The SWI/SNF protein ATRX co-regulates pseudoautosomal genes that have translocated to autosomes in the mouse genome
© Levy et al; licensee BioMed Central Ltd. 2008
Received: 02 April 2008
Accepted: 08 October 2008
Published: 08 October 2008
Pseudoautosomal regions (PAR1 and PAR2) in eutherians retain homologous regions between the X and Y chromosomes that play a critical role in the obligatory X-Y crossover during male meiosis. Genes that reside in the PAR1 are exceptional in that they are rich in repetitive sequences and undergo a very high rate of recombination. Remarkably, murine PAR1 homologs have translocated to various autosomes, reflecting the complex recombination history during the evolution of the mammalian X chromosome.
We now report that the SNF2-type chromatin remodeling protein ATRX controls the expression of eutherian ancestral PAR1 genes that have translocated to autosomes in the mouse. In addition, we have identified two potentially novel mouse PAR1 orthologs.
We propose that the ancestral PAR1 genes share a common epigenetic environment that allows ATRX to control their expression.
The sex chromosomes in modern placental mammals (eutherians) are highly dimorphic but initially evolved from a homologous pair of autosomes . Over millions of years of mammalian evolution, the sex chromosomes have lost most of their homology due to chromosome Y attrition . The remaining homology between the sex chromosomes exists in the pseudoautosomal regions (PARs), located at the ends of the X and Y chromosomes  and was generated when genetic material from the tips of autosomes translocated to the ancient sex chromosomes . Gene dosage between XX females and XY males is usually achieved by the silencing of one X chromosome in every female cell, a process known as X chromosome inactivation (XCI) . Because both males and females have two copies of all PAR genes there is no requirement for dosage compensation and these genes therefore escape this inactivation process .
The α thalassemia mental retardation, X linked (ATRX) protein, transcribed from Xq13.3 belongs to the Sucrose non-fermenting 2 (Snf2) family of enzymes that use the energy of adenosine tri-phosphate (ATP) hydrolysis to disrupt nucleosome stability [13, 14]. Mutations in ATRX result in moderate to profound cognitive deficits, facial dysmorphisms, as well as skeletal and urogenital abnormalities, among other symptoms . The chromatin remodeling properties of ATRX have been demonstrated in vitro . In addition to a conserved ATPase/helicase domain, ATRX has an N-terminal zinc finger ATRX-DNMT3A/B-DNMT3L (ADD) domain that is shared with de novo methyltransferases. Several lines of evidence have also linked ATRX to highly repetitive genomic regions including pericentromeric heterochromatin in mouse and human cells . Moreover, ATRX mutations in humans result in aberrant DNA methylation patterns at several repetitive elements, including ribosomal DNA (rDNA) repeats, subtelomeric repeats and Y-specific satellite repeats . These repetitive sequences usually form heterochromatic structures and seem to be specifically targeted by the ATRX protein.
To assess the role of ATRX in brain development, we previously used Cre-lox P recombination to remove Atrx specifically in the forebrain beginning at E8.5. Loss of ATRX in the embryonic forebrain caused hypocellularity and a reduction in forebrain size and loss of the dentate gyrus .
Genes that are directly regulated by the ATRX protein have not yet been identified in either humans or mice. To identify potential genes that are controlled by ATRX, we performed a screen of gene expression and found that a subset of ancestral PAR1 genes is consistently downregulated in the absence of ATRX in the developing mouse brain. Among them are two potentially novel mouse orthologs of Arse and Asmtl. The only common link between ancestral PAR genes is their adjacent location and shared chromatin environment in the ancestral PAR region. We propose that conserved sequences and/or chromatin features targeted by ATRX were maintained upon translocation of these genes from the PAR1 on the ancestral X chromosome to their current location on mouse autosomes, and allow ATRX to modulate their expression.
Effects of ATRX deletion on forebrain gene expression
The ability of ATRX to remodel chromatin  suggests that ATRX can regulate gene expression. To identify possible gene targets of the ATRX protein in the developing mouse brain, we used the previously described Atrx Foxg1Cre mice that lack ATRX in the forebrain . In this model system, Atrx deletion is achieved by crossing Atrx loxP "floxed" mice to mice that express cyclization recombinase (Cre) under the control of the forebrain-specific forkhead box G1 (Foxg1) promoter . We performed microarray analysis to compare the expression profiles of the Atrx Foxg1Cre and control telencephalon at embryonic day 13.5 (E13.5) (n = 3 pairs) using an Affymetrix mouse genome expression array representing approximately 39,000 transcripts . Only probe sets showing a significant difference (p < 0.05) were included in all subsequent studies. By setting a threshold of 1.5 fold change we identified 202 disregulated probesets, and at a threshold of 2 fold change we identified only 22 altered probe sets. Approximately two-thirds of the probe sets demonstrating altered expression were upregulated (Additional file 1A, B).
