
==== Front
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Cell Rep
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Cell reports
2211-1247

38889007
10.1016/j.celrep.2024.114378
nihpa2011850
Article
An enhancer RNA recruits KMT2A to regulate transcription of Myb
Kim Juhyun 16*
Diaz Luis F. 126
Miller Matthew J. 136
Leadem Benjamin 14
Krivega Ivan 15
Dean Ann 17*
1 Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
2 Oregon Health and Sciences University, Portland, OR 97239, USA
3 University of Iowa Medical School, Iowa City, IA 52242, USA
4 GeneDx, Gaithersburg, MD 20877, USA
5 Sonothera, South San Francisco, CA 94080, USA
6 These authors contributed equally
7 Lead contact
AUTHOR CONTRIBUTIONS

A.D. and I.K. conceived the project. J.K., I.K., and B.L. supervised the experiments. J.K., L.F.D., and M.J.M. performed the experiments. L.F.D., J.K., and A.D. wrote the paper. All authors edited the paper.

* Correspondence: juhyun.kim@nih.gov (J.K.), ann.dean@nih.gov (A.D.)
30 7 2024
23 7 2024
17 6 2024
03 9 2024
43 7 114378114378
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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 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.

Graphical abstract

In brief

Long non-coding RNA Myrlin is transcribed from the murine Myb −81-kb enhancer. Myb enhancer looping is unaffected by Myrlin CRISPRi, but Myb transcription is downregulated. Kim et al. found that Myrlin directly participates in transcription activation by recruiting KMT2A/MLL1 to Myb and allowing RNA Pol II pause release into elongation.
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pmcINTRODUCTION

While the vast majority of the mammalian genome is transcribed, only a small fraction of these transcripts encodes proteins. The functional relevance of most non-coding transcription remains largely unknown. RNA polymerase II (Pol II) non-coding transcripts that are greater than 200 nt in length and lack coding potential are known as long non-coding RNAs (lncRNAs). lncRNAs, much like their protein coding counterparts, can be spliced and polyadenylated, but they are biased toward two-exon transcripts that remain localized to the nucleus.1–3 Nuclear-localized lncRNAs can be involved in gene regulation via interactions with chromatin remodelers, histone-modifying complexes, and transcription factors.4–8 lncRNAs derived from enhancer regulatory regions, known as enhancer RNAs, or eRNAs, have the potential ability to function together with the enhancers from which they are derived to regulate the expression of target genes.9–11

Enhancers increase the transcriptional output of target genes in a cell-type-specific fashion, often bridging substantial genomic distances.12–17 Both active enhancers and genes are marked by histone H3 lysine 4 (H3lys4) methylation. This modification is deposited by the KMT2 (MLL)/COMPASS family of histone methyltransferases, including SET1A/B and four lysine methyltransferase 2 complexes (KMT2A–D).18,19 KMT2A (also known as MLL1) forms a large macromolecular complex with WDR5, menin, RbBP5, ASH2L, and DPY-30 and interacts with the basic transcription machinery, including RNA Pol II.20 KMT2A selectively binds non-methyl CpG DNA through its CXXC domain.21 Additionally, KMT2A adaptor subunit WDR5 promotes the recruitment of KMT2A to genomic targets at a subset of genes.22 The KMT2A complex is recruited by the lncRNA HOTTIP through direct binding with WDR5, establishing H3K4me3 deposition and driving HOXA gene transcription, while HoxBlinc recruits both Set1 and KMT2 complexes to hoxb genes through the SET domain.23,24

The Myb proto-oncogene encodes c-MYB (hereafter, MYB), a critical hematopoietic regulator of cell proliferation and differentiation.25 MYB is a repressor of human fetal hemoglobin (HbF) production.26 Given that the elevation of HbF in adults moderates the symptoms of sickle cell disease and β-thalassemia, MYB is a potential target of therapeutic manipulation. Myb is regulated by microRNAs and by a series of enhancers distributed over more than 100 kb between Myb and the adjacent upstream Hbs1l gene in mice and humans.27–30 In the mouse, five enhancers, −36, −61, −68, −81, and −109 kb, with respect to the Myb transcription start site (TSS), establish proximity with the Myb promoter in an active chromatin hub.28 Repression of Myb, which is required for the terminal differentiation of erythroid cells, is accompanied by the loss of these contacts. The enhancers are occupied by the LDB1 transcription factor complex, which mediates enhancer looping, and reduction of LDB1 using a small hairpin RNA (shRNA)-compromised formation of the Myb enhancer hub.31,32 However, how chromatin looping and transcription activation at the Myb locus are regulated remains unknown.

lncRNAs have been linked to erythropoiesis and the regulation of numerous erythroid genes, including the adult β-globin and fetal γ-globin genes.33–38 We uncovered an lncRNA derived from the murine −81-kb Myb enhancer called Myrlin, for Myb enhancer long intergenic non-coding RNA. The Myrlin transcript was not required for the formation of the Myb enhancer hub. However, Myrlin loss reduced KMT2A/WDR5 recruitment in the Myb locus and decreased CDK9 and RNA Pol II occupancy. Furthermore, Myrlin loss resulted in the pausing of RNA Pol II within the Myb first exon/intron. These results tie the Myb locus lncRNA Myrlin to the detailed mechanism of Myb transcription activation and suggest unexplored avenues that could become therapeutic targets for increasing HbF in β-globin hemoglobinopathies.

RESULTS

The −81-kb murine Myb enhancer contains the TSS for a spliced, long intergenic non-coding RNA

The murine Myb-Hbs1l intergenic region contains previously characterized regulatory enhancers.28 In murine erythroleukemia (MEL) cells and in primary erythroid cells, the five enhancers, located −36, −61, −68, −81, and −109 kb upstream of the Myb promoter, are occupied by the LDB1 complex that includes DNA binding transcription factors GATA1 and TAL1, bridging protein LMO2 and looping protein LDB1 (Figure 1A).28 Chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq data for uninduced MEL cells indicates that RNA Pol II occupies several of the enhancer sites, but active RNA transcription is observed only at the −81-kb enhancer (Figure 1A).28

Rapid amplification of cDNA ends (5′ and 3′ RACE) revealed an unannotated 2-exon transcript at the Myb −81-kb enhancer that exists as two spliced isoforms with a single intron spanning more than 20 kb (Figures 1B and 1C). The primary transcript is 809 nt with a minor 357 nt shorter form attributable to early termination in exon 2. Stranded RNA-seq from induced and uninduced MEL cells publicly sourced from ENCODE indicates that Myb and the −81-kb transcript are divergently transcribed (Figure S1). The transcript has very low coding potential, according to the Coding Potential Assessment Tool (CPAT)39 (Figure 1D). Thus, the transcript qualifies as a lncRNA, and its low abundance (about 10-fold lower than Myb) is consistent with that of an enhancer RNA.40

We named the transcript Myrlin. In uninduced MEL cells Myrlin is primarily nuclear localized, consistent with a potential role in gene regulation (Figure 1E). The expression of Myb decreases upon erythroid cell maturation, which is mirrored by decreases in both Myb and Myrlin upon differentiation of MEL cells by DMSO (Figure 1F). Like Myb, Myrlin is expressed at very low levels in embryonic day E 8.5 yolk sac primitive erythroid cells and then more robustly in E14.5 fetal liver definitive erythroid cells41 (Figure 1G). These findings show that expression of Myb and Myrlin is coordinately regulated in a developmental stage-specific fashion in murine erythroid cells and raises the possibility that Myrlin may play a role in regulating Myb expression.

Reduction of Myrlin reduces Myb expression

To investigate whether Myrlin influences Myb transcription, we transduced MEL cells with lentiviruses incorporating three shRNAs directed against Myrlin. Two shRNAs reduced Myrlin by about 50%, and in both cases, Myb was reduced (Figure S2A). We then transduced primary erythroid progenitor cells isolated from bone marrow with the same shRNA lentiviruses and differentiated the cells toward the erythroid lineage (Figure S2B). Transduction efficiency was low, at about 40%, but the same two shRNAs effective in MEL cells reduced Myrlin modestly, with accompanying decreases in Myb (Figure S2C). The results suggest that Myrlin lncRNA positively regulates Myb transcription.

