Xist lncRNA are the cis acting lncRNAs.

Mary Lyon was the first to describe the X chromosome inactivation (X-inactivation) hypothesis in 1961.  The discovery of the X inactive-specific transcript (Xist) gene in the early 1990s brought a breakthrough in our understanding of the molecular basis for X-inactivation.

Xist was initially identified on the basis of its location in the classically defined master control region for X-inactivation, called the X chromosome inactivation centre (Xic/XIC). The gene was found to be expressed only from the inactive X chromosome and to produce a transcript that does not encode protein, but rather plays a role as a functional RNA that associates in cis with the X chromosome from which it originated. These observations led to the idea that Xist RNA recruits proteins involved in heterochromatinization (Polycomb-group proteins) to trimethylate histone H3 on lysine 27 (H3K27me3), rendering the X chromosome transcriptionally silent (Plath et al., 2003). Targeted disruption of Xist unequivocally showed that it is essential for X-inactivation to occur in cis.

  • Ploycomb-group proteins are the family of proteins first discovered in fruit flies that can remodel chromatin such as epigenetic silencing of HOX genes.
  • Trimethylation of histone H3 at lysine 27 (H3K27me3) is a histone modification with an important role in regulation of gene expression. It is generally associated with low expression levels and known as a repressive mark for transcription. Its function is conserved from animals to plants (Berke et al.,2012)

It has been found that, Xist regulation plays a central role in the initiation of X-inactivation, and its antisense gene, known as Tsix, negatively regulates Xist in cis through chromatin modifications at the onset of X-inactivation

Lee J et al  (in 1999, below link 4) were the first to discover the antisense Tsix to the sense Xist gene. Antisense Tsix gene was found to regulate the Xist gene expression levels though both of the are non coding RNAs.

The dynamic relationship between Xist and Tsix in female cells is as follows:

  1. before the onset of X inactivation, Xist and Tsix are biallelically co-expressed;
  2. at the onset of X inactivation (differentiation), Tsix becomes monoallelic, preceding upregulation of Xist RNA on the future inactive X (species 2);
  3. in the monoallelic form, Tsix RNA associates only with the futureactive X;
  4. once X inactivation is established, Tsix is repressed. Tsix expression is specific to undifferentiated cells regardless of sex, persists briefly at the onset of X inactivation, appears to associate only with the future active X and disappears after X inactivation is established. This profile suggests that Tsix may regulate events at the onset of X inactivation. Its antisense nature raises the possibility that Tsix directly blocks Xist RNA action on the X chromosome.

In order to map the exact arrangement of Tsix and Xist genes in the genome they performed “strand specific RT-PCR of randomly primed cDNA” to determine whether a transcript of either sense or antisense orientation existed at 19 positions. No RT-PCR product was detected at positions 1, 18, 19.  At positions 2−17, where RT-PCR gave products, strand-specific cDNA was used to determine RNA orientation. In the sense orientation (Xist), a PCR product was detected at positions 5−12. In the antisense orientation (Tsix), a PCR product was seen at positions 2−17.


Random Primed Labeling of DNA

This method was developed by Feinberg and Vogelstein (link 5). In this method  random sequence oligonucleotides (primers) are annealed to both strands. Klenow fragment polymerase is then used to extend the oligonucleotides, using three cold nucleotides and one radioactively labeled nucleotide provided in the reaction mixture, to produce a uniformly labeled double-stranded probe.

other useful links:

  1. http://www.nature.com/nrg/journal/v12/n8/full/nrg3035.html
  2. http://rstb.royalsocietypublishing.org/content/368/1609/20110325.abstract
  3. http://www.sciencedirect.com/science/article/pii/S1097276506000505
  4. http://www.nature.com/ng/journal/v21/n4/full/ng0499_400.html
  5. http://link.springer.com/protocol/10.1385%2F0-89603-245-0%3A445
  6. more on Polycomb proteins and H3K27me3 by Bernstein et al., 2006; Zhang et al., 2004