We next compared gene expression patterns in control and Atrx-null forebrain tissue at postnatal day 0.5 (P0.5) (n = 4 pairs). At a threshold of 1.5 fold change, we identified 304 probe sets and at a threshold of 2 fold change, we identified 57 probe sets showing altered transcript levels. When we compared the microarray results at E13.5 and P0.5 we identified 14 common probe sets that were upregulated and 13 that were downregulated more than 1.5 fold, and one increased and three decreased more than 2 fold (Additional file 1A).
We used GeneSpring to identify significantly overrepresented Gene Ontology (GO) categories in the Atrx-null mouse forebrain. Several statistically and biologically significant categories of upregulated genes were related to the immune response. This could be an indirect response to the increased apoptosis that characterizes the Atrx-null forebrain at E13.5 in the developing cortex and to a lesser extent at P0.5 in the hippocampus . In particular, categories and genes involved in phagocytotic clearing of apoptotic cells, such as complement activation , were enriched at both E13.5 and P0.5. Several genes involved in cell adhesion processes were upregulated at P0.5 and, consistent with the abnormal forebrain development described in the Atrx-null forebrain, genes involved in neurogenesis and nervous system development were downregulated at both timepoints (Additional file 2).
Ancestral pseudoautosomal genes are downregulated in the Atrx-null mouse forebrain
Downregulated genes in the ATRX-null forebrain at E13.5 and P0.5.
E13.5 Downregulated Genes
Similar to dehydrogenase/reductase (SDR family) X chromosome ( Dhrsxy ) 1
Colony stimulating factor 2 receptor, alpha, low-affinity (granulocyte-macrophage)
Short stature homeobox 2
Transcription factor 7-like 2, T-cell specific, HMG-box
Gastrulation brain homeobox 2
Similar to Arylsulfatase E ( Arse ) 1
Neurogenic differentiation 4
Paternally expressed 10
Similar to Asmtl (acetylserotonin O-methyltransferase-like) 1
Wnt inhibitory factor 1
P0.5 Downregulated Genes
Colony stimulating factor 2 receptor, alpha, low-affinity (granulocyte-macrophage)
Nuclear receptor subfamily 4, group A, member 2
Similar to Dhrsxy (dehydrogenase/reductase (SDR family) X chromosome 1
Myelin basic protein
Cerebellin 4 precursor protein
Tansient receptor potential cation channel, subfamily C, member 4
Similar to Asmtl (acetylserotonin O-methyltransferase-like) 1
Verification of gene expression changes
Our discovery that the expression of several ancestral PAR1 genes is controlled by ATRX throughout the early developmental period of the mouse brain reveals an unexpected association between the levels of ATRX protein and the expression of these ancestral PAR1 genes.
Identification of a novel ARS family mouse homolog
In humans, a cluster of ARS genes are located approximately 115 kilobases centromeric to the PAR1 region on the X chromosome, but still possess the ability to escape XCI in females . Located outside the PAR1, these genes do not have an identical homolog on the Y chromosome but have pseudogenes, and in the evolutionary past it is believed that they were true pseudoautosomal genes with identical copies on both the X and Y chromosome .
A multiple alignment of amino acid sequences of [GenBank:BE45772] suggested that it is a fragment of the full length ARSE protein, aligning in the middle of the approximately 600 amino acid ARSE protein of multiple other species (Additional file 3). The putative mouse ARSE is 65% identical to rat and 47% identical to human.
Comparisons to available mouse Ars gene family members shows that [GenBank:BE457721] is more similar to Arse genes in rat than to other mouse arylsulfatase family members (Additional file 4), suggesting that we have identified Arse. This data, combined with our ability to specifically amplify this transcript from mouse brain cDNA and also from a commercially available E15 cDNA library (data not shown), indicates that we have likely identified the mouse homologue of a previously unidentified mouse Ars gene rather then a gene fragment from a known mouse family member.
