Epigenetic mechanisms of gene regulation during mammalian ...

[Epigenetics 3:1, 21-27; January/February 2008]; ©2008 Landes Bioscience

Review

Epigenetic mechanisms of gene regulation during mammalian spermatogenesis Ahmad M. Khalil and Claes Wahlestedt* Molecular and Integrative Neurosciences Department (MIND); The Scripps Research Institute; Jupiter, Florida USA

OT D

IST

RIB

Histone acetylation is carried out by histone acetyltransferases and the reverse process, termed deacetylation, is carried out by histone deacetylases;4,12 the methylation of histone proteins is carried out by histone methyltransferases and the reverse process is carried out by histone demethylases.7,13 While a lysine residue can have only one acetyl group, by contrast, a lysine residue can be modified with mono, di or (tri)methylation.4,14 A growing number of enzymes have been identified that are responsible for the modifications of histones including the recently identified histone demethylases13,15 suggesting that histone modifications are highly dynamic. The modification of each histone residue is catalyzed by one or more specific enzyme(s) and some of these enzymes have high specificity for a particular residue. The methylation of H3K4 is carried out by a single enzyme in S. cerevisiae (Set1); by contrast, there are ten different enzymes in mammals that can add a methyl group to H3K4.15 Furthermore, histone modifying enzymes can be tissue specific, region specific, and developmentally regulated. For example, H3K9 can be methylated by Suv39h1, Suv39h2 and G9a. Both Suv39h1 and Suv39h2 (tri)methylate H3K9 at pericentromeric heterochromatin; while Suv39h1 is ubiquitously expressed; Suv39h2 is testis specific.14 By contrast, G9a (di)methylates H3K9 at euchromatic regions of the genome.14 In addition to the core histones, there are histone variants that can also contribute to the epigenetic dynamics of cellular processes. These histone variants can be incorporated into the genome in both a DNA replication dependent and independent manners.16 Histone variants can also be modified by the same chemical modifications as the four core histones. The histone variant H3.3, for example, is enriched with modifications of active chromatin.17 There are also histone variants that show tissue specific expression. During mammalian spermatogenesis there are a number of histone variants that replace the core histones in a stage‑dependent manner suggesting that these histone variants play a role in the process of spermatogenesis.18,19

IEN

CE

.D

ON

The process of spermatogenesis is a unique form of cellular differentiation and is regulated by genetic and epigenetic factors. Recent studies have shown that some epigenetic factors (histone variants and histone modifying enzymes) are exclusively expressed during spermatogenesis; the disruption of these factors leads to spermatogenic defects. Also, during spermatogenesis a key epigenetic process termed meiotic sex chromosome inactivation (MSCI) occurs; MSCI leads to the inactivation of most genes on the X and Y chromosomes; however, the mechanism of MSCI is distinct from X inactivation in female somatic cells. Furthermore, a new class of non-coding RNAs (i.e., piRNAs) has recently been identified which is exclusively expressed during spermatogenesis. This review discusses recent advances in our understanding of epigenetic mechanisms that operate during spermatogenesis with specific emphasis on histone modifications, MSCI and non-coding RNAs. Finally, we propose that spermatogenesis can be a powerful experimental system to dissect epigenetic mechanisms of gene regulation.

UT E

.

Key words: epigenetics, spermatogenesis, meiosis, histone modifications, MSCI, non-coding RNAs

SC

Epigenetics

©

20

08

LA

ND

ES

BIO

Although several definitions for epigenetics have been proposed,1 the term epigenetics refers to changes in gene expression which can be maintained through cell divisions and are not coded in the DNA sequence itself.1,2 Epigenetic mechanisms of gene regulation depend on a variety of factors such as DNA methylation, histone variants and the post‑translational modifications of histone proteins.2,3 Histones are highly conserved proteins which are intimately associated with DNA.4 There are four core histone proteins, histone H4, H3, H2A and H2B; two copies of each of the four core histones are found in the nucleosome which is the fundamental unit of chromatin.5,6 All core histones are subject to posttranslational covalent modifications (e.g., acetylation, methylation and phosphorylation) at specific residues in their N‑termini.7‑9 Histone acetylation is generally associated with transcriptionally active regions of the genome;10,11 however, histone methylation can be associated with active or repressed regions of the genome depending on the residue that becomes methylated. *Correspondence to: Claes Wahlestedt; The Scripps Research Institute; 5353 Parkside Drive RF1-109; Jupiter, Florida 33458 USA; Tel.: 561.799.8905; Fax: 561.799.8958; Email: [email protected] Submitted: 12/03/07; Accepted: 01/10/08 Previously published online as an Epigenetics E-publication: www.landesbioscience.com/journals/epigenetics/article/5555

www.landesbioscience.com

Mammalian Spermatogenesis Spermatogenesis is an elegant form of cellular differentiation and its success is critical for the propagation of species. The process of spermatogenesis takes approximately 85 days in humans and 36 days in the mouse. This process can be divided into three subdivisions: spermatogoniogensis, meiosis and spermiogenesis.20,21 During spermatogoniogenesis, spermatogonia divide mitotically to constantly replenish the pool of cells entering meiosis; cells that

Epigenetics

21

Epigenetic code during spermatogenesis

UT E RIB IST OT D ON

Figure 1. Epigenetic regulation of mammalian spermatogenesis. Cytological preparation of the various stages of mouse spermatogenesis. DNA is stained red with propidium iodide. Arrow identifies the XY body at the pachytene stage of meiosis. A key epigenetic process termed meiotic sex chromosome inactivation (MSCI) which leads to the inactivation of most genes on the X and Y chromosomes occurs during male meiosis. Xist which is repressed in male somatic cells is expressed at the pachytene stage of male meiosis; however, the expression of Xist in other stages of spermatogenesis has not yet been determined. Defects in genes which encode histone variants (e.g., H2AX); histone modifying enzymes (e.g., Meisetz, Jhdm2a) or members of the Piwi subgroup of the argonaute family of proteins (e.g., Mili, Miwi, Miwi2) lead to defects in spermatogenesis.

