The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA (Myrlin) originating from the -81-kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR-Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expressions were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the KMT2A/MLL1 complex. Myrlin CRISPRi compromised KMT2A occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting KMT2A.

SUMMARY The Myb proto-oncogene encodes the transcription factor c-MYB, which is critical for hematopoiesis. Distant enhancers of Myb form a hub of interactions with the Myb promoter. We identified a long non-coding RNA ( Myrlin ) originating from the −81 kb murine Myb enhancer. Myrlin and Myb are coordinately regulated during erythroid differentiation. Myrlin TSS deletion using CRISPR/Cas9 reduced Myrlin and Myb expression and LDB1 complex occupancy at the Myb enhancers, compromising enhancer contacts and reducing RNA Pol II occupancy in the locus. In contrast, CRISPRi silencing of Myrlin left LDB1 and the Myb enhancer hub unperturbed, although Myrlin and Myb expression were downregulated, decoupling transcription and chromatin looping. Myrlin interacts with the MLL1 complex. Myrlin CRISPRi compromised MLL1 occupancy in the Myb locus, decreasing CDK9 and RNA Pol II binding and resulting in Pol II pausing in the Myb first exon/intron. Thus, Myrlin directly participates in activating Myb transcription by recruiting MLL1.

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 β-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 β-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.

Long-range interactions underlie the folding of the genome and the ability of enhancers to connect to their distant target genes. Much of genome folding into topologically associated domains appears to be shared among different types of cells, while enhancer activation of genes is highly cell type specific and underlies differentiation of cells and tissues. High-resolution microscopy and proximity ligation experiments as well as new computational methods are revealing how these fundamentals of nuclear organization can both be accommodated. Here, we review recent work that sheds light on the compelling question of how genomes fold in animal cells. We then consider how information about genome folding informs our understanding of disease and potential therapeutic approaches.

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 β-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.

Chromosome conformation capture (3C) allows for the determination of the proximity in nuclei of DNA sequences that are linearly distant from one another in the genome. Proximity that is above that expected from random interaction provides evidence for potential long-range functional interactions such as between enhancers and their target genes. Many controls are required to convincingly demonstrate increased frequency of interaction between sequences and stringent functional tests must also be applied. Here, we present methodology suitable for 3C experiments that can also be applied as the basis for related 4C, 5C, and Hi-C approaches. These procedures are widely applicable to erythroid cell lines, progenitor cells, and tissues.

Chromatin immunoprecipitation (ChIP) allows determination of the locations to which a select protein is bound in chromatin. Chemical crosslinking of DNA and protein with bi-functional reagents such as formaldehyde and precipitation of the protein with a specific antibody permit PCR amplification (ChIP) or sequencing (ChIP-seq) to identify the bound sites. Here, we present methodology for these approaches that are widely applicable to erythroid cell lines, progenitor cells, and tissues.

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)/beta-globin proximity. CRISPR/Cas9 editing of the beta-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/beta-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/beta-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the beta-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.

An unusual pairing of homologous X chromosomes occurs during X inactivation. A new study in mouse embryonic stem cells shows that telomeres and the telomeric RNA PAR-TERRA are responsible for additional pairwise interactions that guide Xic–Xic pairing.

The EAST protein is a component of nuclear matrix. In the interphase cells, EAST is localized in the extrachromosomal nuclear domain. Increased EAST concentration leads to a change in spatial chromatin structure. However, previously, it was not known whether this protein played any role in the regulation of transcription. This study examines the mechanism of the EAST protein effect on the yellow gene transcription. It was demonstrated that the EAST-dependent repression observed in a model system of the yellow gene could occur not only in the presence of the Su(Hw) insulator sequence but also in the presence of the long terminal repeat sequence of the gypsy retrotransposon. In the LTR, a 92-bp sequence involved in the mechanism of EAST-dependent repression was detected. These results suggest that the found DNA motif is the place of the assembly of a protein complex, which functionally interacts with the EAST protein. This complex either independently suppresses the yellow gene expression in bristles or moves the transgene in the nuclear region with a high concentration of transcription repression factors.

CCCTC-binding factor (CTCF) sites are enriched at the boundaries of topologically associated domains (TADs), but their function within TADs is unclear. Removal of sub-TAD CTCF sites adjacent to the α-globin enhancers is now shown to result in inappropriate activation of neighbouring genes. Intra-TAD enhancer insulation might be broadly important for tissue specificity of enhancers.

