bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2024‒09‒29
twenty-two papers selected by
Connor Rogerson, University of Cambridge
  1. IRF8 defines the epigenetic landscape in postnatal microglia, thereby directing their transcriptome programs.
  2. Structural basis of the human transcriptional Mediator regulated by its dissociable kinase module.
  3. Chromatin protein complexes involved in gene repression in lamina-associated domains.
  4. GenomicLinks: deep learning predictions of 3D chromatin interactions in the maize genome.
  5. Integration of scHi-C and scRNA-seq data defines distinct 3D-regulated and biological-context dependent cell subpopulations.
  6. Activation of automethylated PRC2 by dimerization on chromatin.
  7. Designing realistic regulatory DNA with autoregressive language models.
  8. Selective Enhancer Gain of Function Deregulates MYC Expression in Multiple Myeloma.
  9. Testis-specific H2B.W1 disrupts nucleosome integrity by reducing DNA-histone interactions.
  10. A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.
  11. Nr2f1 enhancers have distinct functions in controlling Nr2f1 expression during cortical development.
  12. Activating mutations remodel the chromatin accessibility landscape to drive distinct regulatory networks in KMT2A-rearranged acute leukemia.
  13. mTOR activity paces human blastocyst stage developmental progression.
  14. Super-silencer perturbation by EZH2 and REST inhibition leads to large loss of chromatin interactions and reduction in cancer growth.
  15. Definition of regulatory elements and transcription factors controlling porcine immune cell gene expression at single cell resolution using single nucleus ATAC-seq.
  16. Single-cell multi-omics map of human fetal blood in Down syndrome.
  17. dHICA: a deep transformer-based model enables accurate histone imputation from chromatin accessibility.
  18. Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation.
  19. Enhanced feature matching in single-cell proteomics characterizes IFN-γ response and co-existence of cell states.
  20. The transition state for coupled folding and binding of a disordered DNA binding domain resembles the unbound state.
  21. Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.
  22. Local volume concentration, packing domains, and scaling properties of chromatin.
  1. Nat Immunol. 2024 Oct;25(10): 1928-1942
    IRF8 defines the epigenetic landscape in postnatal microglia, thereby directing their transcriptome programs.
    Keita Saeki, Richard Pan, Eunju Lee, Daisuke Kurotaki, Keiko Ozato.
      Microglia are innate immune cells in the brain. Transcription factor IRF8 (interferon regulatory factor 8) is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia (DAM)-like genes. Combined analysis of single-cell (sc)RNA sequencing and single-cell transposase-accessible chromatin with sequencing (scATAC-seq) revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Last, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with amyloidβ plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression.
    DOI:  https://doi.org/10.1038/s41590-024-01962-2
  2. Mol Cell. 2024 Sep 19. pii: S1097-2765(24)00734-2. [Epub ahead of print]
    Structural basis of the human transcriptional Mediator regulated by its dissociable kinase module.
    Ti-Chun Chao, Shin-Fu Chen, Hee Jong Kim, Hui-Chi Tang, Hsiang-Ching Tseng, An Xu, Leon Palao, Subash Khadka, Tao Li, Mo-Fan Huang, Dung-Fang Lee, Kenji Murakami, Thomas G Boyer, Kuang-Lei Tsai.
      The eukaryotic transcriptional Mediator comprises a large core (cMED) and a dissociable CDK8 kinase module (CKM). cMED recruits RNA polymerase II (RNA Pol II) and promotes pre-initiation complex formation in a manner repressed by the CKM through mechanisms presently unknown. Herein, we report cryoelectron microscopy structures of the complete human Mediator and its CKM. The CKM binds to multiple regions on cMED through both MED12 and MED13, including a large intrinsically disordered region (IDR) in the latter. MED12 and MED13 together anchor the CKM to the cMED hook, positioning CDK8 downstream and proximal to the transcription start site. Notably, the MED13 IDR obstructs the recruitment of RNA Pol II/MED26 onto cMED by direct occlusion of their respective binding sites, leading to functional repression of cMED-dependent transcription. Combined with biochemical and functional analyses, these structures provide a conserved mechanistic framework to explain the basis for CKM-mediated repression of cMED function.
