Mitotic bookmarking by SWI/SNF subunits.

For cells to initiate and sustain a differentiated state, it is necessary that a 'memory' of this state is transmitted through mitosis to the daughter cells1-3. Mammalian switch/sucrose non-fermentable (SWI/SNF) complexes (also known as Brg1/Brg-associated factors, or BAF) control cell identity by modulating chromatin architecture to regulate gene expression4-7, but whether they participate in cell fate memory is unclear. Here we provide evidence that subunits of SWI/SNF act as mitotic bookmarks to safeguard cell identity during cell division. The SWI/SNF core subunits SMARCE1 and SMARCB1 are displaced from enhancers but are bound to promoters during mitosis, and we show that this binding is required for appropriate reactivation of bound genes after mitotic exit. Ablation of SMARCE1 during a single mitosis in mouse embryonic stem cells is sufficient to disrupt gene expression, impair the occupancy of several established bookmarks at a subset of their targets and cause aberrant neural differentiation. Thus, SWI/SNF subunit SMARCE1 has a mitotic bookmarking role and is essential for heritable epigenetic fidelity during transcriptional reprogramming.

SETDB1-like MET-2 promotes transcriptional silencing and development independently of its H3K9me-associated catalytic activity.

Transcriptionally silenced heterochromatin bearing methylation of histone H3 on lysine 9 (H3K9me) is critical for maintaining organismal viability and tissue integrity. Here we show that in addition to ensuring H3K9me, MET-2, the Caenorhabditis elegans homolog of the SETDB1 histone methyltransferase, has a noncatalytic function that contributes to gene repression. Subnuclear foci of MET-2 coincide with H3K9me deposition, yet these foci also form when MET-2 is catalytically deficient and H3K9me is compromised. Whereas met-2 deletion triggers a loss of silencing and increased histone acetylation, foci of catalytically deficient MET-2 maintain silencing of a subset of genes, blocking acetylation on H3K9 and H3K27. In normal development, this noncatalytic MET-2 activity helps to maintain fertility. Under heat stress MET-2 foci disperse, coinciding with increased acetylation and transcriptional derepression. Our study suggests that the noncatalytic, focus-forming function of this SETDB1-like protein and its intrinsically disordered cofactor LIN-61 is physiologically relevant.

Rad9-mediated checkpoint activation is responsible for elevated expansions of GAA repeats in CST-deficient yeast.

Large-scale expansion of (GAA)n repeats in the first intron of the FXN gene is responsible for the severe neurodegenerative disease, Friedreich's ataxia in humans. We have previously conducted an unbiased genetic screen for GAA repeat instability in a yeast experimental system. The majority of genes that came from this screen encoded the components of DNA replication machinery, strongly implying that replication irregularities are at the heart of GAA repeat expansions. This screen, however, also produced two unexpected hits: members of the CST complex, CDC13 and TEN1 genes, which are required for telomere maintenance. To understand how the CST complex could affect intra-chromosomal GAA repeats, we studied the well-characterized temperature-sensitive cdc13-1 mutation and its effects on GAA repeat instability in yeast. We found that in-line with the screen results, this mutation leads to ∼10-fold increase in the rate of large-scale expansions of the (GAA)100 repeat at semi-permissive temperature. Unexpectedly, the hyper-expansion phenotype of the cdc13-1 mutant largely depends on activation of the G2/M checkpoint, as deletions of individual genes RAD9, MEC1, RAD53, and EXO1 belonging to this pathway rescued the increased GAA expansions. Furthermore, the hyper-expansion phenotype of the cdc13-1 mutant depended on the subunit of DNA polymerase δ, Pol32. We hypothesize, therefore, that increased repeat expansions in the cdc13-1 mutant happen during post-replicative repair of nicks or small gaps within repetitive tracts during the G2 phase of the cell cycle upon activation of the G2/M checkpoint.

Primary high-grade myoepithelial carcinoma of the lung: A study of three cases illustrating frequent SMARCB1-deficiency and review of the literature.

