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The intestinal epithelium undergoes constant regeneration driven by intestinal stem cells. How old age affects the transcriptome in this highly dynamic tissue is an important, but poorly explored question. Using transcriptomics on sorted intestinal stem cells and adult enterocytes, we identified candidate genes, which change expression on aging. Further validation of these on intestinal epithelium of multiple middle-aged versus old-aged mice highlighted the consistent up-regulation of the expression of the gene encoding chemokine receptor Ccr2, a mediator of inflammation and several disease processes. We observed also increased expression of Strc, coding for stereocilin, and dramatically decreased expression of Rps4l, coding for a ribosome subunit. Ccr2 and Rps4l are located close to the telomeric regions of chromosome 9 and 6, respectively. As only few genes were differentially expressed and we did not observe significant protein level changes of identified ageing markers, our analysis highlights the overall robustness of murine intestinal epithelium gene expression to old age.

© The Author(s). 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data., Background: How intestinal epithelial cells interact with the microbiota and how this is regulated at the gene expression level are critical questions. Smarcad1 is a conserved chromatin remodeling factor with a poorly understood tissue function. As this factor is highly expressed in the stem and proliferative zones of the intestinal epithelium, we explore its role in this tissue. Results: Specific deletion of Smarcad1 in the mouse intestinal epithelium leads to colitis resistance and substantial changes in gene expression, including a striking increase of expression of several genes linked to innate immunity. Absence of Smarcad1 leads to changes in chromatin accessibility and significant changes in histone H3K9me3 over many sites, including genes that are differentially regulated upon Smarcad1 deletion. We identify candidate members of the gut microbiome that elicit a Smarcad1-dependent colitis response, including members of the poorly understood TM7 phylum. Conclusions: Our study sheds light onto the role of the chromatin remodeling machinery in intestinal epithelial cells in the colitis response and shows how a highly conserved chromatin remodeling factor has a distinct role in anti-microbial defense. This work highlights the importance of the intestinal epithelium in the colitis response and the potential of microbial species as pharmacological and probiotic targets in the context of inflammatory diseases., This work was supported by BBSRC Institute Strategic Programme Grants (BBS/E/B/000C0404, BBS/E/B/000C0405), a MRC project grant to PVW and MV (MR/N009398/1), a BBSRC-Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP-Brazil Pump-priming award (BB/N013565/1,FAPESP#2015/50379-1), and a Newton Advanced Fellowship from the Royal Society to MARV and PVW (NAF\R1\180116).

The recently discovered histone post-translational modification crotonylation connects cellular metabolism to gene regulation. Its regulation and tissue-specific functions are poorly understood. We characterize histone crotonylation in intestinal epithelia and find that histone H3 crotonylation at lysine 18 is a surprisingly abundant modification in the small intestine crypt and colon, and is linked to gene regulation. We show that this modification is highly dynamic and regulated during the cell cycle. We identify class I histone deacetylases, HDAC1, HDAC2, and HDAC3, as major executors of histone decrotonylation. We show that known HDAC inhibitors, including the gut microbiota-derived butyrate, affect histone decrotonylation. Consistent with this, we find that depletion of the gut microbiota leads to a global change in histone crotonylation in the colon. Our results suggest that histone crotonylation connects chromatin to the gut microbiota, at least in part, via short-chain fatty acids and HDACs., Histone post-translational modifications are known key regulators of gene expression. Here, the authors characterize histone crotonylation at histone H3 lysine 18 in intestinal epithelia and find that it is a highly dynamic cell cycle regulated mark under the regulation of the HDAC deacetylases.