    Keywords:  CDK8; CKM; CTD; IDR; MED12; MED13; MED26; PIC; RNA polymerase II; mediator; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2024.09.001
  3. EMBO J. 2024 Sep 25.
    Chromatin protein complexes involved in gene repression in lamina-associated domains.
    Stefano G Manzo, Abdelghani Mazouzi, Christ Leemans, Tom van Schaik, Nadia Neyazi, Marjon S van Ruiten, Benjamin D Rowland, Thijn R Brummelkamp, Bas van Steensel.
      Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina (NL) and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here, we performed FACS-based whole-genome genetic screens in human cells using LAD-integrated fluorescent reporters to identify such regulators. Surprisingly, the screen identified very few NL proteins, but revealed roles for dozens of known chromatin regulators. Among these are the negative elongation factor (NELF) complex and interacting factors involved in RNA polymerase pausing, suggesting that regulation of transcription elongation is a mechanism to repress transcription in LADs. Furthermore, the chromatin remodeler complex BAF and the activation complex Mediator can work both as activators and repressors in LADs, depending on the local context and possibly by rewiring heterochromatin. Our data indicate that the fundamental regulators of transcription and chromatin remodeling, rather than interaction with NL proteins, play a major role in transcription regulation within LADs.
    Keywords:  Chromatin; Gene Repression; Lamina-associated Domains; Mediator; Nuclear Lamina
    DOI:  https://doi.org/10.1038/s44318-024-00214-1
  4. NAR Genom Bioinform. 2024 Sep;6(3): lqae123
    GenomicLinks: deep learning predictions of 3D chromatin interactions in the maize genome.
    Luca Schlegel, Rohan Bhardwaj, Yadollah Shahryary, Defne Demirtürk, Alexandre P Marand, Robert J Schmitz, Frank Johannes.
      Gene regulation in eukaryotes is partly shaped by the 3D organization of chromatin within the cell nucleus. Distal interactions between cis-regulatory elements and their target genes are widespread, and many causal loci underlying heritable agricultural traits have been mapped to distal non-coding elements. The biology underlying chromatin loop formation in plants is poorly understood. Dissecting the sequence features that mediate distal interactions is an important step toward identifying putative molecular mechanisms. Here, we trained GenomicLinks, a deep learning model, to identify DNA sequence features predictive of 3D chromatin interactions in maize. We found that the presence of binding motifs of specific transcription factor classes, especially bHLH, is predictive of chromatin interaction specificities. Using an in silico mutagenesis approach we show the removal of these motifs from loop anchors leads to reduced interaction probabilities. We were able to validate these predictions with single-cell co-accessibility data from different maize genotypes that harbor natural substitutions in these TF binding motifs. GenomicLinks is currently implemented as an open-source web tool, which should facilitate its wider use in the plant research community.
    DOI:  https://doi.org/10.1093/nargab/lqae123
  5. Nat Commun. 2024 Sep 27. 15(1): 8310
    Integration of scHi-C and scRNA-seq data defines distinct 3D-regulated and biological-context dependent cell subpopulations.
    Yufan Zhou, Tian Li, Lavanya Choppavarapu, Kun Fang, Shili Lin, Victor X Jin.
      An integration of 3D chromatin structure and gene expression at single-cell resolution has yet been demonstrated. Here, we develop a computational method, a multiomic data integration (MUDI) algorithm, which integrates scHi-C and scRNA-seq data to precisely define the 3D-regulated and biological-context dependent cell subpopulations or topologically integrated subpopulations (TISPs). We demonstrate its algorithmic utility on the publicly available and newly generated scHi-C and scRNA-seq data. We then test and apply MUDI in a breast cancer cell model system to demonstrate its biological-context dependent utility. We find the newly defined topologically conserved associating domain (CAD) is the characteristic single-cell 3D chromatin structure and better characterizes chromatin domains in single-cell resolution. We further identify 20 TISPs uniquely characterizing 3D-regulated breast cancer cellular states. We reveal two of TISPs are remarkably resemble to high cycling breast cancer persister cells and chromatin modifying enzymes might be functional regulators to drive the alteration of the 3D chromatin structures. Our comprehensive integration of scHi-C and scRNA-seq data in cancer cells at single-cell resolution provides mechanistic insights into 3D-regulated heterogeneity of developing drug-tolerant cancer cells.
