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The polymerase-associated factor 1 complex (PAF1C) is a key, post-initiation transcriptional regulator of both promoter-proximal pausing and productive elongation catalyzed by RNA Pol II and is also involved in transcriptional repression of viral gene expression during human immunodeficiency virus–1 (HIV-1) latency. Using a molecular docking–based compound screen in silico and global sequencing–based candidate evaluation in vivo, we identified a first-in-class, small-molecule inhibitor of PAF1C (iPAF1C) that disrupts PAF1 chromatin occupancy and induces global release of promoter-proximal paused RNA Pol II into gene bodies. Transcriptomic analysis revealed that iPAF1C treatment mimics acute PAF1 subunit depletion and impairs RNA Pol II pausing at heat shock–down-regulated genes. Furthermore, iPAF1C enhances the activity of diverse HIV-1 latency reversal agents both in cell line latency models and in primary cells from persons living with HIV-1. In sum, this study demonstrates that efficient disruption of PAF1C by a first-in-class, small-molecule inhibitor may have therapeutic potential for improving current HIV-1 latency reversal strategies.

Circadian clocks may mediate lifespan extension by caloric or dietary restriction (DR). We find that the core clock transcription factorClockis crucial for a robust longevity and fecundity response to DR inDrosophila. To identify clock-controlled mediators, we performed RNA-sequencing from abdominal fat bodies across the 24 h day after just 5 days under control or DR diets. In contrast to more chronic DR regimens, we did not detect significant changes in the rhythmic expression of core clock genes. Yet we discovered that DR induced de novo rhythmicity or increased expression of rhythmic clock output genes. Network analysis revealed that DR increased network connectivity in one module comprised of genes encoding proteasome subunits. Adult, fat body specific RNAi knockdown demonstrated that proteasome subunits contribute to DR-mediated lifespan extension. Thus, clock control of output links DR-mediated changes in rhythmic transcription to lifespan extension.

Catalytic-inactivating mutations within the Drosophila enhancer H3K4 mono-methyltransferase Trr and its mammalian homologs, MLL3/4, cause only minor changes in gene expression compared with whole-gene deletions for these COMPASS members. To identify essential histone methyltransferase-independent functions of Trr, we screened to identify a minimal Trr domain sufficient to rescue Trr-null lethality and demonstrate that this domain binds and stabilizes Utx in vivo. Using the homologous MLL3/MLL4 human sequences, we mapped a short ∼80-amino-acid UTX stabilization domain (USD) that promotes UTX stability in the absence of the rest of MLL3/4. Nuclear UTX stability is enhanced when the USD is fused with the MLL4 HMG-box. Thus, COMPASS-dependent UTX stabilization is an essential noncatalytic function of Trr/MLL3/MLL4, suggesting that stabilizing UTX could be a therapeutic strategy for cancers with MLL3/4 loss-of-function mutations.

Objectives Core clock genes regulate tissue-specific transcriptome oscillations that synchronize physiologic processes throughout the body, held in phase by the central circadian rhythm. The central circadian rhythm rapidly dampens with onset of critical illness, but the effect of critical illness on gene expression oscillations is unknown. The objective of this study was to characterize the rhythmicity and phase coherence of core clock genes and the broader transcriptome after onset of critical illness. Design Cross-sectional study. Setting ICUs and hospital clinical research unit. Patients Critically ill patients within the first day of presenting from the community and healthy volunteers. Interventions Usual care (critically ill patients) and modified constant routine (healthy volunteers). Measurements and main results We studied 15 critically ill patients, including 10 with sepsis and five with intracerebral hemorrhage, and 11 healthy controls. The central circadian rhythm and rest-activity rhythms were profiled by continuous wrist actigraphy, and serum melatonin sampled every 2 hours along with whole blood for RNA isolation over 24 hours. The gene expression transcriptome was obtained by RNA sequencing. Core clock genes were analyzed for rhythmicity by cosinor fit. Significant circadian rhythmicity was identified in five of six core clock genes in healthy controls, but none in critically ill patients. TimeSignature, a validated algorithm based on 41 genes, was applied to assess overall transcriptome phase coherence. Median absolute error of TimeSignature was higher in individual critically ill patients than healthy patients (4.90 vs 1.48 hr) and was correlated with encephalopathy severity by Glasgow Coma Scale in critically ill patients (rho, -0.54; p = 0.036). Conclusions Gene expression rhythms rapidly become abnormal during critical illness. The association between disrupted transcriptome rhythms and encephalopathy suggests a path for future work to elucidate the underlying pathophysiology.

