{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Marika Oksanen"],"organism":["Homo sapiens"],"software":["CLAM peak caller","Bowtie2","MACS2","Bismark","Bismark, DSS, ChIPSeeker"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14910"],"description":["This study investigates the molecular mechanisms by which the DNA/RNA-binding protein HNRNPU regulates gene expression during early human neural development. Pathogenic variants in HNRNPU cause a severe neurodevelopmental disorder, but the underlying mechanisms remain poorly understood. To explore the role of HNRNPU in transcriptional and epigenetic regulation, we used human induced pluripotent stem cell (hiPSC)-derived neuroepithelial stem cells (NES) and differentiating neural cells as model systems. We performed formaldehyde crosslinking and ribonucleoprotein immunoprecipitation followed by RNA sequencing (fRIP-seq) to identify RNA targets bound by HNRNPU, whole-genome bisulfite sequencing (WGBS) to assess the impact of HNRNPU silencing on DNA methylation landscapes, and CUT&RUN profiling to map chromatin marks at regulatory regions. Together, these datasets provide a comprehensive view of HNRNPU’s interactions with RNA, and its influence on the epigenetic state of key developmental genes. The data can be used to study RNA–protein interactions, DNA methylation dynamics, and chromatin regulation during neural differentiation. Mass spectrometry data was deposited to PRIDE under accession number PXD061718."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - The libraries were sequenced on NovaSeq X Plus PE150 platform, yielding on average ~20X sequencing depth per sample.","Nucleic Acid Extraction - fRIP-Seq was performed on samples from both CTRLMale and CTRLFemale cell lines at D0 and from CTRLMale at D28. Beads and input samples were subjected to reverse crosslinking. The beads were resuspended to 56 µL Ultra Pure water and 33 µL of reverse crosslinking buffer (PBS--, 6% N-lauroyl sarcosine, 30 mM EDTA, 7.5 mM DTT), 10 mL of Proteinase K and 1 mL of RNaseOUT were added. After incubation (1 h at 42°C then 1 h at 55°C), samples were transferred to 500 μL TRIzol for RNA extraction. Samples in TRIzol reagent (Invitrogen) were phase-separated using chloroform and isopropanol. The aqueous phase was transferred to RNA extraction columns, and the final extraction was carried out with ReliaPrep RNA Cell Miniprep kit (Promega Z6012), including DNA digestion step.","Nucleic Acid Extraction - DNA was extracted after HNRNPU silencing using the PureLink Genomic DNA Mini kit (Thermo Scientific) following the manufacturer’s instructions","Sequencing - sequencing was run with Nextseq 2000, P2, and 100 cycles.","Sequencing - The libraries were sequenced on the NovaSeq X Plus PE150 platform by Novogene (Germany).","Library Construction - sequencing libraries were prepared with 5 ng of enriched DNA using CUTANA CUT&RUN Library Prep Kit (Epicypher, 14-1001) following manufacturer’s recommendations.","Sample Collection - CUT&RUN was performed using CUTANA ChIC/CUT&RUN Kit v5 (Epicypher, 14-1048) following manufacturer’s protocol on extracted nuclei from untreated CTRLMale cells at D0 and D7 time points. Briefly, 5x10^5 nuclei were coupled with Concanavalin A beads with 0.01% Digitonin and incubated overnight at 4°C with 0.5 µg antibody: IgG, H3K4me3, H3K27me3 (all supplied with the kit), HNRNPU (Abcam, ab20666) or TET3 (GeneTex, GTX121453).","Library Construction - The library preparation was performed using Accel-NGS Methyl-Seq DNA Library Kit for illumina (30096, Swift Biosciences, USA).","Nucleic Acid Extraction - Enriched DNA was isolated using SPRI beads supplied in the CUT&RUN kit.","Library Construction - An equal amount of RNA, 18 ng, was used for library preparation with Illumina Stranded Total RNA Prep with Ribo-Zero Plus","Sample Treatment - To silence HNRNPU, NES cells were seeded in 31,500 cells/cm2, left to adhere for 4 h, and transfected with 1 µM Accell SMARTpool siRNA targeting HNRNPU mRNA (Dharmacon E-013501-00-0010) or Accell Nontargeting siRNA (Dharmacon D-001950-01-20) according to manufacturer’s protocol. Transfected NES cells were harvested 72 h later. Cells undergoing differentiation were transfected once in the NES phase, once when switching to differentiation media, once on day 4 of differentiation, and