    DOI:  https://doi.org/10.1038/s41467-024-52440-0
  6. Mol Cell. 2024 Sep 13. pii: S1097-2765(24)00702-0. [Epub ahead of print]
    Activation of automethylated PRC2 by dimerization on chromatin.
    Paul V Sauer, Egor Pavlenko, Trinity Cookis, Linda C Zirden, Juliane Renn, Ankush Singhal, Pascal Hunold, Michaela N Hoehne-Wiechmann, Olivia van Ray, Farnusch Kaschani, Markus Kaiser, Robert Hänsel-Hertsch, Karissa Y Sanbonmatsu, Eva Nogales, Simon Poepsel.
      Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism. Here, we show that human PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans-autoactivation via EED, suggesting a previously unknown mechanism mediating context-dependent activation of PRC2. Our work showcases the molecular mechanism of auto-modification-coupled dimerization in the regulation of chromatin-modifying complexes.
    Keywords:  PRC2; chromatin; cryo-EM; development; epigenetics; gene regulation; histone methyltransferase; trans-activation
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.025
  7. Genome Res. 2024 Sep 25.
    Designing realistic regulatory DNA with autoregressive language models.
    Avantika Lal, David Garfield, Tommaso Biancalani, Gokcen Eraslan.
      Cis-regulatory elements (CREs), such as promoters and enhancers, are DNA sequences that regulate the expression of genes. The activity of a CRE is influenced by the order, composition, and spacing of sequence motifs that are bound by proteins called transcription factors (TFs). Synthetic CREs with specific properties are needed for biomanufacturing as well as for many therapeutic applications including cell and gene therapy. Here, we present regLM, a framework to design synthetic CREs with desired properties, such as high, low, or cell type-specific activity, using autoregressive language models in conjunction with supervised sequence-to-function models. We used our framework to design synthetic yeast promoters and cell type-specific human enhancers. We demonstrate that the synthetic CREs generated by our approach are not only predicted to have the desired functionality but also contain biological features similar to experimentally validated CREs. regLM thus facilitates the design of realistic regulatory DNA elements while providing insights into the cis-regulatory code.
    DOI:  https://doi.org/10.1101/gr.279142.124
  8. Cancer Res. 2024 Sep 23.
    Selective Enhancer Gain of Function Deregulates MYC Expression in Multiple Myeloma.
    Mahshid Rahmat, Kendell Clement, Jean-Baptiste Alberge, Romanos Sklavenitis-Pistofidis, Rohan Kodgule, Charles P Fulco, Daniel Heilpern-Mallory, Katarina Nilsson, David Dorfman, Jesse M Engreitz, Gad Getz, Luca Pinello, Russell Ryan, Irene M Ghobrial.
      MYC deregulation occurs in the majority of multiple myeloma (MM) cases and is associated with progression and worse prognosis. Enhanced MYC expression occurs in about 70% of MM patients, but it is known to be driven by translocation or amplification events in only ~40% of myelomas. Here, we used CRISPR interference (CRISPRi) to uncover an epigenetic mechanism of MYC regulation whereby increased accessibility of a plasma cell-type specific enhancer leads to increased MYC expression. This native enhancer activity was not associated with enhancer hijacking events but led to specific binding of c-MAF, IRF4, and SPIB transcription factors that activated MYC expression in the absence of known genetic aberrations. In addition, focal amplification was another mechanism of activation of this enhancer in approximately 3.4% of MM patients. Together, these findings define an epigenetic mechanism of MYC deregulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.
    DOI:  https://doi.org/10.1158/0008-5472.CAN-24-1440
  9. Nucleic Acids Res. 2024 Sep 26. pii: gkae825. [Epub ahead of print]
    Testis-specific H2B.W1 disrupts nucleosome integrity by reducing DNA-histone interactions.
    Dongbo Ding, Matthew Y H Pang, Mingxi Deng, Thi Thuy Nguyen, Yue Liu, Xulun Sun, Zhichun Xu, Yingyi Zhang, Yuanliang Zhai, Yan Yan, Toyotaka Ishibashi.
