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Cross-talk between DNA methylation and active histone modifications regulates aberrant expression of ZAP70 in CLL.

Amin S, Walsh M, Wilson C, Parker AE, Oscier D, Willmore E, Mann D, Mann J - J. Cell. Mol. Med. (2012)

Bottom Line: Following a direct comparison of ZAP70+ and ZAP70- primary CLLs, we show ZAP70 promoter in expressing CLLs to be associated with a spectrum of active histone modifications, some of which are tightly linked to aberrant DNA methylation in CLL.Cross-talk between histone modifications and reduced DNA methylation culminates in transcriptional de-repression of ZAP70.Moreover, treatment with histone deacetylase (HDAC) and DNA methylation inhibitors results in recovery of ZAP70 expression, which provides a possible explanation for the failure of HDAC inhibitors in CLL treatment and might serve as a cautionary warning for their future use in treatment of this leukaemia.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medical Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.

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Differential global chromatin folding and accessibility in ZAP70+ versus ZAP70− cells. (A) Western blot and RT-PCR for detection of ZAP70− total RNA were prepared from Nalm6 and Namalwa cell lines and used for qRT-PCR detection of ZAP70 (top panel); 30 μg whole cell extract was separated by SDS-PAGE and immunoblotted for ZAP70 (bottom panel). Gels shown are representative of tri-replicate experiments. (B) Native chromatin was isolated from Namalwa or Nalm6 cell lines and digested with micrococcal nuclease for 5 min. with a sample taken every minute. DNA was isolated using phenol–chloroform extraction and separated on 1% agarose gel. (C) Schematic representation of ZAP70 predicted promoter sequence and the position of the primers used to amplify the 1 kb fragment (R1–R4) or the small control fragment (R1). (D) 20 ng of DNA from chromatin digests sampled at 1 min. intervals was used in RT-PCR reactions using ZAP70 R1 sense and ZAP70 R4 antisense primers to amplify R1–R4 fragment (1 kb) or ZAP70 R1 sense and antisense to generate R1 control fragment (200 bp). (E) The R1–R4 amplification as outlined in (C) was carried out using quantitative PCR and values plotted onto a graph ±S.E.M. (n = 3). (F) Difference between the rate of digestion between Nalm6 and Namalwa chromatin were calculated using the ratio between percentage amplification of R1–R4 product in two cell lines at a given time interval.
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fig04: Differential global chromatin folding and accessibility in ZAP70+ versus ZAP70− cells. (A) Western blot and RT-PCR for detection of ZAP70− total RNA were prepared from Nalm6 and Namalwa cell lines and used for qRT-PCR detection of ZAP70 (top panel); 30 μg whole cell extract was separated by SDS-PAGE and immunoblotted for ZAP70 (bottom panel). Gels shown are representative of tri-replicate experiments. (B) Native chromatin was isolated from Namalwa or Nalm6 cell lines and digested with micrococcal nuclease for 5 min. with a sample taken every minute. DNA was isolated using phenol–chloroform extraction and separated on 1% agarose gel. (C) Schematic representation of ZAP70 predicted promoter sequence and the position of the primers used to amplify the 1 kb fragment (R1–R4) or the small control fragment (R1). (D) 20 ng of DNA from chromatin digests sampled at 1 min. intervals was used in RT-PCR reactions using ZAP70 R1 sense and ZAP70 R4 antisense primers to amplify R1–R4 fragment (1 kb) or ZAP70 R1 sense and antisense to generate R1 control fragment (200 bp). (E) The R1–R4 amplification as outlined in (C) was carried out using quantitative PCR and values plotted onto a graph ±S.E.M. (n = 3). (F) Difference between the rate of digestion between Nalm6 and Namalwa chromatin were calculated using the ratio between percentage amplification of R1–R4 product in two cell lines at a given time interval.

Mentions: Gene expression depends on the promoter activity, which is in turn regulated by the accessibility of DNA to the transcription factors. DNA in cells is packaged into chromatin, which can be differentially folded to either aid transcription (in opened conformation) or to prevent it (in closed conformation). There are no previous reports relating to the chromatin state at the ZAP70 regulatory region, so to initially assess the global chromatin structure, we isolated intact, native chromatin from ZAP70− Namalwa and ZAP70+ Nalm6 B cell lines (Fig. 4A). An equivalent amount of chromatin was then digested with MNase for up to 5 min. with sampling every minute (Fig. 4B). Micrococcal nuclease cleaves DNA on the basis of accessibility dictated by chromatin structure; therefore, gene regions with chromatin in relaxed conformation will be digested faster than when chromatin is in a closed, transcriptionally silenced state. DNA isolated from timed digests in both cell lines was used as template in PCRs amplifying the entire ZAP70 promoter (regions R1–R4) (Fig. 4C and D). The ability to amplify 1 kb of DNA at ZAP70 promoter diminished faster in Nalm6 cells as compared with Namalwa (Fig. 4C–E). At 2 min., there was 47% more digested DNA in Nalm6 chromatin relative to Namalwa, which reflects a more accessible, open state of chromatin in the ZAP70 expressing cell line (Fig. 4F). These data support the idea that higher order chromatin structures affect gene expression, whereas both are determined by the type of histone modification associated with the chromatin. We therefore next set out to assess histone modifications at the ZAP70 promoter.


