Sexual differentiation is a fundamental characteristic of all eukaryotes, dictating sex-specific morphology, physiology and behavior. Diploid organisms with heteromorphic sex chromosomes (XX or XY) require regulatory compensation of the X chromosome to maintain correct levels of genetic expression between the sexes, a process termed sex-specific dosage compensation (SSDC). The fruit fly, <i>Drosophila melanogaster</i> dosage compensates by upregulating transcription of most X-linked genes two-fold. Associated with this two-fold up regulation is the male-specific lethal (MSL) complex, a RNA-protein complex comprised of at least five known proteins; MSL1, MSL2, MSL3, males absent on the first (MOF), and maleless (MLE) and two non-translated RNA molecules; roX1 (RNA on the X chromosome) and roX2. The complex modulates the chromatin structure of the male X chromosome via acetylation of H4K16. MOF and MSL3 both exhibit an N-terminal chromodomain, whose function is unclear. The MSL3 chromodomain has been suggested to bind H3K36Me3. Chromodomains are a paradigm of how a single structural fold has evolved in diverse proteins to bind distinct targets. Chromodomains are common to nuclear regulators, and bind diverse targets including histones, DNA, and RNA. They function as recognition motifs of histone post-translational modifications and facilitate the translation of the histone code into a distinct local chromatin structure via recruiting the appropriate chromatin modulating machinery.<p> The goal of this research is to determine the structure of the <i>D. melanogaster</i> MOF and MSL3 chromodomains by X-ray crystallographic and/or nuclear magnetic resonance techniques, to advance our understanding of the structural characteristics of these diverse domains. Here we report the cloning and reproducible expression and purification of the <i>D. melanogaster</i> MOF and MSL3 chromodomains and their Homo sapiens orthologues. The <i>D. melanogaster</i> MOF chromodomain, whose NMR structure was published during this research, has been crystallized. Attempts to solve the crystal structure by molecular replacement, multiple-wavelength anomalous dispersion, and single-wavelength isomorphous replacement are reported.

Sexual differentiation is a fundamental characteristic of all eukaryotes, dictating sex-specific morphology, physiology and behavior. Diploid organisms with heteromorphic sex chromosomes (XX or XY) require regulatory compensation of the X chromosome to maintain correct levels of genetic expression between the sexes, a process termed sex-specific dosage compensation (SSDC). The fruit fly, <i>Drosophila melanogaster</i> dosage compensates by upregulating transcription of most X-linked genes two-fold. Associated with this two-fold up regulation is the male-specific lethal (MSL) complex, a RNA-protein complex comprised of at least five known proteins; MSL1, MSL2, MSL3, males absent on the first (MOF), and maleless (MLE) and two non-translated RNA molecules; roX1 (RNA on the X chromosome) and roX2. The complex modulates the chromatin structure of the male X chromosome via acetylation of H4K16. MOF and MSL3 both exhibit an N-terminal chromodomain, whose function is unclear. The MSL3 chromodomain has been suggested to bind H3K36Me3. Chromodomains are a paradigm of how a single structural fold has evolved in diverse proteins to bind distinct targets. Chromodomains are common to nuclear regulators, and bind diverse targets including histones, DNA, and RNA. They function as recognition motifs of histone post-translational modifications and facilitate the translation of the histone code into a distinct local chromatin structure via recruiting the appropriate chromatin modulating machinery.<p> The goal of this research is to determine the structure of the <i>D. melanogaster</i> MOF and MSL3 chromodomains by X-ray crystallographic and/or nuclear magnetic resonance techniques, to advance our understanding of the structural characteristics of these diverse domains. Here we report the cloning and reproducible expression and purification of the <i>D. melanogaster</i> MOF and MSL3 chromodomains and their Homo sapiens orthologues. The <i>D. melanogaster</i> MOF chromodomain, whose NMR structure was published during this research, has been crystallized. Attempts to solve the crystal structure by molecular replacement, multiple-wavelength anomalous dispersion, and single-wavelength isomorphous replacement are reported.

