## 2 Results
### 3222401L13 Rik Interacts with Npus3 to Control Gene Expression
(**C**) Scheme depicting the significant DNA binding domains (DBD) in the sequence of 3222401L13 Rik determined using the Triplex Domain Finder tool and the sequence motifs where 3222401L13 Rik binds to the promoter of Npas3. (**D**) RNA immunoprecipitation for Npas3, followed by qPCR for 3222401L13 Rik in mouse primary astrocytes (unpaired \(t\)-test; \({}^{\star\ast}p<0.01\)). (**E**) Representative immunofluorescence images showing the transfection of primary astrocytes with Gfp- or Gfp-Npas3-overexpression plasmids. Scale bar: 100 \(\upmu\)m. (**F**) Expression levels of 3222401L13 Rik and 322401L13 Rik and 322401L13 Rik and overexpression of Npas3 in primary astrocytes (One-way ANOVA; \({}^{\star}p<0.05\); \({}^{\star\ast}p<0.01\); \({}^{\star\ast}p<0.0001\); ns = not significant). (**G**) Expression levels of _Glt-1_, _Glast_, and _Nrxn1_ after the simultaneous KD of 3222401L13 Rik_ and overexpression of Npas3 in primary astrocytes (One-way ANOVA; \({}^{\star}p<0.05\); \({}^{\star\ast}p<0.01\); ns = not significant). (**H**) Glutamate uptake of primary astrocytes after the simultaneous KD of 3222401L13 Rik and overexpression of Npas3 (One-way ANOVA; \({}^{\star\ast}p<0.01\); \({}^{\star\ast}\ast\ast\)\(p<0.0001\); ns = not significant). (**I**) Increase in intracellular Ca\({}^{2+}\) levels in response to ATP stimulation after the simultaneous KD of 3222401L13 Rik and overexpression of Npas3 (One-way ANOVA; \({}^{\star\ast}p<0.01\); \({}^{\star\ast}\ast\ast\)\(p<0.001\); ns = not significant). Data are depicted as mean \(\pm\) standard error. NC: negative control. KD: knockdown. These data support a model in which _3222401L13 Rik_ positively regulates _Npas3_ expression, likely by promoting positive autoregulation. Loss of _3222401L13 Rik_ reduces Npas3 levels. To test whether overexpression of _Npas3_ could rescue the functional impairments observed after _3222401L13 Rik_ knockdown, we electroporated primary mouse astrocytes with plasmids containing _Npas3_ and _Gfp_ (Npas3_GFP) under the control of the astrocyte-specific Gfp promoter--to monitor successful transfection--or only Gfp as a control (Gfp_control) (Figure 6E). We then combined this with the transfection of Gapmers to knock down _3222401L13 Rik_ or control Gapmers. While _3222401L13 Rik_ knockdown reduced the expression of Npas3 in astrocytes transfected with Gfp (Figure 6F), we observed significant overexpression of Npas3 in astrocytes electroporated with the Npas3_Gfp construct (Figure 6F). Importantly, knockdown of _3222401L13 Rik_ did not affect _Npas3_ expression in this overexpression setting (Figure 6F), allowing us to test if Npas3 overexpression could rescue the phenotypes associated with loss of _3222401L13 Rik_ function. Consistent with previous data (see Figures 3F and 4A), knockdown of _3222401L13 Rik_ reduced the levels of _Glt-1_, _Glast_, and _Nrxn1_ (Figure 6G) in astrocytes electroporated with the GFP plasmid. However, overexpression of Npas3 reinstated physiological expression levels of all three genes despite the knockdown of _3222401L13 Rik_ (Figure 6G). Similar observations were made for glutamate uptake and intracellular calcium levels. Consistent with previous data (see Figure 4C), glutamate uptake was impaired upon _3222401L13 Rik_ knockdown in astrocytes transfected with Gfp (Figure 6G), while _Npas3_ overexpression could rescue this phenotype (Figure 6H). Finally, we measured intracellular Ca\({}^{2+}\) levels following ATP stimulation. Similar findings were observed for intracellular Ca\({}^{2+}\) levels upon ATP stimulation (Figure 6I). In summary, these data suggest that _3222401L13 Rik_ positively regulates and fine-tunes the expression of Npas3 and that the phenotypes observed upon 3222401L13 Rik knockdown are, at least in part, mediated by loss of Npas3 function.