The Antiviral Drug Efavirenz in Breast Cancer Stem Cell Therapy.

Although many breast cancer therapies show initial success in the treatment of the primary tumour, they often fail to eliminate a sub-population of cells known as cancer stem cells (CSCs). These cells are recognised for their self-renewal properties and for their capacity for differentiation often leading to chemo/radio-resistance. The antiviral drug Efavirenz has been shown to be effective in eliminating triple-negative breast cancer cells, and here we examine its effect on breast CSCs. The effects of Efavirenz on CSCs for several breast cancer cell lines were investigated by examining cellular changes upon drug treatment, including CSC numbers, morphology, RNA/microRNA expression and levels of epithelial/mesenchymal CSC subtypes. Efavirenz treatment resulted in a decrease in the size and number of tumorspheres and a reduction in epithelial-type CSC levels, but an increase in mesenchymal-type CSCs. Efavirenz caused upregulation of several CSC-related genes as well as miR-21, a CSC marker and miR-182, a CSC suppressor gene. We conclude that Efavirenz alters the phenotype and expression of key genes in breast CSCs, which has important potential therapeutic implications.

Cohesin Rad21 mediates loss of heterozygosity and is upregulated via Wnt promoting transcriptional dysregulation in gastrointestinal tumors.

Loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene triggers a series of molecular events leading to intestinal adenomagenesis. Haploinsufficiency of the cohesin Rad21 influences multiple initiating events in colorectal cancer (CRC). We identify Rad21 as a gatekeeper of LOH and a ß-catenin target gene and provide evidence that Wnt pathway activation drives RAD21 expression in human CRC. Genome-wide analyses identified Rad21 as a key transcriptional regulator of critical CRC genes and long interspersed element (LINE-1 or L1) retrotransposons. Elevated RAD21 expression tracks with reactivation of L1 expression in human sporadic CRC, implicating cohesin-mediated L1 expression in global genomic instability and gene dysregulation in cancer.

LINE-1 retrotransposons and let-7 miRNA: partners in the pathogenesis of cancer?

Long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons are insertional mutagens capable of altering the genomic landscape in many ways. Activation of the normally silent LINE-1 retrotransposon is associated with a high level of cancer-associated DNA damage and genomic instability. Studies of LINE-1 have so far focused mainly on changes in gene expression, and our knowledge of its impact on functional non-coding RNAs is in its infancy. However, current evidence suggests that a significant number of human miRNAs originate from retrotransposon sequences. Furthermore, LINE-1 is generally not expressed in normal tissues while its expression is widespread in epithelial cancers. Based on our recent studies, we demonstrate a functional link between aberrant LINE-1 expression and deregulation of let-7 miRNA expression. Since the expression of let-7 is modulated by LINE-1 activity, we discuss possible mechanisms for this effect and how the silencing of LINE-1 activation could provide new therapeutic options for cancer treatment. Based on the deep sequencing of small RNAs in parallel with gene expression profiling in breast cancer cells, we have identified potential pathways linking L1 activity to let-7 processing and maturation and ultimately to the control of stemness in human cancer cells.

Silencing of LINE-1 retrotransposons contributes to variation in small noncoding RNA expression in human cancer cells.

Noncoding RNAs are key players in the maintenance of genomic integrity, particularly in silencing the expression of repetitive elements, some of which are retrotransposable and capable of causing genomic instability. Recent computational studies suggest an association between L1 expression and the generation of small RNAs. However, whether L1 expression has a role in the activation of small RNA expression has yet to be determined experimentally. Here we report a global analysis of small RNAs in deep sequencing from L1-active and L1-silenced breast cancer cells. We found that cells in which L1 expression was silenced exhibited greatly increased expression of a number of miRNAs and in particular, members of the let-7 family. In addition, we found differential expression of a few piRNAs that might potentially regulate gene expression. We also report the identification of several repeat RNAs against LTRs, LINEs and SINE elements. Although most of the repeat RNAs mapped to L1 elements, in general we found no significant differences in the expression levels of repeat RNAs in the presence or absence of L1 expression except for a few RNAs targeting subclasses of L1 elements. These differentially expressed small RNAs may function in human genome defence responses.