Two manifestations of gene regulation-- dosage compensation and ontogenic regulation--were examined in normally positioned and relocated v+ genotypes in Drosophila melanogaster to determine the role of gene position in these control functions. Enzyme assays, used as criteria of gene activity, were performed on various genotypes containing different doses of v+ in normal and relocated positions in male and female flies. The results indicate that although differently positioned v+ genes may specify different tryptophan pyrrolase activities, they still show dosage compensation. In each case, the enzyme activity associated with each gene, either on the X, Y, or third chromosome, is twice as much in males as it is in females. This indicates that dosage compensation is not confined to the gene when located on the X chromosome. In order to determine if the pattern of activity of the gene during ontogeny is altered by relocation, T(l; 3)rasv genotypes and wild type controls were assayed at the same stages of development. The experimental design allowed a comparison of the ontogenic expression of three different genes--v+, Zw, and Pgd--through the activities of their associated enzymes. The results indicate that changing the gene's position may alter its ontogenic expression. Animals with v+ on the third chromosome have a unique peak of tryptophan pyrrolase activity in larvae which is not present in wild type. The activity in this peak is at l east 10 times higher than that observed in 72-hour wild type larvae, in fact, higher than that observed in any normal genotype at any time during development. With the exception of this peak, the developmental curves of enzyme activity are similar, although the relocated genes specify consistently lower enzyme activities than do normally positioned genes. The unique peak is not the result of a general physiological effect since the patterns of Zw and Pgd activity appear to be the same in wild type and translocated v+ genotypes. The relevance of the data to earlier studies and to models for gene regulation is discussed.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-87).

In Drosophila melanogaster, two chromosome-specific targeting and regulatory systems have been described. The male-specific lethal (MSL) complex supports dosage compensation by stimulating gene expression from the male X-chromosome and the protein Painting of fourth (POF) specifically targets and stimulates expression from the heterochromatic 4(th) chromosome. The targeting sites of both systems are well characterized, but the principles underlying the targeting mechanisms have remained elusive. Here we present an original observation, namely that POF specifically targets two loci on the X-chromosome, PoX1 and PoX2 (POF-on-X). PoX1 and PoX2 are located close to the roX1 and roX2 genes, which encode ncRNAs important for the correct targeting and spreading of the MSL-complex. We also found that the targeting of POF to PoX1 and PoX2 is largely dependent on roX expression and identified a high-affinity target region which ectopically recruits POF. The results presented support a model linking the MSL-complex to POF and dosage compensation to regulation of heterochromatin.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Dosage compensation is the key regulatory process employed in <i>Drosophila melanogaster</i> to equalize the level of gene transcripts between the single X chromosome in males (XY) and the two X chromosomes in females (XX). Dimorphic sex chromosomes evolved by the severe degeneration of the Y chromosome, giving rise to an imbalance between the heterogametic sex and the homogametic sex. Vital to the viability of male Drosophila is the dosage compensation complex (DCC), a ribonucleoprotein complex that mediates the precise two-fold transcription of the single male X chromosome. The DCC is comprised of five proteins: male-specific-lethal proteins (MSL) 1, 2, and 3, male absent-on-the-first (MOF), maleless (MLE), and two non-coding RNAs. The complex specifically co-localizes along the male X chromosome in a reproducible manner, resulting in acetylation of lysine 16 of the N-terminal tail of histone H4. The exact mechanism of recruitment and spreading of the DCC along the male X chromosome remains unclear; recent studies propose a multi-step mechanism involving DNA sequence elements, epigenetic marks, and transcription. Understanding how dosage compensation functions provides insight into the interplay between gene regulation and chromatin remodelling. The goal of this project was to better understand how <i>Drosophila</i> MSL1, MSL3, and MOF interact and how their interaction modulates MOFs acetyltransferase activity. Recombinant protein constructs were cloned and over-expressed in a bacterial expression system permitting future structure determination by X-ray crystallography. The dMSL1820-1039 construct consisted of the C-terminal domain, reported to be able to interact with both dMSL3 and dMOF. dMSL3186-512 contained the domain required for the interaction with dMSL1 and dMOF. dMOF371-827 was comprised of the catalytic domain, the CCHC zinc finger, and the chromodomain, as the N-terminal region does not encode any known domains. All three recombinant proteins were successfully cloned, over-expressed, and purified to homogeneity. Recombinant dMOF371-827 was determined to acetylate histones. Interaction studies using GST pull-down assays and size exclusion chromatography determined that dMSL1820-1039 and dMOF371-827 did not interact above background levels. Moreover, size exclusion chromatography revealed dMSL3186-512 and dMOF371-827 did not interact nor did the three recombinant proteins form a stable complex.

