Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells

The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

The Molecular Basis of Lysine Acetylation: Addition, Removal, and Recognition

Acetyltransferases and deacetylases catalyze the addition and removal, respectively, of acetyl groups to the epsilon-amino group of protein lysine residues. This modification can affect the function of a protein through several means, including the recruitment of specific binding partners called acetyl-lysine readers. Acetyltransferases, deacetylases, and acetyl-lysine readers have emerged as crucial regulators of biological processes and prominent targets for the treatment of human disease. This work describes a combination of structural, biochemical, biophysical, cell-biological, and organismal studies undertaken on a set of proteins that cumulatively include all steps of the acetylation process: the acetyltransferase MEC-17, the deacetylase SIRT1, and the acetyl-lysine reader DPF2. Tubulin acetylation by MEC-17 is associated with stable, long-lived microtubule structures. We determined the crystal structure of the catalytic domain of human MEC-17 in complex with the cofactor acetyl-CoA. The structure in combination with an extensive enzymatic analysis of MEC-17 mutants identified residues for cofactor and substrate recognition and activity. A large, evolutionarily conserved hydrophobic surface patch distal to the active site was shown to be necessary for catalysis, suggesting that specificity is achieved by interactions with the alpha-tubulin substrate that extend outside of the modified surface loop. Experiments in C. elegans showed that while MEC-17 is required for touch sensitivity, MEC-17 enzymatic activity is dispensible for this behavior. SIRT1 deacetylates a wide range of substrates, including p53, NF-kappaB, FOXO transcription factors, and PGC-1-alpha, with roles in cellular processes ranging from energy metabolism to cell survival. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an apo form and in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a beta-hairpin structure that complements the beta-sheet of the NAD^+-binding domain, covering an essentially invariant, hydrophobic surface. A comparison of the apo and cofactor bound structures revealed conformational changes throughout catalysis, including a rotation of a smaller subdomain with respect to the larger NAD^+-binding subdomain. A biochemical analysis identified key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain. DPF2 represses myeloid differentiation in acute myelogenous leukemia. Finally, we solved the crystal structure of the tandem PHD domain of human DPF2. We showed that DPF2 preferentially binds H3 tail peptides acetylated at Lys14, and binds H4 tail peptides with no preference for acetylation state. Through a structural and mutational analysis we identify the molecular basis of histone recognition. We propose a model for the role of DPF2 in AML and identify the DPF2 tandem PHD finger domain as a promising novel target for anti-leukemia therapeutics.

The interactions of basic proteins and DNA

I. ELECTROPHORESIS OF THE NUCLEIC ACIDS

A zone electrophoresis apparatus using ultraviolet optics has been constructed to study nucleic acids at concentrations less than 0.004%. Native DNA has a mobility about 15% higher than denatured DNA over a range of conditions. Otherwise, the electrophoretic mobility is independent of molecular weight, base composition or source. DNA mobilities change in the expected way with pH but the fractional change in mobility is less than the calculated change in charge. A small decrease in mobility accompanies an increase in ionic strength. RNA’s from various sources have mobilities slightly lower than denatured DNA except for s-RNA which travels slightly faster. The important considerations governing the mobility of nucleic acids appear to be the nature of the hydrodynamic segment, and the binding of counterions. The differences between electrophoresis and sedimentation stem from the fact that all random coil polyelectrolytes are fundamentally free draining in electrophoresis.

II. THE CYTOCHROME C/DNA COMPLEX

The basic protein, cytochrome c, has been complexed to DNA. Up to a cytochrome:DNA mass ratio of 2, a single type of complex is formed. Dissociation of this complex occurs between 0.05F and 0.1F NaCl. The complexing of cytochrome to DNA causes a slight increase in the melting temperature of the DNA, and a reduction of the electrophoretic mobility proportional to the decrease in net charge. Above a cytochrome:DNA mass ratio of 2.5, a different type of complex is formed. The results suggest that complexes such as are formed in the Kleinschmidt technique of electron microscopy would not exist in bulk solution and are exclusively film phenomena.

III. STUDIES OF THE ELECTROPHORESIS AND MELTING BEHAVIOUR OF NUCLEOHISTONES

Electrophoresis studies on reconstituted nucleohistones indicate that the electrophoretic mobility for these complexes is a function of the net charge of the complex. The mobility is therefore dependent on the charge density of the histone complexing the DNA, as well as on the histone/DNA ratio. It is found that the different histones affect the transition from native to denatured DNA in different ways. It appears that histone I is exchanging quite rapidly between DNA molecules in 0.01 F salt, while histone II is irreversibly bound. Histone III-IV enhances the capacity of non-strand separated denatured DNA to reanneal. Studies on native nucleoproteins indicate that there are no gene-sized uncomplexed DNA regions in any preparations studied.

IV. THE DISSOCIATION OF HISTONE FROM CALF THYMUS CROMATIN

Calf thymus nucleoprotein was treated with varying concentrations of NaCl. The identity of the histones associated and dissociated from the DNA at each salt concentration was determined by gel electrophoresis. It was found that there is no appreciable histone dissociation below 0.4 F NaCl. The lysine rich histones dissociate between 0.4 and 0.5 F NaCl. Their dissociation is accompanies by a marked increase in the solubility of the chromatin. The moderately lysine rich histones dissociate mainly between 0.8 and 1.1 F NaCl. There are two arginine rich histone components: the first dissociates between 0.8 F and 1.1 F NaCl, but the second class is the very last to be dissociated from the DNA (dissociation beginning at 1.0 F NaCl). By 2.0 F NaCl, essentially all the histones are dissociated.

The properties of the extracted nucleoprotein were studied. The electrophoretic mobility increases and the melting temperature decreases as more histones are dissociated from the DNA. A comparison with the dissociation of histones from DNA in NaClO₄ shows that to dissociate the same class of histones, the concentration of NaCl required is twice that of NaClO₄.

Investigations of nonhistone chromosomal proteins

The major nonhistone chromosomal proteins (NHC proteins) are a group of 14-20 acidic proteins associated with DNA in eukaryotic chromatin. In comparisons by SDS gel electrophoresis (molecular weight sieving) one observes a high degree of homology among the NHC protein fractions of different tissues from a given species. Tissue-specific protein bands are also observed. The appearance of a new NHC protein, A, in the NHC proteins of rat liver stimulated to divide by partial hepatectomy and of rat ascites cells suggests that this protein may play a role in preparing the cell for division. The NHC proteins of the same tissue from different species are also very similar. Quantitative but not qualitative changes in the NHC proteins of rat uterus are observed on stimulation (in vivo) with estrogen. These observations suggest that the major NHC proteins play a general role in chromatin structure and the regulation of genome expression; several may be enzymes of nucleic acid and histone metabolism and/or structural proteins analogous to histones. One such enzyme, a protease which readily and preferentially degrades histones, can be extracted from chromatin with 0.7 N NaCl.

Although the NHC proteins readily aggregate, they can be separated from histone and fractionated by ion exchange chromatography on Sephadex SE C-25 resin in 10 M urea-25% formic acid (pH 2.5). Following further purification, four fractions of NHC protein are obtained; two of these are single purified proteins, and the other two contain 4-6 and 4-7 different proteins. These NHC proteins show a ratio of acidic to basic amino acids from 2.7 to 1.2 and isoelectric points from apparently less than 3.7 to 8.0. These isolated fractions appear more soluble and easier to work with than any whole NHC protein preparation.