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, 9 (1), 17033

The Transcriptional Regulator CBX2 and Ovarian Function: A Whole Genome and Whole Transcriptome Approach

Affiliations

The Transcriptional Regulator CBX2 and Ovarian Function: A Whole Genome and Whole Transcriptome Approach

Leila Bouazzi et al. Sci Rep.

Abstract

The chromobox homolog 2 (CBX2) was found to be important for human testis development, but its role in the human ovary remains elusive. We conducted a genome-wide analysis based on DNA adenine methyltransferase identification (DamID) and RNA sequencing strategies to investigate CBX2 in the human granulosa cells. Functional analysis revealed that CBX2 was upstream of genes contributing to ovarian function like folliculogenesis and steroidogenesis (i.e. ESR1, NRG1, AKR1C1, PTGER2, BMP15, BMP2, FSHR and NTRK1/2). We identified CBX2 regulated genes associated with polycystic ovary syndrome (PCOS) such as TGFβ, MAP3K15 and DKK1, as well as genes implicated in premature ovarian failure (POF) (i.e. POF1B, BMP15 and HOXA13) and the pituitary deficiency (i.e. LHX4 and KISS1). Our study provided an excellent opportunity to identify genes surrounding CBX2 in the ovary and might contribute to the understanding of ovarian physiopathology causing infertility in women.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(a) Cytoscape representation of GO-enrichment analysis of CBX2.1 targets. Every dot represents a gene related to the enriched GO‐terms. In green are the GO‐terms over-presented in the developmental process. In blue are the GO‐terms involved in morphogenesis process. In red are all GO-terms related to Molecular Function. The orange colour represents the cluster of genes coding for signalling pathways. Some regulatory processes were over-represented by the yellow colour. The genes presented in the mustard colour were over-represented in response to a stimulus. All data is filtered according to p < 0.05. (b) Cytoscape representation of GO-enrichment analysis of CBX2.2 targets. The green colour represents the GO‐terms which are involved in morphogenesis and differentiation process. In the blue cluster, we found GO‐terms involved in the developmental process. The red colour indicates genes responsible for Molecular Function. The yellow colour represents the regulatory processes. The orange colour is the cluster, which contains genes coding for signalling processes. The genes present in the mustard colour cluster were over-represented in response to a stimulus. All data is filtered according to p < 0.05.
Figure 2
Figure 2
Regulatory network of downstream targets of CBX2.1 in the ovary. We created by the mean of PathwayStudio 11 a network relating the ovarian targets. The genes were found highly interconnected. CBX2.1 targets are SOX9, POF1B, DKK1, ANGPTL4, CYP19A1, DMRT1, KISS1, EMX2, ESR2, POU4F1, FZD7, ESR1, NRG1, BMP2, PTGER2 and FSHR. The interactions of the genes are represented by diverse arrows. Red arrows (T) indicate negative regulations, the green arrows symbolize positive regulations and grey arrows are undefined effects.
Figure 3
Figure 3
Potential role of CBX2.1 and CBX2.2 in the regulation of sex development. The developmental stages plotted in relation to time are indicated as sex determination. We presented male and female factors involved in gonadal development. Green arrows indicate stimulatory effects of CBX2.1 and CBX2.2 on examined downstream factors; the red arrows (T) indicate inhibitory effects.
Figure 4
Figure 4
(a) RT-qPCR analysis of CBX2.1 downstream genes identified by DamID. Relative expression levels (2−ΔΔCt) of the genes were determined after normalization to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). Following CBX2.1 overexpression (Ov Exp. CBX2.1), ESR1, NRG1, BMP2, PTGER2, and FZD7 were found downregulated by CBX2.1 compared to the control set at 1. After silencing the CBX2.1 (si CBX2.1), genes were significantly upregulated except for ESR1 which showed an effect comparable to scrambled sample. All graphs are the average of three independent experiments, error bars represent the standard deviation (SD) from the mean (SEM) and values are expressed as relative to control =1; ***P < 0.001; **P < 0.01 and *P < 0.05. non-significant differences are not indicated. (b) Relative expression levels (2−ΔΔCt) of CBX2.1 related genes. POF1B, DKK1 and SOX9 were upregulated after CBX2.1 forced expression (Ov. Exp CBX2.1). Whereas, when CBX2.1 was silenced (si CBX2.1), SOX9 and POF1B were significantly downregulated. DKK1 did not show any expression change towards the siCBX2.1. All graphs are the average of three independent experiments, error bars represent SD from the mean (SEM), and values are expressed as relative to control =1; ***P < 0.001; **P < 0.01 and *P < 0.05. non-significant differences are not indicated.