Identification of an ASMTL-like gene
[GenBank:AK007409] is the RIKEN cDNA 1810009N02 gene and contains a musculoaponeurotic fibrosarcoma (MAF) domain. A multiple sequence alignment of amino acid sequences was used to further determine the identity of [GenBank:AK007409] (Additional file 5). [GenBank:AK007409] aligns to the N terminus of ASMTL from multiple other species. The N terminal portion of ASMTL also contains a MAF domain. Human ASMTL was generated by a fusion of a duplicated acetylserotonin O-methyltransferase (ASMT) with the bacterial maf gene . While [GenBank:AK007409] contains a MAF domain, it lacks the ASMT domain. However, this is similar to the putative rat ASMTL (Accession [GenBank:NP_001099385]) which also lacks the ASMT domain. The putative mouse ASMTL is 54% identical to rat, and 51% identical to the human protein.
Mutations in the ATRX gene result in profound cognitive deficits, facial dysmorphisms, as well as skeletal and urogenital abnormalities . Global deletion of Atrx in mouse embryonic stem cells results in a growth disadvantage , and conditional loss of Atrx beginning at the 8–16 cell stage leads to embryonic lethality by E9.5 . To bypass early embryonic lethality, we have previously used a conditional approach to delete Atrx in the mouse forebrain beginning at E8.0. These mice have significantly increased cortical progenitor cell apoptosis, causing a reduction in forebrain size and hypocellularity in the neocortex and hippocampus . ATRX is a chromatin remodeling protein  and has been proposed to regulate gene expression by modulating chromatin structure, but gene targets of ATRX have not yet been reported. We used a microarray approach to perform large-scale analysis of gene expression changes in the ATRX-null versus wild type mouse forebrain at E13.5 and P0.5. The fact that relatively few genes display altered expression indicates that ATRX is not a global regulator of gene expression but likely controls specific gene loci. It is not clear at this point if ATRX acts by binding directly to DNA or through other unidentified factors to upregulate the ancestral PAR genes identified in our study. The only target of ATRX identified to date is α globin which is downregulated in patients with germline or somatic ATRX mutations , including α-thalassemia myelodysplastic syndrome , although evidence that ATRX directly binds to the α globin locus is still lacking.
Through global transcriptional profiling we have now identified a distinct group of genes, the ancestral PAR genes, that are controlled by ATRX in the mouse brain. The human PAR1 contains 24 genes, but only 10 of these have been reported in the mouse genome. Arsd/e, Asmtl, Cd99, Csf2ra, Dhrsxy and Shox2 were among the most downregulated genes identified in the ATRX-null embryonic forebrain. Although these genes are unrelated in function, they share a common ancestral location in the PAR1 of the X chromosome millions of years ago. Our findings demonstrate that they have maintained a mechanism of co-regulation that was conserved in evolution and that requires ATRX, even after their dispersal to autosomes in the mouse genome.
The PAR1 region exhibits recombination rates approximately 10 times higher than the rest of the human genome . Consequently, genes in this region undergo rapid evolution leading to high interspecies divergence [9, 31] making positive identification of homologs difficult. Using multiple sequence alignments and phylogenetic analysis we have identified Arsd/e and Asmtl as putative novel mouse ancestral PAR transcripts. Identity between mouse and human sequences are 47%, 40% and 51% for ARSE [GenBank:NM_000047], ARSD [GenBank:NM_001669] and ASMTL [GenBank:NM_004192], respectively, which is similar to what was reported for other PAR1 genes. For example, DHRSXY exhibits 59% protein identity between humans and mice , CD99 46% identity , and 35% for CSF2RA .
ARSD and ARSE are members of the arylsulfatase gene family and are located just outside the human PAR1 in a cluster of four arylsulfatase genes . ARSE gene mutations cause X-linked chondrodysplasia punctata, a disorder characterized by abnormalities in cartilage and bone development . ARSE may therefore play a role in the skeletal defects seen in patients with the ATR-X syndrome if it is also regulated by ATRX in humans. The role of ARSD is unknown and it has no demonstrated sulfatase activity despite its high conservation of the N-terminal domain important for catalytic sulfatase activity . ARSE exhibits a restricted pattern of expression  while ARSD is ubiquitously expressed .
The function of human ASMTL is unknown. The gene was generated by the duplication of the PAR1 gene Asmt which then fused with the bacterial orfE/maf gene . While other ASMT genes involved in the serotonin/N-acetylserotonin/melatonin pathway are expressed specifically in the human brain, pineal gland and retina , ASMTL has a wider expression pattern and may not be involved in this pathway but could still have methyltransferase activity since it retains the necessary domain .