JASPAR motif analysis (https://jaspar.genereg.net/)42 identified a TATA box located −25 nt upstream of the 5′ end of Myrlin as determined by RACE, and a GATA1 binding motif, site of LDB1 complex occupancy, located −56 nt upstream (Figures 2A and S2D). This organization is consistent with the finding that most mouse erythroid-expressed non-coding RNAs are transcribed from conventional promoters regulated by known transcription factors and are regulated by similar Pol II release mechanisms.35,43 To obtain a more stable and robust reduction of Myrlin, we generated several MEL cell lines with small deletions designed to target the TSS of Myrlin (Figures 2A and S2). Three different mutations were obtained, all of which reduced Myrlin and Myb transcription to varying degrees in uninduced MEL cells (Figure 2B). We chose for further study the 17-bp deletion (Δ17, hereafter ΔTSS), which removed most of the sequence between the TATA box and the initiator element and resulted in the strongest reduction of Myrlin and Myb.

To begin to characterize the impairment of Myb transcription in ΔTSS cells, we performed ChIP to detect the occupancy of RNA Pol II. Compared to a control MEL cell line generated with a plasmid lacking a single guide RNA (sgRNA), occupancy of Pol II in ΔTSS cells was reduced at the Myb promoter and at the −81-kb enhancer/Myrlin TSS, as well as at the other enhancer sites (Figure 2C). TBP ChIP revealed that only the −81-kb enhancer was occupied by this member of the preinitiation complex, and in ΔTSS cells, there was a marked reduction (Figure 2D). In accordance with results showing that fetal γ-globin transcription increases when Myb regulatory micro-RNAs are reduced in human cells,27,44–46 after differentiation of ΔTSS MEL cells, there was a several-fold increase in murine embryonic βh1 globin transcription (Figure 2E), consistent with Myb reduction. These results support the idea that Myrlin, transcribed from the −81-kb Myb enhancer locus, is a positive regulator of Myb transcription in MEL cells and in differentiating primary erythroid cells.

Long-range Myb promoter and enhancer contacts are reduced after Myrlin TSS deletion

Myb expression is regulated by long-range interactions between the Myb promoter and LDB1-bound enhancers within the Myb-Hbs1l intergenic region.28 A CTCF site 30 kb downstream of the Myb promoter also participates in the Myb enhancer hub, likely through direct interaction between CTCF and LDB1.47 Moreover, the disruption of this enhancer hub underlies Myb downregulation during erythroid differentiation.28 We used chromatin conformation capture (3C) to determine whether ΔTSS influenced contacts between Myb and its intergenic enhancers. Compared to control cells, ΔTSS cells displayed reduced interaction frequency between the Myb promoter and enhancers, which was particularly evident at the −36-kb and −81-kb enhancer sites and the −30-kb CTCF site (Figure 3A).

The reduction of interaction frequency between Myb and its multiple enhancers by ΔTSS deletion closely resembles the reduced interactions observed upon LDB1 knockdown using an shRNA in MEL cells.28 To investigate further, we carried out ChIP experiments to determine the occupancy of the LDB1 complex after reduction of Myrlin in ΔTSS cells. We observed that diminished long-range interactions in ΔTSS cells correlated with reduced LDB1 and TAL1 across the enhancers but that reduced GATA1 occupancy was only notable at the −81 and −36 enhancers (Figures 3B–3D). The H3K27ac mark, which indicates active enhancers, was not significantly affected in ΔTSS cells (Figure 3E). Together, our results show that the decrease in Myb expression upon deletion of the Myrlin TSS is accompanied by reduced LDB1 complex occupancy across the Myb enhancers and Myb enhancer hub disruption, although the enhancers remain in a potentially active state, retaining the H3K27ac mark.

CRISPRi for Myrlin affects Myb transcription but not enhancer interactions

We next employed CRISPRi as an alternative approach to reduce the Myrlin transcript without altering the sequence context at the Myb −81-kb enhancer. Catalytically dead Cas9 (dCas9) retains the ability to target DNA and can be an adaptable block to transcription elongation when fused to a KRAB repression domain. Using two different guide RNAs (gRNAs) to target dCas9-KRAB to Myrlin exon 1, we observed a 50%–60% reduction in Myrlin transcription leading to a similar drop in Myb transcription, as observed in the Myrlin ΔTSS deletion (Figure 4A).

ChIP localization of histone modifications at Myb enhancers revealed H3K9me3 at the −81-kb enhancer after Myrlin CRISPRi, a signature heterochromatin mark of KRAB-mediated repression (Figure 4B). There was no change in H3K27ac at any enhancer after Myrlin CRISPRi (Figure 4C), similar to what we observed after the Myrlin ΔTSS deletion. However, in contrast to the broad reduction of LDB1 at enhancer sites after Myrlin ΔTSS deletion, LDB1 enhancer occupancy was not significantly reduced by Myrlin CRISPRi (Figure 4D). In addition, 3C experiments revealed relatively little change in interaction frequency between Myb and its enhancers after Myrlin CRISPRi compared to a control clone generated with a non-targeted dCas9-KRAB vector (Figure 4E). Thus, the downregulation of Myb after CRISPRi silencing of Myrlin does not involve loss of the Myb enhancer hub, separating looping and transcription activation at this locus. We conclude that Myb downregulation after Myrlin CRISPRi silencing does not require loss of the Myb enhancer hub, raising the possibility that Myrlin has a direct role in Myb transcription activation.

Myrlin maintains H3K4me3 in the Myb locus through KMT2A-WDR5

To further explore the role of Myrlin in Myb transcription activation, we focused on the Myb promoter and first exon/intron, which contain a CpG island that is highly enriched for H3K4me3 when Myb is active (Figure S3). ChIP-qPCR revealed strong H3K4me3 enrichment across these sequences, which was reduced following Myrlin transcriptional repression by Myrlin CRISPRi (Figure 5A). The KMT2A complex has been shown to deposit H3K4me3 modifications at the Myb locus and activate transcription.48 Therefore, we carried out ChIP for KMT2A complex components KMT2A and WDR5. We found a strong decrease in both the KMT2A and WDR5 enrichment at the Myb locus after Myrlin CRISPRi (Figures 5B and 5C). Interestingly, HOTTIP lncRNA targets the KMT2A/WDR5 complex to promoters of HOXA genes to facilitate gene expression, but currently no known eRNAs have been associated with the KMT2/WDR5 complex in the context of gene regulation.23

The results so far suggest that Myrlin may directly participate in the activation of Myb transcription through recruiting KMT2A. To detect RNA-protein interactions, we performed Myrlin RNA-ChIP and found that the Myrlin transcript interacts with the KMT2A component WDR5 (Figure 5D). RNA-ChIP also detected interactions that were not statistically significant between Myrlin and KMT2A and with menin, another component of the KMT2A complex. Therefore, we performed RNA pulldown using biotinylated Myrlin and blotted against these components of the KMT2A complex. The results confirmed the interaction of Myrlin with WDR5 and further revealed that Myrlin could pull down complex components KMT2A and menin. Biotinylated Myrlin did not pull down SET1a or SET1b, which are members of different COMPASS-like complexes, nor was there any interaction with tubulin (TUB), which served as a negative control (Figure 5E). These results strongly support that Myrlin lncRNA plays a role in KMT2A complex recruitment to promote H3K4me3 deposition and subsequent transcription of Myb and that Myrlin loss compromises this series of events and Myb transcription activation.

RNA Pol II pausing and CDK9 occupancy are affected by CRISPRi of Myrlin

KMT2A complexes can maintain target gene expression by regulating both transcriptional initiation and elongation. KMT2A loss results in the loss of the CDK9, the protein kinase subunit of pTEFb that confers phosphorylation on Pol II ser2 to drive transcription elongation in hematopoietic cells.49,50 Previous studies have shown that inhibition of CDK9 strongly reduced transcription elongation through the Myb gene body and resulted in pausing of Pol II within the Myb first intron.28,51 Using ChIP-qPCR, we found that CDK9 recruitment across the Myb enhancers and in Myb exon 1 and intron 1 was severely diminished after Myrlin CRISPRi (Figure 6A). Subsequent ChIP-qPCR analysis of RNA Pol II localization in the Myb locus revealed decreased occupancy at several Myb enhancers but no significant difference at the Myb promoter (Figure 6B). Interestingly, after Myrlin CRISPRi, RNA Pol II accumulated within the Myb gene body, particularly at Myb exon1/intron 1, which is consistent with the reduction of Myb transcripts.

