACT-3’D: 5’-TGTCCTTCTTGGCCCAGTCAGTTT-3’ and
5’-TAAGAAGCGGCCAAGGTGTCTCTA-3’E: 5’-TAGAGACACCTTGGCCGCTTCTTA-3’ and
5’-CACGGACAACAGCGACATCTACTA-3’ F: 5’-ATGTCCTTTCCCAACAGCTCTCCT-3’ and
5’-AAATATCCAGGGACGGGAACCTCA-3’ G: 5’-TGGTTGGTGCTTGAGTTGGGAAAC-3’ and
5’-TTGCCTGGGTATTGTTGCATTCCC-3’ H: 5’-TCACCGACAGGCAGGTCAAGATTT-3’ and
5’-TGGATGGATGGATGGATGGATGCT-3’- In the PCR reaction, include one cycle of 5 min at 95°C followed by 33 cycles of 95°C for 30 sec, 68°C for 30 sec, 72°C for 30 sec for regions A, B, C, E, F and G. For D, use an annealing temperatures of 66°C, and for H, 60°C. Perform all PCR reactions within the range of linear amplification.
Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873-878 (2004).
Wei, J., Zhai, L., Xu, J. & Wang, H. Role of Bmi1 in H2A Ubiquitylation and Hox Gene Silencing. J. Biol. Chem. 281, 22537-22544 (2006).
Version History
CURRENT STATUS: Posted
Version 1
Posted 08 Oct, 2007
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Associated Publications
Regulation of cell cycle progression and gene expression by H2A deubiquitination
by Joo HY, Zhai L, Yang C, +4
Nature (14 September, 2007)
Method Article
Ubp-M knockdown, RT-PCR and ChIP assays
Hengbin Wang
The University of Alabama at Birmingham
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 08 Oct, 2007
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Posttranslational histone modifications play important roles in regulating chromatin structure and function. One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Although the recent identification of the ubiquitin ligase for histone H2A has revealed important roles for H2A ubiquitination in Hox gene silencing as well as in X inactivation, the enzyme(s) involved in H2A deubiquitination and the function of H2A deubiquitination are not known. In order to identify the deubiquitinase for histone H2A, we developed an in vitro deubiquitination assay employing uH2A (ubiquitinated H2A)-containing mononucleosomes as substrates.
Hela cells
Effectene (Qiagen)
puromycin
siRNA oligonucleotides
lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A]
dilution buffer [1 % Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A]
Ubp-M knock-down cell lines
Ubp-M knock-down cell lines were generated using a vector-based knockdown strategy as previously described (Wang et al., 2004).
- Transfect vectors expressing the hairpin RNAs as listed below into HeLa cells with Effectene (Qiagen).
Select transfected cells in the presence of 2 ug/ml puromycin. 3. Amplify selected clones and analyse the efficiency of Ubp-M knockdown as well as its effect on cellular uH2A levels were analyzed as described (Wang et al., 2004).
The DNA sequence encoding the stem hairpin RNA for Ubp-M: 5’-ACCAGTGCTTAGAGAACTATCTCTTGAATAGTTCTCTAAGCACTG
GT-3’ and 5’-ACCAGTGCTTAGAGAACTATTCAAGAGATAGTTCTCTAAGCA
CTGGT-3’.
For transient knockdown, purchase siRNA oligonucleotides against Ubp-M from Invitrogen in a purified, annealed duplex form and transfect into cells as described (Wei et al., 2006). The sequences for these siRNA are as follows:
siRNA1
5’-CCUCCUGUUCUUACUCUUCAUUUAA-3’
5’-UUAAAUGAAGAGUAAGAACAGGAGG-3’ and
siRNA2
5’-CCGGAAAUCUUAGAUUUGGCUCCUU-3’
5’-AAGGAGCCAAAUCUAAGAUUUCCGG-3’
RNAi resistant Ubp-M constructs
- To construct RNAi resistant Ubp-M constructs, introduce a point mutation into targeting siRNA nucleotide acid sequence but without changing the encoded amino acid.
- Perform site-directed mutagenesis as described (Wang et al., 2004).
