Chipping away at gamma-H2AX foci

The mammalian histone H2AX protein functions as a dosage-dependent genomic caretaker and tumor suppressor. Phosphorylation of H2AX to form gamma-H2AX in chromatin around DNA double strand breaks (DSBs) is an early event following induction of these hazardous lesions. For a decade, mechanisms that regulate H2AX phosphorylation have been investigated mainly through two-dimensional immunofluorescence (IF). We recently used chromatin immunoprecipitation (ChIP) to measure gamma-H2AX densities along chromosomal DNA strands broken in G(1) phase mouse lymphocytes. Our experiments revealed that (1) gamma-H2AX densities in nucleosomes form at high levels near DSBs and at diminishing levels farther and farther away from DNA ends, and (2) ATM regulates H2AX phosphorylation through both MDC1-dependent and MDC1-independent means. Neither of these mechanisms were discovered by previous if studies due to the inherent limitations of light microscopy. Here, we compare data obtained from parallel gamma-H2AX ChIP and three-dimensional IF analyses and discuss the impact of our findings upon molecular mechanisms that regulate H2AX phosphorylation in chromatin around DNA breakage sites

Harvest and Persistence of Wolf Populations: Variable Effects of Harvest on Wolf Packs in the Rocky Mountains

Pubic harvest is a common method used to manage populations of wolves (Canis lupus) in North America. Although wolves appear resilient to the effects of harvest management the influences on demography and pack stability are uncertain. Packs generally drive population dynamics for wolves; thus, we were interested in how harvested populations were maintained and how harvest influenced the abundance and distribution of packs. We used noninvasive genetic data collected in Idaho, USA (2008–2014) and Alberta, Canada (2012–2014) to test whether immigration compensated for harvest mortality and helped maintain population densities. We further fit occupancy models to detection data derived from noninvasive genetic samples and hunter surveys from Alberta, Canada (2012–2014) to test the stability of pack abundance and distribution in a harvested population of wolves. We genetically identified 461 unique wolves across our study areas; 762 hunters reported seeing live wolves in southwestern Alberta. We found our hypothesis that immigration did not compensate for harvest mortality was supported. Density of wolves in the U.S. population declined from 15.49 wolves/1000 km2 (95% credible interval [CRI]: 12.38–18.57) without harvest to 10.20 wolves/1000 km2 (95% CRI: 7.47–12.90) with harvest, whereas the proportion of long-distance immigrants was low and did not change with harvest (ranged 0.01–0.02, SD = 0.1). Density and proportion of immigrants were similar among study areas where harvest occurred. We also found we could not reject our hypothesis that occurrence of packs was generally stable in a harvested population of wolves. The mean annual probability for wolf pack occupancy ranged 0.72–0.74 and the estimated distribution of wolf packs was consistent over time. Model selection indicated harvest did not have a strong effect on pack occurrence but that the probability of detecting a wolf pack was positively associated with the intensity of harvest for wolves. Although immigration did not appear to compensate for harvest mortality, pack occurrence remained generally stable over time, likely due to movement between packs from within the population. Harvest therefore appears to affect within-pack dynamics, but may not directly affect the number and distribution of packs across a population

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre–B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igκ DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-α/δ locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs

Developing a Monitoring Framework to Estimate Wolf Distribution and Abundance in Southwest Alberta

Gray wolf (Canis lupus) populations are difficult to monitor because wolves can be elusive and occur in low densities.  Traditional radiotelemetry-based monitoring methods have limited application when turnover is high within the wolf population and resources to maintain long-term collaring programs are limited.  We worked collaboratively with Alberta Environmental Sustainable Resource Development between 2012 and 2014 to develop techniques for monitoring gray wolf populations in the absence of radiotelemetry in southwest Alberta.  We surveyed potential rendezvous sites and collected DNA samples from wolf scats for genetic analysis and surveyed hunters for wolf sightings made during the hunting seasons. We fit false-positive occupancy models to annual detection data derived from genetic results and hunter surveys with Program PRESENCE.  We found percent forest cover and human density positively influenced pack occupancy whereas detection probabilities varied by survey method, sampling effort, and sampling season.  The model predicted wolf pack occupancy well and distribution and abundance estimates were consistent with agency predictions.  While developing the monitoring framework, questions arose regarding pack turnover and population growth under widespread human harvest.  Previous studies have focused on population recovery following wolf control actions but little emphasis is put on populations that exist under regular harvest.  We will use genetic data to determine how immigration contributes to wolf population trends under a long-term harvest regime and tie this into pack occupancy through colonization and local extinction probabilities.  This will expand the application of our occupancy model and will further clarify how wolf populations respond to long-term regulated harvest

Immigration as a Compensatory Mechanism to Offset Harvest Mortality in Harvested Wolf Populations