Antibiotic-induced dysbiosis is a key factor predisposing intestinal infection by Clostridium difficile. Here, we show that interventions that restore butyrate intestinal levels mitigate clinical and pathological features of C. difficile-induced colitis. Butyrate has no effect on C. difficile colonization or toxin production. However, it attenuates intestinal inflammation and improves intestinal barrier function in infected mice, as shown by reduced intestinal epithelial permeability and bacterial translocation, effects associated with the increased expression of components of intestinal epithelial cell tight junctions. Activation of the transcription factor HIF-1 in intestinal epithelial cells exerts a protective effect in C. difficile-induced colitis, and it is required for butyrate effects. We conclude that butyrate protects intestinal epithelial cells from damage caused by C. difficile toxins via the stabilization of HIF-1, mitigating local inflammatory response and systemic consequences of the infection. Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2012/10653-9, 2013/06810-4, 2014/03002-7, 2015/06134-4, 2017/16280-3, 2018/01753-6]; Funcamp; National Council for Scientific and Technological Development (CNPq); Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) [001]; Newton Advanced Fellowship by the Royal Society; FAPESP [2017/06577-9, 2018/02208-1, 2017/25679-7, 2016/23142-3, 2017/01451-7, 2017/26366-2, 2015/01507-7] This study was supported by Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP; grants 2012/10653-9, 2013/06810-4, 2014/03002-7, 2015/06134-4, 2017/16280-3, and 2018/01753-6), Funcamp, National Council for Scientific and Technological Development (CNPq), Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001, a Newton Advanced Fellowship by the Royal Society (between M.A.R.V. and P.V.-W.). J.L.F., L.P.P., B.K.S., R.O.C., Y.T.M., and T.C. are recipients of fellowships from FAPESP (2017/06577-9, 2018/02208-1, 2017/25679-7, 2016/23142-3, 2017/01451-7, and 2017/26366-2). A.M.T. was supported by a fellowship from FAPESP (2015/01507-7). We thank Juri Kazakevych for help with the RNA sequencing analyses and Maria Teresa Pedrosa Silva Clerici for the formulation of the diets.

Background The microbiota in the human gut are an important component of normal physiology that has co-evolved from the earliest multicellular organisms. Therefore, it is unsurprising that there is intimate crosstalk between the microbial world in the gut and the host. Genome regulation through microbiota-host interactions not only affects the host's immunity, but also metabolic health and resilience against cancer. Chromatin dynamics of the host epithelium involving histone modifications and other facets of the epigenetic machinery play an important role in this process. Scope of review This review discusses recent findings relevant to how chromatin dynamics shape the crosstalk between the microbiota and its host, with a special focus on the role of histone modifications. Major conclusions Host-microbiome interactions are important evolutionary drivers and are thus expected to be hardwired into and mould the epigenetic machinery in multicellular organisms. Microbial-derived short-chain fatty acids (SCFA) are dominant determinants of microbiome–host interactions, and the inhibition of histone deacetylases (HDACs) by SCFA is a key mechanism in this process. The discovery of alternative histone acylations, such as crotonylation, in addition to the canonical histone acetylation reveals a new layer of complexity in this crosstalk., Highlights • Chromatin dynamics of the host epithelium play an important role in host-microbiota crosstalk. • Microbiota-derived short chain fatty acids (SCFA) are a dominant determinant in microbiome–host interaction. • Alternative histone acylations reveal a new layer of complexity in host-microbiota crosstalk.

Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1low cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.

Background Aging is characterized by loss of function of the adaptive immune system, but the underlying causes are poorly understood. To assess the molecular effects of aging on B cell development, we profiled gene expression and chromatin features genome-wide, including histone modifications and chromosome conformation, in bone marrow pro-B and pre-B cells from young and aged mice. Results Our analysis reveals that the expression levels of most genes are generally preserved in B cell precursors isolated from aged compared with young mice. Nonetheless, age-specific expression changes are observed at numerous genes, including microRNA encoding genes. Importantly, these changes are underpinned by multi-layered alterations in chromatin structure, including chromatin accessibility, histone modifications, long-range promoter interactions, and nuclear compartmentalization. Previous work has shown that differentiation is linked to changes in promoter-regulatory element interactions. We find that aging in B cell precursors is accompanied by rewiring of such interactions. We identify transcriptional downregulation of components of the insulin-like growth factor signaling pathway, in particular downregulation of Irs1 and upregulation of Let-7 microRNA expression, as a signature of the aged phenotype. These changes in expression are associated with specific alterations in H3K27me3 occupancy, suggesting that Polycomb-mediated repression plays a role in precursor B cell aging. Conclusions Changes in chromatin and 3D genome organization play an important role in shaping the altered gene expression profile of aged precursor B cells. Components of the insulin-like growth factor signaling pathways are key targets of epigenetic regulation in aging in bone marrow B cell precursors. Electronic supplementary material The online version of this article (10.1186/s13059-018-1489-y) contains supplementary material, which is available to authorized users.