.D

Spermatogenesis‑Specific Histone Modifying Enzymes

.

enter meiosis become committed to a one‑way pathway of cellular differentiation. During meiosis each cell progresses through a series of cytologically identifiable stages (Fig. 1) known as leptotene (chromosomes begin to condense), zygotene (pairing of homologous chromosomes is initiated), pachytene (complete synapsis of homologous chromosomes which undergo recombination), diplotene (homologous chromosomes begin to separate), diakinesis (chromosomes condense), metaphase I (separation of homologous chromosomes) and metaphase II (separation of sister chromatids) (Fig. 1). The germ cells at the end of meiosis produce round spermatids.22 During spermiogenesis, round spermatids go through extensive condensation of their chromatin by replacing most of their histones with transition proteins and then with protamines, and discard most of their cytoplasm.22‑25

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

During spermatogenesis, each spermatocyte undergoes erasure and reestablishment of epigenetic marks to confer a paternal origin of that genome once it is passed to the subsequent generation. This process of epigenetic reprogramming involves, in addition to many other factors, histone modifications and the effecter enzymes that catalyze these modifications (Table 1). Over the past decade intense efforts have been made not only to understand the function of histone modifications but also to identify the enzymes that are responsible for adding these modifications to histone residues. We and others have previously shown that histone modifications are highly dynamic during spermatogenesis26,27 and several histone modifying enzymes have been shown to be expressed only in germ cells. Here, we will discuss some of these histone modifying enzymes and their function during spermatogenesis. The transition of histones to protamines is a crucial step in the maturation of spermatids and is thought to be facilitated by histone H4 hyperacetylation in mammals similar to other species.28,29 Two genes on the human Y chromosome CDY1 and CDY2 (Chromo Domain Y) are expressed exclusively in the testis and have a chromodomain which is implicated in chromatin binding. To that end, Lahn et al. postulated that these genes may code for a novel class of histone acetyltransferases.30 Indeed, the authors found that both human CDY and CDYL and mouse Cdyl display histone aceytltransferase activity preferentially on histone H4 in vitro. Also, mouse Cdyl protein levels become expressed at robust levels beginning at day 25 after birth around the time the first wave of spermatogenesis begins 22

to reach the round spermatid stage and correlate with increased H4 acetylation during spermiogenesis. Importantly, CDY mRNA is absent in human males with infertility.30 These observations suggest that these histone acetyltransferases are strong candidates to be the enzymes responsible for H4 hyperacetylation during mammalian spermiogenesis which need to be validated by the generation of a mouse conditional knockout of the Cdyl enzyme during spermatogenesis. Nonetheless, these results implicate a novel class of histone acetyltransferases in the process of mammalian spermatogenesis. H3K4me3 has the strongest correlation with transcriptional activity in somatic cells among all the histone modifications identified to date.31,32 In 2005, Hayashi et al. identified a meiosis specific histone methyltransferase (Meisetz) that has catalytic activity for H3K4me3 but not H3K4me1 or H3K4me2. In adult male mice, Meisetz is expressed in the testis but not in other tissues, also, the expression of Meisetz initiates at day 10 after birth when the first round of spermatogenesis is underway. Importantly, Meisetz‑/‑ male and female mice are sterile and the weight of the testes in Meisetz‑/‑ adult mice is 75% less than that of wildtype. Histological and cytogenetic examinations of the seminiferous tubules of Meisetz‑/‑ adult mice have shown that meiosis is arrested at the pachytene stage, the phosphorylated H2AX histone variant (g‑H2AX)

Epigenetics

2008; Vol. 3 Issue 1


OT D

IST

RIB

UT E

.

significantly reduced in the round spermatids of the Jhdm2a‑/‑ mice. Using CHIP analysis it was shown that Jhdm2a can associate with the promoters of Prm1and Tnp1, two genes with important function during spermiogenesis. Importantly the levels of H3K9 methylation in the promoters of Prm1 and Tnp1 were significantly increased in the spermatids of Jhdm2a‑/‑ mice which likely contribute to their repression. This study is the first to demonstrate an important role of a histone demethylase in the process of spermatogenesis, and suggest that other histone demethylases, which are yet to be identified, are likely to be important for the completion of mammalian spermatogenesis. Collectively, these studies indicate that histone modifications and the enzymes that catalyze these modifications are crucial for the completion of spermatogenesis, and it is very likely that additional histone modifying enzymes with specific functions in germ cells will be uncovered in the future. Furthermore, the generation of conditional knockouts of these histone modifying enzymes in the testes provides a powerful tool toward understanding the function of distinct histone modifications since conventional knockouts of most histone modifying enzymes results in embryonic lethality.35

Meiotic Sex Chromosome Inactivation (MSCI) During Spermatogenesis Mammalian male somatic cells have transcriptionally active X and Y chromosomes. Furthermore, there is an abundance of X linked genes that are expressed in spermatogonia.36 However, both the X and Y chromosomes undergo transcriptional inactivation during male meiosis beginning shortly prior or at the pachytene stage of meiosis26,37‑39 and they are thought to become active again in the round spermatid stage. During the first stages of meiosis, the X and Y chromosomes occupy random positions in the nucleus. However, by the pachytene stage of meiosis the X and Y chromosomes pair at their homologous pseudoautosomal regions and form the sex body, the chromatin of the sex body is visually more condensed than that of the autosomes. By isolating enriched populations of pachytene spermatocytes and round spermatids, several X and Y linked genes

Term MSCI

Definition

Occurrence

Meiotic sex chromosome inactivation

Male meiosis

Meiotic silencing of unsynapsed chromatin

Meiosis

X‑inactive specific transcript

Female somatic cells

SUV39H1

Suppressor of variegation 3‑9 homolog 1 (histone methyltransferase for H3K9 trimethylation)

Ubiquitously expressed

SUV39H2

Suppressor of variegation 3‑9 homolog 2 (histone methyltransferase for H3K9 trimethylation)

