A modular enhancer mediates SCRT2 repression of ISLET1 in the spinal cord.

How transcriptional programs coordinate the transition from neural progenitors to lineage-committed neurons in the spinal cord remains poorly understood. While much is known about transcription factors acting in the proliferative and differentiated zones, the role of intermediate zone (IZ) factors during lineage specification is less clear. Here, we investigate the function of SCRATCH2 (SCRT2), expressed in the postmitotic cells of the IZ, during dorsal interneuron differentiation. Overexpression of SCRT2 in vivo reduced the number of ISLET1+ dorsal interneurons. Chromatin profiling revealed that SCRT2 primarily binds to intergenic, transcriptionally inactive regions near neurogenic genes. Among these, we identified a conserved regulatory element, ECR4, located between ISLET1 and PARP8. Functional assays showed that ECR4 drives neural transcription and is composed of two subregions: ECR4B, an enhancer activated by ISLET1 and POU4F1, and ECR4A, which contains SCRT2 binding motifs and mediates transcriptional repression. Mutation of the vCES-box, a predicted SCRT2-binding motif within ECR4A, abolished repression, confirming a repressive regulatory interaction. Together, these data support a model in which SCRT2 represses ISLET1 through ECR4 to modulate dI3 lineage specification. These findings identify a novel regulatory mechanism linking intermediate zone transcriptional repression to dorsal interneuron development in the spinal cord.