Novel technologies for emission reduction complement conservation agriculture to achieve negative emissions from row-crop production.

Plants remove carbon dioxide from the atmosphere through photosynthesis. Because agriculture's productivity is based on this process, a combination of technologies to reduce emissions and enhance soil carbon storage can allow this sector to achieve net negative emissions while maintaining high productivity. Unfortunately, current row-crop agricultural practice generates about 5% of greenhouse gas emissions in the United States and European Union. To reduce these emissions, significant effort has been focused on changing farm management practices to maximize soil carbon. In contrast, the potential to reduce emissions has largely been neglected. Through a combination of innovations in digital agriculture, crop and microbial genetics, and electrification, we estimate that a 71% (1,744 kg CO2e/ha) reduction in greenhouse gas emissions from row crop agriculture is possible within the next 15 y. Importantly, emission reduction can lower the barrier to broad adoption by proceeding through multiple stages with meaningful improvements that gradually facilitate the transition to net negative practices. Emerging voluntary and regulatory ecosystems services markets will incentivize progress along this transition pathway and guide public and private investments toward technology development. In the difficult quest for net negative emissions, all tools, including emission reduction and soil carbon storage, must be developed to allow agriculture to maintain its critical societal function of provisioning society while, at the same time, generating environmental benefits.

The transcription factor GATA3 is critical for the development of all IL-7Rα-expressing innate lymphoid cells.

Innate lymphoid cells (ILCs) are critical in innate immune responses to pathogens and lymphoid organ development. Similar to CD4(+) T helper (Th) cell subsets, ILC subsets positive for interleukin-7 receptor α (IL-7Rα) produce distinct sets of effector cytokines. However, the molecular control of IL-7Rα(+) ILC development and maintenance is unclear. Here, we report that GATA3 was indispensable for the development of all IL-7Rα(+) ILC subsets and T cells but was not required for the development of classical natural killer cells. Conditionally Gata3-deficient mice had no lymph nodes and were susceptible to Citrobactor rodentium infection. After the ILCs had fully developed, GATA3 remained important for the maintenance and functions of ILC2s. Genome-wide gene expression analyses indicated that GATA3 regulated a similar set of cytokines and receptors in Th2 cells and ILC2s, but not in ILC3s. Thus, GATA3 plays parallel roles in regulating the development and functions of CD4(+) T cells and IL-7Rα(+) ILCs.

miR-155 activates cytokine gene expression in Th17 cells by regulating the DNA-binding protein Jarid2 to relieve polycomb-mediated repression.

Specification of the T helper 17 (Th17) cell lineage requires a well-defined set of transcription factors, but how these integrate with posttranscriptional and epigenetic programs to regulate gene expression is poorly understood. Here we found defective Th17 cell cytokine expression in miR-155-deficient CD4+ T cells in vitro and in vivo. Mir155 was bound by Th17 cell transcription factors and was highly expressed during Th17 cell differentiation. miR-155-deficient Th17 and T regulatory (Treg) cells expressed increased amounts of Jarid2, a DNA-binding protein that recruits the Polycomb Repressive Complex 2 (PRC2) to chromatin. PRC2 binding to chromatin and H3K27 histone methylation was increased in miR-155-deficient cells, coinciding with failure to express Il22, Il10, Il9, and Atf3. Defects in Th17 cell cytokine expression and Treg cell homeostasis in the absence of Mir155 could be partially suppressed by Jarid2 deletion. Thus, miR-155 contributes to Th17 cell function by suppressing the inhibitory effects of Jarid2.

Transcriptional repression of Gata3 is essential for early B cell commitment.

The mechanisms underlying the silencing of alternative fate potentials in very early B cell precursors remain unclear. Using gain- and loss-of-function approaches together with a synthetic Zinc-finger polypeptide (6ZFP) engineered to prevent transcription factor binding to a defined cis element, we show that the transcription factor EBF1 promotes B cell lineage commitment by directly repressing expression of the T-cell-lineage-requisite Gata3 gene. Ebf1-deficient lymphoid progenitors exhibited increased T cell lineage potential and elevated Gata3 transcript expression, whereas enforced EBF1 expression inhibited T cell differentiation and caused rapid loss of Gata3 mRNA. Notably, 6ZFP-mediated perturbation of EBF1 binding to a Gata3 regulatory region restored Gata3 expression, abrogated EBF1-driven suppression of T cell differentiation, and prevented B cell differentiation via a GATA3-dependent mechanism. Furthermore, EBF1 binding to Gata3 regulatory sites induced repressive histone modifications across this region. These data identify a transcriptional circuit critical for B cell lineage commitment.

