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List of papers

This thesis is based on the following Papers, which are referred to in the text by their Roman numerals.

I Reinius B, Saetre P, Leonard JA, Blekhman R, Merino- Martinez R, Gilad Y, Jazin E*. An evolutionarily conserved sexual signature in the primate brain. PLoS Genet. 2008 June 20; 4(6):e1000100.

II Reinius B*, Jazin E*. Prenatal sex differences in the human brain. Mol Psychiatry. 2009 Nov; 14(11):988-9.

II’ Reinius B*, Jazin E*. mRNA expression of Y-linked transcripts in 12 regions of the prenatal human male brain. Mol Psychiatry. 2009 Nov; 14(11):987. (Featured Image)

III Reinius B*, Shi C, Hengshuo L, Sandhu KS, Radomska KJ, Rosen GD, Lu L, Kullander K, Williams RW, Jazin E. Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse. BMC Genomics. 2010 Nov 3; 11:614.

IV Reinius B*, Johansson MJ, Radomska KJ, Williams RW, Jazin E. Large-scale sex-bias analysis of somatic tissues reveals de-masculinisation of the mouse X-chromosome. 2011. (Manuscript)

V Reinius B, Jazin E*, Kullander K. A new mouse line based on the Gpr101 promoter drives expression of Cre in medium spiny neurons of the striatum. 2011. (Manuscript describing work in progress)

* Corresponding author(s)

Reprints of Papers I-III were made with permission from the publishers.

Additional publications

Lagerström MC, Rogoz K, Abrahamsen B, Persson E, Reinius B, Norde- nankar K, Olund C, Smith C, Mendez JA, Chen ZF, Wood JN, Wallén- Mackenzie A, Kullander K. VGLUT2-dependent sensory neurons in the TRPV1 population regulate pain and itch. Neuron. 2010 Nov 4; 68(3):529- 42.

Contents

Introduction ...... 9 Evolution towards sexually dimorphic brains ...... 9 Background ...... 11 Why study sexual dimorphism? ...... 11 Sexual differentiation and the emergence of X and Y ...... 11 Sexual differentiation of the brain ...... 14 Sex differences in brain anatomy and behaviour ...... 18 Sexually dimorphic gene expression ...... 19 Genes on X and Y ...... 19 Genomic imprinting ...... 20 Sexually dimorphic expression of autosomal genes ...... 21 Aims of the studies...... 23 Paper I ...... 23 Paper II ...... 23 Paper III ...... 23 Paper IV ...... 23 Paper V ...... 24 Methodological considerations ...... 25 Biological specimens ...... 25 Transgenic mice ...... 25 Results and discussion ...... 27 Paper I ...... 27 Paper II ...... 30 Paper III ...... 32 Paper IV ...... 38 Paper V ...... 41 Conclusions ...... 43 Summary in Swedish ...... 45 Sammanfattning ...... 45 Acknowledgements ...... 46 References ...... 48

Abbreviations

BAC Bacterial artificial chromosome c Coding CNS Central nervous system Cre Cyclic recombinase dN Rate of synonymous substitutions dS Rate of non-synonymous substitutions FCG Four core genotype H3K27me3 Tri-methylation at lysine 27 of histone H3 lncRNA Long non-coding RNA macroH2A1 MacroH2A core histone variant 1 myr Million years nc Non-coding NRY Non-recombining region of the Y chromosome TDF Testis determining factor X X-chromosome X(m) Maternally inherited X-chromosome X(p) Paternally inherited X-chromosome Xa Active X-chromosome Xi Inactive X-chromosome XCI X-chromosome inactivation Y Y-chromosome

Gene symbols are not listed.

Introduction

Evolution towards sexually dimorphic brains Reproduction is a fundamental feature of all life. All organisms, from the simplest to the most complex, in all branches of the phylogeny of life, have evolved to efficiently replicate their genomic content. Nearly all vertebrates, and certainly all mammalian species, reproduce sexually and produce two distinct types of gametes: megaspores (ova) and microspores (sperm) [1, 2]. Through evolution, the two reproductive modes within species – females and males – have specialised in delivering only one type of gamete [1]. Since the parental investments required for successful production of offspring differ between the sexes, the optimal strategies during reproduction and life will often not be identical for females and males [2]. This fact constitutes the very basic premise for the evolution of sexual dimorphism. Sexual reproduction opened the doors for new forms of genetic competition and conveyed an acceleration of evolution [3]. It is therefore not surprising that sexual dimorphisms in physiology are extensively evolved in the animal lineages. Some of the most apparent physiological sexual dimorphisms are those of the gonads. However, functional sex differences go far beyond the tissues directly related to reproduction. Indeed, such sex differences can be manifested in anything from colour of coating, weight and size, to metabolism and gene regulation [4]. Just as relevant as the physical sex differences, are the behavioural sex differences. Sex differences are evident in behaviours related to reproduction, but far from exclusively restricted to such functions. Consis- tent sex differences in behavioural phenotype must by necessity be matched by physiological differences in the animal nervous system or elsewhere in the body. Indeed, during the last century, neuroscience has established an enormous catalogue of sex differences in the mammalian brain and behaviour [5]. Recent technological developments have aided the efforts to understand the molecular sex differences that form the foundation of sex-biased neurophysiology [6]. The molecular underpinnings of sexual dimorphism in the brain are truly being revealed. The Papers in this thesis represent my effort to contribute to this progress. My studies provide novel observations regarding the sex-biased expression of genes in the human, non-human primate and mouse brain. For example, the studies demonstrate the first observation of evolutionarily conserved

9 sexual gene expression patterns in the primate brain. I also present a catalogue of Y-linked genes expressed in the human male mid-foetal brain. Of potentially wider significance, are the observations I present regarding the transcriptional regulation of genes and domains on the X-chromosome that undergo so-called “escape” from X-chromosome inactivation (XCI), and thus express from the otherwise silenced copy of the X-chromosome in female cells. More specifically, I provide the first evidence that a class of transcripts, called “long non-coding RNAs” (lncRNAs), are expressed together with protein-coding genes in domains that escape XCI on the mouse X- chromosome. This raises the question whether these lncRNAs might participate in the transcriptional regulation of genes located within or close to XCI- escaping domains. I also show that the mouse X-chromosome is depleted of male-biased genes and enriched with female-biased genes. This suggests that the mouse X-chromosome has undergone both “feminisation” and “de- masculinisation”, which is manifested in the regulation of genes located on this chromosome. By analysing sex-biased gene expression in discrete regions of the mouse brain, I show that the striatum is a remarkably sex-dimorphic brain structure compared to other brain structures included. Indeed, this is demonstrated by expression analysis of two biologically independent striatum data collections. In the last Paper of this thesis, I describe an ongoing work, including the generation and characterisation of a novel transgenic mouse line: “Gpr101- Cre”. This mouse line allows for conditional genetic manipulation, distinc- tively targeted at striatum. Moreover, my initial analysis indicates that Gpr101-Cre drives expression in the striatum during embryonic development, suggesting that Gpr101-Cre could become a useful tool for future investigations of striatal formation.

10 Background

Why study sexual dimorphism?

The heading of this paragraph phrases a relevant question, and a question that I have been requested to answer numerous times during my studies. Fortunately, it is very easy to argue for the relevance of studying sexual dimorphism. In fact, sex affects biology and medicine at every level of organization [5]. Sex differences are observed in the prevalence, course and severity of many common diseases, including cardiovascular diseases, autoim- mune diseases, schizophrenia and Alzheimer’s disease [7]. Therefore, sex is an important variable for understanding the underpinnings of any such ail- ments. Sex is also a crucial factor in many aspects of normal physiology and development, and sex significantly shapes both brain and behaviour [5, 8]. In fact, some differences between the sexes are inherent down to the molecular constituents of each cell. Indeed, studies of sex-specific biological processes have proven fruitful in discovering many cellular and molecular processes.

Sexual differentiation and the emergence of X and Y

In most vertebrates, and in all mammals, the gonadal sex is determined during embryonic development [9]. A common feature in these species is that sexual determination is initiated by the activation of biochemical pathways that lead to the expression of specific genes in the primordial gonads [10, 11]. This gene activation promotes the development of the undifferentiated gonads in a sex-specific manner, resulting in the formation of the female or male internal gonads: ovaries and testes [9]. When sufficiently matured, the female and male internal gonads excrete different levels of sex hormones, which are circulated in the organism. These sex hormones further interact with cellular receptors and modulate of gene expression, resulting in physiological sex differentiation of the external gonads, the brain and other somatic tissues [9]. The initial signal igniting the sex determining reaction pathways varies in vertebrate species [10]. It is likely that the ancient signal was environmental, perhaps comparable to the temperature dependent sex determining mecha-