In less than a decade the U.S. Northern Rocky Mountain gray wolf (Canis lupus) population has experienced large shifts in management practices, from federal protection under the Endangered Species Act to increasingly liberal hunting and trapping seasons in many portions of their range after delisting.  As a result, there is interest in how current wolf management practices will affect this population over time.  Recent research suggests wolf pup recruitment in central Idaho has declined since harvest was initiated, yet wolf densities appear stable in many regions of the state, suggesting other compensatory mechanisms are offsetting the effects of harvest mortality.  Our objective was to evaluate immigration as a compensatory mechanism that may offset the effects of harvest mortality and facilitate population persistence in a heavily harvested wolf population.  Using noninvasively sampled DNA we identified dispersers into two focal study areas in central Idaho prior to and after harvest was initiated.  We measured genetic relatedness within and among wolf packs using three different metrics to assess how immigration has changed with changing management practices.  Our results suggest that at current harvest rates immigration is not acting as a compensatory mechanism to offset the effects of harvest mortality.  Local dispersal may be unaffected by harvest pressure whereas harvest has negative effects on long-distance dispersal.  Our research can help managers consider the effects of immigration on local wolf populations when making harvest management decisions

Dramatically Increased Rearrangement and Peripheral Representation of Vβ14 Driven by the 3′Dβ1 Recombination Signal Sequence

AbstractV(D)J recombination is targeted by short recombination signal (RS) sequences that are relatively conserved but exhibit natural sequence variations. To evaluate the potential of RS sequence variations to determine the primary and peripheral TCRβ repertoire, we generated mice containing specific replacement of the endogenous Vβ14 RS with the 3′Dβ1 RS (Vβ14/3′DβRS). These mice exhibited a dramatic increase in Vβ14+ thymocyte numbers at the expense of thymocytes expressing other Vβs. In addition, the percentage of peripheral Vβ14+ αβ T lymphocytes was similarly increased. Strikingly, this altered Vβ repertoire resulted predominantly from a higher relative level of primary Vβ14/3′DβRS rearrangement to DβJβ complexes, despite the ability of the 3′Dβ1 RS to break B12/23 restriction and allow direct rearrangement of Vβ14/3′DβRS to Jβ segments

Wolf Pack Distribution in Relation to Heavy Harvest in Southwest Alberta

Gray wolf (Canis lupus) populations are difficult to monitor because wolves can be elusive and occur in low densities.  Harvest can further complicate wolf monitoring by affecting wolf behavior, altering pack structure, and potentially reducing probability of detection.  Currently, Montana and Idaho use patch occupancy models to monitor wolves at state-wide scales.  These models were originally developed prior to the initiation of wolf harvest and there is growing concern that current occupancy estimates are becoming less reliable as harvest continues.  Our objectives were to determine whether we could estimate wolf distribution for a heavily harvested wolf population and assess how harvest may be affecting that distribution.  We surveyed potential rendezvous sites and collected DNA samples from wolf scats for genetic analysis and surveyed hunters for wolf sightings in southwestern Alberta from 2012 to 2014. We used a Bayesian approach to fit dynamic occupancy models to the encounter histories while accounting for false-positive detections using JAGS and Program R.  We found both habitat and anthropogenic factors influenced wolf occupancy parameters in southwestern Alberta and detection probability varied by survey method.  Our preliminary results suggest wolf pack distribution is fairly consistent but that source-sink dynamics may be occurring in certain regions of the study area.  Despite heavy harvest pressure, southwestern Alberta appears to maintain a stable wolf population, although this is possibly due to immigration from nearby regions

Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes

Ataxia-telangiectasia mutated (ATM)–deficient lymphocytes exhibit defects in coding joint formation during V(D)J recombination in vitro. Similar defects in vivo should affect both T and B cell development, yet the lymphoid phenotypes of ATM deficiency are more pronounced in the T cell compartment. In this regard, ATM-deficient mice exhibit a preferential T lymphopenia and have an increased incidence of nontransformed and transformed T cells with T cell receptor α/δ locus translocations. We demonstrate that there is an increase in the accumulation of unrepaired coding ends during different steps of antigen receptor gene assembly at both the immunoglobulin and T cell receptor loci in developing ATM-deficient B and T lymphocytes. Furthermore, we show that the frequency of ATM-deficient αβ T cells with translocations involving the T cell receptor α/δ locus is directly related to the number of T cell receptor α rearrangements that these cells can make during development. Collectively, these findings demonstrate that ATM deficiency leads to broad defects in coding joint formation in developing B and T lymphocytes in vivo, and they provide a potential molecular explanation as to why the developmental impact of these defects could be more pronounced in the T cell compartment

The Eδ enhancer controls the generation of CD4−CD8− αβTCR-expressing T cells that can give rise to different lineages of αβ T cells

It is well established that the pre–T cell receptor for antigen (TCR) is responsible for efficient expansion and differentiation of thymocytes with productive TCRβ rearrangements. However, Ptcra- as well as Tcra-targeting experiments have suggested that the early expression of Tcra in CD4−CD8− cells can partially rescue the development of αβ CD4+CD8+ cells in Ptcra-deficient mice. In this study, we show that the TCR Eδ but not Eα enhancer function is required for the cell surface expression of αβTCR on immature CD4−CD8− T cell precursors, which play a crucial role in promoting αβ T cell development in the absence of pre-TCR. Thus, αβTCR expression by CD4−CD8− thymocytes not only represents a transgenic artifact but occurs under physiological conditions

V(D)J Recombination and the Evolution of the Adaptive Immune System

In order for the immune system to generate its vast numbers of receptors, B- and T-cell receptor genes are created by recombining preexisting gene segments. This well- coordinated set of reactions is explaine