最新性别决定研究论文

Avian sex chromosomes: dosage compensation matters Heather A. McQueen & Michael Clinton # Springer Science + Business Media B.V. 2009 Abstract In 2001 it was established that, contrary to our previous understanding, a mechanism exists that equalises the expression levels of Z chromosome genes found in male (ZZ) and female (ZW) birds (McQueen et al. 2001). More recent large scale studies have revealed that avian dosage compensation is not a chromosome-wide phenomenon and that the degree of dosage compensation can vary between genes (Itoh et al. 2007; Ellegren et al. 2007). Although, surprisingly, dosage compensation has recently been described as absent in birds (Mank and Ellegren 2009b), this interpretation is not sup- ported by the accumulated evidence, which indicates that a significant proportion of Z chromosome genes show robust dosage compensation and that a partic- ular cluster of such dosage compensated genes can be found on the short arm of the Z chromosome. The implications of this new picture of avian dosage compensation for avian sex determination are dis- cussed, along with a possible mechanism of avian dosage compensation. Keywords Z chromosome . Dosage compensation . Sex determination .MHMvalley Abbreviations MHM male hypermethylated region DMRT1 doublesex and Mab-3-related transcription factor 1 RT- PCR reverse transcriptase polymerase chain reaction QPCR quantitative RT-PCR. The role of the avian Z and W chromosomes in sex determination is unclear In birds, the sex chromosomes are designated Z and W, and the females possess a heterologous pair of sex chromosomes (ZW) while male sex chromosomes are homologous (ZZ). The chicken Z chromosome is approximately 74Mb long and well represented (with 840 genes identified) in the most recent (May 2006) build of the chicken genome sequence (International Chicken Genome Sequencing consortium 2004, Wahlberg et al. 2007), while the small and highly heterochromatic W chromosome is poorly represented with only 260Kb of sequence available and only 4 genes identified. Despite the shared ancestry between Chromosome Research (2009) 17:687–697 DOI 10.1007/s10577-009-9056-8 Responsible Editor: Edith Heard. H. A. McQueen (*) Institute of Cell Biology University of Edinburgh, West Mains Rd, Edinburgh EH9 3JR, UK e-mail: h.mcqueen@ed.ac.uk M. Clinton Division of Developmental Biology, Roslin Institute and R(D)SVS, Edinburgh University, Midlothian EH25 9PS, UK e-mail: michael.clinton@roslin.ed.ac.uk the Z and W chromosomes, a lack of meiotic pairing along most of the length of the female-specific W chromosome has resulted in its degeneration in both size and sequence content. Lack of recombination between heterologous sex chromosomes is a general phenomenon in the evolution of differentiated sex chromosomes from autosomes, and is thought to be initiated by the isolation of a sex determining gene or genes on one proto-sex chromosome. The identity of the avian sex-determining gene remains unknown and there are two likely mechanisms for sex-determination in birds. A W chromosome based sex-determining mechanism would require a female-specific ovary determining gene located on the W chromosome, while a Z chromosome dosage-based system would be dependent on the number of Z chromosomes present (1 x Z = ovary, 2 x Z = testis). A number of potential avian sex determining genes have been identified and the currently most popular scheme proposes a dosage mechanism of sex determination (reviewed in Ferguson-Smith 2007), which depends on the conserved Z chromosome location of the DMRT1 gene in birds (Nanda et al. 2008). Interest- ingly, the Z and W chromosomes of ratite birds are almost homologous showing minimal W degeneration (Shetty et al. 1999) and are believed to represent an early stage of sex chromosome differentiation. The presence of DMRT1 on the ratite Z chromosome and its absence from the near homomorphic W chromo- some (Shetty et al. 2002) is taken as strong evidence of the sex-determining credentials of this gene. The DMRT1 gene is a provocative but unconfirmed candidate for the key avian sex-determining gene The vertebrate DMRT1 (doublesex and Mab-3-related transcription factor 1) gene is thought to represent an evolutionary conserved sexual regulator owing to a shared ancestry with fly and worm sexual regulators (Raymond et al. 1998). Moreover, DMRT1 has a widespread and common role in sexual differentiation across vertebrate species, often showing temporally appropriate sexually dimorphic expression during differentiation of the male gonads (Raymond et al. 1999). Chicken DMRT1 is located on the Z chromo- some and is expressed at higher levels in male (ZZ) gonads than in female (ZW) gonads, both at and after the suspected point of sex determination on embryonic day 5.5 (Smith et al. 1999). Significantly, DMRT1 expression is elevated in embryonic female gonads ‘masculinised’ by the action of the aromatase inhibitor, fadrozole (Smith et al. 