bims-crepig 2021-09-26 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2021–09–26
28 papers selected by
Connor Rogerson, University of Cambridge, MRC Cancer Unit

SMARCA4 inactivation promotes lineage-specific transformation and early metastatic features in the lung.
Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization.
Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor.
PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation.
NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis.
OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity.
Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.
Tramtrack acts during late pupal development to direct ant caste identity.
Mutational synergy during leukemia induction remodels chromatin accessibility, histone modifications and three-dimensional DNA topology to alter gene expression.
Targeting the IRE1α/XBP1 Endoplasmic Reticulum Stress Response Pathway in ARID1A-Mutant Ovarian Cancers.
Targeting AP-1 transcription factors by CRISPR in the prostate.
DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.
Mutations in non-coding cis-regulatory elements reveal cancer driver cistromes in luminal breast cancer.
Genetic and epigenetic coordination of cortical interneuron development.
The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions.
ATF3 promotes the serine synthesis pathway and tumor growth under dietary serine restriction.
Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.
Differentially expressed genes reflect disease-induced rather than disease-causing changes in the transcriptome.
Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.
LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming.
Suberin plasticity to developmental and exogenous cues is regulated by a set of MYB transcription factors.
HIF-1α hydroxyprolines modulate oxygen-dependent protein stability via single VHL interface with comparable effect on ubiquitination rate.
Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.
Nucleosome assembly protein 1 (NAP-1) is a regulator of histone H1 acetylation.
ATF6 is essential for human cone photoreceptor development.
MYC levels regulate metastatic heterogeneity in pancreatic adenocarcinoma.
CENP-B promotes the centromeric localization of ZFAT to control transcription of non-coding RNA.
Linking nuclear matrix-localized PIAS1 to chromatin SUMOylation via direct binding of histones H3 and H2A.Z.

Cancer Discov. 2021 Sep 24. pii: candisc.0248.2021. [Epub ahead of print]

SMARCA4 inactivation promotes lineage-specific transformation and early metastatic features in the lung.

Carla P Concepcion, Sai Ma, Lindsay M LaFave, Arjun Bhutkar, Manyuan Liu, Lydia P DeAngelo, Jonathan Y Kim, Isabella Del Priore, Adam J Schoenfeld, Manon Miller, Vinay K Kartha, Peter M K Westcott, Francisco J Sanchez-Rivera, Kevin Meli, Manav Gupta, Roderick T Bronson, Gregory J Riely, Natasha Rekhtman, Charles M Rudin, Carla F Kim, Aviv Regev, Jason D Buenrostro, Tyler Jacks.

SMARCA4/BRG1 encodes for one of two mutually exclusive ATPases present in mammalian SWI/SNF chromatin remodeling complexes and is frequently mutated in human lung adenocarcinoma. However, the functional consequences of SMARCA4 mutation on tumor initiation, progression, and chromatin regulation in lung cancer remain poorly understood. Here, we demonstrate that loss of Smarca4 sensitizes CCSP+ cells within the lung in a cell-type dependent fashion to malignant transformation and tumor progression, resulting in highly advanced dedifferentiated tumors and increased metastatic incidence. Consistent with these phenotypes, Smarca4-deficient primary tumors lack lung lineage transcription factor activities and resemble a metastatic cell state. Mechanistically, we show that Smarca4 loss impairs the function of all three classes of SWI/SNF complexes, resulting in decreased chromatin accessibility at lung lineage motifs and ultimately accelerating tumor progression. Thus, we propose that the SWI/SNF complex - via Smarca4 - acts as a gatekeeper for lineage-specific cellular transformation and metastasis during lung cancer evolution.

DOI: https://doi.org/10.1158/2159-8290.CD-21-0248
Nat Commun. 2021 Sep 22. 12(1): 5579

Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization.

Rajesh Sharma, Kyoung-Jae Choi, My Diem Quan, Sonum Sharma, Banumathi Sankaran, Hyekyung Park, Anel LaGrone, Jean J Kim, Kevin R MacKenzie, Allan Chris M Ferreon, Choel Kim, Josephine C Ferreon.

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid-liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.

DOI: https://doi.org/10.1038/s41467-021-25761-7
PLoS Genet. 2021 Sep 24. 17(9): e1009039

Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor.

Yi Kuang, Anna Pyo, Natanel Eafergan, Brittany Cain, Lisa M Gutzwiller, Ofri Axelrod, Ellen K Gagliani, Matthew T Weirauch, Raphael Kopan, Rhett A Kovall, David Sprinzak, Brian Gebelein.

Notch signaling controls many developmental processes by regulating gene expression. Notch-dependent enhancers recruit activation complexes consisting of the Notch intracellular domain, the Cbf/Su(H)/Lag1 (CSL) transcription factor (TF), and the Mastermind co-factor via two types of DNA sites: monomeric CSL sites and cooperative dimer sites called Su(H) paired sites (SPS). Intriguingly, the CSL TF can also bind co-repressors to negatively regulate transcription via these same sites. Here, we tested how synthetic enhancers with monomeric CSL sites versus dimeric SPSs bind Drosophila Su(H) complexes in vitro and mediate transcriptional outcomes in vivo. Our findings reveal that while the Su(H)/Hairless co-repressor complex similarly binds SPS and CSL sites in an additive manner, the Notch activation complex binds SPSs, but not CSL sites, in a cooperative manner. Moreover, transgenic reporters with SPSs mediate stronger, more consistent transcription and are more resistant to increased Hairless co-repressor expression compared to reporters with the same number of CSL sites. These findings support a model in which SPS containing enhancers preferentially recruit cooperative Notch activation complexes over Hairless repression complexes to ensure consistent target gene activation.

