Early Transcriptional Changes Research Articles

Modular Transcriptional Activity Characterizes the Initiation and Progression of Autoimmune Encephalomyelitis

Murine experimental autoimmune encephalomyelitis is a well-established model that recapitulates many clinical and physiopathological aspects of multiple sclerosis (MS). An important conceptual development in the understanding of both experimental autoimmune encephalomyelitis and MS pathogenesis has been the compartmentalization of the mechanistic process into two distinct but overlapping and connected phases, inflammatory and neurodegenerative. However, the dynamics of CNS transcriptional changes that underlie the development and regression of the phenotype are not well understood. Our report presents the first high frequency longitudinal study looking at the earliest transcriptional changes in the CNS of NOD mice immunized with myelin oligodendrocyte glycoprotein 35-55 in CFA. Microarray-based gene expression profiling and histopathological analysis were performed from spinal cord samples obtained at 13 time points around the first clinical symptom (every other day until day 11 and every day onward until day 19 postimmunization). Advanced statistics and data-mining algorithms were used to identify expression signatures that correlated with disease stage and histological profiles. Discrete phases of neuroinflammation were accompanied by distinctive expression signatures, in which altered immune to neural gene expression ratios were observed. By using high frequency gene expression analysis we captured expression profiles that were characteristic of the transition from innate to adaptive immune response in this experimental paradigm between days 11 and 12 postimmunization. Our study demonstrates the utility of large-scale transcriptional studies and advanced data mining to decipher complex biological processes such as those involved in MS and other neurodegenerative disorders.

Exogenous Bcl‐xl fusion protein spares neurons after spinal cord injury

Spinal cord injury (SCI) induces neuronal death, including apoptosis, which is completed within 24 hr at and around the impact site. We identified early proapoptotic transcriptional changes, including upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-xL, Bcl-2, and Bcl-w, using Affymetrix DNA microarrays. Because Bcl-xL is the most robustly expressed antiapoptotic Bcl-2 molecule in adult central nervous system, we decided to characterize better the effect of SCI on Bcl-xL expression. We found Bcl-xL expressed robustly throughout uninjured spinal cord in both neurons and glia cells. We also found Bcl-xL localized in different cellular compartments: cytoplasmic, mitochondrial, and nuclear. Bcl-xL protein levels decreased in the cytoplasm and mitochondria 2 hr after SCI and persisted for 24 hr. To test the contribution of proapoptotic decreases in Bcl-xL to neuronal death, we augmented endogenous Bcl-xL levels by administering Bcl-xL fusion protein (Bcl-xL FP) into injured spinal cords. Bcl-xL FP significantly increased neuronal survival, suggesting that SCI-induced changes in Bcl-xL contribute considerably to neuronal death. Because Bcl-xL FP increases survival of dorsal horn neurons and ventral horn motoneurons, it could become clinically relevant in preserving sensory and motor functions after SCI.

Knockdown of the Transcription Factor PU.1 Causes Characteristic Transcriptional Changes in Hematopoietic Stem Cells Prior to Leukemic Transformation.

Knockdown of the expression of the transcription factor PU.1 in mice results in an initial preleukemic expansion of myeloid progenitors and subsequent transformation to an immature acute myeloid leukemia (AML). Recent reports suggest that functional inactivation of PU.1 might also play a role in human AML. However, the molecular mechanisms underlying the malignant transformation are unknown. Examining the bone marrow of leukemic PU.1 knockdown mice we found an expansion of lin−, c-kit+, Sca1+ hematopoietic stem cells (HSC) as compared to wildtype animals. This finding prompted us to examine the transcriptome of HSC of PU.1 knockdown mice in the initial phase to look for early transcriptional changes underlying the malignant transformation. After lineage-depletion and FACS sorting of lin−, c-kit+, Sca1+ HSC we performed linear amplification of RNA which was then hybridized with Affymetrix Mouse Genome 430 2.0 arrays covering ~39,000 transcripts. Unsupervised hierarchical cluster analysis clearly distinguished PU.1 knockdown and wildtype HSC, demonstrating that reduced PU.1 expression levels lead to characteristic transcriptional changes in the stem cell compartment preceding the malignant transformation. 55 transcripts were upregulated, and 143 transcripts were downregulated in PU.1 knockdown HSC (>1.5-fold, p<0.05), reflecting the role of PU.1 as a transcriptional activator. We corroborated differential expression of 16 genes by quantitative real-time RT-PCR. Among the downregulated genes were c-fes, Bruton's tyrosine kinase, transcription factor EC, and GM-CSF receptor α, demonstrating that those in-vitro targets of PU.1 are also affected in stem cells in vivo. BAFF-R and TACI, two members of the tumor necrosis factor receptor superfamily, receptor tyrosine kinase TEK and toll-like receptor 4 were downregulated, whereas PDGF receptor was upregulated, suggesting disturbed signaling in PU.1 knockdown HSC. We are currently investigating the causal contribution of these deregulated genes to the leukemic phenotype. In summary, our data delineate a distinct transcriptional pattern that precedes the leukemic transformation in PU.1 knockdown HSC, and provide novel insight into PU.1-mediated molecular pathways in vivo.