2003). However, despite the conserved role of DMRT1 as a downstream sexual regulator in male sexual development in many species, no precedent exists for DMRT1 as the key sex- determining gene and the theory of DMRT1 as an avian sex-determining gene hinges on mapping of this gene specifically to the avian Z. Recent mapping data for other ZW species show that DMRT1 double dose can not represent a common mechanism of ZW sex- determination since DMRT1 maps to both Z and W in some lizards (Kawai et al. 2009) and is autosomal in snakes (Matsubara et al. 2006) and ZW turtles (Kawai et al. 2007). Interestingly, the unusual multi-copy sex chromosomes (five XX or XY pairs) of the platypus show some conserved synteny with avian sex chromo- somes (Grutzner et al. 2004; Rens et al. 2007). Here the DMRT1 gene is found on the 5th platypus X chromosome (X5) (Veyrunes et al. 2008), and is present as a double-dose in female platypus and a single dose in males, which contrasts with the situation in birds. Perhaps sex-differential expression of this gene is important for sex determination in these disparate systems but, if so, the primary determinant cannot be double DMRT1 dosage in all cases. One modification of the DMRT1 sex determination theory in birds assigns a role to the Z located non- coding RNA known as MHM (male hypermethylated region), which is only expressed in females (Teranishi et al. 2001). This RNA of unknown function, has been shown to accumulate at the site of transcription which is cytogenetically adjacent to the DMRT1 locus (Teranishi et al. 2001). It has been suggested that MHM RNA represses DMRT1 expression in females as part of the sex determination process (Ferguson- Smith 2007). However, to date there is no evidence of any direct interaction between MHM and DMRT1, nor evidence that MHM has any capacity to silence gene expression. Moreover, sequence information from the most recent build of the chicken genome (May 2006) predicts more than 1Mb of sequence and 11 other genes to lie between the two loci (Fig. 1). The alternative hypothesis to a dosage-based mechanism of sex-determination holds that the W chromosome may carry an ovary-determining gene. The current paucity of sequence information available 688 H.A. McQueen, M. Clinton for the W chromosome confounds full exploration of this possibility and the strongest W candidate to date (the HINTW gene reviewed in Smith 2007) has now been shown to lack dominant sex-determining potential during mis-expression experiments (Smith et al. 2009). A further possibility exists that avian sex determina- tion relies not on sex specific expression differences at a single locus but rather on a different pattern of expression at multiple loci between the two sexes, resulting from the different ZZ versus ZW sex chromosome constitutions. Female avian Z chromosome genes have a reduced gene dosage relative to autosomal and male Z genes The evolution of heteromorphic ZW sex chromosomes in birds has the consequence that all female Z chromosome genes are monosomic. One would expect that such significant aneuploidy would not be easily tolerated (Birchler 2009) and recent studies in mam- mals and insects provide concrete evidence of the need to balance the expression of hemizygous sex chromo- some genes with the expression of autosomes (Cheng and Disteche 2006). Genome-wide microarray expres- sion analysis has recently demonstrated that male mammals achieve global upregulation of expression from their single X chromosome in order to equal the level of expression from autosomal genes, which are present in twice the dose (Nguyen and Disteche 2006a). Similarly for the nematode worm, despite the previously accepted dosage compensation mechanism of “double-down” regulation of expression from the two hermaphrodite X chromosomes, the single male X chromosome is now shown to be up-regulated to give a male X to autosome expression ratio of 1 (Gupta et al. 2006). In addition to, or perhaps as a result of, this sex chromosome to autosome balancing, further modifica- tions occur to reduce sex chromosome expression in homogametic individuals, resulting in equalisation of expression between the sexes. Such modifications in mammals and insects are well studied (reviewed elsewhere in this issue) and the term “dosage compen- sation” is used to describe the normalisation of sex chromosome gene expression that results. The question then arises of whether birds also perform some form of dosage compensation in order to correct for the reduced gene dosage along the single female Z chromosome. Sex chromosome dosage compensation occurs in birds Until relatively recently dosage compensation was widely accepted to be absent in birds, principally Fig. 1 The “MHM valley” region on female chicken Zp showing the positions and dosage compensation status of 61 genes. Genes positions are plotted along the map in kilobases from the Zp telomere, and symbols are attributed according to male to female