DOI: https://doi.org/10.1371/journal.pgen.1009039
Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01201-8. [Epub ahead of print]36(12): 109747

PBRM1 loss in kidney cancer unbalances the proximal tubule master transcription factor hub to repress proximal tubule differentiation.

Xiaorong Gu, Francis Enane, Rita Tohme, Caroline Schuerger, Tomas Radivoyevitch, Yvonne Parker, Eric Zuberi, Bartlomiej Przychodzen, Babal Kant Jha, Daniel Lindner, Brian Rini, Yogen Saunthararajah.

  PBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.

Keywords:  MYC; PAX8; PBRM1; coactivator; corepressor; epithelial differentiation; master transcription factor; nephric lineage; renal cell cancer

DOI:  https://doi.org/10.1016/j.celrep.2021.109747
Mol Cell. 2021 Sep 15. pii: S1097-2765(21)00714-0. [Epub ahead of print]

NSD2 dimethylation at H3K36 promotes lung adenocarcinoma pathogenesis.

Deepanwita Sengupta, Liyong Zeng, Yumei Li, Simone Hausmann, Debopam Ghosh, Gang Yuan, Thuyen N Nguyen, Ruitu Lyu, Marcello Caporicci, Ana Morales Benitez, Garry L Coles, Vladlena Kharchenko, Iwona Czaban, Dulat Azhibek, Wolfgang Fischle, Mariusz Jaremko, Ignacio I Wistuba, Julien Sage, Łukasz Jaremko, Wei Li, Pawel K Mazur, Or Gozani.

  The etiological role of NSD2 enzymatic activity in solid tumors is unclear. Here we show that NSD2, via H3K36me2 catalysis, cooperates with oncogenic KRAS signaling to drive lung adenocarcinoma (LUAD) pathogenesis. In vivo expression of NSD2E1099K, a hyperactive variant detected in individuals with LUAD, rapidly accelerates malignant tumor progression while decreasing survival in KRAS-driven LUAD mouse models. Pathologic H3K36me2 generation by NSD2 amplifies transcriptional output of KRAS and several complementary oncogenic gene expression programs. We establish a versatile in vivo CRISPRi-based system to test gene functions in LUAD and find that NSD2 loss strongly attenuates tumor progression. NSD2 knockdown also blocks neoplastic growth of PDXs (patient-dervived xenografts) from primary LUAD. Finally, a treatment regimen combining NSD2 depletion with MEK1/2 inhibition causes nearly complete regression of LUAD tumors. Our work identifies NSD2 as a bona fide LUAD therapeutic target and suggests a pivotal epigenetic role of the NSD2-H3K36me2 axis in sustaining oncogenic signaling.

Keywords:  CRISPR interference mouse model; H3K36; KRAS; MEK inhibition; NSD2; chromatin; epigenetics; histone methylation; lung adenocarcinoma; lung cancer

DOI:  https://doi.org/10.1016/j.molcel.2021.08.034
Nat Immunol. 2021 Sep 23.

OCT2 pre-positioning facilitates cell fate transition and chromatin architecture changes in humoral immunity.

Ashley S Doane, Chi-Shuen Chu, Dafne Campigli Di Giammartino, Martín A Rivas, Johannes C Hellmuth, Yanwen Jiang, Nevin Yusufova, Alicia Alonso, Robert G Roeder, Effie Apostolou, Ari M Melnick, Olivier Elemento.

During the germinal center (GC) reaction, B cells undergo profound transcriptional, epigenetic and genomic architectural changes. How such changes are established remains unknown. Mapping chromatin accessibility during the humoral immune response, we show that OCT2 was the dominant transcription factor linked to differential accessibility of GC regulatory elements. Silent chromatin regions destined to become GC-specific super-enhancers (SEs) contained pre-positioned OCT2-binding sites in naive B cells (NBs). These preloaded SE 'seeds' featured spatial clustering of regulatory elements enriched in OCT2 DNA-binding motifs that became heavily loaded with OCT2 and its GC-specific coactivator OCAB in GC B cells (GCBs). SEs with high abundance of pre-positioned OCT2 binding preferentially formed long-range chromatin contacts in GCs, to support expression of GC-specifying factors. Gain in accessibility and architectural interactivity of these regions were dependent on recruitment of OCAB. Pre-positioning key regulators at SEs may represent a broadly used strategy for facilitating rapid cell fate transitions.

DOI: https://doi.org/10.1038/s41590-021-01025-w
Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01174-8. [Epub ahead of print]36(12): 109725

Enhancer recruitment of transcription repressors RUNX1 and TLE3 by mis-expressed FOXC1 blocks differentiation in acute myeloid leukemia.

Fabrizio Simeoni, Isabel Romero-Camarero, Francesco Camera, Fabio M R Amaral, Oliver J Sinclair, Evangelia K Papachristou, Gary J Spencer, Michael Lie-A-Ling, Georges Lacaud, Daniel H Wiseman, Jason S Carroll, Tim C P Somervaille.

  Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in ∼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.

Keywords:  FOXC1; Groucho; RUNX1; TLE3; acute myeloid leukemia

DOI:  https://doi.org/10.1016/j.celrep.2021.109725
PLoS Genet. 2021 Sep 22. 17(9): e1009801

Tramtrack acts during late pupal development to direct ant caste identity.

Karl M Glastad, Linyang Ju, Shelley L Berger.