      Multiple testis-specific histone variants are involved in the dynamic chromatin transitions during spermatogenesis. H2B.W1 (previously called H2BFWT) is an H2B variant specific to primate testis with hitherto unclear functions, although its single-nucleotide polymorphisms (SNPs) are closely associated with male non-obstructive infertility. Here, we found that H2B.W1 is only expressed in the mid-late spermatogonia stages, and H2B.W1 nucleosomes are defined by a more flexible structure originating from weakened interactions between histones and DNA. Furthermore, one of its SNPs, H2B.W1-H100R, which is associated with infertility, further destabilizes the nucleosomes and increases the nucleosome unwrapping rate by interfering with the R100 and H4 K91/R92 interaction. Our results suggest that destabilizing H2B.W1 containing nucleosomes might change the chromatin structure of spermatogonia, and that H2B.W1-H100R enhances the nucleosome-destabilizing effects, leading to infertility.
    DOI:  https://doi.org/10.1093/nar/gkae825
  10. Nat Commun. 2024 Sep 27. 15(1): 8268
    A cell type-aware framework for nominating non-coding variants in Mendelian regulatory disorders.
    Arthur S Lee, Lauren J Ayers, Michael Kosicki, Wai-Man Chan, Lydia N Fozo, Brandon M Pratt, Thomas E Collins, Boxun Zhao, Matthew F Rose, Alba Sanchis-Juan, Jack M Fu, Isaac Wong, Xuefang Zhao, Alan P Tenney, Cassia Lee, Kristen M Laricchia, Brenda J Barry, Victoria R Bradford, Julie A Jurgens, Eleina M England, Monkol Lek, Daniel G MacArthur, Eunjung Alice Lee, Michael E Talkowski, Harrison Brand, Len A Pennacchio, Elizabeth C Engle.
      Unsolved Mendelian cases often lack obvious pathogenic coding variants, suggesting potential non-coding etiologies. Here, we present a single cell multi-omic framework integrating embryonic mouse chromatin accessibility, histone modification, and gene expression assays to discover cranial motor neuron (cMN) cis-regulatory elements and subsequently nominate candidate non-coding variants in the congenital cranial dysinnervation disorders (CCDDs), a set of Mendelian disorders altering cMN development. We generate single cell epigenomic profiles for ~86,000 cMNs and related cell types, identifying ~250,000 accessible regulatory elements with cognate gene predictions for ~145,000 putative enhancers. We evaluate enhancer activity for 59 elements using an in vivo transgenic assay and validate 44 (75%), demonstrating that single cell accessibility can be a strong predictor of enhancer activity. Applying our cMN atlas to 899 whole genome sequences from 270 genetically unsolved CCDD pedigrees, we achieve significant reduction in our variant search space and nominate candidate variants predicted to regulate known CCDD disease genes MAFB, PHOX2A, CHN1, and EBF3 - as well as candidates in recurrently mutated enhancers through peak- and gene-centric allelic aggregation. This work delivers non-coding variant discoveries of relevance to CCDDs and a generalizable framework for nominating non-coding variants of potentially high functional impact in other Mendelian disorders.
    DOI:  https://doi.org/10.1038/s41467-024-52463-7
  11. Proc Natl Acad Sci U S A. 2024 Oct;121(40): e2402368121
    Nr2f1 enhancers have distinct functions in controlling Nr2f1 expression during cortical development.
    Zhidong Liu, Athéna R Ypsilanti, Eirene Markenscoff-Papadimitriou, Diane E Dickel, Stephan J Sanders, Shan Dong, Len A Pennacchio, Axel Visel, John L Rubenstein.
      There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis.
    Keywords:  Nr2f1; cortical development; enhancer; epigenomics
    DOI:  https://doi.org/10.1073/pnas.2402368121
  12. Hemasphere. 2024 Sep;8(9): e70006
    Activating mutations remodel the chromatin accessibility landscape to drive distinct regulatory networks in KMT2A-rearranged acute leukemia.
    Qirui Zhang, Ton Falqués-Costa, Mattias Pilheden, Helena Sturesson, Tina Ovlund, Vendela Rissler, Anders Castor, Hanne V H Marquart, Birgitte Lausen, Thoas Fioretos, Axel Hyrenius-Wittsten, Anna K Hagström-Andersson.
