Ulrich Mahlknecht

Histone acetylation modifiers in the pathogenesis of malignant disease.

Chromatin structure is gaining increasing attention as a potential target in the treatment of cancer. Relaxation of the chromatin fiber facilitates transcription and is regulated by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs), which modify the acetylation state of histone proteins and other promoter-bound transcription factors. While HATs, which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, HDACs tend to associate with multisubunit corepressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. HATs and HDACs are known to be involved both in the pathogenesis as well as in the suppression of cancer. Some of the genes encoding these enzymes have been shown to be rearranged in the context of chromosomal translocations in human acute leukemias and solid tumors, where fusions of regulatory and coding regions of a variety of transcription factor genes result in completely new gene products that may interfere with regulatory cascades controlling cell growth and differentiation. On the other hand, some histone acetylation-modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks. In these cases gains and losses of chromosomal material may affect the availability of functionally active HATs and HDACs, which in turn disturbs the tightly controlled equilibrium of histone acetylation. We review herein the recent achievements, which further help to elucidate the biological role of histone acetylation modifying enzymes and their potential impact on our current understanding of the molecular changes involved in the development of solid tumors and leukemias.

Histone Deacetylase 3, a Class I Histone Deacetylase, Suppresses MAPK11-Mediated Activating Transcription Factor-2 Activation and Represses TNF Gene Expression

During inflammatory events, the induction of immediate-early genes, such as TNF-α, is regulated by signaling cascades including the JAK/STAT, NF-κB, and the p38 MAPK pathways, which result in phosphorylation-dependent activation of transcription factors. We observed the direct interaction of histone deacetylase (HDAC) 3, a class I histone deacetylase, with MAPK11 (p38 β isoform) by West-Western-based screening analysis, pull-down assay, and two-hybrid system analysis. Results further indicated that HDAC3 decreases the MAPK11 phosphorylation state and inhibits the activity of the MAPK11-dependent transcription factor, activating transcription factor-2 (ATF-2). LPS-mediated activation of ATF-2 was inhibited by HDAC3 in a time- and dose-dependent manner. Inhibition of HDAC3 expression by RNA interference resulted in increased ATF-2 activation in response to LPS stimulation. In agreement with decreased ATF-2 transcriptional activity by HDAC3, HDAC3-repressed TNF gene expression, and TNF protein production observed in response to LPS stimulation. Therefore, our results indicate that HDAC3 interacts directly and selectively with MAPK11, represses ATF-2 transcriptional activity, and acts as a regulator of TNF gene expression in LPS-stimulated cells, especially in mononuclear phagocytes.

Resistance to Apoptosis in HIV-Infected CD4+ T Lymphocytes Is Mediated by Macrophages: Role for Nef and Immune Activation in Viral Persistence

Apoptosis or programmed cell death may play a critical role in AIDS pathogenesis through depletion of both CD4+ and CD8+ T lymphocytes. Using a reporter virus, a recombinant HIV infectious clone expressing the green fluorescent protein (GFP), apoptosis was measured in productively infected CD4+ T lymphocytes, in the presence and absence of autologous macrophages. The presence of macrophages in the culture increased the frequency of nonapoptotic GFP-positive productively infected CD4+ T lymphocytes. The appearance of nonapoptotic productively infected CD4+ T lymphocytes in the culture required intercellular contacts between macrophages and PBLs and the expression of the HIV Nef protein. The presence of macrophages did not reduce apoptosis when CD4+ T lymphocytes were infected with a GFP-tagged virus deleted for the nef gene. TNF-α (TNF) expressed on the surface of macrophages prevented apoptosis in nef-expressing, productively infected CD4+ T lymphocytes. Similarly, following TNF stimulation, apoptosis was diminished in Jurkat T cells transfected with a nef-expressing plasmid. TNF stimulation of nef-expressing Jurkat T cells resulted in NF-κB hyperactivation, which has been shown to deliver anti-apoptotic signals. Our results indicate that intercellular contacts with macrophages increase the rate of productively infected nonapoptotic CD4+ T lymphocytes. The survival of productively infected CD4+ T lymphocytes requires Nef expression as well as activation by TNF expressed on the surface of macrophages and might participate in the formation and maintenance of viral reservoirs in HIV-infected persons.

Macrophages and T-cell apoptosis in HIV infection: a leading role for accessory cells?

Recent studies indicate that macrophages modulate T-cell apoptosis in HIV infection. Macrophages have been shown to trigger apoptosis of uninfected bystander T cells and to protect HIV-infected T cells from apoptosis. This article raises the possibility that macrophages, via modulation of T-cell apoptosis, play a crucial role in both immune suppression and the formation of viral reservoirs during HIV infection.

When the band begins to play: Histone acetylation caught in the crossfire of gene control

Increasing evidence from recent research suggests a connection between cancer and a deranged equilibrium of histone acetylation, which is maintained by two competing enzymatic activities, histone acetyltransferases (HATs) and histone deacetylases (HDACs). It is our hypothesis that a significant proportion of leukemias and possibly also solid tumors have abnormalities involving HATs or HDACs at the genomic level through genetic mutations or chromosomal alterations. In these cases, altered levels of HATs or HDACs may derange the tightly regulated equilibrium of histone acetylation, which may affect the expression of a broad spectrum of cellular genes. On the other hand, HATs and HDACs may be carried to defined target promoters as cofactors of transcription factor–bound repressor or enhancer complexes and thereby carry out unwanted enzymatic activities in the wrong place at the wrong time. We therefore propose a model for disease being associated with a deranged equilibrium of acetylation that affects histone proteins and promoter‐bound transcription factors. Mol. Carcinog. 27:268–271, 2000.

