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Chromosome Research

, Volume 23, Issue 4, pp 733–752 | Cite as

Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat

  • Debbie M. Figueroa
  • Emily M. Darrow
  • Brian P. Chadwick
Article

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.

Keywords

Macrosatellite DXZ4 X chromosome inactivation Human embryonic stem cells Euchromatin and heterochromatin Long noncoding RNA 

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

Notes

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.

Ethical standards

Experiments performed in this manuscript comply with the current laws of the USA.

Conflict of interest

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.

Supplementary material

10577_2015_9479_MOESM1_ESM.pdf (7.5 mb)
ESM 1 (PDF 7684 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Debbie M. Figueroa
    • 1
    • 2
  • Emily M. Darrow
    • 1
  • Brian P. Chadwick
    • 1
  1. 1.Department of Biological ScienceFlorida State UniversityTallahasseeUSA
  2. 2.NHLBI, National Institutes of HealthBethesdaUSA

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