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Blood, Vol. 113, Issue 20, 4866-4874, May 14, 2009
The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors
Blood Perez-Campo et al.
113: 4866
Supplemental materials for: Perez-Campo et al
Files in this Data Supplement:
- Table S1. Seven mice per genotype at 12 to 14 weeks of age (PDF, 37 KB) -
P values of the significance of the difference in cellularity observed between HAT+∕+ and HAT−∕− mice are indicated.
- Table S2. 12 mice from the same offspring at 14 weeks of age were used to calculate these values (PDF, 56 KB) -
HAT+∕+ n=5, HAT+∕− n=4, and HAT−∕− n=3. P values of the significance of the difference between HAT+∕+ and HAT−∕− mice in percentage of cell populations are indicated.
- Table S3. The fetal liver of 39 mice embryos of 13.5 dpc were used to calculate this values (PDF, 54.9 KB) -
HAT+∕+ n=7, HAT+∕− n=24, and HAT−∕− n=8. P values of the significance of the difference between HAT+∕+ and HAT−∕− mice in percentage of cell populations are indicated.
- Figure S1. Generation of a HAT−∕− MOZ ES cell line (JPG, 136 KB)
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(A) Graphic representation of the MOZ protein showing the different protein domains. H15 domain (H), PHD plant homeodomain-linked zinc finger (P), Basic domain (B), MYST domain containing the nucleosome binding domain (NBD) and the histone acetyl transferase (HAT) domain, glutamate-aspartate rich region (ED), the serine (S) and methionine (M) rich regions and the proline and glutamine (PQ) rich region. Grey bars show regions interacting with Runx1. (B) The arms of the targeting vector were isolated from 129/Ola strain BACs. The final targeting vector consisted of (1) Diphteria Toxin gene for negative selection; (2) a long arm of homology of 4105 bp encompassing exons 9, 10, and exon 11, exon 11 carrying the mutation of the MOZ gene; (3) a LoxP flanked Thymidine-kinase/Neomycine resistance expression cassette and (4) a short arm of homology 1188 bp long. A detailed description of all cloning steps giving rise to the final targeting vector can be obtained directly from F.P.C. B (BamHI), X (XbaI), S (SalI), Sp (SpeI), and A (ApaI). Mouse embryonic stem cells (E14.1, 129/Ola) were electroporated with the ApaI-linearised targeting vector. Clones were screened by PCR for the correct recombination at the level of the short arm of homology. Correctly targeted clones were confirmed at the level of the long arm of homology by Southern blot analysis using the 5′ probe. Out of 384 selected colonies, three correctly targeted clones were identified (+∕−TkNeo). Two positive clones were transiently transfected with a modified Cre recombinase expression vector to excise the TkNeo cassette (+∕−). The second copy of the gene was targeted (−∕−TkNeo) and the TkNeo cassette was removed in the same way (−∕−). Each step of the process was confirmed by Southern blot analysis using either the 5′ or the 3′ probe. Excision of the TkNeo cassette was confirmed by PCR with oligonucleotides F61/F2. (C) Chromatogram representing the sequence obtained from a HAT+∕+ and a HAT−∕− cell line using oligonucleotides F33/F2. (D) Expression of the mutated MOZ protein in HAT+∕+, HAT+∕−, and HAT−∕− embryos. Protein extracts were prepared from 14.5 embryos and the western-blot was revealed with a monoclonal antibody against MOZ.
- Figure S2. Acetylation of free histones or nucleosomes by HAT+∕+ and HAT−∕− MOZ (JPG, 55.9 KB)
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Wild type or mutated (G657E) MYST domain of MOZ was expressed in bacteria and purified. Acetylation of either free histones or nucleosomes by HAT+∕+ MOZ and HAT−∕− MOZ. Additional control consists of MOZ expressed in a baculoviral system. Coomassie staining of the histones is shown.
- Figure S3. Survival curve of MOZ HAT+∕+, HAT+∕−, and HAT−∕− mice (JPG, 32.6 KB)
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Kaplan-Meier analysis of the survival rate of for HAT+∕+, HAT+∕−, and HAT−∕− mice. The survival rate was calculated on a cohort of 118 mice.
- Figure S4. Defective B-cell maturation in HAT−∕− mice (JPG, 77.9 KB)
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Fluorescent activated cell analysis of B-cell populations in bone marrow and spleen of two weeks old (A) and seven months old (B) littermates.
- Figure S5. Antibody response of HAT−∕−, HAT+∕−, and HAT+∕+ mice to DNP-KLH immunisation (JPG, 75.4 KB)
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Two months old HAT−∕−, HAT+∕−, and HAT+∕+ MOZ mice were immunised, bled at week 2, boosted at week 3 and bled at week 4. (A) Relative titers of IgM against DNP in pre-immune (Pre), week 2 (W2), and week 4 (W4) serums. (B) Relative titers of IgG against DNP in pre-immune (Pre), week 2 (W2), and week 4 (W4) serums. The antibody titer of each mouse is represented. Empty rectangles indicate the mean value. (HAT+∕+ n=9, HAT+∕− n=6, HAT−∕− n=4).
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