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ABSTRACT The nuclear matrix, a proteinaceous gel composed of proteins and RNA, is an important nuclear structure that supports chromatin architecture, but its role in human pluripotent stem

cells (hPSCs) has not been described. Here we show that by disrupting heterogeneous nuclear ribonucleoprotein U (HNRNPU) or the nuclear matrix protein, Matrin-3, primed hPSCs adopted

features of the naive pluripotent state, including morphology and upregulation of naive-specific marker genes. We demonstrate that _HNRNPU_ depletion leads to increased chromatin

accessibility, reduced DNA contacts and increased nuclear size. Mechanistically, HNRNPU acts as a transcriptional co-factor that anchors promoters of primed-specific genes to the nuclear

matrix with POLII to promote their expression and their RNA stability. Overall, HNRNPU promotes cell-type stability and when reduced promotes conversion to earlier embryonic states. Access

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WIDESPREAD REORGANISATION OF PLURIPOTENT FACTOR BINDING AND GENE REGULATORY INTERACTIONS BETWEEN HUMAN PLURIPOTENT STATES Article Open access 07 April 2021 B1 SINE-BINDING ZFP266 IMPEDES

MOUSE IPSC GENERATION THROUGH SUPPRESSION OF CHROMATIN OPENING MEDIATED BY REPROGRAMMING FACTORS Article Open access 30 January 2023 RNA IS ESSENTIAL FOR PRC2 CHROMATIN OCCUPANCY AND

FUNCTION IN HUMAN PLURIPOTENT STEM CELLS Article 06 July 2020 DATA AVAILABILITY Sequencing data that support the findings of this study have been deposited in the Gene Expression Omnibus

under accession code GSE242351. Previously published data that were re-analysed here are available as follows. Naive and primed RNA-seq data are from GSE93241 (ref. 80), GSE75868, GSE85689

(ref. 28), PRJNA383735 (ref. 38), PRJNA397941 (ref. 29) and CNP0001454 (ref. 46). Aggregate ChIP-seq data are from Cistrome106; MAR-seq data were from GSE87671 (ref. 12). Other images

supporting the findings of this study are available from Figshare at https://doi.org/10.6084/m9.figshare.24739365 (ref. 115). Data supporting the findings of this study are available from

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references ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (32150710521 and 32270597 to A.P.H., 62375116 to Y.L. and 32270574 to D.L.), Shenzhen

Medical Research Fund (B2302038 to Y.L.), Key Technology Research and Development Program of Shandong Province (2021CXGC010212 to Y.L.), Shenzhen Science and Technology Innovation Program

(JCYJ20220818100416036 and KQTD20200820113012029 to Y.L.), Basic and Applied Basic Research Fund of Guangdong Province (2024A1515011565 to Y.L.) and the Science and Technology Projects in

Guangzhou (2023A03J0045 to D.L.). Additional support was rendered by the Center for Computational Science and Engineering of the Southern University of Science and Technology. We acknowledge

the assistance of SUSTech Core Research Facilities. The H9 TPRX1-GFP cells were a kind gift from M. A. Esteban (Beijing Genomics Institute, Shenzhen), and the EpiSCs were a kind gift from

C. Jiekai (Guangzhou Institutes of Biomedicine and Health, Guangzhou). AUTHOR INFORMATION Author notes * These authors contributed equally: Gang Ma, Xiuling Fu, Lulu Zhou. * These authors

jointly supervised this work: Dongwei Li, Yiming Li, Andrew P. Hutchins. AUTHORS AND AFFILIATIONS * Department of Systems Biology, School of Life Sciences, Southern University of Science and

Technology, Shenzhen, China Gang Ma, Xiuling Fu, Isaac A. Babarinde, Liyang Shi, Jiao Chen, Zhen Xiao, Yu Qiao, Lisha Ma, Yuhao Ou, Yuhao Li, Chen Chang, Boping Deng, Li Sun & Andrew P.

