Fifty shades of Red: the Superhot history of PEV

Preamble to the preamble: this preamble consists of a rather leisurely perambulation through history. If historical context bores you, skip ahead to all of the amazing discoveries which at the time of writing this post I have yet to make. But if, like me, you like to try and climb inside the heads of the scientists who made these seminal discoveries, to try and see things through their eyes, with their knowledge, then read on! Most of the papers are publically available now, and the language is quite convoluted, but worth the effort. These (mostly) dead white males were mental contortionists…imagine how they built models of what was going on in what we now call epigenetics without the resolution our microscopes have today, let alone the knowledge of DNA and molecular biology. These folks are my heroes.

Position Effect Variegation. It just ROLLS off the tongue, doesn’t it? Haven’t you always dreamed of being able to expound knowledgeably about fluffy subjects like this at cocktail parties? Would you not become the life of the party? Doesn’t everyone want to know this stuff?

OK it is probably true that you won’t be pinned wriggling to the wall by people who want to know about position effect variegation (NOTE: they will more likely want to know about CLONING (the Dolly kind), GMO food, climate change and strange sexual practices in the animal kingdom. So make sure you have answers at the ready. Hmm. Subject for later blog.) But, understanding Position Effect Variegation, or PEV, is a very useful way of learning how chromatin structure affects gene expression, part of the revolution in epigenetics, which term has been bandied about of late in the press. PEV is a term coined way back in the second or third decade of last century, when almost anybody reading this (including yours truly, just FYI) were not even embryos; indeed, not even conceptions, real or imagined. Here are a couple of nice reviews of PEV with all the punchlines that include historical context: PMID: 18282501, PMID: 2127578.

Flemming and meIt all began with moose. That is, with German moose, which is to say, moss, a paper published in 1928 by the German Botanist Emil Heitz, who used Moose as a model organism for his cytological studies of the nucleus (for the teutonic among you: In I Jahrb Wiss Botanik, Vol. 69, 762 – 818  Das Heterochromatin der Moose). Cytology then was like molecular biology today – the really good cytologists were rock stars and stud muffins, and Heitz developed clever staining techniques to study the morphological transmogrifications that went on inside the nucleus during the cell cycle. It is remarkable, when you think about it, just what kind of dramatic contortions have to happen when a cell commits to mitosis (or meiosis). Heitz was particularly interested in the interphase nucleus – before all the drama of mitosis begins. Remember, this is 1928 – the chromosome theory of inheritance has been established (Sutton 1903: Biol. Bull 4:231-251) but what ARE chromosomes? Some ideas were bandied about (even DNA on the QT) but their behavior was mesmerizing, as it still is (sometimes I squash onion root tips for fun and stare at them for hours under the scope (Figure 1 – which is my image and which is Flemmings? Walther, that is, not Alexander of Penicillin. For more pretty pictures see here).

Among the most puzzling aspect of chromosomes was their constant appearance and disappearance during the cell cycle – from rod like structures that are easy to count and draw, to an amorphous mushy blur – even today my students don’t always believe me when I tell them the chromosomes are STILL THERE during interphase. (Thank goodness for the candy color chromosome painting techniques…take a look at the stunning 3D imagery in this paper PMID: 15839726). What Heitz noticed, especially looking at chromosomes during early prophase, or later in telophase, was that some portions of the nucleus, in fact the chromosomes themselves, when they began to be visible during early prophase, were diffuse and stained less intensely – he called these regions collectively euchromatic, whereas other portions were stubbornly dark – heterochromatic. One drawing from his 1928 paper is shown in Figure 2. Heitz chromosHeitz has colored the heterochromatin black; note that “m” is perpetually dark – a sex chromosome? He examined chromosomes from multiple plant species, and then insects as well, various species of Drosophila, including D. melanogaster, and his results were both reproducible and independent of artifact.

And so begins a flourishing analysis of the “longitudinal differentiation of chromosomes” – which is to say, how exactly are chromosomes constructed? If you were to dissect a chromosome out of the nucleus, and lay it shimmering in a petri dish, what would you see? Well, nothing of course, because that kind of dissection is actually impossible (almost – here’s a paper about microdissecting fly chromosomes: PMID: 2587252, and human chromosomes: PMID: 1282430) but if you COULD lay out a chromosome in a dish, what would you see? Would the compaction – the degree of coiling – be equal throughout the chromosome or unequal? Unequal, was Heitz’ guess and of course he was right. Chromosomes are highly compacted, compressed in certain regions – the heterochromatic regions around the centromeres and telomeres – and more open and loosely coiled in between – euchromatin. Other remarkable conclusions – Heitz distinguished between constitutive heterochromatin – that which remains condensed apparently throughout the cell cycle – and facultative heterochromatin, that which appears and disappears under different circumstances – notably one of the sex chromosomes (already a heated topic of debate a decade earlier including at least one dead white female – Nettie Stevens – among many dead white males – another story) and the nucleolar region (a prominent nuclear component already observed some seventy years earlier – yet another story: Montgomery 1898).

Heitz was well aware of Thomas Hunt Morgan’s work on Drosophila genetics (in the United States – the beginnings of Drosophila as a genetic model….rare to find any fly people today who aren’t geneticists deep down) and thus could compare his cytological findings with the genetic maps and linkage groups that were being churned out of Morgan’s lab by his army of insufferably prodigious students (Nettie Stevens, Calvin Bridges, Alfred Sturtevant among them). Heterochromatin was presumed to be gene-poor, euchromatin, gene rich, at least for flies. Euchromatin is gene rich across taxa, certainly, but as for heterochromatin, I know from personal experience (my Ph.D. in fact) that heterochromatin is not a hospitable place for genes to be, as we shall see.

And heterochromatin was soon discovered to be responsible for a “group of distinctly peculiar variants” in Drosophila, that variegated, in other words, gave a variable phenotype over a particular tissue. The most dramatic phenotypes involved eye color: an otherwise wild type red eye color gene would manifest as a mottled combination of red and white ommatidia (the individual photoreceptor units in the fruitfly’s compound eye). These mutations appeared among an amazing panoply of mutations induced by radiation – a discovery made by Herman Joseph Muller (author of the above “distinctly peculiar” quote), another of Morgan’s insufferably prodigious former students. Muller generated more mutated strains of flies in a matter of a few weeks using X rays than had been made since research using Drosophila had begun two decades earlier. He published a landmark paper in 1930, cataloging and describing the mutations he found. This paper is special because the peculiar variants had to be viewed in color. No fancy schmanzy microscope cameras in those days so instead, the specimens were drawn, and in the case of Figure 3 and 4, painted, with great accuracy and detail (from J. Genet. July 1930, Vol. 22, Issue 3). Muller's eversportsNote the fly in figure 3 has notched wings – the strain from which she arose originally had wild type (red) eyes, but Muller thought that perhaps the notch mutation resulted from an X-ray induced deficiency that removed both the notch gene (yielding the characteristic dominant wing notching) AND the neighbouring recessive red eye color gene as well (there had been precedents for this). So he crossed flies from this strain with another strain that was mutant for the red eye color gene (had white eyes) and expected to see white-eyed notched-winged progeny, but instead he got this fly – notched wings, but a mottling of red and white ommatidia in the compound eye. Go figure. In Muller’s words: “To the great surprise of the writer, the Notch winged offspring of this cross had neither white nor normal red eyes nor even eyes of any uniform intermediate colour (spelled English style HAH). They had mottled eyes, and exhibited various grades and sizes of lighter and darker areas…” Figure 4 shows details of various mottled eye colour phenotypes – including the more subtle and headache-inducing (I know) variegations of red on red.

