An enhancer RNA recruits KMT2A to regulate transcription of Myb.

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.

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.

Noncoding function of super enhancer derived mRNA in modulating neighboring gene expression and TAD interaction.

Super enhancers are important regulators of gene expression that often overlap with protein-coding genes. However, it is unclear whether the overlapping protein-coding genes and the mRNA derived from them contribute to enhancer activity. Using an erythroid-specific super enhancer that overlaps the Cpox gene as a model, we found that Cpox mRNA has a non-coding function in regulating neighboring protein-coding genes, eRNA expression and TAD interactions. Depletion of Cpox mRNA leads to accumulation of H3K27me3 and release of p300 from the Cpox locus, activating an intra-TAD enhancer and gene expression. Additionally, we identified a head-to-tail interaction between the TAD boundary genes Cpox and Dcbld2 that is facilitated by a novel type of repressive loop anchored by p300 and PRC2/H3K27me3. Our results uncover a regulatory role for mRNA transcribed within a super enhancer context and provide insight into head-to-tail inter-gene interaction in the regulation of gene expression and oncogene activation.

An enhancer RNA recruits MLL1 to regulate transcription of Myb.

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.

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.

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.

Enhancers navigate the three-dimensional genome to direct cell fate decisions.

The activity and selectivity of transcriptional enhancers determine gene expression patterns that enable a zygote to become a complex organism. How enhancers convey regulatory information is a central conundrum in biology. Here, we discuss recent progress provided by rapidly evolving technologies in understanding enhancer-promoter interactions in the context of overall nuclear genome organization.

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.

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.

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.

The mRNA-Binding Protein IGF2BP1 Restores Fetal Hemoglobin in Cultured Erythroid Cells from Patients with ß-Hemoglobin Disorders.

Sickle cell disease (SCD) and ß-thalassemia are caused by structural abnormality or inadequate production of adult hemoglobin (HbA, α2ß2), respectively. Individuals with either disorder are asymptomatic before birth because fetal hemoglobin (HbF, α2γ2) is unaffected. Thus, reversal of the switch from HbF to HbA could reduce or even prevent symptoms these disorders. In this study, we show that insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is one factor that could accomplish this goal. IGF2BP1 is a fetal factor that undergoes a transcriptional switch consistent with the transition from HbF to HbA. Lentivirus delivery of IGF2BP1 to CD34+ cells of healthy adult donors reversed hemoglobin production toward the fetal type in culture-differentiated erythroid cells. Analogous studies using patient-derived CD34+ cells revealed that IGF2BP1-dependent HbF induction could ameliorate the chain imbalance in ß-thalassemia or potently suppress expression of sickle ß-globin in SCD. In all cases, fetal γ-globin mRNA increased and adult ß-globin decreased due, in part, to formation of contacts between the locus control region (LCR) and γ-globin genes. We conclude that expression of IGF2BP1 in adult erythroid cells has the potential to maximize HbF expression in patients with severe ß-hemoglobin disorders by reversing the developmental γ- to ß-globin switch.

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 long-range contacts: The multi-adaptor protein LDB1 is the tie that binds.

The eukaryotic genome is organized at varying levels into chromosome territories, transcriptional compartments and topologically associating domains (TADs), which are architectural features largely shared between different cell types and across species. In contrast, within TADs, chromatin loops connect enhancers and their target genes to establish unique transcriptomes that distinguish cells and tissues from each other and underlie development and differentiation. How these tissue-specific and temporal stage-specific long-range contacts are formed and maintained is a fundamental question in biology. The widely expressed Lim domain binding 1 protein, LDB1, plays a critical role in connecting enhancers and genes by forming complexes with cell-type specificity across diverse developmental pathways including neurogenesis, cardiogenesis, retinogenesis and hematopoiesis. Here we review the multiple roles of LDB1 in cell fate determination and in chromatin loop formation, with an emphasis on mammalian systems, to illuminate how LDB1 functions in normal cells and in diseases such as cancer.

CRISPR/Cas9 offers a new tool for studying the role of chromatin architecture in disease pathogenesis.

A recent study used CRISPR/Cas9 to reveal long-range looping between disease-related genes and their regulatory elements that is mediated by the CCCTC-binding factor (CTCF) in prostate cancer.

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.

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.

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.