Class I histone deacetylases (HDACs) are efficient histone decrotonylases, broadening the enzymatic spectrum of these important (epi-)genome regulators and drug targets. Here, we describe an in vitro approach to assaying class I HDACs with different acyl-histone substrates, including crotonylated histones and expand this to examine the effect of inhibitors and estimate kinetic constants.

The emergence of migratory cell populations within tumours represents a critical early stage during cancer metastasis. We have previously reported one such population, marked by CD66, in cervical cancers. It is unclear what broad mechanisms regulate such migratory populations. Here, we describe the role of a Suv39H1-low heterochromatin state as a driver of cervical cancer migratory populations. Cervical cancer cells sorted based on migratory ability in vitro show low Suv39H1, and Suv39H1 knockdown enhances cell migration. Histopathology shows the emergence of migratory Suv39H1low populations in advanced carcinoma progression. Meta-analysis of data from The Cancer Genome Atlas (TCGA) reveals that Suv39H1-low tumours show migration and CD66 expression signatures, and correlate with lower patient survival. Lastly, genome-wide profiling of migrated populations using RNA-Seq and H3K9me3 ChIP-Seq reveals Suv39H1-linked transcriptome alterations and a broad loss of H3K9me3, suggesting an increase in chromatin plasticity in migrated populations. The understanding of such chromatin based regulation in migratory populations may prove valuable in efforts to develop anti-metastatic strategies.

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.

Introduction The emergence of migratory cell populations within solid tumours is a critical early stage during cancer metastasis. We have previously reported the enhanced migratory properties of one such population, marked by CD66 expression, in cervical cancers. It is not clear what mechanisms drive such migratory populations, and how these cells would retain a memory of initial migration-inducing cues during metastasis. Here, we sought to assess whether an altered heterochromatin state, controlled by chromatin regulator Suv39H1, drives migratory populations in cervical cancer. Material and methods To avoid an assumption regarding the role of an Epithelial to Mesenchymal Transition (EMT) in migration, cervical cancer cell line SiHa was sorted based on migratory ability in vitro . Following this, Suv39H1 function was assessed in vitro using RNAi. These approaches were complemented by a clinicopathological approach, using histopathology of clinical biopsies to examine cancer progression, along with a meta-analysis of RNA-Seq and clinical data obtained from The Cancer Genome Atlas (TCGA). Lastly, to infer putative mechanisms involved, RNA-Seq, confocal imaging and H3K9me3 ChIP-Seq were performed on sorted migrated cell populations. Results and discussions We observe that migrated populations show low Suv39H1, and that Suv39H1 knockdown enhances cell migration. Using histopathology, we observed the proliferative expansion of a Suv39H1 high pool of cells in early stages of clinical progression, followed by the emergence of a sarcomatoid, migratory Suv39H1 low cell population in advanced stages. Additionally, a meta-analysis of TCGA data revealed that low tumoural Suv39H1 also correlated with lower patient survival, and with gene expression signatures of migration, TGF-β signalling, and links with a CD66 cell signature. Lastly, using genome wide profiling and imaging on migrated populations in vitro , we observed that these populations show Suv39H1-linked migratory expression signatures, decondensed nuclei and a broad loss of H3K9me3, particularly across gene promoter elements. Our findings suggest a role for broad Suv39H1-mediated heterochromatin changes in regulating fate plasticity in migrated populations, contrasting with locus-specific heterochromatin changes previously described in EMT-based models. Conclusion Thus, we report a Suv39H1-low heterochromatin state as a driver of migratory cell populations in cervical cancers. The understanding of such drivers may be valuable in efforts to develop anti-metastatic strategies.

