Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome
© McLaughlin and Chadwick; licensee BioMed Central Ltd. 2011
Received: 27 January 2011
Accepted: 13 April 2011
Published: 13 April 2011
Comparative sequence analysis is a powerful means with which to identify functionally relevant non-coding DNA elements through conserved nucleotide sequence. The macrosatellite DXZ4 is a polymorphic, uninterrupted, tandem array of 3-kb repeat units located exclusively on the human X chromosome. While not obviously protein coding, its chromatin organization suggests differing roles for the array on the active and inactive X chromosomes.
In order to identify important elements within DXZ4, we explored preservation of DNA sequence and chromatin conformation of the macrosatellite in primates. We found that DXZ4 DNA sequence conservation beyond New World monkeys is limited to the promoter and CTCF binding site, although DXZ4 remains a GC-rich tandem array. Investigation of chromatin organization in macaques revealed that DXZ4 in males and on the active X chromosome is packaged into heterochromatin, whereas on the inactive X, DXZ4 was euchromatic and bound by CTCF.
Collectively, these data suggest an important conserved role for DXZ4 on the X chromosome involving expression, CTCF binding and tandem organization.
Macrosatellites are a type of variable number tandem repeat (VNTR) that primarily differ from other VNTRs by the size of the individual repeat unit (from 2 to >12 kb) and restriction of the array to one or two locations in the genome . To date, at least eight different macrosatellite arrays have been described in the human genome [1–6], although several others remain largely unexplored .
Among the human macrosatellites, the best characterized is D4Z4, located at the subtelomeric regions of chromosomes 4q35  and 10q26 [7, 8]. D4Z4 consists of a tandem array of 3.3-kb repeat units that can cover hundreds of kilobases at these chromosomal locations . D4Z4 has been a major focus for research since a link was made between the array and facioscapulohumeral muscular dystrophy (FSHD) [6, 9], an autosomal dominant disorder characterized by progressive atrophy of muscles in the face, shoulders and upper arms . In almost all cases, FSHD onset is associated with contraction of the array at 4q35 to ten or fewer repeat units [6, 9]. Contraction alone is not sufficient for disease onset, as pathogenesis is linked to a reduction in the number of D4Z4 repeat units on a defined haplotype termed 4qA161 . Like other macrosatellites [1, 2, 5, 12, 13], D4Z4 is expressed . Each D4Z4 monomer in the array contains an ORF  that encodes a double homeobox protein termed DUX4 . Recent data indicate that transcripts originating from the most distal monomer are stabilized by a poly-adenylation signal located distal to the array [14, 17], a feature that is only found on chromosomes with the 4qA161 haplotype . These advances have focused attention on inappropriate expression of DUX4 as the likely molecular basis of the disease.
Not all macrosatellites are obviously protein coding , which then begs the question of what role they fulfil in genome biology. One such example is the X-linked macrosatellite DXZ4. DXZ4 is a tandem array of 3-kb repeat units located at Xq23 . While several short ORFs are present within a monomer, none have any homology to known proteins and they therefore may simply be a consequence of the high GC DNA sequence content reducing the incidence of stop codons. Identification of the ORF in D4Z4 was facilitated by the presence of the homeobox motif [15, 16], and further supported by conservation of the ORF through to rodents . However, despite no clear ORF within a DXZ4 monomer, the array does adopt intriguing chromatin states in the context of X chromosome inactivation, which is the mammalian form of dosage compensation, a process that balances X-linked gene expression between the sexes by shutting down most gene expression from one of the two X chromosomes in females . Gene silencing at the chosen inactive X chromosome (Xi) is achieved by repackaging the chromosome into facultative heterochromatin, including CpG island DNA hypermethylation [21, 22], acquisition of covalent histone modifications associated with heterochromatin [23–26], and underrepresentation of euchromatic marks, including histone acetylation  and histone H3 dimethylated at lysine 4 (H3K4me2) . Whereas most chromatin on the Xi adopts this