Primary myoepithelial carcinoma of the lung is exceptionally rare and, hence, remained poorly characterized. We present 3 tumors affecting 2 males and 1 female aged 60 to 84 years. Tumor size ranged from 4 to 10 cm. All presented as well circumscribed non-encapsulated peripheral solitary masses. One patient died postoperatively. The other two were lost to follow-up. Histologically, all tumors were high-grade with predominance of myxoid/chordoid (2) and rhabdoid (1) pattern. Immunohistochemistry (IHC) showed reactivity with vimentin, pankeratin, EMA and smooth muscle actin. Two tumors were SMARCB1-deficient (one with additional loss of SMARCA2 and PBRM1). RNA sequencing revealed no gene fusions. Review of reported cases (total: 16) showed that pulmonary myoepithelial carcinoma affects both sexes equally at a median age of 60 years (24-84), presents predominantly as peripheral masses (69%) in the lower lobes (66%) of smokers (70%) with a median size of 6 cm (1.5-13), and originates as high-grade de novo carcinoma. Forty percent of patients died of disease at a median of 12.5 months (0 to 62). Only 40% of patients were disease free at last follow-up (median, 9.5 months). Prominent lobulation and myxoid stroma were frequent histological features. Most tumors displayed variable combinations of epithelioid, spindle, plasmacytoid, clear, ovoid or round cells. Three of 6 tumors subjected to different RNA panels showed EWSR1 rearrangements (fused to PBX1, ZNF444 or to unknown partner). Two of 3 tumors lacking gene fusions were SMARCB1-deficient (both showed secondary EWSR1 FISH abnormalities due to 22q deletion). Primary pulmonary myoepithelial carcinoma is a rare aggressive malignancy that recapitulates its soft tissue and salivary counterpart. Exclusion of metastasis from other primaries is mandatory and can only be achieved by detailed clinical history and imaging.

SWI/SNF-deficient undifferentiated/rhabdoid carcinoma of the gallbladder carrying a POLE mutation in a 30-year-old woman: a case report.

BACKGROUND: Undifferentiated carcinoma of the biliary tract are highly aggressive malignancies. In other organs, a subgroup of undifferentiated carcinoma related to SWI/SNF complex-deficiency have been described. CASE PRESENTATION: A 30-year-old woman presented with rising inflammatory markers (C-reactive protein (CRP)). Ultrasound examination revealed a large tumor of the liver. A computed tomography scan was performed and was primarily interpreted as a tumor-forming liver abscess, possibly caused by gallbladder perforation. Subsequent liver segment resection was performed. Microscopic examination showed an undifferentiated carcinoma with rhabdoid morphology and prominent inflammatory infiltrate in the gallbladder base. With SWI/SNF immunohistochemistry, intact expression of SMARCB1, SMARCA4, ARID1A, but loss of SMARCA2 and PBRM1 was detected. Next-generation-sequencing detected KRAS, PBRM1 and ARID1B mutations, a deleterious splice-site mutation in the POLE-gene and a mutation in the TP53-gene. CONCLUSIONS: We were able to demonstrate loss of SMARCA2 expression and mutations characteristic of an SWI/SNF-deficient carcinoma in a tumor derived from the gallbladder. This is the first reported case of an undifferentiated carcinoma with rhabdoid features in the gallbladder carrying a POLE mutation and SWI/SNF-deficiency of PBRM1 and SMARCA2.

CENP-C functions in centromere assembly, the maintenance of CENP-A asymmetry and epigenetic age in Drosophila germline stem cells.

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)-the epigenetic determinant of centromere identity-is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of 'mitotic drive' that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.

KCTD19 and its associated protein ZFP541 are independently essential for meiosis in male mice.

Meiosis is a cell division process with complex chromosome events where various molecules must work in tandem. To find meiosis-related genes, we screened evolutionarily conserved and reproductive tract-enriched genes using the CRISPR/Cas9 system and identified potassium channel tetramerization domain containing 19 (Kctd19) as an essential factor for meiosis. In prophase I, Kctd19 deficiency did not affect synapsis or the DNA damage response, and chiasma structures were also observed in metaphase I spermatocytes of Kctd19 KO mice. However, spermatocytes underwent apoptotic elimination during the metaphase-anaphase transition. We were able to rescue the Kctd19 KO phenotype with an epitope-tagged Kctd19 transgene. By immunoprecipitation-mass spectrometry, we confirmed the association of KCTD19 with zinc finger protein 541 (ZFP541) and histone deacetylase 1 (HDAC1). Phenotyping of Zfp541 KO spermatocytes demonstrated XY chromosome asynapsis and recurrent DNA damage in the late pachytene stage, leading to apoptosis. In summary, our study reveals that KCTD19 associates with ZFP541 and HDAC1, and that both KCTD19 and ZFP541 are essential for meiosis in male mice.

DOT1L inhibitors block abnormal self-renewal induced by cohesin loss.

