The elevated transcription of ADAM19 by the oncohistone H2BE76K contributes to oncogenic properties in breast cancer
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100374
ISSN1083-351X
AutoresTze Zhen Evangeline Kang, Lina Zhu, Yang Du, Dongbo Ding, Xiaoxuan Zhu, Yi Ching Esther Wan, Jiaxian Liu, Saravanan Ramakrishnan, Landon L. Chan, Siu Yuen Chan, Xin Wang, Haiyun Gan, Junhong Han, Toyotaka Ishibashi, Qing Li, Kui Ming Chan,
Tópico(s)Cancer-related gene regulation
ResumoThe recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect. The recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect. Histones are nuclear proteins required for the packaging of genomic DNA in the form of chromatin. Two copies of each of the core histones H2A, H2B, H3, and H4 with ∼147 bp of DNA form a nucleosome, the basic unit of chromatin (1Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 {Å} resolution.Nature. 1997; 389: 251-260Crossref PubMed Scopus (6884) Google Scholar). The compaction of DNA into the nucleosome, however, creates major obstacles to gene regulatory processes, such as transcription and DNA replication. Nucleosome dynamics, regulated in part by histone posttranslational modifications (PTMs) (2Lawrence M. Daujat S. Schneider R. Lateral thinking : How histone modifications regulate gene expression.Trends Genet. 2016; 32: 42-56Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar), thus play a major role in modulating gene activity by controlling DNA accessibility. Misregulation of histone PTMs has been extensively linked to cancer (3Audia J.E. Campbell R.M. Histone modifications and cancer.Cold Spring Harb. Perspect. Biol. 2016; 8a019521Crossref PubMed Scopus (410) Google Scholar) and was most recently implicated in cancers containing mutations in genes encoding histone H3 (4Wan Y.C.E. Liu J. Chan K.M. Histone H3 mutations in cancer.Curr. Pharmacol. Rep. 2018; 4: 292-300Crossref PubMed Scopus (28) Google Scholar). These include the H3K27M and H3G34R/V mutations found in pediatric gliomas (5Schwartzentruber J. Korshunov A. Liu X.-Y. Jones D.T.W. Pfaff E. Jacob K. Sturm D. Fontebasso A.M. Quang D.-A.K. Tönjes M. Hovestadt V. Albrecht S. Kool M. Nantel A. Konermann C. et al.Driver mutations in histone H3. 3 and chromatin remodelling genes in paediatric glioblastoma.Nature. 2012; 482: 226Crossref PubMed Scopus (1706) Google Scholar, 6Wu G. Broniscer A. Mceachron T.A. Lu C. Paugh B.S. Becksfort J. Qu C. Ding L. Huether R. Parker M. Gajjar A. Dyer M.A. Mullighan C.G. Gilbertson R.J. Mardis E.R. et al.Somatic histone H3 alterations in paediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas.Nat. Genet. 2012; 44: 251-253Crossref PubMed Scopus (1094) Google Scholar) and the H3G34W/L and H3K36M mutations found in sarcomas (7Behjati S. Tarpey P.S. Presneau N. Scheipl S. Pillay N. Van Loo P. Wedge D.C. Cooke S.L. Gundem G. Davies H. Nik-Zainal S. Martin S. McLaren S. Goody V. Goodie V. et al.Distinct H3F3A and H3F3B driver mutations define chondroblastoma and giant cell tumor of bone.Nat. Genet. 2013; 45: 1479-1482Crossref PubMed Scopus (524) Google Scholar). We and others have previously demonstrated the effect of the H3K27-to-M mutation in H3K27 di- and tri-methylation as well as transcriptional misregulation in diffuse intrinsic pontine glioma (DIPG) (8Chan K.-M. Fang D. Gan H. Hashizume R. Yu C. Schroeder M. Gupta N. Mueller S. James C.D. Jenkins R. Sarkaria J. Zhang Z. The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression.Genes Dev. 2013; 27: 985-990Crossref PubMed Scopus (456) Google Scholar, 9Lewis P.W. Müller M.M. Koletsky M.S. Cordero F. Lin S. Banaszynski L.A. Garcia B.A. Muir T.W. Becher O.J. Allis C.D. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma.Science. 2013; 340: 857-861Crossref PubMed Scopus (845) Google Scholar). The H3G34V/R/W/L mutations affect the methylation of lysine 36 in cis while the H3K36M mutation exhibits a dominant negative effect and inhibits the methylation of lysine 36 in trans (9Lewis P.W. Müller M.M. Koletsky M.S. Cordero F. Lin S. Banaszynski L.A. Garcia B.A. Muir T.W. Becher O.J. Allis C.D. Inhibition of PRC2 activity by a gain-of-function H3 mutation found in pediatric glioblastoma.Science. 