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In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells - PubMed

  • ️Fri Jan 01 2016

In Vivo Chromatin Targets of the Transcription Factor Yin Yang 2 in Trophoblast Stem Cells

Raquel Pérez-Palacios et al. PLoS One. 2016.

Abstract

Background: Yin Yang 2 (YY2) is a zinc finger protein closely related to the well-characterized Yin Yang 1 (YY1). YY1 is a DNA-binding transcription factor, with defined functions in multiple developmental processes, such as implantation, cell differentiation, X inactivation, imprinting and organogenesis. Yy2 has been treated as a largely immaterial duplication of Yy1, as they share high homology in the Zinc Finger-region and similar if not identical in vitro binding sites. In contrast to these similarities, gene expression alterations in HeLa cells with attenuated levels of either Yy1 or Yy2 were to some extent gene-specific. Moreover, the chromatin binding sites for YY2, except for its association with transposable retroviral elements (RE) and Endogenous Retroviral Elements (ERVs), remain to be identified. As a first step towards defining potential Yy2 functions matching or complementary to Yy1, we considered in vivo DNA binding sites of YY2 in trophoblast stem (TS) cells.

Results: We report the presence of YY2 protein in mouse-derived embryonic stem (ES) and TS cell lines. Following up on our previous report on ERV binding by YY2 in TS cells, we investigated the tissue-specificity of REX1 and YY2 binding and confirm binding to RE/ERV targets in both ES cells and TS cells. Because of the higher levels of expression, we chose TS cells to understand the role of Yy2 in gene and chromatin regulation. We used in vivo YY2 association as a measure to identify potential target genes. Sequencing of chromatin obtained in chromatin-immunoprecipitation (ChIP) assays carried out with αYY2 serum allowed us to identify a limited number of chromatin targets for YY2. Some putative binding sites were validated in regular ChIP assays and gene expression of genes nearby was altered in the absence of Yy2.

Conclusions: YY2 binding to ERVs is not confined to TS cells. In vivo binding sites share the presence of a consensus binding motif. Selected sites were uniquely bound by YY2 as opposed to YY1, suggesting that YY2 exerts unique contributions to gene regulation. YY2 binding was not generally associated with gene promoters. However, several YY2 binding sites are linked to long noncoding RNA (lncRNA) genes and we show that the expression levels of a few of those are Yy2-dependent.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Yy2 and Mbtps2 expression in ES and TS cells.

(A) Expression of YY2 was detected by indirect immunofluorescence in ES cells and TS cells. YY2 was visualized using αRabbitBiotin followed by StrepAlexa488, nuclei were stained with DAPI (blue). (B) An EtBr stained gel shows PCR products generated with either Yy2 or H2afz-specific primers (as indicated), after amplification of cDNA obtained from ES and TS cells. (C) Expression of Yy2 and cell type-specific markers (or lineage markers) of ES cells (Oct4, Sox2, Nanog) or TS cells (Cdx2, Eomes) was measured by RT-qPCR. Gene expression was normalized to Gapdh and data is represented as the mean ± SEM. (D) Expression of Yy2 and Mbtps2-001 was detected by quantitative RT-PCR in (E14T) ES cells or (TSB7) TS cells (error bars represent SEM). Transcript levels were normalized to H2afz and to the expression in ES cells (100%).

Fig 2
Fig 2. REX1 and YY2 bind RE/ERV elements in both ES and TS cells.

(A) REX1 association to RE in TS cells. Binding was assessed in chromatin immunoprecipitation assays using αREX1 serum followed by quantification of precipitated DNA using real-time qPCR amplification. The figure shows analysis of non-binding reference sites as controls (CGR-A, CGR-B), and Gapdh, several sequences present in the genome as multiple copies (MLV36, γ-satellite) and the ERV elements indicated. Association is represented as percentage bound (relative to purified chromatin extract from the same lysate). Enrichment was calculated as percentage association relative to control chromatin, and is represented as fold binding or enrichment relative to a non-binding reference gene Gapdh. Error bars indicate SEM. (B) As A except for YY2 binding in ES cells. Data on CGR-A, CGR-B and musD are from a single experiment. (C) YY2 binding to Class III elements. Binding of YY2 in ES cells to class III ERV sequences present in the genome as multiple copies (Orr1 and MalR) was assessed by qPCR analysis as described in A. The figure shows fold enrichment relative to a non-binding reference gene Gapdh. Data on CGR-C, musD, Tsix and Spata are from a single experiment.

