Dr. Jim Davie: Introduction

Dr. Jim R. Davie: Introduction to research

The role of the cytoskeleton, nuclear matrix, and chromatin structure in the genesis of cancer.

Backgroud
Introduction
Aberrant nuclear and cellular structures are hallmarks of malignant transformation. Thus it is not surprising that the three-dimensional structure of the cell both affects and is affected by changes in gene expression. Our research investigates the role of the cytoskeleton, nuclear matrix, and chromatin structure in the genesis of cancer. The shape of a cell is governed by a dynamic tissue matrix, which includes extracellular matrix, cytoskeleton and nuclear matrix. Mechanical and chemical signals are transmitted to the nucleus, resulting in alterations in the three-dimensional chromatin organization of genes. The signal transduction pathways affect histone modifications, such as acetylation and phosphorylation, resulting in a relaxed chromatin structure observed in oncogene-transformed cells.

Nuclear Matrix, Chromatin and Cancer

The nucleus is a highly organized, dynamic structure. Chromosomes appear to be compartmentalized into chromosome territories. Proteins involved in replication or transcription assemble as nuclear machines, generating factories that either copy the nuclear DNA or transcribe genes. Within the spaces between the chromosomes are storage sites that contain proteins involved in the processing of the RNA. A dynamic nuclear structure, called the nuclear matrix, orchestrates the organization of nuclear DNA and the placement of the nuclear machines in three-dimensional space.

The nuclear matrix is the nuclear structure that is present following the salt extraction of nuclease-digested nuclei or the electrophoretic removal of chromatin fragments from the nucleus under physiological ionic conditions. The nuclear matrix is the dynamic structural framework of the nucleus comprised of a meshwork of filaments linked to the nuclear lamina proteins.

The chromatin fiber is organized into loop domains. Transcriptionally active genes are found in DNAase I-sensitive chromatin loops that are accessible to transcription factors and transcription machinery. Transcriptionally inactive genes are in higher order, interdigitated chromatin patches. At the base of the loop there are DNA sequences called MARs (matrix attachment regions) that bind to nuclear matrix proteins (Fig. 1).

Cytoskeleton and Nuclear DNA

In eukaryotic cells, the cytoskeleton is composed of actin-containing microfilaments, tubulin- containing microtubules and intermediate filaments that are composed of keratins, desmins, and vimentin. The cytoskeleton is physically associated with molecules involved in chemical signaling events. The cytoskeleton is also associated with the nuclear matrix and may influence directly or indirectly nuclear matrix-DNA interactions, including interactions between the nuclear matrix and transcribed DNA sequences.

We and others have shown that intermediate filaments are associated with nuclear DNA in situ. Intermediate filaments (e.g.,cytokeratins) were cross-linked to DNA in situ by cis-diammine- dichloroplatinum, an agent that preferentially cross-links nuclear matrix proteins to DNA. These results contradict the view that intermediate filaments are found only in the cytoplasm, and strengthen the hypothesis that intermediate filaments exist in the nucleus. These observations provide support for the idea that intermediate filaments influence DNA organization and gene expression (Fig. 1). It is conceivable that intermediate filaments communicate signals from the extracellular matrix to nuclear DNA, resulting in changes in gene expression. Perturbations in intermediate filament composition or structure may alter chromatin organization, resulting in aberrant gene expression and the development of a malignant phenotype.

Histone Modifications

Alteration in expression of specific genes involved in growth regulation can result in malignant transformation. There is an increasing awareness of the role of chromatin structure in the regulation of gene expression and in the genesis or suppression of cancer. The basic repeating unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around a histone octamer. The histone octamer contains two each of the core histones H2A, H2B, H3 and H4 (Fig. 2). Histone H1 associates with the linker DNA that joins nucleosomes. The H1 histones are a group of several subtypes that differ in amino acid sequence. H1 has a tripartite structure consisting of a central globular core and lysine rich N- and C-terminal domains (Fig. 3). These domains interact with linker DNA to stabilize the compaction of chromatin. H1 and the N-terminal domain of H3 have roles in chromatin folding.

The core histones are susceptible to a wide range of post-synthetic modifications, including acetylation, phosphorylation, methylation, ubiquitination, glycosylation, and ADP-ribosylation (Fig. 2). Most modifications occur on the N-terminal basic tail domain. The tail domains of the core histones are involved in transcriptional regulation, replication, and chromatin condensation. There has been considerable interest in histone acetylation and the enzymes catalyzing this reversible process. However, it is important to note that a histone may be multiply modified, e.g., dynamically acetylated H3 is also phosphorylated and methylated.

