Jessica M. Lindvall

Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling

Summary:  Brutons tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X‐linked agammaglobulinemia (XLA) in humans and X‐linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B‐lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full‐length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype–phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering‐RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA‐, Xid‐, and Btk‐knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid‐ and Btk‐KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C‐missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.

Defective Toll-like receptor 9-mediated cytokine production in B cells from Bruton's tyrosine kinase-deficient mice

Brutons tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll‐like receptor 9 (TLR9) activation and the production of pro‐inflammatory cytokines such as interleukin (IL)‐6, tumour necrosis factor (TNF)‐α and IL‐12p40. Our data show that Btk‐deficient B cells respond more efficiently to CpG‐DNA stimulation, producing significantly higher levels of pro‐inflammatory cytokines but lower levels of the inhibitory cytokine IL‐10. The quantitative reverse transcription–polymerase chain reaction (RT‐PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL‐12 family, IL‐27, was significantly increased in Btk‐deficient B cells after CpG‐DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk‐defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.

Expression profiling in transformed human B cells: influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays

We have used both Clontech AtlasTM Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein‐Barr virus (EBV)‐transformed B cells and genes regulated by Brutons tyrosine kinase (Btk). We compared EBV‐transformed wild‐type (WT) B cells from a healthy individual, WT1 and an X‐linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were ≥1.9‐fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down‐regulated in XLA2, indicating their biological role. Higher‐fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real‐time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell‐specific OCT binding factor‐1 (OBF‐1) were induced ≥1.9‐fold in both XLA1 and XLA2 analyzed by AtlasTM filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses.

The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota

BackgroundInnate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood.ResultsWe show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism.ConclusionsThis study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.

A chemical-genetic screen to unravel the genetic network of CDC28/CDK1 links ubiquitin and Rad6–Bre1 to cell cycle progression

Cyclin-dependent kinases (CDKs) control the eukaryotic cell cycle, and a single CDK, Cdc28 (also known as Cdk1), is necessary and sufficient for cell cycle regulation in the budding yeast Saccharomyces cerevisiae. Cdc28 regulates cell cycle-dependent processes such as transcription, DNA replication and repair, and chromosome segregation. To gain further insight into the functions of Cdc28, we performed a high-throughput chemical-genetic array (CGA) screen aimed at unraveling the genetic network of CDC28. We identified 107 genes that strongly genetically interact with CDC28. Although these genes serve multiple cellular functions, genes involved in cell cycle regulation, transcription, and chromosome metabolism were overrepresented. DOA1, which is involved in maintaining free ubiquitin levels, as well as the RAD6–BRE1 pathway, which is involved in transcription, displayed particularly strong genetic interactions with CDC28. We discovered that DOA1 is important for cell cycle entry by supplying ubiquitin. Furthermore, we found that the RAD6–BRE1 pathway functions downstream of DOA1/ubiquitin but upstream of CDC28, by promoting transcription of cyclins. These results link cellular ubiquitin levels and the Rad6–Bre1 pathway to cell cycle progression.

Interaction of Btk and Akt in B cell signaling

Reactive oxygen species (ROS) or reactive oxygen intermediates (ROIs) mediate complex signaling involving multiple pathways. In this report, we demonstrate for the first time that endogenous Brutons tyrosine kinase (Btk) and Akt can interact with each other in DT40 chicken B cells and human Nalm6 B cells and that this interaction is inducible following H2O2 stimulation. This interaction is supported by visualizing the co-localization of Btk and Akt in the perinuclear region and membrane ruffles in COS-7 cells. We have also shown the involvement of phosphatidylinositol 3-kinase (PI 3-K) and Btk in the phosphorylation of Akt following stimulation by hydrogen peroxide (H2O2). Interestingly, Akt phosphorylation was found in the presence of Btk even in the absence of oxidative stress. In addition, we have investigated the involvement of PI 3-K in the MAPKs and ERK and JNK phosphorylation, in the presence or absence of Btk. Phosphorylation of both ERK and JNK increased when the PI 3-K pathway was inhibited and both pathways were modulated positively by Btk. Taken together, based on the study of endogenous conditions, we show the novel interaction of Btk and Akt in H2O2 signaling in B cells.