Variegated phenotypes were at once stable and unstable. They were stable in the sense that once established, they did not revert back to wild type. This contrasted with the findings of other mottled phenotypes in other model organisms at the time, notably, the “mutable loci in Maize” observed as early as 1917 (Emerson PMID: 17245873) and so elegantly dissected by Barbara McClintock in the 1940’s and 50’s (PMID: 15430309). These unstable phenotypes involved transposable elements: pieces of DNA that could move from place to place in the genome. But Morgan’s mottled phenotypes behaved differently. He described them as “eversporting” (PMC1085688), a curious terminological throwback to the history of, well, natural history – even Charles Darwin used this term. “Sports” are varieties, usually very obvious strange variants, described by farmers, gardeners, pigeon fanciers and natural historians, before the genetic basis got hooked up with the term “mutation.” Muller’s “eversporting” mottled variants were unstable within the tissues they affected – so the pattern of mottling varied between individuals carrying the same mutation – but were stable once established in a strain, essentially forever.

Muller made two important observations: these ever sporting variants were recessive to the wild type allele (but dominant to a loss of function allele) and they all correlated with chromosome rearrangements, arising from treatment with X rays (hence he called them “eversporting displacements”). Muller’s expressive efforts to explain how the eye color gene could function then not function as the eye developed in an individual with this phenotype are fascinating – particularly in light of how chromosomes were thought to be constructed – remember – no DNA, no proteins, no sense of how this substance was organized and propagated.

Eversporting mutations were very puzzling to geneticists, which is to say, fascinating to the point of obsession, and I like this conclusion from a paper by Jack Schultz (no relation) and Theodosius Dobzhansky (he of the nothing-in-biology-makes-sense-except-in-the-light-of-evolution adage): “The data presented are consistent with the hypothesis that the behavior of genes is a function of their position in the chromosome, that is, depends upon their relation to other genes.” (PMID: 17246728). So this is what position effect variegation means – a gene’s expression (its phenotype) variegates (varies) depending on its position in the chromosome. Note that this is an epigenetic (“above genetic”) effect: only the location of the gene is different: in modern terms, the sequence of the variegating allele is unchanged.

Thus was born the genetic analysis of chromatin structure. The first reports that the mottling might be in some way connected with heterochromatin came in 1933 (PMC1085890) and then later in 1936 (PMC1076699). The first paper (Gowen and Gay 1933) has a very athletic title: “Eversporting as a function of the Y chromosome in Drosophila”.  Flies are heterogametic – which means they produce gametes with different sex chromosomes (Y-bearing, or X-bearing) just like we do. And like us also, (and indeed many heterogametic species) the Y chromosome is largely heterochromatic. Unlike us, it is also LARGE – almost the same size as the X (the Y chromosome in humans is much smaller than the X) Also very much UNLIKE us, whereas a Y chromosome determines maleness in humans, it is the number of autosomes to sex chromosomes that determines sex in flies. Thus, XY is male in humans and flies (in humans because of the Y, in flies because for any given autosome pair, there is only one X). XX is female in both flies and humans, but XO is female in humans (Turner syndrome), but male in flies (still one X per any given autosome pair), and XXY is FEMALE in flies (2 X’s for any given autosome pair) but male in humans (Kleinfelter Syndrome, in fact). This digression about sex determination is necessary to understand the findings in Gowen and Gay’s 1933 paper.

So here is a quote from Gowen and Gay paper: “On cytological examination of oogonial divisions, mottled-2 females are found to have typical chromosome figures, but every figure from red-eyed females contains an extra chromosome, which looks like a Y” (my emphasis). First, cytological inspection is usually how the kinds of rearrangements producing the eversporting variants were characterized. Second, mottled-2 is one of Muller’s eversporting red and white-eyed mosaic strains. And third, when mottle-eyed female fruitflies are genetically manipulated so they receive a Y chromosome (while retaining their two X’s), their eyes are no longer mottled, but red, like wild type. These females would have genotype w/w[m2]/Y, where w = a loss of function allele in the red eye color gene, w[m2] the rearranged chromosome bearing the eversporting red eye color variant which is wildtype for the red eye gene, but relocated by rearrangement, and Y = the Y chromosome (remember, these flies are female).  Yet these flies looked like they had perfectly normal (wild type) red eye color genes. In other words, the presence of a Y chromosome SUPRESSES the mottled phenotype, making it look MORE wild type (more red eye color pigment). It is almost as if there is something about the Y chromosome that sucks out (in proper parlance – titrates) the variegating substance, whatever it is, away from the red eyed gene locus. But I am getting ahead of myself.

The effect of supernumery Y chromosomes was demonstrated for several genes that variegated, and in 1936 Jack Schultz (no relation) attempted to extend the argument to other so-called “inert” regions of the genome. General understanding of a mechanism was still wanting: did the loss of eye color gene expression result from a loss of the gene itself, or a suppression of the gene’s function? Who knew? Here’s a quote: “…the proportion of “mutant” to wild type tissue, depends upon a quantitative relation between active and “inert” chromosome regions. In addition, however, the “inert” regions are involved in the rearrangements themselves. Thus both the chromosome structure associated with variegation and the extent of variegation are related to the “inert” chromosome regions.” Schultz correctly postulated that variegating genes were interacting in some way with blocks of heterochromatin into which or near which they had been relocated – he and others found that these genes had to be close to a chromosome rearrangement breakpoint that brought them into proximity to the “inert” regions. Also, that there had to be a balance between the active vs inert portions in the nucleus, and that variegation occurs when that balance is upset. This is reminiscent of a mass action model proposed much later (PMID: 3146523). He also proposes, in a footnote, to adopt Heitz’ terminology: “The terminology is obviously outworn: “inert” is certainly a misnomer. Heitz’s use of “heterochromatin” and “euchromatin'”‘ for “inert” and active regions is probably better.” And so it came to pass.

MetropoleThe interwar period – 1920’s and 30’s – is fascinating. Devastating, apocalyptic, hedonistic…some of the greatest literature, art, architecture, music came from this period. (The Lempicka image is a photo of an enormous poster I acquired after attending one of the best exhibits in my life – Montreal 1991 – each room exhibiting art, architecture, design from different major cities during this period). But for the health care, I think I would enjoy a visit to this time (so long as I was provided with enormous wealth).