new configuration, DXZ4 does not. CpG dinucleotides at DXZ4 are hypomethylated [3, 12] and DXZ4 nucleosomes are characterized by H3K4me2 [12, 28] as well as several other euchromatic markers  and the array is bound by the multi-functional zinc finger protein CCCTC binding factor (CTCF) [12, 29]. DXZ4 at the Xi is readily detected as a signal of H3K4me2 within the hypo-H3K4me2 territory of the Xi at interphase [28, 29], and as a distinct signal midway down the long arm of the chromosome at metaphase . Facultative heterochromatin of the Xi is not a homogenous mass of like-chromatin, but is instead composed of at least two different types that occupy reproducible alternating bands along the Xi [30, 31]. DXZ4 resides at the interface of two such bands , and therefore may have some role in organization of the Xi involving CTCF. In sharp contrast, DXZ4 CpG dinucleotides on the active X chromosome (Xa) and in males are hypermethylated [3, 12] and nucleosomes are packaged into constitutive heterochromatin characterized by histone H3 lysine-9 trimethylation (H3K9me3)  and binding of heterochromatin protein 1 gamma (HP1γ) . Therefore, human DXZ4 adopts alternative chromatin states on the Xa and Xi that differ from the surrounding chromosome.
Intriguingly, analysis of D4Z4 chromatin organization in FSHD patients revealed that the contracted allele adopted a more euchromatic organization [32, 33] involving CTCF binding , hence resembling DXZ4 on the Xi . Therefore, these findings highlight how investigating the biology of macrosatellites in general can provide insight into the function of other macrosatellites such as D4Z4 in the context of FSHD.
In order to further understand DXZ4, we sought to identify conserved DNA sequence and chromatin organization for the array by investigating the macrosatellite in other primates, and report our findings here.
DXZ4 is a conserved X-linked macrosatellite repeat in Old World and New World monkeys
Next we sought to determine if DXZ4 is a polymorphic tandem array in Old World monkeys and if DXZ4 sequence conservation was sufficiently high enough to detect DXZ4 in New World monkeys using a human DXZ4 probe. Agarose embedded genomic DNA from a human sample as a hybridization control, one female gorilla, two male macaques, two female macaques and a male and female New World monkey were cut with XbaI and fragments separated by pulsed field gel electrophoresis. Given that there are no recognition sites for XbaI in human DXZ4, genomic DNA will be cut at the first available sites proximal and distal to DXZ4, essentially excising the array intact. Because the copy number of DXZ4 monomers in humans varies between individuals, hybridizing DNA fragments are polymorphic . A Southern blot of the gel was hybridized with the human probe before washing to high stringency (0.2 × SSC, 0.1% SDS at 60°C). A single hybridizing band was detected in all male samples, and two bands in the females, including the New World monkeys (Figure 1b). Three conclusions can be drawn from this result. First, DXZ4 sequence is conserved in primates at least as far as New World monkeys. Second, given the single male band and two female bands, this further supports X-linkage for DXZ4 in primates. Finally, the range of hybridizing fragments (approximately 50 to 350 kb) indicates that DXZ4 is a VNTR in the great apes, Old World monkeys and New World monkeys.
Comparison of the human DXZ4 DNA sequence [GenBank: HQ659140] against the rhesus macaque genome sequence (rheMac2), did not identify a VNTR, but instead identified various broken matches over short intervals and the presence of a large gap in the genome sequence, indicating that like many tandem repeat DNAs in the various early releases of the human genome, the sequence of DXZ4 is poorly assembled in the current build of the macaque genome. In order to further characterize DXZ4 in Old World monkeys, we compared the human DXZ4 sequence to entries in the public databases using BLAST. Several green monkey (Cercopithecus aethiops) and rhesus macaque BAC clones were identified with matches to DXZ4 that were then ordered and obtained. Human DXZ4 is cut once per monomer with HindIII . Digestion of the Old World monkey BACs with HindIII revealed an over representation of a 3-kb fragment in the BAC clones (Figure 1c), supporting the presence of multiple DXZ4 sequences in the clones. The 3-kb HindIII band was excised from the gel and cloned before sequencing.