ratios provided (Itoh et al 2007 supplementary data). Green circles denote dosage compensated genes with ratios of 1.3 or less in any tissue, red diamonds indicate non- compensated genes with ratios of 1.5 or above in all tissues tested and genes intermediate between these two categories are shown as black squares. Expression data was unavailable for DMRT1, DMRT3, and a further 33 annotated genes in this region which are not shown. Locations of the DMRT genes and the MHM gene are taken from the 2.1 build of the chicken genome (accessed 27/11/2008). The red triangle indicates the absence from this sequence of approximately 450 Kb of repetitive MHM sequence. The end of a 9 Kb region showing MHM homology (27,963 kb to 27,972 kb) coincides precisely with an annotated sequencing gap which is presumably approximately 450 Kb long to accommodate the missing MHM sequence. A further 19 gaps exist in the region shown. Pink and red broken lines along the bottom of the diagram indicate that this region is strongly acetylated at H4K16, and painted with non-coding MHM transcripts, specifically on the female Z. Neither the length nor the boundaries of these modified areas are known due to their cytogenetic detection. MHM* indicates that the MHM gene has been confirmed to be affected by the H4K16 acetylation while DMRT1 is unaffected Avian dosage compensation 689 on the basis of limited evidence regarding sex- differential isozyme activity of the Z-linked aconi- tase protein (Baverstock et al. 1982). However, around 2001 this view was reversed when two small- scale studies established that the levels of expression of some Z chromosome genes were equalised in male and female birds, suggesting some form of dosage compensation (McQueen et al. 2001; Kur- oiwa et al. 2002). Using the limited gene sequences and map locations available at that time, we examined transcript levels for a selection of nine genes dispersed along the chicken Z chromosome. Expression levels for all nine genes were analysed by quantitative RT-PCR (QPCR) in early (day 3 and day 4) male and female chick embryos (McQueen et al. 2001). Despite observing very heterogeneous levels of expression between the 12 or more individual male and female birds analysed for each gene, similar averaged levels of expression were found in males and in females for six of these genes (McQueen et al. 2001, Table 1). We concluded that these genes must be subject to some form of dosage compensation but that this regulation did not affect all Z chromosome genes, since at least one of the nine genes was clearly not compensated. Dosage compensation was also independently demonstrated for one of two additional genes studied in 15 day old embryos by a similar technique (Kuroiwa et al. 2002, Table 1). These studies established for the first time that a significant proportion of avian Z chromosome genes were subject to dosage compensation, but gave no real indication as to the extent of this compensation or to the nature of the mechanism involved. Such QPCR studies were advantageous in their ability to detect and present accurate levels of gene expression for individual genes in individual animals and to assess the variability that exists. The small number of genes that were measured could, however, lead to a skewed impression of the extent of dosage compensation. Moreover, while male: female ratios for Z gene expression were measured, the small number of control autosomal genes analysed precluded assessment of average Z chro- mosome: autosome (Z:A) ratios. While comparing expression levels of Z chromosome genes in male and female tissues is useful in terms of monitoring the effects of dosage compensation, the expected role of dosage compensation would not be to achieve parity in Z gene expression between the sexes, but rather to regulate the sex chromosome to autosome ratio within individuals, similar to the recently demonstrated upregulation of the single male X chromosome to autosomal levels in mammals and nematode worms. The most informative measure- ment of the extent of sex chromosome dosage compensation in birds should, therefore, be the global sex chromosome to autosome expression ratio in the heterogametic sex, the female Z:A expression ratio. Average Z:A gene expression ratios are >0.5 but <1 in female birds indicating that dosage compensation is not chromosome-wide Results are now available from a number of medium- and large-scale studies of gene expression in male and female avian tissues giving Z:A expression ratios which provide a more global picture of the extent of dosage compensation in birds. In zebra finch, a medium-scale analysis was performed where 40 Z chromosome genes and 84 autosomal genes were analysed in 4 different adult and newborn tissues by competitive hybridisation with mixtures of male and female cDNA (Itoh et al. 2007). Although the experimental design of this study precluded analysis of Z: A ratios within the separate sexes, some degree