All the work I’ve described took place during this period, and by the time WW2 had begun, Morgan and his (now grown up) prodigies were investigating background and environmental effects that modified the eversporting phenotypes. PEV became a tool to dissect “…the developmental influences that operate in the development of the compound eye” or any other tissue susceptible to variegation (there were lots – wing margins, bristle patterns etc.) Extra heterochromatin, in the form of Y chromosomes for example, we’ve discussed. But a pronounced temperature effect was also observed – higher temperatures suppressed the mottled phenotype (more wild type, more red), lower temps enhanced (more mottled, less red). It is very interesting reading this series of papers – all found in the Carnegie Institution Yearbooks for this period, beautifully digitized here.  Herein, the hypothesis that mottled tissue portions showing the mutant phenotype represent somatic loss of the relevant genes during development was addressed (not the case). So, too, the stoichiometric balance of heterochromatic vs euchromatic factors. Jack Schultz (no relation) undertook a screen for “modifiers of the grade of variegation” reasoning that there might be so-called “interstitial” heterochromatin – pockets of heterochromatin interspersed in the euchromatic arms of the chromosomes, deletion of which would modify variegation as loss and gain of Y chromosomes did. He was right about the interstitial heterochromatin (now called intercalary heterochromatin PMID: 11862456) but more than likely his secondary mutations (14 suppressors and 58 enhancers) that modified PEV were hits in genes we now know encode proteins important in establishing and maintaining heterochromatin. Of especial interest is Schultz’s analysis of the light gene – a mutation that yields a pale brownish red colored eye (as opposed to the normal bright brick red color – we fly pushers can distinguish considerably more than fifty shades of red). This gene is located in heterochromatin, and Schultz observed that it appeared to behave in the opposite way when compared with a euchromatic gene like the wild type red eye-color gene. Schultz discovered a weird phenomenon we still have not adequately explained today, which is that those few unfortunate genes stuck in heterochromatin seem to have adapted to the compressed, transcriptionally repressive environment, indeed they NEED it to function properly, and so what might suppress variegation in euchromatin enhances variegation in heterochromatin, and v.v. (I am reliving prenatal traumas here because this is what I studied for my Ph.D. Here is a classic review: PMID:8825487) There are many tantalizing findings in these Carnegie Institution papers: in addition to environmental effects on PEV and weirdo heterochromatic genes, the authors pondered parent-of-origin effects on PEV (today we may ask: PEV somehow connected to imprinting? PMID: 10101173 – and try this blog for an interesting overview the genetics of imprinting) the nucleic acid vs. protein nature of chromosomes (is heterochromatin maybe connected to RNA? NOT so far from the truth as we shall see) and so on.

But I am getting ahead of myself again. So now let’s skip over the war (such a boon for physicists – less so for Drosophila geneticists) and the paradigm shifting discoveries of DNA structure and the genetic code, and hit the 1960’s, man, where PEV got its groove back. Janice Spofford (a LIVE white female) published the first detailed genetic analysis of PEV (PMID: 6082620, PMID: 17248449) and a classic review (in a book – email me if you would like the reference) that is still used in publications today. Spofford picked up where Schultz and co. left off, mapped and characterized in some detail a third chromosome Suppressor of variegation ( which she called a “Suvar”). The main difference by this point is the preponderance of fabulously useful rearrangements that allowed her to maintain mutant lines, duplicate regions of the genome, move bits of heterochromatin into euchromatin etc. A panoply of what started out as X-ray induced peculiarities now serviceable genetic tools.PEV fig After Spofford’s work, a bit of a hiatus, and then in the 1980’s and 90’s a flurry of enhancer-suppressor screens using chemical and transposable element mutagens (PMID: 6795427, PMID: 2501647, PMID: 8382174 and others).These kinds of screens are very popular with geneticists – the idea being that there are probably secondary mutations that can be induced in the background of a variegating allele that can ENHANCE the mottled phenotype (ie. make it more extreme, more mutant white, less normal red eye pigment) or SUPPRESS the mottled phenotype (more red, less white). Map and identify the secondary mutations, identify the genes involved, and build a more detailed mechanistic picture of what is going on. Figure 5 is my artistic attempt to diagram the effects of these screens, using Muller’s white[mottled 4] rearrangement that moves the normally distal (telomeric) position of the red eye color gene proximally right next to heterochromatin. In this position, as the individual cells differentiate during development in the embryonic eye, the red eye color gene might be shut off by encroaching heterochromatin, (leading to white ommatidia) or escape silencing from the heterochromatin (leading to various distinct shades of red in the adult eye). The result is a mosiac of red(s) and white eye colour. Note this model suggests the heterochromatic border acts something like a tidal front, that ebbs and flows in development. Here are a couple of useful reviews: PMID: 9610404, PMID: 12524009.

By the 1990’s somewhere between 50 and 150 enhancers and suppressors of variegation had been mapped (big range because definitions varied but the point is, MANY genetic changes can modify PEV). Also by this time, the revolution in recombinant DNA technology had taken place, a HUGE tectonic shift for all biological science I am just skipping over here (really important, look here). This revolution meant that it was now possible to isolate the sequences of the genes responsible for modifying PEV, purify the proteins, test for interactions and so on. Huge discoveries, but best of all, these modifiers turn out to be highly conserved throughout taxa. Thus, the obscure eversporting spotty eyes of fruit flies end up providing the groundwork for understanding the epigenetic regulation of gene expression in all eukaryotic organisms, plants and animals.

The field has truly exploded. What I have written to this point, in this very long blog entry, represents the trunk of a massively branching tree of new information. Probably impossible at this point to summarize, but try reading this paragraph out loud in one breath, you might get an idea of how enormous this field has become, and how rapidly it has progressed. So for example: Suvar2-5 encodes a protein called Heterochromatin Protein 1 (PMID: 3099166), that binds to a particular residue on the N-terminal tail of Histone H3 that has been methylated (PMID: 11242053). The methyl group is put there by an enzyme called Suvar3-9, after a histone deacetylase encoded by another Suvar removes an acetyl group from the same amino acid residue, and since HP1 interacts with itself, this chain of events sets up a spreading tendency that compresses chromatin into a heterochromatic state (PMID: 11283354). In fact methylation is one of several post-translational modifications of specific residues in the N-terminal tails of histones that seem to create a kind of chromatin Braille that protein complexes can “read”, compressing the chromatin fiber for silencing (by reducing access to DNA sequences to which transcriptional activators bind) or opening the fiber up, loosening chromatin structure (to facilitate gene expression). These post translational modifications have been collectively dubbed a “histone code”(PMID: 11498575), the interpretation of which helps to explain things like developmental gene expression (how are genes turned on and off in the highly concerted manner required for the differentiation of cells and tissues), transcriptional memory (how does a genome remember which genes are on or off for a given tissue, through the contortions of mitosis), imprinting (parent of origin effects), centromere identity and chromosome segregation, even the early musings that RNA was playing a role have been borne out (though the original hypotheses could not have envisioned RNA interference targeting protein complexes to heterochromatin PMID: 12215653), and all this, the proteins and the mechanisms, all this is conserved – PMID: 11242053, PMID: 10202156, PMID: 8505380, PMID: 10581035 honestly I could go on for several pages with a list of PMIDs but here is a not too ancient review instead: PMID: 17173056.

