{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Lucio Di Filippo"],"organism":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-15688"],"description":["Transcriptional regulation is tightly linked to chromatin organization, with H3K4me3 commonly marking both active and bivalent promoters. In embryonic stem cells (ESC), MLL2 is essential for H3K4me3 deposition at bivalent promoters, which has been proposed to facilitate the induction of major developmental genes during pluripotent cell differentiation. However, prior studies point to a functional discrepancy between the loss of H3K4me3 at bivalent promoters and the largely unaltered transcription of major developmental genes in Mll2-/- cells. In this study, we investigated MLL2-dependent gene regulation in mouse ESC and during their differentiation. Contrary to the prevailing view, we show that MLL2’s primary role is not to oppose Polycomb-mediated repression at the bivalent promoters of developmental genes. Instead, we identify a previously unrecognized regulatory function for MLL2 at the CG-rich 5' untranslated regions (5'UTR) of evolutionarily young LINE-1 (L1) transposable elements (TE). We found that MLL2 binds to the 5’UTR of L1 elements and is critical for maintaining their active state (H3K4me3 and H3K27ac), while preventing the accumulation of repressive H3K9me3. Using both global genomic approaches (i.e. RNA-seq, ChIP-seq and Micro-C) as well as targeted L1 deletions, we demonstrate that these MLL2-bound L1 elements act as enhancers, modulating the expression of neighboring genes in ESC and, more prominently, during differentiation. Together, our findings illuminate novel aspects of MLL2 regulatory function during early developmental transitions and highlight the emerging role of TE as key components of long-range gene expression control."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - The purified DNA was converted into a sequencing library using Illumina-compatible adaptors. Biotin-containing fragments were isolated using streptavidin beads prior to PCR amplification. For each cell type (e.g. WT Day 0), micro-C samples were prepared as two biological replicates, and for each replicate three libraries were prepared.","Nucleic Acid Extraction - The micro-C library was prepared using the Dovetail® Micro-C Kit according to the manufacturer’s protocol. Briefly, the chromatin was fixed with disuccinimidyl glutarate (DSG) and formaldehyde in the nucleus. The cross-linked chromatin was then digested in situ with micrococcal nuclease (MNase). Following digestion, the cells were lysed with SDS to extract the chromatin fragments, and the chromatin fragments were bound to Chromatin Capture Beads. Next, the chromatin ends were repaired and ligated to a biotinylated bridge adapter followed by proximity ligation of adapter-containing ends. After proximity ligation, the crosslinks were reversed, the associated proteins were degraded and the DNA was purified.","Sample Collection - mESC were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM (Thermo Fisher Scientific, 10829018) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, A5256801), 1x antibiotic and antimycotic solution (Sigma-Aldrich, A5955), 1x glutaMAX (Thermo Fisher Scientific, 35050038), 1x non-essential amino acids (NEAA; Thermo Fisher Scientific, 11140035), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985023) and leukemia inhibitory factor (LIF; made in-house). Cells were maintained at 37 °C with 5% CO2. For the multilineage differentiation, cells were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific, 25030024), 0.1 nM β-mercaptoethanol, 1x NEAA, 1x antibiotic and antimycotic solution and 1 µM retinoic acid (RA; Sigma-Aldrich, R2625) for 4 days.","Growth Protocol - mESC were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM (Thermo Fisher Scientific, 10829018) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, A5256801), 1x antibiotic and antimycotic solution (Sigma-Aldrich, A5955), 1x glutaMAX (Thermo Fisher Scientific, 35050038), 1x non-essential amino acids (NEAA; Thermo Fisher Scientific, 11140035), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985023) and leukemia inhibitory factor (LIF; made in-house). Cells were maintained at 37 °C with 5% CO2. For the multilineage differentiation, cells were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific, 25030024), 0.1 nM β-mercaptoethanol, 1x NEAA, 1x antibiotic and antimycotic solution and 1 µM retinoic acid (RA; Sigma-Aldrich, R2625) for 4 days.","Sample Treatment - Starting from a previously described Cre-inducible Mll2-KO mESC line, Mll1-KO mESC were generated via CRISPR editing. Two sg-RNAs were designed flanking the region to-be deleted for each line, using Benchling’s CRISPR tool (https://www.benchling.com/crispr). For each sgRNA, two oligonucleotides were synthesized , annealed and cloned into a CRISPR-Cas9 expression vector (pX330-hCas9-long-chimeric-grna-g2p, provided by Leo Kurian’s lab). mESC were transfected with CRISPR-Cas9 constructs using LipofectamineTM 3000 (Thermo Fisher Scientific, L3000001). For the generation of the double-KO mESC line, cells were treated with (Z)-4-Hydroxytamoxifen (4-OHT; Sigma-Aldrich, H7904) at a final concentration of 800 nM for 48 hours.","Sequencing - Each library was sequenced on an Illumina Novaseq6000 platform to generate 300 million 2x150 bp read pairs/library. Base calling was performed with the instrument software and reads were demultiplexed to FASTQ using standard Illumina procedures."