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Differentially methylated CpG island within human XIST mediates alternative P2 transcription and YY1 binding.

Chapman AG, Cotton AM, Kelsey AD, Brown CJ - BMC Genet. (2014)

Bottom Line: Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing.In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Center, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. andrewgchapman@gmail.com.

ABSTRACT

Background: X-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome, the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates, triggering silencing of the chromosome. In mouse, an alternative Xist promoter, P2 is also the site of YY1 binding, which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation, including absence of a functional antisense regulator Tsix, and absence of strictly paternal inactivation in extraembryonic tissues, prompting us to examine regulatory regions for the human XIST gene.

Results: We demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However, YY1 binding is insufficient to drive P2 expression or establish the DHS, which may require a development-specific factor. Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.

Conclusions: The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST.

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YY1 binding to region of P2 promoter is Xi-specific and upregulates P1 transcription. A) Chromatin immunoprecipitation (ChIP) of YY1 binding to P2 shown relative to input, at P2, a negative control region within XIST, and a positive control region (HCFC1 gene promoter) for female (IMR-90), HT1080XISTi transgenic XIST, and Xi and Xa-containing hybrids. A representative ChIP is shown ± standard deviation of q-PCR triplicates; with replicate experiments showing the same pattern but variable levels of IP relative to input. B) Q-PCR following 72 h of siRNA mediated knockdown of YY1 in IMR-90 female cells demonstrates a decrease in expression of XIST, both upstream (19, 18) and downstream of P2 (200d.2, qXIST5) relative to cells with a control (EGFP) knockdown. Levels of YY1 dropped to less than 20% confirming successful knockdown. C) FISH for XIST RNA following YY1 knockdown. Left panel shows representative IMR-90 female cells treated with lipofectamine alone (Lipo) and right panel shows cells after YY1 knockdown. Arrows indicate location of XIST RNA signal (green) which was substantially reduced after YY1 knockdown.
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Figure 3: YY1 binding to region of P2 promoter is Xi-specific and upregulates P1 transcription. A) Chromatin immunoprecipitation (ChIP) of YY1 binding to P2 shown relative to input, at P2, a negative control region within XIST, and a positive control region (HCFC1 gene promoter) for female (IMR-90), HT1080XISTi transgenic XIST, and Xi and Xa-containing hybrids. A representative ChIP is shown ± standard deviation of q-PCR triplicates; with replicate experiments showing the same pattern but variable levels of IP relative to input. B) Q-PCR following 72 h of siRNA mediated knockdown of YY1 in IMR-90 female cells demonstrates a decrease in expression of XIST, both upstream (19, 18) and downstream of P2 (200d.2, qXIST5) relative to cells with a control (EGFP) knockdown. Levels of YY1 dropped to less than 20% confirming successful knockdown. C) FISH for XIST RNA following YY1 knockdown. Left panel shows representative IMR-90 female cells treated with lipofectamine alone (Lipo) and right panel shows cells after YY1 knockdown. Arrows indicate location of XIST RNA signal (green) which was substantially reduced after YY1 knockdown.

Mentions: We next asked whether P2 activity and hypersensitivity at DHS 200b.1 was correlated with binding of YY1 in vivo using chromatin immunoprecipitation (ChIP). YY1 was found to be enriched at P2 in both female cells and Xi-containing hybrid cells but not Xa-containing hybrid cells, indicating Xi-specific binding (Figure 3A). We also found YY1 binding at P2 in inducible XIST HT1080XISTi cells before induction by DOX, despite these cells lacking a DHS, demonstrating that YY1 binding is insufficient to establish hypersensitivity in the P2 region. Since YY1 binding appeared to be independent of the DHS associated with P2 we wished to address the impact of down-regulating YY1 on the transcription of XIST. After 72 h of siRNA-mediated knockdown of YY1 in IMR90 female cells we saw a 50% decrease in expression of XIST, both upstream and downstream of P2, suggesting that YY1 may be a regulator of transcription at P1 (Figure 3B). By fluorescent in situ hybridization for the XIST RNA we observed only a small focus of expression in 41/86 (48%) of cells and no detectable signal in 35/86 (40%) of cells, with substantial delocalization of the RNA in the remaining 12% of cells (Figure 3C).


Differentially methylated CpG island within human XIST mediates alternative P2 transcription and YY1 binding.