11 nisms found in many reptiles [12, 13]. At some point in the linage of the therian mammals, after their divergence from the monotremes, a dominant genetic male-determining initiation signal emerged on an ordinary autosome [14-16]. This so called “testis determining factor” (TDF), could activate the male sexual differentiation pathway without the originally required environmental stimuli (Figure 1) 1. The advent of TDF was the first step towards a major event in the evolution of mammals: the emergence of the mammalian X- and Y-chromosomes. Alleles increasing male fitness appeared by chance in the chromosomal vicinity of TDF, and suppression of recombination between the nascent X- and Y-chromosomes was favoured by evolution to maintain the linkage between male-beneficial alleles and TDF [15]. Fur- ther accumulation of sexually antagonistic genes (i.e. genes that are beneficial in one sex but detrimental in the other) on the nascent sex chromosomes led to an almost complete segregation of X/Y genetic recombination [17]. X/X recombination continued to occur in the female lineages, and the X- chromosome therefore maintained most of its original genes. The sheltering of Y from recombination, on the other hand, resulted in a stepwise degrada- tion and loss of gene functions in the Y-chromosome in a process known as “Muller’s ratchet” [14, 15, 18, 19]. Loss of gene function on Y favoured the evolution of compensatory up-regulation of singleton homologous X-gene copies in male (XY) cells [20]. Accordingly, the transcriptional output of X- chromosome genes increased approximately two-fold [21, 22]. In turn, this up-regulation resulted in the evolution of “X-chromosome inactivation” (XCI), a mechanism than counter-balanced the X over-expression in females by means of epigenetic silencing of one of the X-chromosomes in XX cells [23, 24]. The stepwise continuation of these processes resulted in the major differences which can be observed in the current mammalian X- and Y- chromosomes; most notably, a small and degraded Y-chromosome, and an X-chromosome of which one copy is transcriptionally silenced in female cells. The intricacies of the X-chromosome do not stop there. As a result of heterogamety in males (XY) and homogamety in females (XX), the X- chromosome is a unique genomic location for sexual conflicts to take place in the selection of sexually antagonistic alleles during evolution. The X- chromosome is on theoretical grounds predicted to be enriched with sexually antagonistic genes, of either female- or male-beneficial type [17, 25, 26]. Further reflections on this subject are however saved for Paper IV.

1 It is plausible that several generations of TDFs might have preceded the current mammalian TDF. For brevity, this discussion is skipped here. For more details see for example: Marshall Graves JA: The rise and fall of SRY. Trends Genet 2002, 18(5):259-264.

Figure 1. Sexual differentiation and the emergence of sex chromosomes. The figure shows a very simplified overview of the processes resulting in sexual differentiation, and the emergence of the X- and Y-chromosomes. A: Ancestral sex determination system. B: The emergence of a dominant genetic testis determining factor (TDF) (the gene Sry in mammals). C: The current mammalian sex determination system, with the male- and female-specific sex chromosome complements (XY or XX).

13 Sexual differentiation of the brain

The female and the male brains represent two modes within a species which can produce striking differences in behaviour. Clearly then, investigating the sexually dimorphic properties of the brain is highly relevant and very intriguing. Developmental processes leading to male and female phenotypes are central for understanding sexual dimorphisms of the nervous system. Landmark studies by Lillie [27], Jost [28] and Phoenix [29] gave rise to the “classical model” of sexual differentiation of the brain [30]. Within the classical model, sexual differentiation of the brain is modelled as a linear process [31]. The classical model dictates that sexual differences in the brain are the direct results of different concentrations of circulating sex hormones in females and males during development and adult life. This process is illus- trated schematically by the black arrows in Figure 2. In mammals, the gonadal sex is determined by the presence or absence of the Y-linked gene Sry [32-34]. This male-specific gene encodes a transcription factor which is active in the primordial gonads (embryonic week 7 in humans and embryonic day 11.5 in mice). The Sry protein ignites a cascade of cellular and developmental processes, involving Sox9 activation, resulting in the development of testes in male embryos [35]. The female foetus, lacks Sry, and therefore produces ovaries. In this context, the female phenotype can be considered to be the default sex [36]. Testes synthesise high levels of testosterone and the ovaries promote an elevation of estradiol and progesterone concentrations. These male and female sex hormones circulate and induce “organisational” effects on genitalia, sperm ducts, brain and other tissues, during critical developmental periods [31, 35]. Testosterone however, does not directly interact with the brain, but is first converted to estradiol by the enzyme p450 aromatase [37, 38]. Estradiol acts via its nuclear receptors, estrogen receptors alpha and beta which work as transcription factors when bound to their ligands, thereby influencing gene expression [39-41]. The male brain thus becomes irreversibly masculinised and defeminised during development, and the female brain becomes permanently feminised [42]. In contrast to these permanent developmental effects stand the reversible “activational” sex hormonal influences on the brain during postnatal life. Experiments in which hormonal levels were manipulated, either by gonadectomy or by chemical interference, showed that some sex-biased behaviours can be either ampli- fied or diminished depending on the type of hormonal manipulation [42, 43]. Thus, the sex hormones contribute with both irreversible and reversible effects to the nervous system. Furthermore, interactions between organisation and activation occur in such ways that the activational effects may require prior organisation to have taken place during development [44]. The classical model provides us with a framework capable of explaining many of the sexual dimorphisms of the brain. However, our current knowl-

14 edge of sex-biased neurobiology has outgrown this simplistic model [31]. Multiple lines of research have contributed to this conclusion. Some of the first evidence came from developmental studies. For instance, male rat embryos were found to weigh more than female rat embryos before sexual differentiation had occurred [45]. Furthermore, studies of embryonic rat brain demonstrated that more tyrosine hydroxylase-immunoreactive cells were present in the female brain than in the male brain prior to the release of gonadal hormones [46]. Other studies showed that male mice that differed genetically only in the origin of their Y-chromosome displayed variable levels of aggression [47, 48], pointing towards the importance of Y-linked genes. Additional evidence came from studies of birds. One classical study involved a rare gynandromorphic zebra finch [49]. This bird was genetically female (ZW) in the left side of its body and genetically male (ZZ) in its right side. It was therefore expected to have equal concentrations of circulating sex hormones in both brain hemispheres. However, the neural song circuit on the right side demonstrated a more masculine phenotype than that on the left side, suggesting that sex chromosomal genes mediated the formation of this sexually dimorphic brain region. The perhaps most outstanding examples of direct sex chromosome influence on brain phenotype come from studies of a mouse model called the “Four Core Genotype” (FCG). In this system, the Sry gene is segregated from the Y-chromosome (Y -Sry), and a translocated functional Sry copy is positioned on an autosomal chromosome [50]. The FCG system allows for the generation of four distinct genotypes: 1) Gonadal male:XY -Sry+Sry, 2) Gonadal female:XY -Sry, 3) Gonadal male:XX+Sry, and 4) Gonadal female:XX. By comparing the phenotypes of these mice, effects of hormones and sex chromosomes can be dissected [51]. The FCG demonstrates XX versus XY differences in behaviour (aggression, parenting, habit formation, nociception, social interactions), gene expression, and susceptibility to disease not mediated by gonadal hormones [52], and the list continues to grow. Bringing the facts above and the results from many other studies together, it is today evident that, although the classical model is a good first approxi- mation, it is clearly incomplete and fails to explain many known sex differences. The classical model must therefore be incorporated into a larger and more complex framework that integrates the contemporary understanding of sex-biased processes and sex-specific genetic features [31, 53]. An outline of such a structure is shown in Figure 2. In contrast to the “feed forward” classical model, a modern theory of sexual differentiation must incorporate the complex interactions of sex chromosomes, sex-specific imprinting, sex- biased gene expression, sex hormones, and sex-specific interactions with environment, to name some of the most central components. In addition, sex-specific compensatory mechanisms which actually act to reduce rather than induce sex-biased phenotypes must be merged into the theory [30]. The modern framework thus more resembles a branched network rather than a

15 linear process, with back-and-forward interactions that altogether shape the sexual dimorphisms of the nervous system. However complex such a framework might appear, several straightfor- ward experimental approaches can be applied to gain more information regarding its structure. One such approach is to catalogue genes with sexual expression differences in neuronal tissues, and to analyse the gene- regulatory mechanisms that govern sexually dimorphic transcription. This is indeed the main strategy of the studies presented in this thesis.

Figure 2. (On the following page). Schematic structure of sexual differentiation of the mammalian brain.2 An overview is shown of important genetic differences, hormonal processes, and parental and environmental influences, skewing the mammalian brain in a male-biased or female-biased manner. The bows connecting “transcriptome” and “brain” with other boxes indicate causal interactions. The black arrows illustrate the main processes of the classical model. The arrows marked “Compensation” in the bottom part of the figure act as reminders of that some sex- specific factors reduce sex-differences. No claims are made that this figure does in any way represent a complete model for sexual differentiation; it is merely a useful structure to base further discussions on.

2 Inspired by the writings of McCarthy MM and Arnold AP: Reframing sexual differentiation of the brain. Nat Neurosci 2011, 14(6):677-683.

