bims-crepig 2024-05-19 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2024–05–19
38 papers selected by
Connor Rogerson, University of Cambridge

Single-cell 3D genome structure reveals distinct human pluripotent states.
CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex.
Inseparable RNA binding and chromatin modification activities of a nucleosome-interacting surface in EZH2.
Genomic context sensitizes regulatory elements to genetic disruption.
G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation.
Single-molecule imaging reveals a direct role of CTCF's zinc fingers in SA interaction and cluster-dependent RNA recruitment.
Enhancer selectivity across cell types delineates three functionally distinct enhancer-promoter regulation patterns.
Disentangling oncogenic amplicons in esophageal adenocarcinoma.
A continuum of zinc finger transcription factor retention on native chromatin underlies dynamic genome organization.
A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.
CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair.
EmptyDropsMultiome discriminates real cells from background in single-cell multiomics assays.
Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.
Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation.
asteRIa enables robust interaction modeling between chromatin modifications and epigenetic readers.
The single-cell chromatin landscape in gonadal cell lineage specification.
H2A.Z chaperones converge on E2F target genes for melanoma cell proliferation.
ChIP-Atlas 3.0: a data-mining suite to explore chromosome architecture together with large-scale regulome data.
RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila.
Metabolic regulator ERRγ governs gastric stem cell differentiation into acid-secreting parietal cells.
Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.
A unified mechanism for PARP inhibitor-induced PARP1 chromatin retention at DNA damage sites in living cells.
Bidirectional epigenetic editing reveals hierarchies in gene regulation.
Learning Micro-C from Hi-C with diffusion models.
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations.
TEENA: an integrated web server for transposable element enrichment analysis in various model and non-model organisms.
Causal gene regulatory analysis with RNA velocity reveals an interplay between slow and fast transcription factors.
A statistical approach for identifying single nucleotide variants that affect transcription factor binding.
FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.
Interpretable online network dictionary learning for inferring long-range chromatin interactions.
GAGE-seq concurrently profiles multiscale 3D genome organization and gene expression in single cells.
Effect of loops on the mean-square displacement of Rouse-model chromatin.
Nuclear actin structure regulates chromatin accessibility.
PAX6 promotes neuroendocrine phenotypes of prostate cancer via enhancing MET/STAT5A-mediated chromatin accessibility.
Endocardial HDAC3 is required for myocardial trabeculation.
Robust differential expression testing for single-cell CRISPR screens at low multiplicity of infection.
Cactus: A user-friendly and reproducible ATAC-Seq and mRNA-Seq analysis pipeline for data preprocessing, differential analysis, and enrichment analysis.
Downregulation of HNF4A enables transcriptomic reprogramming during the hepatic acute-phase response.

Genome Biol. 2024 May 13. 25(1): 122

Single-cell 3D genome structure reveals distinct human pluripotent states.

Niannian Li, Kairang Jin, Bin Liu, Mingzhu Yang, PanPan Shi, Dai Heng, Jichang Wang, Lin Liu.

   BACKGROUND: Pluripotent states of embryonic stem cells (ESCs) with distinct transcriptional profiles affect ESC differentiative capacity and therapeutic potential. Although single-cell RNA sequencing has revealed additional subpopulations and specific features of naive and primed human pluripotent stem cells (hPSCs), the underlying mechanisms that regulate their specific transcription and that control their pluripotent states remain elusive.
RESULTS: By single-cell analysis of high-resolution, three-dimensional (3D) genomic structure, we herein demonstrate that remodeling of genomic structure is highly associated with the pluripotent states of human ESCs (hESCs). The naive pluripotent state is featured with specialized 3D genomic structures and clear chromatin compartmentalization that is distinct from the primed state. The naive pluripotent state is achieved by remodeling the active euchromatin compartment and reducing chromatin interactions at the nuclear center. This unique genomic organization is linked to enhanced chromatin accessibility on enhancers and elevated expression levels of naive pluripotent genes localized to this region. In contradistinction, the primed state exhibits intermingled genomic organization. Moreover, active euchromatin and primed pluripotent genes are distributed at the nuclear periphery, while repressive heterochromatin is densely concentrated at the nuclear center, reducing chromatin accessibility and the transcription of naive genes.
CONCLUSIONS: Our data provide insights into the chromatin structure of ESCs in their naive and primed states, and we identify specific patterns of modifications in transcription and chromatin structure that might explain the genes that are differentially expressed between naive and primed hESCs. Thus, the inversion or relocation of heterochromatin to euchromatin via compartmentalization is related to the regulation of chromatin accessibility, thereby defining pluripotent states and cellular identity.

Keywords:  Chromatin accessibility; Genome structure; Human embryonic stem cells; Naive; Pluripotency; Primed

DOI:  https://doi.org/10.1186/s13059-024-03268-w
Cell Rep. 2024 May 11. pii: S2211-1247(24)00555-2. [Epub ahead of print]43(5): 114227

CUX1 regulates human hematopoietic stem cell chromatin accessibility via the BAF complex.

Weihan Liu, Jeffrey L Kurkewich, Angela Stoddart, Saira Khan, Dhivyaa Anandan, Alexandre N Gaubil, Donald J Wolfgeher, Lia Jueng, Stephen J Kron, Megan E McNerney.

  CUX1 is a homeodomain-containing transcription factor that is essential for the development and differentiation of multiple tissues. CUX1 is recurrently mutated or deleted in cancer, particularly in myeloid malignancies. However, the mechanism by which CUX1 regulates gene expression and differentiation remains poorly understood, creating a barrier to understanding the tumor-suppressive functions of CUX1. Here, we demonstrate that CUX1 directs the BAF chromatin remodeling complex to DNA to increase chromatin accessibility in hematopoietic cells. CUX1 preferentially regulates lineage-specific enhancers, and CUX1 target genes are predictive of cell fate in vivo. These data indicate that CUX1 regulates hematopoietic lineage commitment and homeostasis via pioneer factor activity, and CUX1 deficiency disrupts these processes in stem and progenitor cells, facilitating transformation.

Keywords:  BAF complex; CP: Molecular biology; CP: Stem cell research; CUX1; chromatin remodeler; differentiation; enhancer; epigenetic regulation; hematopoietic stem cells; myeloid neoplasms; pioneer factor; transcription factor

DOI:  https://doi.org/10.1016/j.celrep.2024.114227
Nat Genet. 2024 May 14.

Inseparable RNA binding and chromatin modification activities of a nucleosome-interacting surface in EZH2.

Emma H Gail, Evan Healy, Sarena F Flanigan, Natasha Jones, Xiao Han Ng, Michael Uckelmann, Vitalina Levina, Qi Zhang, Chen Davidovich.

