Anemia-Activated Cis Regulatory Requirements for Erythroid Regeneration

Acute anemia elicits broad transcriptional changes in erythroid progenitors and precursors. Whether anemia-activated transcription and underlying usage of cis-regulatory elements to control gene expression are unique to anemia or overlap with physiological erythropoiesis is an unresolved question regarding anemia response mechanisms. We previously discovered that a cis-regulatory transcriptional enhancer at the Samd14 locus (S14E), defined by a CANNTG-spacer-AGATAA (named E-box-GATA) composite motif and occupied by GATA1 and TAL1 transcription factors, is required for survival in severe anemia. Our working model proposes that the S14E is part of an ensemble of similar anemia-responsive cis-elements promoting erythroid precursor activities. To determine whether loci containing a cohort of S14E-like cis-elements are transcriptionally upregulated in anemia, we conducted single cell RNA-sequencing of Kit+ cells isolated from mouse spleen during the recovery phase of acute anemia. Single-cell regulatory network inference and clustering (SCENIC) analysis - used to predict transcription factors involved in regulatory mechanisms - revealed that anemia-activated transcripts co-expressed with Gata1 and Tal1 were localized in specific populations of erythroid cells. We identified 65 anemia-activated genes associated with GATA1/TAL1-occupied S14E-like cis-elements including Samd14, suggesting common transcriptional mechanisms are involved in restoring homeostasis. Using Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), we found the chromatin accessibility of S14E-like cis-elements associated with synovial sarcoma X-2 interacting protein (Ssx2ip) and tubulin tyrosine ligase-like12 (Ttll12) was significantly increased in anemia. To test S14E-like function in transcriptional activation, we targeted these sequences using sgRNA-guided Cas9 and dCas9-KRAB in mouse and human erythroid cell lines (G1E, K562 and HUDEP-2). Both SSX2IP and TTLL12 S14E-like enhancers were required for maximal gene transcription. These data demonstrate that a cohort of S14E-like cis-elements promote anemia-activated gene expression. Ssx2ip is a poorly studied gene that is upregulated by anemia in erythroid precursors (19.2-fold, p<0.0001). To connect cis-regulatory activation in anemia with erythroid regeneration, we next tested Ssx2ip function in erythroid precursor cells isolated from a mouse model of acute anemia. shRNA mediated knockdown of Ssx2ip decreased the percentage of early stage (CD71lowTer119-) erythroid precursors 4.4-fold (p=0.001). Ssx2ip knockdown decreased burst-forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E) colony forming potential of erythroid precursors by 1.3-fold (p=0.008) and 1.6-fold (p<0.0001), respectively. To investigate a possible role for Ssx2ip in cell proliferation, we conducted BrdU incorporation and cell proliferation assays. These experiments revealed that Ssx2ip is an important determinant of erythroid progenitor cell cycle progression, and proliferation in anemia. Together, we provide rigorous evidence that S14E-like cis-elements associate with anemia-activated genes, activate gene expression, and that the anemia-activated Ssx2ip gene plays important roles in erythroid precursor cells. Our results highlight a common mechanism of gene activation that regulates erythroid regeneration via S14E-like enhancers. Ongoing efforts will establish mechanisms of Ssx2ip activities in erythropoiesis and functional requirements of additional cohorts of S14E-like enhancers involved in gene regulation during anemia recovery. These findings provide a framework to understand anemia-specific transcriptional mechanisms, ineffective erythropoiesis, and anemia recovery. Given the role of Ssx2ip in erythroid regeneration, it is worth considering the pathological implications of this mechanism in acute/chronic anemia and leukemia.