17 Sex differences in brain anatomy and behaviour

The gross anatomical features of the male and female brains are similar but not identical. In many mammals, the overall mean weight and volume of the brain is generally slightly higher in males than in females [8, 54]. In specific regions or nuclei of the brain, sex differences can be more pronounced. For example, in rat, the sexually dimorphic nucleus of the preoptic area is 2.6 times larger in males [55], while the anteroventral periventricular (AVPV) nucleus is 2.2 times larger in females [56]. Both of the named nuclei are significant in regulating copulatory behaviour [11, 57]. However, the sex differences in the mammalian brain are not restricted to brain regions with such obvious sex-biased behaviours. Overemphasising such examples may have contributed to the false impression that a few discrete male or female circuits sit in an otherwise sexually monomorphic brain [31]. On the contrary, sexual dimorphisms can be manifested at multiple levels of brain complexity, including synaptic patterns [58, 59] and neuronal density [60]. In some aspects, the female and male brains are delicately different down to the individual cell. For example, segregated neurons from male rats in culture are more vulnerable to starvation than neurons from females [61]. At the level of behaviour, sex differences are observed in a range of measures, including affiliative behaviour and social bonding [62]; movement and body coordination [63]; eating behaviours [64]; pain [65]; motivation and drug abuse [66]. Sex differences in the neuronal networks facilitating such behaviours are now beginning to surface. A recent example of particular interest in the context of the current thesis (i.e. concerning the striatum connection; see Paper III, IV and V), involved the nigrostriatal system [67]. In this study, the FCG model was used to demonstrate that XX mice have higher Pdyn (prodynorphinin) expression in striatum than XY or X0 mice regardless of their gonadal sex. This suggests that X-linked factors, possibly XCI escaping genes, regulate the difference in striatal Pdyn expression [67]. Gene expression studies can thus provide the genetic markers needed to identify the putative sexually dimorphic neural networks in the mammalian brain that have yet to be uncovered.

18 Sexually dimorphic gene expression

Genes on X and Y All mammalian sex differences must ultimately originate from the sex chromosome complements. This conclusion follows from the fact that any general differences in gene contents between female and male cells are represented exclusively by genes located on the X and Y. Such genes are important candidate factors that may underlie sexual dimorphisms in the central nervous system (CNS) and beyond. Indeed, the igniting actions of the Y- linked Sry lie at the basis of the classical model of mammalian sex differentiation. In addition to hormone-mediated effects, the sex-specific X and Y genes may have intrinsic influences on the nervous system. Female cells have double copies of the X-chromosome while male cells contain a single X-chromosome and a male-specific Y-chromosome. Mam- mals evolved a dosage compensating mechanism to limit the severity that might follow from the female-to-male differences in X-gene copy numbers [68]. In female cells, one of the X-chromosomes is epigenetically inactivated and the transcriptional output from this copy is cancelled. This phenomenon was first hypothesised by Mary Lyon in 1961 [24], and is now known as “X- chromosome inactivation” (XCI). The actual silenced and heterochromatic X-chromosome had however already been observed more than ten years earlier by Murray Barr [69], and was named the “Barr body” in lack of understanding of the wider significance of the finding. The choice of active (Xa) and inactive (Xi) X-chromosome is determined randomly in each cell in early embryonic development [68]. The silenced state, of either maternal or paternal X-chromosome, is thereafter maintained during mitosis in each linage throughout life. XCI is dependent of the master regulator Xist (X- inactive specific transcript) [70]. Xist encodes a long non-coding RNA (lncRNA) expressed exclusively from the silenced X-chromosome, and coats Xi in cis [71]. The inactivation of X is accompanied by the redistribution of various epigenetic marks, including increased DNA methylation, the accumulation of repressive chromatin modifications such as histone H3 lysine 27 trimethylation (H3K27me3) [72-74], and the histon variant macroH2A1 [75]. Marks associated with active transcription are at the same time depleted from Xi [68]. The silence of Xi is however not absolute. Some genes “escape” XCI and therefore transcribe from both X-alleles, producing female-biased RNA levels via biallelic expression [76, 77]. XCI escapee genes are of particular interest because of their plausible roles in feminising phenotypes. Experimen- tal data suggest that as much as 15% of the human X-genes escape X- inactivation [78], while ~3% escapes in mouse [79], which is a main model organism for X-inactivation research. Sex-bias research aside, it is also of

19 medical significance to understand the actions and the regulation of XCI escapee genes since these are important for understanding the physiological conditions associated with Turner’s (X0) and Klinefelter’s (XXY) syndrome [80, 81], to name two examples. It is interesting to note that the cognitive traits often observed in patients with Turner’s syndrome point to the importance of XCI escapee genes in neurobiological processes. Another possible mechanism for sexual differentiation of the brain is via the male-limited expression of genes on the non-recombining region of the Y-chromosome (NRY) [82], where all genes are unique to males. While it is known that several genes on NRY are primarily active in testes [83], it is false to conclude that the Y-chromosome is merely relevant for spermato- genesis, and just as erroneous to conclude that the Y-chromosome is without functionality for brain and behaviour [82]. On the contrary, several Y-genes are expressed in the adult male human brain [84, 85]. Moreover, some proteins encoded on Y have well defined neuronal functions. For example, the hominid-specific brain-expressed PCDH11Y, encodes a member of the pro- tocadherin superfamily, responsible for cell-cell interactions during development of the CNS [82, 86]. Most Y-genes have paralogous genes located on the X-chromosome. However, the expressions patterns, and the levels of expression in brain can be non-equivalent in X/Y paralogous pairs [87]. Moreover, sequence differences between X/Y-pairs may underlie differences in the functional proteins. The importance of Y-genes for sexually dimorphic neurobiology seems to have been overlooked, and therefore this chromosome deserves more attention. Concerning such future investigations, it is important to emphasise that the human Y-chromosome is quite different from the mouse Y- chromosome3. For example, while the human Y encodes 40 protein-coding genes, the mouse Y encodes only 17. In fact, the human Y harbours several primate-specific genes [88]. Hence, it could be problematic to translate some of the conclusions about Y-chromosome functions of the FCG model into inferences about those of the human Y-chromosome.

Genomic imprinting Genomic imprinting is an epigenetic mode of gene regulation resulting in preferential expression of either the maternally or paternally inherited allele [89]. It is believed that imprinting evolved as a result of parental conflicts of optimal energy investment during the generation of offspring [90]. Consis- tent with this hypothesis is that many imprinted genes are expressed in pla- centa, and regulate the delivery of nutrients to the foetus [91]. Additionally, many genes subjected to imprinting are expressed in the brain, and the physiological effects of imprinting clearly go beyond influences on foetal

3 Reviewed via Ensembl release 63, June 2011.

20 development [92]. Imprinting of genes located on the X-chromosome provides a mechanism for sex-specific brain-related differences [93]. Females inherit one maternal and one paternal X-chromosome, while males inherit a single maternal X-chromosome. Paternal imprints on the X-chromosome are therefore present only in female cells. Because of random X-inactivation, the female body is a mosaic of cells with either maternal (m) or paternal (p) imprints on the active X-chromosome: Xa(m)Xi(p) and Xa(p)Xi(m). At the same time, male cells only inherit maternal X-imprints: Xa(m)Y. From this, two main consequences follow: 1) The specific effects of paternal X- imprints are limited to female cells. 2) Because of random X-inactivation in females, males receive a “double dose” of active X-chromosomes with maternal imprints relative to female cells (females: 50% Xa(m) + 50% Xa(p) ; males: 100% Xa(m)) [93, 94]. X-imprinted cognitive effects were first experimentally demonstrated by Skuse and colleagues in 1997 [95]. In their study, a cognitive task was performed by two groups of subjects with Turner’s syndrome, caused by X- monosomy. One group inherited their single X from their father (45,Xa(p)), whereas the other group had inherited their single X from their mother (45,Xa(m)). 45,Xa(m) subjects were impaired on measures of social cogni- tion relative to their 45,Xa(p) counterparts [92, 95]. The first specific X- imprinted genes, besides those related to the paternal XCI (which precedes random XCI), were identified in 2005 [96, 97]. Three imprinted X-linked members of the lymphocyte regulated (Xlr) gene superfamily (Xlr3b, Xlr4b and Xlr4c) were identified by comparing the neural gene expression profiles in 39,X0 mice, in which the parental origin of the single X-chromosome was either paternal (39,Xa(p)) or maternal (39,Xa(m)) [92]. The biological relevance of imprinted X-genes in females and males with normal karyotypes is however unclean. Recent studies reported the first observations of sex-specific autosomal imprinting, using RNA sequencing approaches [98, 99]. This novel concept, involves preferential expression of either maternal of paternal alleles dependent on the sex of the progeny. Its mechanism and physiological significance remain unclear, but the phenomenon certainly opens exciting new avenues for investigation of sexual dimorphism [98].

Sexually dimorphic expression of autosomal genes The first studies to demonstrate sexually dimorphic expression of mammalian autosomal genes described the expression of p450 enzymes, so called “Cyp450s”, in female and male rat liver [100, 101]. The members of the Cyp450 protein family metabolise steroids, including estrogen and testosterone [102]. With the advent of microarray technology it became possible to perform genome-wide screens of sex-biased transcription. Early microarray studies indicated that sexually dimorphic gene expression in the mammalian

21 brain was almost exclusively limited to genes located on the sex chromosomes [84, 103]. However, it is possible that these studies were confounded by technological predicaments of the early generations of microarray platforms, and/or by small sample numbers. The extent of sexually dimorphic gene expression in the mammalian brain was not apparent until 2006, when Yang et al. reported a large-scale analysis of whole brains from more than 150 male and female mice [104]. Their analysis showed that as much as 14% of the brain-expressed genes demonstrated sex-biased levels of expression, with most of these genes being located on autosomal chromosomes. The Yang study also included analyses of liver, adipose, and muscle, and demonstrated an extensive tissue autonomy in the regulation of autosomal sex- biased expression. The sex fold-changes of most autosomal genes in brain were small (<1.2 fold), perhaps explaining why extensive numbers of autosomal sex-biased genes were not reported by the early microarray studies, in which small sample collections had been used. In liver, many autosomal sex differences are regulated by pituitary growth hormone [105, 106], which in turn is regulated by gonadal steroids [107, 108]. It is possible that sex-biased autosomal genes in the brain are also to a large extent regulated by sex hormones, either directly or indirectly, but whether this is the case has so far not been determined. Additional genome- wide analyses of sexually dimorphic gene expression in the mammalian brain are needed to independently resolve the extent of autosomal sex-bias in the brain, and to confirm differential expression of specific autosomal genes.

