CCCTC-binding factor is an upstream regulator of the pluripotency factor Oct4 and functions in active transcription of linc1253 and linc1356 genes in pluripotent cells

The mechanisms that regulate pluripotency are still largely unknown. Here, we show that Telomere Repeat Binding Factor 1 (TRF1), a component of the shelterin complex, regulates the genome-wide binding of polycomb and polycomb H3K27me3 repressive marks to pluripotency genes, thereby exerting vast epigenetic changes that contribute to the maintenance of mouse ES cells in a naïve state. We further show that TRF1 mediates these effects by regulating TERRA, the lncRNAs transcribed from telomeres. We find that TERRAs are enriched at polycomb and stem cell genes in pluripotent cells and that TRF1 abrogation results in increased TERRA levels and in higher TERRA binding to those genes, coincidental with the induction of cell-fate programs and the loss of the naïve state. These results are consistent with a model in which TRF1-dependent changes in TERRA levels modulate polycomb recruitment to pluripotency and differentiation genes. These unprecedented findings explain why TRF1 is essential for the induction and maintenance of pluripotency.

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Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription.

Molecular Biology of the Cell ◽

10.1091/mbc.8.1.145 ◽

1997 ◽

Vol 8 (1) ◽

pp. 145-156 ◽

Cited By ~ 21

Author(s):

H R Junéra ◽

C Masson ◽

G Géraud ◽

J Suja ◽

D Hernandez-Verdun

Keyword(s):

Transcription Unit ◽

Rdna Locus ◽

Variable Number ◽

Ribosomal Genes ◽

S Phase ◽

Similar Amount ◽

Rdna Transcription ◽

Binding Factor ◽

Active Transcription ◽

Upstream Binding Factor

The distribution of the ribosomal genes (rDNA) and the upstream binding factor (UBF), correlatively with their RNA transcripts, was investigated in G1, S-phase, and G2. rDNA was distributed in nucleoli, with alternate sites of clustered and dispersed genes. UBF was found associated with some but not all clustered genes and proportionally more with dispersed genes. It was distributed in several foci that were more numerous and heterogeneous in size during G2 than G1. We suggest that UBF associated with rDNA during S-phase because its nucleolar amount increased during that time and remained stable in G2. 5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole treatment indicated a similar amount of UBF per transcription unit, and consequently heterogeneous size of the UBF foci can represent a variable number of transcription units per foci. Direct visualization of the transcripts demonstrated that only part of UBF is associated with active transcription and that rDNA distribution varied with transcription. We propose that in the same rDNA locus three types of configuration coexist that are correlated with gene activity: 1) clustered genes without UBF; 2) clustered genes with UBF, of which some are associated with transcription; and 3) dispersed genes with UBF and transcription. These results support the hypothesis that rDNA transcription involved several steps of regulation acting successively and locally in the same locus to promote the repressed clustered genes to become actively transcribed dispersed genes.

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Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

Genome Biology ◽

10.1186/s13059-021-02321-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Oriana Genolet ◽

Anna A. Monaco ◽

Ilona Dunkel ◽

Michael Boettcher ◽

Edda G. Schulz

Keyword(s):

Stem Cells ◽

Sex Differences ◽

Embryonic Stem Cells ◽

Genetic Basis ◽

Embryonic Stem ◽

Pluripotent Cells ◽

Screening Approach ◽

Factor Expression ◽

Chromosomal Genes ◽

Pluripotency Factor

Abstract Background X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation. Results To identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression. Conclusions Here, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.

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CTCF is a barrier for 2C-like reprogramming

Nature Communications ◽

10.1038/s41467-021-25072-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Teresa Olbrich ◽

Maria Vega-Sendino ◽

Desiree Tillo ◽

Wei Wu ◽

Nicholas Zolnerowich ◽

...

Keyword(s):

Dna Damage ◽

Transcription Factor ◽

Transcriptional Activation ◽

Binding Sites ◽

Neural Progenitor Cells ◽

Ctcf Binding ◽

Pluripotent Cells ◽

Embryonic Tissues ◽

Binding Factor ◽

Molecular Determinants

AbstractTotipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.

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Chromatin signature of widespread monoallelic expression

eLife ◽

10.7554/elife.01256 ◽

2013 ◽

Vol 2 ◽

Cited By ~ 46

Author(s):

Anwesha Nag ◽

Virginia Savova ◽

Ho-Lim Fung ◽

Alexander Miron ◽

Guo-Cheng Yuan ◽

...

Keyword(s):

Cell Types ◽

Great Accuracy ◽

Monoallelic Expression ◽

Gene Body ◽

Pluripotent Cells ◽

Chromatin Signature ◽

New Approach ◽

Molecular Features ◽

Active Transcription ◽

Clonal Nature

In mammals, numerous autosomal genes are subject to mitotically stable monoallelic expression (MAE), including genes that play critical roles in a variety of human diseases. Due to challenges posed by the clonal nature of MAE, very little is known about its regulation; in particular, no molecular features have been specifically linked to MAE. In this study, we report an approach that distinguishes MAE genes in human cells with great accuracy: a chromatin signature consisting of chromatin marks associated with active transcription (H3K36me3) and silencing (H3K27me3) simultaneously occurring in the gene body. The MAE signature is present in ∼20% of ubiquitously expressed genes and over 30% of tissue-specific genes across cell types. Notably, it is enriched among key developmental genes that have bivalent chromatin structure in pluripotent cells. Our results open a new approach to the study of MAE that is independent of polymorphisms, and suggest that MAE is linked to cell differentiation.

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Identification of X-chromosomal genes that drive global X-dosage effects in mammals

10.1101/2020.03.09.983544 ◽

2020 ◽

Cited By ~ 2

Author(s):

Oriana Genolet ◽

Anna A. Monaco ◽

Ilona Dunkel ◽

Michael Boettcher ◽

Edda G. Schulz

Keyword(s):

Sex Differences ◽

Stem Cell ◽

Target Genes ◽

Embryonic Stem ◽

Adaptor Protein ◽

Induced Pluripotent Stem Cell ◽

Pluripotent Cells ◽

Factor Expression ◽

Chromosomal Genes ◽

Pluripotency Factor

AbstractX-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Here, double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signalling pathway and delaying differentiation. To identify the genetic basis of these sex differences, we have performed a series of CRISPR knockout screens in murine embryonic stem cells to comprehensively identify X-linked genes that cause the female pluripotency phenotype. We found multiple genes that act in concert, among which Klhl13 plays a central role. We show that this E3 ubiquitin ligase substrate adaptor protein promotes pluripotency factor expression, delays differentiation and represses MAPK target genes, and we identify putative substrates. We thus elucidate the mechanisms that drive sex-induced differences in pluripotent cells with implications for gender medicine in the context of induced pluripotent stem cell based therapies.

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A role for upstream binding factor in organizing ribosomal gene chromatin

Biochemical Society Symposium ◽

10.1042/bss0730077 ◽

2006 ◽

Vol 73 ◽

pp. 77-84 ◽

Cited By ~ 14

Author(s):

Jane E. Wright ◽

Christine Mais ◽

José-Luis Prieto ◽

Brian McStay

Keyword(s):

Nucleolar Organizer ◽

Ribosomal Gene ◽

Rna Polymerase I ◽

Nucleolar Organizer Regions ◽

Binding Factor ◽

Upstream Binding Factor ◽

Acrocentric Chromosomes ◽

Polymerase I ◽

Secondary Constrictions ◽

Active Nors

Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus, and during metaphase form novel silver-stainable secondary constrictions, termed pseudo-NORs, that are morphologically similar to NORs.

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