Testis

20

08

XIST

LA

MSUC

Summary of key terms discussed in the text

ND

Table 1

ES

BIO

SC

IEN

CE

.D

ON

becomes inappropriately distributed on the autosomes and there is a significant increase in illegitimate pairing of homologous chromosomes.33 Since the knockout of Meisetz did not affect H3K4me1 or H3K4me2, this is suggested that another one or more H3K4 methyltransferases are responsible for these modifications during meiosis. Also, it is very likely that there are additional histone methyltransferases that operate during gametogenesis yet to be identified. The Suv39h HMTases are encoded by two loci, Suv39h1 and Suv39h2 while both of these genes have an overlapping and ubiquitous expression during embryogenesis; in adult mice Suv39h2 is mainly expressed in the testes.8 Knockout of either Suv39h1 or Suv39h2 does not affect viability or fertility; however, mice mutant for both enzymes displayed (1) hypogonadism and complete spermatogenic failure, (2) nonhomologous interactions and delayed synapsis between the autosomes as well as between the sex chromosomes and the autosomes and (3) lack of H3K9me at pericentromeric heterochromatin in B‑spermatogonia and preleptotene spermatocytes but not at later stages of meiosis indicating that additional methyltransferase(s) are responsible for H3K9me at the later stages of meiosis. Recently, Okada and colleagues have demonstrated that a histone demethylase (Jhdm2a) is important for spermatogenesis.34 First, the authors noted that Jhdm2a is expressed at high levels in the testis at both the mRNA and the protein levels. The expression of Jhdm2a increased approximately 70 folds from day 7 to day 30 in the testis with the highest expression in round spermatids, further the expression of Jhdm2a coincides with the initiation of the first round of spermatogenesis in the mouse. To determine the exact function of Jhdm2a, the authors disrupted the Jhdm2a gene in the mouse. These knockout mice had smaller testes than the wildtype, lower sperm count and were infertile. Closer examination of these mice revealed that they had a decrease in the number of spermatids with some spermatids having multiple tails suggesting that Jhdm2a most likely functions in the later stages of spermatogenesis. Although Jhdm2a‑/‑ mice had some sperm produced, these sperm were immotile and had defects in chromatin condensation. Quantitative RT‑PCR analysis demonstrated that the expression of Tnp1 and Prm1 are

©

G9a

Meisetz H2AX

Non-coding RNAs

histone methyltransferase for H3K9 dimethylation


Meiosis‑induced factor containing PR/SET domain and zinc‑finger (histone methyltransferase for H3K4 trimethylation)

Male and female meiosis

Histone variant for H2A


RNAs that lack protein coding potential


miRNA

Micro RNA (~18‑24 nucleotides)


siRNA

Small interfering RNA (~21‑23 nucleotides)

Exogenous and endogenous

piRNA

Piwi associated RNA (~26‑34 nucleotides)

Spermatogenesis


Epigenetics

23


.

MSCI and Female X Inactivation Differ Mechanistically

OT D

IST

RIB

UT E

Inactivation of one of the two X chromosomes in females to achieve dosage compensation with males which only possess a single transcriptionally active X chromosome occurs during embryonic development and is maintained throughout the life of females.47‑50 This process of embryonic X inactivation is known to require the expression of the Xist gene.47,50 As a reflection of its role in this process, the Xist gene has been found to be expressed in female somatic cells, but not in male somatic cells. However, the Xist gene has been shown to be expressed in males during the pachytene stage of meiosis (Fig. 1).38,51 As this expression pattern correlated with the timing of X inactivation during male meiosis, McCarrey et al. (2002) and Turner et al. (2002) investigated whether or not expression of an intact Xist gene is required for meiotic sex chromosome inactivation.52,53 By investigating expression patterns of sex‑linked genes, as well as changes in chromatin structure associated with these genes and the formation of an XY chromatin body in spermatogenic cells recovered from male knockout mice carrying a single X chromosome from which the Xist gene had been ablated, both groups found that MSCI proceeds normally in the absence of an intact Xist gene.52,53 These results indicate that MSCI is regulated by a different mechanism than the Xist dependent mechanism that regulates embryonic female X inactivation. Furthermore, Xist deficient male mice have normal spermatogenesis suggesting that this transcript does not perform an essential function during spermatogenesis.54 Nevertheless, the expression of Xist during male meiosis remains intriguing. Why does Xist become expressed during male meiosis and escape MSCI? Is Xist involved in the chromatin remodeling that occurs at the XY body during meiosis26 by directing histone modifying enzymes to these chromosomes? Also, further studies will be needed to determine if the mice carrying the ablated Xist gene in the studies reported by McCarrey et al. (2002) and Turner et al. (2002) did not maintain a functional domain of Xist which was sufficient for its function.52,53 DNA methylation is an important epigenetic mark that is highly associated with promoters of inactive genes.55 Therefore, Driscoll and Migeon (1990) examined the levels of DNA methylation in the promoters of housekeeping genes on the human X chromosome during X inactivation in male meiosis and found that these promoter regions remain unmethylated.56 Also, McCarrey et al. (1992) showed that the inactivation of X linked Pgk1 during mouse spermatogenesis is not associated with a change in DNA methylation.37 Collectively, these findings suggested a different mechanism for the inactivation of the X and Y chromosomes during meiosis to the inactivation of the X chromosome in female somatic cells. Given the important role of histone modifications in gene regulation in somatic cells,

.D

Asynapsis of the X and Y Chromosomes Leads to MSCI

Turner et al. examined male carriers of the reciprocal X‑autosome translocation T(X;16)16H which produce a number of different chromosomal associations with varying degree of asynapsis. The authors found that unsynpased autosomal regions which become enriched with g‑H2AX are always Cot‑1 negative suggesting that they are transcriptionally inert.43 The silencing of unpaired chromatin has been supported by another independent study.46 It will be important to determine why these autosomal regions remain unpaired during meiosis, one possibility is that those regions lack homology or there are physical constraints that interfere with pairing.