We have also identified the mouse Shox2 gene as a potential target of ATRX, and we observed that Shox2 expression levels are highly sensitive to ATRX deficiency in the developing mouse brain. Two SHOX genes, SHOX and SHOX2 have been identified in the human genome, on chromosomes X and 3, respectively. Only one mouse homolog has been identified and is mapped to chromosome 3. Like ARSE, SHOX genes are involved in skeletal development: mutations and deletions in SHOX lead to Leri-Weill dyschondrosteosis [38, 39] and non-syndromic idiopathic short stature [40, 41], and deletions cause the short stature phenotype seen in Turner syndrome [41, 42]. SHOX2 is involved in craniofacial and limb development  and SHOX2 mutations lead to cleft palate . Along with ARSE, the SHOX genes provide an intriguing correlation with the skeletal phenotype of ATR-X patients, and future work should address whether these genes are regulated by ATRX in humans.
Future work should focus on identifying the molecular mechanisms by which ATRX can co-regulate this diverse set of genes linked by their ancestral localization in the PAR1 region. This will lead to a better understanding of ATRX function in the regulation of chromatin structure and its effects on gene expression in general.
Mice conditionally deficient for ATRX in the forebrain were generated by crossing Atrx loxP females with heterozygous Foxg1Cre male mice, as previously described . Pregnant females were sacrificed at E13.5, embryos were recovered and yolk sac DNA was genotyped by PCR using the primers 17F, 18R and neo r as described previously . For newborns (P0.5) and juveniles (P17), pups were sacrificed and tail DNA was used for genotyping as previously described .
Total forebrain RNA (10 μg) was isolated from three E13.5 and four P0.5 pairs of littermate-matched ATRX-null and control embryos using the RNeasy Mini kit (Qiagen). cRNA was generated and hybridized to an Affymetrix Mouse Genome 430 2.0 Array at the London Regional genomics Center (London, Canada). For the analysis at E13.5, RNA from two forebrains was pooled for each array. Probe signal intensities were generated using GCOS1.4 (Affymetrix Inc., Santa Clara, CA) using default values for the Statistical Expression algorithm parameters and a Target Signal of 150 for all probe sets and a Normalization Value of 1. Gene level data was generated using the RMA preprocessor in GeneSpring GX 7.3.1 (Agilent Technologies Inc., Palo Alto, CA). Data were then transformed (measurements less than 0.01 set to 0.01), normalized per chip to the 50 th percentile, and per gene to control samples. Probe sets representing Atrx transcripts were removed (10 sets). Remaining probe sets were filtered by fold change of either ≥1.5 or 2 between control and Atrx-null samples, and by confidence level of P < 0.05. Heatmaps were generated using the GeneSpring hierarchical clustering gene tree function. Significantly overrepresented GO categories were determined using GeneSpring: at E13.5 and P0.5, probe sets were filtered by 1.5 fold change, P < 0.05 and categorized as either up or downregulated. Where there were multiple probe sets for a gene, duplicates were removed. P < 0.001 was used as the significance cutoff.
Total RNA was isolated using the RNeasy Mini kit (QIAGEN). First-strand cDNA was synthesized from 3 μg of total RNA using the SuperScript™ II Reverse Transcriptase kit (Invitrogen) with 25 mM dNTPs (GE Healthcare), 1 μL porcine RNAguard (GE Healthcare) and 3 μL random primers (GE Healthcare). PCR reactions were performed on a Chromo4 Continuous Fluorescence Detector in the presence of iQ™ SYBR Green Supermix and recorded using the Opticon Monitor 3 software (Bio-Rad Laboratories, Inc.). Samples were amplified as follows: 95°C for 10 seconds, annealed for 20 seconds, 72°C for 30 seconds (See Additional file 6 for primer sequences and annealing temperatures). After amplification a melting curve was generated, and samples were run on a 1.5% agarose gel (75 V for 1 h) to visualize amplicon purity. Standard curves were generated for each primer pair using three fold serial dilutions of control cDNA. Primer efficiency was calculated as E = [10(-1/slope)-1]*100, where a desirable slope is -3.32 and r2 > 0.99. Samples were normalized to β-actin expression and relative gene expression levels were calculated using GeneX software (Bio-Rad Laboratories, Inc.).
For Arsd/e and Asmtl, the PCR products were gel extracted using the QIAquick Gel Extraction Kit (QIAGEN) according to the manufacturer's instructions and sequenced at the DNA Sequencing Facility at Robarts Research Institute (London, Canada).