Acute myeloid leukemia (AML) is a high-risk malignancy characterized by a diverse spectrum of somatic genetic alterations. The mechanisms by which these mutations contribute to leukemia development and how this informs the use of targeted therapies is critical to improving outcomes for patients. Importantly, how to target loss-of-function mutations has been a critical challenge in precision medicine. Heterozygous inactivating mutations in cohesin complex genes contribute to AML in adults by increasing the self-renewal capacity of hematopoietic stem and progenitor cells (HSPCs) by altering PRC2 targeting to induce HOXA9 expression, a key self-renewal transcription factor. Here we sought to delineate the epigenetic mechanism underpinning the enhanced self-renewal conferred by cohesin-haploinsufficiency. First, given the substantial difference in the mutational spectrum between pediatric and adult AML patients, we first sought to identify if HOXA9 was also elevated in children. Next, using primary HSPCs as a model we demonstrate that abnormal self-renewal due to cohesin loss is blocked by DOT1L inhibition. In cohesin-depleted cells, DOT1L inhibition is associated with H3K79me2 depletion and a concomitant increase in H3K27me3. Importantly, we find that there are cohesin-dependent gene expression changes that promote a leukemic profile, including HoxA overexpression, that are preferentially reversed by DOT1L inhibition. Our data further characterize how cohesin mutations contribute to AML development, identifying DOT1L as a potential therapeutic target for adult and pediatric AML patients harboring cohesin mutations.

Smc3 dosage regulates B cell transit through germinal centers and restricts their malignant transformation.

During the germinal center (GC) reaction, B cells undergo extensive redistribution of cohesin complex and three-dimensional reorganization of their genomes. Yet, the significance of cohesin and architectural programming in the humoral immune response is unknown. Herein we report that homozygous deletion of Smc3, encoding the cohesin ATPase subunit, abrogated GC formation, while, in marked contrast, Smc3 haploinsufficiency resulted in GC hyperplasia, skewing of GC polarity and impaired plasma cell (PC) differentiation. Genome-wide chromosomal conformation and transcriptional profiling revealed defects in GC B cell terminal differentiation programs controlled by the lymphoma epigenetic tumor suppressors Tet2 and Kmt2d and failure of Smc3-haploinsufficient GC B cells to switch from B cell- to PC-defining transcription factors. Smc3 haploinsufficiency preferentially impaired the connectivity of enhancer elements controlling various lymphoma tumor suppressor genes, and, accordingly, Smc3 haploinsufficiency accelerated lymphomagenesis in mice with constitutive Bcl6 expression. Collectively, our data indicate a dose-dependent function for cohesin in humoral immunity to facilitate the B cell to PC phenotypic switch while restricting malignant transformation.

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

OBJECTIVE: Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. CONCLUSIONS: Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.

BAF60a deficiency uncouples chromatin accessibility and cold sensitivity from white fat browning.

Brown and beige fat share a remarkably similar transcriptional program that supports fuel oxidation and thermogenesis. The chromatin-remodeling machinery that governs genome accessibility and renders adipocytes poised for thermogenic activation remains elusive. Here we show that BAF60a, a subunit of the SWI/SNF chromatin-remodeling complexes, serves an indispensable role in cold-induced thermogenesis in brown fat. BAF60a maintains chromatin accessibility at PPARγ and EBF2 binding sites for key thermogenic genes. Surprisingly, fat-specific BAF60a inactivation triggers more pronounced cold-induced browning of inguinal white adipose tissue that is linked to induction of MC2R, a receptor for the pituitary hormone ACTH. Elevated MC2R expression sensitizes adipocytes and BAF60a-deficient adipose tissue to thermogenic activation in response to ACTH stimulation. These observations reveal an unexpected dichotomous role of BAF60a-mediated chromatin remodeling in transcriptional control of brown and beige gene programs and illustrate a pituitary-adipose signaling axis in the control of thermogenesis.

Active thermography for real time monitoring of UV-B plant interactions.

Although Ultraviolet-B (UV-B)-plant interactions have been extensively analysed in the past years, many physiological aspects of the complex plant response mechanisms still need to be elucidated. Depending on the energy dose, this part of the electromagnetic spectrum can induce detrimental or beneficial effects in plant and fruit. In the present work, active thermography is used to analyse in real time the response of plants under different doses of artificial UV-B. In particular, we investigated the temporal variations of the leaf surface temperature (LST) to UV-B exposure by Long Pulse and Lock-in thermography in Epipremnum aureum and in Arabidopsis plants overexpressing or knockout mutants of UVR8, the known UV-B photoreceptor. In both cases, UV-B irradiation triggers a cooling effect, namely a thermal response characterised by a LST lower respect to the initial value. Lock-in thermography demonstrated that the cooling effect is associated with an immediate mobilization and accumulation of water in the leaves. Also, we demonstrated that thermographic responses change according to the different capability of plants to tolerate high UV-B radiation. Our study highlights new physiological and physical aspects of the plants response to UV-B radiation and, more in general, it opens new opportunities for the use of the thermography as smart tool for real-time monitoring of plant environmental interactions.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion.