Hutchins * Department of Biomedical Engineering, Southern University of Science and Technology, Shenzhen, China Lulu Zhou & Yiming Li * Department of Reproductive Medicine, The First

Affiliated Hospital of Xi’an Jiaotong University, Xi’an, China Wenting Yang & Guoqing Tong * Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong

Higher Education Institutes, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Ran Zhang & Dongwei Li Authors * Gang Ma View author publications You can

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author inPubMed Google Scholar CONTRIBUTIONS G.M. performed experiments, designed the project, interpreted data and wrote and revised the paper. X.L.F. performed experiments and revised the

paper. L.L.Z. performed the SMLM experiments and data analysis. L.Y.S. analysed the Hi-C data and performed aggregate ChIP-seq data analysis. I.A.B. and Y.H.L. assisted with the

bioinformatic analysis. W.Y., J.C., Z.X., Y.Q., L.M., Y.O., L.S., B.D. and R.Z. assisted with experiments. C.C. generated plasmids and key reagents. D.L. helped with analysis, revised the

paper and acquired funding. Y.L. supervised the super-resolution imaging, revised the paper and acquired funding. D.L., G.T. and Y.L. interpreted and analysed the findings. A.P.H. supervised

the project, performed some of the analysis, interpreted the data, wrote and revised the paper and acquired funding. All other authors contributed to the interpretation of results,

performed experiments or bioinformatic analysis and helped revise the paper. CORRESPONDING AUTHORS Correspondence to Dongwei Li, Yiming Li or Andrew P. Hutchins. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Cell Biology_ thanks the anonymous reviewers for their contribution to the peer

review of this work. Peer reviewer reports are available. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 NUCLEAR MATRIX GENES FORM A UNIQUE EXPRESSION PATTERN IN PRE-IMPLANTATION EMBRYOS. A Heatmap of the expression nuclear matrix

genes (GO:CC:0016363). Data is from https://github.com/oaxiom/human, and45. B. Boxplot showing the normalized expression of the nuclear matrix genes from panel A in embryonic (panel A,

orange labelled cell types) and somatic cell types (all other colours), as indicated in panel A. Note that the placental tissues were removed from the analysis, as were the hPSC samples, as

they reflect extraembryonic cells and _in vitro_ cells, respectively. Significance is from a two-sided Mann–Whitney U-test. Data is from the re-analysis performed in

https://github.com/oaxiom/human, and45,116. Data includes 3 replicates or more for each cell type or tissue, and the embryonic category contains 16 embryonic cell types or tissues and the

somatic category contains 69 cell types or tissues. C. Bar plot showing the expression of _HNRNPU_ in a selection of human cell types and pre-implantation embryonic stages. Data is from the

re-analysis performed in https://github.com/oaxiom/human, and45,116. D. Bar plot showing the expression of _Hnrnpu_ in a selection of mouse cell types and embryonic stages. Data is from the

re-analysis performed in93. E. Scatter plots showing the expression of _HNRNPU_ versus a selection of genes expressed in early embryonic stages (_DPPA3_, _DPPA5_, _TPRX1_, and _ZSCAN4_) in

various cell types from Extended Data Fig. 1a. Embryonic cell types are marked in orange, and somatic cell types are marked with the indicated colour according to their presumed germ

lineage, as indicated in the key in Extended Data Fig. 1a. Data is from https://github.com/oaxiom/human, and45. Correlation and significance are from Pearson’s correlation coefficient test

(R). F. Scatter plots showing the expression of _HNRNPU_ versus the nuclear matrix proteins _MATR3_ and _SAFB_ in various cell types from Extended Data Fig. 1a. Embryonic cell types are

marked in orange, and somatic cell types are marked according to their presumed germ lineage, as indicated in the key in Extended Data Fig. 1a. Data is from https://github.com/oaxiom/human,

and45. Correlation and significance are from Pearson’s correlation coefficient test (R). Source data EXTENDED DATA FIG. 2 KNOCKDOWN OF _HNRNPU_ CAUSES HPSCS TO ADOPT AN EARLIER NAÏVE-LIKE

EMBRYONIC STATE. A qRT-PCR data showing the RNA levels of _HNRNPU_ in hPSCs transfected with the indicated shRNAs. Data is from one replicate, each in technical triplicate. B. Line plot

showing the population doubling in hPSCs when transfected with the indicated shRNA. Data is from three biological replicates, with one technical replicate each. C. TUNEL (terminal

deoxynucleotidyl transferase-mediated dUTP nick end-labelling) Apoptosis assay in WT hPSCs (H1) or DNase I treated cells as a positive control when transfected with the indicated shRNA.