Enzymes that alter nucleosome structure or position are at the very center of gene and genome regulation, and understanding how, and to what extent, these diverse activities collaborate and control each other to shape the genome for dynamic regulation is a major challenge. A new study provides an important step in this direction by illustrating the cooperative nature of ATP-dependent chromatin-remodeling systems in mammalian cells.

The plasticity of chromatin is governed by multi-subunit protein complexes that enzymatically regulate chromosomal structure and activity. Such complexes include ATP-dependent chromatin remodelling factors that are involved in many fundamental processes such as transcription, DNA repair, replication and chromosome structure maintenance. Because ATP-dependent chromatin remodelling factors play important roles, it is not surprising to find that their functions are regulated in a plethora of ways, including post-translational modifications of their subunits and subunit composition changes. The activity of these enzymes is modulated by many factors, including linker histones, histone variants, histone chaperones, non-histone chromatin constituents such as HMG-proteins and secondary messengers, such as inositolpolyphosphates. Additionally, specific histone modifications and interaction with site-specific transcriptional regulators direct the targeting of these activities. Understanding the network of mechanisms that control ATP-dependent chromatin remodelling will constitute an important challenge towards our understanding of chromatin dynamics.

Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here, we show a new role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of myosin I (Myo1), Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low-phosphate medium compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wild-type but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell., Highlighted Article: The endocytic machinery, including the type 1 myosin Myo1, is required to establish a quiescence response to low-phosphate stress in fission yeast.

During DNA replication, chromatin states have to be accurately transmitted from the parental to the daughter strands for faithful epigenetic inheritance. Chromatin remodelling factors at the replication site are thought to be involved in this process. Recent work adds ATP-dependent nucleosome remodelling factors to this category of enzymes. The WICH complex, consisting of the ISWI-type ATPase SNF2H and the Williams Syndrome Transcription Factor (WSTF), binds to replication foci using PCNA, a key factor in DNA- and chromatin replication and DNA repair, as an interaction platform. Depletion of WSTF results in decreased chromatin accessibility, which is evident already in newly replicated DNA. This leads to heterochromatin formation on a global scale and a decrease in overall transcriptional activity. Here, we propose that WICH, by keeping nucleosomes mobile, provides access to the newly replicated DNA and may thereby create a window of opportunity after DNA replication for rebinding of factors that maintain the epigenetic state, and thus prevents aberrant heterochromatin formation. Our model may provide an explanation for the long-standing observation of a delay in chromatin "maturation" on newly replicated DNA, by connecting this delay with the action of PCNA-bound WSTF-ISWI, and highlights chromatin remodeling shortly after DNA replication as a critical point for regulation.

The histone fold is a structural motif with which two related proteins interact and is found in complexes involved in wrapping DNA, the nucleosome, and transcriptional regulation, as in NC2. We reveal a novel function for histone-fold proteins: facilitation of nucleosome remodeling. ACF1-ISWI complex (ATP-dependent chromatin assembly and remodeling factor [ACF]) associates with histone-fold proteins (CHRAC-15 and CHRAC-17 in the human chromatin accessibility complex [CHRAC]) whose functional relevance has been unclear. We show that these histone-fold proteins facilitate ATP-dependent nucleosome sliding by ACF. Direct interaction of the CHRAC-15/17 complex with the ACF1 subunit is essential for this process. CHRAC-17 interacts with another histone-fold protein, p12, in DNA polymerase ϵ, but CHRAC-15 is essential for interaction with ACF and enhancement of nucleosome sliding. Surprisingly, CHRAC-15/17, p12/CHRAC-17, and NC2 complexes facilitate ACF-mediated chromatin assembly by a mechanism different from nucleosome sliding enhancement, suggesting a general activity of H2A/H2B type histone-fold complexes in chromatin assembly.

