B-cell fate determination requires the action of transcription factors that operate in a regulatory network to activate B-lineage genes and repress lineage-inappropriate genes. However, the dynamics ...and hierarchy of events in B-cell programming remain obscure. To uncouple the dynamics of transcription factor expression from functional consequences, we generated induction systems in developmentally arrested
pre-pro-B cells to allow precise experimental control of EBF1 expression in the genomic context of progenitor cells. Consistent with the described role of EBF1 as a pioneer transcription factor, we show in a time-resolved analysis that EBF1 occupancy coincides with EBF1 expression and precedes the formation of chromatin accessibility. We observed dynamic patterns of EBF1 target gene expression and sequential up-regulation of transcription factors that expand the regulatory network at the pro-B-cell stage. A continuous EBF1 function was found to be required for
promoter activity and for the maintenance of an accessible chromatin domain that is permissive for binding of other transcription factors. Notably, transient EBF1 occupancy was detected at lineage-inappropriate genes prior to their silencing in pro-B cells. Thus, persistent and transient functions of EBF1 allow for an ordered sequence of epigenetic and transcriptional events in B-cell programming.
Lymphopoiesis requires the activation of lineage-specific genes embedded in naive, inaccessible chromatin or in primed, accessible chromatin. The mechanisms responsible for de novo gain of chromatin ...accessibility, known as “pioneer” function, remain poorly defined. Here, we showed that the EBF1 C-terminal domain (CTD) is required for the regulation of a specific gene set involved in B cell fate decision and differentiation, independently of activation and repression functions. Using genome-wide analysis of DNaseI hypersensitivity and DNA methylation in multipotent Ebf1−/− progenitors and derivative EBF1wt- or EBF1ΔC-expressing cells, we found that the CTD promoted chromatin accessibility and DNA demethylation in previously naive chromatin. The CTD allowed EBF1 to bind at inaccessible genomic regions that offer limited co-occupancy by other transcription factors, whereas the CTD was dispensable for EBF1 binding at regions that are occupied by multiple transcription factors. Thus, the CTD enables EBF1 to confer permissive lineage-specific changes in progenitor chromatin landscape.
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•EBF1 induces chromatin accessibility and DNA demethylation in naive chromatin•C-terminal domain (CTD) of EBF1 is required for the regulation of a specific gene set•CTD-dependent genes are involved in B cell fate decision and differentiation•CTD enables EBF1 to access progenitor chromatin at sites with low TF co-occupancy
EBF1 is a key transcription factor for B cell programming in hematopoietic progenitors. Boller et al. show that EBF1 induces lineage-specific accessibility in progenitor chromatin. EBF1 requires its C-terminal domain to access naive chromatin at sites that lack co-occupancy with multiple transcription factors, thereby conferring “pioneer function” upon EBF1.
The transcription factors EBF1 and Pax5 have been linked to activation of the B cell lineage program and irreversible loss of alternative lineage potential (commitment), respectively. Here we ...conditionally deleted Ebf1 in committed pro-B cells after transfer into alymphoid mice. We found that those cells converted into innate lymphoid cells (ILCs) and T cells with variable-diversity-joining (VDJ) rearrangements of loci encoding both B cell and T cell antigen receptors. As intermediates in lineage conversion, Ebf1-deficient CD19(+) cells expressing Pax5 and transcriptional regulators of the ILC and T cell fates were detectable. In particular, genes encoding the transcription factors Id2 and TCF-1 were bound and repressed by EBF1. Thus, both EBF1 and Pax5 are required for B lineage commitment by repressing distinct and common determinants of alternative cell fates.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal ...zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1−/−Prdm1+/gfp
mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1
−/− plasmablasts show a reduced activation of β1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.
Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes ...in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα
Sca1
(PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.
Telomeres are nucleoprotein complexes at the end of linear eukaryotic chromosomes which maintain the genome integrity by regulating telomere length, preventing recombination and end to end fusion ...events. Multiple proteins associate with telomeres and function in concert to carry out these functions. Rap1 interacting factor 1 (Rif1), was identified as a protein involved in telomere length regulation in yeast. Rif1 is conserved upto mammals but its function has diversified from telomere length regulation to maintenance of genome integrity.
