Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1

<p>Abstract</p> <p>Background</p> <p>Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD⁺) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI. In the present study, we examined whether suppression of PARP-1 expression in embryonic fibroblasts derived from PARP-1 knockout mice (PARP-1^-/-) might alter the expression and/or DNA binding properties of Sp1 and NFI. We also explored the possibility that Sp1 or NFI (or both) may represent target proteins of PARP-1 activity.</p> <p>Results</p> <p>Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1^-/-cells. Co-immunoprecipitation assays revealed that PARP-1 physically interacts with Sp1 in a DNA-independent manner, but neither with Sp3 nor NFI, in PARP-1^+/+cells. In addition, <it>in vitro </it>PARP assays indicated that PARP-1 could catalyze the addition of polymer of ADP-ribose to Sp1, which also translated into a reduction of Sp1 binding to its consensus DNA target site. Transfection of the PARP-1 promoter into both PARP-1^+/+and PARP-1^-/-cells revealed that the lack of PARP-1 expression in PARP-1^-/-cells also results in a strong increase in PARP-1 promoter activity. This influence of PARP-1 was found to rely on the presence of the Sp1 sites present on the basal PARP-1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP-1^-/-cells. Subjecting PARP-1^+/+cells to an oxidative challenge with hydrogen peroxide to increase PARP-1 activity translated into a dramatic reduction in the DNA binding properties of Sp1. However, its suppression by the inhibitor PJ34 improved DNA binding of Sp1 and led to a dramatic increase in PARP-1 promoter function.</p> <p>Conclusion</p> <p>Our results therefore recognized Sp1 as a target protein of PARP-1 activity, the addition of polymer of ADP-ribose to this transcription factor restricting its positive regulatory influence on gene transcription.</p

Influence of Sp1/Sp3 expression on corneal epithelial cells proliferation and differentiation properties in reconstructed tissues

PURPOSE : Primary cultured epithelial cells are widely used for the production of tissue-engineered substitutes and are gaining popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often lose their ability to proliferate by progressing toward terminal cell differentiation, a process likely to be determined by altered expression of transcription factors that have functions critical for cell adhesion and differentiation. This study was designed to determine whether the variable life span of primary cultured human corneal epithelial cells (HCECs) might be the consequence of varying expression levels of the well-known transcription factors Sp1 and Sp3 (Sp1/Sp3). METHODS : HCECs were obtained from donor eyes and cultured on irradiated Swiss-3T3. Sp1/Sp3 expression was monitored by Western blot and electrophoretic mobility shift assay (EMSA). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent poly(ADP-ribose) polymerase (rPARP) promoter fused to the CAT reporter gene. HCECs that expressed various levels of Sp1/Sp3 were also used for the production of corneal substitutes. RESULTS : Expression of Sp1/Sp3 was dramatically inconsistent between HCECs isolated from the eyes of different donors. Both factors were highly expressed during one passage and then totally disappeared as cells terminally differentiated. Proper stratification of HCECs on reconstructed tissue substitutes could be obtained only with cells that also had a delayed peak of Sp1/Sp3 expression when cultured in vitro. CONCLUSIONS : Expression of Sp1/Sp3 may represent a good predictor for selecting HCECs that are most likely to proliferate, stratify, and differentiate properly when used for the production of reconstructed corneal substitutes

Transcriptional regulation of the human α6 integrin gene by the transcription factor NFI during corneal wound healing