ON

have been shown to become transcriptionally silenced/repressed at pachytene and are reactivated in round spermatids.39 Currently, there is a debate whether the X and Y chromosomes become reactivated in the round spermatid stage of meiosis. We have previously shown that the phosphorylated form of RNA polymerase II (serine 2) which is associated with transcriptional elongation becomes excluded from the XY body at pachytene; however, it reengages both chromosomes by the round spermatid stage following meiosis.26 Also, Wang et al., examined the expression of eleven X linked and three Y linked genes by RT‑PCR by isolating relatively pure populations of the various stages of spermatogenesis.39 All fourteen sex linked genes examined showed evidence of MSCI, importantly, the authors found that most of these genes undergo postmeiotic reactivation. Also, the reactivation of these genes does not correlate with their proximity to the Xic or Xist.39 Microarray analysis on relatively pure population of spermatids has also shown that X linked genes are expressed in spermatids.40 By contrast, Turner et al. examined round spermatids using Cot1 RNA‑ FISH as a marker for gene expression and found that the X and Y chromosomes to be devoid of Cot1 signal in the majority of the spermatids examined.41 Utilization of microdissection techniques to isolate pure populations of round spermatids followed by qRT‑PCR and/ or performing RNA‑FISH on a large number of sex linked genes during spermatogenesis should provide direct evidence to the extent of postmeiotic sex‑linked gene expression.

20

08

LA

ND

ES

BIO

SC

IEN

CE

At the pachytene stage of meiosis homologous autosomes pair along their entire length; however, since the X and Y chromosomes only share limited homology in their psudoautosomal regions, they remain asynapsed. Recently, Turner et al. have shown, using XYY and mice carrying X‑autosome translocation T(X;16)16H (male mice with two X chromosomes have spermatogonial block), that when the X and Y chromosomes have a pairing partner they fail to undergo MSCI. They assessed MSCI based on examining the distribution of g‑H2AX, and the expression of the Uty (Y chromosome) and Ddx3x (X chromosome) genes by RNA FISH. The authors concluded that the asynapsis of the X and Y chromosomes during mammalian meiosis drives MSCI.41 Spermatocytes that fail to inactivate the X and Y chromosomes are eliminated at the pachytene stage of meiosis; which suggest that MSCI is required for spermatogenesis to proceed.41,42 Turner et al. postulated that one or more X and/or Y linked genes that remain active due to failure in MSCI may be responsible for the elimination of pachytene spermatocytes in human and mice with XYY or X‑autosome translocation. Essentially, failure to inactivate the sex chromosomes could be an underestimated cause of male infertility in humans.41,43

©

Meiotic Silencing of Unsynapsed Chromatin (MSUC) During Spermatogenesis Several studies have shown that Brca1, ATR and g‑H2AX become enriched on the inactive X and Y chromosomes as well as unsynapsed autosomal regions at the pachytene stage of meiosis.43‑45 The localization of ATR and g‑H2AX to unsynpased chromatin is Brca1 dependent as Brca1 mutant mice display defective localization of ATR and g‑H2AX. To determine if unsynapsed autosomal regions become silenced during meiosis, similar to the X and Y chromosomes, 24

Epigenetics



.

UT E

RIB

.D

ON

Since MSCI in male meiosis does not appear to require an intact Xist gene nor DNA methylation,52,53,56 another possibility is that the mechanism for MSCI is mediated by changes in chromatin modifications.26 This is a plausible explanation since the inactivation is transient and need to be reversed at the end of meiosis; therefore, the dynamic nature of chromatin will be very accommodating to this reversible process. The phosphorylated H2AX (g‑H2AX), a histone H2A variant implicated in DNA repair, has been previously shown to accumulate in the XY body at the zygotene‑pachytene transition just prior to the onset of MSCI.44 Spermatocytes in H2AX‑deficient mice fail to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, the authors found that the histone variant macroH2A1.2 fail to preferentially localize with the sex body in the absence of H2AX.44 Recently, it has also been shown that the histone variants H3.1 and H3.2 become devoid of the X and Y chromosomes by mid‑pachytene; by contrast H3.3 when phosphorylated at serine 31 shows high enrichment at the sex chromosomes shortly after MSCI.27 These findings suggest that histone variants have an important role in the process of MSCI which will require further investigations in the future. The inactivation of one of the two X chromosomes in females begins in embryogenesis and is accompanied by chromosome‑wide sequential changes in histone modifications.47 Similarly, we and others have observed sequential changes in histone modifications coinciding with MSCI at pachytene. The X and Y chromosomes become devoid of histone H4 and H3 acetylation, H3K4me3, H3K79me1 and H3K79me2 and become enriched with H3K9me2, H3K9me3.26,27,57 In our study,26 we examined histone H4 acetlyation at lysine 5, 8, 12 and 16; although the X and Y chromosomes become devoid of H4 acetylation during MSCI, the timing of the deacetylation of each residue takes place at different stages depending on the lysine examined. H4K12 and H4K16 deacetylation occurs at pachytene; by contrast, H4K5 and H4K8 deacetylation occur at the diplotene stage and it was preceded by hyperacetylation. These results suggested that different histone deacetylase complexes are being targeted to the XY body to modify each lysine. The significance of the temporal regulation of histone deacetylation during MSCI is not clear, also the significance of H4 hyperacetylation at lysine 5 and 8 of the XY body is intriguing and the enzymes that are required for this process are yet to be identified.26 The XY body becomes deacetylated at H3K9ace and enriched with H3K9me2 by the mid‑pachytene stage of meiosis.26 However, the deacetylation of H3K9 is also preceded by hyperacetylation in early pachytene. Also, in contrast to the inactive X chromosome in female somatic cells, the XY body is enriched with H3K4me2 in comparison to the autosomes during spermatogenesis.58 Although the significance of this enrichment is yet to be determined, one intriguing possibility is that H3K4me2 is serving as an epigenetic mark. H3K4m2 has previously been shown to be an epigenetic mark at X linked genes prior to the onset of X inactivation in female ES cells.59