Polycomb repressive complex 2 (PRC2) interacts with RNA in cells, but there is no consensus on how RNA regulates PRC2 canonical functions, including chromatin modification and the maintenance of transcription programs in lineage-committed cells. We assayed two separation-of-function mutants of the PRC2 catalytic subunit EZH2, defective in RNA binding but functional in methyltransferase activity. We find that part of the RNA-binding surface of EZH2 is required for chromatin modification, yet this activity is independent of RNA. Mechanistically, the RNA-binding surface within EZH2 is required for chromatin modification in vitro and in cells, through interactions with nucleosomal DNA. Contrarily, an RNA-binding-defective mutant exhibited normal chromatin modification activity in vitro and in lineage-committed cells, accompanied by normal gene repression activity. Collectively, we show that part of the RNA-binding surface of EZH2, rather than the RNA-binding activity per se, is required for the histone methylation in vitro and in cells, through interactions with the substrate nucleosome.

DOI: https://doi.org/10.1038/s41588-024-01740-8
Mol Cell. 2024 May 16. pii: S1097-2765(24)00328-9. [Epub ahead of print]84(10): 1842-1854.e7

Genomic context sensitizes regulatory elements to genetic disruption.

Raquel Ordoñez, Weimin Zhang, Gwen Ellis, Yinan Zhu, Hannah J Ashe, André M Ribeiro-Dos-Santos, Ran Brosh, Emily Huang, Megan S Hogan, Jef D Boeke, Matthew T Maurano.

  Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.

Keywords:  enhancer selectivity; gene regulation; genetic engineering; genome writing; genomic regulatory architecture; synthetic regulatory genomics

DOI:  https://doi.org/10.1016/j.molcel.2024.04.013
Mol Cell. 2024 May 16. pii: S1097-2765(24)00330-7. [Epub ahead of print]84(10): 1870-1885.e9

G-quadruplex folding in Xist RNA antagonizes PRC2 activity for stepwise regulation of X chromosome inactivation.

Yong Woo Lee, Uri Weissbein, Roy Blum, Jeannie T Lee.

  How Polycomb repressive complex 2 (PRC2) is regulated by RNA remains an unsolved problem. Although PRC2 binds G-tracts with the potential to form RNA G-quadruplexes (rG4s), whether rG4s fold extensively in vivo and whether PRC2 binds folded or unfolded rG4 are unknown. Using the X-inactivation model in mouse embryonic stem cells, here we identify multiple folded rG4s in Xist RNA and demonstrate that PRC2 preferentially binds folded rG4s. High-affinity rG4 binding inhibits PRC2's histone methyltransferase activity, and stabilizing rG4 in vivo antagonizes H3 at lysine 27 (H3K27me3) enrichment on the inactive X chromosome. Surprisingly, mutagenizing the rG4 does not affect PRC2 recruitment but promotes its release and catalytic activation on chromatin. H3K27me3 marks are misplaced, however, and gene silencing is compromised. Xist-PRC2 complexes become entrapped in the S1 chromosome compartment, precluding the required translocation into the S2 compartment. Thus, Xist rG4 folding controls PRC2 activity, H3K27me3 enrichment, and the stepwise regulation of chromosome-wide gene silencing.

Keywords:  G-quadruplexes; G4; PRC2; Polycomb repressive complex 2; RNA structure; X inactivation; Xist; noncoding RNA

DOI:  https://doi.org/10.1016/j.molcel.2024.04.015
Nucleic Acids Res. 2024 May 14. pii: gkae391. [Epub ahead of print]

Single-molecule imaging reveals a direct role of CTCF's zinc fingers in SA interaction and cluster-dependent RNA recruitment.

Jonas Huber, Nicoleta-Loredana Tanasie, Sarah Zernia, Johannes Stigler.

CTCF is a zinc finger protein associated with transcription regulation that also acts as a barrier factor for topologically associated domains (TADs) generated by cohesin via loop extrusion. These processes require different properties of CTCF-DNA interaction, and it is still unclear how CTCF's structural features may modulate its diverse roles. Here, we employ single-molecule imaging to study both full-length CTCF and truncation mutants. We show that CTCF enriches at CTCF binding sites (CBSs), displaying a longer lifetime than observed previously. We demonstrate that the zinc finger domains mediate CTCF clustering and that clustering enables RNA recruitment, possibly creating a scaffold for interaction with RNA-binding proteins like cohesin's subunit SA. We further reveal a direct recruitment and an increase of SA residence time by CTCF bound at CBSs, suggesting that CTCF-SA interactions are crucial for cohesin stability on chromatin at TAD borders. Furthermore, we establish a single-molecule T7 transcription assay and show that although a transcribing polymerase can remove CTCF from CBSs, transcription is impaired. Our study shows that context-dependent nucleic acid binding determines the multifaceted CTCF roles in genome organization and transcription regulation.

DOI: https://doi.org/10.1093/nar/gkae391
BMC Genomics. 2024 May 16. 25(1): 483

Enhancer selectivity across cell types delineates three functionally distinct enhancer-promoter regulation patterns.

Chengyi Wu, Jialiang Huang.

   BACKGROUND: Multiple enhancers co-regulating the same gene is prevalent and plays a crucial role during development and disease. However, how multiple enhancers coordinate the same gene expression across various cell types remains largely unexplored at genome scale.
RESULTS: We develop a computational approach that enables the quantitative assessment of enhancer specificity and selectivity across diverse cell types, leveraging enhancer-promoter (E-P) interactions data. We observe two well-known gene regulation patterns controlled by enhancer clusters, which regulate the same gene either in a limited number of cell types (Specific pattern, Spe) or in the majority of cell types (Conserved pattern, Con), both of which are enriched for super-enhancers (SEs). We identify a previously overlooked pattern (Variable pattern, Var) that multiple enhancers link to the same gene, but rarely coexist in the same cell type. These three patterns control the genes associating with distinct biological function and exhibit unique epigenetic features. Specifically, we discover a subset of Var patterns contains Shared enhancers with stable enhancer-promoter interactions in the majority of cell types, which might contribute to maintaining gene expression by recruiting abundant CTCF.
CONCLUSIONS: Together, our findings reveal three distinct E-P regulation patterns across different cell types, providing insights into deciphering the complexity of gene transcriptional regulation.

Keywords:  Enhancer selectivity; Enhancer-promoter interactions; Gene regulation patterns across cell types

DOI:  https://doi.org/10.1186/s12864-024-10408-w
Nat Commun. 2024 May 14. 15(1): 4074

Disentangling oncogenic amplicons in esophageal adenocarcinoma.

Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) Consortium

Esophageal adenocarcinoma is a prominent example of cancer characterized by frequent amplifications in oncogenes. However, the mechanisms leading to amplicons that involve breakage-fusion-bridge cycles and extrachromosomal DNA are poorly understood. Here, we use 710 esophageal adenocarcinoma cases with matched samples and patient-derived organoids to disentangle complex amplicons and their associated mechanisms. Short-read sequencing identifies ERBB2, MYC, MDM2, and HMGA2 as the most frequent oncogenes amplified in extrachromosomal DNAs. We resolve complex extrachromosomal DNA and breakage-fusion-bridge cycles amplicons by integrating of de-novo assemblies and DNA methylation in nine long-read sequenced cases. Complex amplicons shared between precancerous biopsy and late-stage tumor, an enrichment of putative enhancer elements and mobile element insertions are potential drivers of complex amplicons' origin. We find that patient-derived organoids recapitulate extrachromosomal DNA observed in the primary tumors and single-cell DNA sequencing capture extrachromosomal DNA-driven clonal dynamics across passages. Prospectively, long-read and single-cell DNA sequencing technologies can lead to better prediction of clonal evolution in esophageal adenocarcinoma.

DOI: https://doi.org/10.1038/s41467-024-47619-4
Mol Syst Biol. 2024 May 14.

A continuum of zinc finger transcription factor retention on native chromatin underlies dynamic genome organization.

Siling Hu, Yangying Liu, Qifan Zhang, Juan Bai, Chenhuan Xu.

  Transcription factor (TF) residence on chromatin translates into quantitative transcriptional or structural outcomes on genome. Commonly used formaldehyde crosslinking fixes TF-DNA interactions cumulatively and compromises the measured occupancy level. Here we mapped the occupancy level of global or individual zinc finger TFs like CTCF and MAZ, in the form of highly resolved footprints, on native chromatin. By incorporating reinforcing perturbation conditions, we established S-score, a quantitative metric to proxy the continuum of CTCF or MAZ retention across different motifs on native chromatin. The native chromatin-retained CTCF sites harbor sequence features within CTCF motifs better explained by S-score than the metrics obtained from other crosslinking or native assays. CTCF retention on native chromatin correlates with local SUMOylation level, and anti-correlates with transcriptional activity. The S-score successfully delineates the otherwise-masked differential stability of chromatin structures mediated by CTCF, or by MAZ independent of CTCF. Overall, our study established a paradigm continuum of TF retention across binding sites on native chromatin, explaining the dynamic genome organization.

Keywords:  CTCF; ChIP; Chromatin Structure; Native Chromatin; Transcription Factor

DOI:  https://doi.org/10.1038/s44320-024-00038-5
Nucleic Acids Res. 2024 May 15. pii: gkae370. [Epub ahead of print]

A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

Robin Kohler, Kurt Engeland.

A-MYB (MYBL1) is a transcription factor with a role in meiosis in spermatocytes. The related B-MYB protein is a key oncogene and a master regulator activating late cell cycle genes. To activate genes, B-MYB forms a complex with MuvB and is recruited indirectly to cell cycle genes homology region (CHR) promoter sites of target genes. Activation through the B-MYB-MuvB (MMB) complex is essential for successful mitosis. Here, we discover that A-MYB has a function in transcriptional regulation of the mitotic cell cycle and can substitute for B-MYB. Knockdown experiments in cells not related to spermatogenesis show that B-MYB loss alone merely delays cell cycle progression. Only dual knockdown of B-MYB and A-MYB causes G2/M cell cycle arrest, endoreduplication, and apoptosis. A-MYB can substitute for B-MYB in binding to MuvB. The resulting A-MYB-MuvB complex activates genes through CHR sites. We find that A-MYB activates the same target genes as B-MYB. Many of the corresponding proteins are central regulators of the cell division cycle. In summary, we demonstrate that A-MYB is an activator of the mitotic cell cycle by activating late cell cycle genes.

DOI: https://doi.org/10.1093/nar/gkae370
Nat Commun. 2024 May 16. 15(1): 4148

CEBPA restricts alveolar type 2 cell plasticity during development and injury-repair.

Dalia Hassan, Jichao Chen.

Cell plasticity theoretically extends to all possible cell types, but naturally decreases as cells differentiate, whereas injury-repair re-engages the developmental plasticity. Here we show that the lung alveolar type 2 (AT2)-specific transcription factor (TF), CEBPA, restricts AT2 cell plasticity in the mouse lung. AT2 cells undergo transcriptional and epigenetic maturation postnatally. Without CEBPA, both neonatal and mature AT2 cells reduce the AT2 program, but only the former reactivate the SOX9 progenitor program. Sendai virus infection bestows mature AT2 cells with neonatal plasticity where Cebpa mutant, but not wild type, AT2 cells express SOX9, as well as more readily proliferate and form KRT8/CLDN4+ transitional cells. CEBPA promotes the AT2 program by recruiting the lung lineage TF NKX2-1. The temporal change in CEBPA-dependent plasticity reflects AT2 cell developmental history. The ontogeny of AT2 cell plasticity and its transcriptional and epigenetic mechanisms have implications in lung regeneration and cancer.

DOI: https://doi.org/10.1038/s41467-024-48632-3
Genome Biol. 2024 May 13. 25(1): 121

EmptyDropsMultiome discriminates real cells from background in single-cell multiomics assays.

Stathis Megas, Valentina Lorenzi, John C Marioni.

  Multiomic droplet-based technologies allow different molecular modalities, such as chromatin accessibility and gene expression (scATAC-seq and scRNA-seq), to be probed in the same nucleus. We develop EmptyDropsMultiome, an approach that distinguishes true nuclei-containing droplets from background. Using simulations, we show that EmptyDropsMultiome has higher statistical power and accuracy than existing approaches, including CellRanger-arc and EmptyDrops. On real datasets, we observe that CellRanger-arc misses more than half of the nuclei identified by EmptyDropsMultiome and, moreover, is biased against certain cell types, some of which have a retrieval rate lower than 20%.

Keywords:  Method; Multiomics; Single-cell

DOI:  https://doi.org/10.1186/s13059-024-03259-x
Proc Natl Acad Sci U S A. 2024 May 21. 121(21): e2405827121

Chromatin regulates alternative polyadenylation via the RNA polymerase II elongation rate.

Joseph V Geisberg, Zarmik Moqtaderi, Kevin Struhl.

  The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.

Keywords:  RNA polymerase II; alternative polyadenylation; chromatin; histone chaperones; transcription elongation

DOI:  https://doi.org/10.1073/pnas.2405827121
Cell Stem Cell. 2024 May 07. pii: S1934-5909(24)00142-5. [Epub ahead of print]

Transcription factor dynamics, oscillation, and functions in human enteroendocrine cell differentiation.

Pratik N P Singh, Wei Gu, Shariq Madha, Allen W Lynch, Paloma Cejas, Ruiyang He, Swarnabh Bhattacharya, Miguel Muñoz Gomez, Matthew G Oser, Myles Brown, Henry W Long, Clifford A Meyer, Qiao Zhou, Ramesh A Shivdasani.