Dynamic Histone Acetylation and the Nuclear Matrix

In avian and mammalian cells, transcriptionally active chromatin regions have core histones undergoing high rates of acetylation and deacetylation, while in repressed chromatin regions the rate of reversible acetylation is slow. Histone acetylation is a dynamic process. Transcriptionally active chromatin has core histones that are rapidly hyperacetylated (t1/2 =5 to 12 min for monoacetylated H4) and rapidly deacetylated (t1/2 = 3 to 7 min). A second population is acetylated (t1/2 = 200-300 min for monoacetylated H4) and deacetylated at a slower rate (t1/2 = 30 min for monoacetylated H4).

Histone acetylation manipulates higher order chromatin and nucleosome structure. The tails of H3 and H4 are important in fiber-fiber interactions, suggesting that acetylation of the H3 and H4 tails will prevent chromatin fibers from interacting with each other. Also, histone acetylation has a profound effect on the solubility of chromatin in 150 mM NaCl or 3 mM MgCl₂. Acetylation of the core histones destabilizes histone-DNA contacts and has a role in maintaining the unfolded structure of transcribed nucleosomes. Thus, dynamic histone acetylation confers instability onto higher order chromatin fiber structures.

Histone Acetyltransferases and Deacetylases

Dr. Dave Allis' group was first to purify a nuclear histone acetyltransferase (HAT A) and to clone its cDNA (Tetrahymena nuclear HAT p55). HAT p55 was found to be homologous to yeast GCN5, a transcriptional adaptor/coactivator with HAT activity. This pivotal discovery told us how HATs were directed to transcribed chromatin regions. A number of transcriptional coactivators with HAT activity have since been identified (http://www.mdanderson.org/~genedev/Bone/hathome.html).

In 1996, Dr. S. Schreiber's group was first to clone a mammalian histone deacetylase (HDAC1). The study revealed that mammalian HDAC1 was related to yeast transcription regulator RPD3, providing a link between transcription regulation and histone deacetylation. Several HDACs have since been reported, including HDAC2 (the mammalian homologue of RPD3) and mammalian HDAC3. Mammalian HDAC1 and HDAC2, but not HDAC3, are in large multiprotein complexes containing mSin3, N-CoR or SMRT (corepressors), SAP18, SAP30, RbAp48, and RbAp46 (Fig. 4).

Several signal transduction pathways are regulated by the HDAC corepressor complex. For example, the Sin3A-N-CoR/SMRT-HDAC1, 2 complex is recruited by unliganded nuclear receptors and the Mad family of bHLH-Zip proteins (Fig. 4). An exciting development is the realization that methyl-CpG- binding protein 2 (MeCP2) binds to Sin3,recruiting the HDAC1/2 complex. These reports suggest that DNA methylation and histone deacetylation are coupled events in the formation of repressive chromatin structures and gene silencing.

We reported that HDAC1 is associated with MAR DNA in human breast cancer cells. These results suggest that HDAC1 may have a role in the organization of nuclear DNA. It is interesting to note that attachment region binding protein (ARBP), a nuclear matrix protein that binds to MARs, is homologous to MeCP2. Thus, N-CoR-Sin3A-HDAC1 complex could be recruited to the nuclear matrix and to MAR-DNA by MeCP2/ARBP (Fig. 1 and Fig. 4).

Several transcription factors can recruit HDAC directly without the assistance of the mSin3, N-CoR and SMRT (Fig. 4). HDAC 1, 2 and 3 bind to YY1. Hypophosphorylated Rb and E2F form a complex with HDAC1. The recruitment of the E2F-Rb-HDAC1 complex is partly responsible for the repression of the cyclin E promoter in G1 phase of the cell cycle.

Aberrant recruitment of histone modifying enzymes is seen in cancer. For example, PML-RAR ( PLZF-RAR) and AML-1-ETO, oncoproteins in acute promyelocytic or myeloid leukemia generated by chromosomal translocations, recruit SMRT-mSin3A-HDAC1 and N-CoR-mSin3A-HDAC1, 2 complexes (Fig. 5). SMRT-mSin3A-HDAC1 complex is recruited by the BTB/POZ domain found in the oncoprotein LAZ3/BCL6.

Relevant Reviews (Publications: Section1)

MICB Home • Scientists • Seminars • Sequencing • Directories • Bookings • Resources • News