Genome-wide expression analysis of cells expressing gain of function mutant D374Y-PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum cholesterol. The possibility that PCSK9 also functions in other pathways needs to be addressed. We have transfected HepG2 cells with mutant D374Y‐PCSK9 or control vector. Gene expression signatures were determined using the Affymetrix GeneChip technology, and the expression pattern of selected genes was confirmed by quantitative real‐time polymerase chain reaction (qRT‐PCR). Data was normalized and analyzed using a model‐based background adjustment for oligonucleotide expression arrays, then filtered based upon expression within treatments group, and subjected to moderated t‐statistics. Five hundred twenty transcripts had altered expression levels between D374Y‐PCSK9 and control vector. Among the 520 probes on our top list, 312 were found to have an assigned Gene Ontology (GO) term, and 96 were found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Genome‐wide expression profiling revealed that “steroid biosynthesis,” “sterol metabolism,” and “cholesterol biosynthsis” were affected by D374Y‐PCSK9. Also, the GO biological process terms “response to stresss,” “response to virus,” “response to unfolded protein,” and “immune response” were influenced by D374Y‐PCSK9. Our results suggest that D374Y‐PCSK9 results in up‐regulation of genes involved in sterol biosynthesis and down‐regulation of stress‐response genes and specific inflammation pathways. J. Cell. Physiol. 217: 459–467, 2008.

Altered DNA methylation of glycolytic and lipogenic genes in liver from obese and type 2 diabetic patients

Objective Epigenetic modifications contribute to the etiology of type 2 diabetes. Method We performed genome-wide methylome and transcriptome analysis in liver from severely obese men with or without type 2 diabetes and non-obese men to discover aberrant pathways underlying the development of insulin resistance. Results were validated by pyrosequencing. Result We identified hypomethylation of genes involved in hepatic glycolysis and insulin resistance, concomitant with increased mRNA expression and protein levels. Pyrosequencing revealed the CpG-site within ATF-motifs was hypomethylated in four of these genes in liver of severely obese non-diabetic and type 2 diabetic patients, suggesting epigenetic regulation of transcription by altered ATF-DNA binding. Conclusion Severely obese non-diabetic and type 2 diabetic patients have distinct alterations in the hepatic methylome and transcriptome, with hypomethylation of several genes controlling glucose metabolism within the ATF-motif regulatory site. Obesity appears to shift the epigenetic program of the liver towards increased glycolysis and lipogenesis, which may exacerbate the development of insulin resistance.

Cdc28 kinase activity regulates the basal transcription machinery at a subset of genes

The cyclin-dependent kinase Cdc28 is the master regulator of the cell cycle in Saccharomyces cerevisiae. Cdc28 initiates the cell cycle by activating cell-cycle–specific transcription factors that switch on a transcriptional program during late G1 phase. Cdc28 also has a cell-cycle–independent, direct function in regulating basal transcription, which does not require its catalytic activity. However, the exact role of Cdc28 in basal transcription remains poorly understood, and a function for its kinase activity has not been fully explored. Here we show that the catalytic activity of Cdc28 is important for basal transcription. Using a chemical-genetic screen for mutants that specifically require the kinase activity of Cdc28 for viability, we identified a plethora of basal transcription factors. In particular, CDC28 interacts genetically with genes encoding kinases that phosphorylate the C-terminal domain of RNA polymerase II, such as KIN28. ChIP followed by high-throughput sequencing (ChIP-seq) revealed that Cdc28 localizes to at least 200 genes, primarily with functions in cellular homeostasis, such as the plasma membrane proton pump PMA1. Transcription of PMA1 peaks early in the cell cycle, even though the promoter sequences of PMA1 (as well as the other Cdc28-enriched ORFs) lack cell-cycle elements, and PMA1 does not recruit Swi4/6-dependent cell-cycle box-binding factor/MluI cell-cycle box binding factor complexes. Finally, we found that recruitment of Cdc28 and Kin28 to PMA1 is mutually dependent and that the activity of both kinases is required for full phosphorylation of C-terminal domain–Ser5, for efficient transcription, and for mRNA capping. Our results reveal a mechanism of cell-cycle–dependent regulation of basal transcription.

DNA damage leads to progressive replicative decline but extends the life span of long-lived mutant animals

Human-nucleotide-excision repair (NER) deficiency leads to different developmental and segmental progeroid symptoms of which the pathogenesis is only partially understood. To understand the biological impact of accumulating spontaneous DNA damage, we studied the phenotypic consequences of DNA-repair deficiency in Caenorhabditis elegans. We find that DNA damage accumulation does not decrease the adult life span of post-mitotic tissue. Surprisingly, loss of functional ERCC-1/XPF even further extends the life span of long-lived daf-2 mutants, likely through an adaptive activation of stress signaling. Contrariwise, NER deficiency leads to a striking transgenerational decline in replicative capacity and viability of proliferating cells. DNA damage accumulation induces severe, stochastic impairment of development and growth, which is most pronounced in NER mutants that are also impaired in their response to ionizing radiation and inter-strand crosslinks. These results suggest that multiple DNA-repair pathways can protect against replicative decline and indicate that there might be a direct link between the severity of symptoms and the level of DNA-repair deficiency in patients.