Research

The problem with sex chromosomes

Fruit flies and humans both have XX females and XY males. Consequentially there are two copies of X-linked genes in females but only one in males. If not corrected this will produce a lethal imbalance in gene products in one sex. Mammalian females compensate by inactivating almost all the genes on one X chromosome. In contrast, male fruit flies double expression from their single X chromosome. Both mechanisms are termed dosage compensation. Although they appear completely dissimilar, striking features are shared. Each modulates gene expression by recruiting a complex composed of protein and RNA to the X chromosome. These complexes act by silencing chromatin (mammals) or modifying chromatin to elevate expression (flies). And each organism must be able to selectively direct these complexes to a single chromosome.

Long non-coding RNA and chromosome identification

Flies encode two dissimilar but functionally redundant long non-coding RNAs, RNA on the X 1 and -2 (roX and roX2). roX RNAs display a striking pattern of localization, binding along the length of the single X chromosome in males. This is explained by the discovery that the long noncoding roX RNAs are an essential component of the dosage compensation complex in flies. Our laboratory has mutated both roX genes and found that single mutations are without obvious phenotype but males with mutations in roX1 and roX2 are lethal and have reduced expression of X-linked genes. Protein components of the complex, normally recruited to X chromosome, mislocalize to autosomal sites in roX1 roX2 males. Female flies do not modulate X chromosome expression and are unaffected.

A)                           B)                       C)

The roX transcripts localize to the X chromosome and are necessary for X chromosome recognition.  A)  In situ hybridization to roX1 detected by alkaline phosphatase (purple) reveals X chromosome binding in a male salivary gland.  B)  Immunodetection of a dosage compensation protein (detected by Texas Red) on a wild type male polytene chromosomes reveal localization to the X chromosome.   C) In roX1 roX2 males localization is no longer exclusive to the X chromosome, but appears at a number of ectopic autosomal sites.

Relevant Publications:

The siRNA pathway participates in X recognition

Some odd observations convinced us that an unknown pathway was contributing to X recognition. A few clues pointed to small RNA. We examined small RNA pathways for a genetic interaction with roX1 roX2 mutants and found that mutation of a single copy of genes in the siRNA pathway enhances roX1 roX2 male lethality. And, when roX1 roX2 males are also deficient in siRNA, recruitment of proteins in the complex is further reduced. The identity of the small RNAs involved, and how they function, is a major focus of our investigations.

Relevant Publications:

A family of satellite repeats produce siRNA and mark the X chromosome

The X chromosome is highly enriched for a family of X-linked repetitive elements, the 1.688 g/cm3 satellite repeats or 1.688X repeats. These are scattered in short, tandem arrays throughout the euchromatic arm of the X, often within or close to genes. Many 1.688X repeats are transcribed from both strands, making them a plausible source of double stranded RNA that is processed into siRNA. We made flies with transgenes that produce 1.688X siRNA to test the idea that these molecules are active in dosage compensation. Remarkably, 1.688X siRNA partially rescues roX1 roX2 mutant males! For example, the roX1SMC17A  roX2Δ chromosome allows <1% male escapers, but 30% of males to emerged as fertile adults when additional siRNA is provided. Furthermore, X chromosome binding by the dosage compensation proteins is partially restored. 1.688X repeats share no identity with the roX genes and 1.688X transgenes that produce long, single stranded RNA do not rescue males.

Small RNAs direct modifications to chromatin at similar regions in many organisms. We hypothesize that 1.688X repeats on the X are sites of siRNA-directed chromatin modification. To test this idea we inserted 1.688X transgenes at autosomal sites and discovered increased expression of autosomal genes near insertion sites in males. In addition, we were able to detect enrichment of a specific mark on chromatin surrounding these insertion sites. These findings confirm that 1.688X repeats mark the X chromosome and suggest that the mechanism involves siRNA-directed chromatin modification.

Hybridization of polytene chromosomes to 1.688X sequence reveals enrichment for these repeats on the X chromosome.

Relevant Publications:

How do 1.688X repeats identify the X chromosome?

Exactly how the 1.688X repeats promote X recognition is a fascinating problem. Several studies have found that the arrangement of the X chromosome in the nucleus contributes to X recognition. If this is the case, architectural proteins that interact with 1.688X repeats may play a role in their activity. To test these ideas we are testing architectural proteins for a role in dosage compensation. We are also looking for evidence that 1.688X repeats contribute to nuclear organization.