22 Aims of the studies

The main aims of the Papers are listed below.

Paper I An evolutionarily conserved sexual signature in the primate brain • To investigate sex-biased transcription genome-wide in the occipital cortex of human, macaque and marmoset. • To identify possible evolutionarily conserved sex-biased gene expression patterns in the primate lineages.

Paper II Prenatal sex differences in the human brain • To identify male-specific Y-linked genes expressed in the mid-foetal human brain.

Paper III Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse • To investigate whether the abundance of sex-biased genes varies between different regions of the mouse brain. • To study patterns in the genomic organisation of sex-biased genes.

Paper IV Large-scale sex-bias expression analysis of somatic tissues reveals de- masculinisation of the mouse X-chromosome • To identify X-chromosome genes sex-biased in somatic tissues. • To investigate whether the X-chromosome is feminised or masculinised in terms of X-gene expression in somatic tissues.

23 Paper V A new mouse line based on the Gpr101 promoter drives expression of Cre in medium spiny neurons of the striatum • To generate a transgenic mouse, expressing Cyclic recombinase (Cre) under the promoter of the X-linked gene Gpr101. • To perform an initial characterisation of Gpr101-Cre expression in the generated mouse line.

24 Methodological considerations

Method sections, describing the experimental procedures of the studies, ac- company each Paper. Since the experiments involved the use of human post- mortem tissues and experimental animals, a section describing the biological specimens is included here.

Biological specimens Biological specimens used in the experiments presented in this thesis were derived from human (Homo sapiens), cynomolgus macaque (Macaca fascicularis), common marmoset (Callithrix jacchus), and house mouse (Mus musculus). The human brain tissues were donated post-mortem samples provided by Stanley Foundation Brain Consortium (Maryland, USA), Maudsley Brain Bank (Institute of Psychiatry, Dept. of Neuropathology, London, UK), and by the Harvard Brain Tissue Resource Center (Massachusetts General Hospital, Massachusetts, USA). Ethical permission for expression studies in human post-mortem tissues was obtained (permit 99277). The macaque samples were controls from a previous research program conducted by Dr. Diana Radu and Dr. Nils Lindefors at the Karolinska Institute, Stockholm, and were a kind gift. These animals had been housed at The Swedish Insti- tute for Infectious Disease Control, Stockholm. The marmoset samples were a donation from Dr. Örjan Lindhe, IMANET, Uppsala, where the animals had been used in the development of PetScan analyses, and they were housed at the facility for laboratory animals, Uppsala University. No primates were killed for the purpose of the experiments in this thesis. The mouse tissues used for quantitative RT-PCR and for primary cell cultures in Paper III were collected from C57BL/6×DBA/2 mice, housed at Uppsala Biomedical Center, Sweden, in agreement with animal research ethical regulations (permit C79/9).

Transgenic mice The mouse line presented in Paper IV was generated by pronuclear injection of a transgenic DNA construct, as described in more detail in the methods section of the paper. In total, three transgenic mouse lines were produced using this procedure: “Gpr101-Cre”(2 founders), ”Trh-Cre”(1 founder) and ”Trhr-Cre” ”(2 founders); under the respective promoters of: G protein-

25 coupled receptor 101 (Gpr101, MGI:2685211, Chr.X: 54,749,845- 54,756,934(-) BAC: RP23-203E19); Thyrotropin releasing hormone (Trh, MGI:98823, Chr.6: 92,192,055-92,194,644(-) BAC: RP23-295F1); and Thy- rotropin releasing hormone receptor (Trhr, MGI:98824, Chr.15: 44,027,681- 44,067,458(+) BAC: RP23-188E14). Only data from the line Gpr101-Cre is presented in this thesis. Pronuclear injections were performed at Uppsala University Transgenic Facility, Department of Medical Biochemistry and Microbiology, BMC. The mice were housed at Uppsala Biomedical Center, Sweden, in agreement with animal research ethical regulations. All experiments involving animals were approved by the appropriate local Swedish ethical committee (permit C79/9).

26 Results and discussion

Paper I

An evolutionarily conserved sexual signature in the primate brain

Setting At the onset of the studies leading to Paper I, sex differences in several pa- rameters of the human brain, such as the relative size of certain brain regions, prevalence of psychiatric disorders and drug abuse, were already well grounded [109-111]. However, there was little empirical evidence of extensive human sex differences in brain gene expression. Early microarray studies of the human brain had suggested that sex-biased expression was essentially limited to a few genes located on the X- and Y-chromosomes [84, 103]. To gain further insight into this issue, we applied an evolutionary approach. We hypothesised that sexual expression dimorphism, important for upholding functional differences between the sexes, might be conserved during the evolution of primates. Using a comprehensive cDNA microarray platform, we quantified female and male brain cortex gene expression in human and in two other species of primates: cynomolgus macaque (Macaca fascicularis), a polygamous old world monkey; and common marmoset (Callithrix jacchus), a monogamous new world monkey.

Main results and significance Genes with conserved sexual expression dimorphism may underlie important differences in neurobiology between the sexes. Paper I provides the first observation of evolutionarily conserved sex-biased gene expression patterns in the primate brain. We identified 85 genes with conserved sex-bias in human and macaque, and 2 genes with conserved sex-bias in all three species. Furthermore, we found that the coding sequences of the conserved sex- biased genes had evolved under strong evolutionary constraint. In addition, hundreds of genes with non-conserved sex-biased expression levels were discovered in human and macaque, while less than ten were found in marmoset. We found that the male-biased genes evolved faster than female- biased genes in coding regions, suggesting differences in selection pressure on male- and female-biased genes.

27 Study description and discussion Human, macaque and marmoset brain samples, from four females and four males in each species, were included in the study (sample origins are described in the section “Methodological considerations”). The brain is an exceptionally heterogeneous tissue, both in terms of structural organisation and gene expression patterns. To reduce variation attributed to biological sampling, we chose to perform our study using a single anatomical region of cortex: the occipital lobe. In addition, we considered cortex to be an interesting tissue to study because of its functions related to higher cognitive processes. At the time of the experiment, the use of DNA microarrays was the best option for genome-wide expression comparisons. The microarrays available were designed using human sequences. A caveat to hybridisation-based techniques in analyses of diverse species is that inter-species differences in gene sequence can result in mismatches between the assayed transcripts and the probes on the microarray. Such mismatches could negatively affect the recorded signal intensities. To circumvent these issues we used human cDNA microarrays (46k probes, 14 621 genes) and performed only within- species competitive hybridisations, using female and male samples from a single species on each array (Figure 1, Paper I). The identification of sex- biased genes was therefore not expected to be significantly biased by the effect of mismatches on hybridisation intensity [112]. To select sex-biased genes within each species, we used a linear model and a penalised F-ratio as ranking statistic. Penalised statistics were used to avoid the selection of genes with small expression differences but high re- producibility, since the selection of such genes is often a problem in the analysis of small microarray sample sizes. As a first result, the analysis showed that hundreds of genes were expressed at different levels in the two sexes in both human and macaque, while less than ten genes were sex-biased in marmoset (Figure 2 and Table 1, Paper I). This indicated extensive sex differences in human and macaque cortex. Furthermore, the amount of genes with sex-biased expression varied substantially between the different species. Interestingly, the marmoset, clearly a monogamous species, showed less sexual gene expression dimorphism than the other species. This observation correlated with the overall trend in marmosets, which are relatively sexually monomorphic in their physical constitution [113]. However, we could only speculate as to whether the low number of sex-biased genes in a single species could be related to that specific organism’s reproductive system. Thereafter we investigated whether any sexual gene expression differences might have been conserved during the evolution of primates. The analysis revealed the most intruiging result of our study. We identified 85 genes with conserved sex differences in human and macaque, and 2 genes

28 with conserved sex differences in all three primates (Figure 4, Paper I). This was the first observation of a conserved sexual gene expression signature in the primate brain. To validate the microarray results with a more sensitive technique, we performed RT-qPCR on 6 of the genes. Altogether, the RT- qPCR supported the array data (Figure 8, Paper I). The conservation of 85 sexually dimorphic gene expression patterns suggested that the genes might have sustained important sex-biased functions during the evolution of primates. We hypothesised that such genes might also have been more conserved at the protein level. Therefore, we analysed the rate of protein evolution using non-synonymous (dN) versus synonymous substitution rates (dS) in the coding gene sequences. Indeed, conserved sex- biased genes showed significantly lower dN/dS ratios than other genes (Figure 6, Paper I). However, brain-expressed proteins had previously been shown to in general evolve under strong constraints [114]. Therefore, we compared the dN/dS ratios of the conserved sex-biased genes with ratios from a refer- ence set of brain-expressed genes. The analysis showed that dN/dS ratios of genes in the conserved sex signature were slightly lower, although the difference was not statistically significant (p=0.16). Earlier studies in Droso- phila melanogaster had showed that dN/dS ratios among male-biased genes were higher than the dN/dS ratios among female-biased genes [115, 116]. We therefore compared dN/dS ratios in genes that were male-biased and female- biased. The analysis showed that sequences of male-biased genes evolved at a rate three times higher than those in female-biased genes (Figure 7, Paper I). This observation might reflect differences in selection pressures on male- and female-beneficial alleles during primate evolution. The result is in agreement with the view that female-biased genes persisted under stronger purifying selection. Alternatively, sequence changes in male-biased genes were under positive selection during primate evolution, perhaps as a result of sexual selection on male-biased traits. The regulatory mechanisms controlling the expression of the evolutionarily conserved sex differences are not known. A first candidate is hormonal regulation, and we therefore investigated the presence of conserved transcription factor binding sites in the promoters of the 85 genes in the conserved sexual signature. This was done by performing a human/macaque sequence alignment and a search for estrogen alpha and androgen cis- response elements (ESR1 and AR) in conserved promoter sequences. We found that 56% of the genes contained at least one ESR1, and 66% contained at least one AR. 26% of the genes contained neither ESR1 nor AR in the investigated regions. A functional analysis would be required to investigate whether the identified conserved ESR1s and ARs are functional, and to elu- cidate whether they might be significant for the regulation of sex-biased gene expression.

