Short expansions in poly-A tracts are associated with a growing number of primarily developmental human disorders, including many involving the central nervous system (5
). Protein folding anomalies resulting in protein aggregation and loss of DNA binding have been proposed to explain how these short expansions in a poly-A tract result in developmental disorders (5
). In this study, we have identified a novel mechanism in which a poly-A tract expansion disrupts the function of a transcription factor by reducing its ability to recruit a cofactor required for normal transcriptional repression at a subset of target sites.
The epilepsy and intellectual disability phenotypes observed in patients with an expansion of the first poly-A tract in ARX
are consistent with this mutation selectively affecting interneurons (19
). The lack of a structural defect (lissencephaly) in patients with this type of mutation also suggests that radial migration is preserved. Two Arx
poly-A tract expansion mouse models recently generated exhibit a partial interneuron phenotype (8
). Both models survive postnatally and exhibit epilepsy, anxiety, learning and memory deficits. One of these models showed a mild loss of parvalbumin-containing interneurons in the cortex at 1 month of age; in contrast, interneurons were greatly reduced in the septum and striatum (8
). In the second model, learning and anxiety abnormalities were observed along with EEG findings of seizures. In this model, reductions in Arx- and calbindin-expressing cortical interneurons along with a reduction in striatal interneurons were identified (9
). These data, along with our data showing a selective interneuron defect in the absence of a radial migration defect, are consistent with the neurologic defects found in patients having no structural defect.
Our data are also consistent with the observed Arx poly-A tract mutation inheritance pattern. Although the in vitro
data indicate that poly-A tract expansion results in nuclear inclusion formation (7
), this mechanism would be expected to have a dominant-negative phenotype, but such a pattern of inheritance is not observed (1
). The lack of inclusions in the mouse models further suggests that this is not the mechanism. Immunohistochemistry for Arx in the brains of Arx
mutant mice showed staining throughout the nucleus, with no evidence of nuclear or cytoplasmic inclusions (data not shown). In addition, TUNEL staining showed no difference at either E11.5 or E18.5 between the brains of Arx
mutant mice and those of wild-type mice (data not shown). However, pathology due to protein misfolding leading to protein dysfunction or to toxic levels of insoluble protein without visible inclusions cannot be ruled out. We have also precluded a DNA-binding mechanism, as has been proposed for poly-A tract expansion mutations in several other proteins (5
). Our results support the idea that poly-A expansion leads to a change in conformation that prevents the recruitment of Tle1 to certain promoter regions (Fig. ). We postulate that additional cofactors will be required in the transcriptional complex associated with ARX. As with other transcription factors, different cofactors will participate in transcriptional regulation at different sites. As proposed in our model, some cofactors will require physical interactions for stabilization of Tle1 for transcriptional repression (e.g. Ebf3
) and others will not (e.g. Lmo1
). Further studies will delineate the complete subset of Arx targets misregulated when the first poly-A is expanded, and investigate the specific functions of these targets in interneurons.
Figure 5. Model of pathological mechanism resulting from expansion of first poly-A tract in Arx. The expansion results in a context-dependent loss of Tle recruitment to promoter sites. The context at the Ebf3 and Shox2 promoters does not allow Tle recruitment with (more ...)
Of considerable interest is the finding that biological function is retained in the dorsal forebrain, but is defective in the ventral forebrain. The deficit caused by the Arx poly-A expansion mutation in the ventral forebrain is in part due to a failure to repress a subset of normal Arx targets resulting from the faulty recruitment of Tle1 to select target sites. Thus, the poly-A expansion leads to a partial loss of function. This conclusion is consistent with the ISSX-like phenotype seen in mice that have Arx
deleted in only a subset of interneurons, suggesting that in humans, this disease may not reflect a complete loss of function of ARX in interneurons (14
) (data not shown). In contrast, most Arx-/Y; Dlx5/6CIG
mice die at or before birth; one interpretation of this finding is that interneuron expression and normal function of Arx is necessary for survival. Given Arx and Dlx5/6 have little overlap in expression outside the central nervous system, this conclusion would seem warranted; however, this does not exclude other central nervous system functions for Arx in Dlx5/6 expressing cells. In addition, while these data demonstrate a context-dependent failure of Arx to function in the ventral forebrain, the rescue of dorsal forebrain function suggests that an expansion in the first poly-A tract does not affect the role of Arx in cortical VZ cells. Alternatively, Tle1 may not be required for repression in the dorsal forebrain, or Arx may be primarily activating in this region.
In contrast to polyglutamine tracts, which can tolerate a broad number of residues with expansions in the hundreds often required to produce disease, poly-A tracts never consist of >20 alanines. Furthermore, they do not undergo the dynamic expansion that leads to the significant generation-to-generation variation in repeat length encoding glutamines (6
). When a tract is expanded beyond its usual length, even if the resulting expanded tract has fewer than 20 alanines, disease results, as seen for nine diseases caused by poly-A tract expansions (5
). Our data suggest a mechanism of disease for the dramatic effect seen in disorders caused by these mutations.
In conclusion, poly-A tract expansions are a relatively newly identified class of mutation that results in human disorders, many of which affect the nervous system. Although several possible pathogenetic mechanisms for a poly-A tract expansion have been proposed, limited in vivo data exist to support these other models. Herein, we present a novel mechanism involving a partial loss of function attributable to a context-dependent ability of Arx to recruit a co-repressor to the transcriptional regulatory complex. These findings are consistent with the phenotypes observed in humans and possibly explain how other poly-A tract expansions may function, although direct testing of these other genes will be required.