These and other things I would love to go on about: the behavior of modifiers has shown that the heterochromatin at telomeres is somehow different from the blocks that surround the centromeres, a phenomenon known as telomeric position effect or TEV (PMID: 11293791, PMID: 12663532). There are modifers of PEV that have led to the discovery of boundary elements in the genome – sequences and protein complexes that control the spread of heterochromatin and effectively divide the genome into different transcriptional domains (PMID: 18082602, PMID: 11700282). There is a dominant PEV allele caused by a chunk of heterochromatin inserted right next to the brown gene (another eye color gene) that exerts its dominant effect by sticking to the wild type brown allele on the other homolog and dragging it into a repressive nuclear compartment (PMID: 8646782, PMID: 7672573). Indeed, another Suvar appears to organize chromosomes in the interphase nucleus (PMID: 11390354), and I am not sure we yet have any idea about those weirdo genes trapped in heterochromatin that require an apparently repressive environment to function (PMID: 16690158). All in all, the arcane intricacies of position effect variegation have unlocked some of the deepest mysteries of epigenetics – how a gene’s position in a chromosome, and that chromosome’s place in the nucleus, and the structure of the nucleus itself work together to orchestrate that magnificent generation of complexity from embryonic protoplam, a story of genetic and epigenetic regulation that continues to unfold today,even as you read this blog.

Drosophila Lamin C mutations: Preamble

I have friends who pick complicated men. I pick complicated genes. As a PhD student, I worked on genes stuck in heterochromatin that tended to be HUGE (hundreds of thousands of base pairs, mostly introns, mostly repetitive DNA).  My last publication (eons ago in 2012) was about a gene that went and quintuplicated itself. For my post doc, I worked on Lamin C, an essential gene nested inside the intron of another essential gene called tout velu. What fun!

Lamin C is a nuclear envelope protein. In multicellular animals, the nucleus has a mesh-like structure that lines the inner surface of the nuclear envelope, made out of filamentous proteins called lamins. Humans have two main types of lamins, A types and B types, and the division of labour between them is still not all that clear, but B types seem to have a predominantly structural role while A type lamins do other things, some of which are very mysterious indeed. Long story short, A type lamins – which in humans are further subdivided into Lamin A and Lamin C – seem to be required inside the nucleus, (as well as the periphery), where they probably play a role controlling nuclear organization and gene expression – very interesting epigenetic stuff. Mutations in A type lamins lead to a very wide range of rare diseases that affect multiple tissues – muscular dystrophies, neuropathies, bone disease – perhaps because when mutant, these lamins upset developmental gene expression programs that commit cells to specific tissues or organs. Or perhaps the tissue is rendered less mechanically sound – a lot of the diseases are muscle disorders, and cardiomyopathies, which are tissues under mechanical stress. Jury is still out, but by far the most unusual disease caused by defects in A type lamins is a premature aging disorder called Hutchinson Gilford Progeria, where children age very rapidly, usually dying before they are teenagers. HBO broadcast a very compelling documentary about the life of one of these unfortunate kids called “Life according to Sam” – although the kid was so amazingly positive and bright that “unfortunate” almost doesn’t seem to apply…

The mechanism of this progeria is still not at all clear. How does a mutant nuclear envelope protein cause rapid and system-wide aging? Do the stem cells fail to replenish themselves? Is differential gene expression messed up? I don’t think any one knows.

So lamins are interesting. They are also very highly conserved  – in animals anyway. Plants seem to have something similar but via a separate evolutionary pathway – a kind of convergent evolution. Among animals, invertebrates tend to have only the B-type lamins, while vertebrates have the A and B types. EXCEPT that Drosophila has two types. There is the B type, in flies called Lamin Dm0 (strange name deriving from its initial discovery involving different forms in the cell cycle) and the A-type called, confusingly, Lamin C. Why Lamin C and not Lamin A? Has to do with something called a prenyl group, and the mechanics of lamin incorporation into the nuclear envelope. It is horrible to explain and I distinctly recall my colleagues’ eyes running and noses bleeding whenever I tried so I won’t go on about it just yet here. Suffice to say that lamins are either prenylated or not, which means they either have a pointy-branchy sticky carbohydrate membrane anchor attached to their C-terminus, or not, because sometimes it gets clipped off post-translationally, which is what happens to human Lamin A. Human Lamin C turns out to be an alternatively spliced version that skips the bit of the gene that encodes the signal to put the anchor on, so it is never prenylated. Drosophila Lamin C is like that too, the gene does not encode a prenyl anchor. And it is true that when you compare the physical location of Lamin C and Lamin Dm0 using confocal microscopy, Lamin C tends to be both at the periphery and in the interior; Lamin Dm0 is more concentrated at the envelope (see figure – sorry no scale bar but the (polytene) nuclei are each about 15-20 microns across – 2 different mags).Lam C & Dm0 confocal

So what is Drosophila Lamin C (dLamin C) doing inside the nucleus? Anything analagous to the human Lamin C?  Are fruit flies just more efficient than humans? No messy alternative splicing or post translational processing to remove the anchor. Turns out you don’t really need Lamin A at all…well, that is if you are a mouse (PMID:16511604).  Maybe the complicated and messy and disease-causing Lamin A is just an evolutionary boo boo that has to get mopped up post-translationally (potentially another example of what I like to call “unintelligent design”. )

Hence, my interest in Lamin C. Well, me and MANY other people. During my post doc I worked on generating a mutation in dLamin C (LamC) which turned out to be a bit tricky because it is nested inside this great big giant essential gene called tout velu (ttv: French for all “all fuzzy” – plays a role in Hedgehog diffusion and pattern formation…another story: PMID:9665133). So making a mutant meant generating a lesion in LamC without disrupting tout velu. Lam C gene regionHere is what it looks like in Flybase: the blue line ticks are 10kb apart so ttv  is a pretty enormous gene.

But that is all ancient history. We made nice mutations and characterized them and it was great fun. There are other mutants about but some of them are iffy – so I wanted to make a new one, using the same method – imprecise excision of a transposon hit. With mutants, the more, the merrier. Most importantly because if you want to use a mutant to study something biological, you have to worry about what else got buggered in the background when you made the mutant, and is that buggery causing the interesting biological effects you are seeing. So duplicating a given experiment with mutations in the same gene but from two different genetic backgrounds is really important.

First thing is to find a fly strain with a P element (transposon) inserted in the right place. There are lots, but finding one that affects LamC without affecting ttv is hard. I highlighted the two best choices below. G00158 is what we used all those years ago during my pos doc (PMID:15965247). They are both in LamC’s first intron, When I started here at WWU I chose EP(2)2199 which is no longer available (probably because it was unhappy and kept dying for reasons that will become clear). So that is where this story begins.