Bioinformatics analysis of novel ancestral PAR genes
Probeset sequences were obtained from the Netaffx website http://www.affymetrix.com/analysis/netaffx and used for BLASTn searches http://www.ncbi.nlm.nih.gov/BLAST. For calculation of interspecies similarity, sequences were obtained from NCBI RefSeq http://www.ncbi.nlm.nih.gov/RefSeq or Ensemble http://www.ensembl.org where RefSeq sequences were not available, and pairwise comparisons made using Jalview .
For generation of trees and sequence alignments, human ARSE (SwissProt P51690, RefSeq NP_000038) and human ASMTL (SwissProt O95671, RefSeq NP_004183) were used as seeds and the GenBank NR database was searched for high-similarity, full-length orthologs and paralogs. Fifty-nine ARSE and twenty-two ASMTL sequences met or exceeded the similarity cutoff, with resultant species spanning the metazoa from anemone and urchin to a diverse set of vertebrates. Sequences were aligned using T-Coffee 5.56  using default parameters. Alignments were manually adjusted via inspection prior to further analysis. Approximate maximum-likelihood trees were built using PHYML 2.4.5  using the WAG model of protein evolution  and a seven-category Gamma-plus-invariant model of rate heterogeneity. All rate parameters were estimated from the data. One hundred bootstrap replicates were performed to assess support for the inferred tree topology. All trees are presented as midpoint-rooted phylograms. Since the given mouse sequences were quite short compared to the full protein length, two sets of trees were built for each family to assess if the mouse sequences were long enough to definitively support their taxonomic clustering. One set utilized a "trimmed" alignment where all alignment columns outside the mouse sequence domain were removed. The trees produced with this trimmed alignment were compared with the set of trees produced from the alignment of the mouse sequences to their respective full-length proteins. For both ARSD/E and ASMTL, very little difference was observed between full-length and trimmed-alignment trees. The trimmed alignments tended to exaggerate sequence divergence and modestly lower bootstrap support levels. Overall topology did not appear significantly different, however, and the text references the full-length sequence phylogeny exclusively.
Cell culture and RNA interference
Neuro-2a cells were grown at 37°C with 5% CO2 in EMEM supplemented with 10% fetal bovine serum (Sigma-Aldrich). For siRNA treatment, 1.5 × 104 cells were plated in a plastic six well dish (Corning Incorporated) on glass coverslips and allowed to grow to 15% confluency (approximately 24 hours). Cultures were transfected using Lipofectamine 2000 (Invitrogen) with 8 nM siATRX (Dharmacon), a non-specific control siRNA (Sigma-Aldrich), or with no siRNA ("Mock") according to the manufacturers' instructions (for siRNA sequences refer to ). Total RNA was extracted from cells after 72 hours, cDNA was generated and qPCR analysis performed as described above. Alternatively, cells were processed for immunofluorescence staining as described below.
Neuro-2a cells were fixed using 3:1 methanol:ethanol, incubated for 1 h with the primary antibody (H300 anti-ATRX, 1:100 dilution; Santa Cruz) followed by the secondary antibody (goat-anti rabbit Alexa 594, 1:1500 dilution; Molecular Probes), then counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min. Coverslips were mounted with Vectashield (Vector Laboratories), Z-stack images were captured using a Leica DMI6000b inverted microscope and Openlab software (v5.0, Improvision) and processed using Volocity software (v4.0, Improvision); deconvolution was performed using iterative restoration set with a confidence limit of 95%.
α thalassemia mental retardation: X linked
X chromosome inactivation
Colony stimulating factor 2 receptor: alpha
dehydrogenase/reductase (SDR family) X chromosome
Atrx Dnmt3a/b Dnmt3L
forkhead box G1
Expressed Sequence Tag
Basic Local Alignment Search Tool nucleotide
BLAST-like Alignment Tool
Small interfering RNA
reverse-transcriptase polymerase chain reaction
short stature homeobox
alpha thalassemia mental retardation: X linked (referring to the syndrome)
We thank Douglas R. Higgs and Richard J. Gibbons for the AtrxloxP mice. We wish to gratefully acknowledge SHARCNET  for providing computational resources. M.L. and D.T were supported by Curtis Cadman Foundation and Natural Sciences and Engineering Research Council of Canada (NSERC) fellowships, respectively. This work was funded by the Canadian Institutes for Health Research (MOP-74748) and NSERC (313403) operating grants to N.G.B. N.G.B. is the recipient of a CIHR New Investigator award.
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