PAR-TERRA directs homologous sex chromosome pairing

Abstract

In mammals, homologous chromosomes rarely pair outside meiosis. One exception is the X chromosome, which transiently pairs during X-chromosome inactivation (XCI). How two chromosomes find each other in 3D space is not known. Here, we reveal a required interaction between the X-inactivation center (Xic) and the telomere in mouse embryonic stem (ES) cells. The subtelomeric, pseudoautosomal regions (PARs) of the two sex chromosomes (X and Y) also undergo pairing in both female and male cells. PARs transcribe a class of telomeric RNA, dubbed PAR-TERRA, which accounts for a vast majority of all TERRA transcripts. PAR-TERRA binds throughout the genome, including to the PAR and Xic. During X-chromosome pairing, PAR-TERRA anchors the Xic to the PAR, creating a 'tetrad' of pairwise homologous interactions (Xic–Xic, PAR–PAR, and Xic–PAR). Xic pairing occurs within the tetrad. Depleting PAR-TERRA abrogates pairing and blocks initiation of XCI, whereas autosomal PAR-TERRA induces ectopic pairing. We propose a 'constrained diffusion model' in which PAR-TERRA creates an interaction hub to guide Xic homology searching during XCI.

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Figure 1: Homologous sex chromosome pairing via the PAR in differentiating XX and XY ES cells.
Figure 2: PAR transcripts produced by the sex chromosomes.
Figure 3: Mapping genomic PAR-TERRA binding sites by CHIRT-seq.
Figure 4: CHIRT-seq: PAR-TERRA RNA binds in cis and in trans throughout the genome.
Figure 5: PAR-TERRA mediates trans PAR pairing of sex chromosomes.
Figure 6: Intrachromosomal interactions between PAR and Xic occur in a PAR-TERRA-dependent manner.
Figure 7: The tetrad as a hub for pairing interactions.
Figure 8: PAR-TERRA is required for proper initiation of XCI.

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Acknowledgements

We are grateful to Y. Jeon and H. Sunwoo for technical advice. This work was funded by a grant from the NIH (R01-GM58839) and Howard Hughes Medical Institute to J.T.L.

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Contributions

H.-P.C. and J.T.L. designed the experiments and analyzed data. H.-P.C. performed experiments, including FISH, CHIRT-seq, TERRA-capture RNA-seq and LNA knockdown. J.E.F. performed the 4C experiment. S.F.P. established the 4C protocol. H.P.C. and H.J.O. optimized CHIRT protocols. F. J., R.S., B.K. and H.P.C. performed bioinformatics analyses. H.P.C. and J.T.L. wrote the manuscript.

Corresponding author

Correspondence to Jeannie T Lee.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 TERRA transcripts localize to the pseudoautosomal regions of sex chromosomes.

a. Left panel: Map of various X-linked loci. Right panel: d4 pairing assays represented in dotplots of inter-allelic distances for the top decile of nuclei with smallest distance for the loci indicated. Normalized distance (ND) = distance /d, where d = nuclear diameter. P values determined using two-tailed student t-test.b. Representative DNA FISH for Atrx and Ngfrap1 at day 4 during ES differentiation of female cells.c. Full distributions of inter-allelic distances for Atrx and Ngfrap1 for the experiments in a,b. n, sample size. ND, normalized distance. Y-axis represents the fraction of total cell population.d. DNA FISH detecting PAR DNA using P34568 sub-probes of BAC RP24-500I4 DNA (Cy3, red), and X chromosomes (FITC labeled X painting probes, green) on metaphase spread in female ES cells. P345678 probes mark at the ends of X chromosomes.e. Electrophoresis of PCR products amplified from BAC RP24-500I4 DNA. The pools of P3, P4, P5, P6, and P8 PCR produces were used for generating P34568 sub-probes to detect PAR DNA in DNA FISH experiments.f. TERRA RNA FISH followed by PAR DNA FISH using P34567 probes, which are subsets of BAC RP24-500I4 DNA. Higher exposures revealed two dominant and multiple smaller TERRA foci in ES cells. DAPI detects nuclear DNA. Lower exposures showed that the dominant TERRA foci were colocalized with PAR DNA.g. Full distributions of inter-allelic distances for Xic, Arhgap6, and Hprt following DNA FISH in d0 and d4 female ES cells. Corresponds to Figure 1a,b. P values determined by the KS test. Mean values indicated by triangles. Biological replicates showed similar results.h. Full distributions of PAR:PAR inter-allelic distances in differentiating male and female ES cells as marked for the experiment in Fig. 1c. P values determined by the KS test. Mean values indicated by triangles.

Supplementary Figure 2 Analysis of PAR-TERRA transcripts.