To further investigate this result, we performed CUT&Tag for the Ser5P and Ser2P phosphorylated forms of RNA Pol II, which represent the initiating and elongating forms of the enzyme, respectively. The genome browser view in Figure 6C illustrates the reduction of both phosphorylated forms of RNA Pol II across the Myb locus after Myrlin CRISPRi. Quantitation of these data shows an approximately 30% reduction in Pol II Ser5P and Ser2P within the Myb gene body and reduced occupancy at each of the enhancers (Figures 6D and 6E). To obtain increased resolution of this result, we analyzed the signals of the shorter (<120 bp) and longer (>270 bp) Pol II Ser5P CUT&Tag fragments, which distinguishes Pol II in the pre-initiation state at the TSS and paused Pol II at peaks up- and downstream of the TSS, respectively52 (Figure 6F). Myrlin CRISPRi reduced the signal for the shorter fragments, suggesting some decrease in Pol II recruitment upon Myrlin loss. At the same time, the signal associated with longer fragments, indicative of paused Pol II, strongly increased across exon/intron 1.

We calculated the pausing index of Pol II Ser5P from the longer fragment data based on the Ser5P ratio at the TSS (exon1) and in the gene body and found an approximately 3-fold higher pausing index in Myb after Myrlin CRISPRi compared to controls (Figure 6G). Similar results were obtained for Pol II pausing in Myb using CUT&Tag peaks without separation by fragment size (Figures S4A and S4B). These results connect Myrlin to the mechanism by which transcription of Myb is regulated, and they suggest that Myrlin, through KMT2A, may help to recruit or stabilize CDK9 at Myb, which is necessary for efficient RNA Pol II elongation through the gene.

KLF1 interacts with Myrlin transcripts within the Myb active chromatin hub

What could be the basis of Myrlin local function in Myb transcription? Myrlin interaction with KMT2A at the actively transcribed Myb promoter raised the question of whether Myrlin is retained within the enhancer hub by tethering to the −81-kb enhancer, adjacent to its TSS. We performed chromatin isolation by RNA immunoprecipitation (ChIRP) experiments to determine Myrlin localization in the Myb locus. Two sets of Myrlin probes (odd and even probes) can specifically capture the Myrlin transcript (Figure 7A). We found prominent peaks for Myrlin at its two exons and at the −81-kb enhancer site, but not at other sites across the locus (Figure 7B). Since the −81-kb enhancer is close to Myrlin exon 1, the signal at that site could have been contributed to by cross-linking of Myrlin bound to exon1. Therefore, we asked whether Myrlin interacted with −81-kb-bound transcription factors, specifically KLF1, which uniquely binds to the −81 site among the enhancers. KLF1 is required for full Myb transcription activation.28 Moreover, transcription factors have recently been documented to commonly bind RNAs.53 Indeed, RNA ChIP for KLF1 revealed interaction with Myrlin, which was confirmed by biotinylated RNA pull-down (Figures 7C and 7D). ChIP-qPCR confirmed occupancy of KLF1 at the −81-kb Myb enhancer, but not at −68 kb, as expected (Figure 7E). KLF1 occupancy was not significantly different after Myrlin CRISPRi. We conclude that KLF1 may contribute to the localization of Myrlin within the Myb enhancer hub.

In summary, structural components contributing to Myb-enhancer interactions within a hub, including the LDB1 complex and KLF1, are present and the hub forms normally when Myrlin is lost through CRISPRi (Figure 7F). However, in the absence of Myrlin, recruitment of KMT2A/WDR5 is reduced, resulting in poor binding of CDK9 and RNA Pol II in the Myb locus. Moreover, RNA Pol II pausing within the Myb first exon/intron and failure to complete Myb transcripts are observed upon Myrlin loss. We conclude that the Myrlin eRNA has important recruitment functions for the KMT2A complex that are required for Myb transcription activation beyond formation of the Myb enhancer hub.

DISCUSSION

Hundreds of lncRNAs are expressed during erythropoiesis, but evidence for their function remains anecdotal.33–35,37 We identified Myrlin, an eRNA, transcribed from the −81-kb murine Myb enhancer, as a positive regulator of Myb. A rigorous paradigm to deduce the function of lncRNA and eRNA loci involves dissection of the functional roles of the RNA transcript versus the act of enhancer transcription, both of which can influence target gene expression and/or chromatin organization.54 To this end, we analyzed the role of the RNA molecule by using Myrlin RNA ChIP and biotinylated Myrlin pull-down assays to document the importance of the transcript per se. We found that Myrlin interacts with KMT2A/WDR5 to promote Myb transcription. In the absence of Myrlin, KMT2A recruitment to Myb and Myb promoter H3K4me3 modification is reduced. In addition, CDK9 is reduced at the Myb promoter, and RNA Pol II accumulates excessively within the Myb first exon/intron. Thus, Myrlin is required for Pol II pause release to promote Myb transcription.

In our initial experiments to reduce Myrlin transcription, we deleted 17 bp of the Myrlin TSS. This resulted in loss of the LDB1 looping complex from all the Myb enhancer sites, disruption of the Myb enhancer hub, and reduction of Myb transcription. The results suggest interdependence among multiple Myb enhancers for formation of the hub. Hubs are understood from single allele interaction experiments to represent close association of all hub sequences within which multiple enhancers show preferential aggregation with one another and with the genes they regulate.55 Enhancer interdependence has been observed at the IgK super-enhancer cluster where deletion of one enhancer affects interactions among the others.56,57 This model comports well with the idea that extrusion of chromatin loops through the action of cohesin complexes and CTCF will bring many points within an extended locus into close proximity where their interactions may be stabilized by specific transcription factors.58,59 Such a hub of interactions and transcription factor density may facilitate the accumulation of transcriptional components such as RNA Pol II, possibly involving liquid-liquid phase separation, favoring transcription.14

The precise role of Myrlin in Myb enhancer hub formation, if any, remains to be clarified, since the LDB1 complex was lost from the enhancer sites in the ΔTSS cells, which is well documented to compromise enhancer looping in this locus.28 Intriguingly, LDB1 binding, and the hub formation were not affected when Myrlin transcription was repressed by Cas9-KRAB, which did not affect LDB1 complex occupancy. It remains unclear why LDB1 is lost from the enhancer sites in ΔTSS cells, but not after Myrlin CRISPRi. One possibility is that the 17-bp deletion of the TSS near the LDB1 complex motif at the −81-kb enhancer distorts the local chromatin architecture sufficiently to reduce LDB1 binding, which then destabilizes binding at the other hub enhancers, and looping. Regardless, losing Myrlin under the circumstances of KRAB repression did not have this effect on LDB1 binding yet still resulted in Myb downregulation, separating looping from transcription activation. Looping in the absence of transcription has been observed at the β-globin locus in erythroid cells and, more generally, in leukemia cells after drug treatment to inhibit BET proteins.60–62 These data support the idea that enhancer looping mechanistically precedes transcription activation.

To explore the difference between looped and transcriptionally active Myb loci versus inactive looped loci, we localized RNA Pol II and found a decrease in Pol II recruitment to the Myb promoter after Myrlin CRISPRi and significant pausing in intron/exon 1. Pol II pausing is correlated with transcriptional repression during terminal erythroid repression.63 Previous work had suggested a Pol II pause region in Myb intron 1 that was proposed to correspond to either a stem-loop-forming sequence followed by a poly (dT) tract 1.7 kb downstream of the Myb TSS or to a CTCF site about 2 kb downstream.28,51 These groups reported that transcription elongation through Myb was inhibited in erythroid cells or in breast cancer cells by CDK9 inhibitors. Our high-resolution CUT&Tag results for Pol II Ser5 and Pol II Ser2 showed localization of paused transcripts across Myb exon 1 and into intron 1, likely encompassing the sites previously suggested as pause sites. We were able to show that Myrlin functions to recruit or stabilize CDK9 in the Myb exon/intron1 Pol II pause region. It has been suggested that interactions of an enhancer with its target promoter can stimulate Pol II pause release, and that this property may be related to CDK9 activity.64–66 Myrlin provides a link between an eRNA and CDK9 at a target gene.