Fig 3
Fig 3. YY2 binding to genomic targets.

(A) Semiquantitative PCR and (B) qPCR to validate YY2 association in TS cells to peaks identified by sequencing (Table 2). (A) EtBr stained gel shows PCR products of the genomic regions immunoprecipitated by YY2 (αYY2). Preimmune serum (PreI) was used as a control. Purified chromatin extract from the lysate was used to confirm amplicons. MQ, reactions without input DNA served as a negative control. Gapdh promoter was used as a reference gene. (B) Association of YY2 in TS cells to potential genomic targets (Table 2) was assessed by locus-specific qPCR analysis after chromatin immunoprecipitation using αYY2, or preimmune serum (PreI) as a control. The Gapdh promoter is included as a negative control. The amount of immunoprecipitated DNA as a percentage of input DNA was recalculated as fold association normalized to the Gapdh promoter. Error bars represent SEM. (C) As B, data is represented directly as a percentage of input DNA. Each panel represents an independent experiment.

Fig 4
Fig 4. Association of YY1 and YY2 to chromatin.

(A) A motif enriched in the genome wide YY2 ChIP-seq peaks is depicted as a sequence logo. Data from the top 20 peaks were analyzed as described in M&M. (B) Association of YY1 (in ES cells) or YY2 (in TS cells) to genomic targets was assessed as described in the legend to Fig 3B, using either αYY1 (left panel) or αYY2 serum (right panel). Codes for genomic targets analyzed refer to a YY1 genomic target (YY1T1) and several YY2 target genes (T5T1, T18T1) from Table 2 (see text for S5T1). The data in the left panel represent the average of three independent experiments ± SEM. The right panel shows the results of a typical experiment out of several performed, except for T18T1, multiple replicates performed.

Fig 5
Fig 5. Expression analysis of genes associated with YY2 binding sites.

(A-C) Gene expression levels in TS cells with attenuated Yy2 levels. Cells were sorted by FACS after transfection with plasmids co-expressing shRNAs and GFP. Expression of either Yy2 itself A, or of putative YY2 target genes B, C was compared between cells expressing a shYy2 sequence (GFP+/ShYy2) and control cells (GFP-). All qPCR data were calculated using Gapdh as a reference gene and values are expressed relative to GFP-negative ES cells from the same experiment (100%). Data shown represent the average of two or more independent experiments ± SEM. No gene expression differences were observed between GFP positive and negative cells transfected with a construct carrying a non-specific shControl sequence. (D) Representation of a lncRNA locus analyzed on Chromosome 4 (GRCm38 Chr4:147018235–147491046). The primers utilized and the direction of transcription of the different transcripts is indicated with arrows and horizontal arrowheads, respectively. The location of the relevant YY2 peak number 2 (Table 2) is indicated with an arrowhead. (E) Localization of the 21 YY2 binding sites identified in TS cells. Peaks are grouped according to the location relative to transcribed annotated features in the database (NCBIM37).

  1. TSS: within 2500 bp of a transcription start site;

  2. O: overlapping feature;

  3. DU: distal and upstream to TSS;

  4. DD: distal from TSS and downstream of transcribed feature;

  5. I: intergenic DNA at 100 kb from nearest annotated feature

(F) Association of peaks with lncRNA genes. The graph shows the YY2 binding sites that map within 5 kilobases with respect to the nearest lncRNA gene (lncRNA within 5 kb), within 100 kilobases with respect to the nearest lncRNA gene (lncRNA within 100 kb) and other sites. (G) Expression levels of lncRNAs associated with YY2 binding sites (indicated in D) in Yy2-attenuated TS cells as described in A-C.

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This work has been supported by grant PI07119 from FIS/CarlosIII, Ministry of Health; PAMER grants, Aragon Health Sciences Institute, Spain; grant PI110/09 from Dpto de CTU (Gobierno de Aragón) and annual grants B77 from the Government of Aragon (Department of Research and Innovation)/European Social Funds. R. Pérez-Palacios and S. Macías-Redondo were supported by PhD fellowships B138/10 and C071/2014, respectively, from the DGA (Aragón, Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.