Lam C P hits

Human Lamins: Preamble

Pallas Eschew ObfuscationI have a big sign in my office: ESCHEW OBFUSCATION. My students often squint at it dubiously, and I reply “exactly.” (You can imagine my reviews on Rate My Prof – which I read for comic relief). Well alas, I am now about to embark on a topic for which obfuscation is almost impossible to eschew. But I love it. My affair with lamins began during my postdoc where I moved out of the treacherous deserts of heterochromatin and into the wider landscape of the nucleus as a whole.  I am always quite peeved by text book images of the nucleus as a grey blob in the middle of the Las Vegas like craziness of the cytoplasm. In fact there is a great deal of organization in the nucleus, enough structure to warrant the term nucleoskeleton, which you don’t hear very often (even WORD doesn’t like it).  Quite apart from the DNA and histones, which fold almost miraculously into chromosomes (getting about two or three meters of DNA into a space that is rarely more than a few millionths of a meter wide) there are specific zones in the nucleus, or workshops where little DNA elves activate or repress particular regions depending on developmental context etc. OK there are no elves, but there is great organizational detail, and something has to hold it all together, through replication, repair and transcription. DNA itself is a good bet for scaffolding – helps explain why there is so much of it outside the gene-encoding bits.  And as I have mentioned elsewhere, actin makes an appearance in the nucleus as well, an essential component of nucleosome remodeling machines. Here is a fairly recent review with nice graphics: PMID: 21450447

Lamins, like actin, belong to that class of proteins that are major building blocks of cell structure. Lamins are intermediate filament proteins, long and stringy, but unlike actin or tubulin, they have no polarity so motors like myosin, kinesin, dynein, can’t walk on them. Also unlike actin and tubulin, intermediate filaments are not dynamically unstable, so once assembled they are STRONG, thus comprising a considerable portion of very familiar body parts – skin, hair…and less obviously to the naked eye, mesh-like structures that hold organelles in place and knit cells together into tissues and organs. Lamins are the less sociable members of the intermediate filament family; they do not live outside the nucleus. They form a mesh-like structure (the nuclear lamina) that underlies and IF evosupports the nuclear envelope. An ever-expanding menagerie of additional proteins interact with lamins, chromatin, the nuclear envelope, making connections with the cytoskeleton to regulate nuclear structure and position. Lamins are also ancestral; all the familiar cytoplasmic intermediate filaments can trace their evolutionary origins to lamins. Figure 1 is a useful figure that summarizes lamin evolution, from a marvellous lab website that has lots of information about intermediate filaments (among other things).

As you might expect considering the ubiquity and importance of structural proteins, mutations in intermediate filaments cause a number of god awful diseases. Here is a review: PMID: 19587450; in fact the whole issue is devoted to intermediate filaments and disease, including diseases arising from mutations in lamins, so-called laminopathies. These are rare, but remarkably numerous and predominantly tissue-specific diseases – perhaps because lamins play important roles in organizing a nucleoskeleton to maintain proper differential gene expression (one hypothesis among many). Among the most distressing of these diseases is Hutchinson Gilford Progeria Syndrome (HGPS) – a disease of rapid, premature aging affecting children, who do not usually live beyond their teens. HBO produced a remarkable documentary about this illness, profiling the life a boy named Sam Berns (“Life according to Sam”) whose parents were doctors, struggled to find a cure or a treatment as their son aged at approximately four times their rate.

Disease always brings a biologist back to earth. The biological puzzles are fun but really the goal has to be finding a way to treat or cure people experiencing the not-so-maternal side of Mother Nature. But to understand how HGPS develops, you have to set aside the human stories for a while, and delve into the rather thorny biochemistry of lamins. Because I have a lot of experience explaining and re-explaining this complex stuff and doling out Kleenexes for all the resulting nose bleeds, I have learned that a just-the-facts-ma’am point form approach is rather useful, so here goes.

  1. Lamins – and the nuclear lamina they form – appear to be confined to metazoan (multicellular) animals. Plants have something similar (PMID: 24808902), but clearly seem to derive from a separate evolutionary origin. The jury is still out on unicellular eukaryotes, but yeasts (S. cerevisiae) do not appear to have lamins. Why not? Dunno.
  2. There are two types of lamins in vertebrates – A types and B types. There is a division of labor between them, but it is not altogether clear what that is. B type lamins are often described as the “house-keeping” lamins, the ones that are used mostly for structural purposes. Indeed, the B types tend to localize more typically at the nuclear envelope, whereas the A types appear to have both a nuclear rim and interior localization.
  3. For the most part (that being the number of genomes that have been investigated) invertebrates make do with B type lamins alone. EXCEPT in Drosophila, where there are two lamin genes – one encoding a putative B type, and the other a putative A type. Why this exception (and is it an exception?) is unknown. Look HERE for a confocal image of A and B type lamin localization in Drosophila.
  4. So what is the difference between A and B type lamins? (Apart from localization). There are some chemical differences, but the most obvious structural difference relates to the C-terminal portion of the protein. Lamins with a particular sequence of amino acids at the C-terminus – a so-called CAAX box– are subjected to post-translational modification: the addition of a hydrophobic membrane anchor consisting of a fatty acid derivative called a prenyl group. CAAX stands for 4 amino acids: cysteine (C-), followed by two aliphatic (i.e., not aromatic, no benzene rings) amino acids (-A-A-), followed by (almost) any amino acid (-X). The prenyl anchor is affixed to the cysteine, and then the –AAX is cut off by a protease, and the resulting exposed hydroxyl group is methylated.  Once prenylated, the B type lamins are incorporated into the nuclear lamina, accounting for the peripheral localization I mentioned earlier. Humans have two B type lamin genes, both encoding CAAX-box containing lamins, both prenylated. Drosophila has one B-type lamin – called Lamin Dm0 – prenylated.
  5. The story is more complicated for A type lamins. Humans have one A type lamin gene, but there are two main transcripts expressed from it. They are both identical except for the very end of the message – one transcript gives rise to a lamin with a CAAX box – this is Lamin A – and the other message has an alternative 3’ exon that does NOT encode a CAAX box – this is Lamin C. In figure 2,  I have tried to show this arrangement by matching up the protein domains and laminopathic mutations with the corresponding genomic and primary transcript regions. The vast majority of mutations are point mutations (only a handful are shown here), causing single amino acid changes, and the majority of diseases are various kinds of muscular dystrophies, which spawned the “mechanical stress hypothesis” – that tissues under mechanical stress are more likely to suffer due to nuclear lamin defects. Lipodystrophies (affecting fat tissue) and premature aging of course do not fall into this category.lamin mut fig
  6. So. Two A type lamins in humans, and one of them is prenylated, like a B type lamin. And therein lies the problem. Or potential problem. Whereas B type lamins are stably prenylated, A type lamins aren’t. What? Yes, here is the weird part. Lamin A is first translated into a protein called prelamin A, which gets prenylated, and incorporated into the nuclear lamina. But then, after Mother Nature goes to ALL the trouble of linking the prenyl anchor (requiring one enzyme), and trimming the –AAX (requiring a second enzyme) and methylating the terminus (a third enzyme) the entire anchor is removed! The final processed protein is called Lamin A. This additional processing seems very wasteful. Does it make any sense?
  7. To make matters worse, if this final cut does not take place, if the prenyl anchor is NOT removed from Lamin A, if Lamin A is now stably incorporated into the lamina, the result is the premature aging disorder HGPS. So here is the puzzle: why does a mutation that produces a stably prenylated A type lamin cause rapid and systemic aging?