We observed that Myrlin interacts with several subunits of the KMT2A complex, including KMT2A, WDR5, and menin, and that Myrlin is important for KMT2A occupancy in the Myb locus and for H3K4me3 modification at the Myb promoter CpG island. Promoter proximal H3K4me3 has recently been linked to RNA Pol II pause release.67 Reduced KMT2A may underlie the loss of CDK9 at the Myb locus, as CDK9 reduction was reported on a global scale after the deletion of KMT2A in hematopoietic stem and progenitor cells.49 In this scenario, the Myrlin eRNA becomes important for Myb transcription after the enhancer hub is formed. We propose that Myrlin interacts with KMT2A and the CDK9 component of pTEFb to recruit RNA Pol II to the locus and ensure efficient elongation through the Myb exon/intron 1 pause region. After Myrlin CRISPR, KMT2A and CDK9 are reduced, and Pol II pausing reduces Myb transcripts. The above putative functions of Myrlin would require its localized presence in the Myb locus/enhancer hub. We explored the potential role of KLF1 in such localization since it binds uniquely to the −81 enhancer, where the Myrlin eRNA is transcribed. Indeed, we documented the interaction of Myrlin and KLF1 by biotinylated RNA pull-down. We propose that this interaction may contribute to Myrlin localization within the Myb enhancer hub, where it can recruit KMT2A to Myb.

One of the most promising strategies for treating sickle cell disease and β-thalassemia is reactivation of HbF in erythroid cells of adult patients. Genome-wide association studies revealed an association between SNPs in the Myb-Hbs1l intergenic region, encompassing several Myb enhancers, that reduce Myb expression and elevate HbF.26,29,30 Two of the SNPs, rs66650371 and rs77755698, are located within the LDB1 complex GATA1/TAL1 compound motif at the human −84-kb MYB enhancer, which is the homolog of the murine −81-kb enhancer.29,68 The SNPs reduce LDB1 binding to the −84-kb enhancer, decrease interaction frequency with the MYB promoter and MYB transcription.29 The effect of the SNPs on interactions of the other enhancers with MYB was not tested in this work, but our results suggest that overall formation of the hub is likely affected.

A non-coding RNA, HMI-LNCRNA, was reported to arise from the −84-kb human MYB enhancer locus.36 Thousands of human lncRNAs have homologs in other species with similar expression patterns but low sequence conservation.35,69 BLATN sequence comparison of HMI-LNCRNA to the mouse genome revealed a small conserved “patch” of 378 nt (85.8% homology) at the 3′ end of HMI-LNCRNA that is shared with the 5′ end of Myrlin, something commonly observed for these poorly conserved lncRNAs69 (Figure S5). The homology patch includes the GATA1/LDB1 complex binding site that mediates enhancer looping in human and mouse erythroid cells, the −81-kb KLF1 binding site, and the Myrlin TATA box and first exon. Knockdown of HMI-LNCRNA had a negative although inconsistent effect on MYB transcription.36 The Myb regulatory functions ascribed to Myrlin in mouse cells remain to be thoroughly investigated for the related transcript in human cells but could represent appealing avenues for manipulation of MYB and HbF to ameliorate the severity of hemoglobinopathies.

Limitations of the study

This work clearly shows that Myrlin eRNA directly participates in activating Myb transcription via KMT2A/MLL1 recruitment in MEL cells. We showed that Myrlin may help to recruit or stabilize CDK9 at Myb through KMT2A, but other molecules could also contribute and should be studied in the future. In murine hematopoietic stem and progenitor cells (HSPCs), we provided evidence that Myrlin reduction negatively affects Myb transcription, but mechanistic studies of Myrlin RNA such as ChIRP and RNA immunoprecipitation will need to be carried out to fully validate Myrlin function in HSPCs. The Myb regulatory functions ascribed to Myrlin in mouse cells also remain to be thoroughly investigated for the potentially related HMI-LNCRNA transcript in human cells.

STAR★METHODS

RESOURCE AVAILABILITY

Lead contact

Further information and request for resources and reagents should be directed to and will be fulfilled by the lead contact, Ann Dean (ann.dean@nih.gov).

Materials availability

Plasmids generated in this study are available by request from the lead contact.

Data and code availability

Sequencing data discussed in this publication are deposited in the Gene Expression Omnibus with accession number GEO: GSE240060.

ENCODE data can be obtained from integrative publication PMID: 22955616; PMC: PMC3439153.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Mice and ethics statement

Yolk sacs from E8.5 and fetal livers from E14.5 were dissected from female C57BL/6J mice and washed in phosphate-buffered saline. Mice were sacrificed between 16 and 18 weeks to collect BM. Mouse protocols were approved by the NIDDK Animal Care and Use Committee in accordance with AALAC specifications.

Cell culture and animals

Control and CRISPR-edited mouse erythroid leukemia (MEL) cell lines were cultured in a 5% CO2 humidified incubator at 37°C in DMEM (Gibco, 11965118) with L-Glutamine (Gibco, A2916801), 10% fetal bovine serum (R&D system, S12450), 1% Penicillin and Streptomycin (Gibco, 15070063) and 1mM sodium pyruvate (Gibco, 11360070) at a density between 1x105 and 1x106 cells/mL. MEL differentiation was induced at a concentration of 2.5 x 105 cells/mL with 2% DMSO (Millipore sigma, D4540) for 4 days.

METHOD DETAILS

CRISPR-Cas9 genome editing of MEL cells

CRISPR gRNAs were designed using GeneTargrter (http://genetargeter.mit.edu/) (see Table S1 for gRNA sequences). gRNAs targeting the Myrlin transcription start site were cloned into the CRISPR-Cas9 and gRNA expression vector pSpCas9(BB)-2A-GFP (PX458) (gift from Feng Zhang, Addgene plasmid #48138) as described.70 MEL cells were transfected with Escort IV lipid transfection reagent (Sigma-Aldrich L3287) according to the manufacturer’s instructions. Fluorescent cells were sorted 48 h later and plated at limiting dilution to isolate clones. Clonal lines were genotyped by PCR using EmeraldAmp GT PCR Master Mix (Takara, RR310A) and target specific primers flanking the Myrlin transcription start site. Deletions were validated by sequencing.

Stable MEL cell clones expressing dCAS9-KRAB were generated using Lenti-dCAS9-KRAB-blast (Gift from Dr. Gary Hon, Addgene plasmid #89567). 2 x 106 of MEL cells were suspended in 100ul of High-Performance Electroporation Solution (BTXpress, 45–0801) with 5–10 μg of plasmid DNA and electroporated with the Gene Pulser Xcell System (Bio-Rad) using 2 pulses at 200 V for 5 ms. Cells were diluted with 100 μL of pre-warmed media and transferred to 2 mL of media in a 12-well culture dish. Cells were selected in 10 μg/mL Blasticydin (Gibco, A1113903) for one week and plated at limiting dilution to isolate clones. Clonal lines were checked for production of S. pyogenes dCAS9 by RT-qPCR. dCAS9-KRAB MEL cells were electroporated as above with gRNAs targeting the Myrlin transcription start site cloned into LentiGuide-puro (gift from Feng Zhang, Addgene plasmid #52963). Cells were selected in 10 μg/mL Blasticydin and 1 μg/mL puromycin (Gibco, A1113803) for one week and plated at limiting dilution to isolate clones. Transfected cells were validated for expression of Myrlin by RT-qPCR.

Isolation and differentiation of HSCs

BM-MNCs were collected from mice. Lin− cells were enriched using EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (Stemcell, 19856) following the protocol provided by the vender. Isolated cells were cultured at a concentration of 1-2 x 106 cells/ml in a 5% CO2 humidified incubator at 37°C in stemPro-34 (ThermoFisher, 10639011) with 1× nutrient supplement, 2 mM L-Glutamine (Gibco, A2916801), 1% penicillin/streptomycin (Gibco, 15070063), 100 μM monothioglycerol (Sigma-Aldrich, M6145), 1 μM dexamethasone (Sigma-Aldrich, D2915), 0.5 U/ml of erythropoietin (AMGEN, NDC55513-126-10), and 100 ng/mL recombinant SCF (R&D Systems, 255-SC-200).

