Dataset Information

Comparison non-targeting hRNA hESCs versus UBE2K shRNA hESCs

ABSTRACT: Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. When compared with their differentiated counterparts, immortal human embryonic stem cells (hESCs) have a unique chromatin architecture and low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess a link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of UBE2K, a ubiquitin-conjugating enzyme. Loss of UBE2K increases the levels of H3K9 trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Concomitantly, loss of UBE2K impairs the ability of hESCs to differentiate into neural progenitors with neurogenic properties. Besides H3K9 trimethylation, we find that UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates both histone H3 levels and H3K9 trimethylation in C. elegans germline. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryo, Embryonic Stem Cell

SUBMITTER: David Vilchez

LAB HEAD: David Vilchez

PROVIDER: PXD018625 | Pride | 2020-06-01

REPOSITORIES: Pride

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			Action	DRS
	20150601_AWH_Vilchez_DilberIrmak_Ube2k1_16.raw	Raw
	20150601_AWH_Vilchez_DilberIrmak_Ube2k1_17.raw	Raw
	20150601_AWH_Vilchez_DilberIrmak_Ube2k1_18.raw	Raw
	20150601_AWH_Vilchez_DilberIrmak_Ube2k1_19.raw	Raw
	20150601_AWH_Vilchez_DilberIrmak_Ube2k1_20.raw	Raw

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Publications

The ubiquitin-conjugating enzyme UBE2K determines neurogenic potential through histone H3 in human embryonic stem cells.

Fatima Azra A Irmak Dilber D Noormohammadi Alireza A Rinschen Markus M MM Das Aniruddha A Leidecker Orsolya O Schindler Christina C Sánchez-Gaya Víctor V Wagle Prerana P Pokrzywa Wojciech W Hoppe Thorsten T Rada-Iglesias Alvaro A Vilchez David D

Communications biology 20200525 1

Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. Human embryonic stem cells (hESCs) have a unique chromatin architecture characterized by low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess the link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that ...[more]

PMID: 32451438

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Project description:X chromosome inactivation (XCI) is a dosage compensation mechanism in female cells to regulate X-linked gene expression. We report here that subcultures from established lines of female hESCs displayed variations (0-100%) in the expression of XCI markers such as XIST RNA coating and enrichment of histone H3 lysine 27 trimethylation (H3K27me3) on inactive X chromosome. Surprisingly, regardless of the presence or absence of XCI markers in different cultures, all female hESCs we examined (H7, H9, and HSF6 cells) exhibit a mono-allelic expression pattern for a majority of X-linked genes. Our results suggest that these established female hESCs have completed XCI during the process of derivation and/or propagation, and the XCI pattern of lines we investigated is already non-random. However, XIST gene expression in subsets of female hESCs is unstable and subject to epigenetic silencing through DNA methylation. Concomitant with the loss of XCI markers including XIST expression and H3K27me3, approximately 12% of X-linked CpG islands become hypomethylated and a subset of previously silenced X-linked alleles are reactivated, resulting a significant elevation of gene expression dosage. Because changes in dosage compensation of X-linked genes could impair somatic cell function, we propose that XCI status should be routinely checked in subcultures of female hESCs, with cultures displaying XCI markers better suited for use in regenerative medicine. Keywords: Genotyping, gene expression and DNA methylation We used Affymetrix Genotyping array for looking for X-linked SNPs with HSF6, H7 and H9 genomic DNA, Agilent Gene expression array for comparing gene expression patten changes between HSF6 hESCs with X-inactiation and HSF6 hESCs without X-inactivation (3 repeats). Finally, mDIP-Chip method was used to detect X-linked CpG island methylation changes differences between hESCs with X-inactivation and hESCs without X-inactivation using Agilent CpG island array (3 repeats, and one of them has dye swap)