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.

HP1s modulate the S-Adenosyl Methionine synthesis pathway in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer in adults. Among the altered pathways leading to HCC, an increasing role is attributed to abnormal epigenetic regulation. Members of the Heterochromatin Protein (HP1) 1 family are key players in chromatin organisation, acting as docking sites for chromatin modifiers. Here, we inactivated HP1α in HepG2 human liver carcinoma cells and showed that HP1α participated in cell proliferation. HP1α-depleted cells have a global decrease in DNA methylation and consequently a perturbed chromatin organisation, as exemplified by the reactivation of transcription at centromeric and pericentromeric regions, eventhough the protein levels of chromatin writers depositing methylation marks, such as EZH2, SETDB1, SUV39H1, G9A and DNMT3A remained unaltered. This decrease was attributed mainly to a low S-Adenosyl Methionine (SAM) level, a cofactor involved in methylation processes. Furthermore, we showed that this decrease was due to a modification in the Methionine adenosyl transferase 2A RNA (MAT2A) level, which modifies the ratio of MAT1A/MAT2A, two enzymes that generate SAM. Importantly, HP1α reintroduction into HP1α-depleted cells restored the MAT2A protein to its initial level. Finally, we demonstrated that this transcriptional deregulation of MAT2A in HP1α-depleted cells relied on a lack of recruitment of HP1ß and HP1γ to MAT2A promoter where an improper non-CpG methylation site was promoted in the vicinity of the transcription start site where HP1ß and HP1γ bound. Altogether, these results highlight an unanticipated link between HP1 and the SAM synthesis pathway, and emphasise emerging functions of HP1s as sensors of some aspects of liver cell metabolism.

PBAF lacking PHD domains maintains transcription in human neutrophils.

The myeloid precursor cell differentiation requires an extensive chromatin remodeling. We show that the level of the PBAF chromatin remodeling complex decreases following the start of differentiation of myeloid precursors, becoming very low in the terminally differentiated peripheral blood (PB) neutrophils where it co-localizes with Pol II on the transcriptionally active chromatin. Previously, we have shown that the PHF10 subunit of the PBAF signature module has four isoforms, two of them (PHF10-P) contain a tandem of C-terminal PHD domains. We found that out of four PHF10 isoforms present in the myeloid precursor cells, only the PHF10-Ss isoform lacking PHD domains, is actively expressed in the PB neutrophils. In particular, the longest of the PHF10 isoforms (PHF10-Pl), which is essential for proliferation, completely disappears in PB neutrophils. In addition, in the myeloid precursors, promoters of neutrophil-specific genes are associated with the PHD-containing isoforms, together with PBAF and Pol II, when these genes are inactive and only during their activation stage. However, at the later stages of differentiation, when neutrophil-specific genes are actively transcribed, PHF10-P isoforms on their promoters are replaced by the PHF10-S isoforms. Evidently, PHD domains of PHF10 are essential for active chromatin remodeling during transcription activation, but are dispensable for the constantly transcribed genes.

C3aR signaling and gliosis in response to neurodevelopmental damage in the cerebellum.

