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Blood, Vol. 113, Issue 11, 2526-2534, March 12, 2009
A myelopoiesis-associated regulatory intergenic noncoding RNA transcript within the human HOXA cluster
Blood Zhang et al.
113: 2526
Supplemental materials for: Zhang et al
Files in this Data Supplement:
- Table S1. Primers for ChIP-PCR and for semi-quantitative and quantitative RT-PCR (PDF, 1.02 MB)
- Figure S1. Map of shRNA and primer sites on HOTAIRM1 (JPG, 94 KB)
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The sequence indicates the target sites of shRNA (in bold italic) for silencing of HOTAIRM1 expression and the binding sites of primers (in shaded italic) used for quantitative RT-PCR.
- Figure S2. HG_U133 transcription profiles of the human HOXA cluster region were assembled for human hematopoietic stem/progenitor cells (HSPC), primary leukocytes, and myeloid cell line NB4 (JPG, 145 KB)
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The HSPC subpopulations, labeled according to the presence (“+”) or absence (“−”) of lineage development markers (CD33, CD34, CD38, or Lin) and low (lo) or high (hi) rhodamine 123 (Rh123) retention, were purified from bone marrow (BM), cord blood (CB), fetal blood (FB), and G-CSF-mobilized peripheral blood (MPB) as indicated. NB4 cells were examined either without treatment or after incubation with ATRA or TPA as indicated. Primary leukocyte populations included total monocytes (MC), adherent monocytes, monocyte-derived dendritic cells (DC), resting neutrophils (PMN) and neutrophils incubated with LPS (10 ng/ml) for 30 or 120 minutes, as indicated. The heat map rows present all genic and intergenic entries of the U133 set within the HOXA gene cluster (arranged from top to bottom in the order of their genomic locations from 3′ to 5′) together with four reference probe sets (two each for ribosomal protein S6 RPS6 and alpha 1b tubulin TUBA1B). The row representing HOTAIRM1 is highlighted with a red arrow on the right margin of the heatmap. Entries with no “present” calls in any sample are blanked out.
- Figure S3. Additional HOTAIRM1 sequence and structure information (JPG, 85.3 KB)
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(A) northern blot hybridization of HOTAIRM1 in HeLa, NB4 and peripheral blood neutrophils, showing the single product of about 0.5 kb; (B) sequence assembly of RACE cDNA clones; the longest open-reading frame prediction is underlined, with bold type indicated the splicing site; (C) the canonical polyadenylation signal (PAS) 18 nt upstream of the 3′ end; (D) a calculated secondary structure of the HOTAIRM1 transcript predicted by the mfold program.
- Figure S4. Conservation of the intergenic region of HOXA1 and HOXA2 among the human, monkey (rhesus macaque), mouse and rat (JPG, 120 KB)
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The level (percentage) of conservation to the corresponding human genome region in each species, as generated by the mVISTA program, are presented as alignments of color-highlighted UCSC Genome Browser tracks (red: genome and query sequence have different bases at this position; orange: the query sequence has an insertion (or genome has a deletion/alignment gap) at this point; blue: the query sequence extends beyond the end of the alignment). Shown are the human reference gene, mRNA, spliced EST, and VISTA curves (color scheme shown on the right top) of conservation in each species. Statistics of conservation in the coding and non-coding intergenic regions are shown at the bottom of the figure.
- Figure S5. Alignment of localized intergenic transcriptional activity in man, compared to the mouse (JPG, 86.3 KB)
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The presence of similarly localized intergenic transcription activity in the mouse is supported by the high-scored BLAT alignment of human HOTAIRM1 to the corresponding intergenic region and by the similarly spliced ESTs in the mouse. The sequence differences are highlighted in color (red: genome and query sequence have different bases at this position; orange: the query sequence has an insertion (or genome has a deletion / alignment gap) at this point; double horizontal lines: both genome and query have an insertion; vertical blue line: insertion at the beginning or end of the query). The lack of conservation at the sequence level and gaps in the alignment are also illustrated by the color-annotated HOTAIRM1 sequence: Matching bases are colored blue and capitalized; light blue bases mark the boundaries of gaps in either sequence.
- Figure S6. Functional effects of shRNA-mediated knockdown of HOTAIRM1 expression (JPG, 54.1 KB)
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Lentivirus-mediated expression of shRNA targeting HOTAIRM1 in ATRA-induced NB4 cells attenuated the induction of genes encoding Beta2 integrins CD11b and CD18, but had no significant impact on CYBB gene induction. Relative expression levels were measured by quantitative real time RT-PCR. The graphs show ratios of the indicated transcript levels to their respective levels in uninduced cells; bars and error lines represent means and standard deviations of the results, normalized to an endogenous reference gene (TUBA1B or RPS6), of at least triplicate experiments for each group.
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