1. Cyclin D–Cdk4 is regulated by GATA-1 and required for megakaryocyte growth and polyploidization
by Andrew G. Muntean, Liyan Pang, Mortimer Poncz, Steven F. Dowdy, Gerd A. Blobel, and John D. Crispino
Blood
Volume 109(12):
June 15, 2007
©2007 by American Society of Hematology

2. Megakaryocytes lacking GATA-1 show defects in both polyploidization and accumulation of cell mass.
Megakaryocytes lacking GATA-1 show defects in both polyploidization and accumulation of cell mass. (A) Ploidy analysis of wild-type and GATA-1 KD bone marrow progenitors differentiated into megakaryocytes for 3 days. The cells were stained with anti-CD41 and DAPI to detect DNA content. Gates indicate the percentage of cells that is 8N or greater. One representative of 3 experiments is shown. (B) Cell size analysis of wild-type and GATA-1 KD cells within the 16N ploidy class were compared in a forward scatter–height histogram. The dotted line indicates the wild-type cells and the solid line, the GATA-1 KD cells. Mean FSC-A for WT and G1KD megakaryocytes was 740 and 668, respectively; confidence in difference is 99.9% based on Kolmogorov-Smimov test. One representative of 3 experiments is shown. (C) mRNA levels were determined by real-time quantitative RT-PCR of RNA isolated from wild-type and GATA-1 KD, BSA gradient–purified megakaryocytes. GATA-1 KD mRNA levels are shown relative to wild type, which were arbitrarily set to one. The means ± 1 SD are shown. Asterisks indicate P values determined by Student t test to be .05 or less. One representative of 2 experiments, each in triplicate, is shown.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology

3. GATA-1 binds and activates the cyclin D1 promoter, while FOG-1 represses its transcription.
GATA-1 binds and activates the cyclin D1 promoter, while FOG-1 represses its transcription. (A) The pGL3-basic construct containing 2 kb of the cyclin D1 promoter upstream of the luciferase gene. Putative GATA-1–binding sites are indicated as diamonds, and dotted vertical lines indicate serial deletions made of the cyclin D1 promoter. D1p-1 is the full 2-kb fragment, and D1p-7 is the shortest 76-bp fragment. (B) Luciferase assay performed by transient transfection of NIH3T3 cells with the indicated micrograms of pcDNA3 vector containing GATA-1, GATA-1s, GATA-1 V205G, and FOG-1. Luciferase levels are shown as fold change relative to cells transfected with reporter construct alone. The total amount of DNA transfected was kept constant by transfection with empty pcDNA3 vector. One representative of 2 experiments, each in triplicate, is shown. (C) Luciferase assay performed as in panel B with luciferase vectors containing mutations in the 3 putative GATA-binding sites within the last 76 base pairs. Mutation of the first GATA site (D1p-7 mut-1) showed reduced response to GATA-1, whereas mutation of 2 other putative GATA sites (D1p-7 mut-2 and D1p-7 mut3) does not alter GATA-1 activation of the cyclin D1 promoter. D1p-1 and D1p-7 are described in panel A. One representative of 3 experiments, each in triplicate, is shown. (D) Chromatin immunoprecipitation assay performed in primary mouse fetal liver–derived megakaryocytes with primer sets directed toward the functional GATA-binding site identified in luciferase assays, as well as, downstream (control primers 1) and upstream (control primers 2) primer sets. Control rat IgG and anti–GATA-1 (N6) antibodies were used for immunoprecipitation and indicate GATA-1 enrichment at the functional GATA-1–binding site in the cyclin D1 promoter. P values were generated using the Student t test and are as follows: *P ≤ .04; #P ≤ .01. One representative of 2 experiments, each in triplicate, is shown. (E) Real-time quantitative PCR data showing expression levels of cyclin D1 mRNA following reconstitution of GATA-1 KD megakaryocytes with the empty MigR1 retroviral vector or containing wild-type GATA-1, GATA-1s, or GATA-1 V205G. Values are shown relative to empty MigR1-infected GATA-1 KD expression levels. P values were generated using the Student t test and are as follows: *P ≤ .006; #P ≤ Changes between expression values of MigR1 and MigR1-G1s were not statistically significant. One representative of 3 experiments, each in triplicate, is shown. For each study in B-E, the means ± 1 SD is shown.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology

4. Ectopic expression of cyclin D1 and Cdk4 results in an increase in polyploidization and cell size of GATA-1 KD megakaryocytes.
Ectopic expression of cyclin D1 and Cdk4 results in an increase in polyploidization and cell size of GATA-1 KD megakaryocytes. (A-D) Ploidy analysis of BSA gradient–purified GATA-1 KD megakaryocytes was performed using flow cytometry of DAPI-stained cells. Cells were doubly infected with the MigR1 containing EGFP and pCS containing hCD25. Cyclin D1 was cloned into MigR1 and Cdk4 was cloned into pCS allowing for double infection and expression of cyclin D1 alone, Cdk4 alone, or cyclin D1 and Cdk4. Double-infected cells were selected through gating of EGFP-positive and CD25 double-positive cells. Bars indicate the percentage of cells that have DNA content of 8N or greater. One representative of 3 experiments is shown. (E-H) Cell size analysis performed using flow cytometry of the infected cells described in panels A-D. Double-positive cells were analyzed using histograms of forward scatter–area to compare cell size. Values for the mean FSC-A are indicated for each population of doubly infected cells. One representative of 3 experiments is shown.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology

5. Ectopic cyclin D1–Cdk4 does not rescue megakaryocyte terminal differentiation. mRNA was extracted from BSA gradient–purified megakaryocytes derived from wild-type fetal livers or G1KD fetal livers infected with cyclin D1 and Cdk4 encoding retroviruses (desc...
Ectopic cyclin D1–Cdk4 does not rescue megakaryocyte terminal differentiation. mRNA was extracted from BSA gradient–purified megakaryocytes derived from wild-type fetal livers or G1KD fetal livers infected with cyclin D1 and Cdk4 encoding retroviruses (described in Figures 3 and 5). Expression levels of β1-tubulin, NF-E2, and PF4 are shown relative to wild-type expression levels. The mean ± 1 SD is shown for each study. As shown, overexpression of cyclin D1, Cdk4, or both does affect the expression of these genes.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology

6. Loss of cyclin D1 alone does not significantly inhibit polyploidization.
Loss of cyclin D1 alone does not significantly inhibit polyploidization. (A) Megakaryocytes were derived from wild-type and cyclin D1−/− bone marrow (BM) and fetal liver (FL). After enrichment on a BSA gradient, cells were stained with DAPI for DNA content and analyzed by flow cytometry. The percentages of cells with DNA content of 8N or more are shown. A reproducible reduction in polyploid cells was observed in the cyclin D1–deficient megakaryocytes. One representative of 2 experiments is shown. (B) Real-time quantitative PCR for cyclins D1, D2, and D3. mRNA was isolated from BSA gradient–purified megakaryocytes generated from wild-type and cyclin D1−/− bone marrow–derived megakaryocytes. Expression of cyclins D1, D2, and D3 were determined using primer sets specific for each transcript. Expression levels are shown relative to wild-type mRNA levels. For each study, the mean ± 1 SD is shown. Asterisks indicate P values determined by Student t test to be of .05 or less. One representative of 2 experiments, each in triplicate, is shown.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology

7. p16 inhibits polyploidization.
p16 inhibits polyploidization. (A) qRT-PCR of mRNA isolated from wild-type and GATA-1 KD megakaryocytes for p16, p21, and p27. GATA-1 KD mRNA levels are shown relative to wild-type expression. P values determined using Student t test. Asterisk indicates P ≤ .01. One representative of 2 experiments, each in triplicate, is shown. For each study, the mean ± 1 SD is shown. (B) Purified, untreated wild-type megakaryocytes were differentiated for 3 days and stained with DAPI. (C,E) Wild-type megakaryocytes were treated with either wild-type TAT-p16 fusion peptide or a charge-matched mutant TAT-p16 peptide at a concentration of 1 μM. Peptide was added 3 times during the second day of differentiation due to instability of the TAT peptides. Cells were fixed and stained with DAPI on day 3 of differentiation. (D,F) Wild-type megakaryocytes were treated with the peptides used in panels C and E at a concentration of 10 μM. Cells were fixed and stained with DAPI on day 3 of differentiation. Gates indicate the percentage of cells at a ploidy class of 8N or greater. One representative of 2 experiments is shown.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology

8. Dual requirements for GATA-1–dependent megakaryocyte development.
Dual requirements for GATA-1–dependent megakaryocyte development. We conclude that GATA-1 controls 2 sets of genes: One group, including cyclin D1, regulates growth and polyploidization. A second class of targets, which includes genes such as NF-E2, PF4, and c-mpl, drives terminal maturation and platelet biosynthesis. The observation that ectopic cyclin D1–cdk4 can cause increased size and DNA content without concomitant expression of NF-E2, PF-4, and c-mpl indicates that the 2 processes (ie, polyploidization and terminal maturation) can be uncoupled from one another.
Andrew G. Muntean et al. Blood 2007;109:
©2007 by American Society of Hematology