Expression, tandem repeat copy number variation and stability of four macrosatellite arrays in the human genome
© Tremblay et al; licensee BioMed Central Ltd. 2010
Received: 8 October 2010
Accepted: 15 November 2010
Published: 15 November 2010
Macrosatellites are some of the largest variable number tandem repeats in the human genome, but what role these unusual sequences perform is unknown. Their importance to human health is clearly demonstrated by the 4q35 macrosatellite D4Z4 that is associated with the onset of the muscle degenerative disease facioscapulohumeral muscular dystrophy. Nevertheless, many other macrosatellite arrays in the human genome remain poorly characterized.
Here we describe the organization, tandem repeat copy number variation, transmission stability and expression of four macrosatellite arrays in the human genome: the TAF11-Like array located on chromosomes 5p15.1, the SST1 arrays on 4q28.3 and 19q13.12, the PRR20 array located on chromosome 13q21.1, and the ZAV array at 9q32. All are polymorphic macrosatellite arrays that at least for TAF11-Like and SST1 show evidence of meiotic instability. With the exception of the SST1 array that is ubiquitously expressed, all are expressed at high levels in the testis and to a lesser extent in the brain.
Our results extend the number of characterized macrosatellite arrays in the human genome and provide the foundation for formulation of hypotheses to begin assessing their functional role in the human genome.
More than half of the human genome is composed of repetitive DNA , a proportion of which are tandem repeats. Tandem repeats are characterized by a given DNA sequence being repeated immediately adjacent to and in the same orientation as the first. In almost all cases the number of repeat units at a given locus vary between individuals and as such they are more commonly known as variable number tandem repeats (VNTRs).
Macrosatellites are among the largest VNTRs in the human genome  and are characterized by individual repeat units of several kilobases (kb) and tend to be specific to a location on one or two chromosomes. Among the human macrosatellites, the best characterized are RS447, an array of 20-103 4.7 kb tandem repeat units on 4p16.1 along with a few copies at 8p23 , D4Z4 on 4q35 and 10q26, an array of as few as 1 to over 100 3.3 kb tandem repeat units [4, 5] and DXZ4, an array of 50-100 copies of a 3-kb repeat unit specifically located at Xq23-24 . The role of some tandem repeat DNA is more obvious due to their location, such as the extensive alpha satellite tandem repeats that define human centromeres  or the tandem repeat DNA at human telomeres . What function macrosatellites have in genome biology is unclear. However, reduction in the size of D4Z4 on 4q35 to fewer than 10 tandem repeat units is associated with onset of the third most common inherited form of muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD) . Numerous models have been proposed to explain how D4Z4 contraction could result in FSHD . One early model suggested that reduction in the size of D4Z4 packaged as heterochromatin could result in a position-effect on adjacent gene expression . Results from several studies testing this model are mixed, with some showing support [11–14], and others not [15–19]. More recently focus has turned to expression of the array itself. Like RS447, that contains an open reading frame (ORF) in each monomer that codes for a novel deubiquitinating enzyme , an ORF is present in D4Z4 monomers that encodes double homeobox 4 (DUX4) , a DNA binding protein  that alters expression levels of myogenic regulators in myoblasts . DUX4 expression can be detected in patient myoblasts  but not control individuals. Recent evidence suggests that detectable levels of DUX4 in patients is due to polyadenylation and stabilization of transcripts originating from the most distal monomer in the array due to a polyadenylation signal located immediately distal to the array , a feature that is found only on chromosomes with the FSHD pathogenic 4qA161 haplotype .
The X-linked macrosatellite DXZ4 is also expressed  and contains several short ORFs , but none show any homology to known proteins. Unlike RS447 and D4Z4, the location of DXZ4 on the X chromosome means that in females it is exposed to the mammalian dosage compensation process X chromosome inactivation (XCI). XCI repackages the chosen inactive X chromosome (Xi) into facultative heterochromatin . Somewhat counter intuitively, DXZ4 adopts a more euchromatic conformation on the Xi while the surrounding chromosome is packaged into heterochromatin, whereas on the active X chromosome (Xa) DXZ4 is packaged into heterochromatin surrounded by euchromatin [25, 27]. The euchromatic Xi form of DXZ4 is bound by the epigenetic organizer protein CTCF  suggesting that on the Xi, DXZ4 is performing a different function than on the Xa. Intriguingly, contracted D4Z4 arrays in FSHD adopt a chromatin configuration similar to DXZ4 on the Xi  including the binding of CTCF  that likely contributes to the disease permissive state.