Lineage-specific transcription factors are critical for long-range enhancer interactions, but direct or indirect contributions of architectural proteins such as CCCTC-binding factor (CTCF) to enhancer function remain less clear. The LDB1 complex mediates enhancer-gene interactions at the β-globin locus through LDB1 self-interaction. We find that an LDB1-bound enhancer upstream of carbonic anhydrase 2 (Car2) activates its expression by interacting directly with CTCF at the gene promoter. Both LDB1 and CTCF are required for enhancer-Car2 looping, and the domain of LDB1 contacted by CTCF is necessary to rescue Car2 transcription in LDB1-deficient cells. Genome-wide studies and CRISPR/Cas9 genome editing indicate that LDB1-CTCF enhancer looping underlies activation of a substantial fraction of erythroid genes. Our results provide a mechanism by which long-range interactions of architectural protein CTCF can be tailored to achieve a tissue-restricted pattern of chromatin loops and gene expression.

Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia.

The β‐hemoglobinopathies are the most common monogenic disorders in humans, with symptoms arising after birth when the fetal γ‐globin genes are silenced and the adult β‐globin gene is activated. There is a growing appreciation that genome organization and the folding of chromosomes are key determinants of gene transcription. Underlying this function is the activity of transcriptional enhancers that increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding or looping that is orchestrated by protein complexes that bind to both sites and stabilize their interaction. We find that enhancer activity can be redirected with concomitant changes in gene transcription. Both targeting the β‐globin locus control region (LCR) to the γ‐globin gene in adult erythroid cells by tethering and epigenetic unmasking of a silenced γ‐globin gene lead to increased frequency of LCR/γ‐globin contacts and reduced LCR/β‐globin contacts. The outcome of these manipulations is robust, pancellular γ‐globin transcription activation with a concomitant reduction in β‐globin transcription. These examples show that chromosome looping may be considered a therapeutic target for gene activation in β‐thalassemia and sickle cell disease.

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and β-thalassemia. Transcription of β-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 β-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on β-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 β-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 β-hemoglobinopathies.

Pairing of homologous alleles is a phenomenon generally associated with imprinted and mono-allelically expressed loci. In this issue, Hogan et al. (2015) examine the earliest steps between pluripotency and lineage commitment in ESCs and find a critical role for transient pairing of Oct4 alleles in exiting the pluripotent state.

Globin gene expression undergoes developmental switching from embryonic (ε) through fetal (γ) to adult (δ and β) genes. Inherited mutations or deletions at the β-gene cause beta-thalassemia. One of the most propitious strategies of treatment for the disease is forced switching from mutated β-gene to unaffected fetal γ-gene expression in adult erythroid cells. Expression of globin genes is regulated by the upstream LCR enhancer. The LCR enhancer loops to globin gene promoters utilizing the LDB1/GATA-1/TAL1/LMO2 protein complex. Additionally histone-modifying enzymes play a significant role in regulation of globin gene expression. G9a methyltransferase, responsible for establishing H3K9me2 histone modification, is involved in repressing fetal and activating adult globin gene expression in mouse erythroid cells. Moreover, inhibition of G9a methyltransferase activity by the synthetic chemical compound UNC0638 activates γ- and represses β-gene expression in adult human hematopoietic precursor CD34(+) cells. Using ex vivo differentiation of primary CD34(+) adult human cells as a model system, we investigated the effect of UNC0638 on switching from β- to γ-globin gene expression, LDB1 complex occupancy and LCR/β-gene promoter looping patterns in adult erythroblast cells.Human peripheral blood CD34(+) progenitor cells from three healthy adult donors were differentiated for 21 days in a three phase serum-free media system. Based upon dose titration studies, 1µM UNC0638 was added to the medium during the most proliferative phase of culture (days 7-14) and compared to control cells grown without UNC0638. Under these conditions, a highly significant 5-fold increase in γ-globin gene expression was observed. UNC0638 treatment also caused a pronounced (3-fold) reduction in β-globin gene expression without substantial change in α-globin. At the end of the culture period, HPLC analyses also demonstrated that UNC0638 treatment resulted in a considerable increase in the cellular fetal hemoglobin (HbF / HbA + HbF: control: 2.9 +/- 1.2%; UNC0638: 30.9 +/- 2.5%, p=0.003).Chromatin immunoprecipitation and chromosome conformation capture assays were utilized to determine if the increase of fetal hemoglobin along with activation of γ-gene expression and concomitant reduction of β-gene expression were associated with epigenetic modification of the β-globin locus. UNC0638 erased H3K9me2 histone modification in the β-globin locus and caused changes in LCR looping from interaction with the β- to the γ-globin gene. Mirroring differences in looping pattern, LDB1 containing protein complex occupancy was significantly increased at the γ-globin gene and decreased at δ- and β-gene promoters.These results support a model whereby G9a establishes conditions preventing activation of γ-gene by interacting with the LCR and facilitating LCR looping with δ- and β-gene promoters and subsequent strong activation of adult globin genes expression during differentiation of adult erythroid progenitor cells. In this view, G9a inhibition represents a promising approach for treatment of β-hemoglobinopathies. Disclosures No relevant conflicts of interest to declare.

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 β-globin locus via LMO2 and is required for looping of the β-globin locus control region (LCR) to the active β-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.