Differential DNA methylation patterns in endo-siRNAs mediated silencing of LINE-1 retrotransposons.

Analyzing differences in DNA methylation is a powerful tool for assessing the effect of endo-siRNAs expression in the human genome. Here, we present a simple genome-wide DNA methylation assay that allows for a precise quantitative analysis of differences in the promoter of human long interspersed nuclear element 1 (LINE-1 or L1) retrotransposons in response to endogenous and exogenous expression of endo-siRNAs. Using the DNA bisulfite modification sequencing, we have optimized the method to detect small changes in heterogeneously methylated L1 repeats at multiple regions across the genome. We also provide guidance for analysis of primary bisulfite sequencing data and interpretation of the methylation status using the Web-based bisulfite sequencing DNA methylation (BISMA) analysis. This refined and reproducible assay can be performed even using a small amount of genomic DNA and is suitable for the analysis of clinical tissue samples.

Exploiting the power of LINE-1 retrotransposon mutagenesis for identification of genes involved in embryonic stem cell differentiation.

Identifying the genes or epigenetic factors that control the self-renewal and differentiation of stem cells is critical to understanding the molecular basis of cell commitment. Although a number of insertional mutagenesis vectors have been developed for identifying gene functions in animal models, the L1 retrotransposition system offers additional advantages as a tool to disrupt genes in embryonic stem cells in order to identify their functions and the phenotypes associated with them. Recent advances in producing synthetic versions of L1 retrotransposon vector system and the optimization of techniques to accurately identify retrotransposon integration sites have increased their utility for gene discovery applications. We have developed a novel episomal, nonviral L1 retrotransposon vector using scaffold/matrix attachment regions that provides stable, sustained levels of retrotransposition in cell cultures without being affected by epigenetic silencing or from some of the common problems of vector integration. This modified vector contains a GFP marker whose expression occurs only after successful gene disruption events and thus the cells with disrupted genes can be easily picked for functional analysis. Here we present a method to disrupt gene function in embryonic stem cells that aid in the identification of genes involved in stem cell differentiation processes. The methods presented here can be easily adapted to the study of other types of cancer stem cells or induced pluripotent stem cells using the L1 retrotransposon as an insertional mutagen.

Inhibition of LINE-1 retrotransposon-encoded reverse transcriptase modulates the expression of cell differentiation genes in breast cancer cells.

Long Interspersed Elements (L1 elements) are biologically active retrotransposons that are capable of autonomous replication using their own reverse transcriptase (RT) enzyme. Expression of the normally repressed RT has been implicated in cancer cell growth. However, at present, little is known about the expression of L1-encoded RT activity or the molecular changes that are associated with RT activity in the development of breast cancer. Here, we report that RT activity is widespread in breast cancer cells. The expression of RT protein decreased markedly in breast cancer cells after treatment with the antiretroviral drug, efavirenz. While the majority of cells showed a significant reduction in proliferation, inhibition of RT was also accompanied by cell-specific differences in morphology. MCF7 cells displayed elongated microtubule extensions that adhered tightly to their substrate, while a large fraction of the T47D cells that we studied formed long filopodia projections. These morphological changes were reversible upon cessation of RT inhibition, confirming their dependence on RT activity. We also carried out gene expression profiling with microarrays and determined the genes that were differentially expressed during the process of cellular differentiation. Genes involved in proliferation, cell migration, and invasive activity were repressed in RT-inhibited cells. Concomitantly, genes involved in cell projection, formation of vacuolar membranes, and cell-to-cell junctions were significantly upregulated in RT-inhibited cells. qRT-PCR examination of the mRNA expression of these genes in additional cell lines yielded close correlation between their differential expression and the degree of cellular differentiation. Our study demonstrates that the inhibition of L1-encoded RT can reduce the rate of proliferation and promote differentiation of breast cancer cells. Together, these results provide a direct functional link between the expression of L1 retrotransposons and the development of breast cancer.