The Williams Syndrome Transcription Factor (WSTF), the product of the WBSCR9 gene, is invariably deleted in the haploinsufficiency Williams-Beuren Syndrome. Along with the nucleosome-dependent ATPase ISWI, WSTF forms a novel chromatin remodeling complex, WICH (WSTF-ISWI chromatin remodeling complex), which is conserved in vertebrates. The WICH complex was purified to homogeneity from Xenopus egg extract and was found to contain only WSTF and ISWI. In mouse cells, WSTF interacts with the SNF2H isoform of ISWI. WSTF accumulates in pericentric heterochromatin coincident with the replication of these structures, suggesting a role for WSTF in the replication of heterochromatin. Such a role is supported by the in vitro activity of both the mouse and frog WICH complexes: they are involved in the assembly of regular spaced nucleosomal arrays. In contrast to the related ISWI-interacting protein ACF1/WCRF180, WSTF binds stably to mitotic chromosomes. As dysfunction of other chromatin remodeling factors often has severe effects on development, haploinsufficiency of WSTF may explain some of the phenotypes associated with this disease.

This review addresses recent developments in the field of ATP-dependent chromatin remodeling factors. These factors use the energy of ATP hydrolysis to introduce superhelical torsion into DNA, which suggests a common mechanistic basis of action. Chromatin remodeling factors function both in transcriptional activation and repression, but they may have roles outside of transcriptional regulation such as DNA repair. A study of the nucleosome dependent ATPase ISWI in yeast illustrates the involvement of ATP-dependent chromatin remodeling in transcriptional repression by setting up inaccessible chromatin structures at promoters. However, factors such as ISWI are also involved in the restructuring of large chromatin domains and even whole chromosomes. Transcriptional regulation by ATP-dependent chromatin remodeling factors occurs in concert with histone modifying enzymes such as histone acetyltransferases and histone deacetylases: In yeast, SWI/SNF targeting is a requirement for histone acetyltransferases activity at promoters that are active at late stages of mitosis, when the chromatin is still condensed. This demonstrates that ATP-dependent remodeling factors facilitate covalent histone modifications. However, they are also regulated by histone modifications and in some circumstances they function in parallel with histone modifications towards the same goal.

Chromatin has shifted into the focus of attention as a key to understanding the regulation of nuclear processes such as transcription. Protein machines have been described that use the energy of ATP to render chromatin dynamic and hence active, but which may also be involved in chromatin assembly. The discovery of three different Drosophila nucleosome remodeling complexes that contain imitation switch (ISWI), an ATPase with a high degree of sequence conservation from yeast to human, points to a central function of this ATPase in chromatin dynamics.

Chromatin structure must be flexible to allow the binding of regulatory proteins and to accommodate different levels of gene activity. Chromatin assembled in a cell-free system derived from Drosophila embryos contains an activity that hydrolyses ATP to render entire nucleosome arrays mobile. Nucleosome movements, most likely their sliding, occurred even in the presence of the linker histone H1. The dynamic state of chromatin in the presence of the activity and ATP globally increased the accessibility of nucleosomal DNA to incoming proteins. This unprecedented demonstration of energy-dependent nucleosome mobility identifies a new principle which is likely to be fundamental to the mechanism of chromatin remodelling and the binding of regulatory proteins.