Scale bar = 100 µm. The experiment was performed twice with similar results. D. Heatmap showing the pair-wise correlation (Pearson’s R) for RNA-seq data generated from hPSCs transfected with

the indicated shRNAs. rp = Repeat. E. qRT-PCR data for a selection of pluripotent and naïve marker genes in hPSCs transfected with shRNAs against _HNRNPU_ or _LUC_ (control). Data is from

three biological replicates, each in technical triplicate. F. qRT-qPCR data for a selection of naïve and primed-specific genes. Data is from three biological replicates, each in technical

triplicate. G. Bar chart showing significantly overrepresented gene ontology biological process terms in the down (left bar chart) and up-regulated (right bar chart) genes defined in Fig.

1e. Significance was determined by go-seq with a Bonferroni-Hochberg (BH)-corrected p-value (q-value) of less than 0.01. H. Volcano plot showing the differentially regulated TEs in the

_HNRNPU_ knockdown cells. Significantly different TEs were classified if their log2 fold change was >0.53 (1.5-fold) and they had a BH-corrected p-value (q-value) less than 0.01. Data is

from three biological replicates. I. qRT-PCR for _HNRNPU_ in primed hPSCs transfected with the indicated shRNA and grown in 4CL medium for the indicated number of days. Data is from one

biological replicate, each in technical triplicate. J. qRT-PCR for a selection of naïve and primed-specific genes in primed hPSCs transfected with the indicated shRNA and grown in 4CL medium

for the indicated number of days. Data is from one biological replicate, each in technical triplicate. K. Immunofluorescence images of KLF4 and HNRNPU in naïve hPSCs grown in 4CL medium for

6 days and transfected with the indicated shRNA. Scale bar = 10 µm. The experiment was performed twice with similar results. L. Grey-scale values for KLF17 was measured in at least 21 cells

from hPSCs grown in 4CL medium for six days transfected with the indicated shRNA. The box represents the first and third quartiles, the midpoint is the median and the whiskers are 1.5x the

interquartile mean. Significance is from a two-sided Welch’s t-test for panels b, e, f, l. EXTENDED DATA FIG. 3 REDUCED _HNRNPU_ PROMOTES CELL TRANSITIONS TO EARLIER EMBRYONIC-LIKE CELL

TYPES. A qRT-PCR for _Hnrnpu_ in primed EpiSCs transfected with the indicated shRNA. Data is from one biological replicates, each in technical triplicate. B. Schematic model of mouse primed

(EpiSC) to naïve (mESC) stem cell transition. C. Dot plot showing the percentages of GFP+ cells (GFP is driven from a Pou5f1::GFP reporter that is only expressed in naïve mESCs) in the

primed-to naïve conversion when transfected with the indicated shRNA. This experiment was performed three times. D. qRT-PCR results for pluripotency genes _Klf2_ and _Klf4_ in the conversion

of primed-to naïve stem cells when transfected with the indicated shRNA. The experiment was performed in biological triplicate. E. qRT-PCR results for naïve-specific genes _Tfcp2l1_ and

_Prdm14_ in the conversion of primed-to naïve stem cells when transfected with the indicated shRNA. The experiment was performed in biological triplicate. F. Schematic model of mouse naïve

mESCs to EpiLCs (Epiblast-like cell) differentiation. G. Percentage of GFP+ percentages cells in the naïve to EpiLC differentiation in cells transfected with the indicated shRNA. This

experiment was performed three times. H. qRT-PCR results in EpiLCs for selected pluripotency or primed-specific genes in cells transfected with the indicated shRNA. The experiment was

performed in biological duplicate. I. GSEA plot showing the enrichment of MORULA-specific genes in the ranked genes when _HNRNPU_ was knocked down. MORULA-specific genes are defined in ref.

116. J. Heatmap of the leading-edge genes specific to MORULA form the GSEA in panel I. Embryo data is from the re-analysis in ref. 116. Data is log2(fold change) relative to sh_LUC_ sample.