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Micro-C data were processed using a custom pipeline implemented in Nextflow v23.04.4, based on the Dovetail Genomics protocol (https://micro-c.readthedocs.io/en/latest/). Reads were aligned with BWA-MEM (v0.7.17-r1188). Valid ligation events were identified using pairtools (v1.0.2) parse (--min-mapq 40, -- walks-policy 5unique, --max-inter-align-gap 30) and duplicates removed using pairtools dedup. BAM files were sorted and indexed using samtools (v1.13).","Data Transformation - Contact matrices were generated with cooler v0.9.2 using cooler cload pairix from pair-sorted, pairix-indexed pairs files. Matrices were balanced with cooler balance (ICE; per-bin weights stored in the weight column). Multi-resolution .mcool files were created with cooler zoomify (v0.9.2)."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"pubmed_abstract":["Transcriptional regulation is tightly linked to chromatin organization, with H3K4me3 commonly marking both active and bivalent promoters. In embryonic stem cells (ESC), MLL2 is essential for H3K4me3 deposition at bivalent promoters, which has been proposed to facilitate the induction of major developmental genes during pluripotent cell differentiation. However, prior studies point to a functional discrepancy between the loss of H3K4me3 at bivalent promoters and the largely unaltered transcription of major developmental genes in  Mll2 -/- cells. In this study, we investigated MLL2-dependent gene regulation in mouse ESC and during their differentiation. Contrary to the prevailing view, we show that MLL2’s primary role is not to oppose Polycomb-mediated repression at the bivalent promoters of developmental genes. Instead, we identify a previously unrecognized regulatory function for MLL2 at the CG-rich 5’ untranslated regions (5’UTR) of evolutionarily young LINE-1 (L1) transposable elements (TE). We found that MLL2 binds to the 5’UTR of L1 elements and is critical for maintaining their active state (H3K4me3 and H3K27ac), while preventing the accumulation of repressive H3K9me3. Using both global genomic approaches (i.e. RNA-seq, ChIP-seq and Micro-C) as well as targeted L1 deletions, we demonstrate that these MLL2-bound L1 elements act as enhancers, modulating the expression of neighboring genes in ESC and, more prominently, during differentiation. Together, our findings illuminate novel aspects of MLL2 regulatory function during early developmental transitions and highlight the emerging role of TE as key components of long-range gene expression control."],"study_type":["Hi-C"],"species":["Mus musculus"],"pubmed_title":["MLL2 facilitates long-range gene regulation through LINE1 elements"],"pubmed_authors":["Lara Zorro Shahidian,  Lucio Di Filippo,  Sarah Malika Robert, Alvaro Rada-Iglesias","Lara Zorro Shahidian","Alvaro Rada-Iglesias","Lucio Di Filippo"],"additional_accession":[]},"is_claimable":false,"name":"MLL2 facilitates long-range gene regulation through LINE1 elements [Micro-C]","description":"Transcriptional regulation is tightly linked to chromatin organization, with H3K4me3 commonly marking both active and bivalent promoters. In embryonic stem cells (ESC), MLL2 is essential for H3K4me3 deposition at bivalent promoters, which has been proposed to facilitate the induction of major developmental genes during pluripotent cell differentiation. However, prior studies point to a functional discrepancy between the loss of H3K4me3 at bivalent promoters and the largely unaltered transcription of major developmental genes in Mll2-/- cells. In this study, we investigated MLL2-dependent gene regulation in mouse ESC and during their differentiation. Contrary to the prevailing view, we show that MLL2’s primary role is not to oppose Polycomb-mediated repression at the bivalent promoters of developmental genes. Instead, we identify a previously unrecognized regulatory function for MLL2 at the CG-rich 5' untranslated regions (5'UTR) of evolutionarily young LINE-1 (L1) transposable elements (TE). We found that MLL2 binds to the 5’UTR of L1 elements and is critical for maintaining their active state (H3K4me3 and H3K27ac), while preventing the accumulation of repressive H3K9me3. Using both global genomic approaches (i.e. RNA-seq, ChIP-seq and Micro-C) as well as targeted L1 deletions, we demonstrate that these MLL2-bound L1 elements act as enhancers, modulating the expression of neighboring genes in ESC and, more prominently, during differentiation. Together, our findings illuminate novel aspects of MLL2 regulatory function during early developmental transitions and highlight the emerging role of TE as key components of long-range gene expression control.","dates":{"release":"2025-10-23T00:00:00Z","modification":"2026-05-27T12:40:51.805Z","creation":"2025-10-09T16:38:31.871Z"},"accession":"E-MTAB-15688","cross_references":{"ENA":["ERP181290"],"EFO":["EFO_0007693","EFO_0002944","EFO_0004170","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184","EFO_0003969"],"doi":["10.1101/2025.08.10.669526"]}}