The establishment of long-lasting immunity against pathogens is facilitated by the germinal center (GC) reaction, during which B cells increase their antibody affinity and differentiate into antibody-secreting cells (ASC) and memory cells. These events involve modifications in chromatin packaging that orchestrate the profound restructuring of gene expression networks that determine cell fate. While several chromatin remodelers were implicated in lymphocyte functions, less is known about SMARCA5. Here, using ribosomal pull-down for analyzing translated genes in GC B cells, coupled with functional experiments in mice, we identified SMARCA5 as a key chromatin remodeler in B cells. While the naive B cell compartment remained unaffected following conditional depletion of Smarca5, effective proliferation during B cell activation, immunoglobulin class switching, and as a result GC formation and ASC differentiation were impaired. Single-cell multiomic sequencing analyses revealed that SMARCA5 is crucial for facilitating the transcriptional modifications and genomic accessibility of genes that support B cell activation and differentiation. These findings offer novel insights into the functions of SMARCA5, which can be targeted in various human pathologies.

• MeSH
• Adenosine Triphosphatases MeSH
• Lymphocyte Activation immunology   MeSH
• B-Lymphocytes * metabolism   immunology   MeSH
• Cell Differentiation * MeSH
• Chromosomal Proteins, Non-Histone * metabolism   genetics   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Immunoglobulin Class Switching genetics   MeSH
• Chromatin Assembly and Disassembly * MeSH
• Germinal Center * immunology   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Chromosomal Proteins, Non-Histone * MeSH
• Smarca5 protein, mouse MeSH Browser

The formation of hematopoietic cells relies on the chromatin remodeling activities of ISWI ATPase SMARCA5 (SNF2H) and its complexes. The Smarca5 null and conditional alleles have been used to study its functions in embryonic and organ development in mice. These mouse model phenotypes vary from embryonic lethality of constitutive knockout to less severe phenotypes observed in tissue-specific Smarca5 deletions, e.g., in the hematopoietic system. Here we show that, in a gene dosage-dependent manner, the hypomorphic allele of SMARCA5 (S5tg) can rescue not only the developmental arrest in hematopoiesis in the hCD2iCre model but also the lethal phenotypes associated with constitutive Smarca5 deletion or Vav1iCre-driven conditional knockout in hematopoietic progenitor cells. Interestingly, the latter model also provided evidence for the role of SMARCA5 expression level in hematopoietic stem cells, as the Vav1iCre S5tg animals accumulate stem and progenitor cells. Furthermore, their hematopoietic stem cells exhibited impaired lymphoid lineage entry and differentiation. This observation contrasts with the myeloid lineage which is developing without significant disturbances. Our findings indicate that animals with low expression of SMARCA5 exhibit normal embryonic development with altered lymphoid entry within the hematopoietic stem cell compartment.

• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Cell Differentiation genetics   MeSH
• Hematopoietic Stem Cells * metabolism   MeSH
• Hematopoiesis * genetics   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH

Smarca5, an ATPase of the ISWI class of chromatin remodelers, is a key regulator of chromatin structure, cell cycle and DNA repair. Smarca5 is deregulated in leukemia and breast, lung and gastric cancers. However, its role in oncogenesis is not well understood. Chromatin remodelers often play dosage-dependent roles in cancer. We therefore investigated the epigenomic and phenotypic impact of controlled stepwise attenuation of Smarca5 function in the context of primary cell transformation, a process relevant to tumor formation. Upon conditional single- or double-allele Smarca5 deletion, the cells underwent both accelerated growth arrest and senescence entry and displayed gradually increased sensitivity to genotoxic insults. These phenotypic characteristics were explained by specific remodeling of the chromatin structure and the transcriptome in primary cells prior to the immortalization onset. These molecular programs implicated Smarca5 requirement in DNA damage repair, telomere maintenance, cell cycle progression and in restricting apoptosis and cellular senescence. Consistent with the molecular programs, we demonstrate for the first time that Smarca5-deficient primary cells exhibit dramatically decreased capacity to bypass senescence and immortalize, an indispensable step during cell transformation and cancer development. Thus, Smarca5 plays a crucial role in key homeostatic processes and sustains cancer-promoting molecular programs and cellular phenotypes.