Chapman AG, Cotton AM, Kelsey AD, Brown CJ - BMC Genet. (2014)

YY1 binding to region of P2 promoter is Xi-specific and upregulates P1 transcription. A) Chromatin immunoprecipitation (ChIP) of YY1 binding to P2 shown relative to input, at P2, a negative control region within XIST, and a positive control region (HCFC1 gene promoter) for female (IMR-90), HT1080XISTi transgenic XIST, and Xi and Xa-containing hybrids. A representative ChIP is shown ± standard deviation of q-PCR triplicates; with replicate experiments showing the same pattern but variable levels of IP relative to input. B) Q-PCR following 72 h of siRNA mediated knockdown of YY1 in IMR-90 female cells demonstrates a decrease in expression of XIST, both upstream (19, 18) and downstream of P2 (200d.2, qXIST5) relative to cells with a control (EGFP) knockdown. Levels of YY1 dropped to less than 20% confirming successful knockdown. C) FISH for XIST RNA following YY1 knockdown. Left panel shows representative IMR-90 female cells treated with lipofectamine alone (Lipo) and right panel shows cells after YY1 knockdown. Arrows indicate location of XIST RNA signal (green) which was substantially reduced after YY1 knockdown.
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Related In: Results  -  Collection

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Figure 3: YY1 binding to region of P2 promoter is Xi-specific and upregulates P1 transcription. A) Chromatin immunoprecipitation (ChIP) of YY1 binding to P2 shown relative to input, at P2, a negative control region within XIST, and a positive control region (HCFC1 gene promoter) for female (IMR-90), HT1080XISTi transgenic XIST, and Xi and Xa-containing hybrids. A representative ChIP is shown ± standard deviation of q-PCR triplicates; with replicate experiments showing the same pattern but variable levels of IP relative to input. B) Q-PCR following 72 h of siRNA mediated knockdown of YY1 in IMR-90 female cells demonstrates a decrease in expression of XIST, both upstream (19, 18) and downstream of P2 (200d.2, qXIST5) relative to cells with a control (EGFP) knockdown. Levels of YY1 dropped to less than 20% confirming successful knockdown. C) FISH for XIST RNA following YY1 knockdown. Left panel shows representative IMR-90 female cells treated with lipofectamine alone (Lipo) and right panel shows cells after YY1 knockdown. Arrows indicate location of XIST RNA signal (green) which was substantially reduced after YY1 knockdown.
Mentions: We next asked whether P2 activity and hypersensitivity at DHS 200b.1 was correlated with binding of YY1 in vivo using chromatin immunoprecipitation (ChIP). YY1 was found to be enriched at P2 in both female cells and Xi-containing hybrid cells but not Xa-containing hybrid cells, indicating Xi-specific binding (Figure 3A). We also found YY1 binding at P2 in inducible XIST HT1080XISTi cells before induction by DOX, despite these cells lacking a DHS, demonstrating that YY1 binding is insufficient to establish hypersensitivity in the P2 region. Since YY1 binding appeared to be independent of the DHS associated with P2 we wished to address the impact of down-regulating YY1 on the transcription of XIST. After 72 h of siRNA-mediated knockdown of YY1 in IMR90 female cells we saw a 50% decrease in expression of XIST, both upstream and downstream of P2, suggesting that YY1 may be a regulator of transcription at P1 (Figure 3B). By fluorescent in situ hybridization for the XIST RNA we observed only a small focus of expression in 41/86 (48%) of cells and no detectable signal in 35/86 (40%) of cells, with substantial delocalization of the RNA in the remaining 12% of cells (Figure 3C).

Bottom Line: Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing.In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medical Genetics, Molecular Epigenetics Group, Life Sciences Center, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada. andrewgchapman@gmail.com.

ABSTRACT

Background: X-chromosome inactivation silences one X chromosome in females to achieve dosage compensation with the single X chromosome in males. While most genes are silenced on the inactive X chromosome, the gene for the long non-coding RNA XIST is silenced on the active X chromosome and expressed from the inactive X chromosome with which the XIST RNA associates, triggering silencing of the chromosome. In mouse, an alternative Xist promoter, P2 is also the site of YY1 binding, which has been shown to serve as a tether between the Xist RNA and the DNA of the chromosome. In humans there are many differences from the initial events of mouse Xist activation, including absence of a functional antisense regulator Tsix, and absence of strictly paternal inactivation in extraembryonic tissues, prompting us to examine regulatory regions for the human XIST gene.

Results: We demonstrate that the female-specific DNase hypersensitivity site within XIST is specific to the inactive X chromosome and correlates with transcription from an internal P2 promoter. P2 is located within a CpG island that is differentially methylated between males and females and overlaps conserved YY1 binding sites that are only bound on the inactive X chromosome where the sites are unmethylated. However, YY1 binding is insufficient to drive P2 expression or establish the DHS, which may require a development-specific factor. Furthermore, reduction of YY1 reduces XIST transcription in addition to causing delocalization of XIST.

Conclusions: The differentially methylated DNase hypersensitive site within XIST marks the location of an alternative promoter, P2, that generates a transcript of unknown function as it lacks the A repeats that are critical for silencing. In addition, this region binds YY1 on the unmethylated inactive X chromosome, and depletion of YY1 untethers the XIST RNA as well as decreasing transcription of XIST.

Show MeSH
Related in: MedlinePlus