Summary Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

The circadian rhythm drives the oscillatory expression of thousands of genes across all tissues, coordinating physiological processes. The effect of this rhythm on health has generated increasing interest in discovering genes under circadian control by searching for periodic patterns in transcriptomic time-series experiments. While algorithms for detecting cycling transcripts have advanced, there remains little guidance quantifying the effect of experimental design and analysis choices on cycling detection accuracy. We present TimeTrial, a user-friendly benchmarking framework using both real and synthetic data to investigate cycle detection algorithms’ performance and improve circadian experimental design. Results show that the optimal choice of analysis method depends on the sampling scheme, noise level, and shape of the waveform of interest; and provides guidance on the impact of sampling frequency and duration on cycling detection accuracy. The TimeTrial software is freely available for download and may also be accessed through a web interface. By supplying a tool to vary and optimize experimental design considerations, TimeTrial will enhance circadian transcriptomics studies.

Significance Determining the state of an individual’s internal physiological clock has important implications for precision medicine, from diagnosing neurological disorders to optimizing drug delivery. To be useful, such a test must be accurate, minimally burdensome to the patient, and robust to differences in patient protocols, sample collection, and assay technologies. TimeSignature is a machine-learning approach to predict physiological time based on gene expression in human blood. A powerful feature is TimeSignature’s generalizability, enabling it to be applied to samples from disparate studies and yield highly accurate results despite systematic differences between the studies. This quality is unique among expression-based predictors and addresses a major challenge in the development of reliable and clinically useful biomarker tests., Circadian clocks play a key role in regulating a vast array of biological processes, with significant implications for human health. Accurate assessment of physiological time using transcriptional biomarkers found in human blood can significantly improve diagnosis of circadian disorders and optimize the delivery time of therapeutic treatments. To be useful, such a test must be accurate, minimally burdensome to the patient, and readily generalizable to new data. A major obstacle in development of gene expression biomarker tests is the diversity of measurement platforms and the inherent variability of the data, often resulting in predictors that perform well in the original datasets but cannot be universally applied to new samples collected in other settings. Here, we introduce TimeSignature, an algorithm that robustly infers circadian time from gene expression. We demonstrate its application in data from three independent studies using distinct microarrays and further validate it against a new set of samples profiled by RNA-sequencing. Our results show that TimeSignature is more accurate and efficient than competing methods, estimating circadian time to within 2 h for the majority of samples. Importantly, we demonstrate that once trained on data from a single study, the resulting predictor can be universally applied to yield highly accurate results in new data from other studies independent of differences in study population, patient protocol, or assay platform without renormalizing the data or retraining. This feature is unique among expression-based predictors and addresses a major challenge in the development of generalizable, clinically useful tests.

The successful treatment of chronic dermal wounds, such as diabetic foot ulcers (DFU), depends on the development of safe, effective, and affordable regenerative tools that the surgeon can rely on to promote wound closure. Although promising, strategies that involve cell-based therapies and the local release of exogenous growth factors are costly, require very long development times, and result in modest improvements in patient outcome. We describe the development of an antioxidant shape-conforming regenerative wound dressing that uses the laminin-derived dodecapeptide A5G81 as a potent tethered cell adhesion-, proliferation-, and haptokinesis-inducing ligand to locally promote wound closure. A5G81 immobilized within a thermoresponsive citrate-based hydrogel facilitates integrin-mediated spreading, migration, and proliferation of dermal and epidermal cells, resulting in faster tissue regeneration in diabetic wounds. This peptide-hydrogel system represents a paradigm shift in dermoconductive and dermoinductive strategies for treating DFU without the need for soluble biological or pharmacological factors.

