biostudies-arrayexpressLucio Di FilippoMus musculusTrailmakerhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17228Transcriptional 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.biostudies-arrayexpressSequencing - Each library was sequenced on an Illumina NovaSeq X platform to a depth of approximately 150 million paired-end reads (2 × 150 bp) per library, with 10% PhiX spike-in added as an internal sequencing control.Nucleic Acid Extraction - Fixed single-cell suspensions were processed using the Parse Biosciences Evercode™ WT Mini scRNA-seq kit. Cells underwent split-pool combinatorial barcoding, in which cells were repeatedly distributed into wells and labeled with unique molecular barcodes. During the first barcoding round, cell-specific barcodes were incorporated and reverse transcription was performed to generate barcoded cDNA from cellular RNA. Subsequent barcoding rounds further labeled cDNA molecules with additional barcode sequences, enabling combinatorial indexing of individual cells. The resulting barcoded cDNA was pooled, amplified, and prepared for next-generation sequencing.Library Construction - Barcoded cDNA was fragmented and processed to generate sequencing-compatible library molecules containing cell-identifying barcode sequences and transcript-derived fragments. Libraries were amplified, purified, and assessed for quality and concentration before sequencing.Sample Collection - 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. Single-cell suspensions were fixed using the Evercode™ Cell Fixation workflow compatible with the Parse Biosciences Evercode WT Mini scRNA-seq kit. Briefly, freshly isolated cells were washed and incubated with the fixation reagents provided in the Parse Biosciences fixation kit to preserve endogenous RNA profiles and stabilize cellular material. Fixed cells were subsequently washed, counted, and stored under recommended conditions prior to whole-transcriptome split-pool combinatorial barcoding library preparation.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Data processing and analysis were performed using the Parse Biosciences' Trailmaker pipeline (Parse Biosciences). FASTQ files were aligned to the mouse reference genome (GRCm39). Each dataset generated from a Parse Biosciences Evercode™ WT Mini scRNA-seq kit,Data Transformation - Quantification and count matrices for each sample were processed using Parse Biosciences' Trailmaker. Cells were filtered to remove background, low-quality, and doublet barcodes based on transcript count distribution. Data normalization was performed using the LogNormalize method within the Seurat framework, followed by dimensionality reduction via principal component analysis. Batch effects across samples were corrected using the Harmony integration method prior to clustering.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XTranscriptional 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.RNA-seq of coding RNA from single cellsMus musculusMLL2 facilitates long-range gene regulation through LINE1 elementsLara Zorro Shahidian, Lucio Di Filippo, Sarah Malika Robert, Alvaro Rada-IglesiasLara Zorro ShahidianAlvaro Rada-IglesiasLucio Di FilippofalseMLL2 facilitates long-range gene regulation through LINE1 elements [scRNA-seq]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.2026-07-12T00:00:00Z2026-07-12T01:00:51.452Z2026-06-25T14:41:13.59ZE-MTAB-17228ERP195728EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_000418410.1101/2025.08.10.669526