TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions

Prasasya, Rexxi D.; Caldwell, Blake A.; Liu, Zhengfeng; Wu, Shaohang; Leu, N. Adrian; Fowler, Johanna M.; Cincotta, Steven A.; Laird, Diana J.; Kohli, Rahul M.; Bartolomei, Marisa S.

doi:10.1101/2023.02.21.529426

Cited by 2 publications

(4 citation statements)

References 113 publications

(228 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition to TEs, imprinted gene DMRs, germ cells, and placenta-specific hypomethylated sites show later DNA methylation loss compared to the rest of the genome. This is due to active DNA demethylation pathways, mediated by TET proteins, that are required for methylation erasure at imprinting control regions ( 39 , 66 ). CGs flanked by A/Ts are more susceptible to aging-associated DNA demethylation ( 51 ).…”

Section: Resultsmentioning

confidence: 99%

“…Gonads were dissected in calcium- and magnesium-free PBS (Gibco) and transferred into 500 μl of 0.25% Trypsin–EDTA (Gibco). Subsequently, the preparation of embryonic germ cells was carried out following the method previously described ( 39 ). For bisulfite mutagenesis, PGCs were snap-frozen for storage at –80°C until further processing.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Lee,

Loo,

Prasasya

et al. 2024

Nucleic Acids Research

View full text Add to dashboard Cite

The Infinium BeadChip is the most widely used DNA methylome assay technology for population-scale epigenome profiling. However, the standard workflow requires over 200 ng of input DNA, hindering its application to small cell-number samples, such as primordial germ cells. We developed experimental and analysis workflows to extend this technology to suboptimal input DNA conditions, including ultra-low input down to single cells. DNA preamplification significantly enhanced detection rates to over 50% in five-cell samples and ∼25% in single cells. Enzymatic conversion also substantially improved data quality. Computationally, we developed a method to model the background signal's influence on the DNA methylation level readings. The modified detection P-value calculation achieved higher sensitivities for low-input datasets and was validated in over 100 000 public diverse methylome profiles. We employed the optimized workflow to query the demethylation dynamics in mouse primordial germ cells available at low cell numbers. Our data revealed nuanced chromatin states, sex disparities, and the role of DNA methylation in transposable element regulation during germ cell development. Collectively, we present comprehensive experimental and computational solutions to extend this widely used methylation assay technology to applications with limited DNA.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Lee,

Loo,

Prasasya

et al. 2024

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…Gonads were dissected in calcium-and magnesium-free PBS (Gibco) and transferred into 500 μl of 0.25% Trypsin-EDTA (Gibco). Subsequently, the preparation of embryonic germ cells was carried out following the method previously described (38). For bisulfite mutagenesis, PGCs were snap-frozen for storage at -80 o C until further processing.…”

Section: Mouse Primordial Germ Cellsmentioning

confidence: 99%

“…In addition to transposable elements, imprinted gene DMRs, germ cells, and placenta-specific hypomethylated sites show later DNA methylation loss compared to the rest of the genome. This is due to active DNA demethylation pathways, mediated by TET proteins, that are required for methylation erasure at imprinting control regions (63,64). CGs flanked by A/Ts are more susceptible to aging-associated DNA demethylation (49).…”

Section: Low-input Beadchip Data Captures the Demethylation Dynamics ...mentioning

confidence: 99%

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Lee,

Loo,

Prasasya

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

The Infinium BeadChip is the most widely used DNA methylome assay technology for population- scale epigenome profiling. However, the standard workflow requires over 200 ng of input DNA, hindering its application to small cell-number samples, such as primordial germ cells. We developed experimental and analysis workflows to extend this technology to suboptimal input DNA conditions, including ultra-low input down to single cells. DNA preamplification significantly enhanced detection rates to over 50% in five-cell samples and ~25% in single cells. Enzymatic conversion also substantially improved data quality. Computationally, we developed a method to model the background signal's influence on the DNA methylation level readings. The modified detectionp-values calculation achieved higher sensitivities for low-input datasets and was validated in over 100,000 public datasets with diverse methylation profiles. We employed the optimized workflow to query the demethylation dynamics in mouse primordial germ cells available at low cell numbers. Our data revealed nuanced chromatin states, sex disparities, and the role of DNA methylation in transposable element regulation during germ cell development. Collectively, we present comprehensive experimental and computational solutions to extend this widely used methylation assay technology to applications with limited DNA.

show abstract

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions

Cited by 2 publications

References 113 publications

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Low-input and single-cell methods for Infinium DNA methylation BeadChips

Contact Info

Product

Resources

About