A key question in the rising field of neuroepigenetics is how behavioral plasticity is established and maintained in the developing CNS of multicellular organisms. Behavior is controlled through systemic changes in hormonal signaling, cell-specific regulation of gene expression, and changes in neuronal connections in the nervous system, however the link between these pathways is unclear. In the ant Camponotus floridanus, the epigenetic corepressor CoREST is a central player in experimentally-induced reprogramming of caste-specific behavior, from soldier (Major worker) to forager (Minor worker). Here, we show this pathway is engaged naturally on a large genomic scale during late pupal development targeting multiple genes differentially expressed between castes, and central to this mechanism is the protein tramtrack (ttk), a DNA binding partner of CoREST. Caste-specific differences in DNA binding of ttk co-binding with CoREST correlate with caste-biased gene expression both in the late pupal stage and immediately after eclosion. However, we find a unique set of exclusive Minor-bound genes that show ttk pre-binding in the late pupal stage preceding CoREST binding, followed by caste-specific gene repression on the first day of eclosion. In addition, we show that ttk binding correlates with neurogenic Notch signaling, and that specific ttk binding between castes is enriched for regulatory sites associated with hormonal function. Overall our findings elucidate a pathway of transcription factor binding leading to a repressive epigenetic axis that lies at the crux of development and hormonal signaling to define worker caste identity in C. floridanus.

DOI: https://doi.org/10.1371/journal.pgen.1009801
Nat Genet. 2021 Sep 23.

Mutational synergy during leukemia induction remodels chromatin accessibility, histone modifications and three-dimensional DNA topology to alter gene expression.

Haiyang Yun, Nisha Narayan, Shabana Vohra, George Giotopoulos, Annalisa Mupo, Pedro Madrigal, Daniel Sasca, David Lara-Astiaso, Sarah J Horton, Shuchi Agrawal-Singh, Eshwar Meduri, Faisal Basheer, Ludovica Marando, Malgorzata Gozdecka, Oliver M Dovey, Aracely Castillo-Venzor, Xiaonan Wang, Paolo Gallipoli, Carsten Müller-Tidow, Cameron S Osborne, George S Vassiliou, Brian J P Huntly.

Altered transcription is a cardinal feature of acute myeloid leukemia (AML); however, exactly how mutations synergize to remodel the epigenetic landscape and rewire three-dimensional DNA topology is unknown. Here, we apply an integrated genomic approach to a murine allelic series that models the two most common mutations in AML: Flt3-ITD and Npm1c. We then deconvolute the contribution of each mutation to alterations of the epigenetic landscape and genome organization, and infer how mutations synergize in the induction of AML. Our studies demonstrate that Flt3-ITD signals to chromatin to alter the epigenetic environment and synergizes with mutations in Npm1c to alter gene expression and drive leukemia induction. These analyses also allow the identification of long-range cis-regulatory circuits, including a previously unknown superenhancer of Hoxa locus, as well as larger and more detailed gene-regulatory networks, driven by transcription factors including PU.1 and IRF8, whose importance we demonstrate through perturbation of network members.

DOI: https://doi.org/10.1038/s41588-021-00925-9
Cancer Res. 2021 Sep 21.

Targeting the IRE1α/XBP1 Endoplasmic Reticulum Stress Response Pathway in ARID1A-Mutant Ovarian Cancers.

Joseph A Zundell, Takeshi Fukumoto, Jianhuang Lin, Nail Fatkhudinov, Timothy Nacarelli, Andrew V Kossenkov, Qin Liu, Joel Cassel, Chih-Chi Andrew Hu, Shuai Wu, Rugang Zhang.

The SWI/SNF chromatin-remodeling complex is frequently altered in human cancers. For example, the SWI/SNF component ARID1A is mutated in more than 50% of ovarian clear cell carcinomas (OCCC), for which effective treatments are lacking. Here, we report that ARID1A transcriptionally represses the IRE1α-XBP1 axis of the endoplasmic reticulum (ER) stress response, which confers sensitivity to inhibition of the IRE1α-XBP1 pathway in ARID1A-mutant OCCC. ARID1A mutational status correlated with response to inhibition of the IRE1α-XBP1 pathway. In a conditional Arid1aflox/flox/Pik3caH1047R genetic mouse model, Xbp1 knockout significantly improved survival of mice bearing OCCCs. Furthermore, the IRE1α inhibitor B-I09 suppressed the growth of ARID1A-inactivated OCCCs in vivo in orthotopic xenograft, patient-derived xenograft, and the genetic mouse models. Finally, B-I09 synergized with inhibition of HDAC6, a known regulator of the ER stress response, in suppressing the growth of ARID1A-inactivated OCCCs. These studies define the IRE1α-XBP1 axis of the ER stress response as a targetable vulnerability for ARID1A-mutant OCCCs, revealing a promising therapeutic approach for treating ARID1A-mutant ovarian cancers. SIGNIFICANCE: These findings indicate that pharmacological inhibition of the IRE1α-XBP1 pathway alone or in combination with HDAC6 inhibition represents an urgently needed therapeutic strategy for ARID1A-mutant ovarian cancers.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-1545
Oncotarget. 2021 Sep 14. 12(19): 1956-1961

Targeting AP-1 transcription factors by CRISPR in the prostate.

Maria Riedel, Huiqiang Cai, Iben C Stoltze, Mikkel H Vendelbo, Erwin F Wagner, Latifa Bakiri, Martin K Thomsen.

