Anton Krumm

The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

ABSTRACT In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-mycpromoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3′Cα region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-mycgenes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

The c-myc Insulator Element and Matrix Attachment Regions Define the c-myc Chromosomal Domain

ABSTRACT Insulator elements and matrix attachment regions are essential for the organization of genetic information within the nucleus. By comparing the pattern of histone modifications at the mouse and human c-myc alleles, we identified an evolutionarily conserved boundary at which the c-myc transcription unit is separated from the flanking condensed chromatin enriched in lysine 9-methylated histone H3. This region harbors the c-myc insulator element (MINE), which contains at least two physically separable, functional activities: enhancer-blocking activity and barrier activity. The enhancer-blocking activity is mediated by CTCF. Chromatin immunoprecipitation assays demonstrate that CTCF is constitutively bound at the insulator and at the promoter region independent of the transcriptional status of c-myc. This result supports an architectural role of CTCF rather than a regulatory role in transcription. An additional higher-order nuclear organization of the c-myc locus is provided by matrix attachment regions (MARs) that define a domain larger than 160 kb. The MARs of the c-myc domain do not act to prevent the association of flanking regions with lysine 9-methylated histones, suggesting that they do not function as barrier elements.

Tumor suppression and transcription elongation: the dire consequences of changing partners

The initiation of transcription has long been thought to be the predominant mechanism of regulating gene expressionto the extent that faulty gene expression in human diseases, including cancer, has been assumed to be due to the disruption of initiation. For example, the inappropriate expression of oncogenes caused by chromosomal translocations that juxtapose oncogenes and regulatory elements (1) has been attributed to alterations in transcription initiation. Similarly, neoplastic transformation associated with the mutation or deletion of regulatory factors (such as the retinoblastoma susceptibility protein Rb or p53) for genes controlling cell cycle progression, development, or cell death has been ascribed to defective initiation. However, this focus on initiation may be shortsighted; in fact, the subsequent step in transcription, elongation, has been recognized as an important control mechanism in prokaryotes for many years, and, in the past decade, the number of eukaryotic genes demonstrated to be regulated by elongation has increased steadily (2). The importance of transcriptional elongation in regulating eukaryotic gene expression is emphasized by three exciting papers in this weeks issue of Science. The work of Duan et al. (3), Aso et al. (4), and Kibel et al. (5) indicates that a transcription elongation factor called Elongin is negatively regulated by the product of the von HippelLindau tumor suppressor gene (VHL) (Fig. 1). In patients with von Hippel-Lindau disease, VHL is defective; the consequent interference with elongation control mechanisms has drastic results. Individuals with germ-line mutations in the VHL gene (6) are predisposed to multiple forms of cancer including renal carcinoma, hemangioblastoma, and pheochromocytoma. That VHL is a key player in the genesis of at least some of these cancers is strengthened by the finding that mutations in this gene occur in nonhereditary (sporadic), as well as hereditary, forms of renal carcinoma (Fig. 2). The

Distinct properties of c-myc transcriptional elongation are revealed in Xenopus oocytes and mammalian cells and by template titration, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), and promoter mutagenesis.

A block to c-myc transcription elongation has been observed in Xenopus oocytes and mammalian cells. Here, we show that the distribution of RNA polymerase II transcription complexes in the c-myc promoter proximal region in Xenopus oocytes is different from that observed previously in mammalian cells. Thus, there are major differences in the c-myc elongation block observed in the two systems. In addition, as first reported for a Xenopus tubulin gene (K. M. Middleton and G. T. Morgan, Mol. Cell. Biol. 10:727-735, 1990). c-myc template titration experiments reveal the existence of two classes of RNA polymerase II transcription complexes in oocytes: one (at low template concentration) that is capable of reading through downstream sites of premature termination, and another (high template concentration) that does not. We show that these classes of polymerases are distinct from those previously identified by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which distinguishes transcription complexes on the basis of transcribed distance, rather than on the basis of differential elongation through sites of premature termination. We also show that mutations that affect the efficiency of initiation of transcription from the c-myc P2 promoter can influence premature termination by at least two mechanisms: TATA box mutations function by the titration effect (decrease in transcription initiation results in a relative decrease in premature termination), while an upstream activator (E2F) site functions by contributing to the assembly of polymerase complexes competent to traverse the downstream sites of premature termination.

Inhibitory Sequences within the Clotting Factor VIII cDNA Block Transcriptional Elongation and Complicate Efforts Toward Gene Therapy for Haemophilia A

The limitations of transfusion therapy for haemophilia have prompted efforts to develop gene therapy for these disorders. The coagulation factors have rather short half-lives, and cannot be adequately replaced to prevent all episodes of bleeding in haemophiliacs [1]. Multiple transfusions are required at times of major trauma or surgery. The benefit of gene therapy would be continuous production of the deficient coagulation factor, preferably after a single treatment which would allow permanent factor replacement.