Knock-down of Myrlin

To knockdown Myrlin in Lin− cells, shRNAs were cloned into SIN40C.SFFV.GFP.miR30n vector (Addgene #169278) and co-transfected with psPAX2 and pMD2.G plasmids into 293FT cell to produce lentivirus following the Addgene protocol. Lin− cells were transduced with lentivirus by spinoculation with Lenti-Boost (SIRION, SB-P-LV-101-10) following the manufacturer’s protocol.

5′ and 3′ rapid amplification of cDNA ends (RACE)

RACE was performed using the FirstChoice RLM-RACE kit (ThermoFisher Scientific, AM1700) following the manufacture′s protocol. Total RNA from MEL cells was extracted and reverse transcribed using the 3′ RACE adapter and the sequence of interest was amplified by nested PCR (3′ RACE). Alternatively, a sample of the same RNA was treated with Calf Intestine Alkaline Phosphatase, then Tobacco Acid Pyrophosphatase and finally ligated to the 5′ RACE adapter. De-capped adapter-ligated RNA was then reverse transcribed, and the sequence of interest was amplified by nested PCR (5′ RACE). PCR products were separated on a 1% agarose gel, purified using the QIAquick gel extraction kit (Qiagen, 28704), and cloned into the pCR4-TOPO vector (Invitrogen, K457502) for sequencing. For RACE primers, see Table S1.

Reverse-transcription qPCR (RT-qPCR)

RNA was isolated from 1x106 MEL cells with the RNeasy Plus kit (Qiagen, 74134). RNA was reverse-transcribed using the Superscript III First-Strand Synthesis System (ThermoFisher Scientific, 18080051) following manufacturer′s instructions. RT-qPCR was performed using the iTaq Universal SYBR Green Supermix (Bio-Rad, 1725120) with the ABI 7900HT (Applied Biosystems). Data was normalized to ActB. For RT-qPCR primers see Table S1 and Stadhouders et al., 2012.28

Chromatin immunoprecipitation (ChIP)

106 of MEL cells per IP were cross-linked with 1% formaldehyde in PBS at room temperature for 10 min. Glycine was added to a final concentration of 0.125 M and sample were incubated at room temperature for 5 min. Cells were washed three times with cold PBS by centrifugation. For LDB1 ChIPmentation cells were double cross-linked. MEL cells were washed three times with cold PBS with 1 mM MgCl2. Disuccinimidyl glutarate (ThermoFisher Scientific, 20593) in DMSO were added to a final concentration of 2 mM and samples were rocked at room temperature for 45 min. Cells were washed three times with PBS by centrifugation and cross-linked with 1% formaldehyde in PBS at room temperature for 10 min, Glycine weas added to a final concentration of 0.125M and sample were incubated at room temperature for 5 min. Cells were washed three times with cold PBS by centrifugation. Cell pellets were snap frozen and thawed on ice on the day of starting the ChIPmentation experiment. Cell pellets were resuspended in 1 mL ChIP sonication buffer (10 mM Tris pH 8.0, 0.25% SDS, 2 mM EDTA) with proteinase inhibitors (Sigma, P8340) and 1 μM phenylmethylsulfonyl fluoride (PMSF) and sonicated by 12 cycles (30% amplitude, 30 s on/30 s off) using Sonifier SFX250 (BRANSON). Then samples were diluted in 1:1.5 ratio with equilibration buffer (10 mM Tris, 233 mM NaCl, 1.66% Triton X-100, 0.166% Na-Deoxycholate, 1 mM EDTA, proteinase inhibitors and 1 μM PMSF). Samples were spun at 14,000g for 10 min at 4°C and supernatant was transferred to a new tube. 1% input was preserved. 3 μg of antibody of interest or isotope-matched IgG was added, and samples were incubated on a rotator overnight at 4°C. On the second day, Pierce ChIP-grade Protein A/G Magnetic Beads (ThermoFisher Scientific, 26162) were washed once with RIPA-LS (10 mM Tris pH 8.0, 140 mM NaCl, 1 mM pH 8.0 EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, proteinase inhibitors and 1 μM PMSF) and added to chromatin. ChIP reactions by adding 25 μL/IP of A/G beads rotated for 2 h at 4°C. Chromatin bound beads were washed twice with ice-cold RIPA-LS, twice with ice-cold RIPA-HS (10 mM Tris pH 8.0, 500 mM NaCl, 1 mM pH 8.0 EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, proteinase inhibitors and 1 μM PMSF), twice with ice-cold RIPA-LiCl (10 mM Tris pH 8.0, 250 μM LiCl, 1 mM pH 8.0 EDTA, 0.5% NP-40, 0.5% Na-Deoxycholate, proteinase inhibitors and 1 μM PMSF) and once with TE (10 mM Tris pH 80, 1 mM EDTA pH 8.0). Beads were resuspended with 48 μL of ChIP elution buffer (10 mM Tris pH 8.0, 300 mM LiCl, 5 mM pH 8.0 EDTA, 0.4% SDS) and 2 μL of proteinase K (20 mg/mL, Invitrogen, AM2546). 0.4% SDS, 300 mM NaCl and 2 μL of proteinase K were added for input samples. Samples were incubated at 55°C for 1 h, then 65°C for 6–10 h. DNA was purified using the ChIP DNA Clean & Concentrator Kit (Zymo Research, D5205) according to the manufacturer’s instructions. Real-time qPCR was performed with published primers28 using the iTaq Universal SYBR Green Supermix. Percent of input was calculated against input. For ChIPqPCR primers, see Table S1.

Chromatin conformation capture assay (3C)

Cells were cross-linked with 2% formaldehyde in PBS at room temperature for 5 min, Glycine weas added to a final concentration of 0.125 M and sample were incubated at room temperature for 5 min. Cells were washed twice with cold PBS and resuspended in lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP40, proteinase inhibitor). After lysis for 30 min on ice, nuclei were collected by centrifugation at 800g for 10 min at 4°C and resuspended in 1.2 X of NEB buffer 2.1 (New England Biolabs, B7202). 1 x 107 nuclei were then solubilized with 0.3% SDS for 1 h at 37°C followed by adding 1.8% of Triton X-100 and incubating 1 h at 37°C. Chromatin was digested with 1000 U of HindIII (New England Biolabs, R0104M) overnight at 37°C. Digested genomic DNA control were taken and stored at −20°C. Restriction enzyme was inactivated using 1.6% of SDS for 25 min at 65°C and chromatin was ligated by adding 4000 U of T4 ligase (New England Biolabs, M0202M) in 1×T4 DNA ligase buffer (New England Biolabs, B0202S) containing 1% Triton X-100 and for 4 h at 16°C followed by additional incubation for 30 min at room temperature. Chromatin was de-cross-linked with a final concentration of 100 μg/mL of proteinase K at 65°C for 6–10 h. Then 0.5 μg/mL of RNase A was treated for 1 h at 37°C. Samples were purified via phenol-chloroform extraction and ethanol precipitation. Relative cross-linking between the Myb promoter and fragments of interest was analyzed by real-time qPCR with published TaqMan probes and primers.28 Ligation products of HindIII digested BAC DNA containing the mouse Myb-Hsbl1 intergenic region were used to determine primer efficiency. Additional primers are listed in Table S1.

Myrlin RNA cellular localization

MEL cells were washed with ice-cold PBS and lysed in hypotonic buffer (25 mM HEPES, 2 mM EDTA, 0.5% Tween 20). Cytoplasmic and nuclear fractions were obtained by centrifugation at 800g for 10 min at 4°C. RNA from the supernatant cytoplasmic material and the nuclear pellet were purified and RNA was reverse transcribed, and cDNA was measured by real-time qPCR. For RT-qPCR primers see Table S1.