      Activating FLT3 and RAS mutations commonly occur in leukemia with KMT2A-gene rearrangements (KMT2A-r). However, how these mutations cooperate with the KMT2A-r to remodel the epigenetic landscape is unknown. Using a retroviral acute myeloid leukemia (AML) mouse model driven by KMT2A::MLLT3, we show that FLT3 ITD , FLT3 N676K , and NRAS G12D remodeled the chromatin accessibility landscape and associated transcriptional networks. Although the activating mutations shared a common core of chromatin changes, each mutation exhibits unique profiles with most opened peaks associating with enhancers in intronic or intergenic regions. Specifically, FLT3 N676K and NRAS G12D rewired similar chromatin and transcriptional networks, distinct from those mediated by FLT3 ITD . Motif analysis uncovered a role for the AP-1 family of transcription factors in KMT2A::MLLT3 leukemia with FLT3 N676K and NRAS G12D , whereas Runx1 and Stat5a/Stat5b were active in the presence of FLT3 ITD . Furthermore, transcriptional programs linked to immune cell regulation were activated in KMT2A-r AML expressing NRAS G12D or FLT3 N676K , and the expression of NKG2D-ligands on KMT2A-r cells rendered them sensitive to CAR T cell-mediated killing. Human KMT2A-r AML cells could be pharmacologically sensitized to NKG2D-CAR T cells by treatment with the histone deacetylase inhibitor LBH589 (panobinostat) which caused upregulation of NKG2D-ligand levels. Co-treatment with LBH589 and NKG2D-CAR T cells enabled robust AML cell killing, and the strongest effect was observed for cells expressing NRAS G12D . Finally, the results were validated and extended to acute leukemia in infancy. Combined, activating mutations induced mutation-specific changes in the epigenetic landscape, leading to changes in transcriptional programs orchestrated by specific transcription factor networks.
    DOI:  https://doi.org/10.1002/hem3.70006
  13. Cell. 2024 Sep 18. pii: S0092-8674(24)00977-2. [Epub ahead of print]
    mTOR activity paces human blastocyst stage developmental progression.
    Dhanur P Iyer, Heidar Heidari Khoei, Vera A van der Weijden, Harunobu Kagawa, Saurabh J Pradhan, Maria Novatchkova, Afshan McCarthy, Teresa Rayon, Claire S Simon, Ilona Dunkel, Sissy E Wamaitha, Kay Elder, Phil Snell, Leila Christie, Edda G Schulz, Kathy K Niakan, Nicolas Rivron, Aydan Bulut-Karslioğlu.
      Many mammals can temporally uncouple conception from parturition by pacing down their development around the blastocyst stage. In mice, this dormant state is achieved by decreasing the activity of the growth-regulating mTOR signaling pathway. It is unknown whether this ability is conserved in mammals in general and in humans in particular. Here, we show that decreasing the activity of the mTOR signaling pathway induces human pluripotent stem cells (hPSCs) and blastoids to enter a dormant state with limited proliferation, developmental progression, and capacity to attach to endometrial cells. These in vitro assays show that, similar to other species, the ability to enter dormancy is active in human cells around the blastocyst stage and is reversible at both functional and molecular levels. The pacing of human blastocyst development has potential implications for reproductive therapies.
    Keywords:  blastoid; development; diapause; dormancy; human; mTOR; pluripotent stem cells
    DOI:  https://doi.org/10.1016/j.cell.2024.08.048
  14. Nat Struct Mol Biol. 2024 Sep 20.
    Super-silencer perturbation by EZH2 and REST inhibition leads to large loss of chromatin interactions and reduction in cancer growth.
    Ying Zhang, Kaijing Chen, Seng Chuan Tang, Yichao Cai, Akiko Nambu, Yi Xiang See, Chaoyu Fu, Anandhkumar Raju, Benjamin Lebeau, Zixun Ling, Jia Jia Chan, Yvonne Tay, Marek Mutwil, Manikandan Lakshmanan, Greg Tucker-Kellogg, Wee Joo Chng, Daniel G Tenen, Motomi Osato, Vinay Tergaonkar, Melissa Jane Fullwood.