BACKGROUND: Conditional ablation of the Smarca5 gene in mice severely impairs the postnatal growth of the cerebellum and causes an ataxic phenotype. Comparative gene expression studies indicated that complement-related proteins were upregulated in the cerebellum of Smarca5 mutant mice. Complement proteins play critical roles within innate immune signaling pathways and, in the brain, are produced by glial cells under both normal and pathological conditions. The C3 complement protein-derived signaling peptide, C3a, has been implicated in contributing to both tissue damage and repair in conditions such as multiple sclerosis and stroke. Here, we investigated whether C3a receptor (C3aR) signaling promoted damage or repair in the developing cerebellum of Smarca5 mutant mice. METHODS: Brain and cerebellum lysates from single Smarca5 conditional knockout (Smarca5 cKO) mice, C3aR1 KO mice, or double mutant mice were used for qRT-PCR and immunoblotting to assess the contribution of C3aR to the Smarca5 cKO brain pathology. Immunohistochemistry was used to characterize alterations to astroglia and phagocyte cells in the developing cerebellum of each of the genotypes. RESULTS: C3aR signaling was observed to limit gliosis and promote granule neuron survival during postnatal cerebellar development. In Smarca5 cKO mice, disorganized astroglia with increased GFAP expression develops concurrently with cerebellar granule neuron loss and phagocyte invasion over the first 10 days following birth. Potential ligand precursors of C3aR-VGF and C3-were found to have upregulated expression and/or altered processing during this time. Phagocytes (microglia and macrophages) in both the control and Smarca5 mutant mice were the only cells observed to express C3aR. Loss of C3aR in the Smarca5 cKO cerebellum resulted in increased numbers of apoptotic cells and early phagocyte invasion into the external granule cell layer, as well as an exacerbated disorganization of the Bergmann glia. The loss of C3aR expression also attenuated an increase in the expression of the efferocytosis-related protein, MerTK, whose transcript was upregulated ~ 2.5-fold in the Smarca5 mutant cerebellum at P10. CONCLUSIONS: This data indicates that C3aR can play an important role in limiting astrogliosis and regulating phagocyte phenotypes following developmental cell loss in the brain.

Sirtuin 7 promotes 45S pre-rRNA cleavage at site 2 and determines the processing pathway.

In humans, ribosome biogenesis mainly occurs in nucleoli following two alternative pre-rRNA processing pathways differing in the order in which cleavages take place but not by the sites of cleavage. To uncover the role of the nucleolar NAD+-dependent deacetylase sirtuin 7 in the synthesis of ribosomal subunits, pre-rRNA processing was analyzed after sirtinol-mediated inhibition of sirtuin 7 activity or depletion of sirtuin 7 protein. We thus reveal that sirtuin 7 activity is a critical regulator of processing of 45S, 32S and 30S pre-rRNAs. Sirtuin 7 protein is primarily essential to 45S pre-rRNA cleavage at site 2, which is the first step of processing pathway 2. Furthermore, we demonstrate that sirtuin 7 physically interacts with Nop56 and the GAR domain of fibrillarin, and propose that this could interfere with fibrillarin-dependent cleavage. Sirtuin 7 depletion results in the accumulation of 5' extended forms of 32S pre-rRNA, and also influences the localization of fibrillarin. Thus, we establish a close relationship between sirtuin 7 and fibrillarin, which might determine the processing pathway used for ribosome biogenesis.

Active chromatin marks drive spatial sequestration of heterochromatin in C. elegans nuclei.

The execution of developmental programs of gene expression requires an accurate partitioning of the genome into subnuclear compartments, with active euchromatin enriched centrally and silent heterochromatin at the nuclear periphery1. The existence of degenerative diseases linked to lamin A mutations suggests that perinuclear binding of chromatin contributes to cell-type integrity2,3. The methylation of lysine 9 of histone H3 (H3K9me) characterizes heterochromatin and mediates both transcriptional repression and chromatin anchoring at the inner nuclear membrane4. In Caenorhabditis elegans embryos, chromodomain protein CEC-4 bound to the inner nuclear membrane tethers heterochromatin through H3K9me3,5, whereas in differentiated tissues, a second heterochromatin-sequestering pathway is induced. Here we use an RNA interference screen in the cec-4 background and identify MRG-1 as a broadly expressed factor that is necessary for this second chromatin anchor in intestinal cells. However, MRG-1 is exclusively bound to euchromatin, suggesting that it acts indirectly. Heterochromatin detachment in double mrg-1; cec-4 mutants is rescued by depleting the histone acetyltransferase CBP-1/p300 or the transcription factor ATF-8, a member of the bZIP family (which is known to recruit CBP/p300). Overexpression of CBP-1 in cec-4 mutants is sufficient to delocalize heterochromatin in an ATF-8-dependent manner. CBP-1 and H3K27ac levels increase in heterochromatin upon mrg-1 knockdown, coincident with delocalization. This suggests that the spatial organization of chromatin in C. elegans is regulated both by the direct perinuclear attachment of silent chromatin, and by an active retention of CBP-1/p300 in euchromatin. The two pathways contribute differentially in embryos and larval tissues, with CBP-1 sequestration by MRG-1 having a major role in differentiated cells.

Mammalian ISWI and SWI/SNF selectively mediate binding of distinct transcription factors.

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair1,2. Although they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.