Initially, when X inactivation in female somatic cells was discovered, it was accepted that the silencing of the X chromosome is complete.60 However, since that discovery several groups have identified a number of genes that escape X inactivation.61‑67 A complete study which examined the vast majority of X linked genes has revealed that 15–25% of genes escape X inactivation.68 Then the question arises how complete is MSCI? Are there genes that escape MSCI in male meiosis? Previously it has been shown that Xist is expressed at pachytene during MSCI.38,51 It is currently accepted that the pseudoautosomal regions escape MSCI in both humans and mice; however, there has been no direct evidence (i.e., gene expression analysis) for it. We have recently shown that H3K4me3 is enriched at regions that escape X inactivation in female somatic cells. Interestingly, we found several regions on the inactive X and Y in male meiosis that are also enriched with H3K4me3 from pachytene to MII.69 We also found that the same regions that are enriched with H3K4me3 are also enriched with the phosphorylated form of RNA polymerase II. These data suggested that there are regions that escape MSCI and the genes that escape MSCI could have a substantial overlap with genes that escape X inactivation in female somatic cells.69 Gene expression analysis using RNA‑FISH at the various stages of meiosis will be instrumental in determining which genes escape MSCI and if those genes are the same as the genes that escape X inactivation in female somatic cells.

IST

Changes in Histone Variants and Histone Modifications Correlate with MSCI

Escape from MSCI

OT D

these modifications along with testis‑specific histone variants are strong candidates for mediating MSCI.

Non-Coding RNAs During Spermatogenesis

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

The textbook model where DNA is transcribed into mRNA which is translated into protein has now become outdated. This is the result of the identification of several classes of RNAs that are not translated into proteins (non-coding RNAs).70 Non-coding RNAs have been classified into several classes based on their size and function (i.e., tRNAs, rRNAs, miRNAs, siRNAs, snoRNAs, snRNAs, piRNAs, natural antisense transcripts and macroRNAs).71 Currently there is an intense interest in uncovering the function and the mechanism of RNA mediated processes. Recently, a long non-coding RNA (macroRNA) was identified in the HOXC locus (HOTAIR).72 The HOTAIR non-coding RNA represses transcription in trans across 40 kb of the HOXD locus by altering the chromatin modifications through enhancement of the PRC2 activity at the HOXD locus.72 It is therefore possible that other non-coding RNAs may also interact with other histone modifying enzymes and chromatin remodeling complexes and recruit them to their targets in a similar manner to HOTAIR. This mechanism could also operate during spermatogenesis where non-coding RNAs can target various histone modifying enzymes to imprinted genes, for example, and contribute to the epigenetic reprogramming that occurs in the male germline. While all classes of non-coding RNAs are expressed during spermatogenesis, piRNAs are exclusively expressed during spermatogenesis in mammals.73,74 piRNAs (26‑34 nucleotides) are several nucleotides longer than siRNAs and miRNAs (18–24 nucleotides),73 interact with Piwi‑family of proteins Miwi, Miwi2 and Mili,75,76 consist of more than 50,000 species and they are produced from discrete loci 50–100 kb in size. While miRNAs and siRNAs biogenesis requires the RNaseIII enzyme DICER,77 piRNAs biogenesis is Dicer independent.73 Furthermore, all Piwi proteins (Miwi, Miwi2


Epigenetics

25


Acknowledgments

UT E

.

spermatogenesis which we are only beginning to uncover.74,76,84 Since spermatogenesis takes place over an extended period of time (36 days in mice and 85 days in humans) and it has distinct stages that can be identified cytologically (Fig. 1), this makes it a powerful experimental system to dissect the order of epigenetics events (i.e., which factors come first, second… etc). Also, since previous attempts to generate conventional mouse knockouts of histone modifying enzymes (e.g., G9a) and RNAi related proteins (e.g., Dicer) resulted in embryonic lethality, the generation of conditional knockouts during spermatogenesis can be a powerful tool for the functional studies of these proteins. Therefore, we propose that spermatogenesis can be a fertile ground for furthering our understanding of epigenetic mechanisms of gene regulation.

IST

RIB

A.K. is the recipient of a postdoctoral fellowship from Conquer Fragile X Foundation. We are grateful to Dr. Daniel J. Driscoll, University of Florida College of Medicine for allowing us to use the images in Figure 1 which were produced in his laboratory by A.K. Reference