These data suggest that tandem repeat copy number, chromatin organization and expression are important features of macrosatellites, and provide some insight into functional attributes of these sequence, in particular the impact of short arrays in FSHD. Indeed, in Saccharomyces cerevisiae, retention of large rDNA tandem repeats are necessary for sister chromatid cohesion and maintenance of genome integrity . Therefore exploring macrosatellite VNTR and organization in the human genome is a high priority. In the current manuscript we describe the organization, expression, tandem repeat copy number polymorphism and stability of four macrosatellite arrays in the human genome.
Characterization of the TAF11-Like macrosatellite array on chromosome 5p15
In order to identify novel human macrosatellite arrays, the UCSC genome browser http://genome.ucsc.edu/ was used with the segmental duplication annotation activated to explore by eye the complete human genome sequence (NCBI36/hg18). The segmental duplication track identifies duplications of 1 kb or greater that share 90% or more DNA sequence identity . Regions annotated as segmental duplications that originate from the same chromosomal interval and displayed a regular repeat masking pattern were selected for further examination. These rough criteria were chosen based on the fact that the well characterized macrosatellite arrays DXZ4, D4Z4 and RS447 were clearly identified using the same parameters.
An individual repeat unit of the array is approximately 3.4 kb and is defined by a BglII restriction enzyme recognition site that cuts once per monomer (Figure 1A). Monomers within the contiguous array share between 99.3-99.7% nucleotide identity based on BLAT alignment results http://genome.ucsc.edu using monomers from genome build hg18, with most variation due to single nucleotide polymorphisms (SNPs) between monomers. The base composition of each monomer is approximately 49.6% GC and a little under a third (31.7%) of each monomer is repeat masked due to a combination of a retrotransposon LTR (MLT1E3), partial Alu repeat (AluSx) and a disrupted DNA transposon (Charlie 2a). A short open reading frame (ORF) resides within each monomer spanning 594 bp, and encodes a predicted TATA binding protein associated factor 11 like protein (TAF11-Like). Three SNPs reside within the ORF in the different monomer sequences represented in the hg18 build. None introduce any frame shifts or premature stop codons and therefore maintain the reading frame of the ORF. One SNP is silent exchanging a glycine codon for another (within the ORF a T-C at nucleotide 24 changing a GGT to a GGC). The other two SNPs result in an amino acid change: a T-G at nucleotide 298 changing TCC to GCC and consequently a seriene to alanine (amino acid 99 within the ORF), and a A-G at nucleotide 581 changing a AAA to an AGA resulting in a lysine to arginine change (amino acid 194 in the ORF).
Copy number variation, meiotic and mitotic instability of the TAF11-Like macrosatellite array
Expression of the TAF11-Like array
Characterization of the chromosome 4 and 19 SST1 macrosatellites
Chromosome 19 contains two SST1 arrays separated by approximately 1 Mb with several partial monomers scattered either side of the more distal array (Figure 5C). Unlike the chromosome 4 array, on chromosome 19 the two SST1 macrosatellites are embedded within a gene rich region of 19q13.12. Intriguingly, most of the genes encode members of the kruppel C2H2-type zinc-finger protein family with two genes proximal, sixteen between and eleven distal of the two SST1 macrosatellites. Monomers of the chromosome 19 SST1 arrays are slightly longer than those at chromosome 4 (2.5 kb v 2.4 kb). Within the proximal or distal chromosome 19 array monomers share >99% sequence identity compared to 97-98% identity between the two arrays based on BLAT results using a single monomer from each array. The orientations of the two arrays are inverted relative to one another and are represented by 17 (proximal array) and 15 monomers (distal array) arranged in tandem (Figure 5D).