2013; 340: 857-861Crossref PubMed Scopus (845) Google Scholar, 10Fang D. Gan H. Lee J.-H. Han J. Wang Z. Riester S.M. Jin L. Chen J. Zhou H. Wang J. Zhang H. Yang N. Bradley E.W. Ho T.H. Rubin B.P. et al.The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas.Science. 2016; 352: 1344-1348Crossref PubMed Scopus (156) Google Scholar, 11Lu C. Jain S.U. Hoelper D. Bechet D. Molden R.C. Ran L. Murphy D. Venneti S. Hameed M. Pawel B.R. Wunder J.S. Dickson B.C. Lundgren S.M. Jani K.S. De Jay N. et al.Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape.Science. 2016; 352: 844-849Crossref PubMed Scopus (239) Google Scholar). Notably, these cancer-associated H3 mutations are all located at or near key lysine residues that are posttranslationally modified and thus affect key regulatory PTMs, subsequently driving oncogenesis through changes in the epigenetic landscape and gene expression profiles. To address whether additional histone mutations exist in other malignancies, we searched for missense mutations that occur in histone encoding genes in cancer patient samples through the cBioPortal database (12Gao J. Aksoy B.A. Dogrusoz U. Dresdner G. Gross B. Sumer S.O. Sun Y. Jacobsen A. Sinha R. Larsson E. Cerami E. Sander C. Schultz N. Integrative analysis of complex cancer genomics and clinical profiles using the cBioPortal.Sci. Signal. 2013; 6pl1Crossref PubMed Scopus (8695) Google Scholar). We identified a couple of novel cancer-associated mutations in genes encoding histone H2B, including the H2BG53D mutation in pancreatic ductal adenocarcinoma (PDAC) we recently reported (13Wan Y.C.E. Leung T.C.S. Ding D. Sun X. Liu J. Zhu L. Kang T.Z.E. Yang D. Zhang Y. Zhang J. Qian C. Huen M.S.Y. Li Q. Chow M.Z.Y. Zheng Z. et al.Cancer-associated histone mutation H2BG53D disrupts DNA--histone octamer interaction and promotes oncogenic phenotypes.Signal. Transduct. Target. Ther. 2020; 5: 1-4Crossref PubMed Scopus (12) Google Scholar, 14Wan Y.C.E. Liu J. Zhu L. Kang T.Z.E. Zhu X. Lis J. Ishibashi T. Danko C.G. Wang X. Chan K.M. The H2BG53D oncohistone directly upregulates ANXA3 transcription and enhances cell migration in pancreatic ductal adenocarcinoma.Signal. Transduct. Target. Ther. 2020; 5: 1-4Crossref PubMed Scopus (5) Google Scholar), and mutations of the Glu76 (E76) residue of histone H2B. Consistent with recent reports (15Nacev B.A. Feng L. Bagert J.D. Lemiesz A.E. Gao J. Soshnev A.A. Kundra R. Schultz N. Muir T.W. Allis C.D. The expanding landscape of ‘oncohistone’mutations in human cancers.Nature. 2019; 567: 473Crossref PubMed Scopus (172) Google Scholar, 16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar), we found the E76 residue to be the most frequently mutated H2B residue in various cancers including breast and lung carcinoma, which contained either Glu76Lys (E76K) or Glu76Gln (E76Q) missense mutations in genes encoding H2B (Table S1). Interestingly, the ectopic overexpression of H2BE76K has been previously shown to enhance colony formation ability in mouse fibroblast and human epithelial cells, suggesting that H2BE76K can confer oncogenic properties (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar, 17Arimura Y. Ikura M. Fujita R. Noda M. Kobayashi W. Horikoshi N. Sun J. Shi L. Kusakabe M. Harata M. Ohkawa Y. Tashiro S. Kimura H. Ikura T. Kurumizaka H. Cancer-associated mutations of histones H2B, H3. 1 and H2A. Z. 1 affect the structure and stability of the nucleosome.Nucleic Acids Res. 2018; 46: 10007-10018PubMed Google Scholar). Within the nucleosome, the H2BE76 residue (equivalent to E73 in Xenopus laevis) interacts directly with the R92 residue of histone H4 through hydrogen bonding at the H2A-H2B dimer–(H3-H4)2 tetramer interface (1Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 {Å} resolution.Nature. 1997; 389: 251-260Crossref PubMed Scopus (6884) Google Scholar). Recent studies showed that the abolishment of this interaction by H2BE76K destabilizes the nucleosome (17Arimura Y. Ikura M. Fujita R. Noda M. Kobayashi W. Horikoshi N. Sun J. Shi L. Kusakabe M. Harata M. Ohkawa Y. Tashiro S. Kimura H. Ikura T. Kurumizaka H. Cancer-associated mutations of histones H2B, H3. 