Distinctive patterns of epigenetic marks are associated with promoter regions of mouse LINE-1 and LTR retrotransposons.

BACKGROUND: The long terminal repeat (LTR) retrotransposons and the non-LTR retrotransposons (LINE-1 or L1) make up more than one-third of the mouse genome. Because of their abundance, the retrotransposons are the major players in genomic structure and function. While much attention has been focused on the biology of retrotransposons, little is known about the chromatin structure of these elements or the potential role of epigenetic marks on the regulation of retrotransposon expression. FINDINGS: Using sequential chromatin immunoprecipitation analysis, we analyzed the cohabitation of several post-translational histone modifications in the promoter regions of mouse L1 and LTR retrotransposons. We show here that the variant histone H2A.Z selectively present in L1 promoters. Notably, H2A.Z and trimethylated histone H3 (H3K9me3) co-localize in the same genomic location of the L1 promoter along with heterochromatin-binding protein HP1α. In contrast, MmERV and intracisternal A-particle (IAP) classes of LTR promoters are enriched with core histone H2A and heterochromatic trimethylated histone H4 (H4K20me3). These distinctive patterns of chromatin modifications are relatively consistent irrespective of cell type. CONCLUSIONS: Chromatin structure regulates the expression of retrotransposons. LINE-1 elements are associated with H2A.Z and HP1α-containing constitutive heterochromatin, while the LTR elements are enriched with H2A and the H4K20me3-type of facultative heterochromatin. Our findings demonstrate that different epigenetic mechanisms operate in the mouse genome to silence different classes of retrotransposons.

Analysis of epigenetic factors in mouse embryonic neural stem cells exposed to hyperglycemia.

BACKGROUND: Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development. METHODS AND FINDINGS: NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers. CONCLUSION/INTERPRETATION: This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.

Prognostic value of LINE-1 retrotransposon expression and its subcellular localization in breast cancer.

Long interspersed nuclear element 1 (L1) belongs to a family of retrotransposons. Expression of the normally repressed L1 retrotransposons has been shown to induce genome instability by creating DNA double-stranded breaks and chromosomal rearrangements through the process of retrotransposition. At present, little is known about the expression of L1-encoded ORF1p and ORF2p which are indispensable for its retrotransposition activity. Given its potentially harmful effects on the genome, we investigated the implications of both ORF1p and ORF2p expression and their subcellular localization in a range of breast cancer cell lines and breast tumor tissues including 15 normal breast tissues, 25 fibroadenomas, 25 ductal carcinomas in situ (DCIS), and 95 invasive cancers. Clinicopathologic parameters and survival outcomes were investigated in association with the cytoplasmic and nuclear expression of ORF1p and ORF2p using univariate and multivariate analysis. High cytoplasmic expression of ORF1p and ORF2p was seen in DCIS tumors, but they were not related with survival outcome. The majority of invasive cancers were found to express both ORF1p and ORF2p in the cytoplasm, while nuclear expression was also seen in a subclass of those invasive cancers in the range of 28-31 %. Tumors with high nuclear expression of ORF1p and ORF2p were more significantly associated with lymph node metastasis (p = 0.001) and the worst patient survival (p < 0.0001) than those with cytoplasmic expression. This is the first study examining the effects of both ORF1p and ORF2p expression in breast cancer tissues. Our observation shows altered expression patterns of ORF1p and ORF2p within invasive cancers, which are related to differences in overall patient survival. The differing patterns of both cytoplasmic and nuclear ORF1p and ORF2p expression indicate that further studies of the biology and function of L1 retrotransposons are required in breast cancer.

