Regulation of Ldb1-dependent erythroid genes by Sp1 and Sp3

Authors

• A Parashar
• VD Myneni
• E Mezey

Abstract

Gene expression during erythropoiesis is closely regulated by transcription factors (TFs), often through protein complexes that bind to DNA. One example is the LIM domain binding 1 (Ldb1) complex that includes DNA binding proteins Gata1 and Tal1, bridge protein Lmo2, and chromatin looping factor Ldb1. The Ldb1 complex binds to erythroid enhancers and a subset of erythroid gene promoters, mediating enhancer-promoter proximity to activate transcription via Ldb1 homodimerization. However, many erythroid Ldb1-dependent gene promoters are not occupied by Ldb1, suggesting that other TFs may orchestrate their interaction with Ldb1 enhancers. Specificity proteins 1 and 3 (Sp1/3) are ubiquitous TFs that regulate erythroid gene promoters and interact with other proteins involved in erythroid differentiation, including Gata1. We deleted Sp1/3 using CRISPR-Cas9 genome editing in MEL cells. RNA-seq demonstrated that Sp1/3 KO dysregulated genes (DEGs) were more abundant in uninduced than induced MEL cells. ChIP-seq revealed candidate genes whose promoters exhibited Sp1/3 occupancy and were Sp1/3 KO DEGs. We integrated these results with Ldb1 enhancer-dependent genes, confirming cooperation between Ldb1 and Sp1/3 in long-distance gene regulation. We determined that full-length Ldb1 interacts with Sp1/3 in MEL cells, which contain Gata1, and HEK293 cells, which do not, suggesting that Ldb1 and Sp1/3 interact directly. We also determined that aa200-285 of Ldb1, including OID and LCCD domains, interact with Sp1/3. We will assess the mechanism of Sp1/3 in regulating Ldb1 enhancer-dependent genes by analyzing which Sp1/3 domains interact with Ldb1. This investigation provides deeper insight into transcriptomic changes during erythroid differentiation dependent on Ldb1.

Scientific Focus Area: Developmental Biology

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