      Human silencers have been shown to regulate developmental gene expression. However, the functional importance of human silencers needs to be elucidated, such as whether they can form 'super-silencers' and whether they are linked to cancer progression. Here, we show two silencer components of the FGF18 gene can cooperate through compensatory chromatin interactions to form a super-silencer. Double knockout of two silencers exhibited synergistic upregulation of FGF18 expression and changes in cell identity. To perturb the super-silencers, we applied combinational treatment of an enhancer of zeste homolog 2 inhibitor GSK343, and a repressor element 1-silencing transcription factor inhibitor, X5050 ('GR'). Interestingly, GR led to severe loss of topologically associated domains and loops, which were associated with reduced CTCF and TOP2A mRNA levels. Moreover, GR synergistically upregulated super-silencer-controlled genes related to cell cycle, apoptosis and DNA damage, leading to anticancer effects in vivo. Overall, our data demonstrated a super-silencer example and showed that GR can disrupt super-silencers, potentially leading to cancer ablation.
    DOI:  https://doi.org/10.1038/s41594-024-01391-7
  15. Genomics. 2024 Sep 24. pii: S0888-7543(24)00165-4. [Epub ahead of print] 110944
    Definition of regulatory elements and transcription factors controlling porcine immune cell gene expression at single cell resolution using single nucleus ATAC-seq.
    Pengxin Yang, Ryan Corbett, Lance Daharsh, Juber Herrera Uribe, Kristen A Byrne, Crystal L Loving, Christopher Tuggle.
      The transcriptome of porcine peripheral blood mononuclear cells (PBMC) at single cell (sc) resolution is well described, but little is understood about the cis-regulatory mechanism behind scPBMC gene expression. Here, we profiled the open chromatin landscape of porcine PBMC that define cis-regulatory elements and mechanism contributing to the transcription using single nucleus ATAC sequencing (snATAC-seq). Approximately 22 % of the identified peaks overlapped with annotated transcription start sites (TSS). Using clustering based on open chromatin pattern similarity, we demonstrate that cell type annotations using snATAC-seq are highly concordant to that reported for sc RNA sequencing (scRNA-seq). The differentially accessible peaks (DAPs) for each cell type were characterized and the pattern of accessibility of the DAPs near cell type markers across cell types was similar to that of the average gene expression level of corresponding marker genes. Additionally, we found that peaks identified in snATAC-seq have the potential power to predict the cell type specific transcription starting site (TSS). We identified both transcription factors (TFs) whose binding motif were enriched in cell type DAPs of multiple cell types and cell type specific TFs by conducting transcription factor binding motif (TFBM) analysis. Furthermore, we identified the putative enhancer or promoter regions bound by TFs for each differentially expressed gene (DEG) with a DAP that overlapped with its TSS by generating cis-co-accessibility networks (CCAN). To predict the regulators of such DEGs, TFBM analysis was performed for each CCAN. The regulator TF-target DEG pairs predicted in this way were largely consistent with the results reported in the ENCODE Transcription Factor Targets Dataset (TFTD). This snATAC-seq approach provides insights into the regulation of chromatin accessibility landscape of porcine PBMCs and enables discovery of TFs predicted to control DEG through binding regulatory elements whose chromatin accessibility correlates with the DEG promoter region.
    Keywords:  FAANG; Immune cells; Pig; Regulatory network; Single nucleus chromatin accessibility; Single-nucleus ATAC; scRNA-seq and snATAC-seq integration
    DOI:  https://doi.org/10.1016/j.ygeno.2024.110944
  16. Nature. 2024 Sep 25.
    Single-cell multi-omics map of human fetal blood in Down syndrome.
    Andrew R Marderstein, Marco De Zuani, Rebecca Moeller, Jon Bezney, Evin M Padhi, Shuo Wong, Tim H H Coorens, Yilin Xie, Haoliang Xue, Stephen B Montgomery, Ana Cvejic.
      Down syndrome predisposes individuals to haematological abnormalities, such as increased number of erythrocytes and leukaemia in a process that is initiated before birth and is not entirely understood1-3. Here, to understand dysregulated haematopoiesis in Down syndrome, we integrated single-cell transcriptomics of over 1.1 million cells with chromatin accessibility and spatial transcriptomics datasets using human fetal liver and bone marrow samples from 3 fetuses with disomy and 15 fetuses with trisomy. We found that differences in gene expression in Down syndrome were dependent on both cell type and environment. Furthermore, we found multiple lines of evidence that haematopoietic stem cells (HSCs) in Down syndrome are 'primed' to differentiate. We subsequently established a Down syndrome-specific map linking non-coding elements to genes in disomic and trisomic HSCs using 10X multiome data. By integrating this map with genetic variants associated with blood cell counts, we discovered that trisomy restructured regulatory interactions to dysregulate enhancer activity and gene expression critical to erythroid lineage differentiation. Furthermore, as mutations in Down syndrome display a signature of oxidative stress4,5, we validated both increased mitochondrial mass and oxidative stress in Down syndrome, and observed that these mutations preferentially fell into regulatory regions of expressed genes in HSCs. Together, our single-cell, multi-omic resource provides a high-resolution molecular map of fetal haematopoiesis in Down syndrome and indicates significant regulatory restructuring giving rise to co-occurring haematological conditions.
