The RNA world strikes again (it never stopped)

Jpx is a long (over 200 nucleotides) nonCoding (for protein that is) RNA (e.g. a lncRNA).  It is an example of the RNA world from which we (presumably) sprang. One of its function is to control another RNA, and a fairly important one at that — namely Xist, which inactivates one of a woman’s two X chromosomes.  The jpx gene is just 10 kiloBases away from that of Xist. Jpx turns on the transcription of Xist which then goes and coats the X chromosome from which it is transcribed, shutting off most of its genes.

One of the mechanisms by which Jpx turns on Xist production is by binding to a protein called CTCF.  CTCF sits on the promoter of the Xist gene until Jpx binds to it displacing CTCF from the promoter.

CTCF is a much better known actor, and along with cohesin is thought to be responsible for the formation of chromosome loops, and the establishment of TADs (topologically associated domains) which are basically loops of chromosomes containing about a million nucleotides with an average of 8 protein coding genes which are coordinately expressed as a result.

That’s fairly impressive.  What happens when you knock out the jpx gene.  [ Cell vol. 184 pp. 6157 – 6173 ’21 ] did just this and all Hell broke loose.  Jpx keeps CTCF from binding promotors, and without jpx thousands of chromosome loops are replaced by others, with downregulation of some 700 protein coding genes.

Again, the RNA world is like some legacy software (think DOS) underlying the latest stuff (think Windows), forgotten but not gone.

Activating a proto-oncogene without mutating it

Many proto-oncogenes have to be mutated to cause cancer. Not so the TAL1, LMO2 genes. They drive blood formation, and are aberrantly activated (e.g. more proteins made from them is expressed) in T cell Acute Lymphoblastic Leukemia (TALL). [ Science vol. 351 pp. 1298- 1299, 1454 – 1458 ’16 ] activated them experimentally using the CRISPR technique, and therein hangs a tale.

Addendum 11 April — LMO2 is well known to gene therapists as early work (2002) using retroviruses inserted randomly in the genome to cure SCID (Severe Combined Immunodeficiency) resulted in TALL in 4kids.  The problem was that the vector integrated in multiple sites all over the genome and one such random site  turned on expression of LMO2.

I’ve written a series of six posts trying to imagine the incredible mass of DNA in a 10 micron nucleus on a human scale — we take it for granted, but it’s far from obvious how this is accomplished — here’s the link to the first — https://luysii.wordpress.com/2010/03/22/the-cell-nucleus-and-its-dna-on-a-human-scale-i/. — just follow the links to the rest.

[ Cell vol. 153 pp. 1187 – 1189, 1281 – 1295 ’13 ] Hi-C and 5C (Carbon Copy Chromosome Conformation Capture) allow determination of chromatin organization and long range chromatin interactions in an unbiased genome wide manner at the megaBase scale. Topologically associated domains (TADs) are the way the genome in the nucleus is organized into megabase to submegaBase sized interacting domains. TADs are conserved between species and are invariant across cell types. [ Call vol. 156 p. 19 ’14 ] They average 700 – 800 kiloBases and are said to contain 5 – 10 protein coding genes and a few hundred enhancers. The expression of genes within a TAD is ‘somewhat correlated’. Some TADs have active genes, while others have repressed genes. Genomic interactions are strong within a domain, but are sharply depleted on crossing the boundary between two TADs.

Well TADs have to be separated from each other. The current thinking is that the boundaries are formed by sites in the DNA which bind the CTCF protein, and possibly cohesin proteins as well. CTCF is a large protein (although maddeningly I can’t seem to find out how many amino acids it has) with a molecular mass of 80 kiloDaltons. It’s DNA binding is quite specific as it contains 11 zinc fingers (each of which can specifically bind a 3 nucleotide stretch of DNA). In addition to binding to DNA it can bind to itself, forming a perfect way to form loops of DNA.

All the Science paper did was to delete a few CTCF binding sites using the CRISPR technique around the two oncogenes and bang — expression increased. Why?  Because the insulation between the TAD containing the genes and adjacent TADs was broken, allowing control of the genes by enhancers in the new and larger TAD that had been previously sequestered in an adjacent TAD.  The deletions were thousands of basepairs away from the coding sequence of the genes themselves.  All very nice, but it’s fairly artificial.

However the paper notes that across a large pan-cancer cohort, there was a 2 fold enrichment for boundary CTCF site mutations.