OT D

1. Bird A. Perceptions of epigenetics. Nature 2007; 447:396‑8. 2. Turner BM. Histone acetylation and an epigenetic code. Bioessays 2000; 22:836‑45. 3. Jaenisch R, Bird A. Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat Genet 2003; 33:245‑54. 4. Rice JC, Allis CD. Histone methylation versus histone acetylation: New insights into epigenetic regulation. Curr Opin Cell Biol 2001; 13:263‑73. 5. Luger K, Rechsteiner TJ, Flaus AJ, Waye MM, Richmond TJ. Characterization of nucleosome core particles containing histone proteins made in bacteria. J Mol Biol 1997; 272:301‑11. 6. Felsenfeld G, Groudine M. Controlling the double helix. Nature 2003; 421:448‑53. 7. Rea S, Eisenhaber F, O’Carroll D, Strahl BD, Sun ZW, Schmid M, Opravil S, Mechtler K, Ponting CP, Allis CD, Jenuwein T. Regulation of chromatin structure by site‑specific histone H3 methyltransferases. Nature 2000; 406:593‑9. 8. Peters AH, O’Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, Weipoltshammer K, Pagani M, Lachner M, Kohlmaier A, Opravil S, Doyle M, Sibilia M, Jenuwein T. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 2001; 107:323‑37. 9. Zhang Y, Reinberg D. Transcription regulation by histone methylation: Interplay between different covalent modifications of the core histone tails. Genes Dev 2001; 15:2343‑60. 10. Jeppesen P, Turner BM. The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression. Cell 1993; 74:281‑9. 11. Litt MD, Simpson M, Gaszner M, Allis CD, Felsenfeld G. Correlation between histone lysine methylation and developmental changes at the chicken beta‑globin locus. Science 2001; 293:2453‑5. 12. Roth SY, Denu JM, Allis CD. Histone acetyltransferases. Annu Rev Biochem 2001; 70:81‑120. 13. Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 2004; 119:941‑53. 14. Sims IIIrd RJ, Nishioka K, Reinberg D. Histone lysine methylation: A signature for chromatin function. Trends Genet 2003; 19:629‑39. 15. Kouzarides T. Chromatin modifications and their function. Cell 2007; 128:693‑705. 16. Tagami H, Ray‑Gallet D, Almouzni G, Nakatani Y. Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 2004; 116:51‑61. 17. McKittrick E, Gafken PR, Ahmad K, Henikoff S. Histone H3.3 is enriched in covalent modifications associated with active chromatin. Proc Natl Acad Sci USA 2004; 101:1525‑30. 18. Govin J, Escoffier E, Rousseaux S, Kuhn L, Ferro M, Thevenon J, Catena R, Davidson I, Garin J, Khochbin S, Caron C. Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis. J Cell Biol 2007; 176:283‑94. 19. Bramlage B, Kosciessa U, Doenecke D. Differential expression of the murine histone genes H3.3A and H3.3B. Differentiation 1997; 62:13‑20. 20. Bellve AR, Cavicchia JC, Millette CF, O’Brien DA, Bhatnagar YM, Dym M. Spermatogenic cells of the prepuberal mouse: Isolation and morphological characterization. J Cell Biol 1977; 74:68‑85. 21. Holstein AF, Schulze W, Davidoff M. Understanding spermatogenesis is a prerequisite for treatment. Reprod Biol Endocrinol 2003; 1:107. 22. Hoyer‑Fender S. Molecular aspects of XY body formation. Cytogenet Genome Res 2003; 103:245‑55. 23. Fuentes‑Mascorro G, Serrano H, Rosado A. Sperm chromatin. Arch Androl 2000; 45:215‑25.

ES

Concluding Remarks

BIO

SC

IEN

CE

.D

ON

and Mili) are essential for male fertility suggesting an important role for piRNAs in mammalian spermatogenesis.76,78,79 Interestingly however, Piwi proteins seem to have a nonoverlapping function since the disruption of each protein results in defects at various stages of spermatogenesis (Fig. 1). Miwi knockout mice display spermatogenic arrest at the beginning of the round spermatid stage,78 however, both Mili and Miwi2 knockout mice have spermatogenic arrest as early as prophase I of meiosis (Fig. 1). Recently, Miwi has been shown to interact with the translational machinery and piRNAs to regulate spermatogenesis;74 and Miwi2 has been shown to play a role in the repression of transposons during spermatogenesis.76,79 These findings suggest an important role for piRNAs and their interacting proteins in the process of mammalian spermatogenesis. Micro RNAs (miRNAs) is another class of small non-coding RNAs that ranges in size from 18–24 nucleotides and function by inhibiting mRNA translation in mammalian cells.80 Over one thousand miRNAs have been identified to date in mammals; however, only a small percentage of these miRNAs targets have been identified. Recently, through the use of miRNAs microarray, Yan et al. were able to show that some miRNAs show differential expression in the testis before and after the initiation of spermatogenesis.81 Also, a miRNA cluster on the X chromosome which is only present in primates has recently been identified; the expression of these primate‑specific miRNAs shows a strong correlation with sexual maturation in rhesus monkey suggesting that these miRNAs may play an important role in regulating gene expression during spermatogenesis.82 Also, another intriguing possibility is that some miRNAs and their related factors are involved in MSUC. Indeed, Costa and colleagues identified a mouse ortholog to the Drosophila high mobility group box protein Maelstrom (Mael) which is associated with the silenced XY body and unsynapsed autosomes in male meiosis.83 The authors found Mael to directly interact with the chromatin remodeler SNF5 and chromatin associated protein SIN3B, furthermore, Mael is found in a complex with Mili and Miwi suggesting a possible role for the miRNA pathway in MSUC. Collectively, these studies suggest an important role for non-coding RNAs and the proteins that associate with them in regulating mammalian spermatogenesis.

©

20

08

LA

ND

The process of spermatogenesis is an elaborate form of cellular differentiation that requires the expression of specific genes and a unique set of epigenetic factors (e.g., histone modifying enzymes, histone variants, and non-coding RNAs). During the process of spermatogenesis several key epigenetic events occur including meiotic sex chromosome inactivation (MSCI), a process that leads to the repression of most genes on the X and Y chromosomes. The inactivation of the X and Y chromosomes during meiosis is intriguing and many questions remain regarding this phenomenon. What is the mechanism of MSCI? What is the significance of inactivating the X and Y chromosomes during meiosis and does it affect male fertility in humans? How many genes escape X and Y inactivation, and if so, are they similar or different from genes that escape X inactivation in female somatic cells? The expression of Xist during male meiosis is also intriguing. Does Xist perform unidentified function in male germ cells? Also, the identification of a special class of non-coding RNAs (i.e., piRNAs) that is exclusively expressed during spermatogenesis suggests that piRNAs play a unique role during 26

Epigenetics



OT D

IST

RIB

UT E

.