So those are the facts, ma’am (or sir). Or some of them, anyway. Here are some more Interesting factoids. The single celled eukaryote Saccharomyces cerevisiae – used in biolabs, breweries and bakeries the world over – does not have lamins, as I have mentioned, but DOES have a very similar prenyl processing pathway. Yeasts come boy and girl versions which look indistinguishable (well…THEY can tell) and they hook up using pheromones. One of these pheromones is processed much like prelamin A – a longer protein is prenylated, trimmed, trimmed, and trimmed again: see Figure 3. In yeast, the prenylated bit is what matters – this is secreted as a mating signal. With Lamin A, though, people tend to focus on the now unprenylated lamin left behind. (Though I have always wondered if there is in fact a function for the prenylated bit. There was ONE paper that tried to address this question but I cannot find it anymore – any information on this would be greatly appreciated). You can see the history of the connection between the pheromone and lamin pathways echoed in the gene nomenclature: the Type I prenylprotease in yeast is called Ste24p (Ste for sterile – mutations first isolated based on a sterility phenotype – no pheromones, no nooky) and Zmpste24 in mammals – for Zinc Metalloprotease ste24.processing

So there you have it, another one of Mother Nature’s retooling specials. She has to make a pheromone, so she trims a precursor in this specific way, and then, in a completely different evolutionary lineage, grabs the same pathway to make an intermediate filament protein fit for…well, fit for what exactly? Why does prelamin A go through all this processing? Where is it going that it needs all this trimming and fussing? So many steps…and perilous when you examine the consequences for disease.

For HGPS, the mutation in the gene is a classic example of why “silent” mutations are not always so silent. The vast majority of HGPS mutations are glycine to glycine changes – GGC to GGT – which at the nucleotide level produces a splicing consensus that is BETTER than the native one, with the result that part of an exon is skipped in frame, and the resulting SHORTER protein (called Progerin) is now missing the proteolytic cleavage recognition site for the Zmpste24 prenyl protease, whose job it is to remove the prenyl anchor. Take a look at Figure 4. progerin figInterestingly, this region in the Lamin A/C gene is susceptible to the same cryptic splicing in otherwise normal cells from aging individuals (PMID: 16645051), perhaps triggering a cascade of events characteristic of normal aging, that are so vastly accelerated in HGPS.

Here is a GREAT review from a lab I like to follow because they do such lovely genetic experiments using a tough model – the knock-in mouse: PMID: 21647293.  These researchers use a great deal of fancy DNA crocheting to design specific A-type lamin variants and engineer them into the mouse germline, to replace the existing, normal A-type lamin gene. Mouse genetics is WAY harder than fly genetics; takes longer, involves a bigger, messier genome and the mice don’t fit well in the vials. In sum, these researchers (and others you will see cited in the references of this review) replaced the A type lamin gene in the mouse with the specific variants and observed the results as described in FIGURE 5 (included here with permission of L. Fong)

lamin mouse KO & KI

So…here are some open questions.

  1. Are Lamin A and Lamin C functionally redundant? Looks like it – since the mouse can get away with a gene solely encoding Lamin A or Lamin C. Flies in this respect are very efficient – their putative A type lamin gene does not encode a CAAX box at all. But there may be specific functions for Lamin A and C in vertebrates yet to be uncovered. Or, the elaborate processing of prelamin A is superfluous. According to the authors of this review: “Given that the prelamin A processing pathway is evolutionarily conserved, we believe that it is overwhelmingly likely that the pathway serves some important function. At this point, however, current evidence suggests that the prelamin A processing pathway is largely dispensable, at least in laboratory mice.” Yes, this elaborate processing pathway appears to be highly conserved in evolution, but does that always imply functional importance? Remember the adage: evolution is not a German engineer making a Mercedes, but a teenager with no money and a broken car. It is equally plausible to me that this messy pathway has been retained because for SOME reason, Mother Nature needs a non prenylated lamin in (mostly) vertebrates (Lamin A or C will do), and from an evolutionary point of view, it is harder (riskier) to mess with the DNA blueprint and get rid of the gene sequences encoding the CAAX box on Lamin A, and easier to use existing (protein) machinery to mop up the mess afterwards, i.e., post translationally. Another example of what I like to call unintelligent design.
  2. I have not discussed other laminopathies here, but do the muscular dystrophies, cardiomyopathies, lipodystrophies and the like occur due to a loss-of-function (LOF) mechanism or a gain-of-function (GOF)? Looks like LOF since the Lamin A/C gene appears to function dominantly in a haploinsufficient manner in these diseases. You can read all about them in the review I mentioned earlier (PMID: 19587450 and papers from the same issue).
  3. Is Progeria solely the result of a GOF or “toxic protein” mechanism, due to the stable prenylation of Lamin A? Maybe. But maybe not. A so-called secondary laminopathy (i.e., not involving lamins directly) was discovered that produces a severe disorder known as Restrictive Dermopathy (RD). This disease is neonatally lethal, and found to be caused by  homozygous recessive mutations in Zmpste24 (PMID: 16297189). RD also arises in humans as a primary laminopathy, with splicing mutations in exon 11 of the Lamin A gene, similar to HGPS (PMID: 15317753). Interestingly though, the RD splicing defect causes a 90 amino acid deletion in Lamin A, while a smaller deletion of 35 amino acids in the same region is associated with an exceptionally rare mild form of HGPS diagnosed in a patient who was 45 years old (PMID: 15121795) (The HGPS deletion is 50 amino acids). Thus, mutations leading to progeroid symptoms can follow an allelic series of varying severity that have in common failure to fully process prelamin A. These observations suggest that loss of the Zmpste24 cleavage site is a factor in disease etiology, but so is the size of the deletion. Also, if the mouse produces an UNPRENYLATED version of progerin (remember, progerin is the shorter version of Lamin A, with the in-frame deletion) the mouse gets Progeria (though not as severe: see part I of Figure 5).

Underneath all these dreadful diseases and complex processing pathways is a central question. If I were writing a grant or a paper, I would use suitably professional yet portentous language, but since I am writing an obscure blog, I will simply ask this: Why does Mother Nature need an unprenylated lamin? And since the consequences of Progerin are so prematurely deadly, and since a similar protein shows up again in normal aging cells, is an unprenylated lamin a key to the secret of eternal youth? Wouldn’t that be nice. And how am I going to incorporate ANY of this stuff into a fly model? Stay tuned!

DMAP1 Project: Preamble

DISCUSSION MAPWARNING: This gene causes many digressions. You might need a discussion map to keep track. Here it is. Figure 1.

DMAP1 is a rather interesting “under the radar” gene. I always like those kinds of genes, or projects, that no one in their right mind would tackle. They represent small spaces in a very crowded research field that people without much cash can try and work on.

I say “under the radar” but that is not true for research using mammalian models. DMAP1 was originally discovered in 2000 (PMID: 10888872) and ever since then has been quite extensively studied in mice and mammalian cell culture. DMAP1 interacts with a variety of other proteins to form transcriptionally repressive complexes that remodel chromatin by modifying histones and DNA (TIP60-p400 AKA NuA4 PMID12963728).  A mouthful of acronyms. In fact, this protein crops up in both signal transduction papers and chromatin papers, both subjects that are THE WORST acronymic offenders. Perhaps it is worth spelling some of them out (hence, the digressions).

