Pax-5 Protein Expression Is Regulated by Transcriptional 3′UTR Editing

The Pax-5 gene encodes a transcription factor that is essential for B-cell commitment and maturation. However, Pax-5 deregulation is associated with various cancer lesions, notably hematopoietic cancers. Mechanistically, studies have characterized genetic alterations within the Pax-5 locus that result in either dominant oncogenic function or haploinsufficiency-inducing mutations leading to oncogenesis. Apart from these mutations, some examples of aberrant Pax-5 expression cannot be associated with genetic alterations. In the present study, we set out to elucidate potential alterations in post-transcriptional regulation of Pax-5 expression and establish that Pax-5 transcript editing represents an important means to aberrant expression. Upon the profiling of Pax-5 mRNA in leukemic cells, we found that the 3′end of the Pax-5 transcript is submitted to alternative polyadenylation (APA) and alternative splicing events. Using rapid amplification of cDNA ends (3′RACE) from polysomal fractions, we found that Pax-5 3′ untranslated region (UTR) shortening correlates with increased ribosomal occupancy for translation. These observations were also validated using reporter gene assays with truncated 3′UTR regions cloned downstream of a luciferase gene. We also showed that Pax-5 3′UTR editing has direct repercussions on regulatory elements such as miRNAs, which in turn impact Pax-5 protein expression. More importantly, we found that advanced staging of various hematopoietic cancer lesions relates to shorter Pax-5 3′UTRs. Altogether, our findings identify novel molecular mechanisms that account for aberrant expression and function of the Pax-5 oncogene in cancer cells. These findings also present new avenues for strategic intervention in Pax-5-mediated cancers.

Receptor Interacting Protein Kinase Pathways Regulate Innate B Cell Developmental Checkpoints But Not Effector Function in Mice

Mutations in the scaffolding domain of Receptor Interacting Protein kinases (RIP) underlie the recently described human autoimmune syndrome, CRIA, characterized by lymphadenopathy, splenomegaly, and autoantibody production. While disease mechanisms for CRIA remain undescribed, RIP kinases work together with caspase-8 to regulate cell death, which is critical for normal differentiation of many cell types. Here, we describe a key role for RIP1 in facilitating innate B cell differentiation and subsequent activation. By comparing RIP1, RIP3, and caspase-8 triple deficient and RIP3, caspase-8 double deficient mice, we identified selective contributions of RIP1 to an accumulation of murine splenic Marginal Zone (MZ) B cells and B1-b cells. We used mixed bone-marrow chimeras to determine that innate B cell commitment required B cell-intrinsic RIP1, RIP3, and caspase-8 sufficiency. RIP1 regulated MZ B cell development rather than differentiation and RIP1 mediates its innate immune effects independent of the RIP1 kinase domain. NP-KLH/alum and NP-Ficoll vaccination of mice doubly deficient in both caspase-8 and RIP3 or deficient in all three proteins (RIP3, caspase-8, and RIP1) revealed uniquely delayed T-dependent and T-independent IgG responses, abnormal splenic germinal center architecture, and reduced extrafollicular plasmablast formation compared to WT mice. Thus, RIP kinases and caspase-8 jointly orchestrate B cell fate and delayed effector function through a B cell-intrinsic mechanism.

Chronic Systemic Curcumin Administration Antagonizes Murine Sarcopenia and Presarcopenia

