Frank Sauer
Assistant Professor of Biochemistry

Regulation of eukaryotic gene expression Biochemistry Molecular Biology Dr. rer. Nat, University of Osnabrück, Germany EMBO Postdoctoral Fellowship, UC Berkeley 1994-1996 Max-Planck Postdoctoral Fellowship, UC Berkeley 1997 VOICE: (951) 827-3616 FAX: (951) 827-4434 EMAIL: frank.sauer@ucr.edu

GOALS

It is our goal to decipher molecular mechanisms underlying regulation of gene expression during different biological processes such as body pattern formation and haematopoiesis in the model organism Drosophila melanogaster. Currently, we focus on three fundamental topics of gene expression. (i) We investigate how posttranslational modifications of histones contribute to transient and epigenetic regulation of gene expression. (ii) We use DNA microarray-based gene expression and DANN; protien interaction analyses to investigate the functional importance of proteins involved in regulation of gene expression and to dissect the molecular mechanisms involved in differentiation/proliferation of Drosophila hemocytes. (iii) We use biochemical approaches to identify new components of signal transduction pathways, which control the activity of transcription factors/coactivators. To tackle theses aims, we use use a combination of various methods in biochemistry, molecular biology, cell biology, genetics, and DNA microarray

PUBLICATIONS

Beisel, C. Imhof, A, Greene, J., Kremmer, E., and Sauer, F. (2002). Histone methylation by the Drosophila epigenetic regulator Ash1. Nature 419, 857-861.

Wassarman, D. A., and Sauer, F. (2001) TAFII250: A transcription toolbox. J. Cell Sci. 114, 2895-2902.

Belz, T., et al. (2001) In vitro assays to study histone ubiquitination. Methods 26, 233-244.

Pham, A.-D. and Sauer, F. (2000). Ubiquitin-activating/conjugating activity of TAFII250, a mediator of activation of gene expression in Drosophila. Science 289, 2357-2360.

CURRENT PROJECTS

i.) Histone Modifications in Transcription

Introduction
One of the most fundamental and demanding tasks of eukaryotic cells is the packaging of the large genomic DNA into the small dimensions of the nucleus. To solve this task, eukaryotic cells package their chromosomal DNA with proteins, mainly histones, into a highly compacted DNA:protein complex, chromatin. Histones (H1, H2A, H2B, H3, and H4) are the basic building blocks of nucleosomes, which represent the smallest structural entity in chromatin. Core-nucleosomes consist of a histone octamer, which contains two copies of each core-histone (H2A, H2B, H3, and H4), around which 146 bp DNA are wrapped. The association of the linker histone H1 with core-nucleosomes stabilizes the interaction of the histone-octamer with DNA and finalizes the formation of the nucleosome. In a not well-understood manner, interactions among and between histones and non-histone proteins culminate in the formation of the highly condensed chromatin structure visible in metaphase chromosomes. Although essential of packaging, the tight association of chromosomal DNA with proteins represents both challenge and opportunity for the execution of DNA-dependent processes. On one hand the regulated interaction of proteins with chromatin during assembly or condensation of chromatin provides the opportunity to establish chromatin structures that prevent the interaction of regulatory proteins with DNA to e.g. silence transcription. Conversely, chromatin represents a challenge for the execution of DNA-dependent processes such as transcription as chromatin may prevent the interactions of regulatory DNA that facilitate transcription as well as other DNA-dependent processes. Therefore, a huge effort in biology has been and is currently devoted to dissect the molecular mechanisms that facilitate 'dynamic structural changes in chromatin' (termed "chromatin dynamics) that accompany DNA-dependent processes such as transcription. In the past ten years, proteins and DNA-elements have been identified that play important roles in chromatin dynamics during transcription (and probably other DNA-dependent events). Among theses components are enzymes that mediate the post-translational modification of histones e.g. ubiquitination, methylation, and acetylation. Preferred targets of the modifications are phylogenetically highly conserved amino acid residues in the NH2- and COOH-terminal tails of the core-histones. These tails protrude from the nucleosome and therefore are accessible for enzymatic modification. It has been proposed that the distinct modification-patterns of histones represent a 'histone code' that determines whether a chromatin region is e.g. transcriptional active or silent. Recent efforts have linked histone acetylation with activation of transcription and deacetylation of transcriptional repression. In addition, the methylation of lysine- and arginine residues in NH2-terminal histone tails and the ubiquitination of COOH-terminal histone tails have been correlated with transcriptional active chromatin structures. However the enzymes that mediate these modifications in the context of transcriptional regulation are only now being discovered. We have identified several enzymes that methylate, phosphorylate or ubiquitinate histones and are currently characterize the functional importance of these enzymes for transcription and other DNA-dependent processes.