Purpose. Wound healing of the corneal epithelium is highly influenced by regulation of integrin gene expression. A recent study demonstrated that laminin (LM), a major constituent of the extracellular matrix (ECM), reduces expression of the human α6 integrin subunit gene by altering the properties of the transcription factor (TF) Sp1. In this work, a target site was identified for the TF nuclear factor I (NFI) on the human α6 gene, and its regulatory influence was characterized in corneal epithelial cells. Methods. Plasmids bearing the α6 promoter fused to the CAT gene were transfected into human (HCECs) and rabbit (RCECs) corneal epithelial cells grown on LM. The DNA-binding site for NFI in the α6 promoter was identified by DNase I footprinting. Expression and DNA binding of NFI was monitored by Western blot, RT-PCR, and electrophoretic mobility shift assays (EMSAs), and its function was investigated through RNAi and NFI overexpression assays. Results. All NFI isoforms were found to be expressed in HCECs and RCECs. Transfection analyses revealed that NFI is a repressor of α6 expression in both types of cells. LM increases expression of NFI, whereas inhibition of each NFI isoform increases promoter activity suggesting that NFI is a key repressor of α6 transcription. In addition, the negative influence of NFI appears to be potentiated by the degradation of Sp1 when cells are grown on LM. Conclusions. Repression of α6 expression therefore contributes to the final steps of corneal wound healing by both reducing proliferation and allowing attachment of the epithelium to the basal membrane

Differential binding of the transcription factors Sp1, AP-1, and NFI to the promoter of the human α5 integrin gene dictates its transcriptional activity

Purpose. Damage to the corneal epithelium results in the massive secretion of fibronectin (FN) shortly after injury and induces the expression of its integrin receptor α5β1. The authors reported previously that FN induces α5 expression in human corneal epithelial cells and rabbit corneal epithelial cells by altering the binding of the transcription factor (TF) Sp1 to a regulatory element from the α5 promoter that it is also flanked by binding sites for the TFs NFI and AP-1. Here, they assessed the function of NFI and AP-1 on α5 gene expression and evaluated the contribution of FN to their overall regulatory influence. Methods. TF binding to the α5 promoter was evaluated in vitro by electrophoretic mobility shift assays and in vivo by ligation-mediated PCR or chromatin immunoprecipitation. TFs expression was monitored by Western blot, whereas their influence was assessed by transfection and RNAi analyses. Results. Coexpression of Sp1, NFI, and AP-1 was demonstrated in all cell types, and each TF was shown to bind efficiently to the α5 promoter. Whereas both AP-1 and Sp1 activated expression directed by the α5 promoter, NFI functioned as a potent repressor of that gene. Interestingly, FN could either promote or repress α5 promoter activity in a cell density–dependent manner by differentially altering the ratio of these TFs. Conclusions. These results suggest that α5 gene expression is likely dictated by subtle alterations in the nuclear ratio of TFs that either repress (NFI) or activate (Sp1 and AP-1) α5 transcription in corneal epithelial cells

Selection of summer feeding sites and food resources by female migratory caribou (Rangifer tarandus) determined using camera collars

Migratory caribou (Rangifer tarandus) is a socioeconomically and culturally key species for northern communities in the Arctic, and most of its populations are experiencing a sharp decline. Female migratory caribou depend on the availability of summer habitat resources to meet the needs associated with lactation and the accumulation of fat reserves to survive when resources are less abundant. Because of the large scales at which habitat and resource data are usually available, information on how female migratory caribou select habitat and resources at fine scales in the wild is lacking. To document selection of summer feeding sites, we equipped 60 female caribou with camera collars from 2016 to 2018. We collected a total of 65,150 10-sec videos between June 1 st and September 1 st for three years with contrasted spring phenology. We determined the selection at the feeding site scale (3 rd scale of Johnson) and food item scale (4 th scale of Johnson) using resource selection probability functions. Wetlands were highly selected as feeding sites in June and July while they were avoided in August. Shrublands were mostly selected in July and August. At the resources scale, lichen, birch, willow, and mushrooms were the most strongly selected resources. Our results provide precise and novel information on habitat selection at feeding sites and food resources selected by female caribou in the wild. This information will help understand foraging patterns and habitat selection behavior of female migratory caribou and will contribute to the management and conservation of its declining populations

Determinants of migration trajectory and movement rate in a long-distance terrestrial mammal

