DLX transcriptional targets have been implicated during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 mutant mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data suggests that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.

DLX transcriptional targets have been implicated during retinal development. The Crx (Cone-Rod homeobox) gene is required for the differentiation and maintenance of cone and rod photoreceptors. Otx2 (Orthodenticle homeobox 2) is a key regulator of photoreceptor cell fate. The Dlx1/Dlx2 mutant mouse retina has a significant reduction of retinal ganglion cells with aberrant Crx expression in the neuroblastic layer and increased retinal Otx2 expression. We hypothesized that the Dlx homeobox genes directly repress Crx and Otx2 expression during retinal development. Expression of CRX demonstrates increased transcript and protein expression in the Dlx1/Dlx2 double knockout retina at E18.5, suggesting that these DLX transcription factors may repress CRX expression. OTX2 expression is increased in the Dlx1/Dlx2 knockout retina at E16.5 suggesting that DLX2 negatively regulates OTX2 expression. The Dlx1/Dlx2 knockout has aberrant and ectopic expression of CRX in the retina along with increased OTX2 expression. Our data suggests that both CRX and OTX2 are transcriptional targets directly repressed by the DLX1 and DLX2.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19274号 / 医博第4038号 / 新制||医||1011(附属図書館) / 32276 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 吉村 長久, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Development of the central nervous system requires the precise coordination of intrinsic genetic programs to instruct cell fate, synaptic connectivity and function. The MEF2 family of transcription factors (TFs) plays many essential roles in neural development; however, the mechanisms of gene regulation by MEF2 in neurons remain unclear. This dissertation focuses on the molecular mechanisms by which MEF2 binds to the genome, activates enhancers, and regulates gene expression within the developing nervous system. We find that one MEF2 family member in particular, MEF2D, is an essential regulator of the development and function of retinal photoreceptors, the primary sensory neurons responsible for vision. Despite being expressed broadly across many tissues, in the retina MEF2D binds to retina-specific enhancers and regulates photoreceptor-specific transcripts, including critical retinal disease genes. Functional genome-wide analyses demonstrate that MEF2D achieves tissue-specific binding and action through cooperation with a retina-specific TF, CRX. CRX recruits MEF2D away from canonical MEF2 binding sites by promoting MEF2D binding to retina-specific enhancers that lack a strong consensus MEF2 binding sequence. MEF2D and CRX then synergistically co-activate these enhancers to regulate a cohort of genes critical for normal photoreceptor development. These findings demonstrate that MEF2D, a broadly expressed TF, contributes to retina-specific gene expression in photoreceptor development by binding to and activating tissue-specific enhancers cooperatively with CRX, a tissue-specific co-factor. A major unresolved feature of MEF2D function in the retina is that the number of MEF2D binding sites significantly exceeds the number of genes that are dependent on MEF2D for expression. We investigated causes of this discrepancy in an unbiased manner by characterizing the activity of MEF2D-bound enhancers genome-wide. We find that many MEF2D-bound enhancers are inactive. Furthermore, less than half of active MEF2D-bound enhancers require MEF2D for activity, suggesting that significant redundancies exist for TF function within enhancers. These findings demonstrate that observed TF binding significantly overestimates direct TF regulation of gene expression. Taken together, our results suggest that the broadly expressed TF MEF2D achieves tissue specificity through competitive recruitment to enhancers by tissue-specific TFs and activates a small subset of enhancers to regulate genes.

AABBSSTTRRAACCTT Phosducin (Pdc) is a highly conserved acidic phosphoprotein, which plays an important role in the regulation of G-protein signalization in intact retina. It binds to Gβγ dimer of heterotrimeric G-protein transducin thereby decreases the pool of available transducin resulting in modulation of signal. Function of phosducin is negatively regulated by its phosphorylation followed by interaction with the 14-3-3 protein. Besides this established way of regulation, we were interested in other putative interaction partners of phosducin, like SUG1 and CRX. SUG1 is a subunit of 26S proteasome with a large scale of biological functions, especially a degradation of many transription factors. Its role in regulation of phosducin is still unclear, but is probably involved in targeting of phosducin to 26S proteasome for its degradation. Subsequently, we prepared four different expression constructs of full-length protein in order to find the best expression and purification strategy. These results suggest that all purified fusion proteins of SUG1 form stable and soluble high molecular weight oligomers. This behaviour was confirmed by dynamic light scattering and analytical ultracentrifugation measurements. In addition, this observation is consistent with previous studies of its bacterial counterpart, PAN...

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18904号 / 医科博第60号 / 新制||医科||4(附属図書館) / 31855 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 吉村 長久, 教授 髙橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Infectious diseases remain a threat to critically ill patients, particularly with the rise of antibiotic-resistant bacteria. Septic shock carries a mortality of up to ∼40% with no compelling evidence of promising therapy to reduce morbidity or mortality. Septic shock survivors are also prone to nosocomial infections. Treatment with toll-like receptor 4 (TLR4) agonists have demonstrated significant protection against common nosocomial pathogens in various clinically relevant models of infection and septic shock. TLR4 agonists are derived from a bacteria cell wall or synthesized de novo, and more recently novel small molecule TLR4 agonists have also been developed. TLR4 agonists augment innate immune functions including expansion and recruitment of innate leukocytes to the site of infection. Recent studies demonstrate TLR4-induced leukocyte metabolic reprogramming of cellular metabolism to improve antimicrobial function. Metabolic changes include sustained augmentation of macrophage glycolysis, mitochondrial function, and tricarboxylic acid cycle flux. These findings set the stage for the use of TLR4 agonists as standalone therapeutic agents or antimicrobial adjuncts in patient populations vulnerable to nosocomial infections.