We have carried out detailed bioinformatic analyses and identified Rif1 homologues in 92 organisms from yeast to human. We identified Rif1 homologues in Drosophila melanogaster, even though fly telomeres are maintained by a telomerase independent pathway. Our analysis shows that Drosophila Rif1 (dRif1) sequence is phylogenetically closer to the one of vertebrates than yeast and has identified a few Rif1 specific motifs conserved through evolution. This includes a Rif1 family specific conserved region within the HEAT repeat domain and a motif involved in protein phosphatase1 docking. We show that dRif1 is nuclear localized with a prominent heterochromatin association and unlike human Rif1, it does not respond to DNA damage by localizing to damaged sites. To test the evolutionary conservation of dRif1 function, we expressed the dRif1 protein in yeast and HeLa cells. In yeast, dRif1 did not perturb yeast Rif1 (yRif1) functions; and in HeLa cells it did not colocalize with DNA damage foci.
Telomeres are maintained by retrotransposons in all Drosophila species and consequently, telomerase and many of the telomere associated protein homologues are absent, including Rap1, which is the binding partner of Rif1. We found that a homologue of yRif1 protein is present in fly and dRif1 has evolutionarily conserved motifs. Functional studies show that dRif1 responds differently to DNA damage, implying that dRif1 may have a different function and this may be conserved in other organisms as well.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chromatin architecture is critical for gene expression during development. Matrix attachment regions (MARs) control and regulate chromatin dynamics. The position of MARs in the genome determines the ...expression of genes in the organism. In this study, we set out to elucidate how MARs temporally regulate the expression of the fibroin heavy chain (FIBH) gene during development. We addressed this by identifying MARs and studying their distribution and differentiation, in the posterior silk glands of Bombyx mori during 5th instar development. Of the MARs identified on three different days, 7.15% MARs were common to all 3 days, whereas, 1.41, 19.27 and 52.47% MARs were unique to day 1, day 5, and day 7, respectively highlighting the dynamic nature of the matrix associated DNA. The average chromatin loop length based on the chromosome wise distribution of MARs and the distances between these MAR regions decreased from day 1 (253.91 kb) to day 5 (73.54 kb) to day 7 (39.19 kb). Further significant changes in the MARs in the vicinity of the FIBH gene were found during different days of 5.sup.th instar development which implied their role in the regulation and expression of the FIBH gene. The presence of MARs in the flanking regions of genes found to exhibit differential expression during 5.sup.th instar development indicates their possible role in the regulation of their expression. This reiterates the importance of MARs in the genomic functioning as regulators of the molecular mechanisms in the nucleus. This is the first study that takes into account the tissue specific genome-wide MAR association and the potential role of these MARs in developmentally regulated gene expression. The current study lays a foundation to understand the genome wide regulation of chromatin during development.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Polycomb group (PcG) proteins maintain expression pattern of genes set early during development. Although originally isolated as regulators of homeotic genes, PcG members play a key role in ...epigenetic mechanism that maintains the expression state of a large number of genes. Polycomb (PC) is conserved during evolution and while invertebrates have one PC gene, vertebrates have five or more homologues. It remains unclear if different vertebrate PC homologues have distinct or overlapping functions. We have identified and compared the sequence of PC homologues in various organisms to analyze similarities and differences that shaped the evolutionary history of this key regulatory protein.
All PC homologues have an N-terminal chromodomain and a C-terminal Polycomb Repressor box. We searched the protein and genome sequence database of various organisms for these signatures and identified approximately 100 PC homologues. Comparative analysis of these sequences led to the identification of a novel insect specific motif and several novel and signature motifs in the vertebrate homologue: two in CBX2 (Cx2.1 and Cx2.2), four in CBX4 (Cx4.1, Cx4.2, Cx4.3 and Cx4.4), three in CBX6 (Cx6.1, Cx6.2 and Cx6.3) and one in CBX8 (Cx8.1). Additionally, adjacent to the chromodomain, all the vertebrate homologues have a DNA binding motif - AT-Hook in case of CBX2, which was known earlier, and 'AT-Hook Like' motif, from this study, in other PC homologues.
