Super-enhancer mediated regulation of adult ß-globin gene expression: the role of eRNA and Integrator.

Super-enhancers (SEs) mediate high transcription levels of target genes. Previous studies have shown that SEs recruit transcription complexes and generate enhancer RNAs (eRNAs). We characterized transcription at the human and murine ß-globin locus control region (LCR) SE. We found that the human LCR is capable of recruiting transcription complexes independently from linked globin genes in transgenic mice. Furthermore, LCR hypersensitive site 2 (HS2) initiates the formation of bidirectional transcripts in transgenic mice and in the endogenous ß-globin gene locus in murine erythroleukemia (MEL) cells. HS2 3'eRNA is relatively unstable and remains in close proximity to the globin gene locus. Reducing the abundance of HS2 3'eRNA leads to a reduction in ß-globin gene transcription and compromises RNA polymerase II (Pol II) recruitment at the promoter. The Integrator complex has been shown to terminate eRNA transcription. We demonstrate that Integrator interacts downstream of LCR HS2. Inducible ablation of Integrator function in MEL or differentiating primary human CD34+ cells causes a decrease in expression of the adult ß-globin gene and accumulation of Pol II and eRNA at the LCR. The data suggest that transcription complexes are assembled at the LCR and transferred to the globin genes by mechanisms that involve Integrator mediated release of Pol II and eRNA from the LCR.

Setd1a and NURF mediate chromatin dynamics and gene regulation during erythroid lineage commitment and differentiation.

The modulation of chromatin structure is a key step in transcription regulation in mammalian cells and eventually determines lineage commitment and differentiation. USF1/2, Setd1a and NURF complexes interact to regulate chromatin architecture in erythropoiesis, but the mechanistic basis for this regulation is hitherto unknown. Here we showed that Setd1a and NURF complexes bind to promoters to control chromatin structural alterations and gene activation in a cell context dependent manner. In human primary erythroid cells USF1/2, H3K4me3 and the NURF complex were significantly co-enriched at transcription start sites of erythroid genes, and their binding was associated with promoter/enhancer accessibility that resulted from nucleosome repositioning. Mice deficient for Setd1a, an H3K4 trimethylase, in the erythroid compartment exhibited reduced Ter119/CD71 positive erythroblasts, peripheral blood RBCs and hemoglobin levels. Loss of Setd1a led to a reduction of promoter-associated H3K4 methylation, inhibition of gene transcription and blockade of erythroid differentiation. This was associated with alterations in NURF complex occupancy at erythroid gene promoters and reduced chromatin accessibility. Setd1a deficiency caused decreased associations between enhancer and promoter looped interactions as well as reduced expression of erythroid genes such as the adult ß-globin gene. These data indicate that Setd1a and NURF complexes are specifically targeted to and coordinately regulate erythroid promoter chromatin dynamics during erythroid lineage differentiation.

Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.

Zinc finger proteins are the most common among families of DNA-binding transcription factors. Designer transcription factors generated by the fusion of engineered zinc finger DNA-binding domains (ZF-DBDs) to effector domains have been valuable tools for the modulation of gene expression and for targeted genome editing. However, ZF-DBDs without effector domains have also been shown to effectively modulate gene expression by competing with sequence-specific DNA-binding transcription factors. Here, we describe the methodology and provide a detailed workflow for the cloning, expression, purification, and direct cell delivery of engineered ZF-DBDs. Using this protocol, ZF-DBDs can be generated with high efficiency in less than 2 weeks. We also describe a nonradioactive method for measuring DNA binding affinity of the purified ZF-DBD proteins as well as a method for direct delivery of the purified ZF-DBDs to mammalian cells.

Activation of Fetal γ-globin Gene Expression via Direct Protein Delivery of Synthetic Zinc-finger DNA-Binding Domains.

Reactivation of γ-globin expression has been shown to ameliorate disease phenotypes associated with mutations in the adult ß-globin gene, including sickle cell disease. Specific mutations in the promoter of the γ-globin genes are known to prevent repression of the genes in the adult and thus lead to hereditary persistence of fetal hemoglobin. One such hereditary persistence of fetal hemoglobin is associated with a sequence located 567 bp upstream of the Gγ-globin gene which assembles a GATA-containing repressor complex. We generated two synthetic zinc-finger DNA-binding domains (ZF-DBDs) targeting this sequence. The -567Gγ ZF-DBDs associated with high affinity and specificity with the target site in the γ-globin gene promoter. We delivered the -567Gγ ZF-DBDs directly to primary erythroid cells. Exposure of these cells to the recombinant -567Gγ ZF-DBDs led to increased expression of the γ-globin gene. Direct protein delivery of ZF-DBDs that compete with transcription regulatory proteins will have broad implications for modulating gene expression in analytical or therapeutic settings.

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range ß-Globin Gene Regulation.

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine ß-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult ß-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult ßmajor-globin gene promoter but did not affect expression of the ßminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult ßmajor-globin gene promoter during erythroid cell differentiation.

Recruitment of transcription complexes to enhancers and the role of enhancer transcription.

Enhancer elements regulate the tissue- and developmental-stage-specific expression of genes. Recent estimates suggest that there are more than 50,000 enhancers in mammalian cells. At least a subset of enhancers has been shown to recruit RNA polymerase II transcription complexes and to generate enhancer transcripts. Here, we provide an overview of enhancer function and discuss how transcription of enhancers or enhancer-generated transcripts could contribute to the regulation of gene expression during development and differentiation.

Methylation changes at NR3C1 in newborns associate with maternal prenatal stress exposure and newborn birth weight.

Early life experiences, including those in utero, have been linked to increased risk for adult-onset chronic disease. The underlying assumption is that there is a critical period of developmental plasticity in utero when selection of the fetal phenotype that is best adapted to the intrauterine environment occurs. The current study is the first to test the idea that extreme maternal psychosocial stressors, as observed in the Democratic Republic of Congo, may modify locus-specific epigenetic marks in the newborn resulting in altered health outcomes. Here we show a significant correlation between culturally relevant measures of maternal prenatal stress, newborn birth weight and newborn methylation in the promoter of the glucocorticoid receptor NR3C1. Increased methylation may constrain plasticity in subsequent gene expression and restrict the range of stress adaptation responses possible in affected individuals, thus increasing their risk for adult-onset diseases.