High-resolution RNA allelotyping along the inactive X chromosome: evidence of RNA polymerase III in regulating chromatin configuration

Ru, Hong; Lin, Bingqing; Lu, Xinyi; Lai, Lan-Tian; Chen, Xin; Sanyal, Amartya; Ng, Huck‐Hui; Zhang, Kun; Zhang, Lifeng

doi:10.1038/srep45460

Cited by 11 publications

(18 citation statements)

References 40 publications

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“…Additionally, we observed a significant enrichment of SINE elements near promoters of escapees and early reactivated genes in cluster 5 (Supplementary Fig. 4h), a property previously described for escapees and genes prone for reactivation after Xistdepletion 70,71 . Moreover, while we did not detect increased expression levels of early genes in NPCs, where they were still transcriptionally inactive (Supplementary Fig.…”

Section: Esrrb Zfp42supporting

confidence: 74%

Chromosome compartments on the inactive X guide TAD formation independently of transcription during X-reactivation

et al. 2021

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A hallmark of chromosome organization is the partition into transcriptionally active A and repressed B compartments, and into topologically associating domains (TADs). Both structures were regarded to be absent from the inactive mouse X chromosome, but to be re-established with transcriptional reactivation and chromatin opening during X-reactivation. Here, we combine a tailor-made mouse iPSC reprogramming system and high-resolution Hi-C to produce a time course combining gene reactivation, chromatin opening and chromosome topology during X-reactivation. Contrary to previous observations, we observe A/B-like compartments on the inactive X harbouring multiple subcompartments. While partial X-reactivation initiates within a compartment rich in X-inactivation escapees, it then occurs rapidly along the chromosome, concomitant with downregulation of Xist. Importantly, we find that TAD formation precedes transcription and initiates from Xist-poor compartments. Here, we show that TAD formation and transcriptional reactivation are causally independent during X-reactivation while establishing Xist as a common denominator.

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Section: Esrrb Zfp42supporting

confidence: 74%

Chromosome compartments on the inactive X guide TAD formation independently of transcription during X-reactivation

et al. 2021

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“…Padlock capture was performed as previously described 1 , 3 , 15 . Briefly, each reaction was performed in 20 µl volume containing 1 unit Ampligase (A3210K, Epicentre), 1 unit Phusion High-Fidelity DNA Polymerase (M0530, New England BioLabs), 1 x Phusion High-Fidelity DNA Polymerase buffer, 10 nM dNTP and 1 ng padlock probe.…”

Section: Methodsmentioning

confidence: 99%

“…The common linker sequence of each padlock probe allows a common PCR primer pair to amplify all the padlock capture products for deep sequencing. It has been shown that a padlock probe library containing tens of thousands of padlock probes worked efficiently 1 , 3 . Compared with other available methods for targeted sequencing, padlock capture is more suitable for population-based carrier screens, as once synthesized, the padlock library can be easily regenerated by PCR, whereby microarrays or RNA baits used for target enrichment in other methods are expensive and non-reusable 4 .…”

Section: Introductionmentioning

confidence: 99%

Cat-D: a targeted sequencing method for the simultaneous detection of small DNA mutations and large DNA deletions with flexible boundaries

Chandola

Zhang

2017

Sci Rep

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We developed a targeted DNA sequencing method that is capable of detecting a comprehensive panel of DNA mutations including small DNA mutations and large DNA deletions with unknown/flexible boundaries. The method directly identifies the large DNA deletions (Cat-D) without relying on sequencing coverage to make the genotype calls. We performed the method to simultaneously detect 10 small DNA mutations in β-thalassemia and 2 large genomic deletions in α-thalassemia from 10 genomic DNA samples. Cat-D was performed on 8 genomic DNA samples in duplicate. The 18 Cat-D samples were combined in one sequencing run. In total, 216 genotype calls were made, and 215 of the genotype calls were accurate. No false negative genotype calls were made. One false positive genotype call was made on one target mutation in one experimental duplicate from a genomic DNA sample. In summary, Cat-D can be developed into a robust, high-throughput and cost-effective method suitable for population-based carrier screens.

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“…Therefore, the XCI status of an X-linked gene can be evaluated by RNA allelotyping of X 129 and X Cast , which provide ample choice of single nucleotide polymorphisms (SNPs). Padlock SNP capture, a high-throughput and high-resolution RNA allelotyping method (19), was performed to profile the XCI status of X linked genes chromosome-wide. The padlock probe library was designed to target 2,969 SNPs covering 1,110 (~55%) of the X-linked genes (19).…”

Section: Atac-seq Was Performed Using Male Es Cell Lines Carrying An mentioning

confidence: 99%

“…Padlock SNP capture, a high-throughput and high-resolution RNA allelotyping method (19), was performed to profile the XCI status of X linked genes chromosome-wide. The padlock probe library was designed to target 2,969 SNPs covering 1,110 (~55%) of the X-linked genes (19). 457 X-linked genes were successfully allelotyped in the experiment.…”

Section: Atac-seq Was Performed Using Male Es Cell Lines Carrying An mentioning

confidence: 99%

The lupus autoantigen La is anXist-binding protein involved inXistfolding and cloud formation

Ding

et al. 2020

Preprint

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Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, the lupus autoantigen La (encoding gene Ssb) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female ES cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. La is a ubiquitous and versatile RNA-binding protein with RNA chaperone activity. Functional dissection of La shows that the RNA chaperon domain plays critical roles in XCI. In mutant cells, Xist transcripts are misfolded and unstable. These results show that La is involved in XCI as an RNA chaperone for Xist. 3 SIGNIFICANCE STATEMENTXist RNA functions as a scaffold to recruit and assemble a protein machinery to epigenetically silence genes along one X chromosome in each female mammalian cell. Here, we devised a FLAG-out system to profile the Xist interactome, and identified the lupus autoantigen La as an Xist-binding protein. The RNA chaperone activity of La is essential for X chromosome inactivation (XCI). When La is downregulated, the folding of Xist RNA is altered, leading to shorter half-life of Xist RNA and compromised Xist cloud formation, which in turn results in XCI defects, including chromatin compaction, chromosome-wide monoallelic gene expression pattern, and death of female ES cells during in vitro differentiation.

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High-resolution RNA allelotyping along the inactive X chromosome: evidence of RNA polymerase III in regulating chromatin configuration

Cited by 11 publications

References 40 publications

Chromosome compartments on the inactive X guide TAD formation independently of transcription during X-reactivation

Chromosome compartments on the inactive X guide TAD formation independently of transcription during X-reactivation

Cat-D: a targeted sequencing method for the simultaneous detection of small DNA mutations and large DNA deletions with flexible boundaries

The lupus autoantigen La is anXist-binding protein involved inXistfolding and cloud formation

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