Reactivation of developmentally silenced globin genes by forced chromatin looping.

Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts ß-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the ß-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total ß-globin synthesis, with a reciprocal reduction in adult ß-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

A Zinc Finger Transcription Factor Faithfully Dedicated to Only a Single Target Gene in Erythroid Cells.

Lan et al. carry out a CRISPR-mediated genetic screen and discover that ZNF410 uniquely regulates the NuRD component CHD4 to repress γ-globin transcription in erythroid cells, establishing a novel fetal hemoglobin regulatory mechanism.

Enhancers, gene regulation, and genome organization.

How transcriptional enhancers function to activate distant genes has been the subject of lively investigation for decades. "Enhancers, gene regulation, and genome organization" was the subject of a virtual meeting held November 16-17, 2020, under sponsorship of the National Cancer Institute (NCI), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) at the National Institutes of Health (NIH). The goal of the meeting was to advance an understanding of how transcriptional enhancers function within the framework of the folded genome as we understand it, emphasizing how levels of organization may influence each other and may contribute to the spatiotemporal specification of transcription. Here we focus on broad questions about enhancer function that remain unsettled and that we anticipate will be central to work in this field going forward. Perforce, we cover contributions of only some speakers and apologize to other contributors in vital areas that we could not include because of space constraints.

Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor.

Chromatin loops juxtapose distal enhancers with active promoters, but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and ß-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the ß-globin promoter in GATA1 null erythroblasts, in which the ß-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the ß-globin promoter substantially activated ß-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.

Long noncoding RNA GATA2AS influences human erythropoiesis by transcription factor and chromatin landscape modulation.

ABSTRACT: Long noncoding RNAs (lncRNAs) are extensively expressed in eukaryotic cells and have been revealed to be important for regulating cell differentiation. Many lncRNAs have been found to regulate erythroid differentiation in the mouse. However, given the low sequence conservation of lncRNAs between mouse and human, our understanding of lncRNAs in human erythroid differentiation remains incomplete. lncRNAs are often transcribed opposite to protein coding genes and regulate their expression. Here, we characterized a human erythrocyte-expressed lncRNA, GATA2AS, which is transcribed opposite to erythroid transcription regulator GATA2. GATA2AS is a 2080-bp long, primarily nucleus-localized noncoding RNA that is expressed in erythroid progenitor cells and decreases during differentiation. Knockout of GATA2AS in human HUDEP2 erythroid progenitor cells using CRISPR-Cas9 genome editing to remove the transcription start site accelerated erythroid differentiation and dysregulated erythroblast gene expression. We identified GATA2AS as a novel GATA2 and HBG activator. Chromatin isolation by RNA purification showed that GATA2AS binds to thousands of genomic sites and colocalizes at a subset of sites with erythroid transcription factors including LRF and KLF1. RNA pulldown and RNA immunoprecipitation confirmed interaction between GATA2AS and LRF and KLF1. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that knockout of GATA2AS reduces binding of these transcription factors genome wide. Assay for transposase-accessible chromatin sequencing (ATAC-seq) and H3K27ac ChIP-seq showed that GATA2AS is essential to maintain the chromatin regulatory landscape during erythroid differentiation. Knockdown of GATA2AS in human primary CD34+ cells mimicked results in HUDEP2 cells. Overall, our results implicate human-specific lncRNA GATA2AS as a regulator of erythroid differentiation by influencing erythroid transcription factor binding and the chromatin regulatory landscape.

Hemogen/BRG1 cooperativity modulates promoter and enhancer activation during erythropoiesis.

Hemogen is a hematopoietic tissue-specific gene that regulates the proliferation and differentiation of hematopoietic cells; however, the mechanism underlying its function in erythropoiesis is unknown. We found that depletion of hemogen in human CD34+ erythroid progenitor cells and HUDEP2 cells significantly reduced the expression of genes associated with heme and hemoglobin synthesis, supporting a positive role for hemogen in erythroid maturation. In human K562 cells, hemogen antagonized the occupancy of corepressors nucleosome remodeling and histone deacetylase (NuRD) complex and facilitated LDB1 complex-mediated chromatin looping. Hemogen recruited SWI/SNF complex ATPase BRG1 as a coactivator to regulate nucleosome accessibility and H3K27ac enrichment for promoter and enhancer activity. To determine whether hemogen/BRG1 cooperativity is conserved in mammalian systems, we generated hemogen-knockout/knockin mice and investigated hemogen/BRG1 function in murine erythropoiesis. Loss of hemogen in embryonic days 12.5 to 16.5 fetal liver cells impeded erythroid differentiation through reducing the production of mature erythroblasts. Chromatin immunoprecipitation sequencing in wild-type and hemogen-knockout animals revealed that BRG1 is largely dependent on hemogen to regulate chromatin accessibility at erythroid gene promoters and enhancers. In summary, the hemogen/BRG1 interaction in mammals is essential for fetal erythroid maturation and hemoglobin production through its active role in promoter and enhancer activity and chromatin organization.

