Expression patterns of different HOXA genes and OS transcripts in adult human tissues and placenta. (A–D) The relative positions of HOXA (including alternative splice forms, marked by “#”) and OS transcripts are schematically drawn above and below the genomic line, respectively. Exons are indicated by black numbered rectangles. (E) Constitutive MIC-2 expression is shown as a control for the PCR, showing that similar amounts of cDNA were present in the RT reactions for each tissue. MIC-2 (CD99) encodes a T-cell surface anti-gene expressed in most human tissues except spermatozoa.

RT-PCR analysis of HOXA genes and OS transcripts in NT2D1 cells. (A) NT2D1 cells were treated with retinoic acid (RA) for 1, 3, 6, and 12 h and 1, 2, 3, and 4 d. Total RNA was extracted from treated and untreated (no RA) cells and analyzed by RT-PCR. Note that HOXA7 is constitutively expressed in NT2 cells. Constitutive MIC-2 expression is shown as the loading control. (B) Analysis of HOXA and ncRNA transcription by real-time RT-PCR. NT2D1 cells were treated with retinoic acid (RA) for 15 and 30 min; 1, 3, 6, and 12 h; and 1, 2, 3, 4, 6, 8, and 10 d. Total RNA was extracted from treated and untreated (no RA) cells and analyzed by RT and PCR amplification on a LightCycler. The cycle threshold numbers of at least two independent experiments are plotted against RA-incubation time. The cycle threshold numbers are reciprocally correlated to the amount of template at the start of linear amplification. MIC-2 expression does not change upon RA treatment (light blue line at threshold cycle 16). The red line indicates threshold cycle 25, which corresponds approximately to a visible amplification product in an ethidium bromide-stained gel. Primer pairs used have comparable amplification efficiencies. The mean values from at least two independent experiments are shown. Error bars indicate the standard deviation.

Histone modifications in the HOXA cluster. ChIP analysis of histone modifications of untreated NT2D1 cells (no RA) and of cells treated for 2 h, 12 h, 1 d, and 4 d with retinoic acid (RA). Chromatin was precipitated with the antisera indicated above the gel photographs in each panel (dK4=histone H3 dimethylated at lysine 4, tK4=histone H3 trimethylated at lysine 4, tK9=histone H3 trimethylated at lysine 9, tK27=histone H3 trimethylated at lysine 27, acH3=acetylated histone H3). Immunopurified DNA was analyzed by PCR using primer pairs near or in the first exon of the indicated transcription units. PCR reactions with 10%, 1%, and 0.1% of the input DNA were loaded to determine the linear range of amplification and to use for normalization during quantification (see Materials and Methods). Below the gel photographs a quantification of two independent ChIP experiments is shown. The values on the y-axis represent the intensities of immunoprecipitated DNA bands as a percentage of the intensities of the input DNA bands. The graphs show the changes in histone modifications after the different periods (untreated, 2 h, 12 h, 1 d, and 4 d) of retinoic acid treatment (dK4=orange 4, tK4=red, tK9=gray, tK27=green, acH3=dark blue). The mean values from at least two independent experiments are shown. Error bars indicate the standard deviation.

Real-time RT-PCR analysis of different HOXA genes and OS transcripts in fetal human tissues. (A) Expression levels of the HOXA genes 1, 2, 3, 4, 6, and 7 and the ncRNAs BG325728, AA489505, BI823151, BE8733499, and AK092154 in human fetal tissues as percentages of MIC-2 expression. Note change of scale above the red line. (B) Expression levels of the HOXA genes 1, 2, 3, 4, and 7 and the ncRNAs BG325728, BI823151, BE8733499, and AK092154 as in A but with adjusted scale to visualize small peaks. Note change of scale above the red line. Primer pairs used have comparable amplification efficiencies. The mean values from at least two independent experiments are shown. Error bars indicate the standard deviation.

In silico analysis of retinoid response elements and CpG islands (RAREs) in the human HOXA cluster. Potential consensus sites for RAR–RXR heterodimers of the DR5 class were identified using the Matinspector software. The 25 RAREs found are shown in A. In bold, conserved nucleotides are indicated (see the consensus at the bottom of the figure); in italics, the 5mer spacer region. In addition, the chromosomal location (according to the UCSC genome browser, assembly March 2006), the strand and the values for core and matrix similarities (from the Matinspector analysis) (Werner 2000) are shown. (B) The location of the potential RAREs in the HOXA cluster (gray ovals) is shown. The first exon of each HOX gene is indicated by a thick black arrow. The two black dotted arrows refer to alternative exons of HOXA3. Arrows below indicate the first exons of the OS transcripts particularly featured in this report. Dotted arrows refer to mapped OS transcripts, not further analyzed in this study (I=BF114958, II=AW449791, III=AI795971, IV=DA748011, V=BF510786; see Table 1). Below CpG islands are shown (dark gray squares).

PRC2 components in the HOXA cluster. PCR analysis with cross-linked and immunoprecipitated DNA from untreated NT2D1 cells and cells treated for 2 h, 12 h, 1 d, and 4 d with retinoic acid (RA). Chromatin was precipitated with the antisera against SUZ(Z)12, EZH2, and POLII (as indicated above the gel photographs in each panel). PCR reactions with 10%, 1%, and 0.1% of the input DNA are loaded to determine the linear range of amplification and to use for normalization during quantification. Below the gel photographs quantitative analyses of the ethidium bromide-stained bands from two independent experiments are shown. The values on the y-axis represent the intensities of immunoprecipitated DNA bands as a percentage of the intensities of the input DNA bands (SU(Z)12=dark blue, EZH2=gray, POLII=red). The mean values from at least two independent experiments are shown. Error bars indicate the standard deviation.