Michael J. Pazin

What's Up and Down with Histone Deacetylation and Transcription?

There is little doubt regarding the biological significance of protein acetylation. Several new and important papers have shown that deacetylases can function in transcriptional repression. These studies additionally suggest many new lines of experimentation. For instance, are core histones and/or HMG proteins the critical downstream targets of the deacetylases? What are the functional consequences of protein acetylation? Why does Rpd3 affect both transcriptional repression and activation? To address some of these questions, it will be important to analyze the expression of endogenous or stably integrated genes rather than transiently transfected templates that are not efficiently packaged into chromatin (note, however, that many of the studies of Sin3 and Rpd3 in yeast have used native endogenous genes). It will also be interesting to investigate whether or not there is a large protein complex containing some or all of the factors shown in Figure 1Figure 1. There are many other questions and issues remaining, as we are in the early stages of understanding the various functions of protein acetylation. In the near future, we can look forward to many more interesting and important discoveries in this area.*To whom correspondence should be addressed.

ACF, an ISWI-Containing and ATP-Utilizing Chromatin Assembly and Remodeling Factor

We describe the purification and characterization of ACF, an ATP-utilizing chromatin assembly and remodeling factor. ACF is a multisubunit factor that contains ISWI protein and is distinct from NURF, another ISWI-containing factor. In chromatin assembly, purified ACF and a core histone chaperone (such as NAP-1 or CAF-1) are sufficient for the ATP-dependent formation of periodic nucleosome arrays. In chromatin remodeling, ACF is able to modulate the internucleosomal spacing of chromatin by an ATP-dependent mechanism. Moreover, ACF can mediate promoter-specific nucleosome reconfiguration by Gal4-VP16 in an ATP-dependent manner. These results suggest that ACF acts catalytically both in chromatin assembly and in the remodeling of nucleosomes that occurs during transcriptional activation.

SWI2/SNF2 and related proteins: ATP-driven motors that disrupt protein-DNA interactions?

For further thought, we have included some additional questions. There has been a significant body of new data on SWI/SNF and related complexes, and there are many interesting and important issues that will likely be clarified in the near future.Is the specificity in the function of the SWI/SNF complex due to targeting of the complex to the appropriate genes (such as through interactions with DNA-bound transcription factors [direct mechanism]), or does the SWI/SNF complex globally facilitate nucleosome mobility in a manner that affects the transcriptional state of only a subset of genes that are sensitive to such changes in chromatin structure (indirect mechanism)?SWI/SNF complex, RSC, and NURF appear to comprise about 4 to 15 polypeptides. What is the function of the polypeptides in SWI/SNF and related complexes that do not possess the conserved NTP-binding motif? For example, are some of the other subunits involved in regulation of the activity of the complex, interactions with other transcription factors, or subcellular localization?Is the SWI/SNF complex an integral component of the RNA polymerase II holoenzyme? There are conflicting data regarding this point (3xCairns, B.R, Lorch, Y, Li, Y, Lacomis, L, Erjument-Bromage, H, Tempst, P, Du, J, Laurent, B, and Kornberg, R.D. Cell. 1996; 87: 1249–1260Abstract | Full Text | Full Text PDF | PubMed | Scopus (479)See all References, 19xWilson, C.J, Chao, D.M, Imbalzano, A.N, Schnitzler, G.R, Kingston, R.E, and Young, R.A. Cell. 1996; 84: 235–244Abstract | Full Text | Full Text PDF | PubMed | Scopus (303)See all References).It has been shown that SWI/SNF complex can facilitate the nucleosome-inhibited binding of GAL4 derivatives to DNA in a mononucleosome. Analogously, NURF can facilitate the nucleosome-inhibited binding of the GAGA factor to DNA in a mononucleosome. In contrast, however, the binding of GAL4 derivatives, GAGA factor, and NF-E2 do not appear to be inhibited by packaging of DNA into extended nucleosome arrays (as opposed to mononucleosomes), even in the absence of ATP-dependent SWI/SNF-like activities (11xPazin, M.J, Kamakaka, R.T, and Kadonaga, J.T. Science. 1994; 266: 2007–2011Crossref | PubMedSee all References, 16xTsukiyama, T and Wu, C. Cell. 1995; 83: 1011–1020Abstract | Full Text PDF | PubMed | Scopus (443)See all References, 1xArmstrong, J.A and Emerson, B.M. Mol. Cell. Biol. 1996; 16: 5634–5644PubMedSee all References). What is the basis for this apparent difference? Are internucleosomal interactions important for the proper functioning of transcription factors?Do transcriptional activation domains participate in the SWI/SNF complex-facilitated binding of factors to chromatin? Studies from different laboratories have led to different conclusions regarding this point. It appears, however, that activation domains can increase the binding of factors to chromatin in vivo. In those instances, is the activation domain directly involved in the binding of the factor to the nucleosome (i.e., does it interact directly with the core histones and/or the DNA), or is it required for cooperative binding with another transcription factor?Lastly, what happens to the nucleosomes upon addition of SWI/SNF complex (or RSC or NURF) and ATP? This process is often referred to as “remodeling.” Is remodeling the dissociation of some or all of the core histones from DNA, is it a conformational change, or is it some other alteration/modification of the nucleosome?*To whom correspondence should be addressed.

Crucial Roles of Sp1 and Epigenetic Modifications in the Regulation of the CLDN4 Promoter in Ovarian Cancer Cells

Claudins form a large family of tight junction proteins that have essential roles in the control of paracellular ion flux and the maintenance of cell polarity. Many studies have shown that several claudin family members are abnormally expressed in various cancers. In particular, CLDN4 (encoding claudin-4) is overexpressed in ovarian cancer. However, although CLDN4 overexpression is well established, the mechanisms responsible for this abnormal regulation remain unknown. In the present study, we delineate a small region of the CLDN4 promoter critical for its expression. This region contains two Sp1 sites, both of which are required for promoter activity. However, because of the ubiquitous expression of Sp1, these sites, although necessary, are not sufficient to explain the patterns of gene expression of CLDN4 in various ovarian tissues. We show that the CLDN4 promoter is further controlled by epigenetic modifications of the Sp1-containing critical promoter region. Cells that overexpress CLDN4 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN4 promoter region, and the reverse is observed in cells that do not express CLDN4. Moreover, the CLDN4-negative cells can be induced to express CLDN4 through treatment with demethylating and/or acetylating agents. Because CLDN4 is elevated in a large fraction of ovarian cancer, the mechanism leading to deregulation may represent a general pathway in ovarian tumorigenesis and may lead to novel strategies for therapy and an overall better understanding of the biology of this disease.

Regulation of the CLDN3 gene in ovarian cancer cells.

The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.