Arsenic and mercury contamination and complex aquatic bioindicator responses to historical gold mining and modern watershed stressors in urban Nova Scotia, Canada.

Beginning in the late-1800s, gold mining activities throughout Nova Scotia, eastern Canada, released contaminants, notably geogenic arsenic from milled ore and anthropogenic mercury from amalgamation, to local environments via surface water flows through tailings fields. We investigated recovery from and legacy effects of the tailings field at the Montague Gold District (~1863-1940) on nearby urban lake ecosystems using geochemical measures and zooplankton remains archived in dated sediment cores from an impact (Lake Charles) and a reference (Loon Lake) lake. Sedimentary levels of total arsenic and total mercury were used to assess mining-related inputs. Arsenic concentrations remain elevated at nearly 300 times above sediment guidelines in Lake Charles surface sediments, due to its upward mobilization from enriched sediment intervals and sequestration by iron oxyhydroxides in surficial sediments. Peak mercury concentrations at Lake Charles were eight times above sediment guidelines during the mining period, and since ~1990 have recovered to levels observed before mining began. Legacy mining impacts at Lake Charles and non-mining related environmental changes in the post-1950 sediments at both lakes have thus combined to structure assemblage compositions of primary consumers. At both lakes, assemblages of pelagic-dominated Cladocera differed (p ≤ 0.05) during the mining period compared to periods before and after mining. Taxon richness differed (p ≤ 0.01) only between the pre- and post-mining periods at mining-impacted Lake Charles and reflects long-term declines of substrate-dwelling littoral taxa. Geochemical and biological recovery have not completely occurred at Lake Charles despite the mine district's closure ~80 years ago. Our findings demonstrate that impacts of ore processing and amalgamation from historical gold mining, combined with recent watershed stressors, continue to affect sedimentary arsenic geochemistry and intermediate trophic levels of nearby, downstream aquatic habitats.

Poly(ADP-ribose)-binding zinc finger motifs in DNA repair/checkpoint proteins.

Post-translational modification (PTM) of proteins plays an important part in mediating protein interactions and/or the recruitment of specific protein targets. PTM can be mediated by the addition of functional groups (for example, acetylation or phosphorylation), peptides (for example, ubiquitylation or sumoylation), or nucleotides (for example, poly(ADP-ribosyl)ation). Poly(ADP-ribosyl)ation often involves the addition of long chains of ADP-ribose units, linked by glycosidic ribose-ribose bonds, and is critical for a wide range of processes, including DNA repair, regulation of chromosome structure, transcriptional regulation, mitosis and apoptosis. Here we identify a novel poly(ADP-ribose)-binding zinc finger (PBZ) motif in a number of eukaryotic proteins involved in the DNA damage response and checkpoint regulation. The PBZ motif is also required for post-translational poly(ADP-ribosyl)ation. We demonstrate interaction of poly(ADP-ribose) with this motif in two representative human proteins, APLF (aprataxin PNK-like factor) and CHFR (checkpoint protein with FHA and RING domains), and show that the actions of CHFR in the antephase checkpoint are abrogated by mutations in PBZ or by inhibition of poly(ADP-ribose) synthesis.

HCLK2 is essential for the mammalian S-phase checkpoint and impacts on Chk1 stability.

Here, we show that the human homologue of the Caenorhabditis elegans biological clock protein CLK-2 (HCLK2) associates with the S-phase checkpoint components ATR, ATRIP, claspin and Chk1. Consistent with a critical role in the S-phase checkpoint, HCLK2-depleted cells accumulate spontaneous DNA damage in S-phase, exhibit radio-resistant DNA synthesis, are impaired for damage-induced monoubiquitination of FANCD2 and fail to recruit FANCD2 and Rad51 (critical components of the Fanconi anaemia and homologous recombination pathways, respectively) to sites of replication stress. Although Thr 68 phosphorylation of the checkpoint effector kinase Chk2 remains intact in the absence of HCLK2, claspin phosphorylation and degradation of the checkpoint phosphatase Cdc25A are compromised following replication stress as a result of accelerated Chk1 degradation. ATR phosphorylation is known to both activate Chk1 and target it for proteolytic degradation, and depleting ATR or mutation of Chk1 at Ser 345 restored Chk1 protein levels in HCLK2-depleted cells. We conclude that HCLK2 promotes activation of the S-phase checkpoint and downstream repair responses by preventing unscheduled Chk1 degradation by the proteasome.

E2f4 regulates fetal erythropoiesis through the promotion of cellular proliferation.

The E2F proteins are major regulators of the transcriptional program required to coordinate cell cycle progression and exit. In particular, E2f4 has been proposed to be the principal family member responsible for the regulation of cell cycle exit chiefly through its transcriptional repressive properties. We have previously shown that E2f4(-/-) mice display a marked macrocytic anemia implicating E2f4 in the regulation of erythropoiesis. However, these studies could not distinguish whether E2f4 was required for differentiation, survival, or proliferation control. Here, we describe a novel function for E2f4 in the promotion of erythroid proliferation. We show that loss of E2f4 results in an impaired expansion of the fetal erythroid compartment in vivo that is associated with impaired cell cycle progression and decreased erythroid proliferation. Consistent with these observations, cDNA microarray analysis reveals cell cycle control genes as one of the major class of genes down-regulated in E2f4(-/-) FLs, and we provide evidence that E2f4 may directly regulate the transcriptional expression of a number of these genes. We conclude that the macrocytic anemia of E2f4(-/-) mice results primarily from impaired cellular proliferation and that the major role of E2f4 in fetal erythropoiesis is to promote cell cycle progression and cellular proliferation.

ETO-2 associates with SCL in erythroid cells and megakaryocytes and provides repressor functions in erythropoiesis.

Lineage specification and cellular maturation require coordinated regulation of gene expression programs. In large part, this is dependent on the activator and repressor functions of protein complexes associated with tissue-specific transcriptional regulators. In this study, we have used a proteomic approach to characterize multiprotein complexes containing the key hematopoietic regulator SCL in erythroid and megakaryocytic cell lines. One of the novel SCL-interacting proteins identified in both cell types is the transcriptional corepressor ETO-2. Interaction between endogenous proteins was confirmed in primary cells. We then showed that SCL complexes are shared but also significantly differ in the two cell types. Importantly, SCL/ETO-2 interacts with another corepressor, Gfi-1b, in red cells but not megakaryocytes. The SCL/ETO-2/Gfi-1b association is lost during erythroid differentiation of primary fetal liver cells. Genetic studies of erythroid cells show that ETO-2 exerts a repressor effect on SCL target genes. We suggest that, through its association with SCL, ETO-2 represses gene expression in the early stages of erythroid differentiation and that alleviation/modulation of the repressive state is then required for expression of genes necessary for terminal erythroid maturation to proceed.

Cell-intrinsic requirement for pRb in erythropoiesis.

Retinoblastoma (Rb) and family members have been implicated as key regulators of cell proliferation and differentiation. In particular, accumulated data have suggested that the Rb gene product pRb is an important controller of erythroid differentiation. However, current published data are conflicting as to whether the role of pRb in erythroid cells is cell intrinsic or non-cell intrinsic. Here, we have made use of an in vitro erythroid differentiation culture system to determine the cell-intrinsic requirement for pRb in erythroid differentiation. We demonstrate that the loss of pRb function in primary differentiating erythroid cells results in impaired cell cycle exit and terminal differentiation. Furthermore, we have used coculture experiments to establish that this requirement is cell intrinsic. Together, these data unequivocally demonstrate that pRb is required in a cell-intrinsic manner for erythroid differentiation and provide clarification as to its role in erythropoiesis.