Geneviève P. Delcuve

Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors.

The zinc-dependent mammalian histone deacetylase (HDAC) family comprises 11 enzymes, which have specific and critical functions in development and tissue homeostasis. Mounting evidence points to a link between misregulated HDAC activity and many oncologic and nononcologic diseases. Thus the development of HDAC inhibitors for therapeutic treatment garners a lot of interest from academic researchers and biotechnology entrepreneurs. Numerous studies of HDAC inhibitor specificities and molecular mechanisms of action are ongoing. In one of these studies, mass spectrometry was used to characterize the affinities and selectivities of HDAC inhibitors toward native HDAC multiprotein complexes in cell extracts. Such a novel approach reproduces in vivo molecular interactions more accurately than standard studies using purified proteins or protein domains as targets and could be very useful in the isolation of inhibitors with superior clinical efficacy and decreased toxicity compared to the ones presently tested or approved. HDAC inhibitor induced-transcriptional reprogramming, believed to contribute largely to their therapeutic benefits, is achieved through various and complex mechanisms not fully understood, including histone deacetylation, transcription factor or regulator (including HDAC1) deacetylation followed by chromatin remodeling and positive or negative outcome regarding transcription initiation. Although only a very low percentage of protein-coding genes are affected by the action of HDAC inhibitors, about 40% of noncoding microRNAs are upregulated or downregulated. Moreover, a whole new world of long noncoding RNAs is emerging, revealing a new class of potential targets for HDAC inhibition. HDAC inhibitors might also regulate transcription elongation and have been shown to impinge on alternative splicing.

Mitotic partitioning of transcription factors

Mitosis is a highly orchestrated process involving numerous protein kinases and phosphatases. At the onset of mitosis, the chromatin condensation into metaphase chromosomes is correlated with global phosphorylation of histone H3. The bulk of transcription is silenced while many of the transcription‐associated proteins, including transcription and chromatin remodeling factors, are excluded from chromatin, typically as a consequence of their phosphorylation. Components of the transcription machinery and regulatory proteins are recycled and equally partitioned between newly divided cells by mechanisms that may involve microtubules, microfilaments or intermediate filaments. However, as demonstrated in the case of Runx2, a subset of transcription factors involved in lineage‐specific control, likely remain associated with their target genes to direct the deposition or removal of epigenetic marks. The displacement and re‐entry into daughter cells of transcription and chromatin remodeling factors are temporally defined and regulated. Reformation of daughter nuclei is a critical time to re‐establish the proper gene expression pattern. The mechanisms involved in the marking and re‐establishment of gene expression has been elucidated for few genes. The elucidation of how the memory of a programmed expression profile is transmitted to daughter cells represents a challenge. J. Cell. Biochem. 105: 1–8, 2008.

Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urea-Triton- and sodium dodecyl sulfate-polyacrylamide gels

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.

Selective Association of Peroxiredoxin 1 with Genomic DNA and COX-2 Upstream Promoter Elements in Estrogen Receptor Negative Breast Cancer Cells

PRDX1 was identified as a protein preferentially crosslinked to DNA in estrogen receptor negative but not in estrogen receptor positive breast cancer cells. In estrogen receptor negative cells, PRDX1 is phosphorylated, binds to NF-κB, and is recruited to COX-2 upstream promoter elements.

Differential compaction of transcriptionally competent and repressed chromatin reconstituted with histone H1 subtypes

Chromatin fragments stripped of H1 histones regain the ability to form higher order structures and aggregates in 0.15 M NaCl following reconstitution with histone H1. However, transcriptionally competent chromatin fragments are resistant to chicken erythrocyte H1/H5 histone-induced 0.15 M NaCl aggregation/precipitation. In this study, we investigated the ability of stripped chromatin fragments reconstituted with one of four histone H1 subtypes (chicken erythrocyte H1, H5, trout liver H1a, H1b) at various stoichiometries to form salt precipitable higher order structures. Our results provide evidence that chicken erythrocyte histone H1 was more effective than histone H5 and trout liver histone H1b better than H1a in forming higher order structures. None of the histone H1 subtypes could render transcriptionally competent chromatin fragments insoluble in 0.15 M NaCl. These results are consistent with the ideas that the histone H1 subtypes differ in their capacities to compact chromatin fiber, and that the alterations in the structure of transcriptionally competent nucleosomes interfere with the capacity of all H1 subtypes to form higher order structures.