1 and H2A. Z. 1 affect the structure and stability of the nucleosome.Nucleic Acids Res. 2018; 46: 10007-10018PubMed Google Scholar). Unlike the H3K27M and H3K36M mutations, which directly alter global levels H3K27 and H3K36 methylation, respectively, H2BE76K does not occur on a posttranslationally modified site and is therefore unlikely to directly affect PTMs (18Huang H. Sabari B.R. Garcia B.A. Allis C.D. Zhao Y. SnapShot: Histone modifications.Cell. 2014; 159: 458Abstract Full Text PDF PubMed Scopus (287) Google Scholar). Instead, the H2BE76K mutation has been suggested to promote gene expression through its nucleosome destabilizing effect (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar). However, the complete picture of how H2BE76K induces transcriptional misregulation remains unclear, as H2BE76K expression has been shown to result in both gene activation and repression (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar). The mechanism of how H2BE76K mediates specific transcriptional changes to promote oncogenic phenotypes also remains to be answered. The average tumor allele frequency of the H2BE76K mutation in tumors (∼20%) suggests that it is an acquired subclonal mutation rather than a germline mutation and is likely to cooperate with other driver mutations in later stages of cancer progression (15Nacev B.A. Feng L. Bagert J.D. Lemiesz A.E. Gao J. Soshnev A.A. Kundra R. Schultz N. Muir T.W. Allis C.D. The expanding landscape of ‘oncohistone’mutations in human cancers.Nature. 2019; 567: 473Crossref PubMed Scopus (172) Google Scholar, 16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar) (Table S1). Supporting this notion, the expression of H2BE76K alone has been shown to have no effect on the normal mammary epithelial cell line MCF10A, but could enhance colony formation ability in the presence of the oncogenic PI3KCA-H1047R mutant (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar). To dissect the molecular mechanism by which H2BE76K promotes breast cancer development, we knock in the H2BE76K mutation in the MDA-MB-231 breast cancer cell line. Using CUT&RUN, we mapped the genome-wide localization of the H2BE76K mutant histone and found that H2BE76K preferentially localizes on gene bodies. We further identify the H2BE76K-enriched gene ADAM19 as a direct target linking H2BE76K to oncogenic phenotypes. Collectively, these findings illustrate the effects of the H2BE76K mutation in transcription misregulation and breast cancer development. The E76 residue of histone H2B is evolutionarily conserved among yeast and higher eukaryotes (Fig. 1A) and is located at the H2B-H4 interface in the center of the histone octamer (Fig. 1B). Recent studies have shown that the H2BE76K mutation destabilizes the nucleosome by disrupting H2B-H4 interactions (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar, 17Arimura Y. Ikura M. Fujita R. Noda M. Kobayashi W. Horikoshi N. Sun J. Shi L. Kusakabe M. Harata M. Ohkawa Y. Tashiro S. Kimura H. Ikura T. Kurumizaka H. Cancer-associated mutations of histones H2B, H3. 1 and H2A. Z. 1 affect the structure and stability of the nucleosome.Nucleic Acids Res. 2018; 46: 10007-10018PubMed Google Scholar). In addition to H2BE76K’s effect on nucleosome stability, we asked whether the mutation might also affect histone modifications and nucleosome–histone chaperone interactions. To address this, FLAG-tagged H2BE76 mutants were expressed in 293T cells followed by FLAG immunoprecipitation (IP). FLAG-IP results confirmed the negative effect of the H2BE76K mutation on H2B-H3/H4 interaction (Fig. 1C). The substitution of H2BE76 to K and Q, as well as to the neutrally charged alanine (A), reduced H2B association with H3 and H4, with the E76K mutant showing the strongest effect. Intriguingly, we also found that the H2BE76K, but not the H2BE76Q mutant, displayed elevated interaction with both the H2A-H2B chaperones nucleosome assembly protein 1-Like 1 and 2 (NAP1L1 and NAP1L2) and reduced interaction with the H2A-H2B chaperone FACT subunit SPT16. While the presence of the mutant H2BE76K did not affect global histone modification levels in transfected 293T cells, the H2BE76K mutation led to a reduced association with H2AK119 ubiquitination (H2AK119ub), a repressive histone mark (19Simon J.A. Kingston R.E. Mechanisms of polycomb gene silencing: Knowns and unknowns.Nat. Rev. Mol. Cell Biol. 2009; 10: 697Crossref PubMed Scopus (0) Google Scholar), and H2BK120 ubiquitination, a histone mark associated with active transcription elongation (20Minsky N. Shema E. Field Y. Schuster M. Segal E. Oren M. Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells.Nat. Cell Biol. 2008; 10: 483-488Crossref PubMed Scopus (272) Google Scholar). We confirmed the reduced H2AK119ub and H2BK120ub levels in fixed mononucleosomes containing H2BE76 mutants (Fig. 1D). Interestingly we also found increased H3K27 acetylation (H3K27ac) levels specifically on H2BE76K-containing nucleosomes. While both H2BE76K and H2BE76Q mutations reduced H2AK119ub and H2BK120ub levels in cis, the two mutations appear to be functionally distinct as H2BE76K, but not the H2BE76Q mutant, protein differentially binds to histone chaperones. We reasoned that since the H2BE76K mutation is an acquired subclonal mutation, normal nontransformed cells might not acquire oncogenic phenotypes when the H2BE76K mutant histone is expressed at a physiological level. To study the effect of H2BE76K mutation in cancer development, we generated knock-in (KI) mutant cell lines using the MDA-MB-231 breast cancer cell line. The CRISPR/Cas9 system was employed to introduce the H2BE76K mutation into an H2B encoding gene to express the H2BE76K mutant at a physiological level mimicking patient conditions (Fig. 1, E and F). To achieve this, we substituted one of the 17 somatic H2B encoding genes, HIST1H2BO, with a wild-type (WT) or E76K mutated H2B through CRISPR/Cas9-mediated homology-directed repair (HDR). We chose to target HIST1H2BO since 1) the H2BE76K mutation was found in this H2B gene in breast cancer (Table S1) and 2) to minimize CRISPR/Cas9 off-target effects, as the sgRNAs targeting this H2B gene had the highest predicted specificity scores. We tagged both the WT and E76K mutant H2B with a FLAG-tag at the C-terminal tail for the assessment of expression levels and the mapping of the genomic distribution of the H2BE76K mutant histones. The targeted editing was confirmed through PCR genotyping with no effects on the top predicted off-target sites and on other H2B genes with high sequence similarity to HIST1H2BO (Fig. S1, A–C, Fig. S2, A and B). Herein, KI cell lines expressing FLAG-WT H2B and FLAG-H2BE76K are referred to as WT and H2BE76K cells, respectively. Global histone modifications levels were unaffected in the mutant cell lines carrying either the WT or E76K mutant H2B (Fig. S3). Cell fractionation assays revealed a small portion of H2BE76K that failed to deposit on the chromatin and remained in the cytosolic fraction; however, the majority of the E76K mutant H2B was still successfully incorporated into the chromatin (Fig. 1G, Fig. S4). We subsequently performed FLAG-IP using the cytoplasmic and mononucleosomes fractions and found that both cytosolic and chromatin-bound H2BE76K protein interacts with NAP1L1 and NAP1L2 (Fig. 1G), similar to the increased H2BE76K-NAP1L1/NAP1L2 interactions observed in 293T cells (Fig. 1, C and D). The H2BE76K-containing nucleosomes additionally displayed decreased H2AK119ub, in agreement with the results of the IP experiments performed with 293T cells (Fig. 1C). While a previous study has shown the transcriptional changes that accompanied the ectopic overexpression of H2BE76K, the exact mechanism underlying H2BE76K’s effect on gene expression remains unclear (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar). To understand H2BE76K’s direct effect on gene expression, we performed CUT&RUN (21Skene P.J. Henikoff S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites.Elife. 