    DOI:  https://doi.org/10.1038/s41586-024-07946-4
  17. Brief Bioinform. 2024 Sep 23. pii: bbae459. [Epub ahead of print]25(6):
    dHICA: a deep transformer-based model enables accurate histone imputation from chromatin accessibility.
    Wen Wen, Jiaxin Zhong, Zhaoxi Zhang, Lijuan Jia, Tinyi Chu, Nating Wang, Charles G Danko, Zhong Wang.
      Histone modifications (HMs) are pivotal in various biological processes, including transcription, replication, and DNA repair, significantly impacting chromatin structure. These modifications underpin the molecular mechanisms of cell-type-specific gene expression and complex diseases. However, annotating HMs across different cell types solely using experimental approaches is impractical due to cost and time constraints. Herein, we present dHICA (deep histone imputation using chromatin accessibility), a novel deep learning framework that integrates DNA sequences and chromatin accessibility data to predict multiple HM tracks. Employing the transformer architecture alongside dilated convolutions, dHICA boasts an extensive receptive field and captures more cell-type-specific information. dHICA outperforms state-of-the-art baselines and achieves superior performance in cell-type-specific loci and gene elements, aligning with biological expectations. Furthermore, dHICA's imputations hold significant potential for downstream applications, including chromatin state segmentation and elucidating the functional implications of SNPs (Single Nucleotide Polymorphisms). In conclusion, dHICA serves as a valuable tool for advancing the understanding of chromatin dynamics, offering enhanced predictive capabilities and interpretability.
    Keywords:  cell-type-specific; deep learning; histone modification prediction; histone modifications; transformer
    DOI:  https://doi.org/10.1093/bib/bbae459
  18. Mol Cell. 2024 Sep 16. pii: S1097-2765(24)00664-6. [Epub ahead of print]
    Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation.
    Maya L Gosztyla, Lijun Zhan, Sara Olson, Xintao Wei, Jack Naritomi, Grady Nguyen, Lena Street, Grant A Goda, Francisco F Cavazos, Jonathan C Schmok, Manya Jain, Easin Uddin Syed, Eunjeong Kwon, Wenhao Jin, Eric Kofman, Alexandra T Tankka, Allison Li, Valerie Gonzalez, Eric Lécuyer, Daniel Dominguez, Marko Jovanovic, Brenton R Graveley, Gene W Yeo.
      RNA interactome studies have revealed that hundreds of zinc-finger proteins (ZFPs) are candidate RNA-binding proteins (RBPs), yet their RNA substrates and functional significance remain largely uncharacterized. Here, we present a systematic multi-omics analysis of the DNA- and RNA-binding targets and regulatory roles of more than 100 ZFPs representing 37 zinc-finger families. We show that multiple ZFPs are previously unknown regulators of RNA splicing, alternative polyadenylation, stability, or translation. The examined ZFPs show widespread sequence-specific RNA binding and preferentially bind proximal to transcription start sites. Additionally, several ZFPs associate with their targets at both the DNA and RNA levels. We highlight ZNF277, a C2H2 ZFP that binds thousands of RNA targets and acts as a multi-functional RBP. We also show that ZNF473 is a DNA/RNA-associated protein that regulates the expression and splicing of cell cycle genes. Our results reveal diverse roles for ZFPs in transcriptional and post-transcriptional gene regulation.
    Keywords:  Cut&Run; RNA binding protein; RNA-seq; Ribo-STAMP; SLAM-seq; ZNF277; ZNF473; eCLIP; multi-omics; zinc finger
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.010
  19. Nat Commun. 2024 Sep 26. 15(1): 8262
    Enhanced feature matching in single-cell proteomics characterizes IFN-γ response and co-existence of cell states.