Increased genomic instability and altered chromosomal protein phosphorylation timing in HRAS‐transformed mouse fibroblasts

The RAS‐mitogen‐activated protein kinase signaling pathway is often deregulated in cancer cells. In metastatic HRAS‐transformed mouse fibroblasts (Ciras‐3), the RAS‐MAPK pathway is constitutively activated. We show here that Ciras‐3 cells exhibit a higher incidence of chromosomal instability than 10T1/2 cells, including higher levels of clonal and nonclonal chromosomal aberrations. Stimulation of serum starved 10T1/2 and Ciras‐3 cells with phorbol esters (TPA) results in the phosphorylation of histone H3 at serine 10 and serine 28. Regardless of the increased genomic instability in Ciras‐3 cells, TPA‐induced H3 phosphorylated at serine 10 and H3 phosphorylated at serine 28 partitioned into distinct nuclear subdomains as they did in the parental cells. However, the timing of the response of the H3 phosphorylation event to TPA induction was delayed in Ciras‐3 cells. Further Ciras‐3 cells, which have a more open chromatin structure, had increased steady state levels of phosphorylated H3 and HMGN1 relative to parental 10T1/2 cells. TPA‐induced H3 phosphorylated at serine 10 and 28 were colocalized with the transcriptionally initiated form of RNA polymerase II in 10T1/2 and Ciras‐3 cells. Chromatin immunoprecipitation assays demonstrated that TPA‐induced H3 phosphorylation at serine 28 was associated with the immediate early JUN promoter, providing direct evidence that this histone post‐translational modification is associated with transcriptionally active genes. Together our results demonstrate the increased genomic instability and alterations in the epigenetic program in HRAS‐transformed cells.

Nucleosomal response, immediate-early gene expression and cell transformation.

Multistep tumorigenesis is a progression of events resulting from alterations in the processing of the genetic information. These alterations result from stable genetic changes (mutations) in tumor suppressor genes and oncogenes (e.g. ras) and potentially reversible epigenetic changes (i.e. modifications in gene function without a change in DNA sequence) (Bird, 2007; Egger et al., 2004; Espino et al., 2005; Hake et al., 2004; Vogelstein and Kinzler, 2004). DNA methylation and protein modifications are two epigenetic mechanisms that are altered in cancer cells (Gal-Yam et al., 2008; Gronbaek et al., 2007). Chromatin modifying enzymes, catalyzing DNA methylation and protein modifications have a central role in the genesis of cancer (Ballestar and Esteller, 2008; Esteller, 2008; Gal-Yam et al., 2008; Gronbaek et al., 2007; Hake et al., 2004; Iacobuzio-Donahue, 2009; Medina and Cespedes, 2008; Momparler, 2003). In this review we will discuss how activation of signal transduction pathways, which are often deregulated in cancer cells, results in alterations in gene expression programming through histone modifications, with a focus on histone H3 phosphorylation.

Mitogen-induced distinct epialleles are phosphorylated at either H3S10 or H3S28, depending on H3K27 acetylation

Upon mitogenic induction of immediate-early genes, phosphorylation of histone H3 at S10 or S28 occurs on different alleles. S28ph depends on CBP/p300-mediated K27ac, whereas H3 acetylated on K9 by PCAF is phosphorylated on S10. The redundant roles of S10ph and S28ph and their random targeting on distinct alleles may enable a fast response.

Epigenetics: Chromatin Organization and Function

Epigenetics refer to processes such as histone post-translational modifications (PTMs), DNA methylation and RNA that regulate gene activity and expression but are not dependent on alterations in DNA sequence. Herein, we review histone PTMs, histone variants and DNA modifications in the functioning of the nucleosome as an epigenetic signalling module. The majority of the human genome is transcribed, with most of the genome producing non-coding RNA, some of which is a component of the nuclear matrix, a dynamic RNA protein nuclear sub-structure. Non-coding RNA and coding RNA are associated with epigenetic modifiers, architectural chromatin proteins, coactivators and corepressors. The impact of changes in DNA sequence (single nucleotide polymorphisms) on the epigenome is discussed.