K. Schematic model of the induction of 8CLCs from primed hPSCs using 4CL and e4CL medium. P3 = passage 3. L. Left panel, representative FACS scatter plots and dotplots on passage 3 in the

conversion of hPSCs to 8CLCs. The y-axis represents _TPRX1_-GFP. NC is negative control (Primed hPSCs), compared to hPSCs on passage 3 in 4CL/e4CL medium transfected with the indicated

shRNA. The experiment was performed twice in biological duplicate. Right panel, Gating strategy for this experiment and all FSC related experiments. M. Percentages of TPRX1-GFP+ cells in

cells grown in 4CL/e4CL medium and transfected with the indicated shRNA. This experiment was performed two times. N. Schematic showing the reprogramming of mouse embryonic fibroblast (MEF)

into iPSCs. O. qRT-PCR for _Hnrnpu_ in MEFs transfected with the indicated shRNA. MEFs were transfected for 5 days. Data is from three biological replicates, each in technical triplicate. P.

AP (alkaline phosphatase) stained images of day 13 reprogramming MEFs transfected with OSKM reprogramming cassette and the indicated shRNA. Scale bar = 5 mm. Q. Number of AP+ colonies at

day 13 of a reprogramming time course, in MEFs transfected with the indicated shRNA. Data is from three biological replicates, each in technical singlicate. R. Representative bright-field

and GFP images of iPSCs transfected with the indicated shRNA. Scale bar = 100 µm. S. Number of GFP+ colonies at day 13 of a reprogramming time course, in MEFs transfected with the indicated

shRNA. Data is from three biological replicates, each in technical singlicate. T. qRT-PCR for selected mesenchymal-epithelial transition (MET) or epithelial-mesenchymal transition (EMT)

genes at day 5 of a reprogramming time course. The experiment was performed once with three technical replicates. Significance is from a two-sided Welch’s t-test for panels c, d, e, g.

EXTENDED DATA FIG. 4 HNRNPU INTERACTS WITH MATR3. A Heatmap of the Co-IP/MS intensity scores and the ‘call’ for present or absent. A protein was considered present if it had at least 1

unique peptide and a minimum intensity of 1,000,000 (Razor+Unique), and an intensity that was at least 2-fold higher than the anti-FLAG control. HNRNPU Co-IP/MS was performed three times,

the MATR3 Co-IP/MS was performed once. The list of proteins used here contains HNRNPU, MATR3, and proteins from the AnimalTFDB and the Epifactors databases91,92. The full list of interacting

proteins can be found in Supplementary Table 3. # indicates the biological repeat number. B. A network where each node is an interacting protein and each edge indicates an interaction with

HNRNPU or MATR3. The network includes all HNRNPU and MATR3 interacting proteins. See also Supplementary Table 3. C. Western blot of reciprocal co-IP for HNRNPU or MATR3. This experiment was

performed twice with similar results. D. Gene ontology analysis of overrepresented terms for proteins detected in the indicated Co-IP/MS. GO analysis was performed using go-seq, without

transcript length correction. A term was considered significant if its BH-corrected p-value (q-value) was less than 0.01. The top 10 terms are shown here, ranked by their q-value. EXTENDED

DATA FIG. 5 COLOUR ASSIGNMENT FOR RATIOMETRIC DUAL-COLOUR 3D SMLM IMAGING OF AF647 AND CF680, VORONOI DIAGRAMS, AND DELAUNAY TRIANGULATION AND KNOCKDOWN OF _MATR3._ A Scatter-plot of the

photons in two channels (transmission and reflection channels) analysed by global fit. The polygons were used to assign single molecules of AF647 and CF680 in different colours. B.

Cross-talk and rejected percentage for AF647 and CF680. C. Workflow for SMLM data filtering and clustering by Voronoi diagrams and Delaunay triangulation (DT) in Grafeo. D. qRT-PCR for

_MATR3_ expression after knockdown of _MATR3_ in hPSCs. Data is from three biological replicates, each in technical triplicate. Significance is from Welch’s t-test. E. Western blot for

HNRNPU and MATR3 in hPSCs transfected with an shRNA targeting _MATR3_. The experiment was performed twice with similar results. F. qRT-PCR for the core pluripotency genes _POU5F1_ and _SOX2_

in the indicated knockdowns. Data is from three biological replicates, each in technical triplicate. Significance is from Welch’s t-test. G. qRT-PCR for selected naïve and primed-specific

genes in the indicated knockdowns. Data is from three biological replicates, each in technical triplicate. Significance is from Welch’s t-test. EXTENDED DATA FIG. 6 HNRNPU IS INCREASED IN

SOMATIC CELLS, AND KNOCKDOWN DOES NOT DISRUPT SOME 3D CHROMATIN STRUCTURES. A Immunofluorescence images of hPSCs and hESF (human embryonic fibroblasts) stained with an antibody against

HNRNPU. Scale bar = 100 µm (top two panels) and 10 µm (bottom six panels). B. Western blot of HNRNPU and b-ACTIN In hPSCs and hESFs. This experiment was performed twice with similar results.