  Enteroendocrine cells (EECs) secrete serotonin (enterochromaffin [EC] cells) or specific peptide hormones (non-EC cells) that serve vital metabolic functions. The basis for terminal EEC diversity remains obscure. By forcing activity of the transcription factor (TF) NEUROG3 in 2D cultures of human intestinal stem cells, we replicated physiologic EEC differentiation and examined transcriptional and cis-regulatory dynamics that culminate in discrete cell types. Abundant EEC precursors expressed stage-specific genes and TFs. Before expressing pre-terminal NEUROD1, post-mitotic precursors oscillated between transcriptionally distinct ASCL1+ and HES6hi cell states. Loss of either factor accelerated EEC differentiation substantially and disrupted EEC individuality; ASCL1 or NEUROD1 deficiency had opposing consequences on EC and non-EC cell features. These TFs mainly bind cis-elements that are accessible in undifferentiated stem cells, and they tailor subsequent expression of TF combinations that underlie discrete EEC identities. Thus, early TF oscillations retard EEC maturation to enable accurate diversity within a medically important cell lineage.

Keywords:  chromatin dynamics; determination of terminal cell fates; hormone producing cells; human enteroendocrine cells; intestinal stem cells; transcription factor oscillation

DOI:  https://doi.org/10.1016/j.stem.2024.04.015
Nucleic Acids Res. 2024 May 16. pii: gkae361. [Epub ahead of print]

asteRIa enables robust interaction modeling between chromatin modifications and epigenetic readers.

Mara Stadler, Saulius Lukauskas, Till Bartke, Christian L Müller.

Chromatin, the nucleoprotein complex consisting of DNA and histone proteins, plays a crucial role in regulating gene expression by controlling access to DNA. Chromatin modifications are key players in this regulation, as they help to orchestrate DNA transcription, replication, and repair. These modifications recruit epigenetic 'reader' proteins, which mediate downstream events. Most modifications occur in distinctive combinations within a nucleosome, suggesting that epigenetic information can be encoded in combinatorial chromatin modifications. A detailed understanding of how multiple modifications cooperate in recruiting such proteins has, however, remained largely elusive. Here, we integrate nucleosome affinity purification data with high-throughput quantitative proteomics and hierarchical interaction modeling to estimate combinatorial effects of chromatin modifications on protein recruitment. This is facilitated by the computational workflow asteRIa which combines hierarchical interaction modeling, stability-based model selection, and replicate-consistency checks for a stable estimation of Robust Interactions among chromatin modifications. asteRIa identifies several epigenetic reader candidates responding to specific interactions between chromatin modifications. For the polycomb protein CBX8, we independently validate our results using genome-wide ChIP-Seq and bisulphite sequencing datasets. We provide the first quantitative framework for identifying cooperative effects of chromatin modifications on protein binding.

DOI: https://doi.org/10.1093/nar/gkae361
BMC Genomics. 2024 May 13. 25(1): 464

The single-cell chromatin landscape in gonadal cell lineage specification.

Hoi Ching Suen, Fanghong Ou, Kai-Kei Miu, Zhangting Wang, Wai-Yee Chan, Jinyue Liao.

  Gonad development includes sex determination and divergent maturation of the testes and ovaries. Recent advances in measuring gene expression in single cells are providing new insights into this complex process. However, the underlying epigenetic regulatory mechanisms remain unclear. Here, we profiled chromatin accessibility in mouse gonadal cells of both sexes from embryonic day 11.5 to 14.5 using single-cell assay for transposase accessible chromatin by sequencing (scATAC-seq). Our results showed that individual cell types can be inferred by the chromatin landscape, and that cells can be temporally ordered along developmental trajectories. Integrative analysis of transcriptomic and chromatin-accessibility maps identified multiple putative regulatory elements proximal to key gonadal genes Nr5a1, Sox9 and Wt1. We also uncover cell type-specific regulatory factors underlying cell type specification. Overall, our results provide a better understanding of the epigenetic landscape associated with the progressive restriction of cell fates in the gonad.

Keywords:  Chromatin accessibility; Gonad development; Regulatory genomics; Sex determination; Single-cell biology; Single-cell sequencing; scATAC-seq

DOI:  https://doi.org/10.1186/s12864-024-10376-1
Genes Dev. 2024 May 14.

H2A.Z chaperones converge on E2F target genes for melanoma cell proliferation.

Sina Jostes, Chiara Vardabasso, Joanna Dong, Saul Carcamo, Rajendra Singh, Robert Phelps, Austin Meadows, Elena Grossi, Dan Hasson, Emily Bernstein.

  High levels of H2A.Z promote melanoma cell proliferation and correlate with poor prognosis. However, the role of the two distinct H2A.Z histone chaperone complexes SRCAP and P400-TIP60 in melanoma remains unclear. Here, we show that individual subunit depletion of SRCAP, P400, and VPS72 (YL1) results in not only the loss of H2A.Z deposition into chromatin but also a reduction of H4 acetylation in melanoma cells. This loss of H4 acetylation is particularly found at the promoters of cell cycle genes directly bound by H2A.Z and its chaperones, suggesting a coordinated regulation between H2A.Z deposition and H4 acetylation to promote their expression. Knockdown of each of the three subunits downregulates E2F1 and its targets, resulting in a cell cycle arrest akin to H2A.Z depletion. However, unlike H2A.Z deficiency, loss of the shared H2A.Z chaperone subunit YL1 induces apoptosis. Furthermore, YL1 is overexpressed in melanoma tissues, and its upregulation is associated with poor patient outcome. Together, these findings provide a rationale for future targeting of H2A.Z chaperones as an epigenetic strategy for melanoma treatment.

Keywords:  H2A.Z; epigenetics; histone chaperones; histone variants; melanoma

DOI:  https://doi.org/10.1101/gad.351318.123
Nucleic Acids Res. 2024 May 16. pii: gkae358. [Epub ahead of print]

ChIP-Atlas 3.0: a data-mining suite to explore chromosome architecture together with large-scale regulome data.

Zhaonan Zou, Tazro Ohta, Shinya Oki.

ChIP-Atlas (https://chip-atlas.org/) presents a suite of data-mining tools for analyzing epigenomic landscapes, powered by the comprehensive integration of over 376 000 public ChIP-seq, ATAC-seq, DNase-seq and Bisulfite-seq experiments from six representative model organisms. To unravel the intricacies of chromatin architecture that mediates the regulome-initiated generation of transcriptional and phenotypic diversity within cells, we report ChIP-Atlas 3.0 that enhances clarity by incorporating additional tracks for genomic and epigenomic features within a newly consolidated 'annotation track' section. The tracks include chromosomal conformation (Hi-C and eQTL datasets), transcriptional regulatory elements (ChromHMM and FANTOM5 enhancers), and genomic variants associated with diseases and phenotypes (GWAS SNPs and ClinVar variants). These annotation tracks are easily accessible alongside other experimental tracks, facilitating better elucidation of chromatin architecture underlying the diversification of transcriptional and phenotypic traits. Furthermore, 'Diff Analysis,' a new online tool, compares the query epigenome data to identify differentially bound, accessible, and methylated regions using ChIP-seq, ATAC-seq and DNase-seq, and Bisulfite-seq datasets, respectively. The integration of annotation tracks and the Diff Analysis tool, coupled with continuous data expansion, renders ChIP-Atlas 3.0 a robust resource for mining the landscape of transcriptional regulatory mechanisms, thereby offering valuable perspectives, particularly for genetic disease research and drug discovery.

