Ivan Krivega

Enhancer and promoter interactions — long distance calls

In metazoans, enhancers of gene transcription must often exert their effects over tens of kilobases of DNA. Over the past decade it has become clear that to do this, enhancers come into close proximity with target promoters with the looping away of intervening sequences. In a few cases proteins that are involved in the establishment or maintenance of these loops have been revealed but how the proper gene target is selected remains mysterious. Chromatin insulators had been appreciated as elements that play a role in enhancer fidelity through their enhancer blocking or barrier activity. However, recent work suggests more direct participation of insulators in enhancer-gene interactions. The emerging view begins to incorporate transcription activation by distant enhancers with large scale nuclear architecture and subnuclear movement.

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 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.

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 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.

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

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

Chromatin looping as a target for altering erythroid gene expression.

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.

A tetrad of chromatin interactions for chromosome pairing in X inactivation

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.

8 – Long-Range Intranuclear Interactions

Abstract 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.

Chromosome Conformation Capture (3C and Higher) with Erythroid Samples

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) with Erythroid Samples

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.

Fetal γ-globin genes are regulated by the BGLT3 long non-coding 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 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.