Abstract
On the male X and female active X chromosome (Xa), the macrosatellite repeat (MSR) DXZ4 is packaged into constitutive heterochromatin characterized by CpG methylation and histone H3 tri-methylated at lysine-9 (H3K9me3). In contrast, DXZ4 on the female inactive X chromosome (Xi), is packaged into euchromatin, is bound by the architectural protein CCCTC-binding factor, and mediates Xi-specific long-range cis contact with similarly packaged tandem repeats on the Xi. In cancer, male DXZ4 can inappropriately revert to a Xi-like state and other MSRs have been reported to adopt alternate chromatin configurations in response to disease. Given this plasticity, we sought to identify factors that might control heterochromatin at DXZ4. In human embryonic stem cells, we found low levels of 5-hydroxymethylcytosine at DXZ4 and that this mark is lost upon differentiation as H3K9me3 is acquired. We identified two previously undescribed DXZ4 associated noncoding transcripts (DANT1 and DANT2) that are transcribed toward DXZ4 from promoters flanking the array. Each generates transcript isoforms that traverse the MSR. However, upon differentiation, enhancer of Zeste-2 silences DANT1, and DANT2 transcription terminates prior to entering DXZ4. These data support a model wherein DANT1 and/or DANT2 may function to regulate constitutive heterochromatin formation at this MSR.
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Abbreviations
- 5-hmC:
-
5′-Hydoxymethylcytosine
- 5-mC:
-
5′-Methylcytosine
- ATCC:
-
American Type Culture Collection
- ATT:
-
Array-traversing transcript
- BiS:
-
Bisulfite sequencing
- CGI:
-
CpG island
- ChIP:
-
Chromatin immunoprecipitation
- CTCF:
-
CCCTC-binding factor
- DANT1:
-
DXZ4-associated noncoding transcript, proximal
- DANT2:
-
DXZ4-associated noncoding transcript, distal
- dsRNA:
-
Double-stranded RNA
- EBOGs:
-
Embryoid body out-growths
- EZH2:
-
Enhancer of Zeste 2
- ESTs:
-
Expressed sequence tags
- FSHD:
-
Facioscapulohumeral muscular dystrophy
- H3K4me2:
-
Histone H3 di-methylated at lysine 4
- H3K9me3:
-
Histone H3 tri-methyated at lysine 9
- hESCs:
-
Human embryonic stem cells
- HGNC:
-
Human Gene Nomenclature Committee
- kb:
-
Kilobases
- LINE:
-
Long interspersed elements
- IncRNA:
-
Long noncoding RNA
- LTR:
-
Long terminal repeats
- mRNA:
-
Messenger RNA
- MSR:
-
Macrosatellite repeat
- ORF:
-
Open reading frame
- oxBiS:
-
Oxidative bisulfite sequencing
- PBS:
-
Phosphate-buffered saline
- PRC2:
-
Polycomb repressive complex 2
- qChIP:
-
Quantitative chromatin immunoprecipitation
- qPCR:
-
Quantitative PCR
- RACE:
-
Rapid amplification of cDNA ends
- RT-PCR:
-
Reverse transcription PCR
- SR:
-
Simple repeat
- SSC:
-
Saline-sodium citrate
- TSS:
-
Transcription start sites
- Xa:
-
Active X chromosome
- XCI:
-
X chromosome inactivation
- Xi:
-
Inactive X chromosome
- XIST:
-
X inactive specific transcript
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Acknowledgments
This work was supported by the National Institutes of Health [GM073120 to B.P.C.] and by a subaward from P01GM085354 to Dr. Stephen Dalton, University of Georgia.
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Experiments performed in this manuscript comply with the current laws of the USA.
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Debbie M. Figueroa declares that she has no conflict of interest.
Emily M. Darrow declares that she has no conflict of interest.
Brian P. Chadwick declares that he has no conflict of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Figueroa, D.M., Darrow, E.M. & Chadwick, B.P. Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat. Chromosome Res 23, 733–752 (2015). https://doi.org/10.1007/s10577-015-9479-3
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DOI: https://doi.org/10.1007/s10577-015-9479-3