The ISWI family protein SMARCA5 contains the ATP-binding pocket that coordinates the catalytic Mg2+ ion and water molecules for ATP hydrolysis. In this study, we demonstrate that SMARCA5 can also possess an alternative metal-binding ability. First, we isolated SMARCA5 on the cobalt column (IMAC) to near homogeneity. Examination of the interactions of SMARCA5 with metal-chelating supports showed that, apart from Co2+, it binds to Cu2+, Zn2+ and Ni2+. The efficiency of the binding to the last-listed metal was influenced by the chelating ligand, resulting in a strong preference for Ni-NTA over the Ni-CM-Asp equivalent. To gain insight in the preferential affinity for the Ni-NTA ligand, QM calculations were performed on model systems and metal-ligand complexes with a limited protein fragment of SMARCA5 containing the double-histidine (dHis) motif. The calculations correlated the observed affinity with the relative stability of the d-block metals to tetradentate ligand coordination over tridentate, as well as their overall octahedral coordination capacity. Likewise, binding free energies derived from model imidazole complexes mirrored the observed Ni-NTA/Ni-CM-Asp preferential affinity. Finally, similar calculations on complexes with a SMARCA5 peptide fragment derived from the AlphaFold structural prediction, captured almost accurately the expected relative stability of the TM complexes, and produced a large energetic separation (~10 kcal∙mol-1) between Ni-NTA and Ni-CM-Asp in favour of the former.
- MeSH
- Adenosine Triphosphatases MeSH
- Chromosomal Proteins, Non-Histone metabolism chemistry MeSH
- Metals chemistry metabolism MeSH
- Quantum Theory MeSH
- Humans MeSH
- Ligands MeSH
- Models, Molecular MeSH
- Chromatin Assembly and Disassembly MeSH
- Protein Binding * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
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
SF3B1 mutations are recurrent in chronic lymphocytic leukemia (CLL), particularly enriched in clinically aggressive stereotyped subset #2. To investigate their impact, we conducted RNA-sequencing of 18 SF3B1MUT and 17 SF3B1WT subset #2 cases and identified 80 significant alternative splicing events (ASEs). Notable ASEs concerned exon inclusion in the non-canonical BAF (ncBAF) chromatin remodeling complex subunit, BRD9, and splice variants in eight additional ncBAF complex interactors. Long-read RNA-sequencing confirmed the presence of splice variants, and extended analysis of 139 CLL cases corroborated their association with SF3B1 mutations. Overexpression of SF3B1K700E induced exon inclusion in BRD9, resulting in a novel splice isoform with an alternative C-terminus. Protein interactome analysis of the BRD9 splice isoform revealed augmented ncBAF complex interaction, while exhibiting decreased binding of auxiliary proteins, including SPEN, BRCA2, and CHD9. Additionally, integrative multi-omics analysis identified a ncBAF complex-bound gene quartet on chromosome 1 with higher expression levels and more accessible chromatin in SF3B1MUT CLL. Finally, Cancer Dependency Map analysis and BRD9 inhibition displayed BRD9 dependency and sensitivity in cell lines and primary CLL cells. In conclusion, spliceosome dysregulation caused by SF3B1 mutations leads to multiple ASEs and an altered ncBAF complex interactome, highlighting a novel pathobiological mechanism in SF3B1MUT CLL.
- MeSH
- Alternative Splicing MeSH
- Leukemia, Lymphocytic, Chronic, B-Cell * genetics pathology metabolism MeSH
- Phosphoproteins * genetics metabolism MeSH
- Humans MeSH
- Mutation * MeSH
- Bromodomain Containing Proteins MeSH
- Chromatin Assembly and Disassembly * MeSH
- RNA Splicing Factors * genetics metabolism MeSH
- Spliceosomes * metabolism genetics MeSH
- Transcription Factors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Chromatin remodeling complexes are required for many distinct nuclear processes such as transcription, DNA replication, and DNA repair. However, the contribution of these complexes to the development of complex tissues within an organism is poorly characterized. Imitation switch (ISWI) proteins are among the most evolutionarily conserved ATP-dependent chromatin remodeling factors and are represented by yeast Isw1/Isw2, and their vertebrate counterparts Snf2h (Smarca5) and Snf2l (Smarca1). In this study, we focused on the role of the Snf2h gene during the development of the mammalian retina. We show that Snf2h is expressed in both retinal progenitors and post-mitotic retinal cells. Using Snf2h conditional knockout mice (Snf2h cKO), we found that when Snf2h is deleted, the laminar structure of the adult retina is not retained, the overall thickness of the retina is significantly reduced compared with controls, and the outer nuclear layer (ONL) is completely missing. The depletion of Snf2h did not influence the ability of retinal progenitors to generate all the differentiated retinal cell types. Instead, the Snf2h function is critical for the proliferation of retinal progenitor cells. Cells lacking Snf2h have a defective S-phase, leading to the entire cell division process impairments. Although all retinal cell types appear to be specified in the absence of the Snf2h function, cell-cycle defects and concomitantly increased apoptosis in Snf2h cKO result in abnormal retina lamination, complete destruction of the photoreceptor layer, and consequently, a physiologically non-functional retina.