Copy number variation and instability of the SST1 macrosatellite arrays
To investigate copy number variation of monomers within the SST1 macrosatellite arrays, agarose embedded DNA from 22 unrelated individuals was digested with XbaI that does not cut within the array and separated by PFGE before Southern blotting and hybridization with a probe specific to the SST1 arrays. Given the high sequence identity between the three arrays, the probe detected both chromosome 4 and 19 SST1 macrosatellites with most individuals showing all 6 alleles (Figure 5E). Alleles ranged from as large as 387 kb to less than 35 kb indicating tandem arrays of between 14 to 154 monomers. In order to determine if large and small alleles are common to both chromosome 19 and/or chromosome 4 SST1 macrosatellite arrays, PFGE was performed on agarose embedded DNA digested with enzymes that specifically cut each monomer within the chromosome 4 array (ApaLI) or chromosome 19 arrays (PvuII) (Figure 5A and 5B). Specifically removing the chromosome 19 arrays with PvuII resulted in small alleles only, whereas most large alleles remained intact and comparable in size upon digestion with ApaLI that specifically removes the chromosome 4 arrays (Figure 5F). These data suggest that large SST1 arrays are more common on chromosome 19 than chromosome 4.
Expression of the SST1 array
According to the UCSC genome browser, a cluster of ESTs match a monomer centered upon the CGI. Primers were designed to amplify across the CGI and were used to detect expression from the SST1 macrosatellite using cDNA prepared from 20 human tissues. Expression was readily detected in all tissues (Figure 4, second row). Several open reading frames exist within chromosome 4 and 19 monomers, but none match any known proteins in the public databases.
Characterization of the PRR20 macrosatellite on chromosome 13
According to the human genome sequence, the five complete monomers are arranged in tandem (Figure 7B). We sought to confirm the size of the array and investigate copy number variation in 11 unrelated individuals. Alleles for the PRR20 array ranged from 5 tandem copies to as many as 20 (Figure 7C) indicating that this is indeed a polymorphic macrosatellite.
Characterization of the ZAV macrosatellite array on human chromosome 9q32
Each tandem repeat unit of the array is 5.3 kb and 60% GC. A little over 28% of each monomer is repeat masked due to the presence of simple microsatellite repeats, an LTR and regions of LINE homology. Two CGIs are found in each repeat unit, one spanning 1.4 kb and the other close to 700 bp. Therefore almost 40% of each monomer, accounting for most of the non-repeat masked region, is a characterized by a CGI. In the current build of the human genome, the array is annotated as 5 repeat units arranged in tandem (Figure 9B). As for the other arrays we examined 22 unrelated individuals to see if the array was polymorphic. Alleles for the array ranged from as small as 20 kb to 168 kb (Figure 9C) indicating that this is indeed a macrosatellite repeat with between 3-31 tandem repeat units and was termed the Z FP37-A ssociated V NTR (ZAV).
One EST aligned with the larger of the two CGIs. We designed primers to this region and looked to see if the array is expressed in any of the 20 human tissue cDNA samples. Clearly the ZAV array is expressed in fetal brain and testis, with weaker signals in whole brain, thymus, uterus and spinal cord (Figure 4, fourth row).
SST1 (Chr4 & 19)
Number of unrelated array alleles
Average array allele size (Median)
178 kb (178 kb)
161 kb (127 kb)
76 kb (67 kb)
71 kb (58 kb)
Range allele size
Inferred monomer copy number
Testis Fetal Brain Whole Brain (Fetal Liver & Prostate)
All tissues examined
Testis (Cerebellum, Fetal Brain, Prostate & Thymus)
Testis Fetal Brain (Whole Brain, Thymus, Uterus & Spinal Cord)
Among the macrosatellites described here, the SST1 array is the only one that is not chromosome specific, but has arrays of significant size 1 Mb apart on 19q13.2 and an additional highly conserved array on chromosome 4q28.3 [2, 33]. A similar situation is true for the FSHD-associated array D4Z4 that has near identical arrays on chromosomes 4q35 and 10q26. Exchange between the 4q and 10q D4Z4 arrays is common in the general population . The subtelomeric location of the arrays results only in the expansion or contraction of an array as well as exchange of the limited DNA sequence from the distal edge of the array to the telomere. Should the chromosome 4 and 19 SST1 arrays recombine this would result in an extensive exchange. While we are aware of no such chromosome 4:19 translocations, it remains a distinct possibility that the two inverted chromosome 19q13 SST1 arrays could undergo intra-chromosomal non-allelic homologous recombination inverting the 1 Mb interval between the arrays, especially given the extensive size and >97% sequence identity between the two macrosatellite arrays.