Animal migrations occur in many taxa and are considered an adaptive response to spatial or temporal variations in resources. Human activities can influence the cost-benefit trade-offs of animal migrations, but evaluating the determinants of migration trajectory and movement rate in declining populations facing relatively low levels of human disturbance can provide new and valuable insights on the behavior of wildlife in natural environments. Here, we used an adapted version of path selection functions and quantified the effects of habitat type, topography, and weather, on 313 spring migrations by migratory caribou (Rangifer tarandus) in northern Québec, Canada, from 2011 to 2018. Our results showed that during spring migration, caribou selected tundra and avoided water bodies, forest, and higher elevation. Higher precipitation and deeper snow were linked to lower movement rates. Weather variables had a stronger effect on the migration trajectories and movement rates of females than males. Duration of caribou spring migration (mean of 48 days) and length (mean of 587 km) were similar in males and females, but females started (22 April) and ended (10 June) spring migrations ca. 6 days earlier than males. Caribou spring migration was influenced by habitat type, topography, and weather, but we also observed that caribou migrations were not spatially constrained. Better knowledge on where and when animals move between their winter and summer ranges can help inform management and land planning decisions. Our results could be used to model future migration trajectories and speed of caribou under different climate change scenarios

Evidence of migratory coupling between grey wolves and migratory caribou

Large‐scale animal migrations influence population and community dynamics along with ecosystem functioning. The migratory coupling concept posits that movement of migrant prey can lead to large‐scale movements of predators. In northern ecosystems, spatial patterns and behavioral responses of grey wolf to spatio‐temporal changes in its primary prey distribution, the migratory caribou, remain poorly documented. We used a long‐term GPS dataset (2011–2021) of 59 wolves and 431 migratory caribou from the declining Rivière‐aux‐Feuilles herd (QC, Canada) to investigate movement patterns and space use of wolves related to caribou seasonal distribution. Wolves home ranges overlapped with areas used by caribou year‐round, especially in May and winter. Wolves exhibited three annual tactics: sedentary (17%), long‐distance migration (> 700 km) between wintering areas and the tundra (36%), and a medium‐distance migration, stopping their northward movement near the treeline (47%). Migratory wolves started spring migration northward earlier than caribou, intercepting their prey on their way to calving grounds, but departed southward for fall migration later than caribou, tracking them on their way back to wintering areas. Wolves near or overlapping areas used by caribou exhibited lower monthly movement rates compared to wolves located further away. Overlap of home range among wolves was higher during migrations and winter but decreased in summer when wolves rear pups and caribou are dispersed on summer grounds. We provide evidence of migratory coupling between grey wolves and migratory caribou, with most wolves adjusting their space use patterns to match their primary prey distribution. Although predation pressure may affect the dynamics of declining caribou herds, the global decline of that prey may in turn impact predators on the long‐term, potentially enhancing intraspecific competition for new resources. Highlighting this migratory coupling is a key step to develop appropriate conservation and management measures for both guilds in the context of large‐scale migratory prey decline

Selection of summer feeding sites and food resources by female migratory caribou (Rangifer tarandus) determined using camera collars

&lt;p&gt;Female migratory caribou (Rangifer tarandus) depend on the availability of summer habitat resources to meet the needs associated with lactation and the accumulation of fat reserves to survive when resources are less abundant. Because of the large scales at which habitat and resource data are usually available, information on how female migratory caribou select habitat and resources at fine scales in the wild is lacking. To document selection of summer feeding sites, we equipped 52 female caribou with camera collars from 2016 to 2018. We collected a total of 65,150 10-sec videos between June 1st and September 1st for three years with contrasted spring phenology. We determined the selection at the feeding site scale (3rd scale of Johnson) and food item scale (4th scale of Johnson) using resource selection probability functions. This data base contains the data of the behaviors observed, habitat used as feeding site, habitat unused has habitat, consumed and unconsumed resources, insect presence and other variables.&lt;/p&gt