Naturally occurring endo-siRNA silences LINE-1 retrotransposons in human cells through DNA methylation.

Long interspersed nuclear element 1 (LINE-1) retrotransposons are mutagens that are capable of generating deleterious mutations by inserting themselves into genes and affecting gene function in the human genome. In normal cells, the activity of LINE-1 retrotransposon is mostly repressed, maintaining a stable genome structure. In contrast, cancer cells are characterized by aberrant expression of LINE-1 retrotransposons, which, in principle, have the potential to contribute to genomic instability. The mechanistic pathways that regulate LINE-1 expression remain unclear. Using deep-sequencing small RNA analysis, we identified a subset of differentially expressed endo-siRNAs that directly regulate LINE-1 expression. Detailed analyses suggest that these endo-siRNAs are significantly depleted in human breast cancer cells compared with normal breast cells. The overexpression of these endo-siRNAs in cancer cells markedly silences endogenous LINE-1 expression through increased DNA methylation of the LINE-1 5'-UTR promoter. The finding that endo-siRNAs can silence LINE-1 activity through DNA methylation suggests that a functional link exists between the expression of endo-siRNAs and LINE-1 retrotransposons in human cells.

An S/MAR-based L1 retrotransposition cassette mediates sustained levels of insertional mutagenesis without suffering from epigenetic silencing of DNA methylation.

L1 is an insertional mutagen that is capable of mediating permanent gene disruption in mammalian genomes. However, currently available L1 retrotransposition vectors exhibit low or unstable transgene expression when expressed in somatic cells and tissues. This restriction limits their potential utility in long-term screening procedures or somatic mutagenesis applications. In this study, we addressed this problem by developing a minicircle, nonviral L1 retrotransposition vector using a scaffold/matrix attachment region (S/MAR) in the vector backbone and evaluated its utility in human cell lines. The S/MAR-based L1 retrotransposition vector provides stable, elevated levels of L1 expression compared to the currently used EBNA1-based L1 vector. In addition, the S/MAR elements effectively mediate sustained levels of L1 retrotransposition in prolonged cell culturing without suffering from epigenetic silencing by DNA methylation or from vector integration problems even in the absence of selection pressure. These findings indicate that the simple inclusion of S/MAR in the vector backbone increased levels of L1 expression and retrotransposition that can be used as an effective tool to generate insertional mutagenesis in large-scale somatic mutagenesis applications in mammalian cells.

The impact of CpG island on defining transcriptional activation of the mouse L1 retrotransposable elements.

BACKGROUND: L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500-3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation, suggesting that only a half of L1 elements are capable of being transcriptionally active. Furthermore, we present experimental evidence that previously unreported CpG islands exist in the promoters of the most active T(F) family of mouse L1 elements. The presence of sequence variations and polymorphisms in CpG islands of L1 promoters that arise from transition mutations indicates that CpG methylation could play a significant role in determining the activity of L1 elements in the mouse genome. CONCLUSIONS: A comprehensive analysis of mouse L1 promoters suggests that the number of transcriptionally active elements is significantly lower than the total number of full-length copies from the three active mouse L1 families. Like human L1 elements, the CpG islands and potentially the transcription factor YY1 binding sites are likely to be required for transcriptional initiation of mouse L1 elements.

Control of chicken CR1 retrotransposons is independent of Dicer-mediated RNA interference pathway.

