Evolution of genomic sequence inhomogeneity at mid-range scales

<p>Abstract</p> <p>Background</p> <p>Mid-range inhomogeneity or MRI is the significant enrichment of particular nucleotides in genomic sequences extending from 30 up to several thousands of nucleotides. The best-known manifestation of MRI is CpG islands representing CG-rich regions. Recently it was demonstrated that MRI could be observed not only for G+C content but also for all other nucleotide pairings (e.g. A+G and G+T) as well as for individual bases. Various types of MRI regions are 4-20 times enriched in mammalian genomes compared to their occurrences in random models.</p> <p>Results</p> <p>This paper explores how different types of mutations change MRI regions. Human, chimpanzee and <it>Macaca mulatta </it>genomes were aligned to study the projected effects of substitutions and indels on human sequence evolution within both MRI regions and control regions of average nucleotide composition. Over 18.8 million fixed point substitutions, 3.9 million SNPs, and indels spanning 6.9 Mb were procured and evaluated in human. They include 1.8 Mb substitutions and 1.9 Mb indels within MRI regions. Ancestral and mutant (derived) alleles for substitutions have been determined. Substitutions were grouped according to their fixation within human populations: fixed substitutions (from the human-chimp-macaca alignment), major SNPs (> 80% mutant allele frequency within humans), medium SNPs (20% - 80% mutant allele frequency), minor SNPs (3% - 20%), and rare SNPs (<3%). Data on short (< 3 bp) and medium-length (3 - 50 bp) insertions and deletions within MRI regions and appropriate control regions were analyzed for the effect of indels on the expansion or diminution of such regions as well as on changing nucleotide composition.</p> <p>Conclusion</p> <p>MRI regions have comparable levels of de novo mutations to the control genomic sequences with average base composition. De novo substitutions rapidly erode MRI regions, bringing their nucleotide composition toward genome-average levels. However, those substitutions that favor the maintenance of MRI properties have a higher chance to spread through the entire population. Indels have a clear tendency to maintain MRI features yet they have a smaller impact than substitutions. All in all, the observed fixation bias for mutations helps to preserve MRI regions during evolution.</p

Cellular Architecture and Cytoskeletal Structures Involved in Cell Haptotaxis

Filopodia play a sensory role in directing motility during embryonic development and axon pathfinding. They also show a low prevalence in cancer cells. Here, I determined whether cultured cells from a rat tracheal epithelial line used filopodia to sense adhesive gradients. Cells exhibited haptotaxis (movement toward the more adhesive surface) when plated on tantalum (Ta) and platinum (Pt) metallic gradients. The gradients were created on glass, and high (H), middle (M), and low (L) positions defined along the gradient. Cell counts in randomly selected fields confirmed that the cells recognized the gradient. To determine whether the prevalence of protrusions differed at the H, M, and L locations, the values of latent factors 4 (filopodia), 5, and 7 were determined. Factor 4 values were high at H and significantly lower at M and L (p\u3c0.05). Cells\u27 ability to form larger protrusions, represented by factors 5 (lamellar distribution) and 7 (nascent neurites), was unaltered across the Ta gradient. The directional cues also appeared to be interpreted within a cell, as shown by analyzing the cells\u27 top (T) side, i.e. the side oriented toward H location, from the bottom (B) side. Factor 4 values at H-T significantly exceed those at M-B and L-T. Trend analysis confirmed a decrease in factor 4 over the gradient with significance of P\u3c0.001 (Ta) and P\u3c0.023 (Pt). On Pt only, factor 5 increased (P\u3c0.0001) as the metal content of the substrate declined. Cdc42 (cell division cycle 42) plays a crucial role in establishing polarity, and is also involved in filopodia formation, cell motility, and directional migration. Since there is some loss of polarity in preneoplastic lesions, the role of Cdc42 in filopodia-mediated sensing was of interest. To determine whether Cdc42 was implicated in reconstruction of cellular architecture and rearrangement of cytoskeletal structures, I tested the role of Cdc42 effectors in gradient sensing. While most effectors bind to Cdc42 at multiple regions, there is usually a short linear stretch of residues that is critical for binding. Peptides representing such stretches of Cdc42 were designed to model its surface and thereby inhibit effector-Cdc42 binding. Using filopodia trend analysis to determine the effect of each peptide, I found that ACK, IQGAP, and Par6 were essential for directional pointing of filopodia. A sequence blocking PKCε interaction with its docking site also prevented pointing activity. Although WASP binds directly to Cdc42, the peptide designed to block this interaction did not affect directional pointing of the filopodia. Introduction of the peptide against PAK gave a paradoxical result. Directional identification mechanisms were intact but the direction of filopodia pointing was reversed. I also studied the relationship between filopodia and ruffles on any given cell, in order to understand the complex processes of cell motility and directional persistence. Ruffle frequency and filopodia were inversely related, and this relationship was independent of the location on the gradient or the peptide introduced into the cells. On tantalum, sites at HT, MT, and LT and on platinum sites at HT, MT, and both LT and LB showed ruffling interacting with filopodia, with a level of significance P\u3c0.025. Despite the tendency for ruffling activity to increase toward the less adhesive end of the gradient, none of the ruffling variables showed a statistically significant trend. However, the data suggest that cells make ruffles at the expense of filopodia regardless of substrate to which they are attached. This inverse relationship was stronger at the top of the cell than at the bottom. Thus it can be confirmed that ruffles and filopodia are inversely related, but ruffling is unlikely to be a mechanism of gradient sensing

