The cell adhesion molecule L1 plays a crucial role in mammalian nervous system development, but regulation of its expression at the transcriptional level is only partly understood. Here, using EMSA and antibody supershift experiments, we showed that the site-specific transcription factor NFI-A specifically interacts with a full NFI recognition site in the first intron of the murine L1
gene. The interaction is very strong, as shown by competition analysis. This high affinity is in accordance with a previous in vitro
study, in which the NFI consensus binding motif was determined by PCR-mediated random site selection [20
]. We also observed an electrophoretic mobility shift using extracts from cells not transfected with NFI-A expression plasmids, probably caused by expression of endogenous NFI proteins. The respective bands were much weaker when NFI-A was overexpressed, either due to a limiting input or due to a down-regulation of endogenous NFI-A by forced NFI-A expression.
ChIP analysis confirmed that the brain-specific isoform of NFI-A (NFI-A bs) binds to the regulatory region of the mouse L1
gene in vivo
as well. By contrast, we did not observe reproducible binding of the ubiquitous NFI-A isoform (NFI-A st) to this genomic region in vivo
, although the in vitro
interaction of NFI-A st with the full binding site in L1
was at least as strong as the one of NFI-A bs. This difference might be caused by the different binding reaction conditions in the two assays. Alternatively, post-translational modifications could be a reason for the different behavior of NFI-A st and NFI-A bs in the two assays, in particular phosphorylation, which has been implied in regulating NFI activity by several studies [21
]. Whereas for EMSA, extracts from CHO cells were used, ChIP was performed using N2A cells. It is thus tempting to speculate that phosphorylation of crucial amino acid residues differs between NFI-A bs and NFI-A st in N2A cells, causing a different affinity to the L1
gene regulatory region. Differences in protein phosphorylation or other modifications might also explain why the apparent molecular weights of NFI-A bs and NFI-A st differed slightly more than one would expect from their amino acid composition. Finally, it should be noted that eight half binding sites for NFI proteins can be found in the 2400 bp immediately upstream of the full site. NFI-A binds to such pentanucleotide sequences with a reduced affinity [20
In order to understand how binding of NFI-A to the L1 regulatory region influences L1 expression, we performed reporter gene assays in mouse neuroblastoma (N2A) cells. NFI-A bs caused a reduction in L1 gene activity to approx. 30% of control level. In this context, it is noteworthy that only the brain-specific isoform significantly interacted with the endogenous L1 gene regulatory region. Moreover, we could nearly abolish repression of L1 transcription by NFI-A bs by deleting NFI-A's transregulatory domain. To our knowledge, these results are the first experimental evidence for a specific role of NFI-A bs in the regulation of a neuronal gene.
How could NFI-A activity at the L1
gene regulatory region be regulated in a physiological context? Several studies suggest that NFI activity is modulated by NFI phosphorylation [21
]. In our case, NFI-A could be inactivated by phosphorylation, leading to enhanced L1 expression. However, there is no direct evidence that phosphorylation affects NFI activity.
Interactions with other site-specific transcription factors might also regulate transactivation/transrepression by NFI-A. NFI proteins physically interact with TTF-1 [24
], Oct-1 [25
], ski [26
], and CBP [27
]. Binding of NFI-A to such factors could alter its inhibitory influence on L1 transcription initiation. Remarkably, the HPD element, which is responsible for stimulation of L1 expression by Pax-6, is located in the same part of the L1
gene as the full NFI binding site identified in our study [6
]. In addition, binding of the homeodomain proteins Barx-2 [6
] and Hoxa-1 [5
] to the L1
gene is also mediated by the HPD element. This might implicate a functional interaction between NFI transcription factors and Pax-6, Barx-2, or Hoxa-1 in regulating L1 expression.