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Epigenetic alterations in the pattern of DNA and histone modifications play a crucial role in cancer development. Analysis of patient samples, however, is hampered by technical limitations in the study of chromatin structure from pathology archives that usually consist of heavily fixed, paraffin-embedded material. Here, we present a methodology [pathology tissue--ChiP (PAT-ChIP)] to extract and immunoprecipitate chromatin from paraffin-embedded patient samples up to several years old. In a pairwise comparison with canonical CHIP, PAT-ChIP showed a high reproducibility of results for several histone marks and an identical ability to detect dynamic changes in chromatin structure upon pharmacological treatment. Finally, we showed that PAT-ChIP can be coupled with high-throughput sequencing (PAT-ChIP-Seq) for the genome-wide analysis of distinct chromatin modifications. PAT-ChIP therefore represents a versatile procedure and diagnostic tool for the analysis of epigenetic alterations in cancer and potentially other diseases. doi/ 10.1073/pnas.1007647107

PML-RAR[alpha] induces a block of hematopoietic differentiation and acute promyelocytic leukemia. This block is based on its capacity to inactivate target genes by recruiting histone deacetylase (HDAC) and DNA methyltransferase activities. Here we report that MBD1, a member of a conserved family of proteins able to bind methylated DNA, cooperates with PML-RAR[alpha] in transcriptional repression and cellular transformation. PML-RAR[alpha] recruits MBD1 to its target promoter through an HDAC3-mediated mechanism. Binding of HDAC3 and MBD1 is not confined to the promoter region but instead is spread over the locus. Knock-down of HDAC3 expression by RNA interference in acute promyelocytic leukemia cells alleviates PML-RAR-induced promoter silencing. We further demonstrate that retroviral expression of dominant-negative mutants of MBD1 in hematopoietic precursors compromises the ability of PML-RAR[alpha] to block their differentiation and thus restored cell differentiation. Our results demonstrate that PML-RAR[alpha] functions by recruiting an HDAC3-MBD1 complex that contributes to the establishment and maintenance of the silenced chromatin state. chromatin | epigenetics | leukemia

A general strategy for inactivation of target proteins is presented, which we have termed 'oligomerization chain reaction.' This technique is based on the fusion of the self-associating coiled-coil (CC) domain of the nuclear factor promyelocytic leukemia (PML) to target proteins that are able to self-associate naturally. Oligomerization through the CC region of promyelocytic leukemia, and through the natural self-associating domain, triggers the oligomerization chain reaction, leading to formation of large molecular weight complexes and functional inactivation of the target. As a test case, we have chosen the oncosuppressor p53, naturally occurring as a tetramer. Fusion of the CC to p53 leads to formation of stable high molecular weight complexes--as shown by size exclusion chromatography--to which wild-type p53 is recruited with high efficiency. CC-p53 chimeras delocalize wild-type p53 to the cytoplasm and inhibit its transcriptional regulatory properties, resulting in a loss of p53 function. We propose that this strategy may be of general application to self-associating factors and represent a complementary approach to currently used functional inactivation-based strategies.

Retinoic acid receptor (RARs) and retinoid X receptor (RXRs) activities were observed during vertebrate development in Xenopus and zebrafish embryos. RAR- and RXR-selective ligands generated genetic expressions similar to those produced by all-trans-retinoic acid. However, RXR-selective ligands activated only an RXR responsive reporter, indicating a variation of liganded RXR activity across different vertebrate species.