FBXO11 Activates Erythroid Gene Transcription By Degrading Heterochromatin-Associated Protein BAHD1

Mature red blood cells (RBC) contain approximately 95% cytosolic hemoglobin for the purpose of blood oxygen transport. This specialized state is achieved during erythropoiesisby regulated gene expression and protein degradation. During late-stage erythropoiesis, ubiquitin ligaseseliminate unnecessary proteins and maintain quality control by degrading unstable proteins, including unpaired hemoglobin subunits. However, ubiquitin ligasesare expressed at all stages of erythropoiesisand the functions of most are unknown. To study ubiquitin ligasesinvolved in RBC formation, we performed a Cas9/single guide (sg) RNA screen for functional ubiquitin-proteasome components in HUDEP-2 cells, an immortalized human cell linethat proliferates as immature erythroblastsand can be induced to undergo terminal maturation. We identified the E3 ubiquitin ligaseFBXO11 as a top-ranked candidate. FBXO11 is a member of the F-box proteinfamily that assembles into a SKP1- CUL1-F-box (SCF) E3 ubiquitin ligasecomplex. Depletion of FBXO11 by 2 different sgRNAs in HUDEP-2 cells inhibited erythroid maturation, as evidenced by reduced hemoglobinization, failure to induce the maturation marker Band3 and persistence of immature cell morphology. In primary human CD34+ cells, suppression of FBXO11 expression by Cas9+ two independent sgRNAs inhibited erythroid maturation, as evidenced by reduced Band3 expression (5.3% vs. 15.6% for non-targeting sgRNA, P We sought to establish how FBXO11 modulates erythropoiesisand erythroid gene expression by identifying the relevant ubiquitinationsubstrate(s). Combined quantitative proteomeanalysis with RNA-seq of FBXO11-depleted HUDEP-2 cells identified several proteins that are upregulated with no change in their corresponding mRNA. We tested whether reduction of these candidate substrates could alleviate the erythroid maturation block conferred by FBXO11 depletion. In FBXO11 gene-disrupted HUDEP-2 cells, suppression of the heterochromatin-associated protein BAHD1 partially rescued hemoglobinization and Band3 expression (4.2% for Cas9+ non-targeting sgRNA vs. 21.7% for Cas9+ BAHD1 sgRNAs, P BAHD1, named after its bromo-adjacent homology domain that interacts with H3K27me3, is part of a transcriptional repressor complex. We showed that BAHD1 and FBXO11 co-immunoprecipitated in cells and that BAHD1 amino (N)-terminal segments of 188 or 240 amino acids were robustly modified with ubiquitinby SCFFBXO11 complex. Chromatin immunoprecipitation-sequencing analysis of BAHD1-V5-expressing WT HUDEP-2 cells showed strong enrichment for BAHD1 occupancy on erythroid gene promoters that were downregulated by FBXO11-deficiency (P Overall, our findings identify FBXO11 as a ubiquitin ligasethat utilizes a novel mechanism to activate erythroid genes during RBC formation. This newly identified pathway may contribute to known activities of FBXO11 as a tumor suppressor and developmental regulator in non-erythroid tissues. Disclosures No relevant conflicts of interest to declare.