  Prostate cancer is the second most diagnosed cancer in men. It is a slow progressing cancer, but when the disease reaches an advanced stage, treatment options are limited. Sequencing analyses of cancer samples have identified genes that can potentially drive disease progression. We implemented the CRISPR/Cas9 technology to simultaneously manipulate multiple genes in the murine prostate and thus to functionally test putative cancer driver genes in vivo. The activating protein-1 (AP-1) transcription factor is associated with many different cancer types, with the proto-oncogenes JUN and FOS being the two most intensely studied subunits. We analyzed expression of FOS and JUNB in human prostate cancer datasets and observed decreased expression in advanced stages. By applying CRISPR/Cas9 technology, the role of these two transcription factors in prostate cancer progression was functionally tested. Our data revealed that loss of either JunB or Fos in the context of Pten loss drives prostate cancer progression to invasive disease. Furthermore, loss of Fos increases Jun expression, and CRISPR inactivation of Jun in this context decreases cell proliferation. Overall, these in vivo studies reveal that JunB and Fos exhibit a tumor suppressor function by repressing invasive disease, whereas Jun is oncogenic and increases cell proliferation. This demonstrates that AP-1 factors are implicated in prostate cancer progression at different stages and display a dual function as tumor suppressor and as an oncogene in cancer progression.

Keywords:  AAV; AP-1; CRISPR; mouse models; prostate cancer

DOI:  https://doi.org/10.18632/oncotarget.27997
Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01171-2. [Epub ahead of print]36(12): 109722

DNA methylation is required to maintain both DNA replication timing precision and 3D genome organization integrity.

Qian Du, Grady C Smith, Phuc Loi Luu, James M Ferguson, Nicola J Armstrong, C Elizabeth Caldon, Elyssa M Campbell, Shalima S Nair, Elena Zotenko, Cathryn M Gould, Michael Buckley, Kee-Ming Chia, Neil Portman, Elgene Lim, Dominik Kaczorowski, Chia-Ling Chan, Kirston Barton, Ira W Deveson, Martin A Smith, Joseph E Powell, Ksenia Skvortsova, Clare Stirzaker, Joanna Achinger-Kawecka, Susan J Clark.

  DNA replication timing and three-dimensional (3D) genome organization are associated with distinct epigenome patterns across large domains. However, whether alterations in the epigenome, in particular cancer-related DNA hypomethylation, affects higher-order levels of genome architecture is still unclear. Here, using Repli-Seq, single-cell Repli-Seq, and Hi-C, we show that genome-wide methylation loss is associated with both concordant loss of replication timing precision and deregulation of 3D genome organization. Notably, we find distinct disruption in 3D genome compartmentalization, striking gains in cell-to-cell replication timing heterogeneity and loss of allelic replication timing in cancer hypomethylation models, potentially through the gene deregulation of DNA replication and genome organization pathways. Finally, we identify ectopic H3K4me3-H3K9me3 domains from across large hypomethylated domains, where late replication is maintained, which we purport serves to protect against catastrophic genome reorganization and aberrant gene transcription. Our results highlight a potential role for the methylome in the maintenance of 3D genome regulation.

Keywords:  3D genome organization; DNA methylation; allele-specific replication; cancer; chromatin; epigenome; replication timing; single-cell sequencing

DOI:  https://doi.org/10.1016/j.celrep.2021.109722
Mol Cancer Res. 2021 Sep 23. pii: molcanres.MCR-21-0471-E.2021. [Epub ahead of print]

Mutations in non-coding cis-regulatory elements reveal cancer driver cistromes in luminal breast cancer.

Samah El Ghamrasni, Rene Quevedo, James Hawley, Parisa Mazrooei, Youstina Hanna, Iulia Cirlan, Helen Zhu, Jeff Bruce, Leslie E Oldfield, S Y Cindy Yang, Paul Guilhamon, Jüri Reimand, Dave Cescon, Susan J Done, Mathieu Lupien, Trevor J Pugh.

Whole genome sequencing of primary breast tumors enabled the identification of cancer driver genes and non-coding cancer driver plexuses from somatic mutations. However, differentiating driver from passenger events among non-coding genetic variants remains a challenge. Herein, we reveal cancer driver cis-regulatory elements linked to transcription factors previously shown to be involved in development of luminal breast cancers by defining a tumor-enriched catalogue of ~100,000 unique cis-regulatory elements from 26 primary luminal ER+PR+ breast tumors. Integrating this catalogue with somatic mutations from 350 publicly available breast tumor whole genomes, we uncovered cancer driver cistromes, defined as the sum of binding sites for a transcription factor, for ten transcription factors in luminal breast cancer such as FOXA1 and ER, nine of which are essential for growth in breast cancer with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of non-coding mutations over cis-regulatory elements concatenated into a functional unit. Implications: Mapping the accessible chromatin of luminal breast cancer led to discovery of an accumulation of mutations within cistromes of transcription factors essential to luminal breast cancer. This demonstrates co-opting of regulatory networks to drive cancer and provides a framework to derive insight into the non-coding space of cancer.

DOI: https://doi.org/10.1158/1541-7786.MCR-21-0471
Nature. 2021 Sep 22.

Genetic and epigenetic coordination of cortical interneuron development.

Kathryn C Allaway, Mariano I Gabitto, Orly Wapinski, Giuseppe Saldi, Chen-Yu Wang, Rachel C Bandler, Sherry Jingjing Wu, Richard Bonneau, Gord Fishell.

