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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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Presence of di- and tri methylated H3K4 and acetylated H3 at ZAP70 promoter is associated with gene expression. (A) Native chromatin obtained from patient samples was digested using monococcal nuclease as outlined in Materials and methods. First sample was taken before the digestion and second following a digestion and spin to remove membranes and debris. Representative picture of pre- and post-digest chromatin is shown. Obtained digested native chromatin contains only mononucleosomes. (B) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti dimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (C) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti trimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (D) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti acetylated H3 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. Statistical analysis – Mann U Whitney where (*), (**) and (***) indicate P < 0.05, P < 0.001 and P < 0.001 respectively.
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fig06: Presence of di- and tri methylated H3K4 and acetylated H3 at ZAP70 promoter is associated with gene expression. (A) Native chromatin obtained from patient samples was digested using monococcal nuclease as outlined in Materials and methods. First sample was taken before the digestion and second following a digestion and spin to remove membranes and debris. Representative picture of pre- and post-digest chromatin is shown. Obtained digested native chromatin contains only mononucleosomes. (B) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti dimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (C) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti trimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (D) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti acetylated H3 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. Statistical analysis – Mann U Whitney where (*), (**) and (***) indicate P < 0.05, P < 0.001 and P < 0.001 respectively.

Mentions: We next wanted to investigate if the distinct histone signatures identified at the ZAP70 promoter in B cell lines are also found between ZAP70+ and/or ZAP70− CLL patient samples. To this end, we obtained lymphocytes from blood of CLL patients; ZAP70 status of the patients was confirmed using flow cytometry. Around 98% of total lymphocytes were found to be CLLs. To eliminate possible contamination of CLLs with other cell types that express ZAP70 (primarily T and NK cells), we utilized negative selection of CD2+ cells by Robosep. We next prepared chromatin from purified ZAP70 expressing and non-expressing CLLs and carried out ChIP assays using antibodies that target active or permissive histone modifications including acetylated H3K14, di-methyl H3K4, tri-methyl H3K4, acetylated H4, acetylated H3 and di-methyl H3K9. We were able to detect the presence of active modifications in regions R1–R4 of ZAP70 promoter in both expressing and non-expressing patient CLLs (Fig. 6A–C). However, there was a significantly higher association of active marks H3K4me2 and H3K4me3 with the promoter in ZAP70+ CLL versus CLL from ZAP70− patients (Fig. 6A and B). We also found a markedly lower association of acetylated H3 in ZAP70− CLL. Therefore, using primary CLL, we were able to recapitulate key discoveries from cell line studies by showing the differential enrichment of H3K4me2 and H3K4me3 in ZAP70+ versus ZAP70− CLL; the most consistent difference in histone modifications between ZAP70 expressing and non-expressing patient samples was acetylation of histone H3, widely considered a modification associated with transcriptional activation [17, 18], which may, at least in part, explain our initial findings (Fig. 1) using HDACi. The enrichment of other active modifications was not statistically significantly different between the CLL patients. Importantly, the presence of active modifications in ZAP70− patient samples confirms a poised chromatin state around the promoter, which resembles the transcriptional state we observed in the ZAP70− Namalwa cell line.


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)

Presence of di- and tri methylated H3K4 and acetylated H3 at ZAP70 promoter is associated with gene expression. (A) Native chromatin obtained from patient samples was digested using monococcal nuclease as outlined in Materials and methods. First sample was taken before the digestion and second following a digestion and spin to remove membranes and debris. Representative picture of pre- and post-digest chromatin is shown. Obtained digested native chromatin contains only mononucleosomes. (B) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti dimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (C) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti trimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (D) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti acetylated H3 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. Statistical analysis – Mann U Whitney where (*), (**) and (***) indicate P < 0.05, P < 0.001 and P < 0.001 respectively.
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Related In: Results  -  Collection

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fig06: Presence of di- and tri methylated H3K4 and acetylated H3 at ZAP70 promoter is associated with gene expression. (A) Native chromatin obtained from patient samples was digested using monococcal nuclease as outlined in Materials and methods. First sample was taken before the digestion and second following a digestion and spin to remove membranes and debris. Representative picture of pre- and post-digest chromatin is shown. Obtained digested native chromatin contains only mononucleosomes. (B) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti dimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (C) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti trimethyl H3K4 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. (D) 100 μg of native chromatin obtained from ZAP70 positive or negative patient CLL cells was incubated with 10 μg of anti acetylated H3 or control antibody; ChIP assay was carried out and R1 to R4 ZAP70 promoter fragments were amplified, n = 8. Statistical analysis – Mann U Whitney where (*), (**) and (***) indicate P < 0.05, P < 0.001 and P < 0.001 respectively.
Mentions: We next wanted to investigate if the distinct histone signatures identified at the ZAP70 promoter in B cell lines are also found between ZAP70+ and/or ZAP70− CLL patient samples. To this end, we obtained lymphocytes from blood of CLL patients; ZAP70 status of the patients was confirmed using flow cytometry. Around 98% of total lymphocytes were found to be CLLs. To eliminate possible contamination of CLLs with other cell types that express ZAP70 (primarily T and NK cells), we utilized negative selection of CD2+ cells by Robosep. We next prepared chromatin from purified ZAP70 expressing and non-expressing CLLs and carried out ChIP assays using antibodies that target active or permissive histone modifications including acetylated H3K14, di-methyl H3K4, tri-methyl H3K4, acetylated H4, acetylated H3 and di-methyl H3K9. We were able to detect the presence of active modifications in regions R1–R4 of ZAP70 promoter in both expressing and non-expressing patient CLLs (Fig. 6A–C). However, there was a significantly higher association of active marks H3K4me2 and H3K4me3 with the promoter in ZAP70+ CLL versus CLL from ZAP70− patients (Fig. 6A and B). We also found a markedly lower association of acetylated H3 in ZAP70− CLL. Therefore, using primary CLL, we were able to recapitulate key discoveries from cell line studies by showing the differential enrichment of H3K4me2 and H3K4me3 in ZAP70+ versus ZAP70− CLL; the most consistent difference in histone modifications between ZAP70 expressing and non-expressing patient samples was acetylation of histone H3, widely considered a modification associated with transcriptional activation [17, 18], which may, at least in part, explain our initial findings (Fig. 1) using HDACi. The enrichment of other active modifications was not statistically significantly different between the CLL patients. Importantly, the presence of active modifications in ZAP70− patient samples confirms a poised chromatin state around the promoter, which resembles the transcriptional state we observed in the ZAP70− Namalwa cell line.

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