Curcumin administration attenuates muscle disuse atrophy, but its effectiveness against aging-induced, selective loss of mass or force (presarcopenia or asthenia/dynopenia), or combined loss (sarcopenia), remains controversial. A new systemic curcumin treatment was developed and tested in 18-month-old C57BL6J and C57BL10ScSn male mice. The effects on survival, liver toxicity, loss of muscle mass and force, and satellite cell responsivity and commitment were evaluated after 6-month treatment. Although only 24-month-old C57BL10ScSn mice displayed age-related muscle impairment, curcumin significantly increased survival of both strains (+20–35%), without signs of liver toxicity. Treatment prevented sarcopenia in soleus and presarcopenia in EDL of C57BL10ScSn mice, whereas it did not affect healthy-aged muscles of C57BL6J. Curcumin-treated old C57BL10ScSn soleus preserved type-1 myofiber size and increased type-2A one, whereas EDL maintained adult values of total myofiber number and fiber-type composition. Mechanistically, curcumin only partially prevented the age-related changes in protein level and subcellular distribution of major costamere components and regulators. Conversely, it affected satellite cells, by maintaining adult levels of myofiber maturation in old regenerating soleus and increasing percentage of isolated, MyoD-positive satellite cells from old hindlimb muscles. Therefore, curcumin treatment successfully prevents presarcopenia and sarcopenia development by improving satellite cell commitment and recruitment.

105 The Epigenetics and Plasticity of CD4+ T Cells in Poultry Health

Marek’s disease (MD) is one of the top priority diseases in chicken. The mechanisms through which immune cells react to virus infection and pathway signals represent the fundamental cell biological question to defeat MD. We hypothesized epigenetic status in immune organs and cells is directly associated with how the Marek’s disease virus (MDV) infection influences intrinsic transcriptional and regulatory networks in MD. To investigate the epigenetics in CD4+ T cells induced by MDV infection, we assembled a multifaceted approach with epigenetics, deep sequencing, and computational methods together to explore the roles of epigenetics in unique inbred lines of chickens. First, a genome-wide transcriptome analysis in the immune organs from resistant line 63 and susceptible line 72 chickens was performed to explore disease resistance mechanisms. MDV infection influences both cytokine-cytokine receptor interaction and cellular development in resistant and susceptible chickens. Second, we examined the epigenetic status of CD4+ T cells induced by MDV infection, including DNA methylations, histone modifications, chromatin accessibility, and 3D chromatin structures. Our results revealed more than 5,000 epigenetic modification changes (FDR< 0.01) and methylation changes (>15,000, FDR< 0.01) caused by MDV infection. Only resistant line 63 chickens could initiate robust adaptive immune responses at the transcription level (>200 genes, FDR< 0.05). The increase in chromatin accessibility (P < 0.001) and chromosome reorganization represented by A/B compartment flipping were related to up-regulated genes induced by MDV infection ten days post-infection in line 63 chickens. Our findings provided a deeper insight into the CD4+ T cell commitment and responses toward viral infection. In particular, the identifications of cis-acting and trans-acting regulations and lipid pathways will improve our understanding of the sequence, structural basis of RNA-protein, and RNA-DNA interactions and serve as the impetus for mechanistic studies to refine the genomic and epigenetic control of MD resistance in poultry.

Master regulators of skeletal muscle lineage development and pluripotent stem cells differentiation

In vertebrates, the skeletal muscles of the body and their associated stem cells originate from muscle progenitor cells, during development. The specification of the muscles of the trunk, head and limbs, relies on the activity of distinct genetic hierarchies. The major regulators of trunk and limb muscle specification are the paired-homeobox transcription factors PAX3 and PAX7. Distinct gene regulatory networks drive the formation of the different muscles of the head. Despite the redeployment of diverse upstream regulators of muscle progenitor differentiation, the commitment towards the myogenic fate requires the expression of the early myogenic regulatory factors MYF5, MRF4, MYOD and the late differentiation marker MYOG. The expression of these genes is activated by muscle progenitors throughout development, in several waves of myogenic differentiation, constituting the embryonic, fetal and postnatal phases of muscle growth. In order to achieve myogenic cell commitment while maintaining an undifferentiated pool of muscle progenitors, several signaling pathways regulate the switch between proliferation and differentiation of myoblasts. The identification of the gene regulatory networks operating during myogenesis is crucial for the development of in vitro protocols to differentiate pluripotent stem cells into myoblasts required for regenerative medicine.