29 Paper II

Prenatal sex differences in the human brain

Setting Male-specific Y-linked transcripts are candidate genetic contributors to the sex-biased functionalities of the human male nervous systems. Roughly 40 protein-coding genes are located on the human Y4, and it is likely that some of them support sex-biased functions in the mature brain as well as during prenatal brain development. A first step in the identification of such genes is to recognise which of the Y transcripts are expressed, and in what brain regions and developmental periods they are transcribed. Therefore, we performed an analysis of Y-chromosome gene expression in human mid-foetal brain.

Main results and significance In Paper II we identify 11 Y-linked genes expressed across several brain regions in a human male foetus during mid-gestation. The identified Y- linked genes are thus candidate contributors to the sexual dimorphisms that may occur during human brain development. Equally important is the recog- nition of Y-linked genes that are not brain expressed. Our data showed no evidence of expression of the testis determining factor SRY in any of the brain regions included in the human prenatal dataset, a gene that has earlier been suggested to directly modulate brain function in male mice [117].

Study description and discussion For this study, I utilized a publicly available whole-genome, exon-level expression dataset from an investigation of alternative splicing and gene expression differences specific to certain regions of the mid-foetal human brain [118]. This high-quality dataset included single channel hybridisations of RNA from the left and the right hemispheres of 12 brain regions, derived from one male (week 18) and three female foetuses (week 19-23). Absolute intensities of individual microarray probes are often unreliable indicators of gene expression levels, especially for inter-probe comparisons. Therefore, to identify Y-genes expressed in the male samples, I used the intensities obtained in females as measurements of the local probe background level for each individual Y-probe, as females do not contain Y-linked genes. To classify expressed Y-genes, I applied the selection criteria of a mean male-to-female fold-changes of at least two. Out of the 26 genes represented on the microarray platform, I identified 11 genes as expressed, namely: RPS4Y1, ZFY, PCDH11Y, PRKY, USP9Y, DDX3Y, UTY,

4 According to Ensembl release 63, June 2011.

30 NLGN4Y, TMSB4Y, TMSB4Y, CYorf15B, and EIF1AY (Figure 1 a, Paper II, and Image, Paper II’). Most of the transcribed Y genes showed ubiquitous expression throughout all of the included brain regions, indicating that brain region specificity in the activation of these genes was limited (Image, Paper II’). On the other hand, the data also presented some support for the view that the rate of transcription might vary in diverse brain regions, at least for some individual genes. For example, the gene PRKY showed lower expression in cerebellum and basal ganglia than in cortex, and ZFY showed evidence of splice- variant-specific regulation among the brain regions included. These observations might suggest that the contribution of all Y genes to sex-biased brain development is not the same throughout the whole male brain. Interestingly, while the testis-determining gene SRY had earlier been discussed in terms of a possible modulator in the adult male brain [119], I found no evidence of SRY expression in any of the brain regions included in the human prenatal dataset. It seemed unlikely that the lack of SRY signals could be attributed to “bad” probes, given that as many as five probes were available for this gene, and none of them presented signals above background level (Figure 1 b, Paper II; and Image, in the top of the chart, Paper II’). Then again, the results did not challenge that SRY could have been brain-expressed in developmental windows outside of week 18, and/or in brain regions not included in the analysis. Another possibility is that SRY mRNA was expressed in a very limited population of cells, diluting the SRY signals in the samples used in the study. All of the 11 identified genes are located in the male-specific region of the human Y-chromosome, and they all have paralogous genes located on the X-chromosome5. Interestingly, only four of them: ZFY, USP9Y, DDX3Y and UTY, have mouse Y-linked orthologues. RPS4Y1, PCDH11Y, CY- orf15B and EIF1AY lack mouse Y-linked, but have mouse X-linked, orthologous genes. NLGN4Y and TMSB4Y, separated by less than 1 Mb distance on the human Y-chromosome, lack homologues in the mouse genome. Expression in human mid-foetal brain of the mentioned genes might therefore point to differences in Y contribution to sex-biased neurobiology in mouse and man. Furthermore, PCDH11Y, which encodes a neuronal cell adhesion protein and is essential for the segmental development and function of the CNS, is one of the rare genes that are specific to the hominoid lineage and absent from other primates [86, 120]. Altogether, the results point towards the importance of Y-linked genes for human brain development.

5 All orthologues and paralogues are discussed according to Ensembl release 63, June 2011.

31 Paper III

Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse

Setting Prior to Paper III, genome-wide investigations of whole adult mouse brain and several non-neuronal tissues had demonstrated that sexually dimorphic gene expression is highly tissues-specific [104]. However, sex-biased gene expression in distinct anatomical sub-structures of the brain had not been thoroughly investigated. Given that the brain is highly compartmentalised, both in terms of functionality and in terms of expression of genes, it seemed likely that the amount and type of genes differentially expressed between the sexes might also vary between separate regions of the brain. To investigate this, we performed a large sex-balanced microarray analysis with hybridisations from striatum, neocortex, hippocampus, eye, and a peripheral tissue, lung, included for comparison.

Main results and significance Paper III showed that the amount of genes with sex-biased expression varied between different brain regions. Specifically, the data demonstrated an order of magnitude increase in the number of sexually dimorphic genes in striatum, as compared to the other brain regions included in the study. The analysis also showed that the sexual expression differences common for several brain regions were mainly attributed to genes located on the X- and Y- chromosome, while autosomal sex-bias was generally restricted to individual brain structures. In addition to answering the basic question regarding the potential existence of brain region specificity in regulation of sex-biased genes, this study provided observations relevant for understanding a much more fundamental aspect of sex-biased transcription: the regulation of genes and domains that escape XCI. The results revealed several female-biased long non-coding RNA (lncRNA) genes, located very close to female-biased protein-coding escapee genes on the mouse X-chromosome. Non-coding, as well as coding transcribed loci, were depleted of the repressive chromatin mark histone H3 lysine 27 tri-methylation (H3K27me3), supporting the view that they escape XCI. Hence, this study provided X-inactivation research community with the first indication that expression of proximal lncRNA may play a role in regulating the epigenetic state of escape domains [76, 121-124]. Furthermore, biallelic expression of a whole new escape domain on the mouse X-chromosome was confirmed by RNA-FISH experiments, adding new genes to the set of less than ten mouse genes that were previously known to escape XCI at that time of the experiments.

32 Study description and discussion To gain access to extensive sex-balanced microarray datasets, I collaborated with groups located at the University of Tennessee and Beth Israel Deacon- ess Medical Center, USA. To my initial disappointment, the microarray hybridisations for the available data had been performed using two different array platforms, namely Illumina Mouse-6v1.1 for striatum and neocortex, and Affymetrix Mouse-430v2.0 for hippocampus, eye and lung. Although these platforms were similar in gene coverage, using two platforms would complicate cross-array comparisons of the results (not to mention provide reviewers with the potential grounds for criticism during the following pub- lication procedures). However, it would later turn out that the combined use of both of these platforms was crucial for the most important outcomes of the study, a point I will return to shortly. After a quality inspection, a total of 456 hybridisations (228 female and 228 male) were included in the analysis, as can be seen in Table 1 (Paper III). To identify differentially expressed genes, I used a two-sided Wilcoxon test followed by the stringent selection criteria of FDR 0.05 based on the Benjamini-Hochberg correction for multiple testing, which corresponded to a maximum raw p-value of 2.3E-4 (the significant genes are found in Addi- tional file 1, Paper III). A first observation was that striatum contained about ten times as many sex-biased genes as did the other included neuronal tissues (Table 1, Paper III). Furthermore, the data showed that striatum harboured sex-biased genes from all chromosomes, whilst sex-biased expression in neocortex, hippocampus and eye was mainly restricted to genes located on the X- and Y-chromosomes. Altogether, these observations indicated that striatum was a more sexually dimorphic tissue than the other included brain regions. Gene ontology analysis of the sex-biased genes in striatum classified association terms such as “Cell-To-Cell Signaling and Interaction”, “Neurological Disease” and “Nervous System Development and Function” as overrepresented (Additional file 2, Paper III). These results supported the view that sexually dimorphic gene expression in striatum resulted in functional brain differences between the sexes. Next, I sought to identify genes that were sex-biased in more than one tissue. I reasoned that such genes could underlie fundamental sex-biased functionalities. Therefore, I determined the overlap of the significant genes in each tissue. I restricted this analysis to within-platform comparisons, because of differences in probe content between the two microarray platforms included, as mentioned previously. This simple approach resulted in a list of 20 genes, as shown in Figure 2 (Paper III). Only 3 out of these 20, were located on autosomal chromosomes: Arid1b (male-biased, chr. 17), Prl (female-biased, chr. 13) and 1700012B15Rik (female-biased, chr. 12). The re- maining transcripts represented 6 male-biased genes on Y and 11 female- biased genes on X. Most Y-genes were well-known sex-biased genes in the

