Brad Lackford

Specificity and Complexity of the Caenorhabditis elegans Innate Immune Response

ABSTRACT In response to infection, Caenorhabditis elegans produces an array of antimicrobial proteins. To understand the C. elegans immune response, we have investigated the regulation of a large, representative sample of candidate antimicrobial genes. We found that all these putative antimicrobial genes are expressed in tissues exposed to the environment, a position from which they can ward off infection. Using RNA interference to inhibit the function of immune signaling pathways in C. elegans, we found that different immune response pathways regulate expression of distinct but overlapping sets of antimicrobial genes. We also show that different bacterial pathogens regulate distinct but overlapping sets of antimicrobial genes. The patterns of genes induced by pathogens do not coincide with any single immune signaling pathway. Thus, even in this simple model system for innate immunity, striking specificity and complexity exist in the immune response. The unique patterns of antimicrobial gene expression observed when C. elegans is exposed to different pathogens or when different immune signaling pathways are perturbed suggest that a large set of yet to be identified pathogen recognition receptors (PRRs) exist in the nematode. These PRRs must interact in a complicated fashion to induce a unique set of antimicrobial genes. We also propose the existence of an “antimicrobial fingerprint,” which will aid in assigning newly identified C. elegans innate immunity genes to known immune signaling pathways.

Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity

The TET family of FE(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) promote DNA demethylation by converting 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), which they further oxidize into 5-formylcytosine and 5-carboxylcytosine. Tet1 is robustly expressed in mouse embryonic stem cells (mESCs) and has been implicated in mESC maintenance. Here we demonstrate that, unlike genetic deletion, RNAi-mediated depletion of Tet1 in mESCs led to a significant reduction in 5hmC and loss of mESC identity. The differentiation phenotype due to Tet1 depletion positively correlated with the extent of 5hmC loss. Meta-analyses of genomic data sets suggested interaction between Tet1 and leukemia inhibitory factor (LIF) signaling. LIF signaling is known to promote self-renewal and pluripotency in mESCs partly by opposing MAPK/ERK-mediated differentiation. Withdrawal of LIF leads to differentiation of mESCs. We discovered that Tet1 depletion impaired LIF-dependent Stat3-mediated gene activation by affecting Stat3s ability to bind to its target sites on chromatin. Nanog overexpression or inhibition of MAPK/ERK signaling, both known to maintain mESCs in the absence of LIF, rescued Tet1 depletion, further supporting the dependence of LIF/Stat3 signaling on Tet1. These data support the conclusion that analysis of mESCs in the hours/days immediately following efficient Tet1 depletion reveals Tet1s normal physiological role in maintaining the pluripotent state that may be subject to homeostatic compensation in genetic models.

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here, we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on OCT4 and WDR5. At the pluripotency genes, Ino80 maintains an open chromatin architecture and licenses recruitment of Mediator and RNA polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network and illustrate the coordination among chromatin remodeler, transcription factor, and histone-modifying enzyme in the regulation of the pluripotent state.

Identification of innate immunity genes and pathways using a comparative genomics approach

To reveal regulators of innate immunity, we used RNAi assays to monitor the immune response when genes are inhibited in Caenorhabditis elegans and mouse macrophages. Genes that altered innate immune responsiveness in C. elegans were validated in murine macrophages, resulting in the discovery of 11 genes that regulate the innate immune response in both systems and the subsequent identification of a protein interaction network with a conserved role in innate immunity regulation. We confirmed the role of four of these 11 genes in antimicrobial gene regulation using available mutants in C. elegans. Several of these genes (acy-1, tub-2, and tbc-1) also regulate susceptibility to the pathogen Pseudomonas aeruginosa. These genes may prove critical to understanding host defense and represent potential therapeutic targets for infectious and immunological diseases.

Identification of Novel Genes That Mediate Innate Immunity Using Inbred Mice

Innate immunity is the first line of defense against microbial infections. Although polymorphisms in toll-like receptors (TLRs) and downstream signaling molecules (CD14, TLR2, TLR4, TLR5, and IRAK4) affect the innate immune response, these variants account for only a portion of the ability of the host to respond to bacteria, fungi, and viruses. To identify other genes involved in the innate immune response, we challenged 16 inbred murine strains with lipopolysaccharide (LPS) systemically and measured serum concentrations of pro-inflammatory cytokines IL-1β, IL-6, and TNFα, and the chemokine KC 6 hr post-treatment. Loci that segregate with strain phenotypes were identified by whole genome association (WGA) mapping of cytokine concentrations. Published gene expression profiles and quantitative trait loci (QTL) were then utilized to prioritize loci and genes that potentially regulate the host response to LPS. Sixteen loci were selected for further investigation by combining WGA analysis with previously published QTL for murine response to LPS or gram negative bacteria. Thirty-eight genes within these loci were then selected for further investigation on the basis of the significance of the identified locus, transcriptional response to LPS, and biological plausibility. RNA interference-mediated inhibition of 4 of 38 candidate genes was shown to block the production of IL-6 in J774A.1 macrophages. In summary, our analysis identified 4 genes that have not previously been implicated in innate immunity, namely, 1110058L19Rik, 4933415F23Rik, Fbxo9, and Ipo7. These genes could represent potential sepsis biomarkers or therapeutic targets that should be further investigated in human populations.