DOI: https://doi.org/10.1093/nar/gkae358
PLoS Genet. 2024 May 17. 20(5): e1011136

RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila.

George J Watase, Yukiko M Yamashita.

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary.

DOI: https://doi.org/10.1371/journal.pgen.1011136
Cell Stem Cell. 2024 May 07. pii: S1934-5909(24)00143-7. [Epub ahead of print]

Metabolic regulator ERRγ governs gastric stem cell differentiation into acid-secreting parietal cells.

Mahliyah Adkins-Threats, Sumimasa Arimura, Yang-Zhe Huang, Margarita Divenko, Sarah To, Heather Mao, Yongji Zeng, Jenie Y Hwang, Joseph R Burclaff, Shilpa Jain, Jason C Mills.

  Parietal cells (PCs) produce gastric acid to kill pathogens and aid digestion. Dysregulated PC census is common in disease, yet how PCs differentiate is unclear. Here, we identify the PC progenitors arising from isthmal stem cells, using mouse models and human gastric cells, and show that they preferentially express cell-metabolism regulator and orphan nuclear receptor Estrogen-related receptor gamma (Esrrg, encoding ERRγ). Esrrg expression facilitated the tracking of stepwise molecular, cellular, and ultrastructural stages of PC differentiation. EsrrgP2ACreERT2 lineage tracing revealed that Esrrg expression commits progenitors to differentiate into mature PCs. scRNA-seq indicated the earliest Esrrg+ PC progenitors preferentially express SMAD4 and SP1 transcriptional targets and the GTPases regulating acid-secretion signal transduction. As progenitors matured, ERRγ-dependent metabolic transcripts predominated. Organoid and mouse studies validated the requirement of ERRγ for PC differentiation. Our work chronicles stem cell differentiation along a single lineage in vivo and suggests ERRγ as a therapeutic target for PC-related disorders.

Keywords:  SPEM; cell specification; regeneration; stomach; tamoxifen; transcription factor

DOI:  https://doi.org/10.1016/j.stem.2024.04.016
Cell Stem Cell. 2024 May 10. pii: S1934-5909(24)00144-9. [Epub ahead of print]

Deciphering lineage specification during early embryogenesis in mouse gastruloids using multilayered proteomics.

Suzan Stelloo, Maria Teresa Alejo-Vinogradova, Charlotte A G H van Gelder, Dick W Zijlmans, Marek J van Oostrom, Juan Manuel Valverde, Lieke A Lamers, Teja Rus, Paula Sobrevals Alcaraz, Tilman Schäfers, Cristina Furlan, Pascal W T C Jansen, Marijke P A Baltissen, Katharina F Sonnen, Boudewijn Burgering, Maarten A F M Altelaar, Harmjan R Vos, Michiel Vermeulen.

  Gastrulation is a critical stage in embryonic development during which the germ layers are established. Advances in sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and their derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho) protein expression during gastruloid differentiation. Our findings revealed many proteins with temporal expression and unique expression profiles for each germ layer, which we also validated using single-cell proteomics technology. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with single-cell RNA sequencing (scRNA-seq) identified a critical role for ZEB2 in mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.

Keywords:  ZEB2; dTAG system; developmental biology; enhancers; gastruloids; germ layers; interaction proteomics; proteomics; somitoids; transcription factors

DOI:  https://doi.org/10.1016/j.stem.2024.04.017
Cell Rep. 2024 May 16. pii: S2211-1247(24)00562-X. [Epub ahead of print]43(5): 114234

A unified mechanism for PARP inhibitor-induced PARP1 chromatin retention at DNA damage sites in living cells.

Petar-Bogomil Kanev, Sylvia Varhoshkova, Irina Georgieva, Maria Lukarska, Dilyana Kirova, Georgi Danovski, Stoyno Stoynov, Radoslav Aleksandrov.

  Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) not only suppress PARP1 catalytic activity but also prolong its association to damaged chromatin. Here, through live-cell imaging, we quantify the alterations in PARP1 dynamics and activity elicited by seven PARPis over a wide range of concentrations to deliver a unified mechanism of PARPi-induced PARP1 chromatin retention. We find that gross PARP1 retention at DNA damage sites is jointly governed by catalytic inhibition and allosteric trapping, albeit in a strictly independent manner-catalytic inhibition causes multiple unproductive binding-dissociation cycles of PARP1, while allosteric trapping prolongs the lesion-bound state of PARP1 to greatly increase overall retention. Importantly, stronger PARP1 retention produces greater temporal shifts in downstream DNA repair events and superior cytotoxicity, highlighting PARP1 retention, a complex but precisely quantifiable characteristic of PARPis, as a valuable biomarker for PARPi efficacy. Our approach can be promptly repurposed for interrogating the properties of DNA-repair-targeting compounds beyond PARPis.

Keywords:  CP: Cancer; CP: Molecular biology; FRAP; PARP inhibitors; PARP1; PARP1 inhibition; PARP1 retention; PARP1 trapping; cancer; laser micro-irradiation; live-cell imaging; mathematical modeling

DOI:  https://doi.org/10.1016/j.celrep.2024.114234
Nat Biotechnol. 2024 May 17.

Bidirectional epigenetic editing reveals hierarchies in gene regulation.

Naomi M Pacalin, Zachary Steinhart, Quanming Shi, Julia A Belk, Dmytro Dorovskyi, Katerina Kraft, Kevin R Parker, Brian R Shy, Alexander Marson, Howard Y Chang.

CRISPR perturbation methods are limited in their ability to study non-coding elements and genetic interactions. In this study, we developed a system for bidirectional epigenetic editing, called CRISPRai, in which we apply activating (CRISPRa) and repressive (CRISPRi) perturbations to two loci simultaneously in the same cell. We developed CRISPRai Perturb-seq by coupling dual perturbation gRNA detection with single-cell RNA sequencing, enabling study of pooled perturbations in a mixed single-cell population. We applied this platform to study the genetic interaction between two hematopoietic lineage transcription factors, SPI1 and GATA1, and discovered novel characteristics of their co-regulation on downstream target genes, including differences in SPI1 and GATA1 occupancy at genes that are regulated through different modes. We also studied the regulatory landscape of IL2 (interleukin-2) in Jurkat T cells, primary T cells and chimeric antigen receptor (CAR) T cells and elucidated mechanisms of enhancer-mediated IL2 gene regulation. CRISPRai facilitates investigation of context-specific genetic interactions, provides new insights into gene regulation and will enable exploration of non-coding disease-associated variants.