33 literature, and were therefore not considered for further studies. Among the X-genes, six were known to escape from, or to be involved in, the regulation of X-inactivation. Specifically there were four escapees: Ddx3x, Eif2s3x, Kdm6a, Kdm5c; and two XCI regulators: Jpx and Xist. The additional five female-biased X-genes: 2010308F09Rik, D330035K16Rik, 5530601H04Rik, 2610029G23Rik and D930009K15Rik; deserved further attention. Only one of these five genes was found to be protein coding, namely 2610029G23Rik. The other four genes encoded long non-coding RNAs, as concluded by an analysis of coding potential based on sequence composition [125], together with searches in non-coding RNA databases and BLASTx in all six frames against protein databases (Table 2, Paper III). An inspection of gene location revealed a striking pattern: The novel female-biased genes, and the known escapees, clustered into adjacent codingc/non-codingnc gene pairs on the X- chromosome: Ddx3xc/2010308F09Riknc, Eif2s3xc/D330035K16Riknc, Kdm5cc/D930009K15Riknc and 2610029G23Rikc/5530601H04Riknc (Figure 3, Paper III). The maximum distance of separation between a pair of coding/non-coding genes was 33kb. Quantitative RT-PCR experiments using independent biological samples altogether validated the female-biased expression of these clusters (Figure 4, Paper III). From the clearly non-random organisation of the genes, I concluded that it was unrealistic that the identification of co-localized female-biased coding and female-biased non-coding genes were independent events. Therefore, the results indicated co- regulation of the genes in each female-biased cluster. The fact that three out of four clusters contained coding genes that escaped X-inactivation, suggested that the female-biased lncRNAs might also transcribe from escaping domains. However, the fourth cluster, 2610029G23Rikc/5530601H04Riknc, lacked previous evidence of escape. Therefore, combined RNA/DNA-FISH experiments were performed on primary female cells, using probes for this novel gene cluster in parallel with probes for Xist. Cell counting indicated biallelic signals for the novel cluster in 80.7% of the cells, demonstrating that genes located therein may indeed escape XCI (Figure 5, Paper III). As a further step, I analysed public data of the X-distribution of a repressive chromatin mark, H3K27me3, an X-inactive histone modification known to be depleted among XCI escaping genes [79]. The data showed that not only coding escapees lacked this repressive mark — the female-biased lncRNA genes were also located in chromosomal zones of H3K27me3 depletion (Figure 6, Paper III). I therefore proposed that lncRNAs transcribe from domains that escape X-inactivation in mouse, suggestive of the intriguing possibility of lncRNA-dependent control functions in XCI escaping domains.

I would here like to make room for a few lines of more personal reflec- tion. If I had followed my initial feelings of disappointment concerning the use of two different array platforms (as mentioned above), and excluded one

34 of the platforms from the analysis, the discovery of the four coding/non- coding escape clusters would likely not have been made (by us). As can be deduced from Figure 2 B (Paper III), the probe performance and probe coverage in the platforms were complementary: while some gene probes were missing, or not performing on one platform, the other platform provided coverage. Therefore, the interpretation of data from both platforms was crucial for the most important outcomes of the study. From this, I realised that all experiments do not have to be perfectly clean cut to in order to lead to most interesting discoveries. But yes, as suggested above, the use of two array platforms was indeed heavily criticised by one reviewer during publi- cation.

Possible roles for lncRNAs in domains that escape X-inactivation The molecular mechanisms controlling escape from X-inactivation are not known. Our study indicated that lncRNAs were expressed in XCI escaping domains, suggestive of lncRNA involvement in the transcriptional regulation of such domains. Because of limitations in time and recourses, it was regret- fully not possible for me to experimentally explore this issue during my PhD studies, but merely to outline some hypothetical models of lncRNA action in XCI escaping domains.

Non-coding RNAs participate in epigenetic regulation but their actions are poorly understood [126-130]. Unlike proteins and small RNAs, lncRNAs can remain tethered to the site of transcription, and therefore uniquely direct allelic regulation [131]. These properties make lncRNAs attractive as candidate molecules for the regulation of XCI escape domains. Three possible roles for lncRNA in escaping domains were suggested in Paper III. These are summarised below. “Activation”: The first role involved transcriptional activation of genes in the escape domain. This could involve local relaxation and opening of the X- inactive heterochromatin, initiated by the expression of non-coding genes (Figure 3 A, below). This sort of action could result in increased accessibility for the transcriptional machineries and transcription factors to the otherwise heterochromatic domain. Evidence for this type of lncRNA-dependent regulation was recently presented in yeast [132-134]. “Movement”: The second role involved lncRNA-dependent movement of the escaping domain (Figure 3 B, below). This movement is expected to be directed away from the X-inactive silent nuclear territory and into a transcriptionally active compartment of the nucleus. Since lncRNA can attach to the site of transcription, one could imagine that lncRNA might remain at- tached to the escape domain and act as a structure to mediate the transloca- tion of the escape cluster towards the active territory. This process would likely require several other factors and possibly anchor points to achieve a “loop out” of the escape domain. Support for lncRNA-mediated chromatin

35 movements had earlier been reported for the regulation of imprinted autosomal alleles, although in that case toward inactive nuclear compartments [135, 136]. “Blocking”: The third role concerned the establishment of chromosomal boundaries between active and inactive domains (Figure 3 C, below). It is likely that elements contained in the border territory of escape domains are needed to restrict the spread of heterochromatin from neighbouring silenced chromosomal regions into to the escape domain. Conversely, such boundary elements might prevent the spread of euchromatin from within the active escape domain out to the inactive regions. Female-biased lncRNA might participate in such roles. Evidence for similar type lncRNA-functionality has been demonstrated in loci involved in organogenesis [137, 138]. “Suppression”: An additional role, not included in Paper III, involves the possibility that the lncRNA might somehow function as a suppressor of expression of the neighbouring genes outside of the escape domains, in contrast to promoting the expression of escapees (Figure 3 D, below). A discussion about this idea was initially included in the manuscript, but was later excluded during revision, as a compromise between the differing opinions of the authors regarding the degree of speculation that was appropriate in the discussion section of the paper. Finally, a “show killing” possibility would be that lncRNAs might escape X-inactivation as a mere side effect of being located close to a functional coding escape gene, and that the identified lncRNAs lack any functional significance whatsoever for the regulation of XCI escaping domains.

There is currently no direct evidence available that favours or disfavours any of the above models. But several approaches could be considered for future testing of the putative lncRNA functionalities. Mutation and partial deletion of the non-coding loci is one possibility. Another alternative is to apply an RNA-silencing approach, using siRNA or locked nucleic acids LNAs in order to block the putative lncRNA functionality without the effort of genetic manipulation. Finally, circular chromosome conformation capture (4C) [139] or Hi-C [140], could be used to identify the possible chromosomal interaction partners of the escape domains. Future experiments carried out by me or other investigators will surely tell if the identified lncRNA genes serve functions in escaping domains.

Figure 3. Possible roles for lncRNAs in domains that escape X-inactivation. The figure shows hypothetical models in which female-biased lncRNA is involved in the transcriptional regulation of escape domains. A “Activation”: The expression of lncRNA (green) promotes transcriptional activation of a coding escapee gene (blue), possibly by relaxation of the proximal chromatin complexes, leading to an increased accessibility for transcriptional machineries, and/or by mediating the recruitment of other transcription factors to the escape domain. Silenced genes in the figure are coloured grey. B “Movement”: lncRNA remains tethered to the nearby chromatin and mediate movement of the escape domain out from the silenced X-inactive compartment into a transcriptionally active nuclear territory. C “Blocking”: lncRNAs in escaping domains mediate the formation of chromosomal boundaries to restrict the spread of heterochromatin from neighbouring silenced regions on the X- chromosome. Conversely, the boundaries might prevent the spread of euchromatin from active into inactive regions. D “Suppression”: lncRNA functions to suppress the expression of genes neighbouring the escape domain, reducing the sex-bias of these genes.

