Search

The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of epigenetic factors that regulate DNA methylation, post-translational histone modifications and chromatin structure. Disturbed epigenetic regulation causes perturbations in the blood cell differentiation program that results in various types of haematopoietic disorders. Thus, accurate epigenetic regulation is essential for functional haematopoiesis. In this study, we used a CRISPR-Cas9 screening approach to identify new epigenetic regulators in myeloid differentiation. We designed a Chromatin-UMI CRISPR guide library targeting 1092 epigenetic regulators. Phorbol 12-myristate 13-acetate (PMA) treatment of the chronic myeloid leukaemia cell line K-562 was used as a megakaryocytic myeloid differentiation model. Both previously described developmental epigenetic regulators and novel factors were identified in our screen. In this study, we validated and characterized a role for the chromatin remodeller CHD2 in myeloid proliferation and megakaryocytic differentiation.

Background Cellular quiescence is a reversible differentiation state during which cells modify their gene expression program to inhibit metabolic functions and adapt to a new cellular environment. The epigenetic changes accompanying these alterations are not well understood. We used fission yeast cells as a model to study the regulation of quiescence. When these cells are starved for nitrogen, the cell cycle is arrested in G1, and the cells enter quiescence (G0). A gene regulatory program is initiated, including downregulation of thousands of genes—for example, those related to cell proliferation—and upregulation of specific genes—for example, autophagy genes—needed to adapt to the physiological challenge. These changes in gene expression are accompanied by a marked alteration of nuclear organization and chromatin structure. Results Here, we investigated the role of Leo1, a subunit of the conserved RNA polymerase-associated factor 1 (Paf1) complex, in the quiescence process using fission yeast as the model organism. Heterochromatic regions became very dynamic in fission yeast in G0 during nitrogen starvation. The reduction of heterochromatin in early G0 was correlated with reduced target of rapamycin complex 2 (TORC2) signaling. We demonstrated that cells lacking Leo1 show reduced survival in G0. In these cells, heterochromatic regions, including subtelomeres, were stabilized, and the expression of many genes, including membrane transport genes, was abrogated. TOR inhibition mimics the effect of nitrogen starvation, leading to the expression of subtelomeric genes, and this effect was suppressed by genetic deletion of leo1. Conclusions We identified a protein, Leo1, necessary for survival during quiescence. Leo1 is part of a conserved protein complex, Paf1C, linked to RNA polymerase II. We showed that Leo1, acting downstream of TOR, is crucial for the dynamic reorganization of chromosomes and the regulation of gene expression during cellular quiescence. Genes encoding membrane transporters are not expressed in quiescent leo1 mutant cells, and cells die after 2 weeks of nitrogen starvation. Taken together, our results suggest that Leo1 is essential for the dynamic regulation of heterochromatin and gene expression during cellular quiescence. Electronic supplementary material The online version of this article (10.1186/s13072-019-0292-7) contains supplementary material, which is available to authorized users.

Background The heat-shock transcription factor 1 (HSF1) has been linked to cell proliferation and survival in cancer and has been proposed as a biomarker for poor prognosis. Here, we assessed the role of HSF1 expression in relation to copy number alteration (CNA) and cancer prognosis. Methods Using 10,287 cancer genomes from The Cancer Genome Atlas and Cbioportal databases, we assessed the association of HSF1 expression with CNA and cancer prognosis. CNA of 8q24.3 was categorized as diploid (reference), deletion (fewer copies), gain (+ 1 copy) and amplification (≥ + 2 copies). Multivariate logistic regression modeling was used to assess 5-year survival among those with a first cancer diagnosis and complete follow-up data (N = 9568), categorized per anatomical location and histology, assessing interaction with tumor stage, and expressed as odds ratios and 95% confidence intervals. Results We found that only 54.1% of all tumors have a normal predicted 8q24.3 copy number and that 8q24.3 located genes including HSF1 are mainly overexpressed due to increased copies number of 8q24.3 in different cancers. The tumor of patients having respectively gain (+ 1 copy) and amplification (≥ + 2 copies) of 8q24.3 display a global increase of 5-year mortality (odds ratio = 1.98, 95% CI 1.22–3.21) and (OR = 2.19, 1.13–4.26) after full adjustment. For separate cancer types, tumor patients with 8q24.3 deletion showed a marked increase of 5-year mortality in uterine (OR = 4.84, [2.75–8.51]), colorectal (OR = 4.12, [1.15–14.82]), and ovarian (OR = 1.83, [1.39–2.41]) cancers; and decreased mortality in kidney cancer (OR = 0.41, [0.21–0.82]). Gain of 8q24.3 resulted in significant mortality changes in 5-year mortality for cancer of the uterus (OR = 3.67, [2.03–6.66]), lung (OR = 1.76, [1.24–2.51]), colorectal (OR = 1.75, [1.32–2.31]) cancers; and amplification for uterine (OR = 4.58, [1.43–14.65]), prostate (OR = 4.41 [3.41–5.71]), head and neck (OR = 2.68, [2.17–3.30]), and stomach (OR = 0.56, [0.36–0.87]) cancers. Conclusions Here, we show that CNAs of 8q24.3 genes, including HSF1, are tightly linked to 8q24.3 copy number in tumor patients and can affect patient outcome. Our results indicate that the integration of 8q24.3 CNA detection may be a useful predictor for cancer prognosis.

