Rebecca Mathew

Elevated and sustained expression of the transcription factors Egr1 and Egr2 controls NKT lineage differentiation in response to TCR signaling

TCR-driven interactions determine the lineage choice of CD4+CD8+ thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we show that TCR-induced Egr2 and Egr1 proteins had elevated and prolonged expression in NKT lineage precursors compared with conventional lineages. ChIP-seq analysis uncovered that Egr2 directly bound and activated the promoter of Zbtb16 which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the Il2rb promoter and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that elevated and persistent Egr2 levels specify the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to instruct a thymic lineage.

Microcephaly Gene Links Trithorax and REST/NRSF to Control Neural Stem Cell Proliferation and Differentiation

Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335 null mice are embryonically lethal, and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that ZNF335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.

Natural killer T (NKT)–B-cell interactions promote prolonged antibody responses and long-term memory to pneumococcal capsular polysaccharides

Significance Antibodies directed against microbial polysaccharides are a critical component of protective immune responses and vaccines. We used nanoparticles coexpressing pneumococcal capsular polysaccharides and a cell wall lipid antigen analog to model NKT–B-cell interactions. Our study demonstrated CD1d-restricted cognate interactions, isotype switch, affinity maturation, and long-term memory, despite the apparent failure of NKT cells to differentiate into follicular helper cells. The findings demonstrate the importance of nonconventional sources of help for B cells responding to polysaccharide antigens. Innate-like natural killer T (NKT) cells critically enhance cell and humoral immunity against infections through recognition of conserved microbial lipid antigens presented by CD1d-expressing antigen-presenting cells, and provision of CD40L and cytokine signals. Whereas NKT cells efficiently licensed dendritic cells to prime potent effector and memory T cells, studies based on model antigens such as alphagalactosylceramide-nitrophenyl conjugates concluded that help to B cells was associated with NKT follicular helper differentiation, but limited to short-term responses without induction of memory. We revisited this surprising conclusion in the context of the extracellular encapsulated pathogen Streptococcus pneumoniae, where recognition of lipid and capsular polysaccharide antigens by NKT cells and B cells, respectively, provide critical host protection. Using liposomal nanoparticles displaying synthetic lipid and polysaccharide antigens to elicit pure and direct NKT–B-cell interactions in vivo, we observed intense and prolonged antibody responses with isotype switch, affinity maturation, and long-lasting B-cell memory, despite modest or absent NKT follicular helper differentiation. Furthermore, conditional ablation of Cd1d demonstrated a requirement for a two-step process involving first cognate interactions with dendritic cells, for NKT cell activation, and then with B cells, for induction of isotype switch and memory. Thus, NKT help to B cells represents both a major arm of antimicrobial defense and a promising target for B-cell vaccines.

BTB-ZF factors recruit the E3 ligase cullin 3 to regulate lymphoid effector programs

The differentiation of several T- and B-cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodelling. Here we report that promyelocytic leukaemia zinc finger (PLZF), the BTB-zinc finger (BTB-ZF) transcription factor directing the innate-like effector program of natural killer T-cell thymocytes, is prominently associated with cullin 3 (CUL3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF transports CUL3 to the nucleus, where the two proteins are associated within a chromatin-modifying complex. Furthermore, PLZF expression results in selective ubiquitination changes of several components of this complex. CUL3 was also found associated with the BTB-ZF transcription factor BCL6, which directs the germinal-centre B cell and follicular T-helper cell programs. Conditional CUL3 deletion in mice demonstrated an essential role for CUL3 in the development of PLZF- and BCL6-dependent lineages. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit CUL3 to alter the ubiquitination pattern of their associated chromatin-modifying complex. We propose that this new function is essential to direct the differentiation of several T- and B-cell effector programs, and may also be involved in the oncogenic role of PLZF and BCL6 in leukaemias and lymphomas.

Multiple layers of transcriptional regulation by PLZF in NKT-cell development.

Significance Natural killer T (NKT) cells are a specialized population of innate-like T cells that acquire their effector program during development under the control of the transcription factor PLZF (promyelocytic leukemia zinc finger, encoded by Zbtb16). To elucidate the mechanisms underlying this unique property of PLZF, we performed ChIP-seq and microarray analysis of NKT cells and PLZF-transgenic T cells, which revealed direct regulation of effector genes and of T-helper–specific transcription factors. Notably, PLZF also bound and repressed Bach2, a global repressor of effector differentiation. Thus, multiple layers of positive and negative regulation coordinate the induction of the innate effector program by PLZF. The transcription factor PLZF [promyelocytic leukemia zinc finger, encoded by zinc finger BTB domain containing 16 (Zbtb16)] is induced during the development of innate and innate-like lymphocytes to direct their acquisition of a T-helper effector program, but the molecular mechanisms involved are poorly understood. Using biotinylation-based ChIP-seq and microarray analysis of both natural killer T (NKT) cells and PLZF-transgenic thymocytes, we identified several layers of regulation of the innate-like NKT effector program. First, PLZF bound and regulated genes encoding cytokine receptors as well as homing and adhesion receptors; second, PLZF bound and activated T-helper–specific transcription factor genes that in turn control T-helper–specific programs; finally, PLZF bound and suppressed the transcription of Bach2, a potent general repressor of effector differentiation in naive T cells. These findings reveal the multilayered architecture of the transcriptional program recruited by PLZF and elucidate how a single transcription factor can drive the developmental acquisition of a broad effector program.