Comparison between human DXZ4 and monomer sequences identified in great ape, lesser ape, Old World monkey and New World monkey
Monomer size (bp)
Percent identity to human
Tandem repeat organization is retained in lemurs, but sequence conservation is restricted to the promoter and CTCF binding region of human DXZ4 in distantly related primates
The ring-tailed lemur sequence was derived from a BAC clone (LB2-162N9) [GenBank: AC133072] within which a 170-kb continuous sequence is assembled. The first 70 kb of this sequence aligns with the 402-bp human DXZ4 sequence 15 times (data not shown), indicating a locally repetitive nature for this interval. Of the remaining 100 kb, multiple extensive single copy matches are made with unique DNA sequences found distal to human DXZ4, indicating that the BAC clone likely contains part of the orthologous ring-tailed lemur DXZ4 array. Comparison of the 170-kb BAC sequence against itself confirmed the presence of a more extensive tandem repeat in the first 70 kb than just the 402-bp sequence. Alignment of a 3-kb sequence from within this interval against the 70-kb sequence clearly demonstrates that the DNA has characteristics of a tandem repeat (Figure 3c). However, unlike human DXZ4 (Tremblay DC et al., in preparation), the sequence is not a perfect tandem array of uninterrupted monomers of a uniform size, but consistent with other primates (Table 1), the 3-kb sequence is characterized by high GC content (62%).
Conserved hypo-methylation of CpG residues on the macaque Xi
Conserved H3K4me2 banding on the macaque Xi
Euchromatic markers are largely absent from the macaque Xi with the exception of a discrete signal within the interphase territory of the chromosome
Xi DXZ4 in macaque is characterized by H3K4me2 and CTCF
To confirm DXZ4 as the site of H3K4me2 and CTCF, chromatin immunoprecipitation was performed on chromatin prepared from male and female rhesus macaque and pig-tailed macaque, along with the heterochromatin marker H3K9me3. Both male and female samples showed the presence of H3K9me3, whereas H3K4me2 and CTCF were readily detected in the female samples from both species of macaque (Figure 7b). The logical interpretation of these data is that DXZ4 in males and on the Xa is characterized by constitutive heterochromatin, whereas DXZ4 on the Xi is characterized, at least in part, by a euchromatic conformation bound by CTCF, consistent with that seen for human DXZ4 .
Macaque DXZ4 is transcribed
To identify functionally important features of DXZ4, we investigated chromatin structure of the array in the Old World monkey macaque, expression and retention of tandem repeat organization, and primary DNA sequence conservation in a variety of closely and distantly related members of the primate lineage.
Our data indicate that DXZ4 in the great apes and in the Old and New World monkeys is a polymorphic tandem-repeat, with array sizes comparable to those observed in humans . DNA sequence data obtained from a BAC clone also provides evidence of tandem arrangement for the orthologous array in ring-tailed lemurs.
DNA sequence analysis reveals 95 to 97% sequence identity to human DXZ4 in the great apes, 90% in the lesser apes, 87% in Old World monkeys and 77% in New World monkeys. All of these primates had a repeat unit size around 3 kb, high GC content (61 to 65%) and a high incidence of CpG (154 to 192 per monomer). Human DXZ4 contains three internal microsatellite repeats that are the only repeat masked portion of a monomer. Through Old World monkeys the [GGGCC] and [CT] repeats are conserved. The [TAAA] repeat sequence is only conserved in chimpanzee and diverges in the other primates. However, all of the other primates have a simple repeat sequence that is enriched in A nucleotides at this location in the monomer, suggesting that retention of A-rich repetitive DNA is important for this region. In the common marmoset (a New World monkey), all three repeat sequences have diverged, but remain repetitive and retain G-rich, C-rich and A-rich sequence composition, respectively. It is generally accepted that DNA sequence composition influences nucleosome positioning . Close examination of predicted nucleosome occupancy  for human DXZ4 using the UCSC genome browser  shows that the [TAAA] and [CT] repeats are strongly inhibitory of nucleosome occupancy, whereas the [GGGCC] repeat sequence resides at a peak of nucleosome occupancy (data not shown), suggesting that these sequences influence the position of nucleosomes in the array. Indeed, microarray hybridization using DNA isolated from human chromatin immunoprecipitated with antibodies to H3K4me2 and H3K9me3 revealed well defined peaks of modified nucleosomes predicted to be approximately every fourth nucleosome in DXZ4 , suggesting that nucleosome distribution within the array is likely well defined. Therefore, it is tempting to suggest that retention of base composition and location of these repeat sequences in primate DXZ4 is necessary to assist in maintaining nucleosome distribution and chromatin structure within the array.