Enhancer looping protein LDB1 regulates hepatocyte gene expression by cooperating with liver transcription factors.

Enhancers establish proximity with distant target genes to regulate temporospatial gene expression and specify cell identity. Lim domain binding protein 1 (LDB1) is a conserved and widely expressed protein that functions as an enhancer looping factor. Previous studies in erythroid cells and neuronal cells showed that LDB1 forms protein complexes with different transcription factors to regulate cell-specific gene expression. Here, we show that LDB1 regulates expression of liver genes by occupying enhancer elements and cooperating with hepatic transcription factors HNF4A, FOXA1, TCF7 and GATA4. Using the glucose transporter SLC2A2 gene, encoding GLUT2, as an example, we find that LDB1 regulates gene expression by mediating enhancer-promoter interactions. In vivo, we find that LDB1 deficiency in primary mouse hepatocytes dysregulates metabolic gene expression and changes the enhancer landscape. Conditional deletion of LDB1 in adult mouse liver induces glucose intolerance. However, Ldb1 knockout hepatocytes show improved liver pathology under high-fat diet conditions associated with increased expression of genes related to liver fatty acid metabolic processes. Thus, LDB1 is linked to liver metabolic functions under normal and obesogenic conditions.

Crystal structure of human LDB1 in complex with SSBP2.

The Lim domain binding proteins (LDB1 and LDB2 in human and Chip in Drosophila) play critical roles in cell fate decisions through partnership with multiple Lim-homeobox and Lim-only proteins in diverse developmental systems including cardiogenesis, neurogenesis, and hematopoiesis. In mammalian erythroid cells, LDB1 dimerization supports long-range connections between enhancers and genes involved in erythropoiesis, including the ß-globin genes. Single-stranded DNA binding proteins (SSBPs) interact specifically with the LDB/Chip conserved domain (LCCD) of LDB proteins and stabilize LDBs by preventing their proteasomal degradation, thus promoting their functions in gene regulation. The structural basis for LDB1 self-interaction and interface with SSBPs is unclear. Here we report a crystal structure of the human LDB1/SSBP2 complex at 2.8-Å resolution. The LDB1 dimerization domain (DD) contains an N-terminal nuclear transport factor 2 (NTF2)-like subdomain and a small helix 4-helix 5 subdomain, which together form the LDB1 dimerization interface. The 2 LCCDs in the symmetric LDB1 dimer flank the core DDs, with each LCCD forming extensive interactions with an SSBP2 dimer. The conserved linker between LDB1 DD and LCCD covers a potential ligand-binding pocket of the LDB1 NTF2-like subdomain and may serve as a regulatory site for LDB1 structure and function. Our structural and biochemical data provide a much-anticipated structural basis for understanding how LDB1 and the LDB1/SSBP interactions form the structural core of diverse complexes mediating cell choice decisions and long-range enhancer-promoter interactions.

Enhancer function: mechanistic and genome-wide insights come together.

Enhancers establish spatial or temporal patterns of gene expression that are critical for development, yet our understanding of how these DNA cis-regulatory elements function from a distance to increase transcription of their target genes and shape the cellular transcriptome has been gleaned primarily from studies of individual genes or gene families. High-throughput sequencing studies place enhancer-gene interactions within the 3D context of chromosome folding, inviting a new look at enhancer function and stimulating provocative new questions. Here, we integrate these whole-genome studies with recent mechanistic studies to illuminate how enhancers physically interact with target genes, how enhancer activity is regulated during development, and the role of noncoding RNAs transcribed from enhancers in their function.

Embryonic erythropoiesis and hemoglobin switching require transcriptional repressor ETO2 to modulate chromatin organization.

The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human ß-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the ß-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

Role of LDB1 in the transition from chromatin looping to transcription activation.