BACKGROUND: Dicer is an RNase III-ribonuclease that initiates the formation of small interfering RNAs as a defence against genomic parasites such as retrotransposons. Despite intensive characterization in mammalian species, the biological functions of Dicer in controlling retrotransposable elements of the non-mammalian vertebrate are poorly understood. In this report, we examine the role of chicken Dicer in controlling the activity of chicken CR1 retrotransposable elements in a chicken-human hybrid DT40 cell line employing a conditional loss-of-Dicer function. RESULTS: Retrotransposition is detrimental to host genome stability and thus eukaryotic cells have developed mechanisms to limit the expansion of retrotransposons by Dicer-mediated RNAi silencing pathways. However, the mechanisms that control the activity and copy numbers of transposable elements in chicken remain unclear. Here, we describe how the loss of Dicer in chicken cells does not reactivate endogenous chicken CR1 retrotransposons with impaired RNAi machinery, suggesting that the control of chicken CR1 is independent of Dicer-induced RNAi silencing. In contrast, upon introduction of a functionally active human L1 retrotransposable element that contains an active 5' UTR promoter, the Dicer-deficient chicken cells show a strong increase in the accumulation of human L1 transcripts and retrotransposition activity, highlighting a major difference between chicken CR1 and other mammalian L1 retrotransposons. CONCLUSION: Our data provide evidence that chicken CR1 retrotransposons, unlike their mammalian L1 counterparts, do not undergo retrotransposition because most CR1 retrotransposons are truncated or mutated at their 5'UTR promoters and thus are not subjected to Dicer-mediated RNAi-silencing control.

Dynamic histone variant exchange accompanies gene induction in T cells.

Changes in chromatin composition are often a prerequisite for gene induction. Nonallelic histone variants have recently emerged as key players in transcriptional control and chromatin modulation. While the changes in chromatin accessibility and histone posttranslational modification (PTM) distribution that accompany gene induction are well documented, the dynamics of histone variant exchange that parallel these events are still poorly defined. In this study, we have examined the changes in histone variant distribution that accompany activation of the inducible CD69 and heparanase genes in T cells. We demonstrate that the chromatin accessibility increases that accompany the induction of both of these genes are not associated with nucleosome loss but instead are paralleled by changes in histone variant distribution. Specifically, induction of these genes was paralleled by depletion of the H2A.Z histone variant and concomitant deposition of H3.3. Furthermore, H3.3 deposition was accompanied by changes in PTM patterns consistent with H3.3 enriching or depleting different PTMs upon incorporation into chromatin. Nevertheless, we present evidence that these H3.3-borne PTMs can be negated by recruited enzymatic activities. From these observations, we propose that H3.3 deposition may both facilitate chromatin accessibility increases by destabilizing nucleosomes and compete with recruited histone modifiers to alter PTM patterns upon gene induction.

Gene knockdown by ecdysone-based inducible RNAi in stable mammalian cell lines.

RNA interference (RNAi) is a powerful tool for the functional analysis of essential genes in the mammalian genome. Here, we present a simple ecdysone-based inducible RNAi approach that allows high induction and adjustable control of short hairpin RNA (shRNA) expression for silencing gene expression in a wide range of mammalian cells. This protocol describes the following: the design and cloning of inducible shRNA; testing and validation of gene knockdown; and methodology for establishing stable cell lines. This step-by-step protocol offers a quick and cost-effective approach for addressing the function of genes essential for cell cycle regulation and development and can be completed in less than 6 weeks.

The nucleosome surface regulates chromatin compaction and couples it with transcriptional repression.

Although it is believed that the interconversion between permissive and refractory chromatin structures is important in regulating gene transcription, this process is poorly understood. Central to addressing this issue is to elucidate how a nucleosomal array folds into higher-order chromatin structures. Such findings can then provide new insights into how the folding process is regulated to yield different functional states. Using well-defined in vitro chromatin-assembly and transcription systems, we show that a small acidic region on the surface of the nucleosome is crucial both for the folding of a nucleosomal template into the 30-nm chromatin fiber and for the efficient repression of transcription, thereby providing a mechanistic link between these two essential processes. This structure-function relationship has been exploited by complex eukaryotic cells through the replacement of H2A with the specific variant H2A.Bbd, which naturally lacks an acidic patch.

H2A.Z contributes to the unique 3D structure of the centromere.