Transcription factor EBF restricts alternative lineage options and promotes B cell fate commitment independently of Pax5.

Alternative lineage restriction and B cell fate commitment require the transcription factor Pax5, but the function of early B cell factor (EBF) in these processes remains mostly unexplored. Here we show that in the absence of EBF, 'expandable' and clonal lymphoid progenitor cells retained considerable myeloid potential. Conversely, ectopic expression of EBF in multipotential progenitor cells directed B cell generation at the expense of myeloid cell fates. EBF induced Pax5 and antagonized expression of genes encoding the transcription factors C/EBPalpha, PU.1 and Id2. Notably, sustained expression of EBF in Pax5-/- hematopoietic progenitor cells was sufficient to block their myeloid and T lineage potential in vivo. Furthermore, in Pax5-/- pro-B cells, higher EBF expression repressed alternative lineage genes. Thus, EBF can restrict alternative lineage 'choice' and promote commitment to the B cell fate independently of Pax5.

Application of ChIP-Seq and related techniques to the study of immune function.

Behaviors observed at the cellular level such as development and acquisition of effector functions by immune cells result from transcriptional changes. The biochemical mediators of transcription are sequence-specific transcription factors (TFs), chromatin modifying enzymes, and chromatin, the complex of DNA and histone proteins. Covalent modification of DNA and histones, also termed epigenetic modification, influences the accessibility of target sequences for transcription factors on chromatin and the expression of linked genes required for immune functions. Genome-wide techniques such as ChIP-Seq have described the entire "cistrome" of transcription factors involved in specific developmental steps of B and T cells and started to define specific immune responses in terms of the binding profiles of critical effectors and epigenetic modification patterns. Current data suggest that both promoters and enhancers are prepared for action at different stages of activation by epigenetic modification through distinct transcription factors in different cells.

Genome-wide analyses of transcription factor GATA3-mediated gene regulation in distinct T cell types.

The transcription factor GATA3 plays an essential role during T cell development and T helper 2 (Th2) cell differentiation. To understand GATA3-mediated gene regulation, we identified genome-wide GATA3 binding sites in ten well-defined developmental and effector T lymphocyte lineages. In the thymus, GATA3 directly regulated many critical factors, including Th-POK, Notch1, and T cell receptor subunits. In the periphery, GATA3 induced a large number of Th2 cell-specific as well as Th2 cell-nonspecific genes, including several transcription factors. Our data also indicate that GATA3 regulates both active and repressive histone modifications of many target genes at their regulatory elements near GATA3 binding sites. Overall, although GATA3 binding exhibited both shared and cell-specific patterns among various T cell lineages, many genes were either positively or negatively regulated by GATA3 in a cell type-specific manner, suggesting that GATA3-mediated gene regulation depends strongly on cofactors existing in different T cells.

Regulation of nucleosome landscape and transcription factor targeting at tissue-specific enhancers by BRG1.

Enhancers of transcription activate transcription via binding of sequence-specific transcription factors to their target sites in chromatin. In this report, we identify GATA1-bound distal sites genome-wide and find a global reorganization of the nucleosomes at these potential enhancers during differentiation of hematopoietic stem cells (HSCs) to erythrocytes. We show that the catalytic subunit BRG1 of BAF complexes localizes to these distal sites during differentiation and generates a longer nucleosome linker region surrounding the GATA1 sites by shifting the flanking nucleosomes away. Intriguingly, we find that the nucleosome shifting specifically facilitates binding of TAL1 but not GATA1 and is linked to subsequent transcriptional regulation of target genes.

Akt1 and Akt2 promote peripheral B-cell maturation and survival.

Although the 3 isoforms of Akt regulate cell growth, proliferation, and survival in a wide variety of cell types, their role in B-cell development is unknown. We assessed B-cell maturation in the bone marrow (BM) and periphery in chimeras established with fetal liver progenitors lacking Akt1 and/or Akt2. We found that the generation of marginal zone (MZ) and B1 B cells, 2 key sources of antibacterial antibodies, was highly dependent on the combined expression of Akt1 and Akt2. In contrast, Akt1/2 deficiency did not negatively affect the generation of transitional or mature follicular B cells in the periphery or their precursors in the BM. However, Akt1/2-deficient follicular B cells exhibited a profound survival defect when forced to compete against wild-type B cells in vivo. Altogether, these studies show that Akt signaling plays a key role in peripheral B-cell maturation and survival.

