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Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.

Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the β-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate β-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult β- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.

GATA-1 controls hematopoietic development by activating and repressing gene transcription, yet the in vivo mechanisms that specify these opposite activities are unknown. By examining the composition of GATA-1–associated protein complexes in a conditional erythroid rescue system as well as through the use of tiling arrays we detected the SCL/TAL1, LMO2, Ldb1, E2A complex at all positively acting GATA-1–bound elements examined. Similarly, the SCL complex is present at all activating GATA elements in megakaryocytes and mast cells. In striking contrast, at sites where GATA-1 functions as a repressor, the SCL complex is depleted. A DNA-binding defective form of SCL maintains association with a subset of active GATA elements indicating that GATA-1 is a key determinant for SCL recruitment. Knockdown of LMO2 selectively impairs activation but not repression by GATA-1. ETO-2, an SCL-associated protein with the potential for transcription repression, is also absent from GATA-1–repressed genes but, unlike SCL, fails to accumulate at GATA-1–activated genes. Together, these studies identify the SCL complex as a critical and consistent determinant of positive GATA-1 activity in multiple GATA-1–regulated hematopoietic cell lineages.

No relevant conflicts of interest to declare.

Gregory: Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment.

Reik: Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment.

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

No relevant conflicts of interest to declare.

Abstract 647

GATA-1 controls hematopoietic development by activating and repressing gene transcription, yet the in vivo mechanisms that specify these opposite activities are unknown. By examining the composition of GATA-1 associated protein complexes in a conditional erythroid rescue system, we detected the SCL/TAL1, LMO2, Ldb1, E2A complex in vivo at all positively acting GATA-1-bound elements examined. ChIP-on-chip tiling arrays for SCL and Ldb1 revealed that chromatin occupancy of SCL and Ldb1 are extremely tightly correlated. Importantly, they showed a strong tendency for co-occupancy with GATA-1. In deed, all regions occupied by GATA-1 that showed enhancer activity were occupied by SCL and Ldb1, independent of the proximity of E-box sequences. Similarly, the SCL complex is present at all activating GATA elements in megakaryocytes and mast cells. In striking contrast, at sites where GATA-1 functions as a repressor, the SCL complex is depleted. A DNA-binding defective form of SCL maintains association with a subset of active GATA elements indicating that GATA-1 is a key determinant for SCL recruitment. Knockdown of LMO2 selectively impairs activation but not repression by GATA-1. ETO-2, an SCL-associated protein with the potential for transcription repression is also absent from GATA-1-repressed genes but, unlike SCL, fails to accumulate at GATA-1 activated genes. Together, these studies identify the SCL complex as a critical and consistent determinant of positive GATA-1 activity in multiple GATA-1-regulated hematopoietic cell lineages.