Chromatin isolation by RNA purification (ChIRP)

The Myrlin probes were designed using the Stellaris Probe Designer version 4.2 and synthesized with 3′ Bio-TEG modification by IDT. LacZ probes were used as a negative control (Millipore, CS216572). ChIRP-qPCR was performed as described (Chu et al., 2012). 2 X 106 of MEL cells per IP were fixed with 1% glutaraldehyde (Sigma-Aldrich, G5882) for 10 min at room temperature. Glycine was added to a final concentration of 0.125 M and sample were incubated at room temperature for 5 min. Cells were washed three times with cold PBS by centrifugation. Cell pellets were snap frozen and thawed on ice on the day of starting the ChIRP experiment. Cell pellets were resuspended in lysis buffer (10 X the mass of pellet, 50 mM Tris pH 7.0, 1% SDS, 10 mM EDTA) with proteinase inhibitors, 0.1 U/μL Superase-in (Invitrogen, AM2694) and 100 mM PMSF, then subjected to sonication with Bioruptor (Diagenode) for 50 min at highest setting with 30 s ON, 45 s OFF pulse intervals. Samples were spun at 16,000 g for 10 min at 4°C and supernatant was transferred to a new tube. Chromatin was snap frozen and thawed on ice on the day of starting the ChIRP experiment. Chromatin is diluted in two times volume of hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris pH 7.0, 1 mM EDTA, 15% Formamide) with proteinase inhibitors, 0.1 U/μL Superase-in and 100 mM PMSF. Pre-designed probes were separated into two groups which are even probe set (probe 2, 4, 6 and 8) and odd probe set (probe 1, 3, 5 and 7) and each probe set (100 pmol/1 mL chromatin) were added to diluted chromatin, which was mixed by end-to-end rotation for 4 h at 37°C. Dynabeads MyOne Streptavidin C1 (Invitrogen, 65001) were washed three times in lysis buffer and resuspended in its original volume. 100 μL of streptavidin beads were added per 100 pmol of probes, and samples were mixed for 30 min at 37°C. Chromatin bound beads were washed five times with 1 mL of pre-warmed wash buffer (2× SSC, 0.5% SDS, 100 mM PMSF) for 5 min at 37°C. At last wash, 100 μL of resuspended beads aliquoted for RNA isolation and 900 μL for DNA fraction. RNA was extracted with the miRNAeasy Mini Kit (Qiagen, 217004), and Superscript III First-Strand Synthesis System was used to reverse transcribe RNA to cDNA. DNA fraction was isolated by phenol-chloroform extraction and ethanol precipitation. qPCR was performed using iTaq Universal SYBR Green Supermix with the ABI 7900HT. Percent of input was calculated against input. For probes see Table S1.

RNA pulldown

Full-length Myrlin RNA was generated using the MEGAscript T7 Kit (Invitrogen, AM1333) according to the manufacturer’s protocol. RNA pulldown was performed using the Pierce Magnetic RNA-Protein Pull-Down Kit (ThermoFisher Scientific, 20164) according to the manufacturer’s protocol. Briefly, 1 μg of biotinylated Myrlin RNA was incubated with 1 mg of precleared protein extracted from MEL cells for 4 h at 4°C. Following this, Streptavidin magnetic beads was added and incubated for an additional 2 h. Finally, proteins were eluted and subjected to western blotting.

Western blot

Proteins were separated on NuPAGE 4%–12% Bis-Tris Gel (Thermo Fisher Scientific, NP0321) and transferred to a PVDF membrane using Trans-Blot Turbo Transfer Pack (Bio-RAD, 1704156). Membrane was blocked in blocking buffer (1 X TBS, 0.1% Tween 20, 5% w/v Nonfat dry milk) for 1 h at room temperature. Primary antibodies in blocking buffer were treated overnight at 4°C. Membranes were washed with TBST three times with shaking for 10 min, incubated with HRP-conjugated secondary antibody in blocking buffer for 1 h at room temperature, and washed three times in TBST. Blots were exposed to SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, 34075) and scanned by Syngene PXi (Syngene). Antibodies are listed in Table S1.

RNA-ChIP

The RNA ChIP-IT Kit (Active Motif, 53024) was used according to the proprietary methods. 106 of MEL cells were collected and cross-linked with 1% formaldehyde at room temperature for 10 min. For KLF1 RNA-ChIP cross-linking was not applied since KLF1 binding site at −81 kb Myb enhancer is close to Myrlin gene location. Nuclei were isolated and sonicated by 10 cycles (30% amplitude, 30 s on/30 s off) using Sonifier SFX250. Immunoprecipitated RNA using antibody of interest or isotope-matched IgG antibody was reverse transcribed using the Superscript III First-Strand Synthesis system and qPCR was performed using iTaq Universal SYBR Green Supermix with the ABI 7900HT.Percent of input was calculated against input. For RT-qPCR primers and antibodies see Table S1.

CUT&Tag library preparation and data processing

CUT&Tag libraries were prepared using the CUT&Tag-IT Assay Kit (Active Motif, 53160) following to the manufacturer′s protocol. 106 of MEL cells were collected for each biological replicate and two replicates were prepared. The MEL cells were bound to Concanavalin A Beads and Incubated with 1:50 rabbit polyclonal Phospho-Rpb1 CTD Ser5 and Ser2 antibody (Cell Signaling 13523 and 13499). Guinea pig a-rabbit antibody was used at 1:100 dilution as secondary antibody. Tagmentation was performed using pA-Tn5 Transposomes at 37°C for 60 min. DNA was purified by DNA Purification Column, then universal i5 primers and uniquely barcoded i7 primers were added to the DNA with 14 cycles of PCR. Individual libraries were purified with SPRI beads and eluted with 20 mL DNA Purification Buffer. The libraries were sequenced on a MiSeq. Antibodies are listed in Table S1.

CUT&Tag data analysis

Raw fastq pairs were preprocessed and removed adapter and low-quality sequences using the cutadapt program (v2.7).71 Preprocessed reads were aligned to the mouse genome available at Gencode M1872 using Bowtie2 (v2.3.5)73 with the settings for Cut&Tag.74 Final reads were retained after removing non-uniquely mapped reads using samtools (v1.9) with “-q 20”75 and duplicated reads using picard (v2.21.4). Peaks were called using MACS2 (version 2.2.7.1).76,77 Browser track files were generated using the deepTools (v3.3.1).78

QUANTIFICATION AND STATISTICAL ANALYSIS

RNA polymerase II (Pol2) pausing index at the Myb locus

For Pol2 abundance over specific regions and Pol2 pausing analysis, bam files were filtered for properly paired reads only with samtools view, and then converted to bedpe format using bedtools bamtobed. The subset of fragments that were shorter than 1000 bp and mapped to chromosomes chr 1–19, X, Y was extracted to generate a bed file of fragments. Subsets of bed files were generated based on the desired size ranges (total, <120 bp, 120–270 bp, >270 bp). Fragments over genomic regions of interest were counted with bedtools intersect. The Pol2 pausing index was calculated as the ratio of the fragments per million (FPM) in the first exon of Myb over the rest of the gene body.

Statistical analysis

As indicated in the figure legends, data values reported in the figures are the mean and standard error of the mean (SEM). GraphPad Prism 8.0 (GraphPad Software) and Excel (Microsoft) were used to perform the statistical analyses. Unpaired Student’s t-test was used for significance test. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001.71,79,80

Supplementary Material

1

ACKNOWLEDGMENTS

We acknowledge computational support from Dr. Lecong Zhou, the NIDDK Genomics Core for sequencing support, and the NIH HPC (Biowulf) for computational support. This work was funded by the Intramural Program of the NIDDK, NIH (DK 075033, to A.D.). We thank Dr. Xiang Guo for kindly providing the mouse tissues.

Figure 1. The Myb −81-kb enhancer is the TSS for the non-coding Myrlin RNA

(A) ENCODE ChIP-seq of LDB1 complex components (GATA1, LDB1, and TAL1), CTCF, and Pol II in the Myb-Hbs1l locus in MEL cells. PolyA RNA-seq is shown in MEL cells. Intergenic LDB1 complex and CTCF binding sites are highlighted (orange and blue vertical bars).

(B) Nested PCR products from 5′ and 3′ RACE in MEL cells. M represents size marker.