Histone ubiquitination
Although ubiquitination of histones had been linked to transcriptionally chromatin structures, the enzymes that ubiquitinate histones during transcription are only now being discovered. Thus, as the first step towards the investigation of the functional connection of histone ubiquitination and regulation of gene expression, we set out to identify proteins that transfer ubiquitin onto histones. As a protein source, we used crude or fractionated Drosophila nuclear extract prepared form 0-12 hour old embryos. To identify histone ubiquitinating enzymes, we used an 'in-gel ubiquitination assay' that is based on the 'in-gel activity assay' used by Allis and colleagues to identify histone acetyl-transferases. In this initial experiment, we could detect a protein with a relative molecular weight of approx. 230 kD that ubiquitinates histones. We used different biochemical approaches to uncover the identity of the putative histone ubiquitinating enzyme. We could show that the enzyme is the coactivator TAFII250, the central subunit of the TFIID complex, which consists of at least 10 different TBP associated factors (TAFIIs) and the TATA-box binding protein (TBP). TAFIIs interact with DNA, general transcription factors and transcription factors to facilitate activation of transcription. Binding of TFIID to promoter regions is thought to represent one of most initial and fundamental steps in transcription. In vitro ubiquitination assays revealed that TAFII250 mono-ubiquitinates histone H1 in vitro. Polyubiquitination of proteins depends on the hierarchic activities of three different proteins: E1, E2 and E3. Our studies indicate that TAFII250 has both ubiquitin-activating- (E1) and ubiquitin-conjugating-activity (E2). This result is conform to other studies suggesting that mono-ubiquitination of proteins requires E1 and E2 only. We have recently identified the cysteine-residue within the E1-domain of TAFII250 that serves as the acceptor of ubiquitin.

Fig 1. Mutation in TAFII250 E1/E2-activity abolish activation of transcription in Drosophila (A to I) Reduction of gene expression in embryos lacking dTAFII250 E1/E2-activity. In situ hybridization showing twist or snail (C expression in dl/+ (A and D) dl/+; TAF250XS-2232- (Band E) and dl/+; TAF250S-625-embryos (C and F). The ventral surface of blastoderm-stage embryos is shown with anterior pointing to the left. (G, H, I) Dark field images of the cuticule body pattern of dl/+ (G), TAF250XS-2232- (H), and dl/+; TAF250S-625- embryos (I) (Pham and Sauer, 2000). To provide evidence for the biological significance of the TAFII250 E1/E2-activity for transcription, we made use of TAF250 alleles, which encode single amino acid exchange point mutations in the putative E1 region of TAFII250. These mutations abolish TAFII250-dependent mono-ubiquitination of H1 in vitro. In Drosophila embryos expressing TAFII250 lacking E1/E2-activity, the transcription of zygotic segmentation genes and the level of mono-ubiquitinated H1 are significantly reduce (Figures 1 and 2). Our studies imply that mono-ubiquitination of H1 by the coactivator TAFII250 may play an important role for the processes directing activation of transcription in metazoan cells.

Fig. 2 H1 is ubiquitinated in Drosophila. "Western-Blot" analysis of H1 isolated from wild type (lanes 1 and 4), homozygous mutant TAFXS-2232 embryos (lanes 2 and 5) or homozygous mutant TAFS625embryos (lanes 3 ad 6). Proteins were detected using anti-H1 (lane 1 to 3) or anti-ubiquitin antibodies (lanes 4 to 6). The position of H1 and mono-ubiquitinated H1 (uH1) is indicated. The position and size (in kilodaltons) of protein standards is indicated to the left.