The NF-κB transcription factors are activated via diverse molecular mechanisms in response to various types of stimuli. A plethora of functions associated with specific sets of target genes could be regulated differentially by this factor, affecting cellular response to stress including an anticancer treatment. Here we aimed to compare subsets of NF-κB-dependent genes induced in cells stimulated with a pro-inflammatory cytokine and in cells damaged by a high dose of ionizing radiation (4 and 10 Gy). The RelA-containing NF-κB species were activated by the canonical TNFα-induced and the atypical radiation-induced pathways in human osteosarcoma cells. NF-κB-dependent genes were identified using the gene expression profiling (by RNA-Seq) in cells with downregulated RELA combined with the global profiling of RelA binding sites (by ChIP-Seq), with subsequent validation of selected candidates by quantitative PCR. There were 37 NF-κB-dependent protein-coding genes identified: in all cases RelA bound in their regulatory regions upon activation while downregulation of RELA suppressed their stimulus-induced upregulation, which apparently indicated the positive regulation mode. This set of genes included a few “novel” NF-κB-dependent species. Moreover, the evidence for possible negative regulation of ATF3 gene by NF-κB was collected. The kinetics of the NF-κB activation was slower in cells exposed to radiation than in cytokine-stimulated ones. However, subsets of NF-κB-dependent genes upregulated by both types of stimuli were essentially the same. Hence, one should expect that similar cellular processes resulting from activation of the NF-κB pathway could be induced in cells responding to pro-inflammatory cytokines and in cells where so-called “sterile inflammation” response was initiated by radiation-induced damage.

TimeSignature’s power is that it is highly accurate across transcriptomic platforms and experimental protocols. TimeSignature can be trained using data from a single platform/study and be applied to independent data without additional processing. Demonstrating this robustness and generalizability required applying TimeSignature to as diverse a set of studies as possible (i.e., refs. 1⇓⇓–4). Because not all studies had melatonin data, we chose to use draw time as a proxy for melatonin phase. Laing et al. (5) criticize this methodology; however, each of the studies (1⇓⇓–4) selected subjects for whom circadian phase was well aligned with local time. We have confirmed that dim-light melatonin onset (DLMO)25%, the time at which 25% of maximum blood melatonin is reached, is strongly correlated with local time in studies with available melatonin data (1⇓–3) (Fig. 1). As expected, training on local time thus accurately predicts melatonin phase (Fig. 2, oTS). Fig. 1. Comparison of circadian and clock … [↵][1]1To whom correspondence should be addressed. Email: rbraun{at}northwestern.edu. [1]: #xref-corresp-1-1