The DNA glycosylase MutY homolog (Myh1) excises adenines misincorporated opposite guanines or 7,8-dihydro-8-oxo-guanines on DNA by base excision repair thereby preventing G:C to T:A mutations. Schizosaccharomyces pombe (Sp) Hst4 is an NAD + -dependent histone/protein deacetylase involved in gene silencing and maintaining genomic integrity. Hst4 regulates deacetylation of histone 3 Lys56 at the entry and exit points of the nucleosome core particle. Here, we demonstrate that the hst4 mutant is more sensitive to H 2 O 2 than wild-type cells. H 2 O 2 treatment results in an SpMyh1-dependent decrease in SpHst4 protein level and hyperacetylation of histone 3 Lys56. Furthermore, SpHst4 interacts with SpMyh1 and the cell cycle checkpoint Rad9-Rad1-Hus1 (9-1-1) complex. SpHst4, SpMyh1, and SpHus1 are physically bound to telomeres. Following oxidative stress, there is an increase in the telomeric association of SpMyh1. Conversely, the telomeric association of spHst4 is decreased. Deletion of SpMyh1 strongly abrogated telomeric association of SpHst4 and SpHus1. However, telomeric association of SpMyh1 is enhanced in hst4 Δ cells in the presence of chronic DNA damage. These results suggest that SpMyh1 repair regulates the functions of SpHst4 and the 9-1-1 complex in maintaining genomic stability.

The protozoan parasite Trypanosoma brucei is responsible for sleeping sickness and alternates between mammal and tsetse fly hosts. Two proteins of the ALBA family associate to mRNA in cytoplasmic granules during starvation stress, are stage regulated, and contribute to trypanosome development in the tsetse fly., The protozoan parasite Trypanosoma brucei is responsible for sleeping sickness and alternates between mammal and tsetse fly hosts, where it has to adapt to different environments. We investigated the role of two members of the ALBA family, which encodes hypothetical RNA-binding proteins conserved in most eukaryotes. We show that ALBA3/4 proteins colocalize with the DHH1 RNA-binding protein and with a subset of poly(A+) RNA in stress granules upon starvation. Depletion of ALBA3/4 proteins by RNA interference in the cultured procyclic stage produces cell modifications mimicking several morphogenetic aspects of trypanosome differentiation that usually take place in the fly midgut. A combination of immunofluorescence data and videomicroscopy analysis of live trypanosomes expressing endogenously ALBA fused with fluorescent proteins revealed that ALBA3/4 are present throughout the development of the parasite in the tsetse fly, with the striking exception of the transition stages found in the proventriculus region. This involves migration of the nucleus toward the posterior end of the cell, a phenomenon that is perturbed upon forced expression of ALBA3 during the differentiation process, showing for the first time the involvement of an RNA-binding protein in trypanosome development in vivo.