collected on day 7 of differentiation.","Sample Collection - Cells were washed with DPBS, dissociated with TrypLE Express (D0) or Accutase and TrypLE Select and incubated with Neural Isolation Enzyme (D28), and resuspended in Wash medium (DMEM/F12+GlutaMAX, 5% FBS, 10 U/mL penicillin/streptomycin). Cells were gently resuspended and spun at 400 × g for 3 min and resuspended in Wash medium or 1 mL of PBS, supplemented with 0.15 % Triton-X and 30 μL RNase A (Sigma, R4642) to degrade all RNA as described earlier(31). Cells were counted, and wash media was added to 5×106 cells/mL concentration. Cells were crosslinked with 0.1% formaldehyde for 10 min with rotation at room temperature, the reaction was stopped with 125 mM glycine for 5 min with rotation at room temperature, and washed twice in cold DPBS with 1x Halt protease inhibitor cocktail (PIC, Thermo Scientific). Cell pellets were stored at −80°C.  Cell lysis and protein immunoprecipitation were performed following an existing protocol (32) with modifications. Shortly, cell pellets were lysed in polysome lysis buffer (PLB, 100 mM KCL, 5 mM MgCl2, 10 mM HEPES (pH 7.0), 0.5% IGEPAL, 1 mM DTT, 100 units/mL RNaseOUT, 1xPIC). The cell lysate was incubated on ice for 10 min before centrifugation at 10, 000 × g at 4°C. The lysate was transferred to a fresh microfuge tube and pre-cleared by incubating with Dynabeads Protein G (Life Technologies) with 25 μL of beads per sample for 30 min at 4°C with slow rotation. For fRIP-Seq, 5% of the sample was removed for input and stored at −80°C. Protein concentrations were measured using Pierce BSA protein assay kit (Thermo Scientific) and an equal protein amount of lysate was divided into two microfuge tubes per replicate, 1.5 and 1 mg for fRIP-Seq and IP-MS, respectively and NT-2 buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.05% IGEPAL, 1 mM DTT, 400 units/mL RNaseOUT, 16.5 mM EDTA) was added to final volume 1 mL. Either the HNRNPU antibody (ab20666, Abcam) or rabbit IgG (12-370, Sigma-Aldrich) was added at a concentration of 7 μg per 1 mg of total protein, and the reactions were incubated for 2 h at 4°C with rotation. Dynabeads protein G was prepared according to the manufacturer’s instructions, and 50 µL per 1 mg of total protein was added to fRIP-Seq and IP-MS tubes, respectively. The incubation was prolonged for 1 h. The beads were washed thrice with 1 mL ice-cold NT-2 buffer, rotating for 10 min at 4°C. After a final wash, the supernatant was removed, and beads were stored at −80°C."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - Reads were aligned to the reference genome (Homo Sapiens CRCh38 Ensembl 109) using Bismark (v0.24.0) with parameters –score_min L,0,-0.2 and -X 700 --dovetail.","Data Transformation - RNA-seq reads were trimmed with bbduk of bbmap (v.39.01) and mapped to Homo Sapiens CRCh38 Ensembl 109 with STAR (38) (v.2.7.9a). Ribosomal reads and PCR duplicates were removed using BEDtools (v.2.31.1) intersecting with CHRCh38_rRNA.bed file (RSeQC) and Samtools (v.1.2) markdup -r, respectively. These files were subjected to CLAM (39) (v1.2.0) preprocessing, after which the peaks were called using only the uniquely mapped reads and bin size 100, read-tagger-method median, --unique-only TRUE, and –pool TRUE. Subsequent peak files were annotated with CLAM peak annotator","Sequence Alignment - Reads were aligned to the Homo Sapiens CRCh38 Ensembl 109 using Bowtie2 (47) (v2.5.4). Up to two valid alignments were reported (-k 2) and the --dovetail option was used.","Data Transformation - Only uniquely mapped reads were retained by removing reads with secondary alignments (XS tag). Reads overlapping ENCODE DAC blacklist regions were excluded using BEDTools (v2.31.0) and duplicate reads were identified and removed as described earlier. Peak calling was performed using MACS2 (48) (v2.2.9.1) with IgG files as control. Broad peaks were identified using the parameters –broad and --broad-cutoff 0.05. To avoid model building, the –nomodel option was applied and read extension and shift were set to 300 bp and 150 bp, respectively. To identify reproducible peaks between biological replicates, peaks called from each replicate were intersected using BEDTools. Peaks were considered reproducible if they overlapped by at least 50% of their length in both