Transgenic analysis of GFAP promoter elements

Abstract

Transcriptional regulation of the glial fibrillary acidic protein gene (GFAP) is of interest because of its astrocyte specificity and its upregulation in response to CNS injuries. We have used a transgenic approach instead of cell transfection to identify promoter elements of the human GFAP gene, since previous observations show that GFAP transcription is regulated differently in transfected cultured cells from in the mouse. We previously showed that block mutation of enhancer regions spanning from bp −1488 to −1434 (the C1.1 segment) and −1443 to −1399 (C1.2) resulted in altered patterns of expression and loss of astrocyte specificity, respectively. This analysis has now been extended upstream to bp −1612 to −1489 (the B region), which previously has been shown especially important for expression. Block mutation of each of four contiguous sequences, which together span the B region, each decreased the level of transgene activity by at least 50%, indicating that multiple sites contribute to the transcriptional activity in a cooperative manner. Several of the block mutations also altered the brain region pattern of expression, astrocyte specificity and/or the developmental time course. Transgenes were then analyzed in which mutations were limited to specific transcription factor binding sites in each of the 4 B block segments as well as in C1.1 and C1.2. Whereas mutation of the conserved consensus AP-1 site unexpectedly had little effect on transgene expression; NFI, SP1, STAT3, and NF-κB were identified as having important roles in regulating the strength of GFAP promoter activity and/or its astrocyte specificity.

Strategies for immunohistochemical protein localization using antibodies: What did we learn from neurotransmitter transporters in glial cells and neurons

“…There are, however, issues with reporter mice also as the transcriptional activity can be influenced by multiple factors. Therefore the expression patterns can be altered depending on how promotor elements are affected (Yeo et al, 2013). Nevertheless, the distribution of fluorescence in reporter lines for the EAAT1, EAAT2 and EAAT4 glutamate transporters Gincel et al, 2007;de Vivo et al, 2010a;de Vivo et al, 2010b) is in good agreement with previous immunocytochemistry Dehnes et al, 1998;Zhou and Danbolt, 2014).…”

Complement Peptide C3a Promotes Astrocyte Survival in Response to Ischemic Stress

“…These results open for the possibility that astrocytes can employ different mechanisms with distinct ontogenetic specificity and dynamics for coping with ischemic stress, and C3a increases astrocyte resilience independently of the engagement of the cytoplasmic intermediate filament system. Whereas it has been recently shown that neither AP-1 nor NF-κB directly regulate GFAP gene expression in astrocytes [62], the role of HIF-1 in the regulation of GFAP in these cells is not known. Notably, mice lacking both GFAP and vimentin showed reduced ERK phosphorylation in the retinas after induced retinal detachment [63], and C3a reduced stromal derived factorinduced ERK phosporylation in neural progenitor cells [16].…”

Neurochemical regulation of the expression and function of glial fibrillary acidic protein in astrocytes

“…In regulating the strength of GFAP gene promoter activity, NF-κB has also been identified as an important TF (Hlavac & VandeVord, 2019;Yeo, Bandyopadhyay, Messing, & Brenner, 2013). NF-κB and nuclear factor-1 from astrocytes bind to the −85to −63-bp promoter segment (Krohn et al, 1999) to regulate GFAP promoter activity distantly.…”