(C) Poly-A RNA-seq and Ldb1 ChIP-seq tracks from ENCODE for MEL at the −81-kb enhancer and downstream Myrlin exon 2.

(D) Prediction of coding potential for Myrlin and select other transcripts as determined by CPAT (see text).

(E) Relative expression of Myrlin in nuclear and cytoplasmic fractions of MEL cells determined by RT-PCR. MALAT1 lncRNA and ActB provided nuclear and cytoplasmic controls, respectively.

(F) Total RNA of uninduced and induced MEL cells was used to determine relative expression of Myrlin and Myb by RT-PCR. Expression was normalized to ActB.

(G) Total RNA of E8.5 yolk sac cells (YS) and E14.5 fetal liver cells (FL) was used to determine relative expression of Myrlin, Myb, and Hbb-b1 by RT-PCR. Expression was normalized to ActB. Error bars indicate SEM of 3 independent biological experiments.

*p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test.

See also Figures S1.

Figure 2. CRISPR deletions at the TSS for Myrlin affect Myb expression

(A) Schematic diagram of the CRISPR-Cas9-mediated 17-bp deletion upstream of the Myrlin TSS. Smaller deletions were within the 17-bp deletion or extended upstream. PCR (below) shows the relative sizes for the 7-, 8-, and 17-bp deletion PCR products using wild-type or mutant gDNA.

(B) Relative expression of Myrlin (exon 1, exon1/2 junction, exon 2) and Myb (exon 2) in MEL CRISPR-Cas9 control cells (no gRNA) and MEL 7-, 8-, and 17-bp CRISPR-Cas9 deletion mutants. Expression was normalized to ActB.

(C) RNA Pol II-ChIP in MEL CRISPR-Cas9 control cells and MEL 17-bp CRISPR-Cas9 deletion mutant (ΔTSS) at the Myb gene promoter and enhancer sites (−36, −61, −68, −71, and −109 kb).

(D) ChIP for TBP at the Myb enhancer sites as in (C) for MEL CRISPR-Cas9 control cells and MEL ΔTSS CRISPR-Cas9 deletion mutant.

(E) Relative expression of Hbb-bh1, Hbb-b1, and Hbb-y in induced MEL CRISPR-Cas9 control cells and MEL ΔTSS CRISPR-Cas9 deletion mutant.

Error bars indicate SEM of 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test.

See also Figure S2.

Figure 3. Chromatin organization of the Myb locus and transcription factor occupancy is affected by reduction of Myrlin in ΔTSS cells

(A) 3C interaction frequency between the enhancers found within the Myb-Hbs1l intergenic region using the Myb promoter as the anchor (black bar) observed for ΔTSS and control cell uninduced MEL cells.

(B) GATA1 occupancy at Myb enhancers in control and ΔTSS uninduced MEL cell lines determined by ChIP-qPCR.

(C) TAL1 occupancy at Myb enhancers in control and ΔTSS uninduced MEL cell lines determined by ChIP-qPCR.

(D) LDB1 occupancy at Myb enhancers in control and ΔTSS uninduced MEL cell lines determined by ChIP-qPCR.

(E) H3K27ac normalized to H3 occupancy at Myb enhancers in control and ΔTSS uninduced MEL cell lines determined by ChIP-qPCR.

Error bars indicate SEM of 3 independent biological experiments. *p < 0.05 and **p < 0.01 by Student’s t test.

Figure 4. CRISPRi targeting of Myrlin compromises Myb transcription but not hub formation

(A) Expression of Myb and Myrlin monitored by RT-qPCR in CRISPRi uninduced MEL cells targeted with dCas9 sgRNA1 or sgRNA3 or without an sgRNA (control).

(B) ChIP-qPCR for H3K9me3 across the Myb locus before and after Myrlin CRISPRi in uninduced MEL cells.

(C) ChIP-qPCR for H3K27ac across the Myb locus before and after Myrlin CRISPRi.

(D) ChIP-qPCR for LDB1 across the Myb locus before and after Myrlin CRISPRi.

(E) 3C interaction frequency between the Myb enhancers using the Myb promoter as the anchor (black bar) observed after Myrlin CRISPRi in uninduced MEL cells targeted with dCas9 sgRNA or without an sgRNA (control).

Error bars indicate SEM of 3 independent biological experiments. *p < 0.05 and **p < 0.01 by Student’s t test.

Figure 5. Myrlin interacts with KMT2A complex

(A) ChIP-qPCR for H3K4me3 across Myb sequences.

(B) ChIP-qPCR for KMT2A at the Myb gene.

(C) ChIP-qPCR for KMT2A complex component WDR5 at the Myb gene.

(D) Myrlin RNA ChIP to detect interaction of Myrlin with KMT2A complex components WDR5, KTM2A, and menin. Glyceraldehyde 3-phosphate dehydrogenase served as control.

(E) RNA pull-down using biotinylated Myrlin and blotting using antibodies to KMT2A components. TUB served as a negative control.

Error bars indicate SEM of 3 independent biological experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by Student’s t test.

See also Figure S3.;

Figure 6. Pol II and CDK9 occupancy within the Myb enhancer hub is affected by CRISPRi targeting of Myrlin

(A) ChIP-qPCR for CDK9 in Myrlin CRISPRi uninduced MEL cells targeted with dCas9-KRAB or without an sgRNA (control).

(B) RNA Pol II occupancy in the Myb promoter/exon 1 region after Myrlin CRISPRi and in control cells.

(C) CUT&Tag for RNA Pol II Ser5 and Ser2 phosphorylated forms.

(D and E) Quantitation of CUT&Tag data showing Pol II Ser5P and Ser2P occupancy in the Myb gene body and at each of the enhancers.

(F) Separate analysis of the shorter (<120 bp) and longer (>270 bp) Pol II Ser5P CUT&Tag fragments displaying Pol II Ser5 occupancy.52 Orange color represents regions of RNA Pol II occupancy shared in both control and CRISPRi conditions. Dotted line represents TSS.

(G) Pausing index calculated for Pol II Ser5 across Myb.

Error bars indicate SEM of 2 independent biological experiments. *p < 0.05 and **p < 0.01 by Student’s t test.

See also Figure S4.

Figure 7. KLF1 interaction with Myrlin contributes to localization within the Myb enhancer hub

(A) Myrlin RNA pull-down was conducted to determine the efficiency of probes for ChIRP.

(B) ChIRP DNA pull-down by Myrlin across Myb and the Myb enhancers.

(C) RNA ChIP for KLF1.

(D) Biotinylated Myrlin pull-down and blotting with KLF1 antibodies. TUB served as a negative control.

(F) Model of the Myb locus enhancer hub. Transcription of Myb is depicted with and without the −81-kb enhancer Myrlin eRNA after Myrlin CRISPRi. Large, shaded circle represents Pol II and LDB1 transcription factor density within the Myb enhancer hub, which is diminished when Myrlin transcription is reduced.

Error bars indicate SEM of 3 independent biological experiments. *p < 0.05 and **p < 0.01 by Student’s t test.