Far-Western based protein–protein interaction screening of high-density protein filter arrays

Even though a rough sketch of the human genome is now available and the number of newly discovered genes, which carry the potential of being biologically and medically relevant is currently greater than ever, only a small proportion has been assigned a biological function. Therefore, enormous attention is now increasingly being drawn towards functional genomics, i.e. the functional characterization of these newly identified sequences. In order to elucidate the role of a particular gene product within its cellular context, we have screened high-density protein filter arrays for protein-protein interactions on the basis of a Far-Western based approach. The methodology described herein easily allows the identification and isolation of cDNAs of proteins, which interact with specific ligands (interacting proteins, antibodies and DNA/RNA sequences), and represents an alternative to tedious conventional protein interaction analyses. Far-Western screening in the context of a whole-genome expression analysis not only facilitates the assignment of biological functions to specific, newly identified protein and DNA sequences, but also is useful in studies that assess the binding capacity of mutant proteins to their interaction partner and in the identification of domains and amino acids involved in known protein-protein interactions. Taken together, we describe an approach that allows the easy and reproducible identification of protein ligands on the basis of a whole-genome expression analysis.

Chromosomal organization and localization of the human histone deacetylase 5 gene (HDAC5)

Histone deacetylases (HDACs) are important participants in the remodeling of chromatin structure and in the regulation of eukaryotic proliferation and differentiation. We have isolated and characterized the human HDAC5 genomic sequence, which spans a region of 39,138 bp and which has one single chromosomal locus. Determination of the exon-intron splice junctions established that HDAC5 is encoded by 26 exons ranging in size from 22 bp (exon 1) to 285 bp (exon 12). Characterization of the 5 flanking genomic region revealed that the human HDAC5 promoter lacks both the canonical TATA and CCAAT boxes. The human HDAC5 mRNA encodes a 1122 aa protein with a predictive molecular mass of 121.9 kDa and an isoelectric point of 5.84. Fluorescence in situ hybridization analysis localized the human HDAC5 gene to chromosome 17q21, a region which is characterized by frequent gains and losses of chromosomal material in several types of cancer.

Chromosomal organization and localization of the human histone deacetylase 9 gene (HDAC9).

Epigenetically mediated modulation of gene promoter function through histone acetylation modifying enzymes, which regulate the acetylation state of histone proteins and other promoter-bound transcription factors, is increasingly appreciated as a key component in the regulation of reversible gene expression. While histone acetyltransferases (HATs), which are frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, histone deacetylases (HDACs) tend to associate with multisubunit corepressor complexes, which result in chromatin condensation and transcriptional repression of specific target genes. We have isolated and characterized the human HDAC9 genomic sequence, which spans a region of 458 kb and which has one single chromosomal locus. Determination of the exon-intron splice-junctions established that HDAC9 is encoded by 23 exons ranging in size from 22 bp (exon 1) to 264 bp (exon 11). Characterization of the 5 flanking genomic region revealed that the human HDAC9 promoter lacks both the canonical TATA and CCAAT boxes; CpG elements are missing. The human HDAC9 open reading frame is 3036 bp long and encodes a 1011 aa protein with a predictive molecular weight of 111.3 kDa and an isoelectric point of 6.41. Fluorescence in situ hybridization analysis localized the human HDAC9 gene to chromosome 7p21, a region which has been associated particularly with the pathogenesis of gynecological tumors.

Assignment of the human histone deacetylase 6 gene (HDAC6) to X chromosome p11.23 by in situ hybridization.

Histone deacetylases (HDACs) play a central role in the modification of chromatin structure and thus in the regulation of transcription and cellular differentiation. Based on structural and functional similarities to the yeast histone deacetylases RPD3, HDA1 and SIR2, mammalian histone deacetylases may be grouped into three categories, class I, II and III HDACs. Based on its homology to HDA1, HDAC6 is a class II histone deacetylase (Grozinger et al., 1999). In view of the fact that the steady-state of histone acetylation and deacetylation plays a key role in the regulation of transcription, alteration of HDAC6 expression levels may affect the expression of specific genes targeted by HDAC6, which in turn may lead to full cellular transformation, malignancy or neurological disease (Mahlknecht and Hoelzer, 2000). Materials and methods

Assignment of the human histone deacetylase 4 gene (HDAC4) to chromosome 2q37.2 by in situ hybridization

Histone deacetylases (HDACs) play a central role in the modification of chromatin structure and thus in the regulation of transcription and cellular differentiation. Based on structural and functional similarities to the yeast histone deacetylases RPD3, HDA1 and SIR2, mammalian histone deacetylases may be grouped into three categories, class I, II and III HDACs. Based on its homology to HDA1, HDAC4 is a class II histone deacetylase (Grozinger et al., 1999; Mahlknecht and Hoelzer, 2000). Through its association with proteins which exhibit kinase activity, HDAC4 links the Ras-MAPK signal transduction pathway to histone deacetylation (Zhou et al., 2000). In addition, HDAC4, which is localized in the cytoplasm and/or the nucleus, is negatively regulated by 14-3-3 proteins, which prevent its nuclear localization (Wang et al., 2000). Materials and methods