An independent investigation using array comparative genome hybridization identified contracted alleles of the TAF11-Like array as a possible contributor to schizophrenia . In one group of families, small alleles (<21 tandem repeat units) segregated with schizophrenia with only one unaffected sibling carrying a small sized allele. However, this association did not extend to other schizophrenia families and therefore linkage of small TAF11-Like arrays to the disease is not statistically significant. Interestingly, the extended analysis to other schizophrenia families made use of quantitative PCR (qPCR) as opposed to PFGE to assess allele size. As the authors acknowledge, the results therefore represent the combined tandem repeat content of both chromosome 5 arrays, potentially masking small alleles. In the present study we also identified small alleles of the TAF11-Like array, with the smallest (~10 tandem repeat units) comparable in size to the pathogenic contracted D4Z4 alleles in FSHD . For these particular individuals, who have alleles of 10 and 81 tandem repeats, qPCR would have indicated approximately 50 tandem repeat units in each chromosome 5 array. This highlights that despite numerous advances in genome research tools, PFGE remains the most effective and reliable means to measure allele size of tandem repeat DNA.
Expression analysis of the four macrosatellite described here revealed that all of the arrays are expressed at high levels in the testis and to a lesser extent in fetal brain and in adult brain (except PRR20). The notable exception is SST1 that is ubiquitously expressed in all tissues examined. Furthermore, expression was independent of a clear ORF as demonstrated by the SST1 and ZAV macrosatellites, although it remains a possibility that either has a short ORF with no homology to known proteins. Expression in the testis is a defining feature of the cancer/testis (CT) genes , many of which are arranged into tandem arrays [38, 39]. Therefore, it is possible that the ZAV, PRR20 or TAF11-Like macrosatellites are novel members of the cancer/testis group, as some CT-genes are expressed in developing neurons as well as testis . Regardless, the strong expression in testis indicates that these arrays are likely packaged into an alternate chromatin state in this organ, most likely in the germ cells, as is the case for some MAGE and GAGE CT genes .
What influence variation in the number of copies of individual tandem repeat units has, if any, on flanking gene expression remains an important question to be addressed, despite the conflicting data regarding D4Z4 [11–19]. For some such as the chromosome 4 SST1 array or the PRR20 array on chromosome 13q21.1 any influence is likely negligible given that the nearest annotated genes are considerable physical distances away. The TAF11-Like array is also some distance from the distal BASP1 gene (210 kb) and therefore is also unlikely to exert any influence on its expression. However, this is making the assumption that influence is 2-dimensional and does not take into account where in 3-dimensional space the arrays are located relative to other genes. In contrast, the 9q32 ZAV array and SST1 arrays at 19q13.12 could conceivably directly influence flanking gene expression. The ZAV array is approximately 2 kb from the proximal ZFP37 gene is composed of GC rich tandem repeat units that each contain two extensive CGIs. Evidence supporting such a notion comes from the fact that the murine homolog Zfp-37 is expressed specifically in the brain and testis , mirroring the human array expression pattern. Therefore, it is tempting to posit that the array in humans is an extensive regulatory element of the ZFP37 gene. The location of the chromosome 19 SST1 arrays are intriguing due to the presence of the 29 zinc-finger genes that reside between and immediately proximal/distal of the two macrosatellites. Of the approximately 800 zinc-finger genes in the human genome, a disproportionate number reside on chromosome 19, with most residing in one of 11 clusters . This is not the only kruppel-type zinc finger cluster on chromosome 19 that is associated with tandem repeat DNA, as an extensive cluster at 19p12 is interspersed with blocks of β-satellite . Kruppel-type zinc fingers are implicated in transcriptional regulation of many crucial processes from development to control of the cell cycle to pluripotency . Despite their close proximity, zinc finger genes within a cluster are differentially expressed  and therefore intricate modes of regulation must be required to tightly regulate the expression of cluster members. We propose that the SST1 array at 19q13.12 has a role in the regulation of the surrounding zinc finger gene cluster.