The TIP60-p400 complex is essentially the same thing as the NuA4 complex – a nucleosome remodeler. (TIP60 nomenclature from flies and humans, NuA4 from yeast). A kind of chromatin snow plough that uses ATP hydrolysis to shift nucleosomes around the DNA, in order to expose sequences that are important for gene regulation. (Always remember that when controlling gene expression in eukaryotes, you have to contend with the occluding effects of chromatin. Here is an excellent review about the eukaryotic chromatin paradigm: an oldie but a goodie PMID: 10412974) Chromatin remodelers are remarkable molecular machines, very complex with multiple subunits (“rabbit dropping” diagrams often used to explain them), and intriguing interactions with actin molecules IN THE NUCLEUS (PMID: 11880634; highly relevant for discussions relating to a putative nucleoskeleton….another story). Here is a nice, comprehensive review about chromatin remodeling in general: PMID: 21358755. Anyhow, this TIP60/NuA4 complex interacts with DMAP1 and another acronym, DNMT1, to function primarily in transcriptional repression by histone modification (PMID: 14978102), heterochromatin formation (PMID: 14665632), imprinting and stem cell pluripotency (PMID: 21383065) and repair of double stranded DNA breaks via the homologous recombination pathway (PMID: 19845771, PMID: 20864525).

So, DMAP1 is a busy molecule. DMAP1 stands for “DNMT1 associated protein 1” (an acronym that stands for another acronym).  DNMT1 is a DNA methyltransferase – if there are two halves to epigenetic regulation (i.e., regulation of gene expression by chromatin structure) – one would be histone modifications (acetylation, phosphorylation, methylation etc) and the other would be DNA modification – exclusively at this time, methylation. Methyl groups are added to the 5’ carbon of the cytosine ring (we are talking vertebrates here). The enzymes that transfer these methyl marks are called methyltransferases, and they are either de novo methyltransferases (creating a new pattern of methyl marks on the DNA) or maintenance methyltransferases (that maintain an existing pattern). DMAP1 was identified as a molecule that interacts with DNA methyltransferase 1 (DNMT1) – a maintenance methyltransferase. So all that epigenetic stuff the NuA4/Tip60 complex does – histone modifications and the like – is presumably engaged in cross talk with DNA modifications via DMAP1 and DNMT1.

Oh dear this is a huge subject which threatens to go off the rails so let me just refer you to an interesting review about DNA methylation patterns across species, what it’s good for, how it might have evolved etc. (PMID: 18463664).

At last, to flies. If you peruse the fly lit, (via Flybase for example) there isn’t much. In fact, until very recently, DMAP1 really only showed up as a putative potential possible something-or-other in one of those interminable (literally) excel spreadsheets in the submerged supplemental iceberg that characterize most publications these days….maybe DMAP1 is involved in cell cycle regulation, maybe Notch signaling, maybe this, maybe that…trouble with these high through-put insert-subject-here papers is that everything gets connected to everything eventually…it is often hard to find the line that separates useful (though expensive) candidate-finding preliminaries from meaninglessness…but I digress again…

Then, recently – about six months ago – DMAP1 shows up in a much more convincing context, as a regulator via chromatin structure of signaling pathways in innate immunity in Drosophila. Yey! Which of course necessitates a brief description of innate immunity. What is innate immunity? Let’s start with immunity. Most of us recognize this to mean our humoral and cellular immune system which detects antigenic substances like viral coat proteins, bacterial cell wall components, pet dander, food allergens (for those of you with peanut sensitivity check out this recent discovery: PMID: 25592987)…this remarkable system is adaptive…when presented with an antigen (literally, an antibody-generating substance) the immune system can recognize it, send cells out to engulf and destroy it, and stick bits of the antigen onto the surface of specific cells that provide a long term reminder of the antigen so the response is quicker and bigger the next time the body is exposed to that antigen. Sometimes the response needs priming (hence, booster shots). Sometimes, people produce an exaggerated response which can be lethal (allergies to bee stings, or certain foods for example). Sometimes people can be attacked by their own immune systems (as in autoimmune disorders like Lupus).

The vertebrate immune system is very complicated. There are a large number of specialized cell types involved, which are part of the blood system, itself built on antibody/antigenic relationships. The genetics of the immune system are wild and crazy…something I really enjoy teaching in my advanced classes, research that garnered a Nobel Prize (Susumu Tonegawa 1987, check out Nobelprize.org). A deeper and more mysterious question relates to the evolution of the immune system…connected perhaps to the unfolding stages of development in metazoans (multicellular organisms) where within one individual, tissues become increasingly differentiated from each other.

What then is innate immunity? In short, whereas the adaptive system I’ve just described is highly specific, innate immunity is non-specific – a first internal line of defense (underneath the skin and mucosal epithelia) that produces a range of antimicrobial peptides and chemicals that enhance an inflammatory response. Mediators of this inflammatory response include the cytokines you might have heard of, which are a little bit like hormones – small molecules that diffuse quickly through the blood stream and into cells. Adaptive immunity and innate immunity work together to mediate a response to injury or infection – innate immunity works fast (matter of hours – think how quickly bacteria reproduce!) while the adaptive system takes longer (days). The word “innate” is also meant to describe the fact that this early response system is shared across major phyla – both vertebrates and invertebrates. But only vertebrates have adaptive immunity. Thank goodness we’re vertebrates.

So fruit flies are protected by an innate immune response. There are two parts to this response. First, a cellular response, where cells circulating in the hemolymph (fly blood) recognize and engulf invading pathogens. Secondly, a humoral response from the fat body (serves as a lymphatic organ among other things) where a signaling cascade releases certain transcription factors from inhibition in the cytoplasm so they can migrate into the nucleus and promote the expression of genes that result in an increased synthesis of immunity-generating peptides that can destroy the invading pathogens.

This business of a ligand-binding-to-a-receptor-and-releasing-a-transcriptional-regulator-from-inhibition is a common backbone for pathways that regulate cytokine synthesis in vertebrates AND innate immunity in flies AND…drumroll please….egg polarity in early fly development. Pardon?  In one of those wonderful twists of evolutionary fate, the same pathway that specifies patterning in the Drosophila egg is used again to regulate immunity. This kind of retooling is not uncommon. It reminds me of two important quotes: one comes from Theodosius Dobzhansky, who famously pointed out that nothing in evolution makes sense except in the light of evolution (American Biology Teacher 35 (3): 125–129). Another quip comes from a colleague who shall remain nameless unless I am instructed otherwise (you know who you are RSH) but I modified it – evolution is not a German engineer making a Mercedes, but in fact a teenager with a broken car and no money. Just gotta get to the next generation, and if you have to jerry-rig it, so be it, deal with it later. Signaling fig smallI have tried to depict the relationships between these signaling pathways in Figure 2, but this is of course grotesquely simplified. Also these pathways probably talk to each other as well – which I didn’t show in my figure: here is a decent review: PMID: 21209287.