• Keywords
• ATAC-seq, MEF, RNA-seq, Smarca5, Snf2h, cell cycle, cell immortalization, homologous recombination, non-homologous end-joining, senescence,
• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Chromatin * MeSH
• Neoplasms * MeSH
• DNA Repair MeSH
• DNA Damage MeSH
• Chromatin Assembly and Disassembly MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Chromatin * MeSH

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

• Keywords
• AML, CRISPR, SMARCA5, SNF2H, leukemia, therapeutic target,
• MeSH
• Adenosine Triphosphatases deficiency   metabolism   MeSH
• Leukemia, Myeloid, Acute * enzymology   genetics   pathology   MeSH
• K562 Cells MeSH
• Chromatids * genetics   metabolism   MeSH
• Chromosomal Proteins, Non-Histone deficiency   metabolism   MeSH
• Gene Knockout Techniques * MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins * deficiency   metabolism   MeSH
• Cell Proliferation * MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Chromosomal Proteins, Non-Histone MeSH
• Neoplasm Proteins * MeSH
• SMARCA5 protein, human MeSH Browser

The imitation switch nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair, and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells accumulated but their maturation toward erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S15Ph °s ) second with CBP/p300 (K376Ac ), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4-hydroxytamoxifen-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis. Stem Cells 2017;35:1614-1623.

• Keywords
• Cell cycle progression, Erythroid differentiation, Fetal liver erythropoiesis, Hematopoietic stem and progenitor cells, Hypoxia, Imitation switch, Smarca5, p53 pathway,
• MeSH
• Adenosine Triphosphatases deficiency   metabolism   MeSH
• Anemia pathology   MeSH
• Cell Differentiation * MeSH
• Cell Cycle MeSH
• Chromosomal Proteins, Non-Histone deficiency   metabolism   MeSH
• Gene Deletion MeSH
• Erythroid Cells cytology   MeSH
• Erythropoiesis MeSH
• Genotype MeSH
• Hematopoietic Stem Cells cytology   metabolism   MeSH
• Hematopoiesis MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Tumor Suppressor Protein p53 metabolism   MeSH
• DNA Damage genetics   MeSH
• Cell Proliferation MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Chromosomal Proteins, Non-Histone MeSH
• RNA, Messenger MeSH
• Tumor Suppressor Protein p53 MeSH
• Smarca5 protein, mouse MeSH Browser

GATA-1 and PU.1 are two important hematopoietic transcription factors that mutually inhibit each other in progenitor cells to guide entrance into the erythroid or myeloid lineage, respectively. PU.1 controls its own expression during myelopoiesis by binding to the distal URE enhancer, whose deletion leads to acute myeloid leukemia (AML). We herein present evidence that GATA-1 binds to the PU.1 gene and inhibits its expression in human AML-erythroleukemias (EL). Furthermore, GATA-1 together with DNA methyl Transferase I (DNMT1) mediate repression of the PU.1 gene through the URE. Repression of the PU.1 gene involves both DNA methylation at the URE and its histone H3 lysine-K9 methylation and deacetylation as well as the H3K27 methylation at additional DNA elements and the promoter. The GATA-1-mediated inhibition of PU.1 gene transcription in human AML-EL mediated through the URE represents important mechanism that contributes to PU.1 downregulation and leukemogenesis that is sensitive to DNA demethylation therapy.

• MeSH
• Leukemia, Erythroblastic, Acute genetics   pathology   MeSH
• Leukemia, Myeloid, Acute genetics   pathology   MeSH
• Cell Differentiation genetics   MeSH
• DNA (Cytosine-5-)-Methyltransferases genetics   metabolism   MeSH
• DNA (Cytosine-5-)-Methyltransferase 1 MeSH
• Transcription, Genetic MeSH
• Histones genetics   MeSH
• Humans MeSH
• DNA Methylation genetics   MeSH
• Promoter Regions, Genetic MeSH
• Proto-Oncogene Proteins biosynthesis   genetics   metabolism   MeSH
• Gene Expression Regulation, Leukemic MeSH
• Trans-Activators biosynthesis   genetics   metabolism   MeSH
• GATA1 Transcription Factor genetics   metabolism   MeSH
• Protein Binding MeSH
• Enhancer Elements, Genetic MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA (Cytosine-5-)-Methyltransferases MeSH
• DNA (Cytosine-5-)-Methyltransferase 1 MeSH
• DNMT1 protein, human MeSH Browser
• GATA1 protein, human MeSH Browser
• Histones MeSH
• proto-oncogene protein Spi-1 MeSH Browser
• Proto-Oncogene Proteins MeSH
• Trans-Activators MeSH
• GATA1 Transcription Factor MeSH