Cancer development is driven by series of events involving mutations, which may become fixed in a tumor via genetic drift and selection. This process usually includes a limited number of driver (advantageous) mutations and a greater number of passenger (neutral or mildly deleterious) mutations. We focus on a real-world leukemia model evolving on the background of a germline mutation. Severe congenital neutropenia (SCN) evolves to secondary myelodysplastic syndrome (sMDS) and/or secondary acute myeloid leukemia (sAML) in 30–40%. The majority of SCN cases are due to a germline ELANE mutation. Acquired mutations in CSF3R occur in >70% sMDS/sAML associated with SCN. Hypotheses underlying our model are: an ELANE mutation causes SCN; CSF3R mutations occur spontaneously at a low rate; in fetal life, hematopoietic stem and progenitor cells expands quickly, resulting in a high probability of several tens to several hundreds of cells with CSF3R truncation mutations; therapeutic granulocyte colony-stimulating factor (G-CSF) administration early in life exerts a strong selective pressure, providing mutants with a growth advantage. Applying population genetics theory, we propose a novel two-phase model of disease development from SCN to sMDS. In Phase 1, hematopoietic tissues expand and produce tens to hundreds of stem cells with the CSF3R truncation mutation. Phase 2 occurs postnatally through adult stages with bone marrow production of granulocyte precursors and positive selection of mutants due to chronic G-CSF therapy to reverse the severe neutropenia. We predict the existence of the pool of cells with the mutated truncated receptor before G-CSF treatment begins. The model does not require increase in mutation rate under G-CSF treatment and agrees with age distribution of sMDS onset and clinical sequencing data., Author summary Cancer develops by multistep acquisition of mutations in a progenitor cell and its daughter cells. Severe congenital neutropenia (SCN) manifests itself through an inability to produce enough granulocytes to prevent infections. SCN commonly results from a germline ELANE mutation. Large doses of the blood growth factor granulocyte colony-stimulating factor (G-CSF) rescue granulocyte production. However, SCN frequently transforms to a myeloid malignancy, commonly associated with a somatic mutation in CSF3R, the gene encoding the G-CSF Receptor. We built a mathematical model of evolution for CSF3R mutation starting with bone marrow expansion at the fetal development stage and continuing with postnatal competition between normal and malignant bone marrow cells. We employ tools of probability theory such as multitype branching processes and Moran models modified to account for expansion of hematopoiesis during human development. With realistic coefficients, we obtain agreement with the age range at which malignancy arises in patients. In addition, our model predicts the existence of a pool of cells with mutated CSF3R before G-CSF treatment begins. Our findings may be clinically applied to intervene more effectively and selectively in SCN patients.

