1. Specific homeodomain-DNA interactions are required for HOX11-mediated transformation
by Bronwyn M. Owens, Yuan-Xiao Zhu, Ting-Chung Suen, Pei-Xiang Wang, Jack F. Greenblatt, Paul E. Goss, and Robert G. Hawley
Blood
Volume 101(12):
June 15, 2003
©2003 by American Society of Hematology

2. HOX11 represses basal transcription of TATA-containing and TATA-less promoters in HeLa cells and C2C12 cells.
HOX11 represses basal transcription of TATA-containing and TATA-less promoters in HeLa cells and C2C12 cells. (A) Transcriptional repression by HOX11 is concentration dependent. Transient transfection and CAT assays were performed in C2C12 cells and HeLa cells using pSV2CAT (2 μg) and indicated amounts of pcDNA3-HOX11. Relative CAT activity, expressed as percentage of control, is indicated. Data shown are representative of 3 independent experiments. (B) HOX11 represses transcription from different promoters. pcDNA3-HOX11 (8 μg) and reporter plasmids (2 μg) containing HSV tk (pBLCAT2), CMV (pCMVCAT), SV40 early, and rat neu (pneuCAT) promoters were cotransfected into C2C12 cells. CAT activity expressed as percentage of control. (C) HOX11 represses SV40 promoter activity in murine cells. pcDNA3-HOX11 (8 μg) and pSV2CAT (2 μg) were cotransfected into NIH3T3 cells and CAT activity measured.
Bronwyn M. Owens et al. Blood 2003;101:
©2003 by American Society of Hematology

3. HOX11-dependent repression may be mediated by interactions with components of the basal general transcription machinery.
HOX11-dependent repression may be mediated by interactions with components of the basal general transcription machinery. (A) Schematic diagram of the SV40 promoter in control and mutant reporter plasmids: enhancer region (En); GC BOX (GC); TATA-like motif (TATA). (B) Deletion (Δ) of cis-regulatory elements, including the enhancer, GC box, and TATA-like motif from the SV40 promoter, failed to abolish HOX11-mediated repression. Relative CAT activities from cotransfection of pFLAG-HOX11 and parental or mutant SV40 reporter plasmids into C2C12 cells (▪) and HeLa cells (□). Transfection assays were repeated at least 3 times. Variability between assays was not more than 15%. (C) HOX11 represses in vitro transcription driven by purified RNA polymerase II holoenzyme. In vitro transcription assays were performed using 0.3 μg reporter plasmid containing the adenovirus major late promoter driving a G-less cassette (G-less) and 5 tandem copies of the Gal4 DNA-binding site.21 Reaction conditions consisted of 5 μg purified polymerase II holoenzyme and 100 ng purified S-tagged HOX11 or PET control protein. Transcription was measured in the absence or presence of purified Gal4-VP16 proteins (50 ng). Radiolabeled RNA was resolved on a 4.5% acrylamide-6M urea denaturing gel and visualized by autoradiography.
Bronwyn M. Owens et al. Blood 2003;101:
©2003 by American Society of Hematology

4. HOX11 constructs and protein expression.
HOX11 constructs and protein expression. (A) Schematic diagram of expression constructs for FLAG-tagged HOX11 WT and mutant proteins. FLAG peptide, Gly/Pro-rich region (Gly/Pro), homeodomain (HD), Gln-rich region (Gln), and PBX interaction motif (PIM; FPWME) are indicated. (B) FLAG-tagged HOX11 proteins were detected in lysates from HeLa cells transiently transfected with pFLAG-HOX11 constructs using immunoprecipitation and Western blotting with an anti–FLAG-M5 antibody (top row, middle row) or by direct Western blotting of lysates from stably transduced NIH3T3 cells using an anti-HOX11 antibody (bottom row). C indicates control.
Bronwyn M. Owens et al. Blood 2003;101:
©2003 by American Society of Hematology

5. Multiple domains of HOX11 contribute to HOX11-mediated transcriptional regulation.
Multiple domains of HOX11 contribute to HOX11-mediated transcriptional regulation. (A) CAT assays were performed to evaluate the contributions of each domain to HOX11 repressor activity. Transient transfections were performed in HeLa cells using pcDNA3-HOX11 (8 μg) and pSV2CAT (2 μg). Thirty-six hours after transfection, CAT activities were determined and normalized to protein expression. The data represent mean values from 3 independent experiments. (B) Point mutations within the homeodomain do not inhibit HOX11 repressor function. HOX11 constructs with point mutations within helix 3 of the homeodomain (Thr47Ile and Lys55Gln) were transiently transfected into HEK 293T cells with a CMV-β-gal reporter plasmid. Data represent 3 independent experiments. (C) Specific HOX11 domains and DNA binding are required for induction of endogenous gene expression. Full-length HOX11 (HOX11) or FLAG-tagged HOX11 (HOX11 WT) and mutant constructs were stably transduced into NIH3T3 cells, and up-regulation of Aldh-1 was measured by Northern blot analysis. β-Actin mRNA served as an internal loading control.
Bronwyn M. Owens et al. Blood 2003;101:
©2003 by American Society of Hematology

6. Establishment and characterization of HOX11-immortalized hematopoietic cell lines.
Establishment and characterization of HOX11-immortalized hematopoietic cell lines. HOX11 constructs were transduced into murine BM progenitors, and cells were maintained in IL-3–containing media until lines were established (> 3 months in culture). Transduction with each construct was performed at least 3 times, with the exception of D1 and M5 (n = 2). Wright-Giemsa–stained cells generated with HOX11 WT (A), D6 (B), and Thr47Ile (C). Original magnification, × 100. (D) Western blot analysis of HOX11 protein expression in representative BM progenitor-derived cell lines using either anti-HOX11 polyclonal antibody (WT, D1, M1, M2, M3, and Thr47Ile mutants) or anti-FLAG antibody (D6 mutant). (E) Southern blot analysis of proviral copy number in 3 independently derived HOX11 WT and HOX11 D6 cell lines. Genomic DNA (10 μg), isolated from 107 cells and digested with EcoRI, was resolved on a 1% agarose gel and hybridized with a neo probe. (F) HOX11 WT cells represent a bipotential monocytic-granulocytic precursor. Three HOX11 WT and HOX11 D6 cell lines were treated with phorbol myristate acetate (PMA; 100 ng/mL). After 72 hours, surface expression of the granulocytic marker Gr-1 and the macrophage marker Mac-1 was determined by immunofluorescence flow cytometric analysis.
Bronwyn M. Owens et al. Blood 2003;101:
©2003 by American Society of Hematology

7. HOX11 mutants are expressed in nuclei of transfected cells.
HOX11 mutants are expressed in nuclei of transfected cells. Transiently transfected HEK 293T cells were cultured on fibronectin-coated coverslips and fixed in 3.7% formaldehyde. Immunohistochemical analysis was performed using 10 μg/mL anti-HOX11 polyclonal antibody and 5 μg/mL fluorescein-linked goat–anti-rabbit secondary antibody and visualized using a fluorescence microscope. Original magnification, × 100. (A) pcDNA3 vector, (B) HOX11 WT, (C) HOX11 H3d, (D) HOX11 M5, (E) HOX11 Thr47Ile, (F) HOX11 Lys55Gln.
Bronwyn M. Owens et al. Blood 2003;101:
©2003 by American Society of Hematology