DOI: https://doi.org/10.1038/s41587-024-02213-3
PLoS Comput Biol. 2024 May 17. 20(5): e1012136

Learning Micro-C from Hi-C with diffusion models.

Tong Liu, Hao Zhu, Zheng Wang.

In the last few years, Micro-C has shown itself as an improved alternative to Hi-C. It replaced the restriction enzymes in Hi-C assays with micrococcal nuclease (MNase), resulting in capturing nucleosome resolution chromatin interactions. The signal-to-noise improvement of Micro-C allows it to detect more chromatin loops than high-resolution Hi-C. However, compared with massive Hi-C datasets available in the literature, there are only a limited number of Micro-C datasets. To take full advantage of these Hi-C datasets, we present HiC2MicroC, a computational method learning and then predicting Micro-C from Hi-C based on the denoising diffusion probabilistic models (DDPM). We trained our DDPM and other regression models in human foreskin fibroblast (HFFc6) cell line and evaluated these methods in six different cell types at 5-kb and 1-kb resolution. Our evaluations demonstrate that both HiC2MicroC and regression methods can markedly improve Hi-C towards Micro-C, and our DDPM-based HiC2MicroC outperforms regression in various terms. First, HiC2MicroC successfully recovers most of the Micro-C loops even those not detected in Hi-C maps. Second, a majority of the HiC2MicroC-recovered loops anchor CTCF binding sites in a convergent orientation. Third, HiC2MicroC loops share genomic and epigenetic properties with Micro-C loops, including linking promoters and enhancers, and their anchors are enriched for structural proteins (CTCF and cohesin) and histone modifications. Lastly, we find our recovered loops are also consistent with the loops identified from promoter capture Micro-C (PCMicro-C) and Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET). Overall, HiC2MicroC is an effective tool for further studying Hi-C data with Micro-C as a template. HiC2MicroC is publicly available at https://github.com/zwang-bioinformatics/HiC2MicroC/.

DOI: https://doi.org/10.1371/journal.pcbi.1012136
Nat Biotechnol. 2024 May 17.

Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations.

C C-S Hsiung, C M Wilson, N A Sambold, R Dai, Q Chen, N Teyssier, S Misiukiewicz, A Arab, T O'Loughlin, J C Cofsky, J Shi, L A Gilbert.

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting one to three genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein-DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.

DOI: https://doi.org/10.1038/s41587-024-02224-0
Nucleic Acids Res. 2024 May 15. pii: gkae411. [Epub ahead of print]

TEENA: an integrated web server for transposable element enrichment analysis in various model and non-model organisms.

Yuzhuo Li, Renzhe Lyu, Shuai Chen, Yejun Wang, Ming-An Sun.

Transposable elements (TEs) are abundant in the genomes of various eukaryote organisms. Increasing evidence suggests that TEs can play crucial regulatory roles-usually by creating cis-elements (e.g. enhancers and promoters) bound by distinct transcription factors (TFs). TE-derived cis-elements have gained unprecedented attentions recently, and one key step toward their understanding is to identify the enriched TEs in distinct genomic intervals (e.g. a set of enhancers or TF binding sites) as candidates for further study. Nevertheless, such analysis remains challenging for researchers unfamiliar with TEs or lack strong bioinformatic skills. Here, we present TEENA (Transposable Element ENrichment Analyzer) to streamline TE enrichment analysis in various organisms. It implements an optimized pipeline, hosts the genome/gene/TE annotations of almost one hundred species, and provides multiple parameters to enable its flexibility. Taking genomic interval data as the only user-supplied file, it can automatically retrieve the corresponding annotations and finish a routine analysis in a couple minutes. Multiple case studies demonstrate that it can produce highly reliable results matching previous knowledge. TEENA can be freely accessed at: https://sun-lab.yzu.edu.cn/TEENA. Due to its easy-to-use design, we expect it to facilitate the studies of the regulatory function of TEs in various model and non-model organisms.

DOI: https://doi.org/10.1093/nar/gkae411
Cell Syst. 2024 May 15. pii: S2405-4712(24)00120-0. [Epub ahead of print]15(5): 462-474.e5

Causal gene regulatory analysis with RNA velocity reveals an interplay between slow and fast transcription factors.

Rohit Singh, Alexander P Wu, Anish Mudide, Bonnie Berger.

  Single-cell expression dynamics, from differentiation trajectories or RNA velocity, have the potential to reveal causal links between transcription factors (TFs) and their target genes in gene regulatory networks (GRNs). However, existing methods either overlook these expression dynamics or necessitate that cells be ordered along a linear pseudotemporal axis, which is incompatible with branching trajectories. We introduce Velorama, an approach to causal GRN inference that represents single-cell differentiation dynamics as a directed acyclic graph of cells, constructed from pseudotime or RNA velocity measurements. Additionally, Velorama enables the estimation of the speed at which TFs influence target genes. Applying Velorama, we uncover evidence that the speed of a TF's interactions is tied to its regulatory function. For human corticogenesis, we find that slow TFs are linked to gliomas, while fast TFs are associated with neuropsychiatric diseases. We expect Velorama to become a critical part of the RNA velocity toolkit for investigating the causal drivers of differentiation and disease.

Keywords:  Granger causality; RNA velocity; corticogenesis; gene regulatory network; graph neural network; regulatory dynamics; transcription factors

DOI:  https://doi.org/10.1016/j.cels.2024.04.005
iScience. 2024 May 17. 27(5): 109765

A statistical approach for identifying single nucleotide variants that affect transcription factor binding.

Nina Baumgarten, Laura Rumpf, Thorsten Kessler, Marcel H Schulz.

  Non-coding variants located within regulatory elements may alter gene expression by modifying transcription factor (TF) binding sites, thereby leading to functional consequences. Different TF models are being used to assess the effect of DNA sequence variants, such as single nucleotide variants (SNVs). Often existing methods are slow and do not assess statistical significance of results. We investigated the distribution of absolute maximal differential TF binding scores for general computational models that affect TF binding. We find that a modified Laplace distribution can adequately approximate the empirical distributions. A benchmark on in vitro and in vivo datasets showed that our approach improves upon an existing method in terms of performance and speed. Applications on eQTLs and on a genome-wide association study illustrate the usefulness of our statistics by highlighting cell type-specific regulators and target genes. An implementation of our approach is freely available on GitHub and as bioconda package.

