HOXAIR: HOX Antisense Intergenic RNA (John L. Rinn 2007) is transcribed in antisense manner with respect to the canonical HOXC genes. Molecular cloning and Northern blot analysis confirmed that HOTAIR is a 2158 nucleotide, spliced, and polyadenylated transcript.

HOXAIR was found to suppress the HOXD complex genes (in trans). This trans activity of HOXAIR was confirmed by the study conducted by Rinn et al., in which the depletion of HOTAIR by RNA interference (siRNA mediated) lead to dramatic transcriptional activation of the HOXD locus on chromosome 2 spanning over 40 Kb, including HOXD8, HOXD9, HOXD10, HOXD11, and multiple ncRNAs. To ensure that this was not an off-target effect of RNA interference, four independent siRNA sequences targeting HOTAIR. Each siRNA depleted HOTAIR ncRNA and led to concomitant HOXD10 activation as determined by quantitative RT-PCR. These observations indicate that HOTAIR ncRNA is required to maintain a transcriptionally silent chromosomal domain in trans on the HOXD locus.

Molecular Mechanism

It was found that HOTAIR enhances the PRC2 (Poly-comb protein complex 2) activity at HOXD locus. To investigate the molecular mechanisms involved in the HOTAIR dependant silencing of the HOXD locus, the group performed chromatin immunoprecipitation to interrogate changes to the HOXD chromatin structure upon depletion of HOTAIR.

  • Polycomb Repressive Complex 2 (PRC2), comprised of H3K27 histone methyl transferase (HMTase) EZH2 and core components Suz12 and EED, initiates this histone modification and subsequently Polycomb Repressive Complex 1 (PRC1) maintains this modification and promotes chromatin compaction (reviewed by (Sparmann and van Lohuizen, 2006)
  • Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell

Previous ChIP-chip experiments indicated that in primary foreskin fibroblasts, the entire HOXD locus was occupied by both Suz12 and H3K27me3. Depletion of HOTAIR followed by ChIP-chip revealed substantial and global loss of H3K27Me3 occupancy over the HOXD locus, with the greatest loss residing in the intergenic region between HOXD4 and HOXD8. HOTAIR depletion also led to a modest but consistent loss of Suz12 occupancy of the HOXD locus. Importantly, occupancy of H3K27me3 and Suz12 across the silent HOXB locus was not affected by HOTAIR depletion in these cells. These results suggest that HOTAIR is selectively required to target PRC2 occupancy and activity to silence transcription of the HOXD locus.