3D Microenvironment-Specific Mechanosensing Regulates Neural Stem Cell Lineage Commitment

While extracellular matrix (ECM) mechanics strongly regulate stem cell commitment, the field′s mechanistic understanding of this phenomenon largely derives from simplified two–dimensional (2D) culture substrates. Here we found a three-dimensional (3D) matrix–specific mechanoresponsive mechanism for neural stem cell (NSC) differentiation. NSC lineage commitment in 3D is maximally stiffness-sensitive in the range of 0.1–1.2 kPa, a narrower and more brain-mimetic range than we had previously identified in 2D (0.75–75 kPa). Transcriptomics revealed stiffness-dependent upregulation of early growth response 1 (Egr1) in 3D but not in 2D. Egr1 knockdown enhanced neurogenesis in stiff ECMs by driving β–catenin nuclear localization and activity in 3D, but not in 2D. Mechanical modeling and experimental studies under osmotic pressure indicate that stiff 3D ECMs are likely to stimulate Egr1 via increases in confining stress during cell volumetric growth. To our knowledge, Egr1 represents the first 3D–specific stem cell mechanoregulatory factor.

Self-Organization Provides Cell Fate Commitment in MSC Sheet Condensed Areas via ROCK-Dependent Mechanism

Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.

EBF1 is expressed in pericytes and contributes to pericyte cell commitment

Early B-cell factor-1 (EBF1) is a transcription factor with an important role in cell lineage specification and commitment during the early stage of cell maturation. Originally described during B-cell maturation, EBF1 was subsequently identified as a crucial molecule for proper cell fate commitment of mesenchymal stem cells into adipocytes, osteoblasts and muscle cells. In vessels, EBF1 expression and function have never been documented. Our data indicate that EBF1 is highly expressed in peri-endothelial cells in both tumor vessels and in physiological conditions. Immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-qPCR) and fluorescence-activated cell sorting (FACS) analysis suggest that EBF1-expressing peri-endothelial cells represent bona fide pericytes and selectively express well-recognized markers employed in the identification of the pericyte phenotype (SMA, PDGFRβ, CD146, NG2). This observation was also confirmed in vitro in human placenta-derived pericytes and in human brain vascular pericytes (HBVP). Of note, in accord with the key role of EBF1 in the cell lineage commitment of mesenchymal stem cells, EBF1-silenced HBVP cells showed a significant reduction in PDGFRβ and CD146, but not CD90, a marker mostly associated with a prominent mesenchymal phenotype. Moreover, the expression levels of VEGF, angiopoietin-1, NG2 and TGF-β, cytokines produced by pericytes during angiogenesis and linked to their differentiation and activation, were also significantly reduced. Overall, the data suggest a functional role of EBF1 in the cell fate commitment toward the pericyte phenotype.

Suspension of Amorphous Calcium Phosphate Nanoparticles Impact Commitment of Human Adipose-Derived Stem Cells In Vitro

Amorphous calcium phosphate (aCaP) nanoparticles may trigger the osteogenic commitment of adipose-derived stem cells (ASCs) in vitro. The ASCs of three human donors are investigated using basal culture medium DMEM to either 5 or 50 µg/mL aCaP nanoparticles suspension (control: no nanoparticles). After 7 or 14 days, stem cell marker genes, as well as endothelial, osteogenic, chondrogenic, and adipogenic genes, are analyzed by qPCR. Free calcium and phosphate ion concentrations are assessed in the cell culture supernatant. After one week and 5 µg/mL aCaP, downregulation of osteogenic markers ALP and Runx2 is found, and averaged across the three donors. Our results show that after two weeks, ALP is further downregulated, but Runx2 is upregulated. Endothelial cell marker genes, such as CD31 and CD34, are upregulated with 50 µg/mL aCaP and a 2-week exposure. Inter-donor variability is high: Two out of three donors show a significant upregulation of ALP and Runx2 at day 14 with 50 µg/mL aCaP compared to 5 µg/mL aCaP. Notably, all changes in stem cell commitment are obtained in the absence of an osteogenic medium. While the chemical composition of the culture medium and the saturation status towards calcium phosphate phases remain approximately the same for all conditions, gene expression of ASCs changes considerably. Hence, aCaP nanoparticles show the potential to trigger osteogenic and endothelial commitment in ASCs.