Cross-talk between DNA methylation and active histone modifications regulates aberrant expression of ZAP70 in CLL.

Amin S, Walsh M, Wilson C, Parker AE, Oscier D, Willmore E, Mann D, Mann J - J. Cell. Mol. Med. (2012)

Differential global chromatin folding and accessibility in ZAP70+ versus ZAP70− cells. (A) Western blot and RT-PCR for detection of ZAP70− total RNA were prepared from Nalm6 and Namalwa cell lines and used for qRT-PCR detection of ZAP70 (top panel); 30 μg whole cell extract was separated by SDS-PAGE and immunoblotted for ZAP70 (bottom panel). Gels shown are representative of tri-replicate experiments. (B) Native chromatin was isolated from Namalwa or Nalm6 cell lines and digested with micrococcal nuclease for 5 min. with a sample taken every minute. DNA was isolated using phenol–chloroform extraction and separated on 1% agarose gel. (C) Schematic representation of ZAP70 predicted promoter sequence and the position of the primers used to amplify the 1 kb fragment (R1–R4) or the small control fragment (R1). (D) 20 ng of DNA from chromatin digests sampled at 1 min. intervals was used in RT-PCR reactions using ZAP70 R1 sense and ZAP70 R4 antisense primers to amplify R1–R4 fragment (1 kb) or ZAP70 R1 sense and antisense to generate R1 control fragment (200 bp). (E) The R1–R4 amplification as outlined in (C) was carried out using quantitative PCR and values plotted onto a graph ±S.E.M. (n = 3). (F) Difference between the rate of digestion between Nalm6 and Namalwa chromatin were calculated using the ratio between percentage amplification of R1–R4 product in two cell lines at a given time interval.
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Related In: Results  -  Collection

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fig04: Differential global chromatin folding and accessibility in ZAP70+ versus ZAP70− cells. (A) Western blot and RT-PCR for detection of ZAP70− total RNA were prepared from Nalm6 and Namalwa cell lines and used for qRT-PCR detection of ZAP70 (top panel); 30 μg whole cell extract was separated by SDS-PAGE and immunoblotted for ZAP70 (bottom panel). Gels shown are representative of tri-replicate experiments. (B) Native chromatin was isolated from Namalwa or Nalm6 cell lines and digested with micrococcal nuclease for 5 min. with a sample taken every minute. DNA was isolated using phenol–chloroform extraction and separated on 1% agarose gel. (C) Schematic representation of ZAP70 predicted promoter sequence and the position of the primers used to amplify the 1 kb fragment (R1–R4) or the small control fragment (R1). (D) 20 ng of DNA from chromatin digests sampled at 1 min. intervals was used in RT-PCR reactions using ZAP70 R1 sense and ZAP70 R4 antisense primers to amplify R1–R4 fragment (1 kb) or ZAP70 R1 sense and antisense to generate R1 control fragment (200 bp). (E) The R1–R4 amplification as outlined in (C) was carried out using quantitative PCR and values plotted onto a graph ±S.E.M. (n = 3). (F) Difference between the rate of digestion between Nalm6 and Namalwa chromatin were calculated using the ratio between percentage amplification of R1–R4 product in two cell lines at a given time interval.
Mentions: Gene expression depends on the promoter activity, which is in turn regulated by the accessibility of DNA to the transcription factors. DNA in cells is packaged into chromatin, which can be differentially folded to either aid transcription (in opened conformation) or to prevent it (in closed conformation). There are no previous reports relating to the chromatin state at the ZAP70 regulatory region, so to initially assess the global chromatin structure, we isolated intact, native chromatin from ZAP70− Namalwa and ZAP70+ Nalm6 B cell lines (Fig. 4A). An equivalent amount of chromatin was then digested with MNase for up to 5 min. with sampling every minute (Fig. 4B). Micrococcal nuclease cleaves DNA on the basis of accessibility dictated by chromatin structure; therefore, gene regions with chromatin in relaxed conformation will be digested faster than when chromatin is in a closed, transcriptionally silenced state. DNA isolated from timed digests in both cell lines was used as template in PCRs amplifying the entire ZAP70 promoter (regions R1–R4) (Fig. 4C and D). The ability to amplify 1 kb of DNA at ZAP70 promoter diminished faster in Nalm6 cells as compared with Namalwa (Fig. 4C–E). At 2 min., there was 47% more digested DNA in Nalm6 chromatin relative to Namalwa, which reflects a more accessible, open state of chromatin in the ZAP70 expressing cell line (Fig. 4F). These data support the idea that higher order chromatin structures affect gene expression, whereas both are determined by the type of histone modification associated with the chromatin. We therefore next set out to assess histone modifications at the ZAP70 promoter.

Bottom Line: Following a direct comparison of ZAP70+ and ZAP70- primary CLLs, we show ZAP70 promoter in expressing CLLs to be associated with a spectrum of active histone modifications, some of which are tightly linked to aberrant DNA methylation in CLL.Cross-talk between histone modifications and reduced DNA methylation culminates in transcriptional de-repression of ZAP70.Moreover, treatment with histone deacetylase (HDAC) and DNA methylation inhibitors results in recovery of ZAP70 expression, which provides a possible explanation for the failure of HDAC inhibitors in CLL treatment and might serve as a cautionary warning for their future use in treatment of this leukaemia.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Medical Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK.

Show MeSH
Related in: MedlinePlus