Cellular Architecture and Cytoskeletal Structures Involved in Cell Haptotaxis

Filopodia play a sensory role in directing motility during embryonic development and axon pathfinding. They also show a low prevalence in cancer cells. Here, I determined whether cultured cells from a rat tracheal epithelial line used filopodia to sense adhesive gradients. Cells exhibited haptotaxis (movement toward the more adhesive surface) when plated on tantalum (Ta) and platinum (Pt) metallic gradients. The gradients were created on glass, and high (H), middle (M), and low (L) positions defined along the gradient. Cell counts in randomly selected fields confirmed that the cells recognized the gradient. To determine whether the prevalence of protrusions differed at the H, M, and L locations, the values of latent factors 4 (filopodia), 5, and 7 were determined. Factor 4 values were high at H and significantly lower at M and L (p\u3c0.05). Cells\u27 ability to form larger protrusions, represented by factors 5 (lamellar distribution) and 7 (nascent neurites), was unaltered across the Ta gradient. The directional cues also appeared to be interpreted within a cell, as shown by analyzing the cells\u27 top (T) side, i.e. the side oriented toward H location, from the bottom (B) side. Factor 4 values at H-T significantly exceed those at M-B and L-T. Trend analysis confirmed a decrease in factor 4 over the gradient with significance of P\u3c0.001 (Ta) and P\u3c0.023 (Pt). On Pt only, factor 5 increased (P\u3c0.0001) as the metal content of the substrate declined. Cdc42 (cell division cycle 42) plays a crucial role in establishing polarity, and is also involved in filopodia formation, cell motility, and directional migration. Since there is some loss of polarity in preneoplastic lesions, the role of Cdc42 in filopodia-mediated sensing was of interest. To determine whether Cdc42 was implicated in reconstruction of cellular architecture and rearrangement of cytoskeletal structures, I tested the role of Cdc42 effectors in gradient sensing. While most effectors bind to Cdc42 at multiple regions, there is usually a short linear stretch of residues that is critical for binding. Peptides representing such stretches of Cdc42 were designed to model its surface and thereby inhibit effector-Cdc42 binding. Using filopodia trend analysis to determine the effect of each peptide, I found that ACK, IQGAP, and Par6 were essential for directional pointing of filopodia. A sequence blocking PKCε interaction with its docking site also prevented pointing activity. Although WASP binds directly to Cdc42, the peptide designed to block this interaction did not affect directional pointing of the filopodia. Introduction of the peptide against PAK gave a paradoxical result. Directional identification mechanisms were intact but the direction of filopodia pointing was reversed. I also studied the relationship between filopodia and ruffles on any given cell, in order to understand the complex processes of cell motility and directional persistence. Ruffle frequency and filopodia were inversely related, and this relationship was independent of the location on the gradient or the peptide introduced into the cells. On tantalum, sites at HT, MT, and LT and on platinum sites at HT, MT, and both LT and LB showed ruffling interacting with filopodia, with a level of significance P\u3c0.025. Despite the tendency for ruffling activity to increase toward the less adhesive end of the gradient, none of the ruffling variables showed a statistically significant trend. However, the data suggest that cells make ruffles at the expense of filopodia regardless of substrate to which they are attached. This inverse relationship was stronger at the top of the cell than at the bottom. Thus it can be confirmed that ruffles and filopodia are inversely related, but ruffling is unlikely to be a mechanism of gradient sensing

Transcriptional profiling of Foxo3a and Fancd2 regulated genes in mouse hematopoietic stem cells

Functional maintenance of hematopoietic stem cells (HSCs) is constantly challenged by stresses like DNA damage and oxidative stress. Foxo factors particularly Foxo3a function to regulate the self-renewal of HSCs and contribute to the maintenance of the HSC pool during aging by providing resistance to oxidative stress. Fancd2-deficient mice had multiple hematopoietic defects including HSC loss in early development and in response to cellular stresses including oxidative stress. The cellular mechanisms underlying HSC loss in Fancd2-deficient mice include abnormal cell cycle status loss of quiescence and compromised hematopoietic repopulating capacity of HSCs. To address on a genome wide level the genes and pathways that are impacted by deletion of the Fancd2 and Foxo3a we performed microarray analysis on phenotypic HSCs (Lin−ckit+Sca-1+CD150+CD48−) from Fancd2 single knockout Foxo3a single knockout and Fancd2−/−Foxo3a−/− double-knockout (dKO) mice. Here we provide detailed methods and analysis on these microarray data which has been deposited in Gene Expression Omnibus (GEO): GSE64215

Persistent response of Fanconi anemia haematopoietic stem and progenitor cells to oxidative stress

<p>Oxidative stress is considered as an important pathogenic factor in many human diseases including Fanconi anemia (FA), an inherited bone marrow failure syndrome with extremely high risk of leukemic transformation. Members of the FA protein family are involved in DNA damage and other cellular stress responses. Loss of FA proteins renders cells hypersensitive to oxidative stress and cancer transformation. However, how FA cells respond to oxidative DNA damage remains unclear. By using an <i>in vivo</i> stress-response mouse strain expressing the <i>Gadd45β</i>-luciferase transgene, we show here that haematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene <i>Fanca</i> or <i>Fancc</i> persistently responded to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. Furthermore, genetic correction of <i>Fanca</i> deficiency almost completely abolished the persistent oxidative stress-induced G₂/M arrest and DNA damage response <i>in vivo</i>. Our study suggests that FA pathway is an integral part of a versatile cellular mechanism by which HSPCs respond to oxidative stress.</p