Transcription factors Fos/Jun and ATF/CREB form cross-family heterodimers, altering their DNA binding specificity. These proteins, which bind to DNA as dimers via the leucine-zipper motif, were previously thought to preferentially bind to distinct DNA sequences (AP-1/TRE for Fos/Jun and ATF/CRE for ATF/CREB). However, this study shows that members of these two families can form selective heterodimers, resulting in distinct DNA binding specificities. This suggests that the Fos/Jun and ATF/CREB families are not as distinct as previously thought and may be grouped into a larger transcription factor superfamily. The study investigated the DNA binding activities of cross-family heterodimers using gel-shift assays with three regulatory elements: ATF/CRE, AP-1, and Enk-2. The results showed that heterodimers formed by ATF/CREB and Fos/Jun proteins had different DNA binding specificities compared to their homodimers. For example, the ATF-2/Jun heterodimer bound more strongly to the CRE site than to the AP-1 or Enk-2 sites, while the ATF-3/Jun heterodimer showed similar binding affinities to all three sites. The ATF-4/Jun heterodimer bound weakly to the CRE site but not to the AP-1 or Enk-2 sites. The ATF-4/Fos heterodimer, however, showed increased binding to the CRE site compared to its homodimers. The study also found that the dimerization specificity of these proteins may be influenced by their leucine-zipper sequences. For example, ATF-4, which behaves similarly to Jun in terms of dimerization, is very similar to Jun in its 4,3 repeat region. In contrast, ATF-3 has features of both the Fos and Jun families. The inability of ATF-3 to form stable dimers with Fos-related proteins may be due to the presence of lysine residues between the fourth and fifth leucines. The findings suggest that protein dimerization plays an important role in transcriptional regulation. The ability of these transcription factors to form heterodimers with different DNA binding specificities provides a mechanism for the involvement of similar regulatory elements in diverse physiological responses that require stimulus-transcription coupling. This study highlights the importance of dimerization in the function of transcription factors and suggests that the distinction between the Fos/Jun and ATF/CREB families may not be as clear as previously thought.Transcription factors Fos/Jun and ATF/CREB form cross-family heterodimers, altering their DNA binding specificity. These proteins, which bind to DNA as dimers via the leucine-zipper motif, were previously thought to preferentially bind to distinct DNA sequences (AP-1/TRE for Fos/Jun and ATF/CRE for ATF/CREB). However, this study shows that members of these two families can form selective heterodimers, resulting in distinct DNA binding specificities. This suggests that the Fos/Jun and ATF/CREB families are not as distinct as previously thought and may be grouped into a larger transcription factor superfamily. The study investigated the DNA binding activities of cross-family heterodimers using gel-shift assays with three regulatory elements: ATF/CRE, AP-1, and Enk-2. The results showed that heterodimers formed by ATF/CREB and Fos/Jun proteins had different DNA binding specificities compared to their homodimers. For example, the ATF-2/Jun heterodimer bound more strongly to the CRE site than to the AP-1 or Enk-2 sites, while the ATF-3/Jun heterodimer showed similar binding affinities to all three sites. The ATF-4/Jun heterodimer bound weakly to the CRE site but not to the AP-1 or Enk-2 sites. The ATF-4/Fos heterodimer, however, showed increased binding to the CRE site compared to its homodimers. The study also found that the dimerization specificity of these proteins may be influenced by their leucine-zipper sequences. For example, ATF-4, which behaves similarly to Jun in terms of dimerization, is very similar to Jun in its 4,3 repeat region. In contrast, ATF-3 has features of both the Fos and Jun families. The inability of ATF-3 to form stable dimers with Fos-related proteins may be due to the presence of lysine residues between the fourth and fifth leucines. The findings suggest that protein dimerization plays an important role in transcriptional regulation. The ability of these transcription factors to form heterodimers with different DNA binding specificities provides a mechanism for the involvement of similar regulatory elements in diverse physiological responses that require stimulus-transcription coupling. This study highlights the importance of dimerization in the function of transcription factors and suggests that the distinction between the Fos/Jun and ATF/CREB families may not be as clear as previously thought.