Our analysis shows that PC is an ancient gene dating back to pre bilaterian origin that has not only been conserved but has also expanded during the evolution of complexity. Unique motifs acquired by each homologue have been maintained for more than 500 millions years indicating their functional relevance in boosting the epigenetic 'tool kit'. We report the presence of a DNA interaction motif adjacent to chromodomain in all vertebrate PC homologues and suggest a three-way 'PC-histoneH3-DNA' interaction that can restrict nucleosome dynamics. The signature motifs of PC homologues and insect specific motif identified in this study pave the way to understand the molecular basis of epigenetic mechanisms.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Summary
Juvenile myelomonocytic leukaemia (JMML) is characterized by gene variants that deregulate the RAS signalling pathway. Children with neurofibromatosis type 1 (NF‐1) carry a defective NF1 ...allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild‐type allele. Here we examined the two‐hit concept in leukaemic cells of 25 patients with JMML and NF‐1. Ten patients with JMML/NF‐1 exhibited a NF1 loss‐of‐function variant in combination with uniparental disomy of the 17q arm. Five had NF1 microdeletions combined with a pathogenic NF1 variant and nine carried two compound‐heterozygous NF1 variants. We also examined 16 patients without clinical signs of NF‐1 and no variation in the JMML‐associated driver genes PTPN11, KRAS, NRAS or CBL (JMML‐5neg) and identified eight patients with NF1 variants. Three patients had microdeletions combined with hemizygous NF1 variants, three had compound‐heterozygous NF1 variants and two had heterozygous NF1 variants. In addition, we found a high incidence of secondary ASXL1 and/or SETBP1 variants in both groups. We conclude that the clinical diagnosis of JMML/NF‐1 reliably indicates a NF1‐driven JMML subtype, and that careful NF1 analysis should be included in the genetic workup of JMML even in the absence of clinical evidence of NF‐1.
Children with neurofibromatosis type 1 (NF‐1) carry a defective NF1 allele in the germline and are predisposed to JMML, which presumably requires somatic inactivation of the NF1 wild‐type allele. We examined the two‐hit concept in leukaemic cells of 25 patients with JMML and clinical diagnosis of NF‐1 and confirmed biallelic NF1 inactivation in all cases but one. Among 16 JMML patients without clinical signs of NF‐1 and no variation in other JMML‐associated driver genes, eight patients exhibited NF1‐inactivating variants (six biallelic, two monoallelic). The data show that the clinical diagnosis of JMML/NF‐1 reliably indicates a NF1‐driven JMML subtype, and that NF1 genetic analysis should be included in the diagnostic workup of JMML even in the absence of clinical evidence of NF‐1.
The rearrangement and expression of the immunoglobulin μ heavy chain (Igh) gene require communication of the intragenic Eμ and 3′ regulatory region (RR) enhancers with the variable (VH) gene ...promoter. Eμ binding of the transcription factor YY1 has been implicated in enhancer-promoter communication, but the YY1 protein network remains obscure. By analyzing the comprehensive proteome of the 1-kb Eμ wild-type enhancer and that of Eμ lacking the YY1 binding site, we identified the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network. We found that MSL2 recruitment depends on YY1 and that gene knockout of Msl2 in primary pre-B cells reduces μ gene expression and chromatin looping of Eμ to the 3′ RR enhancer and VH promoter. Moreover, Mof heterozygosity in mice impaired μ expression and early B cell differentiation. Together, these data suggest that the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication.
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•Quantitative mass spectrometry of the immunoglobulin heavy chain Eμ enhanceosome•The MSL/MOF complex is recruited to the Eμ enhancer via transcription factor YY1•Msl2 inactivation reduces μ expression and YY1-mediated enhancer-promoter looping•Mof heterozygosity in mice impairs μ gene expression and early B cell differentiation
Phongbunchoo et al. perform quantitative mass spectrometric analysis of the immunoglobulin Eμ enhanceosome. They show that the identified MSL/MOF protein complex, a regulator of gene dosage compensation in Drosophila, binds Eμ via transcription factor YY1. MSL/MOF upregulates the expression of the functionally rearranged μ allele by augmenting YY1-mediated enhancer-promoter interactions.
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