C. SMLM images of HNRNPU (stained with an anti-HNRNPU antibody) in primed hPSCs, and 293 T cells. For the upper panel scale bar = 2 µm, for the nuclear periphery, nucleoplasm, and the

z-axis, the scale bar = 200 nm. D. Boxplots showing the density for the HNRNPU clusters (top boxplot) and the localizations density within each cluster per volume for HNRNPU molecules

(bottom boxplot) in primed hPSCs, and 293 T cells. Significance is from a two-sided Welch’s t-test, for this panel and all subsequent panels in this figure. Statistical data relating to

cluster properties were derived from more than 10 cells in 3 independent experiments. The box represents the first and third quartiles, the midpoint is the median and the whiskers are 1.5x

the interquartile mean, for this panel and all subsequent boxplots. E. SMLM images of DNA (stained with an anti-DNA antibody) in primed hPSCs, and 293 T cells. For the upper panel scale bar

= 2 µm, and for the nuclear periphery, nucleoplasm, and the z-axis, the scale bar = 200 nm. F. Boxplots showing the density for the DNA clusters (top boxplot) and the localizations density

within each cluster per volume for HNRNPU (bottom boxplot) in primed hPSCs, and 293 T cells. The box represents the first and third quartiles, the midpoint is the median and the whiskers are

1.5x the interquartile mean. G. SMLM images of HNRNPU (stained with an anti-HNRNPU antibody) in hPSCs transfected with shRNAs targeting _LUC_ or _HNRNPU_. For the upper panel scale bar = 2 

µm, for the nuclear periphery, nucleoplasm, and the z-axis, the scale bar = 200 nm. H. Boxplots showing the density for the HNRNPU clusters (top boxplot) and the localizations density within

each cluster per volume for HNRNPU (bottom boxplot) in primed hPSCs transfected with the indicated shRNA. I. SMLM images of DNA (stained with an anti-DNA antibody) in hPSCs transfected with

a shRNA targeting _LUC_ or _HNRNPU_. For the upper panel scale bar = 2 µm, and for the nuclear periphery, nucleoplasm, and the z-axis, the scale bar = 200 nm. J. Boxplots showing the

density for the DNA clusters (top boxplot) and the localizations density within each cluster per volume for HNRNPU (bottom boxplot) in primed hPSCs transfected with the indicated shRNA. K.

Histogram of TAD size (in megabase pairs) in sh_LUC_ (control) and sh_HNRNPU_ transfected hPSCs. L. Pie charts of A/B compartment reassignment after _HNRNPU_ knockdown. M. Heatmap showing

the Pearson correlation scores for chromosome 2 in hPSCs transfected with the indicated shRNA. The bottom track shows the first principal component, orientated by GC content to assign A/B

compartments. N. Heatmaps showing normalized contact frequency for all of chromosome 2 for hPSCs transfected with the indicated shRNA. O. Differential contact frequency based on the heatmaps

in panel N. EXTENDED DATA FIG. 7 HNRNPU BINDS TO THE NUCLEAR MATRIX. A Genome distribution of the HNRNPU CUT&Tag peaks. Peaks were annotated to the nearest TSS and allocated to bins

either 5’ (negative numbers) or 3’ (positive) kbp relative to the TSS. A random background is shown in grey for comparison. B. Co-correlation heatmap for all hPSC ChIP-seq/CUT&Tag

experiments in primed hPSCs. Data is from Cistrome106, or this study. C. POLII CUT&Tag at the TSS and transcript bodies of housekeeping genes (Defined as genes with an expression

coefficient of variance < 0.25, and a mean expression > 29). Transcripts were scaled to a uniform length between the TSS and TTS, and the flanking 3kbp regions 5’ or 3’ are shown. D.