One of the hallmarks of the cerebral cortex is the extreme diversity of interneurons1-3. The two largest subtypes of cortical interneurons, parvalbumin- and somatostatin-positive cells, are morphologically and functionally distinct in adulthood but arise from common lineages within the medial ganglionic eminence4-11. This makes them an attractive model for studying the generation of cell diversity. Here we examine how developmental changes in transcription and chromatin structure enable these cells to acquire distinct identities in the mouse cortex. Generic interneuron features are first detected upon cell cycle exit through the opening of chromatin at distal elements. By constructing cell-type-specific gene regulatory networks, we observed that parvalbumin- and somatostatin-positive cells initiate distinct programs upon settling within the cortex. We used these networks to model the differential transcriptional requirement of a shared regulator, Mef2c, and confirmed the accuracy of our predictions through experimental loss-of-function experiments. We therefore reveal how a common molecular program diverges to enable these neuronal subtypes to acquire highly specialized properties by adulthood. Our methods provide a framework for examining the emergence of cellular diversity, as well as for quantifying and predicting the effect of candidate genes on cell-type-specific development.

DOI: https://doi.org/10.1038/s41586-021-03933-1
J Biol Chem. 2021 Sep 17. pii: S0021-9258(21)01007-3. [Epub ahead of print] 101205

The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions.

Rajaraman Gopalakrishnan, Fred Winston.

  The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating co-transcriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII interacting factors between wild-type cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1-RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5' ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

Keywords:  Abd1; Saccharomyces cerevisiae; Spt6; mRNA capping; transcription

DOI:  https://doi.org/10.1016/j.jbc.2021.101205
Cell Rep. 2021 Sep 21. pii: S2211-1247(21)01153-0. [Epub ahead of print]36(12): 109706

ATF3 promotes the serine synthesis pathway and tumor growth under dietary serine restriction.

Xingyao Li, Daniel Gracilla, Lun Cai, Mingyi Zhang, Xiaolin Yu, Xiaoguang Chen, Junran Zhang, Xiaochun Long, Han-Fei Ding, Chunhong Yan.

  The serine synthesis pathway (SSP) involving metabolic enzymes phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) drives intracellular serine biosynthesis and is indispensable for cancer cells to grow in serine-limiting environments. However, how SSP is regulated is not well understood. Here, we report that activating transcription factor 3 (ATF3) is crucial for transcriptional activation of SSP upon serine deprivation. ATF3 is rapidly induced by serine deprivation via a mechanism dependent on ATF4, which in turn binds to ATF4 and increases the stability of this master regulator of SSP. ATF3 also binds to the enhancers/promoters of PHGDH, PSAT1, and PSPH and recruits p300 to promote expression of these SSP genes. As a result, loss of ATF3 expression impairs serine biosynthesis and the growth of cancer cells in the serine-deprived medium or in mice fed with a serine/glycine-free diet. Interestingly, ATF3 expression positively correlates with PHGDH expression in a subset of TCGA cancer samples.

Keywords:  ATF3; ATF4; PHGDH; PSAT1; PSPH; p300; serine biosynthesis; serine deprivation; serine metabolism; serine synthesis pathway

DOI:  https://doi.org/10.1016/j.celrep.2021.109706
Nat Commun. 2021 Sep 22. 12(1): 5577

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Huan-Chang Liang, Mariantonia Costanza, Nicole Prutsch, Mark W Zimmerman, Elisabeth Gurnhofer, Ivonne A Montes-Mojarro, Brian J Abraham, Nina Prokoph, Stefan Stoiber, Simone Tangermann, Cosimo Lobello, Jan Oppelt, Ioannis Anagnostopoulos, Thomas Hielscher, Shahid Pervez, Wolfram Klapper, Francesca Zammarchi, Daniel-Adriano Silva, K Christopher Garcia, David Baker, Martin Janz, Nikolai Schleussner, Falko Fend, Šárka Pospíšilová, Andrea Janiková, Jacqueline Wallwitz, Dagmar Stoiber, Ingrid Simonitsch-Klupp, Lorenzo Cerroni, Stefano Pileri, Laurence de Leval, David Sibon, Virginie Fataccioli, Philippe Gaulard, Chalid Assaf, Fabian Knörr, Christine Damm-Welk, Wilhelm Woessmann, Suzanne D Turner, A Thomas Look, Stephan Mathas, Lukas Kenner, Olaf Merkel.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.

DOI: https://doi.org/10.1038/s41467-021-25379-9
Nat Commun. 2021 Sep 24. 12(1): 5647

Differentially expressed genes reflect disease-induced rather than disease-causing changes in the transcriptome.

Eleonora Porcu, Marie C Sadler, Kaido Lepik, Chiara Auwerx, Andrew R Wood, Antoine Weihs, Maroun S Bou Sleiman, Diogo M Ribeiro, Stefania Bandinelli, Toshiko Tanaka, Matthias Nauck, Uwe Völker, Olivier Delaneau, Andres Metspalu, Alexander Teumer, Timothy Frayling, Federico A Santoni, Alexandre Reymond, Zoltán Kutalik.

Comparing transcript levels between healthy and diseased individuals allows the identification of differentially expressed genes, which may be causes, consequences or mere correlates of the disease under scrutiny. We propose a method to decompose the observational correlation between gene expression and phenotypes driven by confounders, forward- and reverse causal effects. The bi-directional causal effects between gene expression and complex traits are obtained by Mendelian Randomization integrating summary-level data from GWAS and whole-blood eQTLs. Applying this approach to complex traits reveals that forward effects have negligible contribution. For example, BMI- and triglycerides-gene expression correlation coefficients robustly correlate with trait-to-expression causal effects (rBMI = 0.11, PBMI = 2.0 × 10-51 and rTG = 0.13, PTG = 1.1 × 10-68), but not detectably with expression-to-trait effects. Our results demonstrate that studies comparing the transcriptome of diseased and healthy subjects are more prone to reveal disease-induced gene expression changes rather than disease causing ones.