Mammalian centromere function depends upon a specialized chromatin organization where distinct domains of CENP-A and dimethyl K4 histone H3, forming centric chromatin, are uniquely positioned on or near the surface of the chromosome. These distinct domains are embedded in pericentric heterochromatin (characterized by H3 methylated at K9). The mechanisms that underpin this complex spatial organization are unknown. Here, we identify the essential histone variant H2A.Z as a new structural component of the centromere. Along linear chromatin fibers H2A.Z is distributed nonuniformly throughout heterochromatin, and centric chromatin where regions of nucleosomes containing H2A.Z and dimethylated K4 H3 are interspersed between subdomains of CENP-A. At metaphase, using the inactive X chromosome centromere as a model, complex folding of this fiber produces spatially positioned domains where H2A.Z/dimethylated K4 H3 chromatin juxtaposes one side of CENP-A chromatin, whereas a region of H2A/trimethyl K9 H3 borders the other side. A second region of H2A.Z is found, with trimethyl K9 H3 at the inner centromere. We therefore propose that H2A.Z plays an integral role in organizing centromere structure.

Inhibition of arginase I activity by RNA interference attenuates IL-13-induced airways hyperresponsiveness.

Increased arginase I activity is associated with allergic disorders such as asthma. How arginase I contributes to and is regulated by allergic inflammatory processes remains unknown. CD4+ Th2 lymphocytes (Th2 cells) and IL-13 are two crucial immune regulators that use STAT6-dependent pathways to induce allergic airways inflammation and enhanced airways responsiveness to spasmogens (airways hyperresponsiveness (AHR)). This pathway is also used to activate arginase I in isolated cells and in hepatic infection with helminths. In the present study, we show that arginase I expression is also regulated in the lung in a STAT6-dependent manner by Th2-induced allergic inflammation or by IL-13 alone. IL-13-induced expression of arginase I correlated directly with increased synthesis of urea and with reduced synthesis of NO. Expression of arginase I, but not eosinophilia or mucus hypersecretion, temporally correlated with the development, persistence, and resolution of IL-13-induced AHR. Pharmacological supplementation with l-arginine or with NO donors amplified or attenuated IL-13-induced AHR, respectively. Moreover, inducing loss of function of arginase I specifically in the lung by using RNA interference abrogated the development of IL-13-induced AHR. These data suggest an important role for metabolism of l-arginine by arginase I in the modulation of IL-13-induced AHR and identify a potential pathway distal to cytokine receptor interactions for the control of IL-13-mediated bronchoconstriction in asthma.

The X and Y chromosomes assemble into H2A.Z-containing [corrected] facultative heterochromatin [corrected] following meiosis.

Spermatogenesis is a complex sequential process that converts mitotically dividing spermatogonia stem cells into differentiated haploid spermatozoa. Not surprisingly, this process involves dramatic nuclear and chromatin restructuring events, but the nature of these changes are poorly understood. Here, we linked the appearance and nuclear localization of the essential histone variant H2A.Z with key steps during mouse spermatogenesis. H2A.Z cannot be detected during the early stages of spermatogenesis, when the bulk of X-linked genes are transcribed, but its expression begins to increase at pachytene, when meiotic sex chromosome inactivation (MSCI) occurs, peaking at the round spermatid stage. Strikingly, when H2A.Z is present, there is a dynamic nuclear relocalization of heterochromatic marks (HP1beta and H3 di- and tri-methyl K9), which become concentrated at chromocenters and the inactive XY body, implying that H2A.Z may substitute for the function of these marks in euchromatin. We also show that the X and the Y chromosome are assembled into facultative heterochromatic structures postmeiotically that are enriched with H2A.Z, thereby replacing macroH2A. This indicates that XY silencing continues following MSCI. These results provide new insights into the large-scale changes in the composition and organization of chromatin associated with spermatogenesis and argue that H2A.Z has a unique role in maintaining sex chromosomes in a repressed state.