Heatmap showing a comparison of MAR-seq in hPSCs (this study) versus MAR-seq in MDAMB231 cells from accession GSE8767112. MAR-seq peaks are centred on their mid-points, and the flanking 2

kbp on either side of the peak centre is shown. E. Genome distribution of MAR-seq peaks in hPSCs (upper panel) or MDADMB231 cells (lower panel), relative to the transcription start site

(TSS). F. Venn diagram of the MAR-seq peaks in hPSCs transfected with shRNAs targeting _LUC_ or _HNRNPU_. G. Pileup heatmap of MAR-seq data for hPSCs transfected with shRNAs targeting _LUC_

or _HNRNPU_. H. Heatmap of the intersection of MAR-seq and HNRNPU CUT&Tag data in hPSCs. I. Proportional bar chart showing the A/T nucleotide percentages in the indicated categories of

HNRNPU or MAR-seq loci, as defined in panel E. A/T frequency was measured using a genomic window of 100 bp centred on the peak summit for each locus. J. Cumulative bar chart of the total

number of chromatin loops significantly up-regulated, unchanged, or downregulated in sh_HNRNPU_ transfected cells compared to sh_LUC_ control cells. Significantly different loops were

determined using DESeq2 based on the weighted normalized pixel value at the loop coordinate. Loops needed to have a q-value (Bonferroni-Hochberg corrected p-value) of less than 0.01 and an

absolute fold change of at least 2. K. Boxplots showing the average loop length for all loops, up- and downregulated loops, and in HNRNPU&MAR-seq, HNRNPU-only and MAR-seq-only loops.

Those loops were defined as at least one end of the loop is inside a HNRNPU or MAR-seq locus. L. Boxplots of the distance of any loop end to the nearest TSS. The top boxplot shows all loops

and up- and downregulated loops. The lower boxplots show only downregulated loops, and loops with at least one end of the loop in inside a HNRNPU or MAR-seq locus. M. Genome view of loops at

the _FGF2_ transcripts on chromosome 4. Loops with one or more ends originating at a HNRNPU&MAR-seq marked locus are indicated. EXTENDED DATA FIG. 8 HNRNPU LINKS CHROMATIN TO THE

NUCLEAR MATRIX TO DIFFERENTIALLY REGULATE CHROMATIN DIRECTLY AND INDIRECTLY. A Heatmap of the MAR-seq, HNRNPU, ATAC-seq, H3K27ac, and H3K4me1 CUT&Tag data in hPSCs or in hPSCs

transfected with the indicated shRNA. B. Pileups of the Hi-C data centred on the HNRNPU or MAR-seq loci, as defined in Extended Data Fig. 7h. The genomic window size is the flanking 500 kbp.

C. Volcano plots showing the differential genome loci for H3K27ac when _HNRNPU_ was knocked down. The three plots are divided based on whether they are bound by HNRNPU or are found in the

MAR-seq data. A locus was considered significantly different if it had a q-value of less than 0.05. D. Radial plot showing the genome distributions of H3K27ac downregulated loci marked by

HNRNPU&MAR-seq. E. Density binding plot of HNRNPU&MAR-seq-bound genes (left plot) or HNRNPU-only-bound genes (right plot) that have decreased H3K27ac upon _HNRNPU_ knockdown. HNRNPU

was determined as bound to a specific gene if it was within 1 kbp of a TSS of a transcribed isoform of that gene. HNRNPU binding density was estimated using a moving window of 1000 genes.

The grey lines are 10 scrambled (random) backgrounds. The black line indicates the mean of the scrambled backgrounds and the red line one standard deviation from the mean. For this and panel

I. F. Gene ontology of the genes that have reduced H3K27ac and are bound by HNRNPU&MAR-seq. G. Volcano plots showing the differential genome loci for ATAC-seq when _HNRNPU_ was knocked

down. The three plots are divided based on whether they are bound by HNRNPU or are found in the MAR-seq data. H. Radial plot showing the genome distributions of loci that have significantly

increased ATAC-seq accessibility and are also marked by HNRNPU&MAR-seq. I. Density binding plot of HNRNPU&MAR-seq-bound genes (left plot) or HNRNPU-only-bound genes (right plot) that

have increased ATAC-seq accessibility upon _HNRNPU_ knockdown. EXTENDED DATA FIG. 9 HNRNPU ANCHORS PRIMED-SPECIFIC GENES TO THE NUCLEAR MATRIX. A Genome view of the MATR3 locus in the hg38

genome. Read density tracks density for MAR-seq and HNRNPU CUT&Tag. Black circles beneath the tracks indicate a binding/enrichment site. Selected _MATR3_ and _SNHG4_ transcript isoforms

are shown. B. Heatmap showing a selection of naïve and primed-marker genes in primed/naive RNA-seq data. Samples are from GSE93241 (ref. 80), GSE75868, GSE85689 (ref. 28), PRJNA383735 (ref.