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
anti-LDB1	Abcam	Cat# ab96799; RRID:AB_10679400	
anti-histone H3	Abcam	Cat# ab1791; RRID:AB_302613	
anti-histone H3K27ac	Abcam	Cat# ab4729; RRID:AB_2118291	
anti-Histone H3K9me3	Abcam	Cat# ab8898; RRID:AB_306848	
anti-Tubulin	Abcam	Cat# ab7291; RRID:AB_2241126	
anti-TBP	Abcam	Cat# ab63766; RRID:AB_1281140	
anti-KLF1	Active Motif	Cat# 61233; RRID:AB_2615069	
anti-SET1a	Bethyl Laboratories	Cat# A700-024-T; RRID:AB_2891825	
anti-Pol II Ser2	Cell signaling	Cat# 13499; RRID:AB_11378081	
anti-Pol II Ser5	Cell signaling	Cat# 13523; RRID:AB_11470388	
anti-WDR5	Cell signaling	Cat# 13105; RRID:AB_2620133	
anti-KMT2A/MLL1 N-Term	Cell signaling	Cat# 14689; RRID:AB_2688009	
anti- KMT2A/MLL1 C-Term	Cell signaling	Cat# 14197; RRID:AB_2688010	
anti-MENIN	Cell signaling	Cat# 6891; RRID:AB_10858216	
anti-SET1b	Cell signaling	Cat# 44922; RRID:AB_2799275	
anti-GAPDH	Cell signaling	Cat# 2118; RRID:AB_561053	
anti-Pol II	Millipore Sigma	Cat# 05–623; RRID:AB_309852	
anti-GATA1	Santa Cruz	Cat# sc-1233; RRID:AB_631559	
anti-TAL1	Santa Cruz	Cat# sc12984; RRID:AB_2199699	
anti-Pol II	Santa Cruz	Cat# sc-900; RRID:AB_2167474	
Normal goat IgG	Santa Cruz	Cat# sc-2028; RRID:AB_737167	
Normal mouse IgG	Santa Cruz	Cat# sc-2025; RRID:AB_737182	
Normal rabbit IgG	Santa Cruz	Cat# sc-2027; RRID:AB_737197	
Chemicals, peptides, and recombinant proteins	
iTaq Universal SYBR Green Supermix	Bio-RAD	Cat# 1725120	
Trans-Blot Turbo Transfer Pack	Bio-RAD	Cat# 1704156	
Nonfat dry milk	Bio-RAD	Cat# 1706404XTU	
High-Performance Electroporation Solution	BTXpress	Cat# 45-0801	
NaCl	Corning	Cat# 46-032-CV	
EDTA	Corning	Cat# 46-034-CI	
HEPES	Corning	Cat# 25-060-CI	
1M Tris-HCl pH 8.0	Corning	Cat# 46-031-CM	
1M Tris-HCl pH 7.5	Corning	Cat# 46-030-CM	
DMEM	Gibco	Cat# 11965118	
L-Glutamine	Gibco	Cat# A2916801	
Penicillin and Streptomycin	Gibco	Cat# 15070063	
Sodium pyruvate	Gibco	Cat# 11360070	
Blasticydin	Gibco	Cat# A1113903	
Puromycin	Gibco	Cat# A1113803	
PBS	Gibco	Cat# 10010023	
Recombinant Proteinase K Solution	Invitrogen	Cat# AM2546	
Superase-in	Invitrogen	Cat# AM2694	
Dynabeads MyOne Streptavidin C1	Invitrogen	Cat# 65001	
SSC	Invitrogen	Cat# AM9765	
LacZ biotinylated probes	Millipore sigma	Cat# CS216572	
Formamide	Millipore sigma	Cat# 344206-M	
DMSO	Millipore sigma	Cat# D4540	
Escort IV lipid transfection reagent	Millipore sigma	Cat# L3287	
Triton X-100	Millipore sigma	Cat# T8787	
Na-Deoxycholate	Millipore sigma	Cat# D6750	
PMSF	Millipore sigma	Cat# 78830	
LiCl	Millipore sigma	Cat# L9650	
NP40	Millipore sigma	Cat# NP40S	
Glycine	Millipore sigma	Cat# 50046	
Monothioglycerol	Millipore sigma	Cat# M6145	
Dexamethasone	Millipore sigma	Cat# D2915	
NEB buffer 2.1	New England Biolabs	Cat# B7202	
HindIII	New England Biolabs	Cat# R0104M	
T4 ligase	New England Biolabs	Cat# M0202M	
T4 DNA ligase buffer	New England Biolabs	Cat# B0202S	
Magnesium chloride	Quality Biological	Cat# 351-033-721EA	
TBS	Quality Biological	Cat# 351-086-131	
20% SDS	Quality Biological	Cat# 351-066-101	
Fetal bovine serum	R&D system	Cat# S12450	
Recombinant SCF	R&D system	Cat# 255-SC-200	
Erythropoietin	AMGEN	Cat# NDC55513-126-10	
EmeraldAmp GT PCR Master Mix	Takara	Cat# RR310A	
NuPAGE 4%–12% Bis-Tris Gel	Thermo Fisher Scientific	Cat# NP0321	
NuPAGE Transfer buffer	Thermo Fisher Scientific	Cat# NP0006-1	
NuPAGE running buffer	Thermo Fisher Scientific	Cat# NP0001	
SuperSignal West Dura Extended Duration Substrate	Thermo Fisher Scientific	Cat# 34075	
Superscript III First-Strand Synthesis System	ThermoFisher Scientific	Cat# 18080051	
Disuccinimidyl glutarate	ThermoFisher Scientific	Cat# 20593	
Halt™ Protease Inhibitor Cocktail	ThermoFisher Scientific	Cat# 78430	
Pierce™ ChIP-grade Protein A/G Magnetic Beads	ThermoFisher Scientific	Cat# 26162	
RNase A	ThermoFisher Scientific	Cat# EN0531	
StemPro-34	ThermoFisher Scientific	Cat# 10639011	
QIAquick gel extraction kit	Qiagen	Cat# 28704	
RNeasy Plus kit	Qiagen	Cat# 74134	
miRNAeasy Mini Kit	Qiagen	Cat# 217004	
ChIP DNA Clean & Concentrator Kit	Zymo Research	Cat# D5205	
Critical commercial assays	
RNA ChIP-IT Kit	Active Motif	Cat# 53024	
CUT&Tag-IT Assay Kit	Active Motif	Cat# 53160	
MEGAscript T7 Kit	Invitrogen	Cat# AM1333	
RLM-RACE kit	ThermoFisher Scientific	Cat# AM1700	
Pierce Magnetic RNA-Protein Pull-Down Kit	ThermoFisher Scientific	Cat# 20164	
EasySep™ Mouse Hematopoietic Progenitor Cell Isolation Kit	Stemcell	Cat# 19856	
Deposited data	
Raw CUTandTag data	This paper	GEO: GSE240060	
Experimental models: Cell lines	
Mouse erythroid leukemia cell	Vakoc et al., 200579	N/A	
Experimental models: Organisms/strains	
C57BL/6J	The Jackson Laboratory	Cat# 000664; RRID:IMSR_JAX:000664	
Oligonucleotides	
Table S1	This paper	N/A	
Recombinant DNA	
pSpCas9(BB)-2A-GFP	Addgene	48138	
Lenti-dCAS9-KRAB-blast	Addgene	89567	
LentiGuide-puro	Addgene	52963	
pCR4-TOPO vector	Invitrogen	K457502	
BAC DNA _Myb	ThermoFisher Scientific	RPCI-23 MM	
SIN40C.SFFV.GFP.miR30n	Addgene	169278	
Software and algorithms	
JASPAR motif analysis	Castro-Mondragon et al., 202242	http://jaspar.binf.ku.dk/	
GeneTargrter	Doench et al., 201680	http://genetargeter.mit.edu/	
Stellaris Probe Designer version 4.2	BIOSEARCH TECHNOLOGIES	https://www.biosearchtech.com/support/tools/design-software/stellaris-probe-designer	
Cutadapt program (v2.7)	Martin., 201171	https://cutadapt.readthedocs.io/en/stable/	
Bowtie2 (v2.3.5)	Langmead. and Salzberg., 201273	http://bowtie-bio.sourceforge.net/index.shtml	
Samtools (v1.9)	Danecek et al., 202175	http://www.htslib.org/	
Picard (v2.21.4)	MIT license	http://broadinstitute.github.io/picard	
MACS2 (version 2.2.7.1)	Zhang et al., 2008,76 Liu., 201476	https://github.com/taoliu/MACS	
DeepTools (v3.3.1)	Ramirez et al., 201478	https://deeptools.readthedocs.io/en/develop/	
Prism 8.0	GraphPad Software	https://www.graphpad.com/scientific-software/prism/	
Adobe Illustrator 2020	Adobe	https://www.adobe.com	

Highlights

Long non-coding RNA Myrlin is transcribed from the murine Myb −81-kb enhancer

CRISPRi of Myrlin separates Myb enhancer looping from transcription activation

Myrlin is necessary for KMT2A/MLL1 recruitment to Myb and for RNA Pol II pause release

Myrlin eRNA directly participates in activating Myb transcription

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2024.114378.

DECLARATION OF INTERESTS

The authors declare no competing interests.
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