Little is known about dosage compensation in autosomal genes. Transcription-level compensation of deletions and other loss-of-function mutations may be a mechanism of dominance of wild-type alleles, a ubiquitous phenomenon whose nature has been a subject of a long debate. We measured gene expression in two isogenic Drosophila lines heterozygous for long deletions and compared our results with previously published gene expression data in a line heterozygous for a long duplication. We find that a majority of genes are at least partially compensated at transcription, both for 1/2-fold dosage (in heterozygotes for deletions) and for 1.5-fold dosage (in heterozygotes for a duplication). The degree of compensation does not vary among functional classes of genes. Compensation for deletions is stronger for highly expressed genes. In contrast, the degree of compensation for duplications is stronger for weakly expressed genes. Thus, partial transcriptional compensation appears to be based on regulatory mechanisms that insure high transcription levels of some genes and low transcription levels of other genes, instead of precise maintenance of a particular homeostatic expression level. Given the ubiquity of transcriptional compensation, dominance of wild-type alleles may be at least partially caused by of the regulation at transcription level.

Chromatin is organized dynamically to accommodate different biological processes. One of the factors required for proper chromatin organization is a group of complexes called condensins. Most eukaryotes have two conserved condensins (I and II) required for chromosome segregation. C. elegans has a third condensin (IDC) that specializes in dosage compensation, a process that down-regulates X gene dosage in XX hermaphrodites to match the dosage in XO males. How the three condensins are regulated is not well understood. Here, I present the discovery and characterization of a novel condensin regulator, SMCL-1. We identified SMCL-1 through purification of a MAP-tagged condensin subunit. Condensins are comprised of SMC ATPases and regulatory CAP proteins; SMCL-1 interacts most abundantly with condensin SMC subunits and resembles the ATPase domain of SMC proteins. Interestingly, the SMCL-1 protein has residues that differ from SMC consensus and potentially render SMCL-1 incapable of hydrolyzing ATP. Worms harboring smcl-1 deletion are viable and show no detectable phenotype. However, deleting smcl-1 in a condensin hypomorph mildly suppresses condensin I and IDC mutant phenotypes, suggesting that SMCL-1 functions as a negative regulator of condensin I and IDC. Consistent with this, overexpression of SMCL-1 leads to condensin loss-of-function phenotypes such as lethality, segregation defects and disruption of IDC localization on the X chromosomes. Homology searches based on the unique ATPase domain of SMCL-1 reveal that SMCL-1-like proteins are present only in organisms also predicted to have condensin IDC. Taken together, we conclude that SMCL-1 is a negative modulator of condensin functions and we propose a role for SMCL-1 in helping organisms adapt to having a third condensin by maintaining the balance among three condensin complexes.