DNA topoisomerases regulate the topological state of the DNA double helix and are key enzymes in the processes of DNA replication, transcription and genome stability. Using the fission yeast model Schizosaccharomyces pombe, we investigate genome wide how DNA topoisomerases I and II affect chromatin dynamics and gene expression in vivo. We show that topoisomerase I activity is directly required for efficient nucleosome disassembly at gene promoter regions. Lack of topoisomerase activity results in increased nucleosome occupancy, perturbed histone modifications and reduced transcription from these promoters. Strong correlative evidence suggests that topoisomerase I cooperates with the ATP-dependent chromatin remodeller Hrp1 in nucleosome disassembly. Our study links topoisomerase activity to the maintenance of open chromatin and regulating transcription in vivo.

Histone acetylation and deacetylation are important for gene regulation. The histone acetyltransferase, Gcn5, is an activator of transcriptional initiation that is recruited to gene promoters. Here, we map genome-wide Gcn5 occupancy and histone H3K14ac at high resolution. Gcn5 is predominantly localized to coding regions of highly transcribed genes, where it collaborates antagonistically with the class-II histone deacetylase, Clr3, to modulate H3K14ac levels and transcriptional elongation. An interplay between Gcn5 and Clr3 is crucial for the regulation of many stress-response genes. Our findings suggest a new role for Gcn5 during transcriptional elongation, in addition to its known role in transcriptional initiation.

The protist Trypanosoma brucei possesses a single Argonaute gene called TbAGO1 that is necessary for RNAi silencing. We previously showed that in strain 427, TbAGO1 knock-out leads to a slow growth phenotype and to chromosome segregation defects. Here we report that the slow growth phenotype is linked to defects in segregation of both large and mini-chromosome populations, with large chromosomes being the most affected. These phenotypes are completely reversed upon inducible re-expression of TbAGO1 fused to GFP, demonstrating their link with TbAGO1. Trypanosomes that do not express TbAGO1 show a general increase in the abundance of transcripts derived from the short retroposon RIME (Ribosomal Interspersed Mobile Element). Supplementary large RIME transcripts emerge in the absence of RNAi, a phenomenon coupled to the disappearance of short transcripts. These fluctuations are reversed by inducible expression of GFP::TbAGO1. Furthermore, we use a combination of Northern blots, RT-PCR and sequencing to reveal that RNAi controls expression of transcripts derived from RHS (Retrotransposon Hot Spot) pseudogenes (RHS genes with retro-element(s) integrated within their coding sequence). Absence of RNAi also leads to an increase of steady-state transcripts from regular RHS genes (those without retro-element), indicating a role for pseudogene in control of gene expression. However, analysis of retroposon abundance and arrangement in the genome of multiple clonal cell lines of TbAGO1−/− failed to reveal movement of mobile elements despite the increased amounts of retroposon transcripts.

Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-ACnp1 nucleosomes., Author Summary Centromeres are unique regions on eukaryotic chromosomes that are essential for chromosome segregation at mitosis and meiosis. Centromere identity and function depends on the presence of specialized chromatin with nucleosomes containing the centromere-specific histone H3 variant CENP-A. Assembly and disassembly of nucleosomes have previously been shown to involve a family of enzymes known as DNA topoisomerases. We show that centromeres are unique in that they are associated with high levels of Top3, but low levels of Top1 and Top2, suggesting that Top3 is particularly important for centromeric DNA topology. Impaired function of Top3 or its partner Rqh1 results in chromosome segregation defects and increased levels of CENP-ACnp1 at centromeres. This role in limiting the levels of CENP-ACnp1 at centromeres is independent of the established role for the Top3-Rqh1 complex in homologous recombination. Therefore, we hypothesize that the Top3-Rqh1 complex exerts this effect by regulating centromere DNA topology, which in turn affects CENP-ACnp1 nucleosome dynamics. Specific removal of negative supercoiling by Top3 could directly have a negative effect on assembly of CENP-ACnp1 nucleosomes with left-handed negative wrapping of DNA and/or act indirectly by inhibiting transcription-coupled CENP-ACnp1 assembly. Alternatively, Top3 may be a factor that promotes formation of CENP-ACnp1 hemisomes with right-handed wrapping of DNA over conventional octamers. This suggests a new role for the Top3-Rqh1 complex at centromeres and may contribute to the understanding of the structural and functional specification of centromeres.

The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1).

Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Kruppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation., Author Summary Chromatin is based on a repetitive structural unit called the nucleosome. However, the regulatory properties of chromatin are mediated by the differences between nucleosomes, due to post-translational modifications or the inclusion of histone variants. These differences are maintained by inheritance through cis-acting epigenetic mechanisms. Here we describe a case where the local character of chromatin is not only determined by cis-acting mechanisms but also negatively regulated in trans. The case involves loading of the histone H2A variant, H2A.Z, into chromatin. We show that H2A.Z in the yeast Schizosaccharomyces pombe is mainly found in genes at the first transcribed nucleosome and is inserted into this nucleosome by the Swr1C remodeling machine. However, Swr1C has a regulatory subunit, Msc1, which is not required for H2A.Z promoter loading but prevents H2A.Z occupancy in the inner centromere and subtelomeric regions. These two specialized regions are neither eu- nor heterochromatin and share certain characteristics, which may predispose them to the aberrant inclusion of H2A.Z and the requirement for trans regulation by Msc1.

Expression profiling, ChiP-CHIP and phenotypic analysis were used to investigate the functional relationships of class III NAD(+)-dependent HDACs (Sirtuins) in fission yeast. We detected significant histone acetylation increases in Sirtuin mutants at their specific genomic binding targets and were thus able to identify an in vivo substrate preference for each Sirtuin. At heterochromatic loci, we demonstrate that although Hst2 is mainly cytoplasmic, a nuclear pool of Hst2 colocalizes with the other Sirtuins at silent regions (cen, mat, tel, rDNA), and that like the other Sirtuins, Hst2 is required for rDNA and centromeric silencing. Interestingly we found specific functions for the fission yeast Sirtuins Hst2 and Hst4 in gene regulation. Hst2 directly represses genes involved in transport and membrane function, whereas Hst4 represses amino-acid biosynthesis genes and Tf2 retrotransposons. A specific role for Hst4 in Tf2 5' mRNA processing was revealed. Thus, Sirtuins share functions at many genomic targets, but Hst2 and Hst4 have also evolved unique functions in gene regulation.

In many eukaryotes, double-stranded (ds) RNA leads to specific degradation of RNA of cognate sequence, a process termed RNA interference (RNAi). Here we used the protozoan Trypanosoma brucei as a model to investigate efficiency and specificity of RNAi generated by expression of long dsRNA of PFRA and PFRC genes, which code for flagellar proteins required for cell motility. Consequences of RNAi were monitored at all three levels: target RNA expression, protein expression and phenotype observation, using population or individual cell analysis. Expression of PFRA dsRNA from an inverted repeat was extremely efficient, knocking down PFRA RNA and PFRA protein, and producing a severe paralysis phenotype. Silencing by expression of PFRA dsRNA using a dual facing promoter system was also very efficient, producing a clear phenotype, although low amounts of PFRA RNA and PFRA protein were detected. Expression via the dual facing promoters of PAR2 dsRNA (83% overall identity with PFRA, including nine blocks of >20 nt total identity) did not produce significant reduction of total amounts of PFRA RNA or PFRA protein. However, individual cell analysis by immunofluorescence revealed that 10–60% cells (depending on subclones) exhibited lower PFRA amounts in their flagellum, producing a reduced-motility phenotype.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture., Author Summary Centromeres are essential to chromatin structures, providing a binding platform for the mitotic spindle. Defects in centromere structure or function can lead to chromosome missegregation or chromosome breakage. This, in turn, can cause cancer in metazoans. Centromeres are defined by specialized chromatin that contains the histone H3 variant CENP-A (also called CenH3, or Cse4 in budding yeast), and transcription over centromeres is tightly controlled. Budding yeast centromeres are composed of a single nucleosome containing the essential Cse4. Loss of one of these specialized centromeric nucleosomes can lead to chromosome missegregation during mitosis followed by cell death. We provide evidence that energy-dependent chromatin remodeling factor Fun30 supports faithful chromosome segregation, especially when centromere structure is challenged by mutation of Cse4 or by forced transcription through centromeres, which disrupt centromere structure. We show that Fun30 binds to centromeres and that loss of Fun30 leads to various defects in centromere chromatin, suggesting a direct role for Fun30 in promoting normal centromere function. Our analysis shows that Fun30 affects nucleosome positioning at many genomic sites, including centromeres, and is required for normal occupancy of histone variant Htz1. In the absence of Fun30, we detect an increase in transcription through centromeres. We suggest that an important function of Fun30 is to limit transcription over centromeres.