Many questions remain about how close association of genes and distant enhancers occurs and how this is linked to transcription activation. In erythroid cells, lim domain binding 1 (LDB1) protein is recruited to the ß-globin locus via LMO2 and is required for looping of the ß-globin locus control region (LCR) to the active ß-globin promoter. We show that the LDB1 dimerization domain (DD) is necessary and, when fused to LMO2, sufficient to completely restore LCR-promoter looping and transcription in LDB1-depleted cells. The looping function of the DD is unique and irreplaceable by heterologous DDs. Dissection of the DD revealed distinct functional properties of conserved subdomains. Notably, a conserved helical region (DD4/5) is dispensable for LDB1 dimerization and chromatin looping but essential for transcriptional activation. DD4/5 is required for the recruitment of the coregulators FOG1 and the nucleosome remodeling and deacetylating (NuRD) complex. Lack of DD4/5 alters histone acetylation and RNA polymerase II recruitment and results in failure of the locus to migrate to the nuclear interior, as normally occurs during erythroid maturation. These results uncouple enhancer-promoter looping from nuclear migration and transcription activation and reveal new roles for LDB1 in these processes.

Fetal γ-globin genes are regulated by the BGLT3 long noncoding RNA locus.

Long noncoding RNAs (lncRNAs) are increasingly being appreciated as participants in regulation of important cellular processes, including transcription. Because lncRNAs are highly cell type specific, they have the potential to contribute to the unique transcriptional repertoire of diverse cells, but underlying mechanisms are unclear. We studied BGLT3, an erythroid lncRNA encoded downstream of Aγ-globin (HBG1). BGLT3 and γ-globin genes are dynamically cotranscribed in erythroid cells in vivo. Deletion of BGLT3 using CRISPR/Cas9 editing shows that it specifically contributes to regulation of γ-globin genes. We used reduction or overexpression of the RNA and inhibition of transcription through the locus by CRISPRi to distinguish functions of the transcript vs the underlying sequence. Transcription of the BGLT3 locus is critical for looping between the γ-globin genes and BGLT3 sequences. In contrast, the BGLT3 transcript is dispensable for γ-globin/BGLT3 looping but interacts with the mediator complex on chromatin. Manipulation of the BGLT3 locus does not compromise γ-globin gene long-range looping interactions with the ß-globin locus control region (LCR). These data reveal that BGLT3 regulates γ-globin transcription in a developmental stage-specific fashion together with the LCR by serving as a separate means to increase RNA Pol II density at the γ-globin promoters.

Enhancers: five essential questions.

It is estimated that the human genome contains hundreds of thousands of enhancers, so understanding these gene-regulatory elements is a crucial goal. Several fundamental questions need to be addressed about enhancers, such as how do we identify them all, how do they work, and how do they contribute to disease and evolution? Five prominent researchers in this field look at how much we know already and what needs to be done to answer these questions.

Insulators organize chromatin: emerging rules of the game.

In this issue of Molecular Cell, Wood et al. (2011) provide mechanistic insight into the regulation of insulators that helps explain how they can organize chromatin in a cell type-specific fashion.

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/ß-globin proximity. CRISPR/Cas9 editing of the ß-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/ß-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/ß-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the ß-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin.

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and ß-thalassemia. Transcription of ß-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult ß-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on ß-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult ß-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of ß-hemoglobinopathies.

The hematopoietic regulator TAL1 is required for chromatin looping between the ß-globin LCR and human γ-globin genes to activate transcription.

TAL1 is a key hematopoietic transcription factor that binds to regulatory regions of a large cohort of erythroid genes as part of a complex with GATA-1, LMO2 and Ldb1. The complex mediates long-range interaction between the ß-globin locus control region (LCR) and active globin genes, and although TAL1 is one of the two DNA-binding complex members, its role is unclear. To explore the role of TAL1 in transcription activation of the human γ-globin genes, we reduced the expression of TAL1 in erythroid K562 cells using lentiviral short hairpin RNA, compromising its association in the ß-globin locus. In the TAL1 knockdown cells, the γ-globin transcription was reduced to 35% and chromatin looping of the (G)γ-globin gene with the LCR was disrupted with decreased occupancy of the complex member Ldb1 and LMO2 in the locus. However, GATA-1 binding, DNase I hypersensitive site formation and several histone modifications were largely maintained across the ß-globin locus. In addition, overexpression of TAL1 increased the γ-globin transcription and increased interaction frequency between the (G)γ-globin gene and LCR. These results indicate that TAL1 plays a critical role in chromatin loop formation between the γ-globin genes and LCR, which is a critical step for the transcription of the γ-globin genes.