A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

Pappas, Jane J.; Toulouse, André; Bradley, W. E. C.

doi:10.1007/s12575-009-9010-3

Cited by 5 publications

(2 citation statements)

References 41 publications

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“…For bisulfite sequencing of 5′ repeats of FWA , bisulfite treatment was the same as described above, but two rounds of semi‐nested PCR were used to amplify the fragment of 5′ repeats. The first‐round PCR cycling conditions were as described previously (Pappas et al. , 2009).…”

Section: Methodsmentioning

confidence: 99%

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Rea¹,

Zheng²,

Chen

et al. 2012

The Plant Journal

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Summary Imprinting, parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, thus resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant shows that the maternal histone H1 allele is required for DME regulation of the MEA, FWA, and FIS2 imprinting in Arabidopsis endosperm while the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in the DME-mediated DNA methylation and gene regulation at imprinted loci.

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Section: Methodsmentioning

confidence: 99%

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Rea¹,

Zheng²,

Chen

et al. 2012

The Plant Journal

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“…Akata cells were subjected to acyclovir treatment for 48 h to suppress EBV replication [ 7 ]. DNA was prepared using a Qiamp kit (Qiagen), and 800 ng of genomic DNA was subject to C to T conversion according to the EZ DNA methylation kit (Zymogen) essentially as described [ 64 ]. Nucleotides 166500–166800 of the Akata virus (KC207813.1 Human herpesvirus 4 strain Akata), surrounding BNLF2a ZRE1, were used to design amplification primers using the following parameters: primer length 24–38, product length 100–350, Tm 55–65 and 1 CpG in first 1/3 of primer.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta

Almohammed

Osborn

Ramasubramanyan

et al. 2018

Journal of General Virology

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The human gamma herpes virus Epstein–Barr virus (EBV) exploits multiple routes to evade the cellular immune response. During the EBV lytic replication cycle, viral proteins are expressed that provide excellent targets for recognition by cytotoxic T cells. This is countered by the viral BNLF2a gene. In B cells during latency, where BNLF2a is not expressed, we show that its regulatory region is embedded in repressive chromatin. The expression of BNLF2a mirrors the expression of a viral lytic cycle transcriptional regulator, Zta (BZLF1, EB1, ZEBRA), in B cells and we propose that Zta plays a role in up-regulating BNLF2a. In cells undergoing EBV lytic replication, we identified two distinct regions of interaction of Zta with the chromatin-associated BNLF2a promoter. We identify five potential Zta-response elements (ZREs) in the promoter that are highly conserved between virus isolates. Zta binds to these elements in vitro and activates the expression of the BNLF2a promoter in both epithelial and B cells. We also found redundancy amongst the ZREs. The EBV genome undergoes a biphasic DNA methylation cycle during its infection cycle. One of the ZREs contains an integral CpG motif. We show that this can be DNA methylated during EBV latency and that both Zta binding and promoter activation are enhanced by its methylation. In summary, we find that the BNLF2a promoter is directly targeted by Zta and that DNA methylation within the proximal ZRE aids activation. The implications for regulation of this key viral gene during the reactivation of EBV from latency are discussed.

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DNA Methylation Analysis of Steroid Hormone Receptor Genes

Hansberg-Pastor

Rodríguez-Dorantes

2014

Methods in Molecular Biology

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Steroid hormone receptors (SHR) are important transcription factors for regulating different physiological and pathological processes. Their altered expression has been strongly associated to cancer progression. Epigenetic marks such as DNA methylation have been proposed as one of the regulatory mechanisms for SHR expression in cancer. DNA methylation occurs at CpG dinucleotides, which form clusters known as CpG islands. These islands are mostly observed at promoter regions of housekeeping genes, and their aberrant methylation in cancer cells is associated with silencing of tumor-suppressor gene expression. SHR genes are characterized for presenting alternative promoters with different CpG island content, which are prone to be methylated. The method of choice for studying DNA methylation is bisulfite sequencing, since it provides information about the methylation pattern at single-nucleotide level. The method is based on the deamination of cytosine residues to uracil after treatment with sodium bisulfite. The converted DNA is amplified by a polymerase chain reaction, cloned, and sequenced. Here, we describe a protocol for bisulfite sequencing suitable for analyzing different CpG regions in SHR genes.

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A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

Cited by 5 publications

References 41 publications

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Mechanism of activation of the BNLF2a immune evasion gene of Epstein-Barr virus by Zta

DNA Methylation Analysis of Steroid Hormone Receptor Genes

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