Conservation of DXZ4 DNA sequence drops off rapidly in the lemurs, galago and tarsier branches of the primate tree. However, sequence homology extends across an approximately 400-bp region of DXZ4 encompassing the bidirectional promoter and binding sites for TFIID and CTCF . Unlike the chromosome 4 macrosatellites RS447  and D4Z4 , which both contain ORFs that are conserved through rodents , DXZ4 does not obviously encode a protein. Therefore, retention of this sequence is very significant, and suggestive of an important role for DXZ4 in primates as a genomic element, involving CTCF binding and transcription. Preliminary analysis of this region throughout mammals (including mouse) indicates the presence of a tandem repeat downstream of PLS3 that shows some homology to this 400-bp sequence, further supporting an important role for this element on the X chromosome (BP Chadwick, unpublished data), and this is now a major focus for our DXZ4 investigation.
Despite a lack of conserved ORFs, DXZ4 is expressed in humans. Most DXZ4 RNA are sense transcripts originating from the Xa, although detectable anti-sense transcription is found specifically in females and therefore likely originates from the Xi . Here we find that DXZ4 is expressed in male and female macaque, although almost all transcription appears to originate form the Xa and no anti-sense transcript was detected. Therefore, expression of DXZ4 is conserved, but the significance of anti-sense transcription remains unclear.
In humans, DXZ4 in males and on the Xa in females is packaged into constitutive heterochromatin characterized by hyper-CpG methylation [3, 12], H3K9me3  and HP1γ . Conversely, DXZ4 on the Xi is packaged, at least in part, into euchromatin characterized by H3K4me2, H3K36me2, and histone acetylation, and is hypomethylated at CpG residues and bound by CTCF [3, 12, 28, 29]. Both forms are expressed despite the contrasting chromatin packaging . Here we show that all of these features are conserved at DXZ4 in the Old World monkey macaque. Furthermore, as has been observed in humans [28, 30, 31], the macaque Xi is characterized by distinct reproducible bands of H3K27me3, with the euchromatic form of DXZ4 located at the distal edge of a major Xq H3K27me3 band. Also consistent with the human Xi , additional distinct H3K4me2 signals reside at the distal edge of other H3K27me3 bands, suggesting that conservation of this arrangement has some role in Xi chromatin organization. Therefore, determining the DNA sequence identity of these chromatin elements is a priority.
These data indicate several conserved features of DXZ4 that are likely important for the organization and function of the array: repeat monomer tandem arrangement; retention of high GC content and CpG incidence; conservation of the internal promoter sequence; conservation of the CTCF binding site; conservation of internal simple repeats; and array expression. Collectively, these features likely contribute to the roles of DXZ4 packaged into constitutive heterochromatin on the Xa and euchromatin bound by CTCF on the Xi. What function DXZ4 has in these contexts remains unclear. However, data from this study highlight important conserved features of DXZ4 that will assist in guiding the formulation of new hypotheses that can be tested to decipher the role of DXZ4 on the X chromosome. Furthermore, elucidation of DXZ4 function on the Xi will likely reveal additional intriguing parallels between the biology of DXZ4 and the contracted form of D4Z4 in FSHD, promoting our appreciation for these enigmatic genomic features.