- Use Primers F1: 5’-AAGGATCCGGAAAGAAACGGACAAAGGGAAAAACTGTTCC-3’ and R1: 5’-TTCTTTTAGGAGCTCCCTGAGTACAGGAGTTTGTGACAA-3’ to amplify the N-terminal portion.
- Use Primers F2: 5’-CAGAACTTGTCACAAACTCCTGTACTCAGGGAGCTCCTAAA-3’ and R2: 5’-GATCTAGATTACAGTATTCTCTCATAAAATAGGAGGTACG-3’ to amplify the C-terminal portion.
- Amplify the full length Ubp-M construct using primers F1 and R2 and inserted into pcDNA3 vector (Invitrogen).
- Verify the mutation by DNA sequencing.
- Perform transfection with Effectene (Qiagen) following the manufacturer’s instruction.
- 72 hrs after transfection, seed and transfect cells with the same plasmid again.
- Collect cells six days after the first transfection and analyze by RT-PCR as described (Wei et al., 2006).
Primers for RT-PCR amplification:
HoxA9: 5’-ACGTGGACTCGTTCCTGCTG-3' and
5’-AGGTTTAATGCCATAAGGCCG-3’HoxB1: 5’-TCAGGCGGTTGACAGCTATG-3’ and
5’-ATGCTGCGGAGGATATGGC-3’HoxC5: 5’-TGTGGGAACTATGGATCGGC-3’ and
5’-ACGGGTAAATCTGTGGCGG-3’HoxD10: 5’-GATTCCTTGATCAGTGCCTGC-3’ and
5’-GCCGAAATGAGTTTGTTGCG-3’GAPDH: 5’-ACCACAGTCCATGCCATCAC-3’ and
5’-TCCACCACCCTGTTGCTGTA-3’ChIP and double ChIP
Perform ChIP and double ChIP as described with modifications as the following 5.
- Lyse cells in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A] and dilute 10 times with dilution buffer [1 % Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.1 mM PMSF, 1 ug/ml aprotinin, 1 ug/ml leupeptin and 1 ug/ml pepstatin A] for immunoprecipitation.
- Perform PCR reactions with the following primers: A: 5'-CTTCATTCAGCTTTGGGCACGCTT-3' and
5'-GGCCTGGTTGAAACAAGCGTTGAA-3'B: 5’-CCCAGAATGCTGAGGCGCTTTAAT-3’ and
5’-ACCACCACTACCACCATGGAAACT-3’C: 5’-TGGTTCTTTAATGAGCCGGACCAC-3’ and
5’-TGTTGCCACACACGGGAAGATACT-3’D: 5’-TGTCCTTCTTGGCCCAGTCAGTTT-3’ and
5’-TAAGAAGCGGCCAAGGTGTCTCTA-3’E: 5’-TAGAGACACCTTGGCCGCTTCTTA-3’ and
5’-CACGGACAACAGCGACATCTACTA-3’ F: 5’-ATGTCCTTTCCCAACAGCTCTCCT-3’ and
5’-AAATATCCAGGGACGGGAACCTCA-3’ G: 5’-TGGTTGGTGCTTGAGTTGGGAAAC-3’ and
5’-TTGCCTGGGTATTGTTGCATTCCC-3’ H: 5’-TCACCGACAGGCAGGTCAAGATTT-3’ and
5’-TGGATGGATGGATGGATGGATGCT-3’- In the PCR reaction, include one cycle of 5 min at 95°C followed by 33 cycles of 95°C for 30 sec, 68°C for 30 sec, 72°C for 30 sec for regions A, B, C, E, F and G. For D, use an annealing temperatures of 66°C, and for H, 60°C. Perform all PCR reactions within the range of linear amplification.
Wang, H. et al. Role of histone H2A ubiquitination in Polycomb silencing. Nature 431, 873-878 (2004).
Wei, J., Zhai, L., Xu, J. & Wang, H. Role of Bmi1 in H2A Ubiquitylation and Hox Gene Silencing. J. Biol. Chem. 281, 22537-22544 (2006).
Associated Publications
Regulation of cell cycle progression and gene expression by H2A deubiquitination
by Joo HY, Zhai L, Yang C, +4
Nature (14 September, 2007)