Egr2-dependent gene expression profiling and ChIP-Seq reveal novel biologic targets in T cell anergy

T cell anergy is one of the mechanisms contributing to peripheral tolerance, particularly in the context of progressively growing tumors and in tolerogenic treatments promoting allograft acceptance. We recently reported that early growth response gene 2 (Egr2) is a critical transcription factor for the induction of anergy in vitro and in vivo, which was identified based on its ability to regulate the expression of inhibitory signaling molecules diacylglycerol kinase (DGK)-α and -ζ. We reasoned that other transcriptional targets of Egr2 might encode additional factors important for T cell anergy and immune regulation. Thus, we conducted two sets of genome-wide screens: gene expression profiling of wild type versus Egr2-deleted T cells treated under anergizing conditions, and a ChIP-Seq analysis to identify genes that bind Egr2 in anergic cells. Merging of these data sets revealed 49 targets that are directly regulated by Egr2. Among these are inhibitory signaling molecules previously reported to contribute to T cell anergy, but unexpectedly, also cell surface molecules and secreted factors, including lymphocyte-activation gene 3 (Lag3), Class-I-MHC-restricted T cell associated molecule (Crtam), Semaphorin 7A (Sema7A), and chemokine CCL1. These observations suggest that anergic T cells might not simply be functionally inert, and may have additional functional properties oriented towards other cellular components of the immune system.

Absolute Intramolecular Distance Measurements with Angstrom-Resolution Using Anomalous Small-Angle X-ray Scattering

Accurate determination of molecular distances is fundamental to understanding the structure, dynamics, and conformational ensembles of biological macromolecules. Here we present a method to determine the full distance distribution between small (∼7 Å radius) gold labels attached to macromolecules with very high-precision (≤1 Å) and on an absolute distance scale. Our method uses anomalous small-angle X-ray scattering close to a gold absorption edge to separate the gold-gold interference pattern from other scattering contributions. Results for 10-30 bp DNA constructs achieve excellent signal-to-noise and are in good agreement with previous results obtained by single-energy SAXS measurements without requiring the preparation and measurement of single labeled and unlabeled samples. The use of small gold labels in combination with ASAXS read out provides an attractive approach to determining molecular distance distributions that will be applicable to a broad range of macromolecular systems.

A negative feedback loop mediated by the Bcl6–cullin 3 complex limits Tfh cell differentiation

Bcl6 and E3 ligase cullin 3 complexes mediate negative feedback regulation during thymocyte development and T cell activation to restrain exaggerated Tfh responses.

A microRNA negative feedback loop downregulates vesicle transport and inhibits fear memory

The SNARE-mediated vesicular transport pathway plays major roles in synaptic remodeling associated with formation of long-term memories, but the mechanisms that regulate this pathway during memory acquisition are not fully understood. Here we identify miRNAs that are up-regulated in the rodent hippocampus upon contextual fear-conditioning and identify the vesicular transport and synaptogenesis pathways as the major targets of the fear-induced miRNAs. We demonstrate that miR-153, a member of this group, inhibits the expression of key components of the vesicular transport machinery, and down-regulates Glutamate receptor A1 trafficking and neurotransmitter release. MiR-153 expression is specifically induced during LTP induction in hippocampal slices and its knockdown in the hippocampus of adult mice results in enhanced fear memory. Our results suggest that miR-153, and possibly other fear-induced miRNAs, act as components of a negative feedback loop that blocks neuronal hyperactivity at least partly through the inhibition of the vesicular transport pathway. DOI: http://dx.doi.org/10.7554/eLife.22467.001

Gold nanocrystal labels provide a sequence–to–3D structure map in SAXS reconstructions

Gold labels provide a sequence–to–low-resolution structure map in SAXS measurements of biological macromolecules. Small-angle x-ray scattering (SAXS) is a powerful technique to probe the structure of biological macromolecules and their complexes under virtually arbitrary solution conditions, without the need for crystallization. While it is possible to reconstruct molecular shapes from SAXS data ab initio, the resulting electron density maps have a resolution of ~1 nm and are often insufficient to reliably assign secondary structure elements or domains. We show that SAXS data of gold-labeled samples significantly enhance the information content of SAXS measurements, allowing the unambiguous assignment of macromolecular sequence motifs to specific locations within a SAXS structure. We first demonstrate our approach for site-specifically internally and end-labeled DNA and an RNA motif. In addition, we present a protocol for highly uniform and site-specific labeling of proteins with small (~1.4 nm diameter) gold particles and apply our method to the signaling protein calmodulin. In all cases, the position of the small gold probes can be reliably identified in low-resolution electron density maps. Enhancing low-resolution measurements by site-selective gold labeling provides an attractive approach to aid modeling of a large range of macromolecular systems.