In traumatic brain injury (TBI) the initial injury phase is followed by a secondary phase that contributes to neurodegeneration. Yet the mechanisms leading to neuropathology in vivo remain to be elucidated. To address this question, we developed a Drosophila head-specific model for TBI, which we term Drosophila Closed Head Injury (dCHI), where well-controlled, non-penetrating strikes are directly delivered to the head of unanesthetized flies. This assay recapitulates many TBI phenotypes, including increased mortality, impaired motor control, fragmented sleep, and increased neuronal cell death. To discover novel mediators of TBI, we used glial targeted translating ribosome affinity purification in combination with RNA sequencing. We detected significant changes in the transcriptome at various times after TBI including in genes involved in innate immunity within 24 hours after TBI. To test the in vivo functional role of these changes, we examined TBI-dependent behavior and lethality in mutants of the master immune regulator NF-κB and found that while lethality effects were still evident, changes in sleep and motor function were substantially reduced. These studies validate a new head-specific model for TBI in Drosophila and identify glial immune pathways as candidate in vivo mediators of TBI effects.Traumatic brain injury (TBI) is one of the leading causes of death and disability in the developed world [1-3]. Yet the underlying mechanisms that lead to long term physical, emotional, and cognitive impairment remain unclear.Unlike in most forms of trauma, a large percentage of people killed by traumatic brain injuries do not die immediately but rather days or weeks after the insult [4]. TBI consists of a primary and a secondary phase. The primary brain injury is the result of an external mechanical force, resulting in damaged blood vessels, axonal shearing [5], cell death, disruption of the blood– brain barrier, edema, and the release of damage associated molecular patterns (DAMPs) and excitotoxic agents [6]. In response, local glia and infiltrating immune cells upregulate cytokines (tumor necrosis factor α) and interleukins (IL-6 and IL-1β) that drive post-traumatic neuroinflammation [7-10]. This secondary injury develops over a much longer time course, ranging from hours to months after the initial injury and is the result of a complex cascade of metabolic, cellular and molecular processes [11-13]. Neuroinflammation is beneficial when it is promoting clearance of debris and regeneration [14] but can become harmful, mediating neuronal death, progressive neurodegeneration, and neurodegenerative disorders [15-18]. The mechanisms underlying these opposing outcomes are largely unknown, but are thought to depend of the location and timing of the neuroinflammatory response [19, 20]. It remains to be determined what the relative roles of TBI-induced neuroinflammation and other TBI-induced changes are in mediating short and long-term impairments in brain function in vivo.To study the mechanisms that mediate TBI pathology in vivo over time, we employ the fruit fly Drosophila melanogaster, a model organism well suited to understanding the in vivo genetics of brain injury. Despite considerable morphological differences between flies and mammals, the fly brain operates on similar principles through a highly conserved repertoire of neuronal signaling proteins, including a large number of neuronal cell adhesion receptors, synapse-organizing proteins, ion channels and neurotransmitter receptors, and synaptic vesicle-trafficking proteins [21]. This homology makes Drosophila a fruitful model to study neurodegenerative disorders [22], including ALS [23], Alzheimer’s disease [24], Huntington’s disease [25] and Parkinson’s disease [26].Trauma-induced changes in glial gene expression are a highly conserved feature of both mammalian [27, 28] and Drosophila glia [29-32] (reviewed in [33]). In Drosophila, glia are able to perform immune-related functions [32, 34]. Ensheathing glia can act as phagocytes and contribute to the clearance of degenerating axons from the fly brain [29, 31, 35]. The Drosophila innate immune system is highly conserved with that of mammals and consists primarily of the Toll, Immunodeficiency (Imd) and Janus Kinase protein and the Signal Transducer and Activator of Transcription (JAK-STAT) pathways, which together combat fungal and bacterial infections [36, 37]. Dysregulation of cerebral innate immune signaling in Drosophila glial cells can lead to neuronal dysfunction and degeneration [38, 39], suggesting that changes in glia cells could underlie secondary injury mechanisms in our Drosophila model of TBI.Existing Drosophila TBI models [40, 41] deliver impacts to the entire body, not just the head, and thus, one cannot definitively attribute ensuing phenotypes to TBI. To remove the confound of bodily injury, we have developed a novel, head-specific Drosophila model for TBI, Drosophila Closed Head Injury (dCHI). Here we show that by delivering precisely controlled, non-penetrating strikes to an unanesthetized fly’s head, we can induce cell death and increased mortality in a dose-dependent manner. In addition, TBI results in impaired motor control and decreased, fragmented sleep. Impaired motor control persists for many days after TBI while the sleep phenotype disappears after three days. These TBI-induced behavioral phenotypes do not occur in mutants lacking the master immune regulator NF-κB Relish (Rel), even though TBI-induced mortality is greatly induced in these mutants. In wild type flies, TBI results in changes in glial gene expression, where many immune related genes are upregulated 24 hours after injury. Together, these results establish a platform where powerful Drosophila genetics can be utilized to study the complex cascade of secondary injury mechanisms that occur after TBI in order to genetically disentangle its beneficial and detrimental effects.

Oligonucleotide microarrays belong to the basic tools of molecular biology and allow for simultaneous assessment of the expression level of thousands of genes. Analysis of microarray data is however very complex, requiring sophisticated methods to control for various factors that are inherent to the procedures used. In this article we describe the individual steps of a microarray experiment, highlighting important elements and factors that may affect the processes involved and that influence the interpretation of the results. Additionally, we describe methods that can be used to estimate the influence of these factors, and to control the way in which they affect the expression estimates. A comprehensive understanding of the experimental protocol used in a microarray experiment aids the interpretation of the obtained results. By describing known factors which affect expression estimates this article provides guidelines for appropriate quality control and pre-processing of the data, additionally applicable to other transcriptome analysis methods that utilize similar sample handling protocols. Reviewers: This article was reviewed by Dr. Janet Siefert, Dr. Leonid Hanin, and Dr. I King Jordan.