- MeSH
- Adenosine Triphosphatases * metabolism MeSH
- Cell Nucleus metabolism MeSH
- Chromatin * metabolism MeSH
- Chromosomal Proteins, Non-Histone * metabolism MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Cell Proliferation MeSH
- Chromatin Assembly and Disassembly * MeSH
- Retina MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Previous studies have demonstrated an involvement of chromatin-remodelling SWI/SNF complexes in the development of prostate cancer, suggesting both tumor suppressor and oncogenic activities. SMARCD1/BAF60A, SMARCD2/BAF60B, and SMARCD3/BAF60C are mutually exclusive accessory subunits that confer functional specificity and are components of all known SWI/SNF subtypes. To assess the role of SWI/SNF in prostate tumorigenesis, we studied the functions and functional relations of the SMARCD family members. Performing RNA-seq in LnCAP cells grown in the presence or absence of dihydrotestosterone, we found that the SMARCD proteins are involved in the regulation of numerous hormone-dependent AR-driven genes. Moreover, we demonstrated that all SMARCD proteins can regulate AR-downstream targets in androgen-depleted cells, suggesting an involvement in the progression to castration-resistance. However, our approach also revealed a regulatory role for SMARCD proteins through antagonization of AR-signalling. We further demonstrated that the SMARCD proteins are involved in several important cellular processes such as the maintenance of cellular morphology and cytokinesis. Taken together, our findings suggest that the SMARCD proteins play an important, yet paradoxical, role in prostate carcinogenesis. Our approach also unmasked the complex interplay of paralogue SWI/SNF proteins that must be considered for the development of safe and efficient therapies targeting SWI/SNF.
- MeSH
- Receptors, Androgen * genetics metabolism MeSH
- Chromosomal Proteins, Non-Histone genetics metabolism MeSH
- Humans MeSH
- Prostatic Neoplasms * genetics MeSH
- Gene Expression Regulation MeSH
- Chromatin Assembly and Disassembly genetics MeSH
- Signal Transduction MeSH
- Transcription Factors metabolism MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Male germ cells experience a drastic chromatin remodeling through the nucleo-histone to nucleo-protamine (NH-NP) transition necessary for proper sperm functionality. Post-translational modifications (PTMs) of H4 Lys5, such as acetylation (H4K5ac), play a crucial role in epigenetic control of nucleosome disassembly facilitating protamine incorporation into paternal DNA. It has been shown that butyrylation on the same residue (H4K5bu) participates in temporal regulation of NH-NP transition in mice, delaying the bromodomain testis specific protein (BRDT)-dependent nucleosome disassembly and potentially marking retained nucleosomes. However, no information was available so far on this modification in human sperm. Here, we report a dual behavior of H4K5bu and H4K5ac in human normal spermatogenesis, suggesting a specific role of H4K5bu during spermatid elongation, coexisting with H4K5ac although with different starting points. This pattern is stable under different testicular pathologies, suggesting a highly conserved function of these modifications. Despite a drastic decrease of both PTMs in condensed spermatids, they are retained in ejaculated sperm, with 30% of non-colocalizing nucleosome clusters, which could reflect differential paternal genome retention. Whereas no apparent effect of these PTMs was observed associated with sperm quality, their presence in mature sperm could entail a potential role in the zygote.
- MeSH
- Acetylation MeSH
- Chromatin * metabolism MeSH
- Histones metabolism MeSH
- Humans MeSH
- Mice MeSH
- Nucleosomes * metabolism MeSH
- Protein Processing, Post-Translational MeSH
- Protamines metabolism MeSH
- Chromatin Assembly and Disassembly MeSH
- Semen metabolism MeSH
- Spermatids metabolism MeSH
- Spermatogenesis physiology MeSH
- Spermatozoa metabolism MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
This Special Issue highlights the advantages of using combined approaches to explore chromatin molecular complexes [...].
- MeSH
- Chromatin * genetics MeSH
- Genome * MeSH
- Chromatin Assembly and Disassembly MeSH
- Publication type
- Editorial 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.
Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.
- MeSH
- Chromatin chemistry immunology MeSH
- Child MeSH
- Host Cell Factor C1 genetics immunology MeSH
- Heterozygote MeSH
- Histones genetics immunology MeSH
- Infant MeSH
- Humans MeSH
- Mutation, Missense * MeSH
- Adolescent MeSH
- Loss of Function Mutation * MeSH
- Tumor Suppressor Proteins deficiency genetics immunology MeSH
- Neurodevelopmental Disorders genetics immunology pathology MeSH
- Child, Preschool MeSH
- Proteasome Endopeptidase Complex genetics immunology MeSH
- BRCA1 Protein genetics immunology MeSH
- Gene Expression Regulation MeSH
- Chromatin Assembly and Disassembly genetics immunology MeSH
- Family MeSH
- T-Lymphocytes immunology pathology MeSH
- Ubiquitin Thiolesterase deficiency genetics immunology MeSH
- Ubiquitin genetics immunology MeSH
- Ubiquitination MeSH
- Ubiquitin-Protein Ligases genetics immunology MeSH
- Germ-Line Mutation * MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.
- MeSH
- Algorithms MeSH
- beta Catenin chemistry metabolism MeSH
- Cell Differentiation MeSH
- Chromatin metabolism MeSH
- Phenotype MeSH
- Transcription, Genetic * MeSH
- Homeostasis MeSH
- Hyperplasia MeSH
- JNK Mitogen-Activated Protein Kinases metabolism MeSH
- Stem Cells metabolism MeSH
- RNA, Messenger genetics metabolism MeSH
- Mutation genetics MeSH
- Mutant Proteins metabolism MeSH
- Mice MeSH
- Organoids metabolism MeSH
- Cell Proliferation MeSH
- Chromatin Assembly and Disassembly MeSH
- Base Sequence MeSH
- Signal Transduction MeSH
- Intestinal Mucosa cytology MeSH
- Transcription Factors metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