53. Turner JM, Mahadevaiah SK, Elliott DJ, Garchon HJ, Pehrson JR, Jaenisch R, Burgoyne PS. Meiotic sex chromosome inactivation in male mice with targeted disruptions of Xist. J Cell Sci 2002; 115:4097‑105. 54. Marahrens Y, Panning B, Dausman J, Strauss W, Jaenisch R. Xist‑deficient mice are defective in dosage compensation but not spermatogenesis. Genes Dev 1997; 11:156‑66. 55. Mann JR, Szabo PE, Reed MR, Singer‑Sam J. Methylated DNA sequences in genomic imprinting. Crit Rev Eukaryot Gene Expr 2000; 10:241‑57. 56. Driscoll DJ, Migeon BR. Sex difference in methylation of single‑copy genes in human meiotic germ cells: Implications for X chromosome inactivation, parental imprinting, and origin of CpG mutations. Somat Cell Mol Genet 1990; 16:267‑82. 57. Namekawa SH, Park PJ, Zhang LF, Shima JE, McCarrey JR, Griswold MD, Lee JT. Postmeiotic sex chromatin in the male germline of mice. Curr Biol 2006; 16:660‑7. 58. Khalil AM, Driscoll DJ. Histone H3 lysine 4 dimethylation is enriched on the inactive sex chromosomes in male meiosis but absent on the inactive X in female somatic cells. Cytogenet Genome Res 2006; 112:11‑5. 59. O’Neill LP, Randall TE, Lavender J, Spotswood HT, Lee JT, Turner BM. X‑linked genes in female embryonic stem cells carry an epigenetic mark prior to the onset of X inactivation. Hum Mol Genet 2003; 12:1783‑90. 60. Lyon MF. Gene action in the X‑chromosome of the mouse (Mus musculus L.). Nature 1961; 372‑3. 61. Migeon BR, Shapiro LJ, Norum RA, Mohandas T, Axelman J, Dabora RL. Differential expression of steroid sulphatase locus on active and inactive human X chromosome. Nature 1982; 299:838‑40. 62. Brown CJ, Ballabio A, Rupert JL, Lafreniere RG, Grompe M, Tonlorenzi R, Willard HF. A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature 1991; 349:38‑44. 63. Disteche CM. Escape from X inactivation in human and mouse. Trends Genet 1995; 11:17‑22. 64. Lingenfelter PA, Adler DA, Poslinski D, Thomas S, Elliott RW, Chapman VM, Disteche CM. Escape from X inactivation of Smcx is preceded by silencing during mouse development. Nat Genet 1998; 18:212‑3. 65. Greenfield A, Carrel L, Pennisi D, Philippe C, Quaderi N, Siggers P, Steiner K, Tam PP, Monaco AP, Willard HF, Koopman P. The UTX gene escapes X inactivation in mice and humans. Hum Mol Genet 1998; 7:737‑42. 66. Disteche CM. Escapees on the X chromosome. Proc Natl Acad Sci USA 1999; 96:14180‑2. 67. Disteche CM, Filippova GN, Tsuchiya KD. Escape from X inactivation. Cytogenet Genome Res 2002; 99:36‑43. 68. Carrel L, Willard HF. X‑inactivation profile reveals extensive variability in X‑linked gene expression in females. Nature 2005; 434:400‑4. 69. Khalil AM, Driscoll DJ. Trimethylation of histone H3 lysine 4 is an epigenetic mark at regions escaping mammalian X inactivation. Epigenetics 2007; 2:114‑8. 70. Engstrom PG, Suzuki H, Ninomiya N, Akalin A, Sessa L, Lavorgna G, Brozzi A, Luzi L, Tan SL, Yang L, Kunarso G, Ng EL, Batalov S, Wahlestedt C, Kai C, Kawai J, Carninci P, Hayashizaki Y, Wells C, Bajic VB, Orlando V, Reid JF, Lenhard B, Lipovich L. Complex Loci in human and mouse genomes. PLoS Genet 2006; 2:47. 71. Wahlestedt C. Natural antisense and non-coding RNA transcripts as potential drug targets. Drug Discov Today 2006; 11:503‑8. 72. Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, Chang HY. Functional demarcation of active and silent chromatin domains in human HOX loci by non-coding RNAs. Cell 2007; 129:1311‑23. 73. Girard A, Sachidanandam R, Hannon GJ, Carmell MA. A germline‑specific class of small RNAs binds mammalian Piwi proteins. Nature 2006; 442:199‑202. 74. Grivna ST, Pyhtila B, Lin H. MIWI associates with translational machinery and PIWI‑interacting RNAs (piRNAs) in regulating spermatogenesis. Proc Natl Acad Sci USA 2006; 103:13415‑20. 75. Aravin AA, Sachidanandam R, Girard A, Fejes‑Toth K, Hannon GJ. Developmentally regulated piRNA clusters implicate MILI in transposon control. Science 2007; 316:744‑7. 76. Carmell MA, Girard A, van de Kant HJ, Bourc’his D, Bestor TH, de Rooij DG, Hannon GJ. MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev Cell 2007; 12:503‑14. 77. Carmell MA, Hannon GJ. RNase III enzymes and the initiation of gene silencing. Nat Struct Mol Biol 2004; 11:214‑8. 78. Deng W, Lin H. miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev Cell 2002; 2:819‑30. 79. Kuramochi‑Miyagawa S, Kimura T, Ijiri TW, Isobe T, Asada N, Fujita Y, Ikawa M, Iwai N, Okabe M, Deng W, Lin H, Matsuda Y, Nakano T. Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 2004; 131:839‑49. 80. Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004; 116:281‑97. 81. Yan N, Lu Y, Sun H, Tao D, Zhang S, Liu W, Ma Y. A microarray for microRNA profiling in mouse testis tissues. Reproduction 2007; 134:73‑9. 82. Zhang R, Peng Y, Wang W, Su B. Rapid evolution of an X‑linked microRNA cluster in primates. Genome Res 2007; 17:612‑7. 83. Costa Y, Speed RM, Gautier P, Semple CA, Maratou K, Turner JM, Cooke HJ. Mouse MAELSTROM: The link between meiotic silencing of unsynapsed chromatin and microRNA pathway? Hum Mol Genet 2006; 15:2324‑34. 84. Megosh HB, Cox DN, Campbell C, Lin H. The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr Biol 2006; 16:1884‑94.