Materials and methods
Human female telomerase immortalized retinal pigment epithelial cells (hTERT-RPE1) were obtained from Clontech (Mountain View, CA, USA). Human male lymphoblastoid cell line GM06992 was obtained from the Coriell Cell Repositories (Coriell Institute for Medical Research), as were the following primate primary fibroblast cells: rhesus macaque (M. mulatta) AG08305 (male), and AG08312 (female); pig-tailed macaque (M. nemistrina) AG07921 (male), and AG08452 (female); common squirrel monkey (Saimiri sciureus) AG05311 (female); black-handed spider monkey (Ateles geoffroyi) AG05352 (male). Cells were maintained according to Coriell's recommendations. Female gorilla (Gorilla gorilla) lymphoblast cells  were a gift from H Willard. Culture media (RPMI for lymphoblasts, and DMEM for fibroblasts), fetal bovine serum and supplements were all obtained from Invitrogen Corp (Carlsbad, CA, USA).
Metaphase chromosome preparation
In order to enrich for cells in metaphase, growth media of rhesus macaque primary fibroblasts was supplemented with colcemid (Invitrogen) to 25 ng/ml before returning cells to the incubator for an additional hour. Cells were harvested and resuspended in 37°C 75 mM KCl for 15 minutes. To this, one-sixth volume of fixative (three parts methanol to one part acetic acid) was applied before pelleting the cells. Cells were washed an additional six times with fixative, pelleting cells between each wash. Fixed cells were dropped from approximately 30 cm onto cleaned microscope slides resting on damp paper towels on top of a 37°C heat block. Slides were dried at room temperature for an additional 24 hours before use.
Pulsed field gel electrophoresis, Southern blotting and hybridization
Agarose embedded genomic DNA from primate cells were prepared essentially as described .
Agarose embedded DNA was digested with XbaI (NEB, Ipswich, MA, USA). Each plug was first equilibrated in 300 μl of 1 × digest buffer at room temperature for 20 minutes, before replacement of buffer with 100 μl of 1 × digest buffer containing 200 units of restriction enzyme. Digests were performed overnight at 37°C. Plugs were loaded onto a 1.0% agarose gel prepared using pulsed field certified agarose (Biorad, Hercules, CA, USA) in 0.5 × TBE. DNA was separated at 13°C in 0.5 × TBE and conditions selected to separate 100 to 400 kb using the auto algorithm function of the CHEF Mapper (Biorad). Markers were loaded in the outer lanes (NEB, MidRange PFG Markers I and II). The gel was then rinsed with water before staining with ethidium bromide (1 μg/ml) at room temperature for 30 minutes. The gel was washed twice with water for 15 minutes each and an image captured. The gel was then treated with 0.25 M HCl for 15 minutes before denaturing for 30 minutes (1.5 M NaCl, 0.5 M NaOH). DNA was transferred to Hybond-N+ (GE Healthcare, Piscataway, NJ, USA) overnight by standard Southern blotting . The membrane was rinsed with 2 × SSC before baking at 120°C for 30 minutes.
A DXZ4 probe was prepared by PCR amplification of regions of human DXZ4 with the following oligonucleotides: CAGGCAGAAATGAGCACCAC and TGGTGGCGGCCATGATCTG (485 bp); ACCAGGCAAACTGCCCAAG and TTCTGGTTTGTCAGGAAGGC (550 bp); ACCCTGTCCTTGGCAGATG and GTTGGACGTAGGCCAGGTG (491 bp); GCCTACGTCACGCAGGAAG and CCAGCGGAAAGTCCATGGG (402 bp); CACTTGGGAGACTCCTGAAC and TGTCCCCGAGGTTGTCTTG (485 bp); TCTCTCGCCCACTTCTACTG and GAGTCGATGGGCCTCTTAG (530 bp). The PCR products were cleaned (Qiagen, Valencia, CA, USA) before labeling with DIG-11-dUTP by random priming (Roche, Basel, Switzerland). The probes were tested for specificity and detection of the anticipated DNA fragment size on a Southern blot of EcoRI digested total genomic DNA.