37 Paper IV

Large-scale sex-bias expression analysis of somatic tissue reveals de- masculinisation of the mouse X-chromosome

Setting Heterogamety (XY) in males and homogamety in females (XX) make the X- chromosome a unique genomic location for genes with sex-biased functions. The mammalian X-chromosomes have been under selection in males about 1/3 of the evolutionary time, while 2/3 of the evolutionary time was spent in females. On this basis, is expected that female-beneficial dominant genes should be enriched on the X-chromosome [17, 25, 26]. At the same time, since only one copy of X is present in male cells, any recessive allele that gives males reproductive advantages would be immediately available for positive selection in male individuals [17, 25, 26]. Additionally, because of the dual copies of X in female cells, recessive female-beneficial and recessive female-deleterious alleles are masked from sex-specific selection when the allele-frequencies of such traits are low. Rice therefore suggested that genes expressed preferentially in males are likely to be accumulated on the X-chromosome [25, 26]. Empirical evaluations of sex-biased mammalian X- chromosome gene expression are needed to address these issues. Curiously, studies in Drosophila melanogaster revealed an underrepresentation of male-biased genes located on X [141]. This was found both in intact and in gonadectomized flies, suggesting de-masculinisation had taken place [141]. However, mouse studies resulted in partially contradictory conclusions [26]. Based on the preferential expression of X-genes in reproductive-related tissue, both feminisation and masculinisation of the mouse X-chromosome has been suggested [142, 143]. In any case, sex-biased physiology and extensive sex-biased gene expression occur beyond gonadal tissues [104]. Therefore, hoping to illuminate new aspects of the subject, we performed a high- resolution investigation of X-chromosome-wide sex-biased gene expression in several non-reproductive tissues.

Main results and significance Paper IV showed that male-biased genes expressed in mouse kidney and liver were significantly underrepresented on the X-chromosome, while on the contrary, female-biased genes were overrepresented on X. These results suggested that de-masculinisation of the X-chromosome occurred in terms of expression in non-reproductive tissues. We found that the contributions from genes previously known to escape X-inactivation could not explain the overrepresentation of female-biased genes. This indicated that many novel tissue- specific escapee genes were present in our gene lists. Alternatively, additional female-skewed gene-regulatory mechanisms were required to explain

38 the observed overrepresentation of female-biased X-genes. By RNA-FISH experiments, one novel candidate escapee, Tmem29, demonstrated biallelic expression in a limited percentage of cells (7%). In addition, our study provided more support for our earlier finding that non-coding loci transcribed in domains that escaped X-inactivation in mouse [144]. We identified three novel female-biased non-coding genes apparently positioned in the transition-region between active and inactive chromosomal domains near the well- known coding escapee Kdm5c. Also in Paper IV, an analysis of gene age showed that sex-biased X-genes are generally evolutionary old genes (>100 million years). These results have implications for understanding both the regulatory and the evolutionary processes controlling mammalian X-chromosomes.

Study description and discussion Previous studies [104, 144] had indicated that most sexual gene expression differences in mouse were small in magnitude (generally below 1.2 fold). To obtain sufficient statistical power and the resolution required to detect small expression differences, the current study clearly required large expression datasets. Therefore, I initiated a second collaboration with our colleagues at the University of Tennessee, USA. This partnership provided me with access to data from a total of 728 Affymetrix Mouse-430v2.0 microarray hybridisations with male and female RNA from six tissues: kidney, liver, lung, striatum, eye and hippocampus. Using nonparametric statistics (two-sided Wilcoxon test, p<0.001), my analysis identified sex-biased genes in each tissue, as reported in Table 1, Paper IV. Large tissue differences in the amount of sex-biased genes were observed both for X-located genes and for autosomal genes, while the number of Y-genes was essentially constant. Kidney and liver were clearly the most sexually dimorphic among the tissues included. While not emphasised as a key point in Paper IV, I found it interesting that striatum demonstrated large numbers of sex-biased genes (Table 1, Paper IV). This corroborated the conclusion in Paper III, that striatum is an especially sexually dimorphic brain structure in terms of its gene expression, since these striatum datasets were biologically and technically independent. I found that the magnitudes of sexual expression differences were generally small for X-linked genes compared to the fold-changes observed among many autosomal genes (Fig- ure 1, Paper IV), while probes for the single X-gene Xist (“master regulator” of XCI) showed large fold-changes, as expected. These observations might have reflected either an evolutionary constraint on sexual expression bias of genes on the X-chromosome, or constraints in the gene-regulatory mechanisms producing the X-gene expression differences. To investigate whether sex-biased genes might be clustered on specific chromosomal segments on X, I studied the within-chromosome distribution

39 of sex-biased genes (Figure 2, Paper IV). This distribution did not markedly diverge from what was expected based on the overall gene density on X. Sex-biased expression may indicate a previously resolved sexual antago- nism [145]. Therefore, to address the questions regarding potential feminisation or masculinisation of the mouse X-chromosome, I compared the frequencies of sex-biased genes on X and on autosomes. This led to the perhaps most interesting observation made in the study. I found that male-biased genes were significantly underrepresented on X relative to the frequencies on autosomal chromosomes in the most sexually dimorphic tissues: kidney and lung (Figure 3 A, Paper IV). At the same time, I observed that female- biased genes were overrepresented on X, as previously reported [104]. Put together, these two observations might suggest that both de-masculinisation (decrease in number of male-biased genes) and feminisation (increase in number of female-biased genes) of the X-chromosome occurred during evolution. Next, I explored the possibility that genes previously shown to escape X- inactivation might have caused the overrepresentation of female-biased genes on X. However, I reached the conclusion that the contribution of known escapees could not explain the large number of female-biased genes observed (Figure 3 B, Paper IV). This indicated that many novel, and possibly tissue-specific, genes were present in our gene lists. Alternatively, additional sex-skewed gene-regulatory mechanisms must have been present to produce the observed female-skewed population of genes on the X. To identify transcripts with robust sex-bias in more than one tissue, I determined the overlap of significant probes in each tissue. This resulted in a list of 94 female-biased and 33 male-biased genes (Table 2, Paper IV). An exciting finding in this list was the protrusion of four female-biased non- coding RNA genes located directly downstream of the coding XCI escapee Kdm5c. One of them had already been discovered in the studies leading to Paper III. The other three non-coding genes were novel, expanding the boundaries of the female-biased Kdm5c cluster. This provided additional support for my previous proposal that non-coding RNA transcribed in escaping domains [144]. Further, based on public data on chromatin immunopre- cipitation of two X-inactive repressive histon marks, I predicted that the three novel female-biased non-coding loci were situated in the transition- region between active and inactive chromosomal domains (see Figure 6 together with the female-biased cluster marked “f” in Table 2 A, Paper IV). The list in Table 2 A (Paper IV) contains candidate genes for tissue- specific escape from XCI, particularly for specificity in kidney and liver cells. As a first step, to explore the escape properties of these genes, RNA- FISH was performed on five candidate genes using a fibroblasts cell culture derived from embryonic female skin. This analysis confirmed that one of these genes, Tmem29, escaped XCI in fibroblasts (Figure 4, Paper IV). The other four tested genes showed consistent monoallelic expression. The pos-

40 sibility remains that these genes escape XCI in cell types other than skin fibroblasts. It has previously been suggested that genes on the X-chromosome with female-biased function (defined by high expression in female reproductive tissues) are generally evolutionarily old genes, while genes with male-biased function (defined by high expression in male reproductive tissues) are enriched among young genes (<50 myr) [146]. I investigated whether the same conclusions could hold for X-genes with sex-biased expression in non- reproductive tissues, using the same gene dating as Zhang et al. In line with the results of Zhang et al., I found that that most of the female-biased X- genes are indeed evolutionarily old (>100 myr; Figure 5, Paper IV). How- ever, I also observed a significant abundance of evolutionarily old male- biased genes in one of my datasets (kidney). It is at this stage difficult to draw any general conclusions from this gene-age data, and therefore I leave these results as curious observations.

Paper V

A new mouse line based on the Gpr101 promoter drives expression of Cre in medium spiny neurons of the striatum

Setting Transgenic mouse lines that allow for specific genetic manipulation of sexually dimorphic nuclei of the brain would be valuable for dissecting the sexually dimorphic properties of such brain regions. With the aim to construct a mouse expressing Cyclic recombinase (Cre), specifically in amygdala and hypothalamus, we designed a transgene in which Cre was placed under the promoter of the X-linked gene Gpr101.

Main results, significance and brief discussion Using a recombination strategy, we successfully inserted Cre into the first coding exon of the Gpr101 gene in a bacterial artificial chromosome (BAC). Pronuclear injection of the Gpr101-Cre gene construct resulted in the generation of 41 mice, of which two founder individuals carrying the Gpr101- Cre transgene were identified. To characterise the activity of Cre in the generated mice, we performed crossings with a reporter line (tdTomato Ai14), expressing red fluorescent protein in cell lineages in which Cre had, at any time, been activated. Surprisingly, an analysis of the progeny from these crossings indicated intensive red fluorescence most intensively in the striatum of adult mice. Fluorescence was at the same time absent in the expected regions of the amygdala and hypothalamus, wherein endogenous Gpr101 mRNA was expressed. The results showed that the Gpr101-Cre transgene

41 lost the original ability of the Gpr101 promoter to drive expression in these brain regions. In addition, our results suggested that Gpr101 is expressed transiently in the striatum during development. If Gpr101-Cre would be confirmed to express in striatal progenitors by future experiments, Gpr101- Cre mice may prove to be useful a tool for studying the development and differentiation of the cellular networks of the striatum.