38), PRJNA397941 (ref. 29), and CNP0001454 (ref. 46). C. Volcano plot of differentially regulated genes in the naïve and primed states (as defined in panel B). Significantly differentially

regulated genes were defined as those genes that had a Bonferroni-Hochberg corrected p-value (q-value) of <0.01 and a fold change of at least 1.5. D. Genome contact falloff line plots.

Contact frequency plotted as a function of genome distance for all chromosomes. E. Aggregate heatmaps of the Hi-C data centred on the TSSs of naïve or primed-specific genes in hPSCs

transfected with shRNAs targeting _LUC_ or _HNRNPU_. The genomic window size is the flanking 500 kbp. F. Genome view and pileup of the MAR-seq data in hPSCs for a selection of

primed-specific genes. Read density tracks density for MAR-seq (black) and HNRNPU (red) CUT&Tag are shown. Black circles beneath the tracks indicate a binding/enrichment site. G. Genome

view and pileup of the MAR-seq data in hPSCs for a selection of naive-specific genes. Read density tracks density for MAR-seq (black) and HNRNPU (red) CUT&Tag are shown. Black circles

beneath the tracks indicate a binding/enrichment site. H. POLII CUT&Tag at the TSS and transcript bodies of primed-specific and naïve-specific genes (As defined in panel C). A transcript

was considered bound if an HNRNPU binding site was within 500 bp of the TSS. Transcripts were scaled to a uniform length between the TSS and TTS, and the flanking 3kbp regions 5’ or 3’ are

shown. EXTENDED DATA FIG. 10 HNRNPU IS BOUND TO PRIMED-SPECIFIC AND SOMATIC RNAS. A Pearson correlation heatmap for the pair-wise Pearson R scores for the RIP-seq data generated in this

study. # indicates the biological replicate number. B. Volcano plot showing the HNRNPU-bound RNAs. HNRNPU-bound RNA was defined as an RNA significantly enriched in the anti-HNRNPU RIP-seq

data versus the anti-IgG RIP-seq data. The volcano plot is shown for hPSCs. Selected RNAs are indicated. Significantly enriched genes were defined as those genes that had a

Bonferroni-Hochberg corrected p-value (q-value) of <0.01 and a fold change of > 1.5. C. Venn diagram showing the overlap of transcripts bound by HNRNPU in hPSCs and 293 T cells. D. Bar

chart showing significantly enriched gene ontology terms for the HNRNPU-bound RNAs in hPSC RIP-seq data. E. Genome view and pileup of the RIP-seq data in hPSCs and 293 T cells at the _OTX2_

genome locus. F. Line plot showing the log2(fold change) relative to hour 0 in hPSCs treated with Actinomycin D to block transcription. The line plots show primed-specific RNAs either bound

(right plot) or not bound (left plot) by HNRNPU. The line shows mean of all transcripts in class and the upper and lower thin lines are the 90% confidence intervals. Significance is from a

Mann–Whitney U-test of sh_LUC_ versus sh_HNRNPU_. SUPPLEMENTARY INFORMATION REPORTING SUMMARY PEER REVIEW FILE SUPPLEMENTARY TABLE 1 Lists of shRNA and primer sequences used in this study.

SUPPLEMENTARY TABLE 2 Lists of significantly differentially regulated genes in this study. SUPPLEMENTARY TABLE 3 Proteins detected in the Co-IP–MS experiments. SUPPLEMENTARY TABLE 4 RNAs

bound by HNRNPU identified in the RIP-seq in hPSCs and 293T cells. SOURCE DATA SOURCE DATA FIG. 1 Uncropped western blots. SOURCE DATA EXTENDED DATA FIG. 1 Statistical source data. RIGHTS

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ma, G., Fu, X., Zhou, L. _et al._ The nuclear matrix stabilizes primed-specific genes in human pluripotent stem cells. _Nat Cell Biol_ 27,

232–245 (2025). https://doi.org/10.1038/s41556-024-01595-5 Download citation * Received: 06 December 2023 * Accepted: 11 December 2024 * Published: 09 January 2025 * Issue Date: February

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