Figure 5
Figure 5
(a) Relative expression levels (2−ΔΔCt) of the RNA-seq of CBX2.1 downstream genes. ANGPTL4, NTRK1, CYP19A1, DMRT1, EMX2, ESR2 and KISS1 were upregulated after CBX2.1 overexpression (Over Exp). CBX2.1 silencing assay (siCBX2.1) reduced significantly the genes: ANGPTL4, DMRT1, ESR2 and KISS1. NTRK1, CYP19A1 and EMX2 gene did not show any expression in response to siCBX2.1. All graphs are the average of three independent experiments, error bars represent SD from the mean (SEM), and values are expressed as relative to control =1; ***P < 0.001; **P < 0.01; *P < 0.05. non-significant differences are not indicated. (b) Effect of CBX2.2 on DamID downstream targets: AKRC1, TGFα, AMIGO2 and RSPO3. Gene expression levels showed a substantial downregulation after CBX2.2 overexpression (Ov. Exp). The silencing of CBX2.2 (si CBX2.2) significantly stimulated the expression of AMIGO2, RSPO3 and AKR1C1 genes compared to scrambled siRNA. TGBβ2, NTRK2, FZD5 and SOX4 genes were significantly upregulated by CBX2.2. In the siCBX2.2 samples, NTRK2, FZD5 and TGFβ2 expression levels were found to be negatively regulated. TGFα and SOX4 expressions showed no effect relative to the scrambled sample. All graphs are the average of three independent experiments, error bars represent SD from the mean (SEM), and values are expressed as relative to control =1; ***P < 0.001; **P < 0.01; *P < 0.05. non-significant differences are not indicated.
Figure 6
Figure 6
RT-qPCR analysis of RNA-seq downstream genes of CBX2.1. After CBX2.1 overexpression (Ov. Exp), the relative expression of BMP2 was downregulated compared to the control (empty vector). CBX2.1 knocking down induced the relative expression of BMP2 and LHX4. However, POF1B and FSHR were significantly decreased. Forced expression of CBX2.1 does not impact LHX4. All graphs are the average of three independent experiments, error bars represent SD from the mean (SEM), and values are expressed as relative to control =1; ***P < 0.001; **P < 0.01; *P < 0.05. non-significant differences are not indicated.
Figure 7
Figure 7
Relative expression levels (2−ΔΔCt) of the RNA-seq downstream genes of CBX2.2. CBX2.2 forced expression (Over Exp) positively regulated BMP15. Whereas, silencing (si) CBX2.2 (siCBX2.2) resulted in the downregulation of the same gene. Under overexpression (Over Exp) of CBX2.2, the genes TEX14 and BMP10 were significantly downregulated. Expressions of MAP3K15 and HOXA13 were comparable to the control. Silencing CBX2.2 (siCBX2.2) significantly enhanced the genes TEX14, BMP10, MAP3K15 and HOXA13. All graphs are the average of three independent experiments, error bars represent SD from the mean (SEM), and values are expressed as relative to control =1; ***P < 0.001; **P < 0.01; *P < 0.05. non-significant differences are not indicated.

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References

    1. Richards JS, Pangas SA. The ovary: basic biology and clinical implications. The Journal of Clinical Investigation. 2010;120:963–972. doi: 10.1172/JCI41350. - DOI - PMC - PubMed
    1. Berta P, et al. Genetic evidence equating SRY and the testis-determining factor. Nature. 1990;348:448–450. doi: 10.1038/348448a0. - DOI - PubMed
    1. Biason-Lauber A, Konrad D, Navratil F, Schoenle EJ. A WNT4 mutation associated with Mullerian-duct regression and virilization in a 46,XX woman. N Engl J Med. 2004;351:792–798. doi: 10.1056/NEJMoa040533. - DOI - PubMed
    1. Parma P, et al. R-spondin1 is essential in sex determination, skin differentiation and malignancy. Nat Genet. 2006;38:1304–1309. doi: 10.1038/ng1907. - DOI - PubMed
    1. Crisponi L, et al. The putative forkhead transcription factor FOXL2 is mutated in blepharophimosis/ptosis/epicanthus inversus syndrome. Nat Genet. 2001;27:159–166. doi: 10.1038/84781. - DOI - PubMed
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