a. PAR transcripts resemble TERRA RNA in Northern blotting analysis. Top panel: Map of sub-BAC probes. Northern blot analysis of PAR-TERRA in ES cells using TERRA-specific or PAR-specific oligo probes (27k, 29k, 34k and 47k).b. Read statistics for TERRA-capture RNA-seq. After TERRA capture, cDNA libraries were generated using either random primers or telomeric-repeat primers. These tables are related to Figure 2f.c. Overwhelming majority of (TTAGGG)n-containing RNAs originate from sex chromosomes. Mapped fragment (frag.) counts for TERRA-capture RNA-seq experiments for various chromosomes. The X and Y are grouped together because (i) X and Y PARs are nearly identical, (ii) the highly repetitive nature of the PARs makes sequence assembly nearly impossible at the present time, and (iii) more PAR sequences have been assembled on the Y than on the X. See Panel d below.d. Mapped fragment counts for TERRA-capture RNA-seq experiments for various chromosomes. “+ strand” indicates the forward (Watson) strand. “– strand “ indicates the reverse (Crick) strand. The X and Y reads are separated. See also Panel c above. However, it should be noted that these allelic calls on the X and Y PARs are not fully reliable because (i) X and Y PARs are nearly identical, (ii) the highly repetitive nature of the PARs makes sequence assembly nearly impossible at the present time, and (iii) more PAR sequences have been assembled on the Y than on the X. Thus, the Y-PAR has more mapped reads, but this should be regarded as a technical artifact. RNA FISH signal densitometry suggests the X and Y produce similar amounts of PAR-TERRA in vivo, as XX and XY ES cells have similarly intense PAR-TERRA foci.e. PAR DNA contains long telomeric repeats. Diagrams of PAR BAC clones: RP24-500I4 and RP24-338D22, consist of PAR consensus sequence, GACA simple repeats and telomeric repeats.f. PAR transcripts continue to transcribe into telomeric repeats. The Tophat alignment of TERRA-capture RNA-seq show that PAR-TERRA transcripts are aligned to the junction of GACA and telomeric repeats in PAR BAC DNA (RP24-500I4).

Supplementary Figure 3 PAR-TERRA CHIRT-seq analysis.

a. Quantitative PCR showing the enrichment of PAR DNA following CHIRT using oligo probes TERRA-AS (AS) that targets to TERRA transcripts or sense probes. Various detergents (0.1% NP40, or 0.1% SDS, or 0.1% N-lauryol Sarcosine) were added separately during the final DNA elution. NP40 retains RNaseH activity better than other detergents in CHIRT elution.b. Quantitative PCR showing the enrichment of PAR-TERRA transcripts following CHIRT using oligo probes TERRA-AS (AS) that targets to TERRA transcripts. U1, U6 and GAPDH RNAs were not enriched in TERRA-CHIRT.c. Scatterplot comparing log2 coverages of biological replicates for PAR and TERRA CHIRT-seq analysis in ES cells. Pearson’s r shown. Replicate 1 (Rep1) was normalized with input. Replicate 2 (Rep2) was normalized to RNaseA pre-treated control.d. Read statistics for two biological replicates of the PAR and TERRA CHIRT-seq analysis. TER = TERRA.e. RT-qPCR shows that PAR-TERRA expression is higher in ES cells than in MEF. Data are normalized to GAPDH levels.

Supplementary Figure 4 Correlation of PAR-TERRA binding in cells of various differentiation stages.

Scatterplot analysis comparing Log2 coverages of TERRA and PAR CHIRT between ES cells of various differentiation days and MEFs. Pearson’s r shown. CHIRT results were normalized to input.

Supplementary Figure 5 TERRA depletion also disrupts PAR–PAR pairing between ChrX and ChrY in male ES cells.

a. TERRA knockdown disrupts inter-PAR pairing in male ES cells. Full distributions of inter-PAR distances in d4 ES cells after TERRA versus control (Scr) knockdown. P values determined by the KS test. Mean values indicated by triangles.b. Dotplot of inter-PAR distances for the top decile of nuclei shown in a. P values determined using two-tailed student t-test.

Supplementary Figure 6 Interaction between endogenous and transgenic PAR competes away endogenous PAR–PAR pairing.

a. DNA FISH detecting PAR DNA using P34568 sub-probes of BAC RP24-500I4 DNA (Cy3, red), and Arhgap6 (FITC, green) and P1 (Cy5, cyan blue) PAR transgenic male ES cells. Arhgap6 marks at the end of ChrX. P1 marks transgenic site.b. Copy number determination by FISH densimetry. Quantification by DNA FISH for PAR signals. Transgenic PAR signals are distinguished by colocalization with P1 signals. The transgene signal is 3-4 times lower, indicating that the copy number does not exceed the repeat copy number at the endogenous PAR locus. Mean ratio =3.4 (endogenous versus transgenic PAR). n, sample size.c. DNA FISH to assess pairing between endogenous PARs in d4 transgenic ES cells. PARs detected using P34568 sub-probes of BAC RP24-500I4 DNA (Cy5, cyan blue). The endogenous PARs are distinguished by coincident signals with ChrX_end (RP24-283L17, green; located between Arhgap6 and Mid1) or Y-linked Sry (Cy3, red).d. Full distributions of endogenous PAR:PAR (X-Y) distances in transgenic cells on d4 of differentiation. n, sample size.e. Dotplot of inter-allelic distances for the top decile of transgenic and parental nuclei shown for the experiment shown in c,d. P values determined using two-tailed student t-test. Note loss of pairing between endogenous PARs due to interference by the PAR transgene.

Supplementary Figure 7 TERRA and the subtelomeric region on Chr3.

a. RNA-seq tracks of Wls, a subtelomeric gene on Chromosome 3. Shown are RNA-seq tacks for whole transcriptome (total) and the TERRA-captured fraction. Note that telomeric (TTAGGG)n repeats are distal to Wls, whose transcription extends into the repeats.b. Quantitative RT-PCR shows that Wls expression is impaired by TERRA depletion, but not by PAR depletion in ES cells. When treated with TERRA LNA, Wls transcription is reduced. When treated with PAR LNA, there is no reduction. Thus, PAR LNA affects PAR-TERRA sites, not sites associated with autosomal TERRA. PAR, TERRA, and Scr LNAs were administered for 12 hours prior to RT-qPCR. *, P< 0.05, determined by student t-test.

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Chu, H., Froberg, J., Kesner, B. et al. PAR-TERRA directs homologous sex chromosome pairing. Nat Struct Mol Biol 24, 620–631 (2017). https://doi.org/10.1038/nsmb.3432

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