of male over-expression was detected for 36 Z located genes which argues against a chromosome wide system of tight dosage compensation. A larger scale analysis of chicken gene expression by the same authors used affymetrix chicken genome microarrays containing more than 16,000 probes of which around 5% represented Z chromosome genes. Gene expression was measured in brain, liver and heart from 20 male and 20 female day 14 embryos, with five sets of pooled samples from each sex being hybridised separately. Male: female expression ratios were established for indi- vidual genes, and used to generate an average male: female ratio of 1.2–1.4 for the Z-chromosome, while the average male:female ratio for autosomal genes was close to 1.0. In contrast, mammals were reported to show male:female expression ratios close to 1.0 for both autosomal and X-linked genes. Since the category of Z located genes showing male biased expression was shown to include housekeeping genes, the authors concluded that their results did 690 H.A. McQueen, M. Clinton not simply reflect a preferential Z location for sex- biased genes, but instead provides evidence of “ineffective” dosage compensation of avian sex chromosomes (Itoh et al. 2007). However, the authors noted that the chicken Z:A ratios, which were consistently higher in males (0.92–1.08) than females (0.70–0.87), were nevertheless within the range of X:A ratios reported for mammals which does indeed suggest balancing of Z and A gene expression in birds (Itoh et al. 2007). In a separate study, chicken affymetrix arrays were hybridised with day 18 embryonic heart, gonad and brain samples from each of 4 male and 4 female birds. Sexually dimorphic expression was reported for just under a quarter of all genes, of which 25% were located on the Z chromosome (Ellegren et al. 2007). Male:female expression ratios were calculated for individual genes and the mean fold-change of Z gene expression in male versus female somatic tissues was reported as 1.42 (although a subsequent study by the same authors reported a reduced average male: female expression ratio of 1.24 in embryonic and adult brain (Mank and Ellegren 2009a)). Autosomal genes displayed no such sex-specific differences with a mean fold-change close to 1.0 (Ellegren et al. 2007). In contrast to the results of Itoh, Ellegren and colleagues described significantly elevated female A: Z ratios, (corresponding to low Z:A ratios) quoting a 39% higher level of gene expression for autosomal versus Z chromosome genes in somatic tissue. This appears to be at odds with data presented in additional file 7 (Ellegren et al. 2007) which seems to show hybridisation intensities for the female Z chromosome that are in the same range as those from autosomes. The authors also noted that the overall average level of expression for dosage compensated Z chromosome genes tends to be lower than average levels of autosomal gene expression and conclude that this reflects down-regulation of male expression rather than up-regulation of female expression (Ellegren et al. 2007). This is a surprising finding given our assumption that the Z: A ratio would principally be a problem for the heterogametic females where upre- gulation of expression from the single Z chromosome might be expected. It is clear from both studies that a significant number of genes across the avian Z are expressed at a higher level (although not normally 2-fold) from the two male Z chromosomes than from the single female Z chromosome (Itoh et al. 2007; Ellegren et al. 2007). This strongly argues against global all-inclusive dosage compensation across the avian Z and suggests that many genes are not compensated, or only partially compensated. Importantly, both papers pres- Table 1 Dosage compensation status of 10 chicken Z genes showing male to female ratios derived from 3 independent studies Gene Position on the chromosome m:f ratio cytogenetic sequence (kb) Zov3 (p2.1) 14,147-14,166 1.351 Follistatin (p2.2–2.3) 15,391-15,398 1.351 Brm(Smarca2) (p1.2) 27,123-27,225 1.231, 1.13, 1.34, 1.25 VLDL (p1.2–1.3) 27,352-27,367 0.781, 1.03, 0.94, 0.65 CHDZ 50,156-50,203 1.232 ChrnB/ACHB3 (q1.3) 52,500-52,510 1.331 AldoB (q1.5) 63,699-63,708 0.941, 0.93, 0.94, 1.05 ScII (q1.5–1.6) 65,031-65,032 2.241 Ggtb2/B4galt1 (q1.5–1.6) 68,712-68,722 1.391, 2.092, 1.93, 1.94, 1.75 Irebp (q1.6) 69,043-69,081 0.831, 1.13, 1.14, 1.05 Average: 1.271 1 day 3 and day 4 whole embryos analysed by QPCR (McQueen et al. 2001), 2 day 15 embryo fibroblasts analysed by QPCR (Kuroiwa et al. 2002), 3 day 14 embryonic brain analysed by microarray analysis (Itoh et al. 2007), 4 day 14 embryonic heart analysed by microarray analysis (Itoh et al. 2007), 5 day 14 embryonic liver analysed by microarray analysis (Itoh et al. 2007) Avian dosage compensation 691 ent graphs representing the spread of male: female expression ratios which have an obvious bimodal appearance rather than a continuous spread from low to high value