<p>Abstract</p><p>DNA replication is an integral part of the cell cycle. Every time a cell divides, the entire genome has to be copied once and only once in a timely manner. In order to accomplish this, DNA replication begins at many points throughout the genome. These start sites are called origins of replication, and they are initiated in a temporal manner throughout S phase. How these origins are selected and regulated is poorly understood. Saccharomyces cerevisiae and Schizosaccharomyces pombe have autonomously replicating sequences (ARS) that can replicate plasmids extrachromosomally and function as origins in the genome. Metazoans, however, have shown no evidence of ARS activity.</p><p>DNA replication is a multistep process with several opportunities for regulation. Potential origins are marked with the origin recognition complex (ORC), a six subunit complex. In S. cerevisiae, ORC binds to the ARS consensus sequence (ACS), but no sequence specificity is seen in S. pombe or in metazoans. Therefore, factors other than sequence play a role in origin selection.</p><p>In G1, the pre-replicative (pre-RC) complex assembles at potential origins. This involves the recruitment of Cdc6 and Cdt1 to ORC, which then recruits MCM2-7 to the origin. In S phase, a subset of these pre-RC marked origins are initiated for replication. These origins are not fired simultaneously; instead, origins are fired in a temporal manner, with some firing early, some firing late, and some not firing at all.</p><p>The temporal firing of origins leads to wide regions of the genome being copied at different times during S phase. , which makes up the replication timing profile of the genome. These regions are not random, and several correlations between replication timing and both transcriptional activity and chromosomal landscape. Regions of the genome with high transcriptional activity tend to replicate earlier in S phase, and it is well know that the gene rich euchromatin replicates earlier than the gene poor heterochromatin. Additionally, areas of the genome with activating chromatin marks also replicate earlier than regions with repressive marks. Though many correlations have been observed, no single mark or transcriptional player has been shown to directly influence replication timing.</p><p>We mapped the replication timing profiles of three cell lines derived from Drosophila melanogaster by pulsing cells with the nucleotide analog bromodeoxyuridine (BrdU), enriching for actively replicating DNA labeled with BrdU, sequencing with high throughput sequencing and mapping the sequences back to the genome. We found that the X chromosome of the male cell lines replicated earlier than the X chromosome in the female cell line or the autosomes. We were then able to compare the replication timing profiles to data sets for chromatin marks acquired through the modENCODE (model organism Encyclopedia Of DNA Elements). We found that the early replicating regions of the male X chromosomes correlates with acetylation of lysine 16 on histone 4 (H4K16).</p><p>Hyperacetylation of H4K16 on the X chromosome in males is a consequence of dosage compensation in D. melanogaster. Like many organisms, D. melanogaster females have two X chromosomes while males have one. To compensate for this difference, males upregulate the genes on the X chromosome two-fold. This upregulation is regulated by the dosage compensation complex (DCC), which is restricted to the X chromosome. This complex includes a histone acetyl transferase, MOF, which acetylates H4K16. This hyperacetylation allows for increased transcription of the X chromosome. </p><p>We hypothesized that the activities of the DCC and the hyperacetylation of H4K16 also influences DNA replication timing. To test this, I knocked down components of the DCC (MSL2 and MOF) using RNAi. Cells were arrested in early S phase with hydroxyurea, released, and pulsed with the nucleotide analog EdU. The cells were arrested in metaphase and labeled for H4K16 acetylation and EdU. We found that male cells were preferentially labeled with EdU on the X chromosome, which corresponded with H4k16 acetylation. When the DCC was knocked down, H4K16 acetylation was lost along with preferential EdU labeling on the X chromosome. These results suggest that the DCC and H4K16 acetylation are necessary for early replication of the X chromosome. Additionally, early origin mapping of different cell lines showed that while ORC density does not differ between male and female cell lines, early origin usage is increased on the X chromosome of males, suggesting that this phenomenon is regulated at the level of activation, not pre-RC formation. Other experiments in female cell lines have been unclear about whether the DCC and subsequent H4K16Ac is sufficient for early X replication. However, these results are exciting because this is, to our knowledge, the first mark that has been found to directly influence replication timing.</p><p>In addition to these timing studies, I attempted to design a new way to map origins. A consequence of unidirectional replication with bidirectional replication fork movement is Okazaki fragments. These are short nascent strands on the lagging strand of replicating DNA. Because these fragments are small, we can isolate them by size and map them back to the genome. Okazaki density could tell us about origin usage and any directional preferences of origins. The process proved to be tedious, and although they mapped back with a higher density around ORC binding sites than randomly sheared DNA, little information about origin usage was garnered from the data. Additionally, the process proved difficult to repeat.</p><p>In these studies, we examined the replication timing program in D. melanogaster. We found that the male X chromosome replicates earlier in S phase, and this early replication is regulated by the DCC. However, it is unclear if the change in chromatin landscape directly influences replication or if the replication program is responding to other dosage compensation cues on the X chromosome. Regardless, we have found one the first conditions in which a mark directly influences the DNA replication timing program.&#8195;</p> / Dissertation