42 Conclusions

This thesis summarises my studies of sexually dimorphic gene expression in mammalian somatic tissues. In particular, together with my colleagues, I have explored sexually dimorphic gene expression in the brains of humans, non-human primates, and mice, using microarray approaches. These studies now provide us with a number of novel observations regarding the nature of sex-biased genes. I have presented genes with conserved sex differences in mRNA expression in the occipital cortex among three primates: human (Homo sapiens), cynomolgus macaque (Macaca fascicularis) and common marmoset (Cal- lithrix jacchus). Specifically, 85 genes showed conserved sex-biased expression patterns in human and macaque, while only 2 genes showed conserved sex-bias in all three primates. This finding implies the existence of biological sex differences in the human cortex, and further, it unveils the existence of conserved sexual signatures in the primate cortex. The identified conserved sexually dimorphic genes might underlie important functional sex differences in the brains of humans and macaques. I have described 11 Y-chromosome genes that are expressed in the human male brain during week 18 of embryonic development. Interestingly, the sex determination gene SRY, shows no evidence of expression in male week 18 brain in my dataset. These results are of importance for understanding sexual dimorphisms that occur in the human brain during prenatal brain development. The perhaps most important outcome of my studies is the discovery that long non-coding RNAs are expressed in domains on the mouse X- chromosome that undergo escape from X-inactivation. This finding suggests that long non-coding RNAs could be involved in the transcriptional regulation of the escaping domains. Within this thesis I hypothesise about four possible models of such gene regulatory roles. My studies have also revealed that there is considerable variation in the amount of sex-biased genes harboured within different brain regions. Spe- cifically, my analysis has shown that the striatum is highly sexually dimorphic, with an order of magnitude increase in the number of sex-biased genes in this brain region as compared to the other studied brain regions. Future investigations of the sexually dimorphic properties of the striatum could be assisted by the list of genes with sex-biased expression in striatum presented in this thesis.

43 Some observations of importance for understanding the evolutionary processes shaping the mammalian X-chromosome have been presented. In particular, I found that the X-chromosome is enriched with female-biased genes, and at the same time, it is significantly depleted of male-biased genes, as shown by an analysis of X-gene expression in somatic tissues from female and male mice. This suggests that the X-chromosome has undergone both feminisation and de-masculinisation through evolution. Finally, I have presented the construction of transgenic mice, with the aim to express Cyclic recombinase (Cre) in sexually dimorphic brain regions. One of the three mouse lines created, Gpr101-Cre, expresses Cre in medium spiny neurons of the striatum. This mouse model might in the future become a useful tool that could be employed for developmental studies of the neuronal circuits of the striatum. Each of these studies lays down its own piece into the complex jigsaw puzzle that is the understanding of sexual dimorphisms of the mammalian brain. Combined efforts, applying multiple perspectives, and working within different layers of biological complexity, will be needed to complete this puzzle. And that concludes my thesis.

44 Summary in Swedish

Sammanfattning Ämnet som avhandlas är könsskillnader i genuttryck i däggdjurshjärnor. I avhandlingen presenterar jag resultaten från fem studier av människa, makakapa, marmosetapa och mus. I den första studien undersöker jag könsskill- nader i genuttryck i hjärnans cortex hos primater. Resultaten från denna studie visar att hundratals gener är uttryckta i olika mängd i hjärnan hos de två könen i människa och i makakapa. Mer intressant är att evolutionärt bevarade könsskillnader identifieras. 85 gener som har samma könsskillnader i makakapa såväl som i människa presenteras. Könsskillnaden i genuttryck hos dessa gener tycks därför vara evolutionärt bevarade genom utvecklings- linjerna mellan människa och makakapa. I den andra studien undersöks vilka av de gener som är belägna på mannens Y-kromosom som uttrycks i hjärnan hos under vecka 18 av fosterutvecklingen. Av de 26 gener som undersöks registreras uttryck av 11 gener i fostrets hjärna. Identifieringen av hjärnut- tryckta gener som är belägna på Y-kromosomen är viktig för att förstå dessa geners roll under hjärnans utveckling. I den tredje studien undersöks huruvi- da vissa regioner av hjärnan är mer eller mindre sexuellt dimorfa mätt i anta- let gener med könsskillnader som kan registreras i varje hjärnregion. En genuttrycksanalys av olika regioner av musens hjärna visar att hjärnregionen striatum innehåller cirka tio gånger så många gener med könsskillnader i uttrycksnivå som cortex och hippocampus. Ett annat viktigt fynd som gjor- des i denna studie är upptäckten av att en klass av genregulatoriska moleky- ler, så kallade icke-kodande RNA, möjligtvis är inblandade i regleringen av vissa kromosomala domäner belägna på X-kromosomen. I den fjärde studien undersöker jag aspekter av X-kromosomens evolution. Studien visar att det finns en överrepresentation av gener med högre uttrycksnivå hos honor bland gener belägna på X-kromosomen. Samtidigt är gener med högre ut- trycksnivå i hanar underrepresenterade på X-kromosomen. Studien visar också att gener med könsskillnader i uttrycksnivå på X-kromosomen oftast är evolutionärt gamla gener. De flesta av dessa gener uppkom för över 100 miljoner år sedan. Det är ännu oklart vad denna observation kan ha för vida- re betydelse. I den femte studien presenteras en transgen mus som uttrycker enzymet ”Cre” i hjärnregionen striatum. Med hjälp av denna mus kan man studera funktionen hos striatum. Det återstår att se om man också kan studera den embryonala utvecklingen av striatum med hjälp av denna transgena mus.

45 Acknowledgements

I would like to express my gratitude towards a number of researchers, students, friends and foundations...

First of all, to my main supervisor Elena Jazin: Thank you for all your mentorship and friendship. It has been absolutely terrific to work in your laboratory! Thanks for all the good times we shared. I would like to thank you in particular for giving me the freedom to follow up many of my own ideas and projects, while at the same time providing the support of a senior investigator. Next, to my second supervisor Klas Kullander: You are really an exemplar of a PI, and a great source of inspiration. Thank you for your everlasting patience and for always taking your time when I came knocking at your door. Special ac- knowledgment goes to my opponent Professor Art Arnold, taking his time to travel all the way from UCLA for my dissertation. It is an absolute privilege for me to have the opportunity to present my thesis before one of the unquestionably most influential figures in the field – about whom else would one find a paper titled “State-of-the art (Arnold) behavioral neuroendocrinology” [147]? Peter Saetre: I learned a lot from you. Thanks for providing sensible perspectives to both research and data, and thank you for inviting me to spend wonderful times with your family. Reinald Fundele: Thank you for always having your door open, and for the many good scientific discussions. I also enjoyed hearing about your amazing wilderness trips, which was inspiring for me. Rolf Ohlsson: You and your group provided such a vibrant research climate to our department during my first year of PhD studies. Thank you for that. To all the members of the Ohlsson group: Thanks for fantastic seminar sessions and likewise for great Friday pub sessions. Chandrasekhar Kanduri: Many thanks for providing the MEFs for the FISH experiment in Paper IV and for good discussions. To the people at the Department of Organismal Biology: Thanks for fantastic fika sessions and for many other things… and special tributes go to Rose-Marie Löfberg and Helena Malmikumpu: Our department would be in complete chaos without you. Thanks for all your care. Barbro, Carina and Calle: Thanks for being there, providing a pleasant atmosphere; and thanks Calle for your great lectures in Zoology. Per Ahlberg: For being a source of inspiration at the department. Lina, Julia, Eva: I have really enjoyed your company. Martin: I wish you all the best during you PhD studies. Thanks for all your help and for great coffee. Katarzyna: Thank you for your help with cell culturing and for nice company in the office space. Best of luck to you in your research! Vincent: Thanks for sharing DVDs with British comedy. I am very thankful to Susana M. Chuva de Sousa Lopes for sending cheerful emails with pep talk during my two final weeks of writing. Robert Williams, Yoav Gilad, Jennifer Leonard, Ran Blekhman, Roxana Merino-Martinez, Glenn Rosen, Lu Lu: are acknowledged for successful collaborations. Liu and Chengxi: are acknowledged for successful collaborations and for friendship. Kuljeet: For being such a good and interesting fellow. I wish that we had worked in the same group during our PhD periods. Let’s collaborate on some interesting project in the near future. Kali: My other great Indian friend; thank you for being such a caring guy. Let’s throw together that mouse paper now! I would also like to thank Sulena for the great Indian dinners. To all the other Drs and students of the Kullander lab: Thank you for being such a great team... and a special

46 thanks goes to Anders, Martin, Katarzyna, Christiane, Malin, Chetan, Karin and Richardson: for fun times in the lab, for all your help with many practicalities, and for countless fascinating conversations. Tatjana: Thanks for the three-martini lunches. Graham Budd: For checking the English in one of the papers, and for being an intriguing character. Dominic: Is acknowledge for the ”the” in the title of this thesis. Gabriel: I could fill this page with declarations of friendship and gratitude towards you, but I will only say: Lass uns Tanzen! Mao: Thanks for yummy dinners. Carlborg: For being my number one Risk opponent during these years. Tobbe, Camsund, Silver: For all the fun we have together. Barbara: Thanks for the many wonderful dinners, and for all the other good things that you do. Lars and Caroli- na: For lunches, dinners, and for many hours of great company. Till Källmänningarna, Myrenbergar- na, Reiniusarna: Tack för allt! To my dear family; Ulf, Eva, Magnus, Eva-Lill, Vilgot, Signe, Staf- fan, Ellen, Edith: Thank you for always providing support and love. I have missed you so much each time when I could not get off from work to visit. Finally, my beloved Viveca: I would not have made it without you!

I acknowledge the following foundations for providing financial support:

Marcus Borgström’s Foundation

Nils von Hoofsteen’s Zoological Foundation

Liljewalchs’ Foundation

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