DOI: https://doi.org/10.1038/s41467-021-25805-y
PLoS Biol. 2021 Sep 22. 19(9): e3001394

Interplay between the EMT transcription factors ZEB1 and ZEB2 regulates hematopoietic stem and progenitor cell differentiation and hematopoietic lineage fidelity.

Jueqiong Wang, Carlos Farkas, Aissa Benyoucef, Catherine Carmichael, Katharina Haigh, Nick Wong, Danny Huylebroeck, Marc P Stemmler, Simone Brabletz, Thomas Brabletz, Christian M Nefzger, Steven Goossens, Geert Berx, Jose M Polo, Jody J Haigh.

The ZEB2 transcription factor has been demonstrated to play important roles in hematopoiesis and leukemic transformation. ZEB1 is a close family member of ZEB2 but has remained more enigmatic concerning its roles in hematopoiesis. Here, we show using conditional loss-of-function approaches and bone marrow (BM) reconstitution experiments that ZEB1 plays a cell-autonomous role in hematopoietic lineage differentiation, particularly as a positive regulator of monocyte development in addition to its previously reported important role in T-cell differentiation. Analysis of existing single-cell (sc) RNA sequencing (RNA-seq) data of early hematopoiesis has revealed distinctive expression differences between Zeb1 and Zeb2 in hematopoietic stem and progenitor cell (HSPC) differentiation, with Zeb2 being more highly and broadly expressed than Zeb1 except at a key transition point (short-term HSC [ST-HSC]➔MPP1), whereby Zeb1 appears to be the dominantly expressed family member. Inducible genetic inactivation of both Zeb1 and Zeb2 using a tamoxifen-inducible Cre-mediated approach leads to acute BM failure at this transition point with increased long-term and short-term hematopoietic stem cell numbers and an accompanying decrease in all hematopoietic lineage differentiation. Bioinformatics analysis of RNA-seq data has revealed that ZEB2 acts predominantly as a transcriptional repressor involved in restraining mature hematopoietic lineage gene expression programs from being expressed too early in HSPCs. ZEB1 appears to fine-tune this repressive role during hematopoiesis to ensure hematopoietic lineage fidelity. Analysis of Rosa26 locus-based transgenic models has revealed that Zeb1 as well as Zeb2 cDNA-based overexpression within the hematopoietic system can drive extramedullary hematopoiesis/splenomegaly and enhance monocyte development. Finally, inactivation of Zeb2 alone or Zeb1/2 together was found to enhance survival in secondary MLL-AF9 acute myeloid leukemia (AML) models attesting to the oncogenic role of ZEB1/2 in AML.

DOI: https://doi.org/10.1371/journal.pbio.3001394
Cell Death Differ. 2021 Sep 23.

LIN28A enhances regenerative capacity of human somatic tissue stem cells via metabolic and mitochondrial reprogramming.

Kelvin Pieknell, Yanuar Alan Sulistio, Noviana Wulansari, Wahyu Handoko Wibowo Darsono, Mi-Yoon Chang, Ji-Yun Ko, Jong Wook Chang, Min-Jeong Kim, Man Ryul Lee, Sang A Lee, Hyunbeom Lee, Gakyung Lee, Byung Hwa Jung, Hyunbum Park, Geun-Ho Kim, Doory Kim, Gayoung Cho, Chun-Hyung Kim, Dat Da Ly, Kyu-Sang Park, Sang-Hun Lee.

Developing methods to improve the regenerative capacity of somatic stem cells (SSCs) is a major challenge in regenerative medicine. Here, we propose the forced expression of LIN28A as a method to modulate cellular metabolism, which in turn enhances self-renewal, differentiation capacities, and engraftment after transplantation of various human SSCs. Mechanistically, in undifferentiated/proliferating SSCs, LIN28A induced metabolic reprogramming from oxidative phosphorylation (OxPhos) to glycolysis by activating PDK1-mediated glycolysis-TCA/OxPhos uncoupling. Mitochondria were also reprogrammed into healthy/fused mitochondria with improved functional capacity. The reprogramming allows SSCs to undergo cell proliferation more extensively with low levels of oxidative and mitochondrial stress. When the PDK1-mediated uncoupling was untethered upon differentiation, LIN28A-SSCs differentiated more efficiently with an increase of OxPhos by utilizing the reprogrammed mitochondria. This study provides mechanistic and practical approaches of utilizing LIN28A and metabolic reprogramming in order to improve SSCs utility in regenerative medicine.

DOI: https://doi.org/10.1038/s41418-021-00873-1
Proc Natl Acad Sci U S A. 2021 09 28. pii: e2101730118. [Epub ahead of print]118(39):

Suberin plasticity to developmental and exogenous cues is regulated by a set of MYB transcription factors.

Vinay Shukla, Jian-Pu Han, Fabienne Cléard, Linnka Lefebvre-Legendre, Kay Gully, Paulina Flis, Alice Berhin, Tonni G Andersen, David E Salt, Christiane Nawrath, Marie Barberon.

  Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine-tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93), each of which is individually regulated by these two signals and is sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing leads to a dramatic reduction in suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips) through a compensatory mechanism. Through the functional analysis of these four MYBs, we generated plants allowing unbiased investigation of endodermal suberin function, without accounting for confounding effects due to Casparian strip defects, and were able to unravel specific roles of suberin in nutrient homeostasis.

Keywords:  ABA; CIF; endodermis; root; suberin

DOI:  https://doi.org/10.1073/pnas.2101730118
J Mol Biol. 2021 Sep 16. pii: S0022-2836(21)00477-0. [Epub ahead of print] 167244

HIF-1α hydroxyprolines modulate oxygen-dependent protein stability via single VHL interface with comparable effect on ubiquitination rate.

Wenguang He, Sarah Batty-Stuart, Jeffrey E Lee, Michael Ohh.

  The basic molecular mechanism underlying mammalian oxygen-dependent regulation of hypoxia-inducible factor (HIF) via the von Hippel-Lindau E3 ubiquitin ligase is well established. The principal step in this critical cellular process is the hydroxylation of either or both of the two conserved proline residues P402 and P564 within the oxygen-dependent degradation domain (ODD) of HIF-1α subunit via prolyl hydroxylases, which is necessary for binding VHL. However, the significance of the two prolines has remained unclear considering that only one hydroxyproline is sufficient for the recruitment of VHL. Here, we show using biophysical analyses that both hydroxyprolines bind to the same interface on VHL with similar affinity; VHL binding affinity to HIF-1α ODD remains relatively unchanged regardless of whether the ODD contains one or two hydroxyprolines; ODD with two hydroxyprolines can accommodate two VHLs; and the rate of in vitro ubiquitination of ODD with one hydroxyproline via VHL E3 ligase is comparable to the rate observed with ODD containing two hydroxyprolines. However, the two hydroxyprolines show distinct contributions to the intracellular stability of HIF-1α ODD. These results demonstrate for the first time that the graduated HIF-1α stability profile observed over a range of oxygen tension is not attributed to the binding of or ubiquitination via VHL per se, but is likely due to the preceding events such as the efficacy of oxygen-dependent prolyl hydroxylase-mediated hydroxylation of HIF-1α.

Keywords:  HIF-1α; Hydroxyproline; Hypoxia; ODD; Oxygen; Ubiquitination; VHL

DOI:  https://doi.org/10.1016/j.jmb.2021.167244
Nat Commun. 2021 Sep 21. 12(1): 5557

Spatial regulation by multiple Gremlin1 enhancers provides digit development with cis-regulatory robustness and evolutionary plasticity.

Jonas Malkmus, Laurène Ramos Martins, Shalu Jhanwar, Bonnie Kircher, Victorio Palacio, Rushikesh Sheth, Francisca Leal, Amandine Duchesne, Javier Lopez-Rios, Kevin A Peterson, Robert Reinhardt, Koh Onimaru, Martin J Cohn, Aimée Zuniga, Rolf Zeller.

Precise cis-regulatory control of gene expression is essential for normal embryogenesis and tissue development. The BMP antagonist Gremlin1 (Grem1) is a key node in the signalling system that coordinately controls limb bud development. Here, we use mouse reverse genetics to identify the enhancers in the Grem1 genomic landscape and the underlying cis-regulatory logics that orchestrate the spatio-temporal Grem1 expression dynamics during limb bud development. We establish that transcript levels are controlled in an additive manner while spatial regulation requires synergistic interactions among multiple enhancers. Disrupting these interactions shows that altered spatial regulation rather than reduced Grem1 transcript levels prefigures digit fusions and loss. Two of the enhancers are evolutionary ancient and highly conserved from basal fishes to mammals. Analysing these enhancers from different species reveal the substantial spatial plasticity in Grem1 regulation in tetrapods and basal fishes, which provides insights into the fin-to-limb transition and evolutionary diversification of pentadactyl limbs.

DOI: https://doi.org/10.1038/s41467-021-25810-1
J Biochem. 2021 Sep 22. pii: mvab098. [Epub ahead of print]

Nucleosome assembly protein 1 (NAP-1) is a regulator of histone H1 acetylation.

Mitsuhiro Yoneda, Kiyoshi Yasui, Takeya Nakagawa, Naoko Hattori, Takashi Ito.

  Acetylation of histone H1 is generally considered to activate transcription, whereas deacetylation of H1 represses transcription. However, the precise mechanism of the acetylation is unknown. Here, using chromatography, we identified nucleosome assembly protein 1 (NAP-1) as having inhibitory activity against histone H1 acetylation by acetyltransferase p300. We found that native NAP-1 interacts with H1 in a Drosophila crude extract. We also found that it inhibits the deacetylation of histone H1 by histone deacetylase 1 (HDAC1). The core histones in nucleosomes were acetylated in a GAL4-VP16 transcriptional activator-dependent manner in vitro. This acetylation was strongly repressed by hypoacetylated H1 but to a lesser extent by hyperacetylated H1. Consistent with these findings, a micrococcal nuclease assay indicated that hypoacetylated H1, which represses activator-dependent acetylation, was incorporated into chromatin, whereas hyperacetylated H1 was not. To determine the contribution of NAP-1 to transcriptional regulation in vivo, we compared NAP-1 knockdown (KD) with coactivator CREB-binding protein (CBP) KD using RNA sequencing in Drosophila Schneider 2 cells. Most genes were downregulated rather than upregulated by NAP-1 KD, and those downregulated genes were also downregulated by CBP KD. Our results suggest that NAP-1 plays a role in transcriptional regulation by fine-tuning the acetylation of histone H1.

