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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.

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.

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.

The hemoglobinopathies, such as β-thalassemia and sickle cell anemia (SCA), are characterized by mutations of the β-globin gene resulting in either decreased or functionally abnormal hemoglobin (Hb) production. As bone marrow transplant is the only curative option for these patients, there is a strong need for new therapeutic approaches. Both β-thalassemia and SCA represent ideal targets for gene therapy since introduction of a normal β-globin gene can ameliorate the phenotype, as we and others have shown previously. Overcoming the developmental silencing of the fetal γ-globin gene represents an additional approach for the treatment of hemoglobinopathies. Here, we directly compare a recently established approach to activate the γ-globin gene using forced chromatin looping with pharmacologic approaches to raise γ-globin expression.

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

The β-genes undergo developmental activation and silencing in part by competing for their upstream enhancer, the locus control region (LCR). In adult erythroid cells, the LCR contacts the β-globin gene promoter by forming a loop that precludes interaction with the embryonic and fetal β-type globin genes. Reversing this developmental gene expression switch in favor of embryonic/fetal genes has therapeutic implications for patients with hemoglobinopathies. Here we employed a forced chromatin looping approach to activate the silenced murine embryonic βh1-globin gene and the human fetal γ-globin gene in adult erythroid cells. We have previously shown that forced recruitment via artificial zinc finger proteins of Ldb1, a protein necessary for long-range chromatin interactions at the β-globin locus, can trigger chromatin loop formation and transcription initiation. Here, we designed a zinc finger protein targeting the βh1 promoter, fused it to the self-association domain of Ldb1 (βh1-Ldb1), and introduced it into an adult murine erythroid cell line that normally produces nearly 100% adult β-globin. βh1-Ldb1 expression activated βh1-globin transcription up to 3000-fold accounting for ∼20% of total β-globin expression. βh1-Ldb1 similarly increased expression of βh1-globin in fetal liver derived primary erythroid cells. These results are striking given the degree to which murine embryonic globin genes are normally repressed. To test whether the activity βh1-Ldb1 was due to a looped interaction of the βh1 promoter with the LCR, we introduced βh1-Ldb1 into fetal liver derived erythroblasts from mice in which the LCR had been deleted. βh1-Ldb1 was virtually inactive in the absence of the LCR, demonstrating the dependence on the LCR and, by inference, long range looping of βh1-Ldb1 function. We next extended this approach to the human β-globin locus in an effort to activate expression of the fetal γ-globin gene in adult erythroid cells. Ldb1 was fused to a previously described γ-globin promoter binding zinc finger protein, GG1, to generate GG1-Ldb1. Introduction of GG1-Ldb1 into adult primary human erythroid cells strongly activated γ-globin expression with a concomitant reduction in β-globin transcription. Strikingly, γ-globin accounted for nearly 90% of total β-type globin transcription. Furthermore, fetal hemoglobin expression was nearly pan-cellular as determined by flow cytometry. These results demonstrate the power of forced chromatin looping to reprogram developmental regulation of gene expression, and provide a novel proof of concept for activating the γ-globin gene for the benefit of patients with hemoglobinopathies.

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

Abstract 280This icon denotes a clinically relevant abstract

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

Abstract 2598

The mammalian β-globin locus is under the coordinated control of multiple transcription factors to ensure the correct expression of the globin genes during development. The distal β-globin locus control region (LCR) physically interacts with β-like globin promoters to form developmentally dynamic chromatin loops. The hematopoietic transcription factor GATA-1 and its associated cofactor Ldb1 bind to the LCR and the β-globin promoter and are essential for loop formation and β-globin expression. However, the molecular basis of chromatin looping and its cause-effect relationship with transcriptional activation are unclear. Here, we examined whether Ldb1 is an effector of GATA-1 during loop formation. Specifically, we tested whether artificial tethering of Ldb1 to the endogenous β-globin promoter and LCR can substitute for GATA-1 function. Ldb1 was fused to artificial zinc finger proteins (ZFP) designed to bind to the LCR and β-globin promoter. Ldb1-ZFPs were introduced pairwise into murine GATA-1 null erythroid cells in which the β-globin locus is relaxed and transcriptionally silent. In vivo binding of the Ldb1-ZFPs to their targets was verified by ChIP assay. Strikingly, expression of Ldb1-ZFPs but not Ldb1 or ZFPs alone led to substantial activation of β-globin transcription in the absence of GATA-1. Moreover, chromosome conformation capture experiments showed that Ldb1-ZFPs triggered physical association between the LCR and the β-globin promoter. Recruitment of RNA polymerase II (Pol II) and its phosphorylation at serine 5 are critical LCR-dependent regulatory steps in β-globin transcription. We found that Ldb1-ZFP expression facilitated Pol II recruitment at the β-globin promoter and serine 5 phosphorylation to the same level as GATA-1-expressing erythroid cells. This is consistent with an Ldb1-ZFP-induced LCR-β-globin promoter chromatin loop. In concert, these results indicate that Ldb1 is a critical effector for GATA-1 by mediating enhancer-promoter loops. In broader terms, our results suggest that chromatin loop formation can be sufficient for gene activation in the absence of an essential transcription factor. We are currently in the process of examining whether targeting of the LCR to embryonic and fetal globin genes can be used to activate them in adult cells. Targeted chromatin loop formation may provide a method to activate fetal or adult hemoglobin expression in individuals with β-thalassemia or sickle cell anemia.No relevant conflicts of interest to declare.

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.

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.