So NOW can we please get back to DMAP1? Yes! Because about six months ago, a paper was published that I think convincingly points to a biological function for this gene in flies. Here is the paper: PMID: 24947515. In sum, these researchers showed that DMAP1 interacts with Relish which if you take a look at my Figure 2 is in one of these related pathways that regulates innate immunity. The same researchers had earlier published a high through-put proteomic paper in which they identified hundreds of potential interactors with components of these signaling pathways and this DMAP1 paper represents a detailed confirmation of this specific interaction (by other methods – QPCR and coimmunoprecipitation analyses) and takes a stab at a mechanism by using a genetic approach – knocking down DMAP1 expression first in cell culture and then in living flies. They also discover connections with other chromatin remodelers – notably the Brahma complex (NO NOT ANOTHER DIGRESSION) and suggest that DMAP1 may be involved in modulating the specificity of the response from these pathways in development. This is a signal transduction thing – multiple inputs get integrated and turned into specific responses. DMAP1 may play a role in the specification of response. The mechanism is still pretty vague (isn’t it always…don’t you love it when a reviewer sends back a 23 page manuscript with seventeen expensive experiments and asks “please establish a mechanism beyond all reasonable doubt. Have a nice day).

The notable thing for ME about this paper concerns the lengths they had to go through in order to knock down DMAP1 in flies. What they needed was a nice clean mutation in the gene. There isn’t one. Well, there are P element hits but they are all in the wrong place. These researchers used transgenic RNAi and the UAS GAL4 system combined with an inducible inhibitor to control when and where DMAP1 was knocked down (I’ll explain the UAS GAL4 system later – another nose bleeder). I always liked this gene because…well it’s under the radar as I said, but also because I am curious about what a DNA methylation associated protein is doing in flies, since flies do not have the same degree of methylation as vertebrates (see digressions above).

I want to make a mutation in this gene. But LOOK at the configuration of the one potentially useful P hit that’s available (highlighted in yellow): it is in the 5’ UTR of the upstream gene (Figure 3). If I try to make an excision, I’ll probably knock out the upstream gene CG33785, not DMAP1. No wonder there is no mutant. Ah! The perfect project for a masochistic geneticist!

Genomic region for DMAP1

I guess I could do a screen with a mutagen but I have no idea of phenotype. Well that’s not strictly true – like the authors of this paper, I used an RNAi stock from Vienna (PMID:17625558) and a ubiquitous driver and it was lethal. I also had three different RNAi lines for the upstream gene and with the same ubiquitous driver, the flies were perfectly viable. So lethality is likely a phenotype. So something like an EMS screen over a deficiency is a possibility, but there is another way too, which makes use of the P element. And for this I have enlisted the collaborative support of a colleague at SFU, also a masochist, who also teaches genetics, but she teaches a LAB as well as a lecture, so can enlist the awesome power of undergraduates to do the boring stuff (undergrads: it is good for you).  In fact, she has been doing this for some time now– using the P element in the upstream gene in a male recombination scheme to select for imprecise excisions in the direction of DMAP1. She has incorporated this experiment into several semesters of her genetics lab course. We now have several lines that we are going to characterize. I will let my colleague talk about the screens, and the results, but I can tell you that there are viability AND fertility issues, and just this weekend I yanked out the ovaries from heterozygous females and their escaper (homozygous mutant) sibs and as you can see (Figure 4), the ovaries are pretty messed up. Apparently this line doesn’t even lay eggs – I am not surprised.

ovaries mutant and wt 02

The liberating thing about putting all this into a blog that well over three people might read is that I can make wild leaps in possibly the wrong direction! Here is a wild leap. The microscopy supports a role for DMAP1 in these retooled pathways for immunity and the specification of egg polarity! Sounds GREAT doesn’t it!

Leaps in the wrong direction should be enjoyed briefly, but then, bubbles must be burst (to mix metaphors). We have yet to do the following VERY basic things:

  1. Confirm lesions in DMAP1 (PCR)
  2. Confirm no effect on upstream gene (QPCR? Rescue?)
  3. Determine if the original P mutant has a background lethal or sterile that is causing this effect

I should point out that I also pulled out testes from escaper males and they don’t look right either. So once we have a better idea about the lesion, we also have to:

  1. Determine penetrance and expressivity of sterility
  2. Lethal phase analysis
  3. At this point the sky becomes the theoretical limit but cash flow may bring that down a tad. Should also point out that the chances of doing any of this in order are pretty remote.

STAY TUNED!

Remodelling the sperm nucleus: Preamble

What on earth is a needle nucleus??? Well it is the shape of the nucleus in a sperm head. Yes, when studying this subject one can talk loudly about SPERM in coffee shops.

Drosophila sperm are particularly unusual. They have very long tails. How long? VERY long. In many species, way longer than the actual body of the fly. Just google “Drosophila bifurca” for an extreme example. In Drosophila melanogaster, the sperm are almost as long as the male’s body, about 1.8mm. Blokes usually feel a bit uncomfortable thinking about this, but for flies, this appears to be an evolutionary strategy where sperm SIZE is traded against sperm NUMBER. Added to which, when females are mated, the sperm is stored in the female’s body, where individual spermatids will “compete” for numerous fertilization events, a mysterious process involving some female control, and modification of female behaviour by accessory proteins that get transferred along in the seminal fluids.

testes picHonestly I could talk about fly sperm all day, which can attract the wrong kind of attention. But here is some even more amazing stuff about fly sperm – how it develops. The sperm forms in the testes, no surprise there. In flies, the testes are very long, coiled structures held inside the body. At the anterior tip, there is a little niche of stem cells, and every time one divides into two, one daughter cell remains in the stem cell niche, while the other undergoes four mitoses, leading to 2/\4 or 16 cells, but all the cells in the group remain attached to each other by cytoplasm, and the collection is housed in a cyst. If you look at the testes under the microscope, you can see groups of cysts containing the cells in different stages of development. Eventually the sixteen early sperm cells (called spermatagonia which sounds like a far off and enchanted place) undergo meiosis (they are gametes, after all), so the cyst at this point contains 16 X 4 or 64 cells. This is a very cool stage where if you are lucky you can actually see the spindles of the dividing cells. Then a yet more remarkable thing happens – the cells start to elongate very dramatically. First all the mitochondria coalesce into structures that look like another nucleus under the scope, and collars of actin form at the base of the nucleus. Then the actin collars squeeze the cytoplasm down the elongating tail, with the mitochondria right along behind, so that at the end, that single celled blob right up at the top of the testis is now a very, very long but still single cell, with the heads of the sperm near the base of the testis and the bundled tails trailing tidily all the way back to the top. A remarkable set of morphogenetic changes in one cell.

needle nuclei summary figThe nucleus of each sperm cell also undergoes a tremendous change. It becomes long and thin, looks exactly like a needle under the scope when stained with DAPI to label the DNA. The genome packaging in sperm is about as compressed as it can possibly be – even histones are a problem and they get chucked and replaced with protamines – ancient histone-like proteins that are conserved across taxa, a universal (?) sperm chromatin protein.

So what happens to the nuclear envelope when the nucleus changes from a blob into a needle? Dunno yet.  But seems like the sperm nucleus is a great model for trying to figure this out. Hmmm……