replicates (-f 0.5 -r -u). The resulting consensus peak sets were used for downstream analyses. The peaks were annotated using ChIPSeeker R package (v1.38.0) using default settings.","Data Transformation - Cytosine methylation levels were extracted using Bismark_methylation_extractor (--no-overlap). Differentially methylated regions were identified using DSS software with parameters smoothing.span=200, minlen = 50, minCG=3, dis.merge=100, pct.sig=0.5 and p.threshold=1x10-5. A significant DMR was defined as methylation difference > |0.1|. DMRs were annotated using ChIPseeker (v1.38.0), using the default priority and promoters defined as +-2000 bp."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X","NextSeq 2000"],"pubmed_abstract":["<h4>ABSTRACT</h4>  HNRNPU is an RNA-binding protein with diverse roles in transcriptional and post-transcriptional regulation. Pathogenic genetic variants of HNRNPU cause a severe neurodevelopmental disorder (NDD), but the underlying molecular mechanisms are unclear. Here, we comprehensively investigate the HNRNPU molecular interactome by integrating protein-protein interaction (PPI) mapping, RNA target identification, and genome-wide DNA methylation profiling in human neuroepithelial stem cells and differentiating neural cells. We identified extensive HNRNPU-centered networks, including a strong association with the mammalian SWI/SNF chromatin-remodelling complex, and uncovered a previously unrecognized role in translation. We present evidence that HNRNPU associates with mRNAs encoding proteins important for neuronal development, including several linked to NDDs. Silencing HNRNPU reprogrammed methylation dynamics at regulatory regions, particularly at active and bivalent promoters of neurodevelopmental transcription factors. Integrative analysis across PPI, RNA, and methylome datasets identified 19 converging genes at all three molecular levels, including NDD genes within the SWI/SNF complex and RNA-processing machinery such as  SYNCRIP . Together, these data showcase HNRNPU as a central coordinator of RNA metabolism and epigenetic remodelling during neural differentiation, linking RNA-binding, chromatin organization, and DNA methylation to the pathogenesis of HNRNPU-related NDDs."],"study_type":["RIP-seq"],"species":["Homo sapiens"],"pubmed_title":["Molecular interactome of HNRNPU reveals regulatory networks in neuronal differentiation and DNA methylation"],"pubmed_authors":["Marika Oksanen","Marika Oksanen, Francesca Mastropasqua, Krystyna Mazan-Mamczarz, Jennifer L. Martindale, Xuan Ye, Abishek Arora, Nirad Banskota, Myriam Gorospe, Kristiina Tammimies"],"additional_accession":[]},"is_claimable":false,"name":"HNRNPU RNA targets and DNA methylation changes during neural differentiation","description":"This study investigates the molecular mechanisms by which the DNA/RNA-binding protein HNRNPU regulates gene expression during early human neural development. Pathogenic variants in HNRNPU cause a severe neurodevelopmental disorder, but the underlying mechanisms remain poorly understood. To explore the role of HNRNPU in transcriptional and epigenetic regulation, we used human induced pluripotent stem cell (hiPSC)-derived neuroepithelial stem cells (NES) and differentiating neural cells as model systems. We performed formaldehyde crosslinking and ribonucleoprotein immunoprecipitation followed by RNA sequencing (fRIP-seq) to identify RNA targets bound by HNRNPU, whole-genome bisulfite sequencing (WGBS) to assess the impact of HNRNPU silencing on DNA methylation landscapes, and CUT&RUN profiling to map chromatin marks at regulatory regions. Together, these datasets provide a comprehensive view of HNRNPU’s interactions with RNA, and its influence on the epigenetic state of key developmental genes. The data can be used to study RNA–protein interactions, DNA methylation dynamics, and chromatin regulation during neural differentiation. Mass spectrometry data was deposited to PRIDE under accession number PXD061718.","dates":{"release":"2026-02-02T00:00:00Z","modification":"2026-02-02T02:01:40.604Z","creation":"2025-03-07T17:24:48.343Z"},"accession":"E-MTAB-14910","cross_references":{"EFO":["EFO_0002944","EFO_0004170","EFO_0005310","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184","EFO_0003969"],"doi":["10.1101/2025.02.19.638869"]}}