We have compared chicken erythrocyte linker histones H1 and H5 binding to a synthetic four-way DNA junction. Each histone binds to form a single complex, with an affinity which permits competition against a large excess of linear duplex DNA. The affinity of H5 is higher than that of H1. The globular domain from either protein will also bind strongly, but in this case multiple binding occurs. Binding of intact H1 is inhibited by cations: Mg2+ and spermidine are very effective, Na+ much less so. This inhibition is not likely to be a general ion-competition effect, for Mg2+ is much less effective in inhibiting the binding of H1 to linear DNA. Instead, the inhibition of binding may be due to ion-dependent changes in the conformation of the four-way junction, which are known to occur under similar conditions. These results strongly suggest that the angle formed between the arms of the DNA junction could be a major determinant in the interaction of H1 with DNA crossovers.

The histone modification repertoire has recently been expanded. Dot1p is a new type of methyltransferase that methylates lysine 79 in the histone H3 core only in its nucleosomal context and has a possible role in marking open chromatin regions.

Disruption of chromatin organization during replication poses a major challenge to the maintenance and integrity of genome organization. It creates the need to accurately reconstruct the chromatin landscape following DNA duplication but there is little mechanistic understanding of how chromatin based modifications are restored on newly synthesized DNA. ATP-dependent chromatin remodeling activities serve multiple roles during replication and recent work underscores their requirement in the maintenance of proper chromatin organization. A new component of chromatin replication, the SWI/SNF-like chromatin remodeler SMARCAD1, acts at replication sites to facilitate deacetylation of newly assembled histones. Deacetylation is a pre-requisite for the restoration of epigenetic signatures in heterochromatin regions following replication. In this way, SMARCAD1, in concert with histone modifying activities and transcriptional repressors, reinforces epigenetic instructions to ensure that silenced loci are correctly perpetuated in each replication cycle. The emerging concept is that remodeling of nucleosomes is an early event imperative to promote the re-establishment of histone modifications following DNA replication.

Chromatin remodeling complexes can translocate nucleosomes along the DNA in an ATP-dependent manner. Here, we studied autofluorescent protein constructs of the human ISWI family members Snf2H, Snf2L, the catalytically inactive Snf2L+13 splice variant, and the accessory Acf1 subunit in living human and mouse cells by fluorescence microscopy/spectroscopy. Except for Snf2L, which was not detected in the U2OS cell line, the endogenous ISWI proteins were abundant at nuclear concentrations between 0.14 and 0.83 μM. A protein interaction analysis showed the association of multimeric Snf2H and Acf1 into a heterotetramer or higher-order ACF complex. During the G1/2 cell cycle phase, Snf2H and Snf2L displayed average residence times

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

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.

Alterations of chromatin structure play an important role in gene regulation. One way of doing so involves ATP-dependent chromatin remodelling enzymes that act as molecular machines coupling ATP-hydrolysis to structural changes of the nucleosome. Several recent studies shed important insights into the mechanism of these factors and indicate that they couple DNA translocation within the nucleosome to DNA loop propagation through the nucleosome. This reaction causes the movement of a nucleosome with respect to a given DNA sequence and also drives its disassembly. It is becoming clear that the biology of these factors is very complex considering the plethora of known ATP-dependent nucleosome remodelling factors and their many, in part overlapping functions and varied ways of regulation and targeting. Finally, nucleosome remodelling may only be one aspect of the function of these enzymes, because they may impart or regulate higher order levels of chromatin organization. The importance of these enzymes for normal growth and development is illustrated by disorders and neoplasias linked to mutations of those factors or their misregulation. Given that these enzymes have such profound roles in gene expression and cell proliferation, they may constitute important drug targets for clinical applications in the future

Dynamic changes of chromatin structure control DNA-dependent events, including DNA replication. Along with DNA, chromatin organization must be replicated to maintain genetic and epigenetic information through cell generations. Chromatin remodelling is important for several steps in replication: determination and activation of origins of replication, replication machinery progression, chromatin assembly and DNA repair. Histone chaperones such as the FACT complex assist DNA replication within chromatin, probably by facilitating both nucleosome disassembly and reassembly. ATP-dependent nucleosome remodelling enzymes of the SWI/SNF family, in particular imitation switch (ISWI)-containing complexes, have been linked to DNA and chromatin replication. They are targeted to replication sites to facilitate DNA replication and subsequent chromatin assembly.