Hybridization was performed overnight at 60°C using Expresshyb (Clontech). Blots were washed the following day at 60°C using two 8-minute washes in 2 × SSC, 0.1% SDS followed by one wash of 8 minutes in 0.2 × SSC, 0.1% SDS. The probe was detected using anti-DIG-alkaline phosphatase, blocking, wash and detection buffers according to the manufacturer's instructions (Roche). Signals were detected by exposure to photographic film (Kodak).
BAC clone analysis
Human BAC clone 2272M5 was obtained from Invitrogen. Human BAC clone RP11-268A15 was obtained from the Children's Hospital and Research Center at Oakland (CHRCO), as were the following green monkey (C. aethiops) and macaque BAC clones: macaque (M. mulatta) BAC clones from the CHORI-250 library - CH250-131A6 and CH250-345N15; green monkey BAC clones from the CHORI-252 library - CH252-257K14, CH252-445A22, CH252-338G16 and CH252-199I1. Individual DXZ4 monomers from BAC clones CH250-131A6 and CH252-338G16 were generated by first performing a HindIII digestion on the BAC clone DNA, gel purifying the 3-kb fragment and cloning into calf intestinal alkaline phosphatase (NEB) treated HindIII cut pBluescript-II (Agilent Technologies, Santa Clara, CA, USA). Inserts were sequenced on both strands using T7, MM-F1 CCTCTTGATGGCAGTATTGC, MM-F2 CCTGGCCAGCATAGGTCAG, MM-F3 AGAGGCGGCAAGAGAAATGC, SP6, MM-R1 TTGTCAGGAAGGCAGGCTAG, MM-R2 ACATCGGGTTTCCGTCACAG and MM-R3 ATCCAACTTCCACCTCAACG.
DNA and RNA FISH
For DNA FISH, probes of human BAC clones RP11-268A15 and 2272M5 were labeled with Spectrum Orange or Spectrum Green by nick translation according to the manufacturer's instructions (Abbott Molecular, Abbott Park, IL, USA), followed by ethanol precipitation in the presence of 25 μg of human Cot-1 DNA (Invitrogen) and resuspension in 0.1 ml of Hybrisol VII (MP Biomedicals, Solon, OH, USA). A 1:1 mix of the two probes was denatured at 75°C for 4 minutes, and repetitive sequences blocked at 37°C for 30 minutes before being applied directly to the slide, covered with cover glass, sealed with rubber cement and hybridized for 16 hours at 37°C. Slides were washed at 37°C twice in 50% formamide, 2 × SSC for 8 minutes each, then once in 2 × SSC for 8 minutes before adding ProLong Gold antifade containing DAPI (Invitrogen).
For RNA FISH, a direct-labeled Spectrum Green probe of human XIST exon 1 was prepared as described above and used with a Spectrum Red rhesus macaque 131A6 DXZ4 3-kb subclone probe. Fibroblasts were grown directly on slides before fixing and extracting in 4% formaldehyde, 0.1% Triton-X100 1 × phosphate buffered saline for 10 minutes at room temperature. Slides were washed for 2 minutes each in 1 × phosphate buffered saline before dehydration through 70% and 100% ethanol for 2 minutes each and then air-drying. A 1:1 mix of the BAC and XIST probes was denatured at 72°C for 5 minutes before placing at 37°C for 30 to 60 minutes to block repetitive elements. The probe was applied onto cells and sealed with a coverslip and rubber cement at 37°C for 16 hours in a humidified chamber. Slides were washed twice at room temperature in 50% formamide, 2 × sodium citrate sodium chloride (SSC), followed by 3 minutes at 37°C in 50% formamide 2 × SSC and one wash of 3 minutes at 37°C in 2 × SSC before addition of ProLong Gold antifade containing DAPI (Invitrogen). Control RNA FISH used a Spectrum Red MIC2 BAC clone RP11-1151O1 from Invitrogen.