    Karl K Krull, Syed Azmal Ali, Jeroen Krijgsveld.
      Proteome analysis by data-independent acquisition (DIA) has become a powerful approach to obtain deep proteome coverage, and has gained recent traction for label-free analysis of single cells. However, optimal experimental design for DIA-based single-cell proteomics has not been fully explored, and performance metrics of subsequent data analysis tools remain to be evaluated. Therefore, we here formalize and comprehensively evaluate a DIA data analysis strategy that exploits the co-analysis of low-input samples with a so-called matching enhancer (ME) of higher input, to increase sensitivity, proteome coverage, and data completeness. We assess the matching specificity of DIA-ME by a two-proteome model, and demonstrate that false discovery and false transfer are maintained at low levels when using DIA-NN software, while preserving quantification accuracy. We apply DIA-ME to investigate the proteome response of U-2 OS cells to interferon gamma (IFN-γ) in single cells, and recapitulate the time-resolved induction of IFN-γ response proteins as observed in bulk material. Moreover, we uncover co- and anti-correlating patterns of protein expression within the same cell, indicating mutually exclusive protein modules and the co-existence of different cell states. Collectively our data show that DIA-ME is a powerful, scalable, and easy-to-implement strategy for single-cell proteomics.
    DOI:  https://doi.org/10.1038/s41467-024-52605-x
  20. Nucleic Acids Res. 2024 Sep 24. pii: gkae794. [Epub ahead of print]
    The transition state for coupled folding and binding of a disordered DNA binding domain resembles the unbound state.
    Mikhail Kuravsky, Conor Kelly, Christina Redfield, Sarah L Shammas.
      The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable α-helices upon target DNA binding. Here, we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state. The residue closest to the dimerization domain is largely folded within both unbound and transition states; dimerization apparently propagates additional helical propensity into the basic region. The results are consistent with electrostatically-enhanced DNA binding, followed by rapid folding from the folded zipper outwards. Fly-casting theory suggests that protein disorder can accelerate binding. Interestingly however, we did not observe higher association rate constants for mutants with lower levels of residual structure in the unbound state.
    DOI:  https://doi.org/10.1093/nar/gkae794
  21. Elife. 2024 Sep 25. pii: e89725. [Epub ahead of print]13
    Arp2/3 complex activity enables nuclear YAP for naïve pluripotency of human embryonic stem cells.
    Nathaniel Paul Meyer, Tania Singh, Matthew L Kutys, Todd G Nystul, Diane L Barber.
      Our understanding of the transitions of human embryonic stem cells between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of human embryonic stem cells (hESCs) as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for the actin ring, to establish uniform cell mechanics within naïve colonies, promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.
    Keywords:  cell biology; human; regenerative medicine; stem cells
    DOI:  https://doi.org/10.7554/eLife.89725
  22. Elife. 2024 Sep 27. pii: RP97604. [Epub ahead of print]13
    Local volume concentration, packing domains, and scaling properties of chromatin.
    Marcelo A Carignano, Martin Kroeger, Luay M Almassalha, Vasundhara Agrawal, Wing Shun Li, Emily M Pujadas-Liwag, Rikkert J Nap, Vadim Backman, Igal Szleifer.
      We propose the Self Returning Excluded Volume (SR-EV) model for the structure of chromatin based on stochastic rules and physical interactions. The SR-EV rules of return generate conformationally defined domains observed by single-cell imaging techniques. From nucleosome to chromosome scales, the model captures the overall chromatin organization as a corrugated system, with dense and dilute regions alternating in a manner that resembles the mixing of two disordered bi-continuous phases. This particular organizational topology is a consequence of the multiplicity of interactions and processes occurring in the nuclei, and mimicked by the proposed return rules. Single configuration properties and ensemble averages show a robust agreement between theoretical and experimental results including chromatin volume concentration, contact probability, packing domain identification and size characterization, and packing scaling behavior. Model and experimental results suggest that there is an inherent chromatin organization regardless of the cell character and resistant to an external forcing such as RAD21 degradation.
    Keywords:  SR-EV; chromatin; none; physics of living systems; theory
    DOI:  https://doi.org/10.7554/eLife.97604
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