Dynamic changes of chromatin structure control DNA-dependent events, including DNA replication. Along with DNA, chromatin organization must be replicated to maintain genetic and epigenetic information through cell generations. Chromatin remodelling is important for several steps in replication: determination and activation of origins of replication, replication machinery progression, chromatin assembly and DNA repair. Histone chaperones such as the FACT complex assist DNA replication within chromatin, probably by facilitating both nucleosome disassembly and reassembly. ATP-dependent nucleosome remodelling enzymes of the SWI/SNF family, in particular imitation switch (ISWI)-containing complexes, have been linked to DNA and chromatin replication. They are targeted to replication sites to facilitate DNA replication and subsequent chromatin assembly.

Nucleosome-remodelling factors are key facilitators of chromatin dynamics. At the level of single nucleosomes, they are involved in nucleosome-repositioning, altering histone–DNA interactions, disassembly of nucleosomes, and the exchange of histones with variants of different properties. The fundamental nature of chromatin dictates that nucleosome-remodelling affects all aspects of eukaryotic DNA metabolism, but much less is known about the functional interactions of nucleosome-remodelling factors with folded chromatin fibres. Because remodelling machines are abundant constituents of eukaryotic nuclei and, therefore, have ample potential to interact with chromatin, they might also affect higher-order chromatin architecture. Recent observations support roles for nucleosome-remodelling factors at the supra-nucleosomal level.

Chromatin structures have to be precisely duplicated during DNA replication to maintain tissue-specific gene expression patterns and specialized domains, such as the centromeres. Chromatin remodeling factors are key components involved in this process and include histone chaperones, histone modifying enzymes and ATP-dependent chromatin remodeling complexes. Several of these factors interact directly with components of the replication machinery. Histone variants are also important to mark specific chromatin domains. Because chromatin remodeling factors render chromatin dynamic, they may also be involved in facilitating the DNA replication process through condensed chromatin domains.

Chromatin structures have to be precisely duplicated during DNA replication to maintain tissue-specific gene expression patterns and specialized domains, such as the centromeres. Chromatin remodeling factors are key components involved in this process and include histone chaperones, histone modifying enzymes and ATP-dependent chromatin remodeling complexes. Several of these factors interact directly with components of the replication machinery. Histone variants are also important to mark specific chromatin domains. Because chromatin remodeling factors render chromatin dynamic, they may also be involved in facilitating the DNA replication process through condensed chromatin domains.

Chromatin states have to be faithfully duplicated during DNA replication to maintain cell identity. It is unclear whether or how ATP-dependent chromatin-remodelling factors are involved in this process. Here we provide evidence that the Williams syndrome transcription factor (WSTF) is targeted to replication foci through direct interaction with the DNA clamp PCNA, an important coordinator of DNA and chromatin replication. WSTF, in turn, recruits imitation switch (ISWI)-type nucleosome-remodelling factor SNF2H to replication sites. These findings reveal a novel recruitment mechanism for ATP-dependent chromatin-remodelling factors that is fundamentally different from the previously documented targeting by sequence-specific transcriptional regulators. RNA-interference-mediated depletion of WSTF or SNF2H causes a compaction of newly replicated chromatin and increases the amount of heterochromatin markers, including HP1beta. This increase in the amount of HP1beta protein is mediated by progression through S phase and is not the result of an increase in HP1beta mRNA levels. We propose that the WSTF-ISWI complex has a role in the maintenance of chromatin structures during DNA replication.

