LECTURE 13: DOSAGE COMPENSATION Reading: Ch. 12, p. 431-2 Supplementary Reading: Turner Ch. 12, p. 249-267; 271-2 (on reserve in Biosciences Library; don't worry about the details) Problems: Ch. 12, problems 12.19 – 12.21

Last time, we ended lecture with a discussion of trisomies and monosomies of the autosomes and sex chromosomes in humans. We discussed that trisomy of the X is much less severe than that of the autosomes. Most trisomies and all monosomies of the autosomes are embryonic lethals. We discussed that monosomy of the X leads to XO females that are viable, but usually sterile.

So, aneuploidy for the autosomes is more severe than aneuploidy for the X. Now we’ll discuss dosage compensation, which helps explain why this is true.

Brief review of sex determination in fruit flies and humans:

TABLE 3.1 XXX XX XXY XO XY XYY OY Drosophila Dies Normal Normal Sterile male Normal Normal Dies female female male male Humans Nearly Normal Kleinfelter Turner Normal Normal or Dies normal female male female male nearly female (sterile); (sterile); normal tall, thin male

In humans, it is the presence or absence of the Y that makes the difference; a person carrying a Y will look like a male; a person with one X and lacking a Y (XO) will look female. Drosophila also use an XX female, XY male strategy of sex determination, but it is the ratio of X chromosomes to autosomes (non sex chromosomes) that makes the difference. Flies with X:A = 1 are female, flies with X:A = 0.5 are male, and flies with X:A ratios between 0.5-1 are "intersex".

What we’ll talk about today is how the difference in number of X chromosomes between males and females of both species is compensated for by the organism. That is, the activity of X-linked genes must be equalized in the two sexes. Although humans and fruit flies both use a XX female / XYmale strategy for sex determination, they use two different mechanisms for dosage compensation. In human females, one of the X-chromosomes is inactivated in each somatic cell. In fruit flies, transcription of the X chromosome is hyperactivated in males versus females.

X-Chromosome Inactivation in mammals As we mentioned last time, aneuploidy for the X chromosome (either extra or missing sex chromosomes) is much less severe than aneuploidy of autosomal genes. In 1961, Mary Lyon proposed the X chromosome inactivation hypothesis to explain dosage compensation in mammals. Her observations of X-linked coat color genes in mice led her to propose that the amount of products derived from X-linked genes were equalized in the sexes by inactivating one of the X chromosomes in females. Inactive X chromosomes can be seen in interphase nuclei as darkly staining heterochromatic masses. These masses are called Barr bodies after the cytologist who discovered them. XX females have one Barr body per cell, XXX females have 2 Barr bodies per cell, and XXY Klinefelter males have one Barr body per cell (Barr bodies are not observed in XY males). This is why X chromosome aneuploidy can be tolerated; all but one of the extra X chromosomes is unactivated. Why do individuals aneuploid for the X have any phenotype at all? As we’ll discuss, not all of the genes on the X are inactivated. A possible explanation is that in Klinefelter’s Syndrome males (XXY), these genes are expressed at two times the levels normally expressed in XY males. Turner’s Syndrome (XO) may be explained by the absence of X re- activation. Normally, when female germ cells start to make oocytes and enter meiosis, the inactive X becomes active, so that developing oocytes have two functional doses of X chromosome gene and every oocyte receives an active X. Developing oocytes in Turner’s syndrome females only have one dose of X chromosome genes.

At the 500-1000 cell stage (postfertilization day 16 in humans), each embryonic cell randomly decides to inactivate either the paternally-derived X or the maternally-derived X. This “decision” is inherited by all of the descendents of the original cell. Thus, all females are mosaic! There is an X-linked mutation that causes anhidrotic ectodermal displasia (absence of sweat glands). Females heterozygous for the mutation display large patches of skin lacking sweat glands; this is because the decision to inactivate the X is made randomly at early stages of development, leading to mitotic clones that eventually become patches of normal and mutant skin. If the X- linked gene you are following is a recessive lethal or a disease gene, you can imagine that the ratio of cells (and the cell type) carrying the wild-type allele and those carrying the mutant allele is what is important.

Another way to detect mosaicism is by looking at electrophoretic variants of X-linked enzymes such as glucose 6-phosphate dehydrogenase (G6PD). When a sample of tissue from a female carrying two different variants A and B is examined, we see both variants. However, if we look at which variants are expressed in a single cell (by isolating single cells and culturing them), we see that that cell will express either A or B, but not both. In 1963, these data were presented as direct evidence in favor of X chromosome inactivation.

Now we know even more details. There are three basic steps: (1) X chromosome counting, (2) selection of the X to inactivate, and (3) the process of X-inactivation. X chromosome inactivation begins at a particular site called the X Inactivation Center and then spreads to adjacent regions towards the ends of the chromosome. The chromosome becomes heterochromatic. Heterochromatin is highly condensed chromosomal regions that stain darkly. Not all genes are inactivated – about a dozen genes escape inactivation. Most of these are active on the active X chromosome, but one of these genes is inactive on the active X, and instead active on the heterochromatic X. This latter gene is part of the XIC and is called XIST (for X inactive specific transcript). An X chromosome must have Xist in order to be inactivated. If Xist is missing from one X chromosome, then the other X must be inactivated. Deletion of the Xist gene abolishes a chromosome’s capacity for X inactivation. If Xist is inserted into an autosome, it can induce partial inactivation on that autosome. The Xist gene transcribes a large RNA molecule that has no open reading frames, thus makes no protein. Instead in makes a long 17 Kb RNA that decorates the inactive X chromosome, perhaps preventing transcription of most genes. What might influence what X is inactivated? The question is still being actively investigated, but there is evidence that another transcript called Tsix (also part of the XIC) may be involved. Tsix is also a non-coding RNA and part of the Tsix transcript is "antisense" to the Xist transcript. Tsix is expressed only very early during the X-inactivation process. If an X chromosome carries a deletion of the Tsix promoter (therefore does not express Tsix), then that chromosome is always chosen for inactivation.

The model is this: before the choice, Xist and Tsix are expressed from both X chromosomes. The Xist transcript is unstable however, so it does not accumulate. As the choice is made, Tsix transcription is decreased from one chromosome (the pre-Xi chromosome, [Xi meaning the inactive X]). This correlates with stabilization of Xist transcripts from the pre-Xi chromosome. Then, Xist transcripts begin to coat the Xi chromosome. Soon after, Xist and Tsix are both no longer transcribed from the active X. (See Figure 12.6 in Turner). Recently, another Xic region, named Xite, which positively regulates Tsix expression has been identified. Deletion of a region that includes Xite (and the 5’ end of Tsix) causes the X chromosome containing the deletion to become silenced, even in male cells where there is only one X chromosome. Some models suggest that this region of the Xic binds a “blocking factor” produced in limiting quantities from an autosome. The hypothesis is that the amount of blocking factor produced is only enough to bind one X chromosome and binding protects that X from becoming silenced. The other X chromosome(s) is/are then selected for silencing.

Dosage compensation in other animals: The purpose of dosage compensation is to equalize expression of the X chromosome in males and females, but the mechanism of how this is accomplished is different in different organisms. It is even clear that the mechanism may have differences among mammals (humans versus mice for example). Like previously mentioned, dosage compensation in fruit flies is achieved by hyper-activating transcription of the single X chromosome is in males versus females. In the worm C. elegans, where XX gives hermaphrodites and XO gives males, dosage compensation is achieved by reducing transcription on each X of hermaphrodites by half.