Histone methylation
Distinct histone methylation patterns have been correlated to both transcriptional active a silent chromatin structures. Enzymes that methylate lysine-residues in histones during the process of transcriptional activation remain opaque. To date, we have isolated seven distinct enzymatic activities, which methylate histones isolated from Drosophila nuclear extracts. One of the identified histone methyltransferases (HMT) is the epigenetic activator "absent small and homeotic discs' (ASH1), which is a member of the trithorax-Group of epigenetic regulators of gene expression. ASH1 methylates histone H3 and H4 in poly-nucleosomes and histone octamers. Deletion analyses indicate that the SET-domain and adjacent cysteine-rich regions (termed pre- and post-SET domains) of ASH1 mediates methylation of H3 and H4. In contrast to the epigenetic repressor SUV39H1, which methylates lysine-residue 9 in H3, ASH1 methylates lysine 4 (H3-K4) and 9 (H3-K9) of H3 and lysine 20 (K20) of H4. We have identified point mutations in the ASH1 SET-domain, which abolish ASH1 HMT-activity in vitro. In Drosophila the same point mutations abrogate ASH1-mediated activation of gene expression. 'Chromatin immunoprecipitation' experiments indicate that ASH1 methylates H3-K4, H3-K9 and H4K20 in the promoter region of artificial and natural target genes in Drosophila cells and imaginal discs. In mutant ASH1 proteins, which lack HMT-activity, transcriptional activation and histone methylation were not detectable. In conclusion, our results imply that a trivalent histone methylation pattern placed by ASH1 plays an important role for epigenetic activation of transcription (Beisel et al., 2002).

Figure 2 ASH1 methylates H3-K4, H3-K9, and H4-K20. a, Microsequencing of radiolabeled polynucleosomal H3. b, HMTase-assays using ASH1(SET) and wild type or mutant H3-peptides (amino acids 1-20). H3(1-20)K4 peptides that contain H3-K4 only, H3(1-20)K9 peptides H3-K9 only, and H3(1-20)L4/L9 peptides contain L instead of K4 and K9. c, Western blot analyses using anti-dimethylated-H4-K20 monoclonal antibody [dim(H4-K20)] of HMTase-assays containing recombinant H4. d, Western blot analyses as in (c) except that reactions were programmed with polynucleosomes or histone core-octamers as indicated. e, Top panel: Schematic representation of mutant ASH1DN proteins. Bottom panel: Coomassie-blue stained SDS-gel (top) and fluorogram (bottom) of HMTase-assays programmed with the indicated ASH1-derivatives and polynucleosomes. f, Top panel: Schematic representation of the N18/15 reporter gene containing the 4 kb regulatory element of the bxd-region, which contains three TRX response elements (TRE), fused to the Drosophila mini-white gene (white). Bottom panel: Photographs of eyes from N18/15/+ (top), N18/15/ash110 (middle) and N18/15/ash121 heterozygotes (bottom).

Histone phosphorylation
We have identified enzymes that specifically phosphorylate histones. We are in the process to dissect the role and function of these enzymes for regulation of transcription.

ii.) Gene Expression Profiling by DNA Microsrray
In collaboration with the groups of Dr. Jörg Hoheisel and Dr. Renato Paro (Heidelberg, Germany) we have established a novel Drosophila DNA microarray that contains probes for 21.000 putative open reading frames in the Drosophila genome. In collaboration with the group of Dr. Istvan Ando (Hungary Academy of Sciences, Szeged, Hungary) we will use this microarray to establish gene expression profiles that accompany hemocyte differentiation/proliferation in Drosophila to identify the key regulators of this process. In addition, we will use the same approach to dissect the molecular basis of immune response in Drosophila (for example in response to the introduction of an egg planted by a parasitic wasp) and the leukemia-like hyperproliferation of hemocytes in response to mutations in signal transduction pathways. In addition, we will use the microarray to investigate the functional relevance of identified regulators of transcription for regulation of gene expression in the context of the entire Drosophila genome. For example, we want to elucidate which and how many genes are regulated by the E1/E2-activity of TAFII250. This may provide clues whether e.g. this particular enzymatic activity controls the expression of gene clusters, which control specific biological programs.

iii.) JAK/STAT pathway
We have isolated new interaction partners of the Drosophila JAK/STAT signal transduction pathway, phylogenetically highly conserved signal transduction pathway, and are in the process to functionally characterize the identified proteins in vitro and in Drosophila.
STATs (signal transducers and activators of transcription) are cytoplasmic latent transcription factors, whose nuclear translocation and transcriptional activity is regulated by the JAK/STAT-pathways. The binding of ligands to membrane bound receptors triggers a kinase-cascade that ultimately results in the phosphorylation of STATs. Phosphorylated STATs can form homo- or heterodimers which, in contrast to monomers, enter the nucleus and activate the expression of specific target genes. Genetics experiments provide circumstantial evidence that Drosophila JAK (HOPSCOTCH) does not only interact with STAT to transmit its signal to the level of gene expression but also communicates with other factors. In this screen, we have identified proteins that interact with HOPSCOTCH. We are in the process to characterize these proteins and their role and function in JAK-dependent signal transduction. These studies may elucidate how JAKs probably by regulating the activity of transcription factors/coactivators control the execution of biological processes.