The mechanism by which the eukaryotic DNA-replication machinery penetrates condensed chromatin structures to replicate the underlying DNA is poorly understood. Here we provide evidence that an ACF1-ISWI chromatin-remodeling complex is required for replication through heterochromatin in mammalian cells. ACF1 (ATP-utilizing chromatin assembly and remodeling factor 1) and an ISWI isoform, SNF2H (sucrose nonfermenting-2 homolog), become specifically enriched in replicating pericentromeric heterochromatin. RNAi-mediated depletion of ACF1 specifically impairs the replication of pericentromeric heterochromatin. Accordingly, depletion of ACF1 causes a delay in cell-cycle progression through the late stages of S phase. In vivo depletion of SNF2H slows the progression of DNA replication throughout S phase, indicating a functional overlap with ACF1. Decondensing the heterochromatin with 5-aza-2-deoxycytidine reverses the effects of ACF1 and SNF2H depletion. Expression of an ACF1 mutant that cannot interact with SNF2H also interferes with replication of condensed chromatin. Our data suggest that an ACF1-SNF2H complex is part of a dedicated mechanism that enables DNA replication through highly condensed regions of chromatin.

Eukaryotic chromosomes are organized inside the nucleus in such a way that only a subset of the genome is expressed in any given cell type, but the details of this organization are largely unknown. SATB1 ('special AT-rich sequence binding 1'), a protein found predominantly in thymocytes, regulates genes by folding chromatin into loop domains, tethering specialized DNA elements to an SATB1 network structure. Ablation of SATB1 by gene targeting results in temporal and spatial mis-expression of numerous genes and arrested T-cell development, suggesting that SATB1 is a cell-type specific global gene regulator. Here we show that SATB1 targets chromatin remodelling to the IL-2Ralpha ('interleukin-2 receptor alpha') gene, which is ectopically transcribed in SATB1 null thymocytes. SATB1 recruits the histone deacetylase contained in the NURD chromatin remodelling complex to a SATB1-bound site in the IL-2Ralpha locus, and mediates the specific deacetylation of histones in a large domain within the locus. SATB1 also targets ACF1 and ISWI, subunits of CHRAC and ACF nucleosome mobilizing complexes, to this specific site and regulates nucleosome positioning over seven kilobases. SATB1 defines a class of transcriptional regulators that function as a 'landing platform' for several chromatin remodelling enzymes and hence regulate large chromatin domains.

The chromatin accessibility complex (CHRAC) was originally defined biochemically as an ATP-dependent ‘nucleosome remodelling’ activity. Central to its activity is the ATPase ISWI, which catalyses the transfer of histone octamers between DNA segments in cis. In addition to ISWI, four other potential subunits were observed consistently in active CHRAC fractions. We have now identified the p175 subunit of CHRAC as Acf1, a protein known to associate with ISWI in the ACF complex. Interaction of Acf1 with ISWI enhances the efficiency of nucleosome sliding by an order of magnitude. Remarkably, it also modulates the nucleosome remodelling activity of ISWI qualitatively by altering the directionality of nucleosome movements and the histone ‘tail’ requirements of the reaction. The Acf1–ISWI heteromer tightly interacts with the two recently identified small histone fold proteins CHRAC-14 and CHRAC-16. Whether topoisomerase II is an integral subunit has been controversial. Refined analyses now suggest that topoisomerase II should not be considered a stable subunit of CHRAC. Accordingly, CHRAC can be molecularly defined as a complex consisting of ISWI, Acf1, CHRAC-14 and CHRAC-16.

The ISWI ATPase of Drosophila is a molecular engine that can drive a range of nucleosome remodelling reactions in vitro. ISWI is important for cell viability, developmental gene expression and chromosome structure. It interacts with other proteins to form several distinct nucleosome remodelling machines. The chromatin accessibility complex (CHRAC) is a biochemical entity containing ISWI in association with several other proteins. Here we report on the identification of the two smallest CHRAC subunits, CHRAC-14 and CHRAC-16. They contain histone fold domains most closely related to those found in sequence-specific transcription factors NF-YB and NF-YC, respectively. CHRAC-14 and CHRAC-16 interact directly with each other as well as with ISWI, and are associated with functionally active CHRAC. The developmental expression profiles of both subunits suggest specialized roles in chromatin remodelling reactions in the early embryo for both histone fold subunits.