2017; 6e21856Crossref PubMed Scopus (566) Google Scholar) to map the genomic localization of the FLAG-tagged WT H2B and H2BE76K mutant histones in WT and H2BE76K cell lines, respectively (Fig. 2A, Fig. S5). Histone H2A CUT&RUN was performed to assess nucleosomal occupancy and additionally acts as an internal control for the FLAG CUT&RUN (Fig. S5A). Intriguingly, we found that the genomic distribution of FLAG-H2BE76K was markedly different compared with that of FLAG-WT H2B. While the major portion of FLAG-WT H2B peaks were localized in intergenic regions, more than 70% of FLAG-H2BE76K peaks are located in genic regions (Fig. 2B). Profiles of average FLAG CUT&RUN signals show that FLAG-H2BE76K is specifically enriched on gene body regions, downstream of the transcription start site (TSS) (Fig. 2C). The increased genic FLAG-H2BE76K enrichment was not attributed to nucleosomal occupancy changes, as the average H2A CUT&RUN signals were comparable between the WT and H2BE76K mutant cells (Fig. S5). A previous report on H2BE76K’s effect on chromatin accessibility showed that ectopic H2BE76K overexpression promoted chromatin accessibility and gene expression in MCF10a cells (16Bennett R.L. Bele A. Small E.C. Will C.M. Nabet B. Oyer J.A. Huang X. Ghosh R.P. Grzybowski A.T. Yu T. Zhang Q. Riva A. Lele T.P. Schatz G.C. Kelleher N.L. et al.A mutation in histone H2B represents a new class of oncogenic driver.Cancer Discov. 2019; 9: 1438-1451Crossref PubMed Scopus (40) Google Scholar). Interestingly, however, our genome-wide chromatin accessibility using ATAC-seq (Assay for transposase-accessible chromatin sequencing) (22Buenrostro J.D. Giresi P.G. Zaba L.C. Chang H.Y. Greenleaf W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position.Nat. Methods. 2013; 10: 1213Crossref PubMed Scopus (3185) Google Scholar) showed that H2BE76K-enriched genes, as exemplified at the CHKA, KMT5B, LRP5, PPP6R3, and MRPL21 loci, did not display apparent chromatin accessibility changes in our H2BE76K mutant cells (Fig. 2A). We further performed differential occupancy analysis on genes and found significant FLAG-H2BE76K enrichment (Benjamini–Hochberg (BH) adjusted p < 0.05, log2 fold change (log2FC) > 0.5) in more than 2000 genes (Fig. S5C). We validated the FLAG CUT&RUN results by chromatin immunoprecipitation (ChIP)-qPCR and confirmed H2BE76K enrichment patterns on selected genes in chromosome 11 highlighted in Figure 2A (Fig. S6). Analysis of genome-wide differential distribution patterns between WT H2B and H2BE76K further revealed the specific gain of H2BE76K enrichment in promoter (16.2%) and genic (82.9%) regions, whereas the lost H2B occurred equally across promoter (35.7%), genic (38.7%), and intergenic (25.7%) regions (Fig. 2D). Collectively, these results show that H2BE76K is redistributed to and preferentially localizes in genic regions. To further investigate the effect of the H2BE76K mutation on gene expression, we analyzed the transcriptomes of the KI cell lines using RNA-sequencing (RNA-seq). The H2BE76K cell lines exhibited a distinct gene expression profile with 228 significantly upregulated and 493 downregulated genes (BH adjusted p < 0.05, absolute log2FC > 0.25) from two independent experiments comparing two H2BE76K KI clones and two isogenic WT clones (Fig. 3A, Fig. S7). Importantly, we found that some cancer-associated pathways such as cell adhesion and cell proliferation were significantly enriched in genes showing upregulation by gene ontology (GO) analysis. In contrast, cancer-related pathways such as focal adhesion assembly, cell signaling as well as response to hypoxia were also significantly enriched in downregulated genes (Fig. 3B). We performed RT-qPCR and confirmed the differential expression of several cancer driver genes (23Bailey M.H. Tokheim C. Porta-Pardo E. Sengupta S. Bertrand D. Weerasinghe A. Colaprico A. Wendl M.C. Kim J. Reardon B. Ng P.K.-S. Jeong K.J. Cao S. Wang Z. Gao J. et al.Comprehensive characterization of cancer driver genes and mutations.Cell. 2018; 173: 371-385.e18Abstract Full Text Full Text PDF PubMed Scopus (944) Google Scholar): increased mRNA expression of oncogenes MYC and PIK3CG, as well as decreased expression of tumor suppressor genes CDKN1A and EPHA2, was observed in the H2BE76K mutant cells (Fig. 3C). To investigate the direct role of H2BE76K in gene misregulation, we analyzed H2BE76K’s genomic localization in relation to gene expression changes (Fig. 3D, Fig. S8, A and B). Intriguingly, we observed that genes upregulated in H2BE76K mutant cells exhibited increased H2BE76K enrichment (enrichment over FLAG-WT H2B) (Fig. 3D). As represented by H2A CUT&RUN, total nucleosomal occupancy on the upregulated genes was comparable between WT and H2BE76K mutant cells (Fig. S9, C–E). More importantly, gene set enrichment analysis (GSEA) revealed a positive correlation between upregulated genes and H2BE76K enrichment, suggesting a relationship between H2BE76K enrichment and increased transcription (Fig. 3E). Figure 3F shows the 78 upregulated genes with the most significant H2BE76K enrichment (Table S2). The role of the H2BE76K mutation in cancer was further characterized by studying the oncogenic properties of the H2BE76K mutant cells. The H2BE76K mutant cell lines formed bigger colonies than the WT isogenic lines and one of the H2BE76K mutant lines had moderate increased proliferation rates (Fig. S9, A and B). RNA-seq results showed that the expression of genes that control cellular phenotypes was affected (Fig. 3B), which might directly contribute to the increased clonogenicity of the H2BE76K mutant cells. Indeed, several upregulated genes in the H2BE76K mutant cells, such as WNT7B and PTN, are involved in promoting metastasis-associated tumor cell phenotypes, including increased clonogenicity (24De P. Carlson J.H. Wu H. Marcus A. Leyland-Jones B. Dey N. Wnt-beta-catenin pathway signals metastasis-associated tumor cell phenotypes in triple negative breast cancers.Oncotarget. 2016; 7: 43124Crossref PubMed Scopus (42) Google Scholar, 25Huang P. Ouyang D. Chang S. Li M. Li L. Li Q. Zeng R. Zou Q. Su J. Zhao P. Pei L. Yi W.-J. Chemotherapy-driven increases in the CDKN1A/PTN/PTPRZ1 axis promote chemoresistance by activating the NF-κB pathway in breast cancer cells.Cell Commun. Signal. 2018; 16: 92Crossref PubMed Scopus (24) Google Scholar) (Fig. S9C). To identify H2BE76K target genes that promote colony formation in the H2BE76K mutant cells, we depleted multiple H2BE76K target genes individually using lentiviral short hairpin RNAs (shRNAs). Intriguingly, we found that the knockdown of one of the H2BE76K-enriched genes, ADAM19 (Fig. 4, A and B, Figs. S9A and S10), reduced the sizes of colonies formed by the H2BE76K cells to the sizes of WT colonies treated with nontargeting (NT) shRNA (Fig. 4, C and D) with minimal effect on proliferation (Fig. S9). ADAM19 belongs to the ADAM (a disintegrin and metalloproteinase) family that comprises transmembrane and secreted proteins and is found to be highly expressed in various human carcinomas including breast invasive carcinoma (BRCA) (Fig. S11). Conversely, the overexpression of ADAM19 significantly increased the sizes of colonies formed by the WT cells (Fig. 4, E–G). These results demonstrate that ADAM19 upregulation promotes the colony formation ability of the H2BE76K cells. We next investigated the mechanism of ADAM19’s upregulation in the H2BE76K mutant cells. As H2BE76K destabilizes the nucleosome, its enrichment on gene bodies may provide a more permissive environment for RNA polymerase II (Pol II) progression. As Pol II recruitment is a key step of transcription activation, we first examined Pol II occupancy on ADAM19 by ChIP-qPCR. We monitored Pol II recruitment by treating the cells with 5,6-dichlorobenzimidazole1-β-D-ribofuranoside (DRB) to inhibit the positive transcription elongation factor-b (P-TEFb) kinase. DRB treatment inhibits initiating Pol II from entering productive elongation while allowing elongating complexes to complete transcription, effectively arresting Pol II surrounding the TSS. Pol II recruitment to ADAM19 was comparable between WT and H2BE76K cells, suggesting an alternative mechanism driving ADAM19’s upregulation (Fig. 5A). To further examine the effect of H2BE76K on ADAM19 transcription, we monitored in vivo Pol II transcription elongation by transiently inhibiting transcription elongation with DRB (26Singh J. Padgett R.A. Rates of in situ transcription and splicing in large human genes.Nat. Struct. Mol. Biol. 2009; 16: 1128Crossref PubMed Scopus (316) Google Scholar) (Fig. 5B). As DRB’s inhibitory effect on transcription is reversible, transcription elongation resumes upon its removal. We quantifie
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