Katherine L. Dunn

The insulator binding protein CTCF associates with the nuclear matrix.

Nuclear DNA is organized into chromatin loop domains. At the base of these loops, matrix-associated regions (MARs) of the DNA interact with nuclear matrix proteins. MARs act as structural boundaries within chromatin, and MAR binding proteins may recruit multiprotein complexes that remodel chromatin. The potential tumor suppressor protein CTCF binds to vertebrate insulators and is required for insulator activity. We demonstrate that CTCF is associated with the nuclear matrix and can be cross-linked to DNA by cisplatin, an agent that preferentially cross-links nuclear matrix proteins to DNA in situ. These results suggest that CTCF anchors chromatin to the nuclear matrix, suggesting that there is a functional connection between insulators and the nuclear matrix. We also show that the chromatin-modifying enzymes HDAC1 and HDAC2, which are intrinsic nuclear matrix components and thought to function as corepressors of CTCF, are incapable of associating with CTCF. Hence, the insulator activity of CTCF apparently involves an HDAC-independent association with the nuclear matrix. We propose that CTCF may demarcate nuclear matrix-dependent points of transition in chromatin, thereby forming topologically independent chromatin loops that may support gene silencing.

Stimulation of the Ras-MAPK pathway leads to independent phosphorylation of histone H3 on serine 10 and 28

The Ras-mitogen activated protein kinase (Ras-MAPK) pathway plays an integral role in the formation of human malignancies. Stimulation of this pathway results in phosphorylation of histone H3 at serines 10 and 28 and expression of immediate-early genes. Phosphorylated (serine 10) H3, which is also acetylated on lysine 14, is associated with immediate-early genes. In this report, we investigated the relationship between these two H3 phosphorylation events in parental and ras-transformed fibroblasts. Immunoblot analyses of two-dimensional gel patterns demonstrated that all three H3 variants were phosphorylated after stimulation of the Ras-MAPK pathway and during mitosis. Following stimulation of the Ras-MAPK pathway, H3 phosphorylated on serines 10 and 28 was excluded from regions of highly condensed chromatin and was present in increased levels in ras-transformed cells. Although H3 phosphorylated at serine 10 or 28 was dynamically acetylated, H3 phosphorylated at serine 28 had a higher steady state of acetylation than that of H3 phosphorylated at serine 10. When visualized with indirect immunofluorescence, most foci of phosphorylated serine 28 H3 did not co-localize with foci of H3 phosphorylated on serine 10 or phosphoacetylated on serine 10 and lysine 14, suggesting that these two phosphorylation events act separately to promote gene expression.

Nuclear organization and chromatin dynamics--Sp1, Sp3 and histone deacetylases.

Regulation of gene expression involves the coordinated activities and interplay between chromatin remodeling factors and transcription factor recruitment. Histone acetyltransferases, histone deacetylases, histone kinases, histone phosphatases, histone methyltransferases, histone demethylases and ATP-dependent chromatin remodeling complexes mediate chromatin remodeling and are components of a complex epigenetic network regulating gene expression during development and differentiation. Transcription factors play key roles in the recruitment of histone modifying enzymes and chromatin remodeling complexes to specific gene promoters. Sp1 and Sp3 are two transcription factors that are expressed in all mammalian cells and are involved in the regulation of genes involved in most cellular processes. Remodeling of chromatin is a necessary event in preparing the gene for transcription. In this review we will cover the organization and remodeling of chromatin, with a focus on dynamic histone acetylation and the histone deacetylase enzymes. The structure and function of transcription factors Sp1 and Sp3 will be presented. The role of these factors in the regulation of the estrogen responsive trefoil factor 1 gene will be highlighted. In the analyses of the factors involved in the regulation of the expression of a specific gene, the chromatin immunoprecipitation assay in which the protein factor of interest is cross-linked to DNA with formaldehyde is an essential tool. The limitations of this assay in cancer cells in which genomic instability is rampant are discussed.

Histone modifications as a platform for cancer therapy

Tumorigenesis and metastasis are a progression of events resulting from alterations in the processing of the genetic information. These alterations result from stable genetic changes (mutations) involving tumor suppressor genes and oncogenes (e.g., ras, BRAF) and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. Mutations of genes coding for proteins that directly or indirectly influence epigenetic processes will alter the cells gene expression program. Epigenetic mechanisms often altered in cancer cells are DNA methylation and histone modifications (acetylation, methylation, phosphorylation). This article will review the potential of these reversible epigenetic processes as targets for cancer therapies.

Chromatin organization and nuclear microenvironments in cancer cells

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. However, our knowledge of events resulting in changes in nuclear shape and chromatin organization in cancer cells is limited. Nuclear matrix proteins, which include lamins, transcription factors (Sp1) and histone modifying enzymes (histone deacetylases), and histone modifications (histone H3 phosphorylation) have roles in organizing chromatin in the interphase nucleus, regulating gene expression programs and determining nuclear shape. Histone H3 phosphorylation, a downstream target of the Ras‐mitogen activated protein kinase pathway, is involved in neoplastic transformation. This article will review genetic and epigenetic events that alter chromatin organization in cancer cells and the role of the nuclear matrix in determining nuclear morphology. J. Cell. Biochem. 104: 2004–2015, 2008.

Abnormalities of chromatin in tumor cells

Nuclear morphometric descriptors such as nuclear size, shape, DNA content and chromatin organization are used by pathologists as diagnostic markers for cancer. Tumorigenesis involves a series of poorly understood morphological changes that lead to the development of hyperplasia, dysplasia, in situ carcinoma, invasive carcinoma, and in many instances finally metastatic carcinoma. Nuclei from different stages of disease progression exhibit changes in shape and the reorganization of chromatin, which appears to correlate with malignancy. Multistep tumorigenesis is a process that results from alterations in the function of DNA. These alterations result from stable genetic changes, including those of tumor suppressor genes, oncogenes and DNA stability genes, and potentially reversible epigenetic changes, which are modifications in gene function without a change in the DNA sequence. DNA methylation and histone modifications are two epigenetic mechanisms that are altered in cancer cells. The impact of genetic (e.g., mutations in Rb and ras family) and epigenetic alterations with a focus on histone modifications on chromatin structure and function in cancer cells are reviewed here.

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.