Macaque genomic DNA was isolated from rheusus macaque and pig-tailed macaque cells using the Blood and Cell Culture DNA Midi-Kit (Qiagen), and bisulfite modified DNA prepared using the EpiTect Bisulfite kit (Qiagen) according to the manufacturer's recommendations. A 621-bp fragment of DNA was PCR amplified from bisulfite-modified DNA using the following primer pair: forward, GGGTATTAGGTAAATTGTTTA; reverse, CCATCCCAAAAACATAATTAAAA. PCR products were TA cloned into pDrive (Qiagen) and individual clones sequenced with M13R.
Immunofluorescence on interphase cells and metaphase chromosomes
Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA). Mouse monoclonal anti-H3K27me3 (ab6002) was obtained from Abcam (Cambridge, MA, USA). Secondary antibodies were obtained from Jackson ImmunoResearch Laboratories Inc. and Invitrogen (West Grove, PA, USA). Cell staining and preparation of metaphase chromosomes was performed essentially as described . Images were collected using a Zeiss Axiovert 200M fitted with an AxioCam MRm and images managed using AxioVision 4.4 software (Carl Zeiss Microimaging, Inc.).
Chromatin immunoprecipitation using rhesus macaque and pig-tailed macaque cells was performed essentially as described . Antibodies were obtained from Millipore: anti-CTCF (07-729), anti-H3K4me2 (07-030) and anti-H3K9me3 (07-523). Rabbit serum negative control was obtained from EMD (Gibbstown, NJ, USA). Immunoprecipitated DNA was PCR amplified using MM-F1 and MM-R1 (sequences given above).
RT-PCR, strand-specific RT-PCR and quantitative RT-PCR
Macaque total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen). Residual genomic DNA was removed by pre-treating the RNA with DNaseI (Invitrogen) for 20 minutes at room temperature, before heat inactivating the DNaseI at 70°C in the presence of 2.5 mM EDTA for 15 minutes. cDNA was prepared using 1 μg of total RNA with or without M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's instructions.
cDNA was amplified using Taq polymerase (NEB) with the following cycle: 95°C for 2 minutes, followed by 35 cycles of 95°C for 20 seconds, 58°C for 20 seconds, 72°C for 30 seconds. Amplification used the following primers: MM-F1 and MM-R1; MM-F2 and MM-R2; MM-F3 and MM-R3 (sequences given above).
Strand-specific cDNA was prepared essentially as described above except random hexamers were replaced with a strand-specific primer and an additional control was included of reverse transcriptase without primer. Anti-sense cDNA was primed with MM-F4 (TGACCAAGAGGTCAAAGGCG), whereas sense-strand cDNA was primed with MM-R2. cDNA was assessed with MM-F2 and MM-R2 or MM-F4 and MM-R4 (GTTGGACGTAGGCCAGGTG).
qRT-PCR was performed in triplicate four independent times using random primed cDNA with MM-F2 and MM-R4 using DyNAmo SYBR Green qPCR (NEB) on a CFX96 (Biorad).
bacterial artificial chromosome
CCCTC binding factor
fluorescence in situ hybridization
facioscapulohumeral muscular dystrophy
histone H3 dimethylated at lysine 4
histone H3 trimethylated at lysine 4
histone H3 trimethylated at lysine 9
histone H3 trimethylated at lysine 27
histone H3 dimethylated at lysine 36
heterochromatin protein 1 gamma
open reading frame
quantitative reverse transcription polymerase chain reaction
reverse transcription polymerase chain reaction
sodium citrate sodium chloride
variable number tandem repeat
active X chromosome
inactive X chromosome
X inactive specific transcript.
This work was supported by a grant from the National Institute of General Medical Sciences to BPC (NIH R01 GM073120). We are grateful to Hunt Willard for use of the gorilla cell line. We are also grateful to Rani Dhanarajan for assistance with subcloning green monkey and rhesus macaque DXZ4 monomers, and thank Jonathan Dennis for his helpful comments and discussion.
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