Keywords:  Computational bioinformatics; Computational mathematics; Genomics

DOI:  https://doi.org/10.1016/j.isci.2024.109765
Nat Commun. 2024 May 16. 15(1): 4097

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Teena Bhakuni, Pieter R Norden, Naoto Ujiie, Can Tan, Sun Kyong Lee, Thomas Tedeschi, Yi-Wen Hsieh, Ying Wang, Ting Liu, Amani A Fawzi, Tsutomu Kume.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

DOI: https://doi.org/10.1038/s41467-024-48134-2
PLoS Comput Biol. 2024 May 16. 20(5): e1012095

Interpretable online network dictionary learning for inferring long-range chromatin interactions.

Vishal Rana, Jianhao Peng, Chao Pan, Hanbaek Lyu, Albert Cheng, Minji Kim, Olgica Milenkovic.

Dictionary learning (DL), implemented via matrix factorization (MF), is commonly used in computational biology to tackle ubiquitous clustering problems. The method is favored due to its conceptual simplicity and relatively low computational complexity. However, DL algorithms produce results that lack interpretability in terms of real biological data. Additionally, they are not optimized for graph-structured data and hence often fail to handle them in a scalable manner. In order to address these limitations, we propose a novel DL algorithm called online convex network dictionary learning (online cvxNDL). Unlike classical DL algorithms, online cvxNDL is implemented via MF and designed to handle extremely large datasets by virtue of its online nature. Importantly, it enables the interpretation of dictionary elements, which serve as cluster representatives, through convex combinations of real measurements. Moreover, the algorithm can be applied to data with a network structure by incorporating specialized subnetwork sampling techniques. To demonstrate the utility of our approach, we apply cvxNDL on 3D-genome RNAPII ChIA-Drop data with the goal of identifying important long-range interaction patterns (long-range dictionary elements). ChIA-Drop probes higher-order interactions, and produces data in the form of hypergraphs whose nodes represent genomic fragments. The hyperedges represent observed physical contacts. Our hypergraph model analysis has the objective of creating an interpretable dictionary of long-range interaction patterns that accurately represent global chromatin physical contact maps. Through the use of dictionary information, one can also associate the contact maps with RNA transcripts and infer cellular functions. To accomplish the task at hand, we focus on RNAPII-enriched ChIA-Drop data from Drosophila Melanogaster S2 cell lines. Our results offer two key insights. First, we demonstrate that online cvxNDL retains the accuracy of classical DL (MF) methods while simultaneously ensuring unique interpretability and scalability. Second, we identify distinct collections of proximal and distal interaction patterns involving chromatin elements shared by related processes across different chromosomes, as well as patterns unique to specific chromosomes. To associate the dictionary elements with biological properties of the corresponding chromatin regions, we employ Gene Ontology (GO) enrichment analysis and perform multiple RNA coexpression studies.

DOI: https://doi.org/10.1371/journal.pcbi.1012095
Nat Genet. 2024 May 14.

GAGE-seq concurrently profiles multiscale 3D genome organization and gene expression in single cells.

Tianming Zhou, Ruochi Zhang, Deyong Jia, Raymond T Doty, Adam D Munday, Daniel Gao, Li Xin, Janis L Abkowitz, Zhijun Duan, Jian Ma.

The organization of mammalian genomes features a complex, multiscale three-dimensional (3D) architecture, whose functional significance remains elusive because of limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we introduce genome architecture and gene expression by sequencing (GAGE-seq), a scalable, robust single-cell co-assay measuring 3D genome structure and transcriptome simultaneously within the same cell. Applied to mouse brain cortex and human bone marrow CD34+ cells, GAGE-seq characterized the intricate relationships between 3D genome and gene expression, showing that multiscale 3D genome features inform cell-type-specific gene expression and link regulatory elements to target genes. Integration with spatial transcriptomic data revealed in situ 3D genome variations in mouse cortex. Observations in human hematopoiesis unveiled discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level. GAGE-seq provides a powerful, cost-effective approach for exploring genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

DOI: https://doi.org/10.1038/s41588-024-01745-3
Phys Rev E. 2024 Apr;109(4-1): 044502

Effect of loops on the mean-square displacement of Rouse-model chromatin.

Tianyu Yuan, Hao Yan, Mary Lou P Bailey, Jessica F Williams, Ivan Surovtsev, Megan C King, Simon G J Mochrie.

Chromatin polymer dynamics are commonly described using the classical Rouse model. The subsequent discovery, however, of intermediate-scale chromatin organization known as topologically associating domains (TADs) in experimental Hi-C contact maps for chromosomes across the tree of life, together with the success of loop extrusion factor (LEF) model in explaining TAD formation, motivates efforts to understand the effect of loops and loop extrusion on chromatin dynamics. This paper seeks to fulfill this need by combining LEF-model simulations with extended Rouse-model polymer simulations to investigate the dynamics of chromatin with loops and dynamic loop extrusion. We show that loops significantly suppress the averaged mean-square displacement (MSD) of a gene locus, consistent with recent experiments that track fluorescently labeled chromatin loci. We also find that loops reduce the MSD's stretching exponent from the classical Rouse-model value of 1/2 to a loop-density-dependent value in the 0.45-0.40 range. Remarkably, stretching exponent values in this range have also been observed in recent experiments [Weber et al., Phys. Rev. Lett. 104, 238102 (2010)0031-900710.1103/PhysRevLett.104.238102; Bailey et al., Mol. Biol. Cell 34, ar78 (2023)1059-152410.1091/mbc.E23-04-0119]. We also show that the dynamics of loop extrusion itself negligibly affects chromatin mobility. By studying static "rosette" loop configurations, we also demonstrate that chromatin MSDs and stretching exponents depend on the location of the locus in question relative to the position of the loops and on the local friction environment.

DOI: https://doi.org/10.1103/PhysRevE.109.044502
Nat Commun. 2024 May 15. 15(1): 4095

Nuclear actin structure regulates chromatin accessibility.

Buer Sen, Zhihui Xie, Michelle D Thomas, Samantha G Pattenden, Sean Howard, Cody McGrath, Maya Styner, Gunes Uzer, Terrence S Furey, Janet Rubin.

Polymerized β-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

DOI: https://doi.org/10.1038/s41467-024-48580-y
J Exp Clin Cancer Res. 2024 May 15. 43(1): 144

PAX6 promotes neuroendocrine phenotypes of prostate cancer via enhancing MET/STAT5A-mediated chromatin accessibility.

Nan Jing, Xinxing Du, Yu Liang, ZhenKeke Tao, Shijia Bao, Huixiang Xiao, Baijun Dong, Wei-Qiang Gao, Yu-Xiang Fang.

   BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a lethal subset of prostate cancer which is characterized by neuroendocrine differentiation and loss of androgen receptor (AR) signaling. Growing evidence reveals that cell lineage plasticity is crucial in the failure of NEPC therapies. Although studies suggest the involvement of the neural transcription factor PAX6 in drug resistance, its specific role in NEPC remains unclear.
METHODS: The expression of PAX6 in NEPC was identified via bioinformatics and immunohistochemistry. CCK8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay were used to illustrate the key role of PAX6 in the progression of in vitro. ChIP and Dual-luciferase reporter assays were conducted to confirm the binding sequences of AR in the promoter region of PAX6, as well as the binding sequences of PAX6 in the promoter regions of STAT5A and MET. For in vivo validation, the xenograft model representing NEPC subtype underwent pathological analysis to verify the significant role of PAX6 in disease progression. Complementary diagnoses were established through public clinical datasets and transcriptome sequencing of specific cell lines. ATAC-seq was used to detect the chromatin accessibility of specific cell lines.
RESULTS: PAX6 expression was significantly elevated in NEPC and negatively regulated by AR signaling. Activation of PAX6 in non-NEPC cells led to NE trans-differentiation, while knock-down of PAX6 in NEPC cells inhibited the development and progression of NEPC. Importantly, loss of AR resulted in an enhanced expression of PAX6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the MET/STAT5A signaling pathway. Through ATAC-seq, we found that a high expression level of PAX6 elicited enhanced chromatin accessibility, mainly through attenuation of H4K20me3, which typically causes chromatin silence in cancer cells.
CONCLUSION: This study reveals a novel neural transcription factor PAX6 could drive NEPC progression and suggest that it might serve as a potential therapeutic target for the management of NEPC.

Keywords:  Lineage plasticity; Neuroendocrine prostate cancer; PAX6; STAT5A

DOI:  https://doi.org/10.1186/s13046-024-03064-1
Nat Commun. 2024 May 16. 15(1): 4166

Endocardial HDAC3 is required for myocardial trabeculation.

Jihyun Jang, Mette Bentsen, Ye Jun Kim, Erick Kim, Vidu Garg, Chen-Leng Cai, Mario Looso, Deqiang Li.

Failure of proper ventricular trabeculation is often associated with congenital heart disease. Support from endocardial cells, including the secretion of extracellular matrix and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of extracellular matrix and growth factors is initiated and regulated by endocardial cells. We find that genetic knockout of histone deacetylase 3 in the endocardium in mice results in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identifies significant downregulation of extracellular matrix components in histone deacetylase 3 knockout endocardial cells. Secretome from cultured histone deacetylase 3 knockout mouse cardiac endothelial cells lacks transforming growth factor ß3 and shows significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which is remarkably rescued by transforming growth factor ß3 supplementation. Mechanistically, we identify that histone deacetylase 3 knockout induces transforming growth factor ß3 expression through repressing microRNA-129-5p. Our findings provide insights into the pathogenesis of congenital heart disease and conceptual strategies to promote myocardial regeneration.

DOI: https://doi.org/10.1038/s41467-024-48362-6
Genome Biol. 2024 May 17. 25(1): 124

Robust differential expression testing for single-cell CRISPR screens at low multiplicity of infection.

Timothy Barry, Kaishu Mason, Kathryn Roeder, Eugene Katsevich.

Single-cell CRISPR screens (perturb-seq) link genetic perturbations to phenotypic changes in individual cells. The most fundamental task in perturb-seq analysis is to test for association between a perturbation and a count outcome, such as gene expression. We conduct the first-ever comprehensive benchmarking study of association testing methods for low multiplicity-of-infection (MOI) perturb-seq data, finding that existing methods produce excess false positives. We conduct an extensive empirical investigation of the data, identifying three core analysis challenges: sparsity, confounding, and model misspecification. Finally, we develop an association testing method - SCEPTRE low-MOI - that resolves these analysis challenges and demonstrates improved calibration and power.

DOI: https://doi.org/10.1186/s13059-024-03254-2
Genomics. 2024 May 10. pii: S0888-7543(24)00079-X. [Epub ahead of print] 110858

Cactus: A user-friendly and reproducible ATAC-Seq and mRNA-Seq analysis pipeline for data preprocessing, differential analysis, and enrichment analysis.

Jérôme Salignon, Lluís Millan-Ariño, Maxime Garcia, Christian G Riedel.

  The ever decreasing cost of Next-Generation Sequencing coupled with the emergence of efficient and reproducible analysis pipelines has rendered genomic methods more accessible. However, downstream analyses are basic or missing in most workflows, creating a significant barrier for non-bioinformaticians. To help close this gap, we developed Cactus, an end-to-end pipeline for analyzing ATAC-Seq and mRNA-Seq data, either separately or jointly. Its Nextflow-, container-, and virtual environment-based architecture ensures efficient and reproducible analyses. Cactus preprocesses raw reads, conducts differential analyses between conditions, and performs enrichment analyses in various databases, including DNA-binding motifs, ChIP-Seq binding sites, chromatin states, and ontologies. We demonstrate the utility of Cactus in a multi-modal and multi-species case study as well as by showcasing its unique capabilities as compared to other ATAC-Seq pipelines. In conclusion, Cactus can assist researchers in gaining comprehensive insights from chromatin accessibility and gene expression data in a quick, user-friendly, and reproducible manner.

Keywords:  ATAC-Seq; Data integration; Enrichment analysis; Pipeline; Reproducible; User-friendly; mRNA-Seq

DOI:  https://doi.org/10.1016/j.ygeno.2024.110858
Commun Biol. 2024 May 16. 7(1): 589

Downregulation of HNF4A enables transcriptomic reprogramming during the hepatic acute-phase response.

Charlotte Ehle, Aishwarya Iyer-Bierhoff, Yunchen Wu, Shaojun Xing, Michael Kiehntopf, Alexander S Mosig, Maren Godmann, Thorsten Heinzel.

The hepatic acute-phase response is characterized by a massive upregulation of serum proteins, such as haptoglobin and serum amyloid A, at the expense of liver homeostatic functions. Although the transcription factor hepatocyte nuclear factor 4 alpha (HNF4A) has a well-established role in safeguarding liver function and its cistrome spans around 50% of liver-specific genes, its role in the acute-phase response has received little attention so far. We demonstrate that HNF4A binds to and represses acute-phase genes under basal conditions. The reprogramming of hepatic transcription during inflammation necessitates loss of HNF4A function to allow expression of acute-phase genes while liver homeostatic genes are repressed. In a pre-clinical liver organoid model overexpression of HNF4A maintained liver functionality in spite of inflammation-induced cell damage. Conversely, HNF4A overexpression potently impaired the acute-phase response by retaining chromatin at regulatory regions of acute-phase genes inaccessible to transcription. Taken together, our data extend the understanding of dual HNF4A action as transcriptional activator and repressor, establishing HNF4A as gatekeeper for the hepatic acute-phase response.

DOI: https://doi.org/10.1038/s42003-024-06288-1