Background The NF-κB and IRF transcription factor families are major players in inflammation and antiviral response and act as two major effectors of the innate immune response (IIR). The regulatory mechanisms of activation of these two pathways and their interactions during the IIR are only partially known. Results Our in silico findings report that there is cross-regulation between both pathways at the level of gene transcription regulation, mediated by the presence of binding sites for both factors in promoters of genes essential for these pathways. These findings agree with recent experimental data reporting crosstalk between pathways activated by RIG-I and TLR3 receptors in response to pathogens. Conclusions We present an extended crosstalk diagram of the IRF - NF-κB pathways. We conclude that members of the NF-κB family may directly impact regulation of IRF family, while IRF members impact regulation of NF-κB family rather indirectly, via other transcription factors such as AP-1 and SP1. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1511-7) contains supplementary material, which is available to authorized users.

We present an integrated dynamical cross-talk model of the epithelial innate immune reponse (IIR) incorporating RIG-I and TLR3 as the two major pattern recognition receptors (PRR) converging on the RelA and IRF3 transcriptional effectors. bioPN simulations reproduce biologically relevant gene-and protein abundance measurements in response to time course, gene silencing and dose-response perturbations both at the population and single cell level. Our computational predictions suggest that RelA and IRF3 are under auto- and cross-regulation. We predict, and confirm experimentally, that RIG-I mRNA expression is controlled by IRF7. We also predict the existence of a TLR3-dependent, IRF3-independent transcription factor (or factors) that control(s) expression of MAVS, IRF3 and members of the IKK family. Our model confirms the observed dsRNA dose-dependence of oscillatory patterns in single cells, with periods of 1–3 hr. Model fitting to time series, matched by knockdown data suggests that the NF-κB module operates in a different regime (with different coefficient values) than in the TNFα-stimulation experiments. In future studies, this model will serve as a foundation for identification of virus-encoded IIR antagonists and examination of stochastic effects of viral replication. Our model generates simulated time series, which reproduce the noisy oscillatory patterns of activity (with 1–3 hour period) observed in individual cells. Our work supports the hypothesis that the IIR is a phenomenon that emerged by evolution despite highly variable responses at an individual cell level.

Background The NF-κB family plays a prominent role in the innate immune response, cell cycle activation or cell apoptosis. Upon stimulation by pathogen-associated patterns, such as viral RNA a kinase cascade is activated, which strips the NF-κB of its inhibitor IκBα molecule and allows it to translocate into the nucleus. Once in the nucleus, it activates transcription of approximately 90 genes whose kinetics of expression differ relative to when NF-κB translocates into the nucleus, referred to as Early, Middle and Late genes. It is not obvious what mechanism is responsible for segregation of the genes’ timing of transcriptional response. Results It is likely that the differences in timing are due, in part, to the number and type of transcription factor binding sites (TFBS), required for NF-κB itself as well as for the putative cofactors, in the Early versus Late genes. We therefore applied an evolutionary analysis of conserved TFBS. We also examined whether transcription dynamic was related to the presence of AU-rich elements (ARE) located in 3′UTR of the mRNA because recent studies have shown that the presence of AREs is associated with rapid gene induction. We found that Early genes were significantly enriched in NF-κB binding sites occurring in evolutionarily conserved domains compared to genes in the Late group. We also found that Early genes had significantly greater number of ARE sequences in the 3′UTR of the gene. The similarities observed among the Early genes were seen in comparison with distant species, while the Late genes promoter regions were much more diversified. Based on the promoter structure and ARE content, Middle genes can be divided into two subgroups which show similarities to Early and Late genes respectively. Conclusions Our data suggests that the rapid response of the NF-κB dependent Early genes may be due to both increased gene transcription due to NF-κB loading as well as the contribution of mRNA instability to the transcript profiles. Wider phylogenetic analysis of NF-κB dependent genes provides insight into the degree of cross-species similarity found in the Early genes, opposed to many differences in promoter structure that can be found among the Late genes. These data suggest that activation and expression of the Late genes is much more species-specific than of the Early genes.