Resolution of unique Sca-1highc-Kit- lymphoid-biased progenitors in adult bone marrow.

We have identified a distinctive lymphoid-restricted progenitor population in adult mouse bone marrow based on a unique c-Kit(-)Sca-1(high)Flt3(+) AA4(+) surface phenotype. These cells are highly lymphoid biased and rapidly generate B and T cells after adoptive transfer. However, whereas previously described lymphoid progenitors such as common lymphoid progenitors express TdT and relatively high levels of RAG2, and are enriched for cells with an active V(D)J recombinase, Flt3(+) AA4(+) cells within the c-Kit(-)Sca-1(high) bone marrow fraction are TdT(-), are RAG2(low), and do not display evidence for ongoing or past recombinase activity. Furthermore, unlike common lymphoid progenitors that readily generate B cells upon stimulation with IL-7, c-Kit(-)Sca-1(high)Flt3(+) precursors do not express abundant levels of the IL-7R, and require costimulation with Flt3 ligand and IL-7 to generate B cells in vitro. Moreover, these findings suggest that hematopoietic stem cells in adults generate an array of lymphoid-biased progenitor populations characterized by distinct gene expression and cytokine response profiles.

Transcriptional regulation of early B cell development.

All blood cell types including mature B cells derive from pluripotent hematopoietic stem cells. The developmental cues responsible for guiding multipotent cells to the B cell fate remain to be fully elucidated. During recent years, it has become clear that firm commitment to the B cell fate requires the active suppression of differentiation potentials for alternative fates. Through the work of our laboratory and many others, it is now apparent that early B cell development and B-lineage commitment is controlled by a complex interplay between specific cytokine receptors and a variety of transcription factors. Whereas the transcription factor Pax5 has been touted as the chief transcriptional regulator of B-lineage commitment, our recent studies suggest that the B cell fate is established through the concerted action of several transcription factors including Early B cell Factor-1 (EBF). Notably, we recently found that EBF is able to suppress myeloid and T cell differentiation when introduced into multipotent Pax5(null/null) progenitors. Past work has also established that EBF expression is connected to the activity of the receptor for the cytokine IL-7. Therefore, the IL-7/EBF pathway plays a key and non-redundant role in establishing the B cell fate. This work provides a provisional model for understanding the molecular basis for B cell developmental biology. Furthermore, because aging leads to a decline in early B cell development and reduced IL-7 responsiveness, this work establishes a conceptual framework for understanding how and why aging leads to the loss of early B cell precursors.

Histone demethylases UTX and JMJD3 are required for NKT cell development in mice.

BACKGROUND: Natural killer (NK)T cells and conventional T cells share phenotypic characteristic however they differ in transcription factor requirements and functional properties. The role of histone modifying enzymes in conventional T cell development has been extensively studied, little is known about the function of enzymes regulating histone methylation in NKT cells. RESULTS: We show that conditional deletion of histone demethylases UTX and JMJD3 by CD4-Cre leads to near complete loss of liver NKT cells, while conventional T cells are less affected. Loss of NKT cells is cell intrinsic and not due to an insufficient selection environment. The absence of NKT cells in UTX/JMJD3-deficient mice protects mice from concanavalin A-induced liver injury, a model of NKT-mediated hepatitis. GO-analysis of RNA-seq data indicates that cell cycle genes are downregulated in UTX/JMJD3-deleted NKT progenitors, and suggest that failed expansion may account for some of the cellular deficiency. The phenotype appears to be demethylase-dependent, because UTY, a homolog of UTX that lacks catalytic function, is not sufficient to restore their development and removal of H3K27me3 by deletion of EZH2 partially rescues the defect. CONCLUSIONS: NKT cell development and gene expression is sensitive to proper regulation of H3K27 methylation. The H3K27me3 demethylase enzymes, in particular UTX, promote NKT cell development, and are required for effective NKT function.

H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation.

Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy, and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers, and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a general facilitator that generates access for a variety of complexes, both activating and repressive.