The wealth of information made available from the human genome project has provided an invaluable tool to assist in the identification and characterization of complex repetitive DNA elements such as the macrosatellites. Our results more than double the number of characterized macrosatellite arrays in the human genome and further highlight our lack of understanding as to the role of these unusual sequences in genome biology despite their direct relevance to disease. We anticipate that further characterization of these arrays will reveal the functional significance of macrosatellites arrays, explaining why they are retained in our genome.
Lymphoblastoid cell lines of CEPH family members and the individuals used in the variation panels were obtained from the Coriell Institute for Medical Research http://www.coriell.org/. Cells were maintained according to Coriell recommendations. Culture media (RPMI), fetal bovine serum and supplements were all obtained from Invitrogen corp.
Approximately 4 × 107 cells were resuspended in 1 ml of L-buffer (100 mM EDTA [8.0], 10 mM Tris-HCl [8.0], 20 mM NaCl), before mixing 1:1 with 1.0% (w/v) molten low-melt agarose (Biorad). The cell mixture was transferred to plug moulds (Biorad) with ~80 ul of the cell suspension per plug (approximately 1.6 × 106 cells/plug). Plugs were allowed to set at 4°C for 10 minutes before transfer to 10 volumes of L-buffer containing 1% (w/v) sarkosyl and 1 mg/ml Proteinase-K (Roche) and incubating overnight at 50°C. Plugs were rinsed with water before three washes of one hour each with 50 volumes of TE [8.0]. Plugs were incubated at 50°C for 30 minutes in 10 volumes of TE [8.0] supplemented with 80 ug/ml PMSF (Roche). Plugs were rinsed once more with water before three additional hour-long washes in 50 volumes of TE [8.0] at room temperature before storage at 4°C.
Pulsed field gel electrophoresis
Agarose embedded DNA was digested with the restriction enzymes given in the legend for the appropriate data figures. All enzymes were obtained from NEB. Each plug was first equilibrated in 300 ul of 1× digest buffer at room temperature for 20 minutes, before replacement of buffer with 100 ul 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) in 0.5 × TBE. DNA was separated at 14°C in 0.5 × TBE according to the time and conditions determined by the auto algorithm function of the CHEF Mapper (Biorad). Markers were loaded in the outer lanes (NEB, MidRange PFG Markers I and II).
Southern blotting & hybridization
At the end of the PFGE run, the gel was rinsed with water before staining with ethidium bromide (1 ug/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) overnight by standard Southern blotting . The membrane was rinsed with 2 × SSC before baking at 120°C for 30 minutes.
Macrosatellite specific probes were prepared by PCR amplification using primers listed in table 1. The PCR products were cleaned (Qiagen) before labelling with DIG-11-dUTP by random priming (Roche). 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 manufacturers instructions (Roche). Signals were detected by exposure to photographic film (Kodak).
Reverse Transcription PCR
Human tissue total RNA was obtained from Clontech (636643). 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. Complementary DNA was prepared using 1 ug of total RNA with or without MMLV reverse transcriptase (Invitrogen) according to the manufacturers instructions.
cDNA was amplified using Taq polymerase (NEB) with the following cycle: 95°C for 2 minutes, followed by 35 cycles of 95°C 20 seconds, 58°C 20 seconds, 72°C 30 seconds. Primers used for amplification and size of anticipated product are listed in Table 1.
This work was supported in part by a grant from the National Institutes of Health (GM073120, B.P.C).
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