Fip1 regulates mRNA alternative polyadenylation to promote stem cell self‐renewal

mRNA alternative polyadenylation (APA) plays a critical role in post‐transcriptional gene control and is highly regulated during development and disease. However, the regulatory mechanisms and functional consequences of APA remain poorly understood. Here, we show that an mRNA 3′ processing factor, Fip1, is essential for embryonic stem cell (ESC) self‐renewal and somatic cell reprogramming. Fip1 promotes stem cell maintenance, in part, by activating the ESC‐specific APA profiles to ensure the optimal expression of a specific set of genes, including critical self‐renewal factors. Fip1 expression and the Fip1‐dependent APA program change during ESC differentiation and are restored to an ESC‐like state during somatic reprogramming. Mechanistically, we provide evidence that the specificity of Fip1‐mediated APA regulation depends on multiple factors, including Fip1‐RNA interactions and the distance between APA sites. Together, our data highlight the role for post‐transcriptional control in stem cell self‐renewal, provide mechanistic insight on APA regulation in development, and establish an important function for APA in cell fate specification.

The Caenorhabditis elegans Germ Line Regulates Distinct Signaling Pathways to Control Lifespan and Innate Immunity

The relationship between the mechanisms that control an organisms lifespan and its ability to respond to environmental challenges are poorly understood. In Caenorhabditis elegans, an insulin-like signaling pathway modulates lifespan and the innate immune response to bacterial pathogens via a common mechanism involving transcriptional regulation by the DAF-16/FOXO transcription factor. The C. elegans germ line also modulates lifespan in a daf-16-dependent manner. Here, we show that the germ line controls the innate immune response of C. elegans somatic cells to two different Gram-negative bacteria. In contrast to the insulin-like signaling pathway, the germ line acts via distinct signaling pathways to control lifespan and innate immunity. Under standard nematode culture conditions, the germ line regulates innate immunity in parallel to a known p38 MAPK signaling pathway, via a daf-16-independent pathway. Our findings indicate that a complex regulatory network integrates inputs from insulin-like signaling, p38 MAPK signaling, and germ line stem cells to control innate immunity in C. elegans. We also confirm that innate immunity and lifespan in C. elegans are distinct processes, as nonoverlapping regulatory networks control survival in the presence of pathogenic and nonpathogenic bacteria. Finally, we demonstrate that the p38 MAPK pathway in C. elegans is activated to a similar extent by both pathogenic and nonpathogenic bacteria, suggesting that both can induce the nematode innate immune response.

The THO complex regulates pluripotency gene mRNA export and controls embryonic stem cell self-renewal and somatic cell reprogramming.

Embryonic stem cell (ESC) self-renewal and differentiation are governed by a broad-ranging regulatory network. Although the transcriptional regulatory mechanisms involved have been investigated extensively, posttranscriptional regulation is still poorly understood. Here we describe a critical role of the THO complex in ESC self-renewal and differentiation. We show that THO preferentially interacts with pluripotency gene transcripts through Thoc5 and is required for self-renewal at least in part by regulating their export and expression. During differentiation, THO loses its interaction with those transcripts due to reduced Thoc5 expression, leading to decreased expression of pluripotency proteins that facilitates exit from self-renewal. THO is also important for the establishment of pluripotency, because its depletion inhibits somatic cell reprogramming and blastocyst development. Together, our data indicate that THO regulates pluripotency gene mRNA export to control ESC self-renewal and differentiation, and therefore uncover a role for this aspect of posttranscriptional regulation in stem cell fate specification.

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.

Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

Embryonic stem cell (ESC) identity and self‐renewal is maintained by extrinsic signaling pathways and intrinsic gene regulatory networks. Here, we show that three members of the Ccr4‐Not complex, Cnot1, Cnot2, and Cnot3, play critical roles in maintaining mouse and human ESC identity as a protein complex and inhibit differentiation into the extraembryonic lineages. Enriched in the inner cell mass of blastocysts, these Cnot genes are highly expressed in ESC and downregulated during differentiation. In mouse ESCs, Cnot1, Cnot2, and Cnot3 are important for maintenance in both normal conditions and the 2i/LIF medium that supports the ground state pluripotency. Genetic analysis indicated that they do not act through known self‐renewal pathways or core transcription factors. Instead, they repress the expression of early trophectoderm (TE) transcription factors such as Cdx2. Importantly, these Cnot genes are also necessary for the maintenance of human ESCs, and silencing them mainly lead to TE and primitive endoderm differentiation. Together, our results indicate that Cnot1, Cnot2, and Cnot3 represent a novel component of the core self‐renewal and pluripotency circuitry conserved in mouse and human ESCs. STEM CELLS 2012;30:910–922