©

20

08

LA

ND

ES

BIO

SC

IEN

CE

.D

ON

24. Handel MA. The XY body: A specialized meiotic chromatin domain. Exp Cell Res 2004; 296:57‑63. 25. Meistrich ML, Trostle‑Weige PK, Lin R, Bhatnagar YM, Allis CD. Highly acetylated H4 is associated with histone displacement in rat spermatids. Mol Reprod Dev 1992; 31:170‑81. 26. Khalil AM, Boyar FZ, Driscoll DJ. Dynamic histone modifications mark sex chromosome inactivation and reactivation during mammalian spermatogenesis. Proc Natl Acad Sci USA 2004; 101:16583‑7. 27. van der Heijden GW, Derijck AA, Posfai E, Giele M, Pelczar P, Ramos L, Wansink DG, van der Vlag J, Peters AH, de Boer P. Chromosome‑wide nucleosome replacement and H3.3 incorporation during mammalian meiotic sex chromosome inactivation. Nat Genet 2007; 39:251‑8. 28. Christensen ME, Dixon GH. Hyperacetylation of histone H4 correlates with the terminal, transcriptionally inactive stages of spermatogenesis in rainbow trout. Dev Biol 1982; 93:404‑15. 29. Oliva R, Mezquita C. Histone H4 hyperacetylation and rapid turnover of its acetyl groups in transcriptionally inactive rooster testis spermatids. Nucleic Acids Res 1982; 10:8049‑59. 30. Lahn BT, Tang ZL, Zhou J, Barndt RJ, Parvinen M, Allis CD, Page DC. Previously uncharacterized histone acetyltransferases implicated in mammalian spermatogenesis. Proc Natl Acad Sci USA 2002; 99:8707‑12. 31. Mikkelsen TS, Ku M, Jaffe DB, Issac B, Lieberman E, Giannoukos G, Alvarez P, Brockman W, Kim TK, Koche RP, Lee W, Mendenhall E, O’Donovan A, Presser A, Russ C, Xie X, Meissner A, Wernig M, Jaenisch R, Nusbaum C, Lander ES, Bernstein BE. Genome‑wide maps of chromatin state in pluripotent and lineage‑committed cells. Nature 2007; 448:553‑60. 32. Barski A, Cuddapah S, Cui K, Roh TY, Schones DE, Wang Z, Wei G, Chepelev I, Zhao K. High‑resolution profiling of histone methylations in the human genome. Cell 2007; 129:823‑37. 33. Hayashi K, Yoshida K, Matsui Y. A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature 2005; 438:374‑8. 34. Okada Y, Scott G, Ray MK, Mishina Y, Zhang Y. Histone demethylase JHDM2A is critical for Tnp1 and Prm1 transcription and spermatogenesis. Nature 2007; 450:119-23. 35. Tachibana M, Sugimoto K, Nozaki M, Ueda J, Ohta T, Ohki M, Fukuda M, Takeda N, Niida H, Kato H, Shinkai Y. G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. Genes Dev 2002; 16:1779‑91. 36. Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X‑linked genes expressed in spermatogonia. Nat Genet 2001; 27:422‑6. 37. McCarrey JR, Berg WM, Paragioudakis SJ, Zhang PL, Dilworth DD, Arnold BL, Rossi JJ. Differential transcription of Pgk genes during spermatogenesis in the mouse. Dev Biol 1992; 154:160‑8. 38. Salido EC, Yen PH, Mohandas TK, Shapiro LJ. Expression of the X‑inactivation‑associated gene XIST during spermatogenesis. Nat Genet 1992; 2:196‑9. 39. Wang PJ, Page DC, McCarrey JR. Differential expression of sex‑linked and autosomal germ‑cell‑specific genes during spermatogenesis in the mouse. Hum Mol Genet 2005; 14:2911‑8. 40. Nguyen DK, Disteche CM. Dosage compensation of the active X chromosome in mammals. Nat Genet 2006; 38:47‑53. 41. Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev Cell 2006; 10:521‑9. 42. Homolka D, Ivanek R, Capkova J, Jansa P, Forejt J. Chromosomal rearrangement interferes with meiotic X chromosome inactivation. Genome Res 2007; 17:1431‑7. 43. Turner JM, Mahadevaiah SK, Fernandez‑Capetillo O, Nussenzweig A, Xu X, Deng CX, Burgoyne PS. Silencing of unsynapsed meiotic chromosomes in the mouse. Nat Genet 2005; 37:41‑7. 44. Fernandez‑Capetillo O, Mahadevaiah SK, Celeste A, Romanienko PJ, Camerini‑Otero RD, Bonner WM, Manova K, Burgoyne P, Nussenzweig A. H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis. Dev Cell 2003; 4:497‑508. 45. Turner JM, Aprelikova O, Xu X, Wang R, Kim S, Chandramouli GV, Barrett JC, Burgoyne PS, Deng CX. BRCA1, histone H2AX phosphorylation, and male meiotic sex chromosome inactivation. Curr Biol 2004; 14:2135‑42. 46. Baarends WM, Wassenaar E, van der Laan R, Hoogerbrugge J, Sleddens‑Linkels E, Hoeijmakers JH, de Boer P, Grootegoed JA. Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol Cell Biol 2005; 25:1041‑53. 47. Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E. Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 2004; 303:644‑9. 48. Huynh KD, Lee JT. Inheritance of a preinactivated paternal X chromosome in early mouse embryos. Nature 2003; 426:857‑62. 49. Avner P, Heard E. X‑chromosome inactivation: Counting, choice and initiation. Nat Rev Genet 2001; 2:59‑67. 50. Migeon BR. X chromosome inactivation: Theme and variations. Cytogenet Genome Res 2002; 99:8‑16. 51. McCarrey JR, Dilworth DD. Expression of Xist in mouse germ cells correlates with X‑chromosome inactivation. Nat Genet 1992; 2:200‑3. 52. McCarrey JR, Watson C, Atencio J, Ostermeier GC, Marahrens Y, Jaenisch R, Krawetz SA. X‑chromosome inactivation during spermatogenesis is regulated by an Xist/Tsix‑independent mechanism in the mouse. Genesis 2002; 34:257‑66.


Epigenetics

27