Characterisation of early transcriptional changes involving multiple signalling pathways in the Mla13 barley interaction with powdery mildew ( Blumeria graminis f. sp. hordei )

Suppression subtractive hybridisation was used to isolate 21 cDNAs ( bmi1- bmi21) up-regulated 1-5 h post-inoculation (hpi) in a barley ( Hordeum vulgare L. cv. Pallas) near-isogenic line (NIL) P11 ( Mla13) challenged with either avirulent or virulent isolates of Blumeria graminis f. sp. hordei. Transcriptional changes at these time-points are crucial for the Mla-mediated hypersensitive response [W.R. Bushnell and Z. Liu (1994) Physiol Mol Plant Pathol 44:389-402]. Seven sequences were up-regulated by 1 hpi, when the pathogen has formed only the primary germ tube. Some transcripts were similar to genes with a role in regulating programmed cell death in animals, including NF kappaB and oxysterol-binding protein. Moreover, bmi7, similar to rice resistance gene Xa21, was rapidly up-regulated in both compatible and incompatible interactions, but was then down-regulated by 5 hpi in the virulent interaction. Only nine of the transcripts were up-regulated in mlo5 resistance in cv. Pallas NIL P22, confirming differential pathway induction between Mla13 and mlo5. However, eight sequences up-regulated in the Mla13 response in P11 were already highly elevated in uninoculated mlo5 mutant P22, suggesting that they may be negatively regulated by wild-type Mlo. Regulation of bmi sequences was investigated using salicylic acid, methyl jasmonate, ethylene, H(2)O(2), abscisic acid, wounding and a glucan elicitor. No single stimulus up-regulated all genes, suggesting either combinations of these stimuli, or additional stimuli, are involved in early Mla13 and mlo5 resistances. Whereas H(2)O(2) up- or down-regulated 17 of the transcripts detected in Northern analyses, salicylic acid stimulated only down-regulation of 5 transcripts.

Effects of stimulus frequency and intensity on c-fos mRNA expression in the adult rat auditory brainstem.

Induction of the cellular fos gene (c-fos) is one of the earliest transcriptional changes observed following neuronal excitation. Although not an activity marker in the strict electrophysiological sense, many neurons in the central nervous system increase their c-fos expression after periods of sustained stimulation at physiological levels of intensity. In the present study, induction of c-fos mRNA expression was examined in the auditory brainstem after 1 hour of continuous free-field acoustic stimulation. Sprague-Dawley rats were exposed to pure tones of 2, 8, 16, or 32 kHz or half-octave noise bands centered on 2, 8, or 32 kHz at 80-120 dB SPL. Stimulation-induced c-fos mRNA expression was evident at all levels of the auditory brainstem, and this expression was intensity dependent. In some brain areas, induced expression manifested a clear tonotopic organization, i.e., in dorsal, posteroventral, and anteroventral cochlear nuclei, and in the medial nucleus of the trapezoid body. The inferior colliculus exhibited multiple tonotopic representations. The dorsal nucleus of the lateral lemniscus had a crude tonotopy. Although expression was present, tonotopy was not evident in periolivary nuclei or in the ventral or intermediate nuclei of the lateral lemniscus. Free-field diotic stimulation did not induce c-fos mRNA expression in the medial or lateral superior olivary nuclei. Expression was induced in the lateral superior olive by dichotic stimulation (after a unilateral cochlear ablation), and that expression was tonotopically organized. The results suggest that stimulation-induced c-fos mRNA expression can be an effective way of mapping neuronal activity in the central auditory system under both normal and pathological conditions.

Induction of nerve growth factor-induced gene-B (NGFI-B) as an early event in the cyclic adenosine monophosphate response of dog thyrocytes in primary culture.

We investigated the induction of nerve growth factor-induced gene-B (NGFI-B) in dog thyrocytes in primary culture stimulated by different agents. The dog NGFI-B complementary DNA (cDNA) was cloned from a cDNA library of dog thyrocytes and used to study, by Northern blotting, the level of NGFI-B messenger RNA (mRNA) in those cells. We have shown that TSH and forskolin, which both induce proliferation and differentiation of the thyroid cells by activation of the protein kinase A pathway, lead to a strong and transient expression of two NGFI-B mRNA species, which differ in the length of the poly(A) tail. In contrast, 12-O-tetradecanoyl-13-phorbol-acetate (TPA) and epidermal growth factor, which induce proliferation and dedifferentiation of those cells by activation of the protein kinase C and the protein tyrosine kinase cascade, respectively, lead to a weaker expression of NGFI-B mRNA. In parallel, we studied the transactivation capacity of NGFI-B in the same cell system by transient transfection of a chloramphenicol acetyl transferase reporter construction containing a NGFI-B-dependent synthetic promoter. The highest transactivation was observed after forskolin stimulation, whereas transactivation after TPA stimulation was weak and no significant transactivation was observed after epidermal growth factor stimulation. Taken together, these results show that NGFI-B is an immediate early gene product that is mainly induced by the cAMP-dependent pathway in dog thyrocytes. Moreover they suggest that NGFI-B expression could be one of the early transcriptional changes induced specifically by this cascade and leading to differentiation and/or proliferation of these cells.