(A) Site-specific transcription of the HOXA locus. Left: The hybridization intensity of 50,532 probes that tile the human HOXA locus for each of the 11 samples (numbered in circles). The intensity of each probe is displayed as the log₂ of the ratio of the individual probe intensity divided by the average intensity of all 301,027 probes on the array. The log₂ ratio of each probe was averaged over a 100 bp window; red and green bars indicate expression above or below the array mean, respectively. Genomic locations of protein-coding HOX genes are displayed as brown boxes. Right: Anatomic origins of the 11 fibroblast samples with respect to the developmental axes. (B) Transcribed regions were identified by contiguous signals on tiling array, then compared with Refseq sequence to define genic [exonic (pink color) and intronic (blue)] and intergenic transcribed regions (purple). Each predicted HOX exon or intron was named HOXn or int-HOXn, respectively. Intergenic transcribed regions were named as nc-HOXn where n is the HOX paralog located 3′ to the ncRNA on the HOX coding strand. (C) Summary of transcribed regions in all four HOX loci defining the number of HOX genic, intronic, and ncRNA transcribed regions.

Occupancy of Suz12, H3K27me3, and pol II versus transcriptional activity over ~100 kb of the HOXA locus for primary lung (top) or foot (bottom) fibroblasts (Fb). For chIP data, the log₂ ratio of ChIP/Input is plotted on the Y-axis. For RNA data, the hybridization intensity on a linear scale is shown. Dashed line highlights the boundary of opposite configurations of chromatin modifications and intergenic transcription.

(A) RNA expression profiles of HOXD locus (top), HOXC locus surrounding HOTAIR (bottom left), and a control region on chromosome 22 (bottom right) following transfection of siRNA targeting GFP (siGFP) or a pool of four siRNAs targeting HOTAIR (siHOTAIR). Intensities of RNA hybridized to the tiling array from the siGFP and the siHOTAIR transfections are plotted on a linear scale in blue and red respectively. *, genes with significant increased transcription.
(B) qRT-PCR measuring the relative abundance of the HOTAIR transcript in the primary foreskin samples show in (A).
(C, D) qRT-PCR measuring the relative abundance of the HOTAIR (C) and HOXD10 (D) transcripts after depletion of four individual siRNAs to HOTAIR and the pool.

(A) Immunoprecipitation of Suz12 retrieves endogenous HOTAIR. Nuclear extracts of foot or foreskin fibroblasts were immunoprecipiated by IgG (lanes 1, 3, 5), anti-Suz12 (lanes 2, 4), or anti-YY1 (lane 6). Co-precipitated RNAs were detected by RT-PCR using primers for HOTAIR (rows 1 and 2) or U1 small nuclear RNA (row 3). To demonstrate that the HOTAIR band was not due to DNA contamination, each RT-PCR was repeated without reverse transcriptase (-RT, row 2). Immunoprecipitation of Suz12 and YY1 were successful as demonstrated by IP-western using the cognate antibodies (row 4). RT-PCR of nuclear extracts demonstrated equal input RNAs (row 5).
(B) In vitro transcribed HOTAIR retrieves PRC2 subunits. Immunoblot analysis of the indicated proteins is shown; five percent of input extract (5 μg) was loaded as input control.
(C) Model of long ncRNA regulation of chromatin domains via histone modification enzymes. Transcription of ncRNAs in cis may increase the accessibility of TrxG proteins such as ASH1 or MLL or directly recruit them, leading to H3K4 methylation and transcriptional activation of the downstream HOX gene(s). In contrast, recruitment of PRC2 is programmed by ncRNAs produced in trans, which targets PRC2 activity by yet incompletely defined mechanisms to target loci. PRC2 recruitment leads to H3K27 methylation and transcriptional silencing of neighboring HOX genes.

(A) HOX-encoded transcripts differentially expressed along the proximal-distal axis. Sixty transcribed regions (12 HOX genes and 48 ncRNAs) were differentially expressed (P<0.05, Student’s t-test) between distal fibroblast samples (foot, finger, foreskin, and prostate) and all other cells. Expression level of each transcribed region above or below the global median is denoted by the color scale (3 fold to 0.3 fold on linear scale or +1.6 to −1.6 on log₂ scale). Transcribed regions were ordered by their position along the chromosome, and samples were hierarchically clustered by similarity of expression of these 60 transcripts. The evolutionary origin of HOX paralogs to fly ultrabithorax (UBX) or antennapedia (Antp) are indicated by blue and yellow boxes, respectively.
(B) HOX encoded transcripts differentially expressed along the anterior-posterior anatomic division. A total of 92 transcripts (6 HOX genes, 86 ncRNAs) were differentially expressed (P<0.05, Student’s t-test) in anterior or posterior primary fibroblast cultures (above or below the umbilicus). Expression of each ncRNA is represented as in (A).
(C) Enriched sequence motifs in HOX ncRNA based on their pattern of expression (p<10⁻⁹). Logograms of sequence motifs enriched in the primary sequences of ncRNAs over non-transcribed HOX sequences, or in ncRNAs with distal, proximial, or posterior patterns of expression are shown. ncRNAs expressed in anterior anatomic sites did not share a primary sequence motif more than expected by chance.

(A) Genomic location of HOTAIR at the boundary of two chromatin domains. ChIP-chip and RNA expression on tiling array are as shown in Fig. 3.
(B) Strand specific RT-PCR shows exclusive expression of HOTAIR from the strand opposite to HOXC genes (bottom). Primers for reverse transcription (P-RT) and PCR (P-PCR) were designed to specifically target either the top (primers F1–F3) or bottom strand (primer R1) of HOTAIR.
(C) Northern blot analysis of HOTAIR in lung and foreskin fibroblast RNA.
(D) Size-fractionated small RNA was probed with pools of oligonucleotides spanning HOTAIR (sets #1–3), full length antisense HOTAIR (CDS), or a probe against miRNA let7a.
(E) Posterior and distal expression of HOTAIR in human fibroblasts as measured by qRT-PCR. The site of origin of each fibroblast sample is indicated by the sample number on the anatomic cartoon. “A” is derived from the scalp. The relative abundance of HOTAIR in each position, relative to scalp (most anterior) is shown on the X-axis.
(F) Whole mount in situ hybridization using HOTAIR sense (bottom strand) or antisense (top strand) probes in embryonic day 10.5 whole mount embryos. (top panels) and the hind limb and tail (bottom left and right panels, respectively). Expression of HOTAIR in posterior hindlimb (arrowhead) and tail (arrow) are highlighted.

(A) Change in H3K27me3 ChIP-chip signal over the HOXD locus caused by depletion of HOTAIR compared to control siRNA against GFP. The location of HOXD genes are indicated by brown boxes.
(B) ChIP of H3K27me3 and Suz12 of select promoters across the HOXD locus after siRNA treatment targeting GFP or HOTAIR. Bottom: quantitation of ChIP assays (mean ± standard error).