Keywords:  linker histone H1histone acetylationhistone deacetylase 1 (HDAC1)NAP-1p300

DOI:  https://doi.org/10.1093/jb/mvab098
Proc Natl Acad Sci U S A. 2021 Sep 28. pii: e2103196118. [Epub ahead of print]118(39):

ATF6 is essential for human cone photoreceptor development.

Heike Kroeger, Julia M D Grandjean, Wei-Chieh Jerry Chiang, Daphne D Bindels, Rebecca Mastey, Jennifer Okalova, Amanda Nguyen, Evan T Powers, Jeffery W Kelly, Neil J Grimsey, Michel Michaelides, Joseph Carroll, R Luke Wiseman, Jonathan H Lin.

  Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

Keywords:  ATF6 signaling; achromatopsia; cone photoreceptors; retinal organoids; stem cell biology

DOI:  https://doi.org/10.1073/pnas.2103196118
Cancer Discov. 2021 Sep 22. pii: candisc.1826.2020. [Epub ahead of print]

MYC levels regulate metastatic heterogeneity in pancreatic adenocarcinoma.

Ravikanth Maddipati, Robert J Norgard, Timour Baslan, Komal S Rathi, Amy Zhang, Asal Saeid, Taku Higashihara, Feng Wu, Angad Kumar, Valli Annamalai, Saurav Bhattacharya, Pichai Raman, Christian A Adkisson, Jason R Pitarresi, Maximilian D Wengyn, Taiji Yamazoe, Jinyang Li, David Balli, Michael J LaRiviere, Tuong-Vi C Ngo, Ian W Folkert, Ian D Millstein, Jonathan Bermeo, Erica L Carpenter, John C McAuliffe, Maja H Oktay, Rolf A Brekken, Scott W Lowe, Christine A Iacobuzio-Donahue, Faiyaz Notta, Ben Z Stanger.

The degree of metastatic disease varies widely amongst cancer patients and impacts clinical outcomes. However, the biological and functional differences that drive the extent of metastasis are poorly understood. We analyzed primary tumors and paired metastases using a multi-fluorescent lineage-labeled mouse model of pancreatic ductal adenocarcinoma (PDAC) - a tumor type where most patients present with metastases. Genomic and transcriptomic analysis revealed an association between metastatic burden and gene amplification or transcriptional upregulation of MYC and its downstream targets. Functional experiments showed that MYC promotes metastasis by recruiting tumor associated macrophages (TAMs), leading to greater bloodstream intravasation. Consistent with these findings, metastatic progression in human PDAC was associated with activation of MYC signaling pathways and enrichment for MYC amplifications specifically in metastatic patients. Collectively, these results implicate MYC activity as a major determinant of metastatic burden in advanced PDAC.

DOI: https://doi.org/10.1158/2159-8290.CD-20-1826
J Biol Chem. 2021 Sep 18. pii: S0021-9258(21)01016-4. [Epub ahead of print] 101213

CENP-B promotes the centromeric localization of ZFAT to control transcription of non-coding RNA.

Shuhei Ishikura, Kazumasa Yoshida, Sayuri Hashimoto, Kazuhiko Nakabayashi, Toshiyuki Tsunoda, Senji Shirasawa.

  The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of non-coding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mechanisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Co-immunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription.

Keywords:  CENP-B; DNA binding protein; ZFAT; centromere; noncoding RNA; protein-protein interaction; transcription regulation; zinc finger

DOI:  https://doi.org/10.1016/j.jbc.2021.101213
J Biol Chem. 2021 Sep 16. pii: S0021-9258(21)01002-4. [Epub ahead of print] 101200

Linking nuclear matrix-localized PIAS1 to chromatin SUMOylation via direct binding of histones H3 and H2A.Z.

Zhaosu Chen, Yunpeng Zhang, Qingqing Guan, Huifang Zhang, Jing Luo, Jialun Li, Wei Wei, Xiang Xu, Lujian Liao, Jiemin Wong, Jiwen Li.

  As a conserved post-translational modification, SUMOylation has been shown to play important roles in chromatin-related biological processes including transcription. However, how the SUMOylation machinery associates with chromatin is not clear. Here, we present evidence that multiple SUMOylation machinery components, including SUMO E1 proteins SAE1 and SAE2 and the PIAS (protein inhibitor of activated STAT) family SUMO E3 ligases, are primarily associated with the nuclear matrix rather than with chromatin. We show using nuclease digestion that all PIAS family proteins maintain nuclear matrix association in the absence of chromatin. Importantly, we identify multiple histones including H3 and H2A.Z as directly interacting with PIAS1, and demonstrate that this interaction requires the PIAS1 SAP (SAF-A/B, Acinus, and PIAS) domain. We demonstrate that PIAS1 promotes SUMOylation of histones H3 and H2B in both a SAP domain- and an E3 ligase activity-dependent manner. Furthermore, we show that PIAS1 binds to heat shock-induced genes and represses their expression, and that this function also requires the SAP domain. Altogether, our study reveals for the first time the nuclear matrix as the compartment most enriched in SUMO E1 and PIAS family E3 ligases. Our finding that PIAS1 interacts directly with histone proteins also suggests a molecular mechanism as to how nuclear matrix-associated PIAS1 is able to regulate transcription and other chromatin-related processes.

Keywords:  PIAS1; SUMO; chromatin; histone SUMOylation; nuclear matrix

DOI:  https://doi.org/10.1016/j.jbc.2021.101200